Drug-regulatable, inducible cytokine expression

Abstract

Disclosed are nucleic acids comprising a nucleotide sequence encoding a polypeptide comprising a cytokine, or a functional portion of the cytokine; a transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence; and an immunomodulatory imide drug (IMiD)-degradable degron, wherein the nucleotide sequence encoding the polypeptide comprises a promoter operatively associated with the nucleotide sequence encoding polypeptide. Also disclosed are related recombinant expression vectors, viral particles, host cells, populations of cells, pharmaceutical compositions, methods of treating or preventing cancer, and methods of enhancing the immune response to a cancer.

Description

DRUG-REGULATABLE, INDUCIBLE CYTOKINE EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 652,871, filed May 29, 2024.STATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number ZIA- BC-010984 by the National Institutes of Health. National Cancer Institute. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 32,955 Byte Extensible Markup Language (XML) file named “773102_ST26.xml,’?dated May 28. 2025.BACKGROUND OF THE INVENTION

[0004] Adoptive cell therapy can be an effective treatment for cancer in some patients. However, obstacles to the overall success of adoptive cell therapy still exist. For example, the anti-tumor activity of the transferred T cells can, in some cases, decrease following adoptive transfer. Clinical applications of adoptive cell therapy may also be limited by toxicity induced by high levels of circulating cytokines. Despite considerable research in the field of adoptive cell therapy, there still exists a need for improved methods and products for producing cells for adoptive cell therapy and treating and / or preventing cancer.BRIEF SUMMARY OF THE INVENTION

[0005] An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising: a cytokine, a functional portion of the cytokine, a T-cell regulatory protein, or a functional portion of the T-cell regulatory’ protein; atransmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence; and an immunomodulatory imide drug (IMiD)-degradable degron, wherein the nucleotide sequence encoding the polypeptide comprises a promoter operatively associated with the nucleotide sequence encoding polypeptide.

[0006] Further aspects of the invention provide related nanoparticles, recombinant expression vectors, viral particles, polypeptides, host cells, populations of cells, pharmaceutical compositions, methods of treating or preventing cancer, and methods of enhancing an immune response against a cancer.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figure 1A is a schematic illustrating the binding of lenalidomide (LEN) to cereblon (CRBN), a E3 ubiquitin (Ub) ligase, which leads to poly ubiquitination and degradation of single-chain IL- 12 containing the degron sequence. P40 and P35 are the subunits of the single-chain IL- 12.

[0008] Figure IB is a schematic illustrating green fluorescent protein (GFP) reporter constructs for testing degron activity. The constructs contained the P40 subunit of IL- 12, GFP. and a degron sequence, with (P40-GFP-TM-D) or without (P40-GFP-D) a transmembrane (TM) domain.

[0009] Figure 1C is a graph showing the percentage of GFP positive cells measured following culture of Jurkat cells (transduced with P40-GFP-TM-D or P40-GFP-D) in the presence or absence of LEN.

[0010] Figure ID is a graph showing the percentage of IL-12 secreting cells detected following treatment of transduced cells with the indicated doses (nM) of LEN. The cells had been transduced with single-chain IL- 12 in a secreted form without GFP or a TM domain and were intracellularly stained.

[0011] Figure IE is a graph showing the amount of IL-12 (pg / ml) secreted by T cells cultured alone or upon stimulation with TYK-nu, with or without LEN treatment. The T cells were transduced with a vector encoding secreted IL- 12 or IL- 12 with a IMiD-degradable degron and a TM domain (abbreviated as IL-12TM-D).

[0012] Figure IF is a schematic illustrating that co-culture of T cells co-transduced with (i) a TCR targeting the p53R175Hmutation (hereafter referred to as the p53 TCR) and (ii) IL- 12TM-D with target human ovarian cancer cells, TYK-nu, leads to activation of the p53 TCRand subsequent induction of transcriptional activity of NF AT to ultimately express IL-12TM- D.

[0013] Figure 1G is a graph showing the percentage of IL- 12 positive cells measured following co-culture of target cells with effector cells in the presence or absence of LEN. Target cells were HLA-A2+ T2 cells pulsed with the mutant p53R175Hpeptide (R175H minimal epitope (ME)) or DMSO control. Effector cells were Jurkat cells transduced with the p53 TCR and membranous IL-12 (i.e., IL-12TM) with or without the degron. Jurkat cells cultured alone or with T2 cells treated with DMSO served as a control. Jurkat cells transduced with the p53 TCR alone also served as a control.

[0014] Figure 1H is a graph showing the percentage of IL-12 positive cells measured following co-culture of target cells with effector cells in the presence (+) or absence (-) of LEN. Target cells were TYK-nu (p53R175H+) or 4259 patient-derived xenograft colon cancer cells (PDX; p53220C+). Effector cells were T cells transduced with a p53R175HTCR and membranous IL-12 (i.e., IL-12TM) with or without the degron. T cells cultured alone served as a control.

[0015] Figure II is a time response curve showing the normalized number of IL-12+ cells measured following LEN (1 pM) treatment of Jurkat cells (transduced with IL-12TM-D in the MSGV vector in reverse orientation) cultured for the indicated durations.

[0016] Figure 1J is a dose response curve showing the percentage of IL-12+ cells measured following treatment of Jurkat cells (transduced with IL-12TM-D in the MSGV vector in reverse orientation) with the indicated concentrations of LEN.

[0017] Figures IK is a graph showing the normalized number of tumor cells measured following co-culture of TYK-nu ovarian cancer cells with p53 TCR-transduced T cells with or without IL-12TM-D or IL-12TM expression (MSGV). Tumor cells cultured alone served as a control.

[0018] Figure 2A shows schematics of the MSGV-NFAT-IL12-TM-degron-reverse (MSGV) and SERS-NFAT-IL12-TM-degron (SERS) constructs.

[0019] Figure 2B is a graph showing normalized mean fluorescence intensity (MFI) of IL- 12 PE as a surrogate for IL- 12 expression measured following transduction of cells with IL-12TM-D in the MSGV vector or the SERS vector.

[0020] Figure 2C is a graph showing the percentage of IL-12 positive cells detected following culture of effector cells in the presence or absence of TYK-nu cells and in thepresence or absence of LEN. Effector cells were healthy donor T cells transduced with the p53 TCR alone or with IL-12TM-D in the SERS vector or the MSGV vector.

[0021] Figures 2D-2E are graphs showing the normalized tumor cell counts measured at the indicated number of hours following co-culture of target cells with effector cells.Effector cells were healthy donor T cells transduced with the p53 TCR and IL-12TM-D in the SERS vector or the MSGV vector (2D) or with a TCR targeting KRASG12D(KRAS TCR) and IL-12TM-D (2E). Target cells were TYK-nu cells (2D) or 4069 PDX cells (2E). T cells transduced with an empty vector (Mock) served as a control. Healthy donor T cells transduced with the TCR only (no IL-12TM-D) also served as a control.

[0022] Figure 2F is a time response curve showing the normalized number of IL- 12+ cells measured following LEN (1 pM) treatment of T cells (transduced with IL-12TM-D in the SERS vector) cultured for the indicated durations.

[0023] Figure 2G is a dose response curve showing the percentage of IL- 12+ cells measured following treatment of T cells (transduced with IL-12TM-D in the SERS vector) with the indicated concentrations of LEN.

[0024] Figure 3A is a graph showing the normalized number of IL-12 positive cells detected following co-culture of target cells with effector cells in the presence of the indicated concentrations (nM) of LEN. Target cells were HLA-A2+ T2 cells pulsed with the mutant p53R175Hpeptide. Effector cells were p53 TCR+ Jurkat cells transduced with wildtype (WT) IL-12TM-D or the indicated mutated IL-12TM-D constructs. p53 TCR+ Jurkat cells transduced with membrane-bound single chain IL-12 with no degron served as a control.

[0025] Figure 3B is a graph showing the normalized number of IL-12 positive cells detected following co-culture of target cells with effector cells in the presence of the indicated concentrations (nM) of LEN. Target cells were TYK-nu cells. Effector cells were human healthy donor T-cells co-transduced with the p53 TCR and IL-12TM-D without mutations (WT) or with the L1F, I20L, or L22R mutation.

[0026] Figure 3C is a graph showing the number of normalized IL-12 positive cells detected following co-culture of target cells with effector cells at the indicated number of hours after LEN treatment. Target cells were TYK-nu cells. Effector cells were human healthy donor T-cells co-transduced with the p53 TCR and IL-12TM-D without mutations (WT) or with the L22R mutation.

[0027] Figure 3D is a graph showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells. Target cellswere TYK-nu cells. Effector cells were human healthy donor T-cells transduced with the p53 TCR only or co-transduced with TL-12TM-D without mutations (WT) or with the L22R mutation. T cells transduced with an empty vector (Mock) served as a control.

[0028] Figures 4A-4B show graphs showing the percentage of 4-1BB positive cells, as a marker for T-cell activation (4A) and percentage of IL- 12 positive cells (4B) measured following co-culture of target cells with effector cells in the absence or presence of the indicated concentration (nM) of LEN. Target cells were an autologous 3795 melanoma cell line (3795 TC) or an irrelevant tumor (TYK-nu). Effector cells were the tumor infiltrating lymphocyte (TIL) infusion product (RX) used in the treatment of Patient 3795 transduced with either an empty vector (EV) or IL-12TM-D. Effector cells cultured alone (T alone) and target cells non-specifically stimulated with anti-CD3 / anti-CD28 antibody-conjugated beads served as controls.

[0029] Figure 4C is a graph showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells at an effector to target ratio of 4: 1 . Target cells were an autologous 3795 melanoma cell line (3795 TC). Effector cells were the TIL infusion product used in the treatment of Patient 3795 transduced with either an empty vector (EV) or IL-12TM-D. Target cells cultured alone (tumor alone) served as a control. Target cells cultured with irrelevant TIL (In RX) served as a control.

[0030] Figure 4D is a graph showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells. Target cells were autologous colorectal cancer PDX cells. Effector cells were T cells transduced with the 4391 TCR (KRASG12V-specific) alone or co-transduced with IL-12TM-D. Target cells cultured alone (tumor alone) served as a control.

[0031] Figure 4E is a graph showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells. Target cells were human pancreatic HP AC cells (KRASG12D+). Effector cells were T cells transduced with the 4095-9A TCR (KRASG12D-specific) alone or co-transduced with IL-12TM-D.Target cells cultured alone (tumor alone) served as a control.

[0032] Figures 4F-4G show graphs showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells. Target cells were CD19-low NALM6 clone Z (MFI 1,114) (4F) or CD 19-intermediate NALM6 clone 11 (MFI 5,675) (4G). Effector cells were T cells transduced with the anti-CD 19 CAR alone or co-transduced with IL-12TM-D. Target cells cultured alone (NALM6 alone) served as a control.

[0033] Figures 4H-4I show graphs showing the normalized number of tumor cells counted at the indicated number of hours following co-culture of target cells with effector cells. Target cells were unmodified NALM6 cells that were wild-type for CD 19 expression (MFI 56,420) (4H) or CD19 knock-out NALM6 cells (MFI 174) (41). Effector cells were T cells transduced with the anti-CD19 CAR alone or co-transduced with IL-12TM-D. Target cells cultured alone (NALM6 alone) serv ed as a control.

[0034] Figure 5A is a schematic illustrating the methods employed in the experiments described for Figures 5B-5D. “D” is "‘Day.” “POM7’ is pomalidomide.

[0035] Figure 5B shows dot plots illustrating the percentage of effector cells expressing 4-1BB and IL-12 following culture alone or co-culture with TYK-nu cells, as measured by flow cytometry, pre-gated for p53 TCR and CD8. Effector cells were T cells co-transduced with the p53 TCR and IL-12TM-D and were tested on the day of mouse injection for functional validation. The measurement was conducted on the day of T-cell injection into tumor-bearing mice.

[0036] Figure 5C shows graphs showing the expression level of IL-12 (of mTCR positive, CD8 positive cells) of T cells from tumor-bearing mice treated with effector cells and either PBS or POM. Effector cells were T cells co-transduced with the p53 TCR and IL- 12TM-D. Exogenously expressed TCRs, including the p53 TCR, were modified to harbor murine TCR constant region sequences for enhanced pairing, and were flow cytometrically detected using a murine TCR beta (mTCR) antibody. The experiment was carried out twice. Results are shown for both experiments. PBS served as a control.

[0037] Figure 5D is a graph showing the correlation between 4-1 BB and IL-12 expression by TILs from tumor-bearing mice treated with effector cells and PBS. Effector cells were T cells co-transduced with the p53 TCR and IL-12TM-D. y = 0.3807x + 1.5666; R2= 0.8925.

[0038] Figure 5E is a schematic illustrating the methods employed in the experiments described for Figures 5F-5I. “D” is “Day.”

[0039] Figures 5F-5G show graphs showing the mean tumor size (mm2) measured at the indicated number of days post-adoptive cell transfer (ACT) of tumor-bearing mice treated with 5e6 effector T cells per mouse (5F) or 15e6 effector T cells per mouse (5G). Effector Tcells were T cells transduced with the p53 TCR alone, or co-transduced with the p53 TCR and IL-12TM-D. T cells that were mock-transduced (Mock) served as a control.

[0040] Figures 5H-5I show graphs showing the probability of survival measured at the indicated number of days post- ACT of tumor-bearing mice treated with 5e6 effector T cells per mouse (5H) or 15e6 effector T cells per mouse (51). Effector T cells were T cells transduced with an empty vector (Mock), the p53 TCR alone, or co-transduced with the p53 TCR and IL-12TM-D.

[0041] Figure 6 is a graph showing the percentage of IL-12 positive cells (of CD8 positive, mTCR positive cells) measured following transduction of T cells with IL-12TM (no degron) or IL-12TM-D. Transduction was performed using SERS as a vector. N=8. Fold difference: 2.39, P<0.001.

[0042] Figure 7 is a graph showing the percentage of IL-12 positive cells (of CD8 positive, mTCR positive cells) measured following transduction of T cells with IL-12TM (no degron) or IL- 12TM-D. Transduction was performed using MSGV as a vector. N=18. Fold difference: 1.57, P=0.016.

