Novel regulators of il-2 secretion for enhanced t-cell immunotherapy

Abstract

Described herein are methods to promote endogenous IL-2 production in a cell, cells and cell populations comprising modifications to enhance IL-2 production in the cell, and methods of use.

Description

Atty. Dkt. No.: 121384-0289NOVEL REGULATORS OF IL-2 SECRETION FOR ENHANCED T-CELL IMMUNOTHERAPYCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 714,633 filed October 31, 2024, which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number CA277507 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] The present invention relates generally to the field of T-cell immunotherapy.

[0005] The pivotal role of IL-2 signaling in regulating the proliferation, differentiation, and memory development of CD8+ T cells underscores a significant, yet unaddressed, opportunity to enhance immunotherapeutic efficacy across various cancer types. Although high-dose IL-2 is indispensable for promoting T cell expansion in Tumor-Infiltrating Lymphocyte (TIL) therapy, its broader clinical utility is curtailed by severe toxicities, such as capillary leak syndrome and end-organ dysfunction. Emerging evidence suggests that autocrine IL-2 signaling, particularly during secondary viral infections, is critical for memory T cell expansion, thereby indicating that exogenous IL-2 supplementation alone might be insufficient for optimal cellular immunotherapy.

[0006] Thus, there is an unmet need for a T cell therapy that can function without exogenous IL-2.-1-4870-3559-0385.3Atty. Dkt. No.: 121384-0289SUMMARY OF THE INVENTION

[0007] To refine T cell-mediated antitumor responses, Applicant has developed cellular therapies that endogenously enable T cells to produce IL-2. Applicant has developed methods to promote endogenous IL-2 production in a T cell, to reduce or remove the need for exogenously applied IL-2.

[0008] Applicant has circumvented the toxicity issue by enabling T cells to autonomously produce IL-2, which sustains their proliferative and cytotoxic functions over extended periods. This self-sufficiency diminishes the toxicity associated with high systemic IL-2 levels but also enhances the overall durability and effectiveness of the T cell therapeutic response.

[0009] Described herein are methods of immunotherapy comprising enhancing IL-2 production in a subject in need thereof concurrently with administering immunotherapy to the subject in need thereof. In some embodiments at least one regulator of IL-2 production is knocked down or knocked out in a T cell population administered to the subject in need, thereby enabling increased endogenous production of IL-2 in the cell. The at least one regulator of IL-2 production may be selected from HRH2, IL1R11, PTGER2, OR13D1, MC1R, LOXL2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, TBKBP1, RASA4, ACKR4, ITGB1, and AKR1A1.

[0010] Also described herein is a T cell comprising at least one modification that enhances IL-2 production. In some aspects the modification is a gene knockdown or knockout. In some aspects a gene selected from HRH2, IL1R11, PTGER2, OR13D1, MC1R, LOXL2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, TBKBP1, RASA4, ACKR4, ITGB1, and AKR1 Al is knocked down or knocked out. In one aspect the T cell is a CD8+ cell or a tumor infiltrating lymphocyte. In some aspects the T cell has been engineered to include a T cell receptor, a synthetic agonist receptor, and / or a chimeric antigen receptor.

[0011] Also described herein is a population of T cells comprising at least one modification that enhances IL-2 production.-2-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[00121 Further described herein are compositions and kits comprising the T cell or T cell population described herein. The composition may include an additional therapeutic agent. The kit may comprise the composition and instructions for use.

[0013] Also provided herein are methods to promote longevity, growth, or expansion in the T cell or T cell population described herein, comprising growing the T cell or T cell population in a culture medium, wherein exogenous IL-2 is not included in the culture medium.[00141 Also provided herein are methods to promote longevity, growth, or expansion in the T cell or T cell population described herein, comprising growing the T cell or T cell population in a culture medium, wherein a low or reduced amount of exogenous IL-2 is included in the culture medium, and wherein the low or reduced dose is determined in proportion to the dose that would be administered to T cell that does not comprise at least one modification that enhances IL-2 production.[00151 Further provided herein are methods to promote longevity, growth, or expansion of a T cell population in a subject in need, comprising administering to the subject the T cell population described herein, wherein exogenous IL-2 is not concurrently or consecutively administered to the subject in need.

[0016] Still further provided herein are methods to promote longevity, growth, or expansion of a T cell population in a subject in need, comprising administering to the subject the T cell population described herein, wherein a low or reduced dose of exogenous IL-2 is concurrently or consecutively administered to the subject in need. The low or reduced dose administered with the T cell comprising a modification to enhance IL-2 production is lower than the dose that would be administered to a subject with a T cell or T cell population that does not comprise at least one modification that enhances IL-2 production.[0017| Still further provided herein are methods to inhibit the growth of cancer and / or increase the survival rate of a subject in need comprising administering to the subject the T cell, T cell population, or composition described herein to the subject in need, wherein exogenous IL-2 is not concurrently or consecutively administered to the subject in need.-3-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[0018| Still further provided herein are methods to inhibit the growth of cancer and / or increase the survival rate of a subject in need comprising administering to the subject the T cell, T cell population, or composition described to the subject in need, wherein a low or reduced dose of exogenous IL-2 is concurrently or consecutively administered to the subject in need. The low or reduced dose administered with the T cell comprising a modification to enhance IL-2 production is lower than the dose that would be administered to a subject with to a T cell or T cell population that does not comprise at least one modification that enhances IL-2 production.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGURE 1. Schematic Diagram Illustrating the Workflow for Identifying Positive Regulators of IL-2 Secretion in Human Primary Cytotoxic T Cells: This figure outlines the systematic approach employed to pinpoint genetic elements that enhance IL-2 production in primary human cytotoxic T cells, utilizing a comprehensive genome-wide CRISPR-Cas9 knockout screen.|'<)020] FIGURE 2. Next-Generation Sequencing Results from the Genome-Wide CRISPR Screen in Human Primary Cytotoxic T Cells.

[0021] FIGURE 3. Fold Change in IL-2 Secretion in Knockout Cell Groups Compared to Control Group. This figure quantifies the changes in IL-2 secretion between knockout cell groups and the control group (Primary human CD8+ cells). The analysis, based on data from three independent experiments (n=3), utilizes a one-way ANOVA to statistically evaluate the differences in IL-2 secretion levels across the groups.

[0022] FIGURE 4. Longevity Assay of Knockout and Control Cell Groups. This figure depicts the results of a longevity assay comparing the survival of knockout cell groups with a control cell group (Primary human CD8+ cells), without IL2 addition to the media. The data, derived from three replicates (n=3), are analyzed using a two-way ANOVA to assess the impact of gene knockout on cell survival over time.

[0023] FIGURE 5A shows volcano plot showing differential gene expression. Genes above the significance threshold (FDR = 0.05) are colored blue and considered significantly-4-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 differentially expressed, while non- significant genes are shown in gray. FIGURE 5B shows distribution of sgRNAs targeting selected genes across samples, demonstrating that each gene is targeted by four distinct sgRNAs. FIGURE 5C shows Gene Ontology (GO) enrichment analysis of significantly enriched sgRNAs, highlighting the top immune regulatory pathways.

[0024] FIGURE 6A shows Enzyme-linked immunosorbent assay (ELISA) quantification of IL-2, IFN-y, and TNF-a in primary human CD8+T cells following targeted knockout of IL-2 regulatory genes. Data are mean ± SD of n = 3 replicates. FIGURE 6B shows long-term survival analysis of knockout and control T cell populations cultured in the absence of exogenous IL-2 in knockout cells. Data are mean ± SD of n = 3 replicates. FIGURE 6C shows flow cytometric profiling of canonical exhaustion markers under IL-2-deprived conditions. FIGURE 6D shows T cell cytotoxicity after co-killing of 48 hours or 72 hours via prolonged co-culture cytotoxicity assays using A375 melanoma cells. FIGURE 6E shows flow cytometric profiling of degranulation markers and cytotoxic markers under IL-2- deprived conditions. FIGURE 6F shows differential gene expression analysis via RNA sequencing of LOXL2-deficient CD8+T cells. FIGURE 6G shows Gene set enrichment analysis (GSEA) in LOXL2-deficient CD8+T cells.