[0043] Figure 8A is a schematic of repeat co-culture between T cells expressing the p53 TCR with or without IL-12TM-D expression and TYK-nu cells as target tumor cells. Five hundred thousand T cells were co-cultured with an equal number of irradiated TYK-nu cells with or without 100 nM lenalidomide. At days 0. 7 and 14. T cells were analyzed using flow cytometry and single-cell CITE-seq.

[0044] Figure 8B is a graph showing quantified TIGIT levels measured by flow cytometry' (N=3). The experiment was reproduced using T cells from a different healthy donor. Statistical analysis by two-tailed Student’s t-test. ***p<0.00I.

[0045] Figure 8C is a graph showing the results of an in vitro TYK-nu killing assay. Healthy-donor T cells expressing the p53 TCR with or without IL-12TM-D following 2 week-long co-culture with or without 100 nM lenalidomide in Fig. 8 A were co-cultured another time with TYK-nu cells. Equal numbers of TYK-nu cells and T cells were plated (n=8). Statistical analysis by two-way ANOVA. ***p<0.001.

[0046] Figure 9A is a schematic illustrating the methods employed in the experiments described for Figures 9B-9D.

[0047] Figs. 9B-9D are graphs showing the mean tumor size (mm2) measured at the indicated number of days post-adoptive cell transfer (ACT) of tumor-bearing mice treated with 0.5e6 effector T cells per mouse (9B), 1.5e6 effector T cells per mouse (9C), or 5e6effector T cells per mouse (9D). Effector T cells were T cells transduced with the p53 TCR alone, p53 TCR + IL-12TM, or p53 TCR + IL-12TM-D. is number of tumor-free mice. “NS” means “not significant.”

[0048] Figure 10A is a schematic illustrating the methods employed in the experiments described for Figures 10B-10C.

[0049] Figures 1 OB- IOC are graphs showing the mean tumor size (mm2) (10B) and probability of survival (IOC) measured at the indicated number of days post-adoptive cell transfer (ACT) of tumor-bearing mice treated with vehicle, mock-transduced T cells or T cells transduced with the anti-mutant KRAS TCR alone, anti-mutant KRAS TCR + IL-12TM, or IL-12TM-D alone.DETAILED DESCRIPTION OF THE INVENTION

[0050] Aspects of the invention provide nucleic acids and polypeptides which provide drug-regulatable, inducible, and cell membrane-bound cytokine (e g., interleukin (IL)-12) (or T-cell regulator ' protein) expression. The inventive nucleic acids and polypeptides may provide any one or more of a variety of advantages. For example, the inventive nucleic acids and polypeptides may. advantageously, reduce or avoid the systemic toxicity which has been associated with previous clinical applications of IL-12 (Zhang et al., Clin. Cancer Res., 21 : 2278-2288 (2015)). The inventive nucleic acids and polypeptides may reduce or eliminate systemic secretion of the cytokine (or T-cell regulatory protein), provide local expression or delivery of the cytokine (or T-cell regulatory protein), and / or provide drug-regulatable expression of the cytokine (or T-cell regulatory protein). The inventive nucleic acids advantageously may provide drug and target-antigen dependent regulation of cytokine (or T- cell regulatory' protein) expression. The combination of NFAT-inducible cytokine (or T-cell regulatory protein) expression and a drug-regulatable degron may provide complete or near complete regulation of cytokine (or T-cell regulatory protein) expression with minimal or no “leaky” (uncontrolled) expression. Moreover, cells expressing the inventive nucleic acids or polypeptides may provide enhanced function (e.g., increased anti-tumor efficacy) as compared to, for example, unmodified T cells or cells transduced with an antigen-specific receptor alone.

[0051] Addition of a degron to the NFAT-inducible, membrane-bound cytokine (or T-cell regulatory protein) may also provide a further unexpected but practical advantage: compared to NFAT-inducible membrane-bound cytokine (or T-cell regulatory protein) without thedegron. the inventive nucleic acids may provide more effective transduction of cytokine (or T-cell regulatory protein) into T cells. Accordingly, it may be beneficial to regulate cytokine (or T-cell regulatory protein) expression during transduction and expansion of the number of T cells by drug treatment to minimize the toxicity of "leaky" (uncontrolled) cytokine (or T- cell regulatory protein) expression without the degron. Expression of the inventive nucleic acids may be effectively induced upon T-cell activation, and drug treatment may rapidly and potently turn off cytokine (or T-cell regulatory protein) expression in vitro and in vivo. The inventive nucleic acids and polypeptides may provide effective and safe expression of cytokine (or T-cell regulatory protein) to enhance the efficacy of ACT.

[0052] An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising: a cytokine, a functional portion of the cytokine, a T-cell regulators' protein, or a functional portion of the T-cell regulatory protein; a transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence; and an IMiD -degradable degron, wherein the nucleotide sequence encoding the polypeptide comprises a promoter operatively associated with the nucleotide sequence encoding polypeptide.

[0053] In an aspect of the invention, the nucleic acid comprises a nucleotide sequence of Formula I:5 -P-Rlm-C-R2n-T-Ll-D-3‘(Formula I), wherein:P is the promoter;C is the cytokine, the functional portion of the cytokine, a T-cell regulatory protein, or a functional portion of the T-cell regulatory protein;T is the transmembrane-intracellular amino acid sequence or the transmembrane amino acid sequence; each one of R1and R2is. independently 1 to 3 amino acid residues; each one of m and n is, independently, 0 or 1;L1is a first linker sequence; and D is the IMiD- degradable degron.

[0054] T-cell regulatory proteins are those that stimulate or inhibit one or more of T cell development, activation, and function. In an aspect of the invention, the T-cell regulatory protein is an immune checkpoint molecule. Immune checkpoint molecules are ligand-receptor pairs expressed on immune cells that inhibit or stimulate the immune response. In an aspect of the invention, the T-cell regulatory protein is a stimulatory immune checkpoint molecule. For example, the stimulatory immune checkpoint molecule may be a member of the tumor necrosis factor (TNF) receptor superfamily. In an aspect of the invention, the stimulatory immune checkpoint molecule is CD 122, CD 137, CD27, CD28. CD40, GITR, ICOS, or 0X40. In another aspect of the invention, the T-cell regulatory protein is an inhibitory immune checkpoint molecule. For example, the inhibitory immune checkpoint molecule may be A2AR, A2BR, B7-H4, BTLA, CD276, CTLA-4, IDO, KIR, LAG3, N0X2, PD-1, SIGLEC7. SIGLEC9, TIM-3, TIGIT, TET2, CD39, CD103 or VISTA.

[0055] In an aspect of the invention, the cytokine is a single-chain cytokine or a functional portion of a single-chain cytokine. The cytokine may be, for example, IL-2, single chain IL-7, single chain IL-12, IL-15, IL-18, tumor necrosis factor alpha (TNF-a), or interferon-gamma (IFN-y). In a preferred aspect of the invention, the cytokine is a single chain cytokine including one or more of the subunits, e.g., all of the subunits, of the cytokine on a single polypeptide chain.

[0056] In a preferred aspect, the cytokine is single chain IL-12. IL-12 is a heterodimeric cytokine composed of covalently linked p35 and p40 subunits and is produced by activated innate immune cells. IL- 12 enhances the cytotoxic activity of natural killer (NK) cells and T cells. For example, IL-12 enhances cytotoxic activity by inducing the production of effector cytokines, e.g., IFN-y, TNF-a, and / or granulocyte macrophage colony stimulating factor (GM-CSF). IL-12 signals via the receptor chains IL12R01 and IL12R02. IL12R01 binds specifically to IL-12 p40, while IL-12R02 binds IL-12 p35. Upon binding of IL-12 p35 and p40 to their respective receptor chains, JAK2 and TYK2 mediate phosphorylation of STAT4. IL- 12 mediates signaling via phosphorylated (p) STAT4. In a preferred aspect of the invention, the inventive nucleic acid encodes a single polypeptide chain comprising both of IL-12 p40 and IL-12 p35.

[0057] In an aspect of the invention, the nucleotide sequence encodes an IL- 12 p40 amino acid sequence. The IL- 12 p40 amino acid sequence may be a human IL- 12 p40 amino acid sequence. Human IL- 12 p40 amino acid sequences include Genbank Accession Nos: AAD56386.1, NP_002178.2, AAM34792.1, AAG32620.1, and AAL05890.1. In an aspect of the invention, the human IL- 12 p40 amino acid sequence is at least 85%, at least 90%, at least 91%. at least 92%. at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In apreferred aspect the human IL- 12 p40 amino acid sequence comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO: 1 .

[0058] In an aspect of the invention, the nucleotide sequence encodes an IL- 12 p35 amino acid sequence. The IL-12 p35 amino acid sequence may be a human IL-12 p35 amino acid sequence. Human IL-12 p35 amino acid sequences include Genbank Accession Nos: NP_000873.2, NP 001341511.1, NP_001341512. L AAC05211.1, and AAD16432.1. In an aspect of the invention, the human IL-12 p35 amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. In a preferred aspect, the human IL- 12 p35 amino acid sequence comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO: 3.

[0059] In an aspect of the invention, the single chain IL- 12 comprises an amino acid sequence of Formula II:S^-NELLS N2(Formula II), wherein: each one of S1and S2is, independently, a signal sequence; each one of p and q is, independently, 0 or 1; each one of N1and N2is an IL-12 p40 amino acid sequence and the other is an IL-12 p35 amino acid sequence; andL2is a second linker sequence.

[0060] In an aspect of the invention, each of S1and S2of Formula II is, independently, a signal sequence. The signal sequence is not limited and may be any sequence which facilitates the translocation of the encoded polypeptide to the cell membrane. Examples of signal sequences are human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence, human prolactin signal sequence, and human IgE signal sequence. Examples of signal sequences include MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 19) and MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 20). Without being bound to a particular theory or mechanism, while it is believed that the signal sequence(s) may facilitate expression of the polypeptide, the presence of the signal sequence(s) in the expressed polypeptide may not be necessary in order for the polypeptide to function. In an aspect of the invention, upon expression of the polypeptide by the cell, the signal sequence(s) may be cleaved off of the polypeptide. In aspects of the invention, the IL- 12 subunit at the aminoterminus of the cytokine may comprise an amino terminal signal sequence, while the IL- 12 subunit at the carboxyl terminus of the cytokine may lack an amino terminal signal sequence. Accordingly, in an aspect of the invention, in Formula II, p is 1 and q is 0.

[0061] In an aspect of the invention, in Formula II, N2is the IL- 12 p40 amino acid sequence and N1is the IL-12 p35 amino acid sequence. In a preferred aspect, in Formula II, N1is the IL- 12 p40 amino acid sequence and N2is the IL- 12 p35 amino acid sequence.

[0062] The functional portion of the cytokine (or T-cell regulatory protein) can be any portion comprising contiguous amino acids of the cytokine (or T-cell regulatory protein) of which it is a part, provided that the functional portion specifically binds to the respective cytokine receptor (or T-cell regulatory protein receptor). The term "functional portion7’ when used in reference to a cytokine (or T-cell regulatory protein), refers to any part or fragment of the cytokine (or T-cell regulatory protein), which part or fragment retains the biological activity of the cytokine (or T-cell regulatory protein) of which it is a part (the parent cytokine or parent T-cell regulatory protein). Functional portions encompass, for example, those parts of a cytokine (or T-cell regulatory protein) that retain the abi hty to specifically bind to the respective cytokine receptor (or T-cell regulatory protein receptor), activate the downstream targets of the cytokine (or T-cell regulatory7protein), enhance the cytotoxic activity of immune cells (e.g.. NK cells and T cells), and / or induce the production of effector cytokines, e.g., IFN-y. TNF-a, and / or GM-CSF, to a similar extent, the same extent, or to a higher extent, as the parent cytokine (or parent T-cell regulatory protein). The biological activity of the functional portion of the cytokine (or functional portion of the T-cell regulatory7protein) may be measured using assays known in the art. In reference to the parent cytokine (or parent T-cell regulatory protein), the functional portion can comprise, for instance. 60%. 70%, 80%, 90%, 95%, or more, of the parent cytokine (or parent T-cell regulatory protein).

[0063] In an aspect of the invention, the nucleic acid further comprises a nucleotide sequence encoding a transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence. The transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be any transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence which binds the cytokine (or T-cell regulatory7protein) to the cell membrane.

[0064] In an aspect of the invention, the transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence comprises a B7-1 transmembrane- intracellular amino acid sequence, a B7-2 transmembrane-intracellular amino acid sequence,a CD8a transmembrane-intracellular amino acid sequence, a CD28 transmembrane- intracellular amino acid sequence, a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, a CD8a transmembrane amino acid sequence, a human leukocyte antigen (HLA) transmembrane amino acid sequence, or a CD28 transmembrane amino acid sequence. The HLA transmembrane amino acid sequence may be that which is encoded by any HLA gene, for example, HL A- A, HLA-B, HLA-C, HLA-DRB L HLA- DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, or HLA-DPB1.

[0065] The transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be a human transmembrane-intracellular amino acid sequence or a human transmembrane amino acid sequence, respectively. In an aspect of the invention, the transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human B7-1 transmembrane-intracellular amino acid sequence of SEQ ID NO: 4.

[0066] In an aspect of the invention, the nucleic acid further comprises a nucleotide sequence encoding an IMiD-degradable degron. A degron is a protein motif that is recognized by proteolytic machineries, such as the ubiquitin-proteasome complex, to cause the degradation of a protein or polypeptide. A degron functions as a degradation signal.

[0067] In an aspect of the invention, the IMiD- degradable degron comprises one or more substrates of cereblon (CRBN). CRBN is a ubiquitin-ligase that binds to IMiDs. IMiDs include, for example, thalidomide and thalidomide analogs, such as lenalidomide and pomalidomide. When bound to an IMiD, CRBN targets the CRBN substrate for degradation. Accordingly, the degron provides an OFF-switch that employs a CRBN substrate as an IMiD- responsive target that induces degradation. Lenalidomide (and some of its analogs) are FDA- approved for the treatment of myelodysplastic syndromes and multiple myeloma. The known safety profile of lenalidomide and human origins of the degron may facilitate clinical translation of this degron.

[0068] In an aspect of the invention, the degron comprises a CRBN substrate or a portion of a CRBN substrate that is capable of IMiD-inducible binding to CRBN. Examples of CRBN substrates include IKZF1, IKZF3, CKlalpha, ZFP91, GSPT1, MEIS2, GSS, E4F1, ZN276, ZN517, ZN582, ZN653. ZN654, ZN692, ZN787. and ZN827. Further examples IMiD- degradable degrons are described in, e.g., US 2021 / 0040166.