[0025] FIGURE 7A illustrates the molecular regulation of IL-2 secretion following LOXL2 knockout (lower panel) or without LOXL2 knockout (upper panel) in primary human CD8+T cells. FIGURE 7B shows protein expression before (WT) and after LOXL2 knockout (LOXL2-KO) measured by western blot. FIGURE 7C shows treatment with a NFKB and HIF-loc inhibitor leads to increased IL-2 secretion in both control and LOXL2-deficient T cells. Data are mean ± SD of n = 3 replicates. FIGURE 7D shows cytotoxicity of edited T cells and un-edited T cells against human primary skin cells under 1% O2 hypoxic condition. Data are mean ± SD of n = 3 replicates. FIGURE 7E shows cytotoxicity of edited T cells and un-edited T cells against Nalm6 leukemia, SKOV3 and SKBR3 breast cancer cell lines under 1% O2 hypoxic condition. Data are mean ± SD of n = 3 replicates. FIGURE 7F shows upregulated CD4 helper-like marker genes expression in LOXL2-deficient CD8+T cells revealed by RNA sequencing, and their alignment with published RNA sequencing profile of human CD4+ T cells (R=0.84, P value=0).-5-4870-3559-0385.3Atty. Dkt. No.: 121384-0289

[0026] FIGURE 8A illustrates the in vivo experimental workflow used to assess tumor suppression and survival outcomes following adoptive T cell transfer. FIGURE 8B shows tumor growth kinetics in KPC-bearing immunocompetent mice treated with vehicle control, wild-type (WT) CD8+T cells, LOXL2-knockout T cells (20% ACT dosage), or MC1R- knockout T cells (20% ACT dosage). Data are mean ± SD of n = 3 replicates. FIGURE 8C shows Kaplan-Meier survival analysis in KPC-bearing immunocompetent mice receiving LOXL2 or MClR-knockout T cells Data are mean ± SD of n = 3 replicates. FIGURE 8D shows flow cytometric analysis of tumor-infiltrating lymphocytes in tumors of KPC-bearing immunocompetent mice treated with knockout T cells relative to wild type and control groups. Data are mean ± SD of n = 5 replicates. FIGURE 8E shows tumor growth kinetics in CT26-bearing immunocompetent mice treated with vehicle control, wild-type (WT) CD8+T cells, or LOXL2-knockout T cells (20% ACT dosage). Data are mean ± SD of n = 3 replicates. FIGURE 8F shows Kaplan-Meier survival analysis in CT26-bearing immunocompetent mice receiving LOXL2-knockout T cells. FIGURE 8G shows flow cytometric analysis of tumor-infiltrating lymphocytes shows altered immune cell composition and reduced exhaustion marker expression in tumors of CT26-bearing immunocompetent mice treated with knockout T cells relative to WT and control groups. Data are mean ± SD of n = 5 replicates. FIGURE 8H shows tumor growth kinetics and body weight monitoring in KPC-bearing immunocompetent mice treated with vehicle control, wild-type (WT) CD8+T cells, or LOXL2-knockout (KO) CD8+T cells at 50% adoptive cell transfer (ACT) dosage.DETAILED DESCRIPTION

[0027] Unless otherwise specified “a” or “an” means one or more.

[0028] All numeric values should be treated as having the term “about” placed before a specific numeric value.

[0029] As used herein, the term “about” placed before a specific numeric value may mean ±20% of the numeric value; ±18% of the numeric value, ±15% of the numeric value; ±12% of the numeric value; ±8% of the numeric value; ±5% of the numeric value; ±3% of the numeric value; ±2% of the numeric value; ±1% of the numeric value or ±0.5% of the numeric value.-6-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[0O3O| The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0032] The term “subject” refers includes but is not limited to a subject at risk of an immune disorder, or autoimmune response, disorder or disease, as well as a subject that has already developed an immune disorder, or autoimmune response, disorder or disease. Such subjects, include mammalian animals (mammals), such as a non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans. Subjects include animal disease models, for example, mouse and other animal models of immune disorders, or autoimmune response, disorder or disease known in the art.

[0033] Treatment of cancer or an infection, immune disorder, or autoimmune response, disorder or disease can be at any time during the cancer or an infection, immune disorder, or autoimmune response, disorder or disease. Certain embodiments of the present disclosure can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the methods described herein as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by cancer or an infection, immune disorder, or autoimmune response, disorder or disease, or an adverse symptom, condition or complication associated with or caused by cancer or an infection, immune disorder, or autoimmune response, disorder or disease. Thus, a method can be practiced one or more times (e.g., 1-10, 1-5 or 1-3 times) an hour, day, week, month, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. A non-limiting dosage schedule is 1-7 times per week,-7-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.

[0034] Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies, e.g. a mouse, and the amount treatment or agent disclosed herein administered in an amount that is determined to be effective.[00351 Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has previously had cancer or an infection, immune disorder, or autoimmune response, disorder or disease, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, the treatment protocol and compositions, the clinical endpoint desired, the occurrence of previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0036] In the methods of the invention, the route, dose, number and frequency of administrations, treatments, and timing / intervals between treatment and disease development can be modified. In certain embodiments, a desirable treatment of the present disclosure will elicit robust, long-lasting immunity against cancer or an infection, immune disorder, or autoimmune response, disorder or disease. Thus, in certain embodiments, disclosure methods, uses and compositions provide long-lasting immunity to cancer or an infection, immune disorder, or autoimmune response, disorder or disease.[0037J A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0038] The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed-8-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0039] The term “protein,” “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0040] As used herein, the term “T cell,” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T- cells may either be isolated or obtained from a commercially available source. “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gammadelta T cells.

[0041] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0042] Modes for Carrying out the Disclosure-9-4870-3559-0385.3Atty. Dkt. No.: 121384-0289

[0043] Engineered T cell, T cell populations, and compositions

[0044] Provided herein are T cells comprising at least one modification that enhances IL-2 production. In some aspects, the modification is a knockdown or knockout of a gene in the T cell. In some aspects, the modification is a knock-in of a gene in the T cell. In yet other aspects, the modification is the insertion of a gene into the T cell.

[0045] In some aspects, the modification is a knockdown or knockout of a gene selected from HRH2, IL1RL1, PTGER2, OR13D1, MC1R, I. OXI.2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, and AKR1A1 is knocked down or knocked out (FIGURES 2 and 5A).

[0046] In some aspects the modification is a knockdown or knockout of a gene selected from LOXL2, MC1R, OR13D1, PTGER2, IL1RL1 andHRH2 (FIGURES 2, 3, and 4).

[0047] As used herein, HRH2 refers to histamine receptor 2 (histamine receptor H2). Nonlimiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NP_001124527.1 or NM_001367711.1 each of which is incorporated by reference herein in its entirety.[0048| As used herein, IL1RL1 refers to interleukin 1 receptor like 1. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NP_003847.2 or NM_003856.4 each of which is incorporated by reference herein in its entirety.

[0049] As used herein PTGER2 refers to prostaglandin E receptor 2 Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_013082.1, NP_000947.2, or NM_000956.4, each of which is incorporated by reference herein in its entirety.

[0050] As used herein OR13D1 refers to olfactory receptor family 13 subfamily D member 1. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_001004484.2 or NP_001004484.2, each of which is incorporated by reference herein in its entirety.-10-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[00511 As used herein MC1R refers to melanocortin 1 receptor. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_012026.1, NP_002377.4, or NM_002386.4, each of which is incorporated by reference herein in its entirety.

[0052] As used herein LOXL2 refers to lysyl oxidase like 2. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_002318.3 or NP_002309.1, each of which is incorporated by reference herein in its entirety.

[0053] As used herein PHC2 refers to polyhomeotic homolog 2. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_001330488.2 or NP_001317417.1, each of which is incorporated by reference herein in its entirety.[0 54| As used herein KRTAP5-5 refers to keratin associated protein 5-5. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_001001480.3 or NP_001001480.2 , each of which is incorporated by reference herein in its entirety.

[0055] As used herein HGC6.3 refers to uncharacterized LOC100128124. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NR 171011.1, each of which is incorporated by reference herein in its entirety.

[0056] As used herein LVRN refers to laeverin, also known as aminopeptidase Q. Nonlimiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_173800.5 or NP_776161.3, each of which is incorporated by reference herein in its entirety.

[0057] As used herein ABHD14B refers to ab hydrolase domain containing 14B. Nonlimiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_001146314.2 or NP_001139786.1, each of which is incorporated by reference herein in its entirety.-11-4870-3559-0385.3Atty. Dkt. No.: 121384-0289

[0058] As used herein AKR1A1 refers to aldo-keto reductase family 1 member Al. Nonlimiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NM_001202413.2 or NP_001189342.1, each of which is incorporated by reference herein in its entirety.