[0069] In an aspect of the invention, the degron comprises an amino acid sequence that is at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. The full-length degron sequence of SEQ ID NO: 6 was derived from two substrates of CRBN. namely ZFP91 and IKZF3. The components of the full-length degron sequence of SEQ ID NO: 6 are shown in Table 6 in Example 8.

[0070] It has also been discovered that a mutation, e.g., an amino acid substitution, in the degron sequence enhances the IMiD-degradable degradation of the polypeptide. As shown in Example 8. the L22R mutation enhanced the IMiD-degradable degradation of the polypeptide. Analysis of an alignment of all of the known substrate sequences of CRBN showed that I, K, L, and R are the four most common amino acids at position 22. Accordingly, it is believed that L22I or L22K mutation may also enhance the IMiD- degradable degradation of the polypeptide. In an aspect of the invention, the IMiD- degradable degron comprises an amino acid sequence at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in Table 1.TABLE 1

[0071] In an aspect of the invention, the nucleic acid further comprises a nucleotide sequence encoding one or more (e.g., first and second or more) linker sequences. The composition of the linker sequence is not particularly limited and may be any linker sequence which joins the components of the polypeptide together. Examples of linker sequences are described, e.g., in Chen et a ., Ad Drug Deliv. Rev., 65(10): 1357-69 (2013). One example of a linker sequence is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21). In an aspect of the invention, the polypeptide encoded by the nucleic acid comprises no linker sequences.

[0072] The length of the linker sequence is not limited and may be from 5 to 50 amino acid residues, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36. 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. or 50 amino acid residues, or a range between any two of the foregoing values. For example, in an aspect of the invention, the linker sequence L1of Formula I comprises 5 to 50, 10 to 40, 10 to 30, or 10 to 20 naturally occurring amino acid residues.

[0073] In some aspects of the invention, the linker sequence is a non-cleavable linker sequence. In a preferred aspect, the linker sequence is a flexible linker. In an aspect of the invention, the flexible linker comprises, consists, or consists essentially of glycine and serine residues. In this regard, the linker sequence may consist of from 5 to 50 amino acid residues independently selected from glycine and serine. For example, the flexible linker maycomprise, consist, or consist essentially of one or more units of Ge (SEQ ID NO: 22), Gs (SEQ ID NO: 23), GeS (SEQ ID NO: 2), GsS (SEQ ID NO: 24), G4S (SEQ ID NO: 25) and G3S (SEQ ID NO: 26) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of one or more units of GeS (SEQ ID NO: 2), GsS (SEQ ID NO: 24), G4S (SEQ ID NO: 25) and G3S (SEQ ID NO: 26)). For example, the flexible linker may comprise, consist, or consist essentially of (G4S)3 (SEQ ID NO: 27), GeS (SEQ ID NO: 2) or (GS)4GG (SEQ ID NO: 5).

[0074] In an aspect of the invention, the linker sequence is a rigid linker. In an aspect of the invention, the rigid linker comprises, consists, or consists essentially of glutamic acid, alanine, leucine, and lysine residues. In this regard, the linker sequence may consist of from 5 to 50 amino acid residues independently selected from glutamic acid, alanine, leucine, and lysine. For example, the rigid linker may comprise, consist, or consist essentially of one or more units of EA3K (SEQ ID NO: 28) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 units of EA3K (SEQ ID NO: 28)). Further examples of rigid linkers include, but are not limited to, A(EA3K)4ALEA(EA3K)4A (SEQ ID NO: 29) and AEA3KEA3KA (SEQ ID NO: 30). In another example, the linker sequence may consist of from 5 to 50 amino acid residues independently selected from alanine and proline. For example, the rigid linker may comprise, consist, or consist essentially of (PA)2P (SEQ ID NO: 31) or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 units of AP.

[0075] In an aspect of the invention, a first linker sequence may be positioned (a) between (i) the cytokine (or T-cell regulatory protein) and (ii) the transmembrane- intracellular amino acid sequence or transmembrane amino acid sequence, (b) between (i) the cytokine (or T-cell regulatory protein) and (ii) the degron, or (c) between (i) the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence and (ii) the degron. In a preferred aspect, the first linker sequence is positioned between the (i) the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence and (ii) the degron.

[0076] In an aspect of the invention, a second linker sequence may be positioned between subunits of the cytokine (or T-cell regulatory protein). For example, a linker sequence may be positioned betw een the IL- 12 p35 subunit and the IL- 12 p40 subunit.

[0077] The nucleotide sequence may, optionally, encode one or more (e.g., one or two or more) spacer sequences. Accordingly, in an aspect of the invention, each one of R1and R2of Formula I is. independently. I to 3 amino acid residues. The amino acid residues of the spacer sequence is not particularly limited and may include any amino acid residue(s). Forexample, a first spacer sequence (R1of Formula I) may be MA (start codon and Kozak sequence), and a second spacer sequence (R2of Formula 1) may be CGR or AAA (amino acid residues associated with the insertion of a cloning site). The spacer sequence(s) may be present or absent. For example, a first spacer sequence including a Kozak sequence may enhance expression of the polypeptide, but might not be necessary to obtain expression of the polypeptide. Accordingly, in an aspect of the invention, each one of m and n of Formula I is, independently, 0 or 1.

[0078] In an aspect of the invention, the nucleotide sequence encoding the polypeptide comprises a promoter operatively associated with the nucleotide sequence encoding polypeptide. The selection of promoters, e.g.. native, normative, strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter may be selected to provide transient expression, stable expression, or both. Also, the promoter may be selected to provide constitutive expression or inducible expression. For example, it may be desirable to employ a constitutive promoter to provide constitutive expression of cytokines that can be used to grow cells (e.g. IL-2).

[0079] In an aspect of the invention, the nucleic acid encodes for inducible expression of the polypeptide. In this regard, the nucleic acid may comprise an inducible promoter operatively associated with the nucleotide sequence encoding the polypeptide. The inducible promoter may be one which activates the expression of the polypeptide only when the cell (e.g., an antigen-specific receptor expressed by the cell) is specifically stimulated by an antigen and / or the cell (e.g., the calcium signaling pathway of the cell) is non-specifically stimulated by, e.g.. phorbol myristate acetate (PMA) / Ionomycin. For example, the nucleic acid may comprise a NFAT-responsive element linked to a minimal human IL-2 promoter operatively associated with the nucleotide sequence encoding the polypeptide, as described, for example, in Zhang et al., Mol. Ther., 19(4):751-9 (2011); Zhang et al., Clin. Cancer Res., 21(10): 2278-88 (2015); and U.S. Patent No. 8,556,882 or nuclear factor kappa B (NF-kappa B) responsive element. In this regard, the polypeptide may be expressed only when the cell (e.g., an antigen-specific receptor expressed by the cell) is specifically stimulated by an antigen and / or the cell (e.g., the calcium signaling pathw ay of the cell) is non-specifically stimulated by, e.g.. phorbol myristate acetate (PMA) / Ionomycin. Accordingly, the expression of the polypeptide may be controlled to occur only when and where it is needed, e.g., in the presence of cancer or at a tumor site. In an aspect of the invention, the NF AT-responsive element linked to a minimal human IL-2 promoter comprises the nucleotide sequence of SEQ ID NO: 8.

[0080] The terms “nucleic acid” and “polynucleotide,” as used herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, double- and single-stranded RNA, and double-stranded DNA-RNA hybrids. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and / or capped polynucleotides. In an aspect of the invention, the nucleic acid is complementary DNA (cDNA). In an aspect of the invention, the nucleic acid is an mRNA encoding the inventive polypeptide. Such an rnRNA may be directly injected into isolated cells or into a patient.

[0081] The term “nucleotide,” as used herein, refers to a monomeric subunit of a polynucleotide that has a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are typically derivatives of purine or pyrimidine, though aspects of the invention include the use of naturally and non-naturally occurring base analogs. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though aspects of the invention include the use of naturally and non-naturally occurring sugar analogs. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like). Methods of preparing polynucleotides are within the ordinary skill in the art (Green and Sambrook, Molecular Cloning: A Laboratory Manual, (4th Ed.) Cold Spring Harbor Laboratory Press, New York (2012)).

[0082] In some aspects, the nucleotide sequence may be codon optimized. Without being bound to a particular theory' or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by' tRNA that is more readily available within a cell, thus increasing translation efficiency. Codon optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0083] In an aspect of the invention, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the full length polypeptide amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or any one of the full-length polypeptide amino acid sequences set forth in Table 2.TABLE 2

[0084] The cytokine (or T-cell regulatory protein), or a functional portion of the cytokine (or functional portion of the T-cell regulatory protein), transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence, and degron may be arranged in any order. For example, in an aspect of the invention: i. the cytokine (or T-cell regulatory’ protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), may be positioned at the amino terminus of the polypeptide, the degron may be positioned at the carboxyl terminus of the polypeptide, and the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned between the cy tokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), and the degron; ii. the cytokine (or T-cell regulatory’ protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), may be positioned at theamino terminus of the polypeptide, the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned at the carboxyl terminus of the polypeptide, and the degron may be positioned between the cytokine (or T-cell regulator ' protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), and the transmembrane- intracellular amino acid sequence or transmembrane amino acid sequence; iii. the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned at the amino terminus of the polypeptide, the degron may be positioned at the carboxyl terminus of the polypeptide, and the cytokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory' protein), may be positioned between the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence and the degron; iv. the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned at the amino terminus of the polypeptide, the cy tokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), may be positioned at the carboxyl terminus of the polypeptide, and the degron may be positioned between the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence and the cytokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein); v. the degron may be positioned at the amino terminus of the polypeptide, the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned at the carboxyl terminus of the polypeptide, and the cytokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory' protein), may be positioned between the degron and the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence; or vi. the degron may be positioned at the amino terminus of the polypeptide, the cytokine (or T-cell regulatory' protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein), may be positioned at thecarboxyl terminus of the polypeptide, and the transmembrane-intracellular amino acid sequence or transmembrane amino acid sequence may be positioned between the degron and the cytokine (or T-cell regulatory protein), or functional portion of the cytokine (or functional portion of the T-cell regulatory protein).

[0085] Another aspect of the invention provides a nanoparticle comprising any of the inventive nucleic acids described herein. Nanoparticles are spherical, polymeric particles and may range in size from 10 to 500 nm.

[0086] In an aspect of the invention, the nucleic acid is carried in a recombinant expression vector. Accordingly, an aspect of the invention provides a recombinant expression vector comprising any of the inventive nucleic acids described herein with respect to other aspects of the invention.

[0087] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally -occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant expression vector can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both ty pes of linkages. Preferably, the non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector. The vector may contain regulatory nucleic acid sequences which provide for expression of the inventive nucleic acid.

[0088] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series, the pBluescript series, the pET series, the pGEX series, and the pEX series (Takara Bio USA, Inc., Mountain View, CA). Bacteriophage vectors, such as GT10, ZGT I 1 , ZZapII. ZEMBL4, and ZNM 1 149, also can be used. Examples of animal expressionvectors useful in the context of the invention include pEUK-Cl. pMAM, and pMAMneo (Takara Bio USA, Inc.).

[0089] In some aspects, the recombinant expression vector is a viral vector. Suitable viral vectors include, without limitation, lentiviral, retroviral, alphaviral, vaccinial, adenoviral, adeno-associated viral, herpes viral, and fowl pox viral vectors, and preferably have a native or engineered capacity to transform T cells. An example of a retroviral vector is the MSGV1 retroviral vector. In an aspect of the invention, the recombinant expression vector is a self-inactivating (SIN) vector. An example of a retroviral SIN vector is the SERS retroviral vector. In an aspect of the invention, the recombinant expression vector is a transposon. In a preferred aspect of the invention, the recombinant expression vector is a retroviral vector, a lentiviral vector, or a transposon.

[0090] The recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, (4th Ed.) Cold Spring Harbor Laboratory’ Press, New York (2012). Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 p plasmid, X, SV40, bovine papilloma virus, and the like.

[0091] The recombinant expression vector can comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.

[0092] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy' metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the recombinant expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0093] Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art andinclude, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase, and nitroreductase.

[0094] In aspects of the invention, expression of the polypeptide in “reverse orientation” in the recombinant expression vector may be useful for reducing or eliminating uncontrolled cytokine (or T-cell regulatory protein), e.g., IL- 12, expression. In this regard, in an aspect of the invention, the recombinant expression vector comprises a vector backbone comprising a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein the nucleotide sequence encoding the polypeptide is positioned in between the 5’ LTR and the 3’ LTR, wherein the 5’ LTR and the 3’ LTR are each positioned in 5 ' to 3' orientation, and wherein the nucleotide sequence encoding the polypeptide is positioned in 3’ to 5’ orientation relative to the 5‘ LTR and the 3’ LTR. In other aspects of the invention, the polypeptide may be positioned in the recombinant expression vector in “forward orientation.” In this regard, in an aspect of the invention, the recombinant expression vector comprises a vector backbone comprising a 5’ LTR and a 3‘ LTR. wherein the nucleotide sequence encoding the polypeptide is positioned in between the 5’ LTR and the 3’ LTR, wherein the 5’ LTR and the 3’ LTR are each positioned in 5’ to 3’ orientation, and wherein the nucleotide sequence encoding the polypeptide is positioned in 5’ to 3’ orientation relative to the 5’ LTR and the 3’ LTR.

[0095] Another aspect of the invention provides a viral particle comprising any of the inventive nucleic acids or the recombinant expression vectors described herein. Examples of viral particles include, but are not limited to, adeno-associated virus (AAV), adenovirus, baculovirus, retrovirus, lentivirus, foamy virus, herpes virus, Moloney murine leukemia virus, Vaccinia virus, and hepatitis virus. Such viral particles may, for example, be directly injected into isolated cells or into a patient.

[0096] The inventive nucleic acids and recombinant expression vectors may be introduced into host cells using transfection, transformation, transduction, electroporation, a transposon, or a genome editing technique. In an aspect of the invention, the genome editing technique to introduce the nucleic acids and recombinant expression vectors uses a zinc finger nuclease, transcription activator-like effector nuclease (TALENs), a CRISPR / Cas system, or engineered meganuclease.