[0059] According to some aspects the T cell is selected from a CD3+ cell, CD8+ cell, CD4+ cell, regulatory T cell, a gamma delta T cell (ybT), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Th 17 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, or a naive T (TN) cell.

[0060] In one aspect the T cell is a CD8+ cell.

[0061] In one aspect the T cell has been engineered to include a T cell receptor (TCR). IN one aspect the T cell has been engineered to include a synthetic agonist receptor (SAR). In one aspect the T cell has been engineered to include a chimeric antigen receptor (CAR).

[0062] In some aspects the T cell is a tumor infiltrating lymphocyte (TIL). In some aspects the TIL has been engineered to have enhanced tumor homing ability, longevity, cytotoxicity, and / or trafficking into the tumor. In some aspects the TIL has been engineered to include at least one gene selected from NeoR or Neo, TNFa, TRAIL, IL2, IL- 12, CXCR2, or CXCR1. In one aspect the TIL has been engineered to have a knockdown or knockout of the PD-1 gene.

[0063] As used herein, NeoR or Neo refers to aminoglycoside 3 '-phosphotransferase (bacterial gene for neomycin-resi stance). A non-limiting exemplary sequence of aminoglycoside 3 '-phosphotransferase or the underlying gene or functions thereof may be found under UniProt P00552 (K. pneumoniae) which is incorporated by reference herein in its entirety.-12-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[0O64| As used herein, TNFa refers to tumor necrosis factor alpha. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_007462.1 or NP_000585.2, each of which is incorporated by reference herein in its entirety.

[0065] As used herein, TRAIL refers to tumor necrosis factor superfamily member 10 (also known as TNF-related apoptosis inducing ligand TRAIL). Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NP_001177871.1 or NM_001190942.2, each of which is incorporated by reference herein in its entirety.

[0066] As used herein, IL2 refers to interleukin 2. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_016779.1 or NP_000577.2, each of which is incorporated by reference herein in its entirety.[0067| As used herein, IL-12 refers to interleukin 12. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_033022.2 (IL-12A) or NG_009618.1 (IL-12B), each of which is incorporated by reference herein in its entirety.

[0068] As used herein, CXCR2 refers to C-X-C motif chemokine receptor 2. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_052975.1 or NP_001161770.1, each of which is incorporated by reference herein in its entirety.] 0069] As used herein, CXCR1 refers to C-X-C motif chemokine receptor 1. Non-limiting exemplary sequences of this protein or the underlying gene or functions thereof may be found under NCBI NG_011814.1 or NP_000625.1, each of which is incorporated by reference herein in its entirety.

[0070] In still other aspects the TIL has been engineered to include, or have altered expression, of a different gene.-13-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[00711 Further described herein a T cell population comprising a T cell as described herein. In some aspects the T cell population is heterogeneous. In other aspects the T cell population is homogeneous.

[0072] Also described herein is a composition which comprises the T cell or T cell population described herein.

[0073] In some aspects, the composition further comprises an additional therapeutic agent.

[0074] In some aspects the composition further comprises a bispecific antibody, wherein the bispecific antibody, wherein the bispecific antibody can recognize both the T-cell and a tumor.[0075[ Also described herein is a kit which comprises the T cell, T cell population, or composition described herein and instruction for use. Kits may optionally include additional components.

[0076] Composition

[0077] Compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of a component or a combination as described herein, can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. The component or combination can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the component or combination provided herein into preparations which can be used pharmaceutically.]0078[ The component or combination of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), oral, by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g, urethral suppository) or topical routes of administration (e.g, gel, ointment, cream, aerosol, etc.) and can be formulated in suitable dosage unit formulations-14-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.10079] In one embodiment, this technology relates to a composition comprising a component or a combination as described herein and a carrier.

[0080] In another embodiment, this technology relates to a pharmaceutical composition comprising a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0081] In another embodiment, this technology relates to a pharmaceutical composition comprising a therapeutically effective amount of a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0082] As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0083] The pharmaceutical compositions for the administration of a component or a combination as disclosed herein can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy. The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, each component provided herein is included in an amount-15-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 sufficient to produce the desired effect. For example, pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation. In one aspect, administration is intraperitoneal.[00841 For topical administration, the component or the combination can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.

[0085] Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.[0086| Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.[0087| Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use. To this end, the component or the combination provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.[0088 [ For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0089] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or-16-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films, or enteric coatings.

[0090] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., com starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan. The pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions.