[0097] Genome editing techniques can modify gene expression in a target cell by inserting, replacing, or removing DNA in the genome using an artificially engineered nuclease. Examples of such nucleases may include zinc finger nucleases (ZFNs) (Gommans et al., J. Mol Biol.., 354(3): 507-519 (2005)), TALENs (Zhang et al., Nature Biotechnol. 29:149-153 (2011)). the CRISPR / Cas system (Cheng et al., Cell Res., 23: 1163-71 (2013)), and engineered meganucleases (Riviere et al., Gene Ther., 21 (5): 529-32 (2014)). The nucleases create specific double-stranded breaks (DSBs) at targeted locations in the genome, and use endogenous mechanisms in the cell to repair the induced break by homologous recombination (HR) and nonhomologous end-joining (NHEJ).

[0098] Another aspect of the invention provides a polypeptide encoded by the any of the nucleic acids described herein. The term "polypeptide," as used herein, includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds.

[0099] In an aspect of the invention, the polypeptide comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or any one of the full-length polypeptide amino acid sequences set forth in Table 2.

[0100] Included in the scope of the invention are functional variants of the polypeptides described herein, including the cytokines (or T-cell regulator}' proteins), transmembrane- intracellular amino acid sequences, transmembrane amino acid sequences, degrons, and linkers described herein. The term “functional variant’" as used herein refers to a polypeptide, cytokine (or T-cell regulatory protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker having substantial or significant sequence identity or similarity7to a parent polypeptide, cytokine (or T-cell regulatory7protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker, respectively, which functional variant retains the biological activity of the polypeptide, cytokine (or T-cell regulatory7protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker, respectively, of which it is a variant. Functional variants encompass, for example, those variants of the IL-12 described herein (the parent IL- 12) that retain the ability to specifically bind to the respective IL- 12 receptor chain, activate the downstream targets of IL- 12, and / or enhance the cytotoxic activity7of immune cells (e.g., NK. cells and T cells), and / or induce the production of effector cytokines, e.g., IFN-y, TNF-a, and / or GM-CSF, to a similar extent, the same extent, or to a higher extent, as the parent IL-12. In reference to the parent polypeptide, cytokine (or T-cell regulatory protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker, the functional variant can, for instance, be at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical in amino acid sequence to the parent polypeptide, cytokine (or T-cell regulatory' protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker.

[0101] A functional variant can, for example, comprise the amino acid sequence of the parent polypeptide, cytokine (or T-cell regulatory protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker with at least one conservative amino acid substitution. Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent polypeptide, cytokine (or T-cell regulatory protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent polypeptide, cytokine (or T-cell regulatory' protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker.

[0102] Amino acid substitutions of the polypeptide, cytokine (or T-cell regulatory protein), transmembrane-intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai. etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta- branched side-chain (e.g., He, Thr. and Vai), an amino acid with anaromatic side-chain substituted for another amino acid with an aromatic side chain (e.g.. His, Phe, Trp, and Tyr), etc.

[0103] The polypeptide, cytokine (or T-cell regulatory protein), transmembrane- intracellular amino acid sequence, transmembrane amino acid sequence, degron, or linker can consist essentially of the specified amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not materially change the biological activity of the functional variant.

[0104] Another aspect of the invention provides a host cell comprising any of the recombinant expression vectors described herein. Still another aspect of the invention provides a host cell expressing any of the nucleic acids described herein or the one or more polypeptides described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokary otic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a A. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokary otic cell, e.g., a DH5a cell. For purposes of producing polypeptide(s) encoded by the inventive nucleic acids, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell, a natural killer (NK) cell, or an NK T cell.

[0105] For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary' T cell, or a T cell from a cultured T cell line, e.g., Jurkat. SupTl. etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+double positive T cells, CD4+helper T cells, e.g., Thi and Th2 cells, CD4+Tcells. CD8+T cells (e.g.. cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e g., central memory T cells and effector memory T cells), naive T cells, gamma delta (y5) T cells, alpha beta (aft) T cells, and the like. In an aspect of the invention, the T cell is a CD39 CD69’ T cell. For example, the inventive polypeptide can be expressed in TILs to potentiate ACT using TILs that are autologous to the patient.

[0106] In an aspect of the invention, the host cell comprises (e.g., expresses) an antigenspecific receptor. In a preferred aspect, the antigen-specific receptor has antigenic specificity for a cancer antigen. The phrases “antigen-specific'’ and “antigenic specificity,” as used herein, mean that the antigen-specific receptor can specifically bind to and immunologically recognize an antigen, or an epitope thereof, such that binding of the antigen-specific receptor to antigen, or the epitope thereof, elicits an immune response.

[0107] The term “cancer antigen,” as used herein, refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or overexpressed by a tumor cell or cancer cell, such that the antigen is associated with the tumor or cancer. The cancer antigen can additionally be expressed by normal, non-tumor, or non- cancerous cells. However, in such cases, the expression of the cancer antigen by normal, non-tumor, or non-cancerous cells is not as robust as the expression by tumor or cancer cells. In this regard, the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non- tumor, or non-cancerous cells. Also, the cancer antigen can additionally be expressed by cells of a different state of development or maturation. For instance, the cancer antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host. Alternatively, the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host. Examples of cancer antigens include, but are not limited to, mesothelin, CD 19, CD22, CD30, CD70, CD276 (B7H3), gplOO, MART-1, Epidermal Grow th Factor Receptor Variant III (EGFRVIII), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), TRP-1, TRP-2, tyrosinase, human papillomavirus (HPV) 16 E6, HPV 16 E7, HPV 18 E6, HPV 18 E7, KK- LC-1, NY-BR-1, NY-ESO-1 (also known as CAG-3), SSX-2, SSX-3, SSX-4, SSX-5, SSX-9, SSX-10, MAGE-A1, MAGE-A2, BRCA, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, HER-2, etc. In an aspect of the invention, the cancer antigen may be a mutated antigen that is expressed or overexpressed by tumor or cancer cells and which is not expressed by normal, non-tumor, ornon-cancerous cells. Examples of such cancer antigens may include, but are not limited to, mutated KRAS and mutated p53. T cells having antigenic specificity for a cancer antigen may, advantageously, reduce or avoid cross-reactivity with normal tissues such as, for example, that which may occur using T cells having antigenic specificity for minor histocompatibility antigens.

[0108] The cancer antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor. For example, the cancer antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumor, or non-cancer cells.

[0109] In an aspect of the invention, the antigen-specific receptor is an exogenous T cell receptor (TCR). By "exogenous" is meant that the TCR is not native to (naturally -occurring on) the T cell. The exogenous TCR may be a recombinant TCR. A recombinant TCR is a TCR which has been generated through recombinant expression of one or more exogenous TCR a-, P-, y-, and / or 5-chain encoding genes. A recombinant TCR can comprise polypeptide chains derived entirely from a single mammalian species, or the recombinant TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species. For example, the TCR can comprise a variable region derived from a human TCR, and a constant region of a mouse TCR such that the TCR is “murinized.” Any exogenous TCR having antigenic specificity for a cancer antigen may be useful in the inventive methods and compositions. The TCR generally comprises two polypeptides (i.e., polypeptide chains), such as an a-chain of a TCR, a -chain of a TCR, a y- chain of a TCR, a 5-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The cancer antigen-specific TCR can comprise any amino acid sequence, provided that the TCR can specifically bind to and immunologically recognize a cancer antigen or epitope thereof. Examples of exogenous TCRs that may be useful in the inventive methods and compositions include, but are not limited to, those disclosed in, for example, U.S. Patents 7,820,174; 7,915,036; 8,088,379; 8,216,565; 8,431,690; 8,613,932;8,785,601; 9.128,080; 9,345,748; 9,487,573; 9,822,162; 10.611,815; 11,306,131; 11.352,410; 11.434.272; and 11,939,365. each of which is incorporated herein by reference.

[0110] In an aspect of the invention, the antigen-specific receptor is a chimeric antigen receptor (CAR). Typically, a CAR comprises the antigen binding domain of an antibody, e.g., a single-chain variable fragment (scFv), fused to the transmembrane and intracellular domains of a TCR. Thus, the antigenic specificity of a TCR of the invention can be encoded by a scFv which specifically binds to the cancer antigen, or an epitope thereof. Any CAR having antigenic specificity for a cancer antigen may be useful in the inventive methods and compositions. Examples of CARs that may be useful in the inventive methods and compositions include, but are not limited to, those disclosed in, for example, U.S. Patents 8,465,743; 9.266,960; 9,765,342; 9,359,447; 9,868,774; 11.236,161; and 11,951.131, each of which is incorporated herein by reference.[OHl] In an aspect of the invention, the antigen-specific receptor is an endogenous TCR. In some aspects, the T cell comprising the endogenous TCR does not comprise (e.g., express) a CAR or an exogenous TCR. In other aspects, a T cell comprising an endogenous cancer antigen-specific TCR can also be transformed, e.g., transduced or transfected, with one or more nucleic acids encoding an exogenous (e.g., recombinant) TCR or other recombinant receptor (e.g., CAR). Such exogenous receptors, e.g., TCRs, can confer specificity' for additional antigens to the transformed T cell beyond the antigens for which the endogenous TCR is naturally specific. This can, but need not, result in the production of T cells having dual antigen specificities.

[0112] Also provided by an aspect of the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g.. a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one aspect of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0113] In an aspect of the invention, the inventive nucleic acids, recombinant expression vectors, polypeptide(s), host cells, and populations thereof may be isolated or purified. The term "isolated," as used herein, means having been removed from its natural environment. The term "purified," as used herein, means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least 50%, can be greater than 60%, 70% or 80%, 90% or can be 100%.

[0114] The inventive nucleic acids, recombinant expression vectors, viral particles, polypeptide(s), host cells (and populations thereof) (hereinafter, “inventive cytokine materials”) may be included in a composition, such as a pharmaceutical composition. In this regard, an aspect of the invention provides a pharmaceutical composition comprising any of the inventive cytokine materials described herein and a pharmaceutically acceptable carrier.

[0115] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the administration of cells. Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0116] The choice of carrier may be determined in part by the particular method used to administer the particular inventive cytokine material. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or interperitoneal administration. More than one route can be used to administer the inventive cytokine material, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0117] Preferably, the inventive cytokine material is administered by injection, e g., intravenously. A suitable pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w / v of NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an aspect, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0118] For purposes of the invention, the dose, e.g.. number of inventive host cells administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the number of inventive host cells administered should be sufficient to bind to a cancer antigen or treat or prevent cancer in a period of from about 2 hours or longer, e.g.. 12 to 24 or more hours, from the time of administration. In certain aspects, the time period could be even longer. The number of inventive host cells administered will be determined by, e.g., the efficacy of the particular population of host cells (e.g., T cells) to be administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e g., human) to be treated.

[0119] Many assays for determining an administered number of inventive host cells are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or one or more cytokines such as, e.g., IFN-y and IL-2 is secreted upon administration of a given number of such T cells to a mammal among a set of mammals of which is each given a different number of the T cells, could be used to determine a starting number to be administered to a mammal. The extent to which target cells are lysed or cytokines such as, e.g., IFN-y and IL-2 are secreted upon administration of a certain number can be assayed by methods known in the art. Secretion of cytokines such as. e.g., IL- 2, may also provide an indication of the quality (e.g., phenotype and / or effectiveness) of a T cell preparation.

[0120] The number of inventive host cells administered also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular population of cells. Typically, the attending physician will decide the number of cells with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, route of administration, and the severity' of the condition being treated. By way of example and not intending to limit the invention, the number of cells to be administered can be 10 x 106to 10 x 1011cells per infusion, 10 x 109cells to 10 x 1011cells per infusion, or 10 x 107to 10 x 109cells per infusion.

[0121] It is contemplated that the inventive cy tokine materials can be used in methods of treating or preventing cancer in a mammal. In this regard, an aspect of the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the inventive cytokine materials or pharmaceutical compositions described herein in an amount effective to treat or prevent cancer in the mammal.

[0122] It is also contemplated that the inventive cytokine materials can be used in methods of enhancing an immune response against a cancer in a mammal. In this regard, an aspect of the invention provides a method of enhancing an immune response against a cancer in a mammal, comprising administering to the mammal any of the inventive cytokine materials or pharmaceutical compositions described herein in an amount effective to enhance the immune response against the cancer in the mammal.

[0123] One or more additional therapeutic agents can be coadministered to the mammal. By “coadministering” is meant administering one or more additional therapeutic agents and the inventive cytokine material sufficiently close in time such that the inventive cytokine material can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the inventive cytokine material can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the inventive cytokine material and the one or more additional therapeutic agents can be administered simultaneously. Additional therapeutic agents that may enhance the function of the inventive cytokine material may include, for example, one or more cytokines or one or more antibodies (e.g., antibodies that inhibit PD-1 function). An exemplary therapeutic agent that can be co-administered with the inventive cytokine material is IL-2. Without being bound to a particular theory or mechanism, it is believed that IL-2 may enhance the therapeutic effect of the inventive populations of host cells. In an aspect of the invention, the inventive methods may further comprise administering to the mammal (i) an antigen-specific receptor, a nucleic acid comprising a nucleotide sequence encoding the antigen-specific receptor, a recombinant expression vector comprising the nucleic acid comprising a nucleotide sequence encoding the antigen-specific receptor, a host cell comprising the recombinant expression vector, a population of cells comprising the host cell, or (ii) a pharmaceutical composition comprising (i), wherein the antigen-specific receptor has antigenic specificity for an antigen of the cancer.

[0124] An aspect of the invention further comprises lymphodepleting the mammal prior to administering the inventive cytokine material. Examples of lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0125] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizesas having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset or recurrence of the cancer, or a symptom or condition thereof.

[0126] In an aspect of the invention, for purposes of the inventive methods, wherein populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal. However, the administered cells need not be allogeneic or autologous to the mammal. Accordingly, in some aspects, the administered cells are not allogeneic or autologous to the mammal.

[0127] With respect to the inventive methods, the cancer can be any cancer, including any of leukemia (e.g., B cell leukemia), sarcomas (e.g., sy novial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.. Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head-neck cancer, acute lymphocytic cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral canty, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer, lary nx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, oropharynx, ovarian cancer, pancreatic cancer, penis, peritoneum, rectum, omentum, and mesentery cancer, pancreas, pharynx cancer, prostate cancer, rectal cancer, renal cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, vagina, and urinary bladder cancer.