[0091] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.

[0092] Kit

[0093] The kit components, (e.g., reagents) can be packaged in a suitable container. The kit can also comprise, or alternatively consist essentially of, or yet further consist of, e.g., a-17-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise, or alternatively consist essentially of, or yet further consist of components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

[0094] As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.

[0095] Methods of use

[0096] Also describe herein are methods of immunotherapy. In one embodiment, the method comprises, consists of, or consists essentially of enhancing IL-2 production in a subject in need thereof concurrently with administering immunotherapy to the subject in need thereof. In one aspect, IL-2 production is enhanced in a subject by administering a T cell or T cell population with enhanced IL-2 production. In one aspect, IL-2 production is enhanced in the T cell or T cell population by knocking down or knocking out at least one regulator of IL-2 production, thereby enabling increased endogenous IL-2 production in the cell or cell population.

[0097] According to some aspects, the at least one regulator of IL-2 production is selected from HRH2, IL1R11, PTGER2, OR13D1, MC1R, LOXL2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, TBKBP1, RASA4, ACKR4, ITGB1, and AKR1A1.

[0098] Also described herein are methods of use of the T cell comprising at least one modification that enhances IL-2 production, T cell population, or composition described herein.-18-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[00991 In one embodiment, provided is a method to promote longevity, growth, or expansion of a T cell population grown in vitro in culture medium, the method comprising, consisting of, or consisting essentially of growing the T cell population described herein in culture medium, wherein exogenous IL-2 is not included in the culture medium (FIGURE 3).

[0100] In one embodiment, provided herein in a method to promote longevity, growth, or expansion in the T cell population grown in vitro in culture medium, the method comprising, consisting of, or consisting essentially of growing the T cell population in a culture medium, wherein a low or reduced amount of exogenous IL-2 is included in the culture medium, and wherein the low or reduced dose is determined in proportion to the dose that would be administered to a T cell that does not comprise at least one modification that enhances IL-2 production.[01011 In another embodiment, provided is a method to promote longevity, growth, or expansion of a T cell population in a tumor microenvironment or tumor draining lymph node of a subject in need, the method comprising, consisting of, or consisting essentially of administering to the subject in need the T cell, T cell population, or composition described herein, wherein exogenous IL-2 is not administered concurrently or consecutively to the administration of the T cell or T cell population in the subject in need (FIGURE 4).

[0102] In another embodiment, provided herein is a method to promote longevity, growth, or expansion of a T cell population described herein, in a tumor microenvironment or tumor draining lymph node of a subject in need, the method comprising, consisting of, or consisting essentially of administering to the subject in need the T cell, T cell population, or composition described herein, wherein a low or reduced dose of IL-2 is administered concurrently or consecutively to the administration of the T cell, T cell population, or composition. The low or reduced dose administered with the T cell comprising a modification to enhance IL-2 production is lower than the dose that would be administered to a subject with a T cell or T cell population that does not comprise at least one modification that enhances IL-2 production.

[0103] In yet another embodiment, provided is a method to inhibit the growth of a cancer and / or increase the survival rate of a subject in need, the comprising, consisting of, or-19-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 consisting essentially of administering to the subject in need the T cell, T cell population, or composition described herein, wherein exogenous IL-2 is not administered concurrently or consecutively to the administration of the T cell population in the subject in need.

[0104] Still further provided herein are methods to inhibit the growth of cancer and / or increase the survival rate of a subject in need comprising consisting of, or consisting essentially of administering to the subject the T cell, T cell population, or composition described herein to the subject in need, wherein a low or reduced dose of exogenous IL-2 is concurrently or consecutively administered to the subject in need. The low or reduced dose administered with the T cell comprising a modification to enhance IL-2 production is lower than the dose that would be administered to a subject with a T cell or T cell population that does not comprise at least one modification that enhances IL-2 production.[0105 [ In some aspects, the T cell, T cell population, or composition described herein are administered to the subject in need consecutively or concurrently with an additional therapeutic agent.

[0106] In one aspect, the subject is a mammal, optionally a human.