[0128] The inventive cytokine materials may also be useful during the extraction and growth of T cells, i.e., during expansion of the numbers of cells and growth cycles, for example, to activate them. An aspect of the invention provides a method of culturing T cells in vitro, the method comprising introducing any of the inventive nucleic acids, nanoparticles, or recombinant expression vectors described herein into isolated T cells, thereby expressing the nucleic acid by the host cells. The method may further comprise expanding the number of T cells. In an aspect of the invention, expanding the number of T cells comprisesexpanding the number of T cells in the presence of one or both of (a) one or more cytokines and (b) one or more non-specific T cell stimuli. Examples of non-specific T cell stimuli include, but are not limited to, one or more of irradiated allogeneic feeder cells, irradiated autologous feeder cells, anti-CD3 antibodies (e.g.. 0KT3 antibody), anti -4- IBB antibodies, and anti-CD28 antibodies. Exemplary cytokines that may be useful for expanding the numbers of cells include interleukin (IL)-2, IL-7, IL-21, IL-18, and IL-15.

[0129] Expansion of the numbers of cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; and U.S. Patent Application Publication No. 2012 / 0244133. In an aspect of the invention, the numbers of cells are expanded by physically contacting the cells with one or more non-specific T cell stimuli and one or more cytokines. For example, expansion of the numbers of cells may be carried out by culturing the cells with OKT3 antibody, IL-2, and feeder PBMC (e g., irradiated allogeneic PBMC).

[0130] The following examples further illustrate the invention but, of course, should not be constmed as in any way limiting its scope.EXAMPLES

[0131] The following materials and methods were employed in the experiments described in Examples 1-17.Construction of the IL-12TM-D and the P40-GFP reporter plasmid

[0132] Human single-chain IL- 12 with a G6S linker and B7 transmembrane with anNF AT promoter was prepared. The lenalidomide degron (Jan et al.. Set. Transl. Med., 13(575) (2021)) was attached to the C terminus of IL-12 with a linker (GSGSGSGSGG) (SEQ ID NO: 5). The entire cassette was codon-optimized and inserted into the SERS backbone using Ncol and BamHI in a forward orientation and into the MSGV backbone using Notl in a reverse orientation. P40-GFP reporter was derived from the single chain IL- 12 construct by replacing the P35 portion with EGFP. with or without the B7 transmembrane domain. Oligonucleotide generation and cloning were performed by Genscript (custom synthesis and cloning, NJ, USA).Introduction of single amino acid substitutions to improve the lenalidomide degron ’s sensitivity to lenalidomide treatment

[0133] A logos plot was created using sequences of known substrates of CRBN (Jan et al., Sci. Transl. Med., 13(575) (2021)) when bound to lenalidomide using WebLogo (ver. 2.8.2, weblogo.berkeley.edu / ). Based on the frequencies of certain amino acids at each position, 9 different mutants each with single amino acid substitution were generated using site-directed mutagenesis (Genscript, custom service). The mutagenesis and testing were performed using IL-12TM-D in the SERS vector.Tumor cell lines

[0134] Jurkat (Paria et al., J. Immunother., 44(1): 1-8 (2021)) TYK-nu (Kim et al., Cancer Immunol. Res., 10(8):932-946 (2022)), T2 (Kim et al., Cancer Immunol. Res., 10(8):932-946 (2022)), 4391 PDX (Levin et al., J. Immunother. Cancer, 12(5)) (2024)), 4069 PDX (Levin et al., J. Immunother. Cancer, 12(5)) (2024)) and NALM6 (wild-ty pe or modified to preclude CD19 expression) (Majzner et al., Cancer Discov., 10(5):702-723 (2020)) cells were previously described. 3795 melanoma cell line was established as described (Dudley et al., J. Immunother ., 26(4):332-42 (2003)). All the above tumor cell lines were cultured in RPMI1640 media (Cat# 11875093) containing 10% FBS (Cat# SH30071.03HI, Cytiva, MA), 1% sodium pyruvate (Cat# 11360070), 1% Glutamax (Cat# 35050061), 1% MEM non- essential amino acids (Cat# 11140050), 55 pM 2-mercaptoethanol (Cat# 21985023), and 1% Pen / Strep (Cat# 15140122) (all of the above from Gibco, USA except for FBS). 293 GP cells were maintained with D10 media (DMEM (Cat# 10564011, Gibco) containing 10% FBS, 1% Glutamax and 1% Pen / Strep).Primary cell cultures

[0135] Melanoma TIL infusion products were generated as described previously (Goff et al., J. Clin. Oncol.. 34(20):2389-97 (2016)). TILs were maintained in 50 / 50 media [1640 media containing 10% human serum (Cat# HS1021HI, Valley Biomedical, USA or Cat# 100- 512-100, GemimBio, CA, USA), 1% Glutamax, 12.5 mM HEPES (Cat# 15630080, Gibco), 1% Pen / Strep, and 5 pg / mL gentamicin (Cat# 120-099-661, Quality7Biological, USA) mixed with AIM-V (Cat# 0870112BK, Gibco) at 1 to 1 ratio] supplemented with 3000 lU / mL IL-2 (Aldesleukin, National drug code: 76310-0022-01, Clinigen, USA). Healthy -donor peripheralblood mononuclear cells were obtained as described previously (Kim et al., Cancer Immunol. Res., 10(8):932-946 (2022)) and maintained in 50 / 50 media supplemented with 300 lU / mL IL-2.Retroviral transduction of TCR / CAR and IL- 12TM-D

[0136] Various neoantigen-specific TCRs (Kim et al., Cancer Immunol. Res., 10(8):932- 946 (2022); Levin et al., Clin. Cancer Res., 27(18):5084-5095 (2021)) and CD19 CAR (Kochenderfer et al.. Blood, 116(20):4099-102 (2010) were previously described. Retroviral transduction of TCRs / CAR into Jurkat cells and human primary T cells were conducted as previously described (Kim et al., Cancer Immunol. Res., 10(8):932-946 (2022)).Transduction of IL-12TM-D was performed with the following modifications. Twelve- million 293 GP cells were seeded in a 15 cm poly-D-lysine-treated plate one day before transfection, and transfection of the IL-12TM-D plasmid (30 pg) and the RD114 envelope plasmid (22 pg) were performed using Lipofectamine 2000 (Cat. 11668019, Thermo Fisher Scientific, MA, USA). On the next day following transfection, media of 293 GP cells were replaced with 20 rnL of fresh D10 media. Two days later, viral supernatant was collected, filtered and used either freshly or after preservation at -80 °C. When primary T cells were transduced with both TCR / CAR or IL-12TM-D, the volumetric ratio of TCR / CAR and the IL-12TM-D viral supernatant w as 1 :2. TILs w ere transduced by stimulating TILs for 48 hours using TransAct (Cat# 130-111-160, Miltenyi Biotech, Germany)Antibodies, flow cytometry and fluorescence-activated cell sorting (FACS)

[0137] T cells following a co-culture with target tumor cells or T2 cells as antigen presenting cells were stained with antibodies specific for the following human markers: CD8 FITC (1 :20, Cat# 555634), IL-12 (P70) PE (1 :10, Cat# 130-103-672, Miltenyi Biotec), 4- 1BB APC (clone 4B4-1; 1 :20, Cat# 550890, RRID:AB_398477), CD4 APC-Cy7 (Clone RPA-T4; 1:20, Cat# 557871) and CD3 BUV496 (Clone UCHT1; 1 :20, Cat# 612940) (all from BD Biosciences, USA except for IL-12 PE). T cells engineered to express a TCR with murine TCRA / TCRB constant region sequences were stained with mTCRP (1 :40, Cat# 562839, BD Biosciences). Intracellular staining of IL-12 w as performed using Cytofix / Cy toperm™ Fixation / Permeabilization Kit (Cat# 554714, BD Biosciences) according to the manufacturer's instructions. Flow cytometric analysis of naive / stem cell markers and inhibitory markers were performed using following antibodies: panel 1: CD3APC-Cy7 (SK7; 1:25, Cat# 341090. BD Biosciences) , CD8 PE-Cy7 (clone RPA-T8; 1 :40, Cat# 560917, BD Biosciences), CD4 F1TC (clone RPA-T4; 1 :20, Cat# 555346, BD Biosciences), CD39 PE (clone Al; 1 :40, catalog no. 328208, BioLegend), CD69 BV650 (clone FN50; 1:25, Cat# 563835, BD Biosciences), PD-1 BV421 (clone EH12.1, BD Biosciences, Cat# 562516) and mTCR-APC (Clone H57-597; 1:20. Cat# 553174, BD Biosciences). Panel 2: CD62L BV421 (clone DREG-56; 1:50, Cat# 304828, BioLegend), CD8 BV650 (clone RPA-T8, 1:20, Cat# 301042, BioLegend), TIM3 BB515 (clone FN50; 1 :20, Cat# 565568, BD Biosciences), TIGIT PE-Cy7 (clone A15153G, 1:20, Cat# 372714, BioLegend), CD45RO APC (Clone UCHL1; 1 :20, Cat# 559865, BD Biosciences), CD4 APC-H7 (Clone SK3; 1 :20, Cat# 641398. BD Biosciences) and mTCR-PE (Clone H57-597; 1 :20, Cat# 553172, BD Biosciences). Analytic flow cytometry was performed on FACSymphony (BD Biosciences, USA) with analysis by FLOWJO software (ver 10.10.0, BD Biosciences). All cells were gated via lymphocytes (FSC and SSC) and live cells by exclusion of cells stained with propidium iodide (Cat# P1304MP. Thermo Fisher Scientific).ACT of human cancer cells in NSG mice

[0138] Animal experiments were approved by the Institutional Animal Care and Use Committees of the NCI and performed in accordance with the National Institutes of Health guidelines. Six-week-old female NSG mice (NOD-Prkdcem26Cd52I12rgem26Cd22 / NjuCrl, also known as NCG) were obtained from Charles River Laboratories (MA, USA). ACT of xenograft models using TYK-nu cells was performed as previously described (Kim et al., Cancer Immunol. Res., 10(8):932-946 (2022)) and as in Fig. 5A, 5E and 9A. ACT of xenograft models using 4391 PDX cells was performed as previously described (Levin et al. J. Immunother. Cancer 12:e008645 (2024)) and as in Fig. 10A. Following tumor cellinoculation, healthy-donor PBLs genetically engineered with the p53 or KRAS TCR and IL- 12TM-D were intravenously injected into tumor bearing NSG mice. Three daily doses of 180,000 IU of recombinant human IL-2 (Aldesleukin, Clinigen) in 500 pl of PBS were injected intraperitoneally following the T-cell transfer. Tumor growth was measured once or twice a w eek, and tumor size w as calculated as the product of two perpendicular measurements. All experiments were conducted in a blinded manner. Sample sizes of 5 mice per experimental arm were used. Two-way ANOVA was used for statistical analysis of tumor growth betw een experimental arms using Prism 10 softw are. Log-rank test w as performed for statistical analysis of mouse survival using Prism 10 software.Repeat co-culture of II.-S2TM-I) T cells with TYK-nu cells

[0139] Five hundred thousand T cells were co-cultured with an equal number of irradiated TYK-nu cells (20,000 rad) with or without 100 nM lenalidomide. Additional irradiated TYK-nu cells were added every two to three days to the culture for a total of six times. At days 0, 7 and 14, 50,000 T cells were collected and analyzed using flow cytometry and at day 14, 100,000 cells were used for single-cell CITE-seq as described in other sections.Single-cell CITE sequencing

[0140] For CITE-seq staining, a panel of 137 TotalSeq-C barcoded antibodies (Cat# 399905, BioLegend) was used. At the end of repeat-coculture, 100,000 T cells were washed and stained with a mixture of TotalSeq-C antibodies for 30 min. on ice according to the manufacturer’s instructions. Cells were then washed twice before chromium capture.

[0141] Cells were resuspended in PBS at 5x105 cells / ml, and loaded onto a CHROMIUM CONTROLLER system (10X Genomics, USA) for single-cell sample preparation. One channel per reaction was used to prepare each sample for sequencing following the manufacturer’s protocol. Briefly, 10,000 T cells per channel were loaded on the CHROMIUM CONTROLLER system with the targeted cell recovery' of 6,000-8,000 single cells. The single-cell cDNA samples were first universally amplified by a 16-cycle PCR reaction using a thermocycler (ABI Veriti, Applied Biosystems, Inc., USA) and the CHROMIUM NEXT GEM Single Cell 5’ Reagent Kits V2 (Cat# PN-1000265, 10X Genomics) according to the manufacturer’s instructions. The amplified whole transcriptome cDNA and feature barcoded fragments were purified per the manufacture’s protocol. The whole transcriptomes (GEX) next-generation sequencing (NGS) and feature barcoded (FB) libraries were processed according to manufacturer’s protocol. The GEX and FB libraries were sequenced using the Illumina NEXTSEQ 2000-P3 (300cycle) kit (Readl: 26bp, Indexl: lObp, Index2: lObp, Read2: 90bp).Bioinformatic analysis for single-cell sequencing data

[0142] Single-cell transcriptome sequencing data were initially processed using Cell Ranger pipelines (v7.0.1; 10X Genomics, USA). The demultiplexed sequencing data were mapped to the human reference genome, GRCh38, along with the transduced p53 TCRsequence. Gene expression matrices were generated from the unique molecular identifier- collapsed read counts on individual cell barcodes. These matrices included cells and annotated genes, utilizing error-corrected hdf5 reads. Low-quality cells and doublets were filtered out based on feature distribution.

[0143] Further downstream analyses were conducted using the R package Seurat v4.Cells that expressed fewer than 250 genes and genes with fewer than three read counts across all cells were excluded from the analysis. For each library, antibody data (ADT) were adjusted to account for non-specific binding using isotype control counts and then normalized with the dsb R package (Mule et al., Nat. Commun.. 13( 1 ):2099 (2022)).

[0144] A common normalized gene expression matrix was created using Seurat's "SCT" function. The data were standardized by regressing out highly variable and mitochondrial genes, followed by normalization and scaling. All TRAV / TRBV genes were excluded to remove endogenous TCR expression as a potential source of clustering bias before processing with Seurat.

[0145] Uniform Manifold Approximation and Projection (UMAP) plots were generated based on the clustered principal components (PCs). Cluster markers were identified through differential gene expression analysis for each cluster using default parameters. The FeaturePlot function was utilized to display various genes of interest on the UMAP.

[0146] Single-cell gene set enrichment analysis (scGSEA) was performed using the fgsea R package (Korotkevich et al., bioRxiv, 060012 (2021), which utilizes a rank -based gene signature metric to compute the expression score of a gene list about all other genes using single-cell RNA (scRNA) expression data. In brief, normalized scRNA gene expression matrices with barcodes were used as input alongside 80 single-cell gene set lists or gene sets derived from previous studies (Lowery et al., Science, 375(6583):877-884 (2022)).EXAMPLE 1

[0147] This example demonstrates that LEN treatment regulates the expression of membrane-bound IL- 12 but not secreted IL- 12.

[0148] To achieve the ability' to pharmacologically regulate the expression of IL-12, the lenalidomide-degradable degron (also referred to as “lenalidomide degron”) was conjugated with single chain human IL- 12. This semi-synthetic degron was created by mixing and matching degron sequences from substrates of cereblon (CRBN) (Jan et al., Sci. Transl. Med., 13 (2021)). Lenalidomide (LEN) binds to CRBN to modulate its specificity7for targetsubstrates and can lead to degradation of proteins of interest (POI) containing the degron sequence (Figs. 1 A-1B).

[0149] The regulation of IL-12 was tested in both the secreted form and membrane-bound form of IL-12. Among the various transmembrane (TM) domains, the B7-1 TM, which showed the least amount of IL-12 shedding into the cultured media (Zhang et al.. J. Immunother. Cancer, 8(l):e000210 (2020)), was selected. A green fluorescent protein (GFP) reporter for the degron activity was constructed. As a surrogate for the single-chain IL- 12, the p35 subunit of IL-12 was replaced with GFP (Fig. 1C). Jurkat cells were transduced with the reporter genes with or without a TM domain. Following stimulation of the Jurkat cells for GFP induction, with LEN treatment, a significant reduction in GFP signal was observed only in the format containing a TM domain (Fig. 1C).

[0150] Single-chain IL- 12 with the degron in a secreted form without GFP did not show any reduction upon LEN treatment even at the high dose of 10 pM (Fig. ID). These results suggest that CRBN may not be able to access or degrade IL-12 if it is packaged for secretion (i.e without a TM domain).

[0151] Incorporating a TM domain can be advantageous in that it will lead to minimal systemic release of IL-12. To this end, single-chain IL-12 was conjugated with the degron and the B7-1 TM domain (abbreviated as IL-12TM-D). IL-12TM-D included the following components, in order from the amino terminus to the carboxyl terminus: the IL- 12 p40 subunit, a first linker, the IL-12 p35 subunit, CGR, the B7-1 TM domain, a second linker, and the degron. The amino acid sequences of the components of IL-12TM-D are show n in Table 3. The “MA” at the N-terminus of the IL- 12 p40 subunit was introduced to enhance expression of the polypeptide.TABLE 3

[0152] The full-length amino acid sequence of IL-12TM-D is shown in Table 4.TABLE 4

[0153] When expressed in human T cells, relative to the secreted form of IL-12, membranous IL-12 did not lead to an increase in the IL-12 level in the culturing media (Fig. IE).EXAMPLE 2

[0154] This example demonstrates the inducibility of NFAT-driven IL-12TM-D expression.

[0155] Given that the lenalidomide degron would lead to constitutive expression IL-12, inducible expression of IL-12 could minimize the need to treat patients continuously with LEN. which can be impractical and costly. In addition to conjugating single-chain IL-12 with the degron and the B7-1 TM domain (abbreviated as IL-12TM-D), the IL-12TM-D of Example 1 w as incorporated downstream of an NFAT-inducible promoter (also referred to as “NF AT promoter”) to make IL-12TM-D expression inducible in response to T-cell activation. The NF AT promoter had the nucleotide sequence of SEQ ID NO: 8.

[0156] To test the activity of the degron and the inducibility of NFAT-driven IL-12TM-D expression, a p53 neoantigen model (Kim et al., Cancer Immunol. Res., 10: 932-946 (2022)) was used. A TCR specific for p53R175H(referred to as “p53 TCR”) restricted by human leukocyte antigen (HLA)-A2 was co-transduced with IL-12TM-D. Co-culture with human ovarian cancer cells, TYK-nu, that naturally expressed the p53R175Hneoantigen and HLA-A2, or A2+T2 cells pulsed with the p53R175Hminimal epitope, led to activation of the p53 TCR and subsequent induction of transcriptional activity of NF AT to ultimately express IL-12TM- D (Fig. IF). TYK-nu cells express a cognate mutant p53 epitope, which can activate T cells expressing a mutant-p53-reactive TCR and, in turn, the expression of IL-12 or IL-12TM-D.EXAMPLE 3

[0157] This example demonstrates the effect of LEN treatment on the inducibility of IL- 12TM-D expression.The inducibility of IL-12TM-D expression and the effect of LEN treatment were tested. Jurkat cells were transduced with the p53 TCR, sorted and transduced with membranous IL- 12 with or without the degron. These transduced cells were then co-cultured with HLA-A2+T2 cells pulsed with the mutant p53R175Hpeptide or DMSO control. Without the p53R175Hminimal epitope (ME) (amino acid sequence: HMTEVVRHC) (SEQ ID NO: 32), low level IL-12 was detected, suggesting leaky expression of IL-12 under the NF AT promoter without TCR activation (Fig. 1G and Table 5 A). When co-cultured with p53R175H-pulsed T2 cells, significant induction of membrane IL- 12 expression was detected (Fig. 1G). Upon treatmentwith LEN, near complete reduction of IL-12 expression was noted (Fig. 1G). Membranous IL- 12 without the degron (IL-12TM) did not respond to LEN treatment (Fig. 1 G).TABLE 5 A

[0158] Next, the regulation of IL- 12 expression of p53 TCR-engineered primary T cells was tested in co-cultures with human tumor cell lines, TYK-nu and 4259 patient-derived xenograft colon cancer cells (PDX). The 4259 PDX line, which expressed a p53Y220Cmutation, served as a negative control. Healthy donor-derived T cells were transduced with the p53 TCR in conjunction with either IL-12TM or IL-12TM-D. Both IL-12TM and IL- 12TM-D expression were induced at the modest level when co-cultured with p53R175H+TYK- nu cells but not with 4259 PDX cells (Fig. 1H). Lenalidomide treatment effectively downregulated the expression of IL-12TM-D in the T cells co-cultured with TYK-nu cells.

[0159] Next, Jurkat cells were activated for 24 h with T2 cells pulsed with p53R175HME followed by LEN (1 pM) treatment for the duration of Oh, 15min, 30min. Ih. 3h. 6h, 12h or 24h. IL-12 expression was measured by flow cytometry. Expression of membrane-bound IL-12 rapidly decreased upon LEN treatment (Table 5B; Fig. II).TABLE 5B

[0160] Next, Jurkat cells (transduced with IL-12TM-D in the MSGV vector in reverse onentation) were co-cultured with T2 cells pulsed with p53R175HME for 24 h with varying concentrations of LEN (0~10 pM) treatment. The maximum plasma concentration of LEN in multiple myeloma patients with 25 mg per day dosing is 1.9 pM. IL-12 expression was measured by flow cytometry. Expression of membrane-bound IL-12 expression decreased at a concentration achievable with oral LEN treatment (Table 5C; Fig. 1 J). The half-life was measured to be 1.876 hours (Fig. II) and the IC50 was measured to be 14.76 nM (Fig. 1J).TABLE 5C

[0161] These data demonstrated that IL-12TM-D under the transcriptional control of the NF AT promoter was highly inducible and that LEN treatment effectively abrogated the expression of IL-12TM-D.EXAMPLE 4

[0162] This example demonstrates that IL-12TM-D expression enhances tumor killing, which can be blunted by LEN treatment.

[0163] Using TYK-nu human ovarian cancer cells that naturally expressed p53R175Hand HLA-A2 as a model, the killing efficacy of p53 TCR-transduced T cells with or without IL- 12TM-D or IL-12TM expression was measured. 5,000 TYK-nu cells were co-cultured with 5,000 T cells for 72 h and the number of live cells were measured at every 3 h. Tumor cellscultured alone and T cells transduced with an empty- vector (mock) served as controls. IL- 12TM-D expression enhanced tumor killing (Fig. I K).EXAMPLE 5

[0164] This example demonstrates that transduction efficiency of the IL-12TM-D construct is higher with a SERS vector compared to that which is observed with a lentiviral vector or an MSGV vector.

[0165] To express IL-12TM-D in an inducible fashion with minimal leaky expression, two platforms were considered. A gamma retroviral MSGV vector had intact 5’ long terminal repeats (LTR), which served as a viral promoter, and could therefore induce downstream gene expression even in the presence of the NF AT promoter. Expression of IL- 12 in a reverse orientation was used in the previous clinical trial to reduce the leaky IL-12 expression (Zhang et al.. Clin. Cancer Res.. 21 :2278-2288 (2015)) (Fig. 2A).

[0166] Alternatively, a lentiviral vector or a self-inactivating retroviral vector (SERS) had a truncated 5’ LTR, which could no longer serve as a viral promoter, which allow ed insertion of the NF AT promoter and IL-12TM-D in a forward orientation (Fig. 2A).

[0167] Expression of IL-12TM-D in the lentiviral, MSGV, or SERS vector w as compared. The result showed that lentiviral expression of IL-12TM-D was functional, but inferior to MSGV or SERS driven expression of IL-12TM-D. Increasing the viral titer (from a MOI (Multiplicity- of Infection) of 5 to an MOI of 10) did not improve the transduction efficiency.

[0168] Next, the transduction efficiency of IL-12TM-D was compared between MSGV and SERS, as measured by cell surface expression of IL-12 following T-cell stimulation. When stimulated against TYK-nu cells, the transduction efficiency of IL-12TM-D was significantly higher with the SERS vector as compared to that which was observed with the MSGV vector (Figure 2C). The expression level of IL-12 based on median fluorescence intensity (MFI) was also significantly higher with the SERS vector as compared to that which was observed with the MSGV vector (Fig. 2B) (n=8, fold difference of 5.7, p=0.016).EXAMPLE 6

[0169] This example demonstrates that IL-12TM-D expressed by SERS self-inactivating y -retrovirus show s rapid and sensitive degradation of IL- 12 by LEN treatment.

[0170] Healthy donor PBL transduced with SERS-IL-12 were used to investigate the degradation kinetics of SERS-IL-12. Similar to the MSGV-induced IL-12 expression, the degradation of IL-12 expressed by SERS was rapid and highly sensitive to lenalidomide treatment (Figs. 2F-2G). The half-life was measured to be 3.714 h (Fig. 2F), and the IC50 was measured to be 1.121 nM (Fig. 2G).EXAMPLE 7

[0171] This example demonstrates that the ability of p53 TCR-transduced cells to kill cancer cells is higher with IL-12TM-D in a SERS vector as compared to that which was observed with IL-12TM-D in an MSGV vector.

[0172] T cells transduced with IL-12TM-D in the SERS vector or the MSGV vector of Example 5 were functionally tested. Healthy donor T cells (from 7 different donors) transduced with the p53 TCR with or without IL-12TM-D were co-cultured with TYK-nu cells continuously for 120 hours (h). At the time of the experiment, transduction efficiency with the SERS vector was higher at 59% as compared to 25. 1% with the MSGV vector. The killing ability of the p53 TCR-transduced cells was significantly higher with the SERS vector as compared to that which was observed with the MSGV vector (Fig. 2D). These results indicate that anti -tumor efficacy of T cells was enhanced in an IL-12-level dependent manner (Fig. 2D).

[0173] Next, the functional difference between the SERS vector and the MSGV vector was compared in a different neoantigen model, namely a TCR specific for KRASG12Drestricted by HLA-A11 (Levin et al., Clin. Cancer Res., 27: 5084-5095 (2021)) against autologous patient derived xenograft tumor cells (4069 PDX). At the time of co-culture, the transduction efficiency of IL-12TM-D with the SERS vector and the MSGV vector was 60.6% and 19.8%, respectively. In this model, the T cells transduced with the SERS vector showed significantly better killing of the PDX cells as compared to the MSGV vector (Fig. 2E).

[0174] Based on the more robust transduction of IL-12TM-D and its functional enhancement with the SERS vector, the SERS vector was used as the primary' expression platform in subsequent experiments (unless specified otherwise).EXAMPLE 8

[0175] This example demonstrates that a L22R mutation in the lenalidomide degron enhances the degradation of IL-12TM-D without impairing the function of IL-12TM-D.

[0176] Although high transduction efficiency of IL-12TM-D was achieved with the SERS vector, incomplete degradation of IL-12TM-D was noted upon LEN treatment (Fig. 2C).

[0177] It was hypothesized that the lenalidomide degron sequence could be further modified to achieve more complete IL-12TM-D degradation. The lenalidomide degron sequence was derived from two know n substrates of CRBN: the 5’ half from ZFP91 and 3’ half from IKZF3. The components of the full-length lenalidomide degron sequence of SEQ ID NO: 6 are shown in Table 6.TABLE 6

[0178] The ZFP91 and IKZF3-second components of the degron sequence of SEQ ID NO: 6 together provide the amino acid sequence of: LQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 15).

[0179] Nine candidate single amino acid substitutions w ere selected based on the abundance of amino acids among the library of known substrate sequences of CRBN (Table 7). In Table 7. “WT’?is wild-type, and asterisks indicate the nine selected candidate single amino acid substitutions. The position numbers of the amino acid residues provided in Table 7 correspond to the amino acid residue position numbers of the combined ZFP91 and IKZF3- second components of the degron sequence of SEQ ID NO: 15. For example, the leucine at position number 1 of Table 7 corresponds to the leucine at position number 1 of SEQ ID NO: 15, even though this leucine is located at position 17 of the degron sequence of SEQ ID NO: 6 and at position 610 of the full-length IL-12TM-D of SEQ ID NO: 7. The glycine at position number 14 of Table 7 corresponds to the glycine at position number 14 SEQ ID NO: 15, even though this glycine is located at position 30 of the degron sequence of SEQ ID NO: 6 and at position 623 of the full-length 1L-12TM-D of SEQ ID NO: 7.TABLE 7

[0180] The mutated IL-12TM-D or the WT IL-12TM-D were expressed in p53 TCR+Jurkat cells. Subsequently, the Jurkat cells were subjected to co-culture with HLA-A2+T2 cells pulsed with the p53R175HME in the presence of varying concentration of LEN. Some mutations, such as T9Q and L16M, led to reduced sensitivity to LEN. However, the L22R mutation appeared to enhance the degradation of IL-12TM-D (Fig. 3A).

[0181] Next, healthy human donor T cells were co-transduced with the p53 TCR and IL- 12TM-D without mutations or with the L1F, I20L, or L22R mutations. IL-12 degradation upon LEN treatment was significantly improved with the L22R mutation relative to the WT (Fig. 3B). When TC50 was calculated, the TC50 for the WT (28.94 nM) was 1 57-fold higher than that for the L22R mutant (18.39 nM) (Table 8).TABLE 8

[0182] When the kinetics of IL-12TM-D degradation were measured, the L22R mutant did not show a significant difference to the WT (Fig. 3C). The half-life measured with respect to the WT and the L22R mutant was 4.005 and 3.981 hours, respectively.

[0183] The amino acid sequences of the LR22 IKZF3-second component, the L22R lenalidomide-degradable degron, and the full-length L22R IL-12TM-D are set forth in Table 9. The mutated amino acid residue is underlined in Table 9.TABLE 9EXAMPLE 9

[0184] This example demonstrates that there is no difference in the tumor killing ability of TCR positive cells expressing the L22R mutant IL-12TM-D as compared to those expressing the WT IL-12TM-D.

[0185] Whether the L22R mutant IL-12TM-D of SEQ ID NO: 14 is functionally different compared to the WT IL-12TM-D of SEQ ID NO: 7 was tested. In a tumor killing assay using TYK-nu cells, no difference in the tumor killing ability of T cells expressing the WT and the L22R mutant was observed, indicating the introduction of the L22R mutation does not impact the function of IL-12TM-D (Fig. 3D). All subsequent expenments were carried out using IL- 12TM-D with the L22R mutant, unless specified otherwise.EXAMPLE 10

[0186] This example demonstrates that TILs transduced with IL-12TM-D express IL-12 in vitro upon co-culture with target cancer cells.

[0187] The functional consequences of IL-12TM-D expression in TILs were assessed. The TIL infusion product that was used to treat a cancer patient (Patient 3795) was transduced with either an empty vector or WT IL-12TM-D (SEQ ID NO: 7). When cocultured with the autologous 3795 melanoma cell line, activation of T cells measured by 4- 1 BB upregulation was noted in both the empty -vector control and the IL-12TM-D-expressing TILs. Expression of IL-12 was seen only in the IL-12TM-D-transduced TIL (Figs. 4A-4B).EXAMPLE 11

[0188] This example demonstrates that IL-12TM-D enhances target cancer cell killing in vitro when expressed in TILs.

[0189] The effect of WT IL-12TM-D (SEQ ID NO: 7) expression on anti-tumor functions of T cells was tested using various models.

[0190] The TILs of Example 10 were used to conduct a tumor killing assay. Significant killing of the autologous melanoma cells was seen with the autologous TILs transduced with the empty vector relative to the tumor alone control or the tumor cells treated with irrelevant TILs. However, expression of IL-12TM-D led to more significant killing than the empty vector control (Fig. 4C).

[0191] Next, the functional impact of IL-12TM-D expression was tested in two different KRAS neoantigen models.

[0192] A TCR specific for KRASG12Vrestricted by HLA-C01 :02 (Levin et al., Clin. Cancer Res., 27: 5084-5095 (2021)) (4391 TCR) was co-transduced with IL-12TM-D. Transduced cells were used in a tumor killing assay against autologous colorectal cancer PDX cells. The TCR alone condition showed significant tumor cell killing, but IL-12TM-D expression further enhanced the killing of the PDX cells (Fig. 4D).

[0193] A different TCR specific for KRASG12Dpresented by HLA-C08:02 was initially identified in a colorectal cancer patient, who showed durable clinical responses following a TIL treatment (Tran et al., N. Engl. J. Med., 375:2255-2262 (2016)). Among the three TCRs identified in this patient's TIL that shared the same mutant KRAS specificity and HL A restriction, the TCR used in this experiment (4095-9A TCR) was the one that lacked in vivopersistence (Lu et al., Cancer Immunol. Res., 7: 1824-1836 (2019)). The TCR alone condition did not result in any significant tumor cell killing (Fig. 4E). Only when co-transduced with IL-12TM-D did the KRASG12D-specific TCR lead to significant tumor killing of HP AC cells (human pancreatic cancer cells that naturally expressed KRASG12Dand HLA-C08:02).

[0194] Lastly, the effect of IL-12TM-D expression was tested in a well-characterized CD 19 chimeric antigen receptor (CAR) model. It was previously reported that CD 19 complete loss or down regulation was one of the prominent tumor-escape mechanisms (Majzner et al., Cancer Discov.. 10:702-723 (2020)). In this report, CD19 was knocked out ofNALM6 leukemia cells expressing high levels of endogenous CD 19 and subsequently CD 19 was exogenously re-expressed to generate CD 19 low or intermediate cell clones.

[0195] Using these CD19-low (“NALM6-Z”) and intermediate (“NALM6-H”) clones, it was examined whether expression of IL-12TM-D could enhance the effector function of a CAR to address antigen downregulation as an escape mechanism. An anti-CD19 CAR with the intracellular CD28 domain (Kochenderfer et al.. Blood, 116: 4099-4102 (2010)) was cotransduced with IL-12TM-D. When co-cultured with unmodified CD19+NALM6, robust T- cell activation (measured by 4- IBB upregulation) and expression of IL- 12 were noted (Table 10). Against NALM6-11 and NALM6-Z clones, 4-1BB and IL-12 levels w ere reduced in a CD19-level dependent manner (Table 10). Anti -tumor function of the anti-CD19 CAR with or without IL-12 were assessed against unmodified NALM6, NALM6-11 and NALM6-Z. Regardless of whether IL-12 was expressed or not, the anti-CD19 CAR was effective in killing unmodified NALM6 cells (Fig. 4H). In the case of NALM6-11 (CD19-intermediate) and NALM6-Z (CD19-low) cells, co-expression of IL-12TM-D and the anti-CD19 CAR w-as significantly more efficient in killing NALM6- 11 and NALM6-Z cells as compared to the anti-CD19 CAR alone (Figs. 4F, 4G). Regardless of IL-12TM-D expression, no significant killing of CD19 knock-out clones w as observed, indicating that antigen specificity of CD19 CAR was not affected by IL-12TM-D expression. (Fig. 41). The data suggested that IL- 12TM-D expression may potentiate the function of CAR-T cell therapies to prevent tumor escape due to downregulation of CD 19.TABLE 10EXAMPLE 12

[0196] This example demonstrates that the expression of IL-12TM-D can be effectively regulated in adoptively transferred T cells.

[0197] To study the regulation of WT IL-12TM-D (SEQ ID NO: 7) expression in vivo, immunoincompetent NOD-scid IL2Rgammanul1(NSG) mice were subcutaneously implanted with TYK-nu cells (Fig. 5A). At four weeks, the tumor reached 50-100 mm2in size, which was expected to maximize T-cell activation for the tumor-infiltrating T cells. T cells cotransduced with the p53 TCR and IL-12TM-D were injected intravenously. On the day of T- cell injection, the T cells were subjected to an overnight co-culture with TYK-nu cells to evaluate the expression of IL-12TM-D in vitro (Fig. 5B).

[0198] On day 6 and 7 post-T-cell injection, the mice were treated with pomalidomide (POM) (30 mg / kg) or PBS as a vehicle twice (once daily) by oral gavage. PBS was a vehicle used to dilute POM. POM is an analogue of thalidomide that is also approved for the treatment of multiple myeloma and myelodysplastic syndromes. POM was chosen for the in vivo experiment because it has a longer plasma in vivo half-life than LEN. Two hours after the second POM treatment, tumors and peripheral blood were harvested. Blood serum analysis revealed a significant increase in serum IFN-y level, which was unaffected by pomalidomide treatment. IL-12 level was detected at a low level (-0. 1 pg / mL) in the mice that received T cells expressing both the p53 TCR and IL-12TM-D, which was also unchanged by pomalidomide treatment. To minimize enzymatic shedding of IL-12TM-D. the tumors were mechanically homogenized. Tumor-infiltrating T cells were isolated for analysis. Flow cytometric analysis showed that the T cells from the PBS-treated mice expressed IL-12TM-D at a higher level as compared to those from the POM-treated mice (3.3 fold, p<0.001; Fig. 5C).

[0199] Although POM-treatment led to significantly reduced IL-12TM-D expression, the absolute level of IL-12TM-D induction in the T cells from the PBS-treated mice in Fig. 5C appeared low relative to the in vitro co-culture result in Fig. 5B. When T-cell activation measured by 4-1BB was compared to the IL-12 level, linear correlation was observed (Fig.5D). These data indicated that at the time of the tumor harvest, maximal T-cell activation was not achieved or the tumor-infdtrating T cells were exhausted and could not express high levels of 4-1BB and hence IL-12TM-D.EXAMPLE 13A

[0200] This example demonstrates that IL-12TM-D enhances the anti -tumor function of adoptively transferred T cells in a pre-clinical mouse model.

[0201] 50 NSG mice were subcutaneously injected with 2 million TYK-nu cells 2 weeks before T-cell injection. PBS (vehicle control) or T cells transduced with p53 TCR alone, p53 TCR + IL-12TM (in the MSGV vector in reverse orientation), or p53 TCR + IL-12TM-D (in the MSGV vector in reverse orientation) were tested. 3 dose levels were tested: 0.5, 1.5 or 5x106T cells per mouse were injected (Fig. 9A). Healthy-donor PBLs were transduced, enriched for CD8 and p53 TCR and expanded in a GREX plate (6 well) for 17 days.

[0202] On the day of T-cell injection into mice, the T cells were co-cultured with TYK- nu cells, and expression of IL-12 was measured by flow cytometry (Table 11).TABLE 11

[0203] T cells expressing p53 TCR and IL-12TM-D showed enhanced anti-tumor efficacy relative to TCR alone at 1.5 or 5 million cells dose levels (Figs. 9B-9D). In this experiment, expression of IL-12TM did not lead to enhanced tumor regression, possibly due to low transduction efficiency.EXAMPLE 13B

[0204] This example demonstrates that IL-12TM-D potentiates the anti-tumor function of adoptively transferred T cells in a pre-clinical mouse model.

[0205] Next, the anti-tumor efficacy of IL-12TM-D expression using the SERS vector was evaluated using TYK-nu cells as a model. NSG mice were subcutaneously implanted with TYK-nu cells. T cells were transduced as a mock or with the p53 TCR alone or with both the p53 TCR and IL-12TM-D. Eighteen days after the tumor-cell injection, the tumorbearing mice were administered T cells either at 5 million cells per mouse or 15 million cells per mouse (Fig. 5E).

[0206] At the dose level of 5 million cells per mouse, mice treated with T cells expressing both the p53 TCR and IL-12TM-D showed significantly less tumor growth as compared to those treated with the mock T cells or T cells expressing the p53 TCR alone (Fig. 5F).

[0207] At 15 million cells per mouse, the mice that received the T cells expressing the p53 TCR alone showed significant tumor control relative to those who received the mock- transduced T cells. However, the mice that received the T cells expressing both the p53 TCR and IL-12TM-D showed even better tumor control than the p53 TCR alone group (Fig. 5G). All five mice were cured of tumors (Fig. 5G). This improved tumor control translated into long-term survival: among the mice treated with the p53 TCR and IL-12TM-D, three out of five mice treated at the 5 million cell dose and all five mice treated at the 15 million cell dose survived long term, whereas none of the mice treated with the p53 TCR alone achieved longterm survival (Fig. 5H-5I).EXAMPLE 14

[0208] This example demonstrates that IL-12TM-D provides more efficient transduction as compared that which is observed with IL-12TM (without the degron sequence).

[0209] IL-12 has a significant anti-proliferative effect on T cells (Zhang et al., Clin.Cancer Res., 21: 2278-2288 (2015)). Because the expression of the IL-12-degron can be downregulated during and after transduction by LEN treatment. T cells can be preserved from the anti-proliferative effect of high-level IL-12 expression, leading to higher IL-12 transduction efficiency than IL-12TM without a degron.

[0210] T cells were transduced with a self-inactivating retroviral vector (SERS) encoding IL-12TM (no degron) or WT IL-12TM-D (SEQ ID NO: 7). The transduced cells were cultured in the presence of LEN. The percentage of IL-12 positive cells was measured. Transduction was significantly more efficient (2.39-fold higher, p<0.001) with IL-12TM-D as compared to that which was observed with IL-12TM (Fig. 6).

[0211] T cells were transduced with a SERS vector encoding IL-12TM (no degron) or WT IL-12TM-D (SEQ ID NO: 7). IL-12 positivity was measured following rapid expansion using OKT3 (anti-CD3 antibody), high-dose IL-2 and feeders. IL- 12 with the degron maintained significantly higher levels of IL- 12 positivity than IL- 12 without the degron. These data also support that down-regulation of IL- 12 during T-cell rapid expansion can protect T cells from the anti-proliferative effect of IL- 12.EXAMPLE 15

[0212] This example demonstrates that cells transduced with MSGV encoding IL-12TM- D achieve higher IL-12 expression than those transduced with MSGV encoding IL-12TM.

[0213] T cells were transduced with a MSGV encoding IL-12TM (no degron) or WT IL- 12TM-D (SEQ ID NO: 7). The percentage of IL-12 positive cells was measured. Cells transduced with IL-12TM-D demonstrated higher (1.57-fold) IL-12 expression following rapid expansion of the number of transduced cells as compared to cells transduced with IL- 12TM (Fig. 7), indicating in an MSGV setting, IL-12TM-D can be more effectively transduced than IL-12TM.EXAMPLE 16

[0214] This example demonstrates the adjustment of IL-12TM-D expression to reduce or prevent exhaustion associated with high-level IL- 12 expression.

[0215] Although IL- 12 has been regarded as a bona fide proinflammatory cytokine, sustained and high levels of IL-12 expression can cause T-cell exhaustion (Gerhardt et al., J. Immunol., 210(10): 1598-1606 (2023); Yang et al., J. Clin. Invest., 122(4): 1271-82 (2012)). It was hypothesized that low concentrations of lenalidomide can reduce IL- 12 expression to prevent excessive differentiation or exhaustion of T cells without compromising the efficacy of IL-12TM-D. Using the p53 TCR and TYK-nu cells, it was tested whether repeated induction of IL-12TM-D expression could enhance T-cell exhaustion. First, lenalidomide concentrations were titrated to limit IL- 12 expression to <50% of the maximum level achieved without lenalidomide treatment. When treated with 100 nM lenalidomide, T cells from two healthy donors, co-transduced w ith IL-12TM-D and the p53 TCR, showed a 60- 80% reduction in IL-12 expression relative to untreated cells. These T cells were then subjected to six rounds of co-culture with TYK-nu cells with tw o to three-day intervals over 14 days. At days 0, 7 and 14, an aliquot of cells was harvested for flow cytometric analysisof exhaustion markers and single-cell indexing of transcriptomes and epitopes by sequencing (CITE-seq) (Stoeckius et al., Nat. Methods, 14(9): 865-868 (2017)). CITE-seq was used to complement transcript-based measurement (conventional single-cell RNA sequencing) of expression changes in exhaustion-associated molecules, some of which were surface proteins. Using flow cytometry, TIGIT (T cell immunoreceptor with Ig and ITIM domains), TIM-3 (T- cell immunoglobulin and mucin-domain containing-3, also known as HAVCR2), PD-1 (programmed cell death protein 1), CD39 (ENTPD1), and CD69 were examined as markers for T-cell exhaustion as well as CD62L as a naive / stem-like T cell marker (Fig. 8A). TIGIT levels, when normalized to that of day 0, significantly increased at day 14 in T cells expressing the p53 TCR and IL-12TM-D relative to the T cells expressing p53 TCR alone (Fig. 8B). This trend was reversed by co-treatment of 100 nM lenalidomide (Fig. 8B). Other markers of exhaustion did not show significant changes.

[0216] At day 14 after repeat co-culture, T cells were subjected to CITE-seq. A total of 22,973 cells were analyzed using Uniform Manifold Approximation and Projection (UMAP) based on transcriptome data. Seventeen distinct clusters were identified and all the clusters contained T cells positive for the p53 TCR expression except for clusters 10 and 16. Based on gene expression by transcriptome and by cell-surface expression (measured by barcoded antibodies), clusters 0 and 3 showed high levels of exhaustion-associated genes, such as TIGIT and TOX along with low levels of genes associated with T-cell sternness, such as KLF2 and SLAMF6 (CD352) as well as cytotoxicity-related genes, such as PRF1 and GZMH, indicating pronounced exhaustion of these clusters of cells at the expense of sternness and effector functions. In contrast, cluster 1 and 11 exhibited the opposite profde with reduced exhaustion and increased sternness and effector functions as well as proliferation (MKI67, TOP2) relative to clusters 0 and 3. Single-cell gene set enrichments analyses revealed that cluster 0 and 3 bore similarity with CD39+exhausted cells (Oh et al., Cell, 181(7): 1612-1625 el3 (2020)) and terminally differentiated cells (Krishna et al., Science, 370(6522): 1328-1334 (2020)), respectively, while cluster 1 and 11 showed gene expression similar to that of proliferating T cells (Oh et al.. Cell, 181(7): 1612-1625 el3 (2020); Oliveira et al., Nature, 596(7870):! 19-125 (2021)). Among the 4 tested conditions, clusters 0 and 3 were largely composed of T-cells transduced with IL-12TM-D without lenalidomide, which were greater than cells without IL-12TM-D transduction or lenalidomide treatment or cells transduced with IL-12TM-D and treated with low-dose lenalidomide. On the other hand, cluster 1 was largely populated with IL-12TM-D LEN" or IL-12TM-D+LEN+cells, which were greater than IL-12TM-D LEN" cells in frequencies. Similarly, cluster 11 contained predominantly IL-12TM-D LEN+cells. These data demonstrated that uncontrolled expression of IL-12 could enhance T-cell exhaustion, which was prevented by low-dose lenalidomide treatment. Finally, in an in vitro killing assay using p53 TCR-expressing T cells after 14 days of repeat co-culture, cells that had been treated with low-dose lenalidomide exhibited significantly enhanced tumor cell-killing abilities compared to T cells without lenalidomide treatment, possibly due to reduced exhaustion and increased effector function (Fig. 8C). Taken together, these data indicate that uncontrolled expression IL- 12TM-D can enhance T-cell exhaustion, and low-dose lenalidomide can fine-tune IL-12TM- D expression to minimize exhaustion, ultimately improving IL-12TM-D’s anti -tumor efficacy.EXAMPLE 17

[0217] This example demonstrates that tumor regression and survival are observed in mice treated with cells transduced with both a KRASG12V-reactive TCR and IL-12TM-D.

[0218] The in vivo efficacy of IL-12TM-D was tested in a different xenograft ACT model. Colorectal cancer 4391 PDX cells (KRASG12V+) were subcutaneously injected into NSG mice. Ten million healthy -donor T cells expressing the KRASG12V-reactive TCR and IL-12TM-D, along with other control conditions, were intravenously injected (Fig. 10A). In this experiment, KRASG12V-reactive TCR alone did not regress the tumor, possibly due to low CD8 cell proportions. However, T cells expressing both the KRASG12V-reactive TCR and IL-12TM-D led to complete tumor regression and long-term survival (Fig. 10B-10C).

[0219] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0220] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of twoor more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms ‘'comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary’ language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0221] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising: a cytokine, a functional portion of the cytokine, a T-cell regulatory protein, or a functional portion of the T-cell regulatory protein; a transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence; and an immunomodulatory imide drug (IMiD)-degradable degron, wherein the nucleotide sequence encoding the polypeptide comprises a promoter operatively associated with the nucleotide sequence encoding polypeptide.

2. The nucleic acid of claim 2. comprising a nucleotide sequence of Formula I;5 ’ -P-R'm-C-R r-L'-DT'(Formula I), wherein:P is the promoter;C is the cytokine, the functional portion of the cytokine, a T-cell regulatory protein, or a functional portion of the T-cell regulatory protein;T is the transmembrane-intracellular amino acid sequence or the transmembrane amino acid sequence; each one of R1and R2is, independently 1 to 3 amino acid residues; each one of m and n is. independently. 0 or 1;L1is a first linker sequence; andD is the IMiD-degradable degron.

3. The nucleic acid of claim 2. wherein L1comprises 5 to 50 naturally occurring amino acid residues.

4. The nucleic acid of any one of claims 1-3, wherein the T-cell regulatory protein is an immune checkpoint molecule.

5. The nucleic acid of any one of claims 1-3, wherein the T-cell regulatory protein is a stimulatory immune checkpoint molecule.

6. The nucleic acid of claim 5. wherein the stimulatory immune checkpoint molecule is a member of the tumor necrosis factor (TNF) receptor superfamily.

7. The nucleic acid of claim 5, wherein the stimulatory immune checkpoint molecule is CD122, CD137, CD27, CD28, CD40, GITR, ICOS, or 0X40.

8. The nucleic acid of any one of claims 1-3, wherein the T-cell regulatory protein is an inhibitory immune checkpoint molecule.

9. The nucleic acid of claim 8. wherein the inhibitory immune checkpoint molecule is A2AR. A2BR, B7-H4. BTLA. CD276, CTLA-4, IDO, KIR, LAG3, N0X2, PD-1, SIGLEC7, SIGLEC9, TIM-3, TIGIT, TET2, CD39, CD 103 or VISTA.

10. The nucleic acid of any one of claims 1-3, wherein the cytokine is IL-2, single chain IL-7, single chain IL-12. IL-15, IL-18, tumor necrosis factor alpha (TNF-a), or interferon-gamma.

11. The nucleic acid of any one of claims 1-3, wherein the cytokine is single chain IL- 12.

12. The nucleic acid of claim 1 1, wherein the single chain IL-12 comprises an amino acid sequence of Formula II:S'p-N’-LVS N2(Formula II), wherein: each one of S1and S2is, independently, a signal sequence; each one of p and q is, independently, 0 or 1; each one of N1and N2is an IL-12 p40 amino acid sequence and the other is an IL-12 p35 amino acid sequence; andL2is a second linker sequence.

13. The nucleic acid of claim 12, wherein N1is the IL-12 p40 amino acid sequence and N2is the IL-12 p35 amino acid sequence.

14. The nucleic acid of any one of claims 1-13, wherein the transmembrane- intracellular amino acid sequence or transmembrane amino acid sequence is a B7-1 transmembrane-intracellular amino acid sequence, a B7-2 transmembrane-intracellular amino acid sequence, a CD8a transmembrane-intracellular amino acid sequence, a CD28 transmembrane-intracellular amino acid sequence, a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, a CD8a transmembrane amino acid sequence, a human leukocyte antigen (HLA) transmembrane amino acid sequence, or a CD28 transmembrane amino acid sequence.

15. The nucleic acid of any one of claims 1-14, wherein the IMiD-degradable degron comprises an amino acid sequence at least 90% identical to QKGNLLRHIKXHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 16), wherein X at position 11 of SEQ ID NO: 16 is A, C. D, E, F, G, H, I, K, L, M, N, P, Q, R. S, T, V, W, or Y.

16. The nucleic acid of any one of claims 1-15, wherein the IMiD-degradable degron comprises an amino acid sequence at least 90% identical to QKGNLLRHIKXHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 16), wherein X at position 11 of SEQ ID NO: 16 is I. K, L, or R.

17. The nucleic acid of any one of claims 1-16 wherein the IMiD-degradable degron comprises the amino acid sequence of QKGNLLRHIKRHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 12).

18. The nucleic acid of claim 1, comprising a nucleotide sequence encoding an amino acid sequence at least 90% identical to SEQ ID NO: 18, wherein X at position 631 of SEQ ID NO: 18 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S. T, V, W, or Y.

19. The nucleic acid of claim 1, comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 14.

20. The nucleic acid of any one of claims 1-19, wherein the nucleic acid is DNA.

21. The nucleic acid of any one of claims 1-19, wherein the nucleic acid is RNA.

22. A nanoparticle comprising the nucleic acid of any one of claims 1-21.

23. A polypeptide encoded by the nucleic acid of any one of claims 1-21.

24. A recombinant expression vector comprising the nucleic acid of any one of claims 1-21.

25. The recombinant expression vector of claim 24, wherein the recombinant expression vector is a retroviral vector, a lentiviral vector, or a transposon.

26. The recombinant expression vector of claim 24, wherein the recombinant expression vector is a self-inactivating vector.

27. The recombinant expression vector of any one of claims 24-26, wherein the recombinant expression vector comprises a vector backbone comprising a 5’ long terminal repeat (LTR) and a 3?LTR, wherein the nucleotide sequence encoding the polypeptide is positioned in between the 5’ LTR and the 3’ LTR, wherein the 5’ LTR and the 3’ LTR are each positioned in 5’ to 3’ orientation, and wherein the nucleotide sequence encoding the polypeptide is positioned in 3’ to 5 ’ orientation relative to the 5’ LTR and the 3’ LTR.

28. A viral particle comprising the nucleic acid of any one of claims 1-21, the nanoparticle of claim 22. or the recombinant expression vector of any one of claims 24-27.

29. An isolated host cell comprising the recombinant expression vector of claim 24- 27.

30. An isolated host cell expressing the nucleic acid of any one of claims 1-21, the nanoparticle of claim 22. or the polypeptide of claim 23.

31. The host cell of claim 29 or 30, wherein the host cell comprises an antigenspecific receptor.

32. The host cell of claim 31, wherein the antigen-specific receptor is a chimeric antigen receptor (CAR).

33. The host cell of claim 31, wherein the antigen-specific receptor is an endogenous T cell receptor (TCR).

34. The host cell of claim 33, wherein the host cell is a tumor infiltrating lymphocyte (TIL).

35. The host cell of claim 31, wherein the antigen-specific receptor is an exogenous TCR.

36. The host cell of any one of claims 31-35, wherein the antigen-specific receptor has antigenic specificity for a cancer antigen.

37. An isolated population of host cells comprising the host cell of claim 29-36.

38. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleic acid of any one of claims 1-21, the nanoparticle of claim 22, the polypeptide of claim 23, the recombinant expression vector of any one of claims 24-27, the viral particle of claim 28, the host cell of any one of claims 29-36, or the population of cells of claim 37.

39. The nucleic acid of any one of claims 1-21, the nanoparticle of claim 22. the polypeptide of claim 23, the recombinant expression vector of any one of claims 24-27, theviral particle of claim 28, the host cell of any one of claims 29-36, the population of cells of claim 37, or the pharmaceutical composition of claim 38 for use in the treatment of cancer in a mammal.

40. The nucleic acid of any one of claims 1-21, the nanoparticle of claim 22, the polypeptide of claim 23, the recombinant expression vector of any one of claims 24-27, the viral particle of claim 28, the host cell of any one of claims 29-36, the population of cells of claim 37, or the pharmaceutical composition of claim 38 for use in the enhancement of an immune response against cancer in a mammal.

41. The nucleic acid, nanoparticle, polypeptide, recombinant expression vector, viral particle, host cell, population of cells, or pharmaceutical composition for the use of claim 39 or 40, further comprising (i) an antigen-specific receptor, a nucleic acid comprising a nucleotide sequence encoding the antigen-specific receptor, a recombinant expression vector comprising the nucleic acid, a host cell comprising the recombinant expression vector, a population of cells comprising the host cell, or (ii) a pharmaceutical composition comprising (i), wherein the antigen-specific receptor has antigenic specificity for an antigen of the cancer.

42. A method of culturing T cells in vitro. the method comprising: introducing the nucleic acid of any one of claims 1-21, the nanoparticle of claim 22, or the recombinant expression vector of any one of claims 24-27 into isolated T cells, thereby expressing the nucleic acid by the host cells.

43. The method of claim 42, further comprising expanding the number of T cells.

44. The method of claim 43, wherein expanding the number of T cells comprises expanding the number of T cells in the presence of (a) one or more cytokines and (b) one or more non-specific T cell stimuli.

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