[0017] In one aspect the cancer is a solid tumor. In one aspect the cancer is selected from a small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0108] In one aspect, administration of the T cell, T cell population, or composition is parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intramedullary and intrathecal), intraperitoneal, or intratumoral.[0109[ In one aspect, in the methods described herein, the T cell, T cell population, or composition are applied in a therapeutically effective amount. The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological-20-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.| OLIO] Experimental 01.11] This study is primarily focused on elucidating the molecular regulators of IL-2 secretion in cytotoxic T cells via comprehensive genome-wide CRISPR knockout screens. The goal is to pinpoint the specific factors that modulate IL-2 secretion, which could transform the landscape of IL-2 -based therapies by enhancing both their safety and efficacy.[0112 { Applicant aims to identify and target druggable regulators of IL-2 secretion within therapeutic T cells, ultimately leading to the development of novel immunotherapeutic strategies that foster autonomous IL-2 production, thereby minimizing systemic toxicities associated with exogenous IL-2 administration.

[0113] The genome-wide CRISPR screen is meticulously planned to encompass approximately seven cellular doublings. Activation of CD8+ T cells is initiated one day before viral transduction. Post-activation, these cells are transduced with the Brunello Human CRISPR Knockout Pooled Library lentivirus (Addgene #73179) at an MOI below 0.3.Following transduction, the cells undergo a selection process with puromycin (2 pg / ml) for 48 hours to eliminate any cells that have not been successfully transduced.

[0114] The cells are subsequently isolated utilizing a FACSARIA 4-Laser Sorter (Flow Cytometry Core, Northwestern), employing a fluorescence-based IL-2 Secretion Assay (Miltenyi Biotec #130090763) to identify IL-2 secreting T cells. Eight days after the initiation of the screening process, the cells are harvested, and genomic DNA is extracted for nextgeneration sequencing analysis. See FIGURE 1.-21-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[0115| This analysis aims to elucidate the impact of various genetic disruptions on IL-2 secretion in human primary CD8+ T cells. Applicant has commenced in vitro validations of the top 14 candidate regulators implicated in modulating IL-2 secretion. The in vitro validation was focused on assessing the longevity of cells without exogenous supply of IL-2 after generating mutant cells. Further, the cells were tested for their capacity to secrete IL-2[0116| The ultimate goal of this research is to pioneer a novel T-cell therapy that intrinsically produces IL-2, thereby reducing the reliance on exogenous IL-2 administration, which is often associated with significant toxicities and adverse effects in current immunotherapeutic regimens.

[0117] To this end, 6 hits characterized by Applicant include: LOXL2, MC1R, OR13D1, PTGER2, IL1RL1 cmdHRH2.

[0118] Mechanistic hypotheses on selected hits:

[0119] MC1R

[0120] Melanocortin 1 Receptor (MC1R), traditionally associated with skin pigmentation, appears to modulate IL-2 secretion in immune cells. Upon activation by its ligand, a- melanocyte-stimulating hormone (a-MSH), MC1R stimulates an increase in cyclic AMP (cAMP) levels through the activation of the Gas protein and adenylate cyclase. This surge in cAMP activates Protein Kinase A (PKA), which has a known role in inhibiting the NF-KB signaling pathway (Takahashi, N. et al 2002) — a critical regulator of IL-2 gene transcription. PKA achieves this by phosphorylating IKB, leading to its stabilization and preventing NF-KB from translocating to the nucleus where it would typically enhance IL-2 transcription. Further investigation will probe how MClR’s signaling impacts IL-2 levels, providing a novel link between MC1R activation and the modulation of an essential cytokine involved in T-cell activation and immune response regulation.

[0121] LOXL2

[0122] Previous research has demonstrated that LOXL2 is crucial in modulating the extracellular matrix (ECM), which in turn can affect TGF-P activation. Notably, Barry--22-4870-3559-0385.3Atty. Dkt. No.: 121384-0289Hamilton et al. (2010) have shown that the inhibition of LOXL2 leads to decreased TGF-P signaling, suggesting that LOXL2 may facilitate the activation of latent TGF-P, which is known to suppress IL-2 production in T cells. Given the pivotal role of TGF-P in immune regulation, particularly in inhibiting IL-2 production (Das L et al., 2008), this may explain the appearance of this factor as a strong hit in the screen.[0123| HRH2[01241 HRH2 (Histamine Receptor H2) inhibition enhances IL-2 secretion in T cells, potentially due to enhanced NF-KB signaling. It has been established that HRH2 inhibitors can decrease NF-KB activity, which is associated with reduced production of pro- inflammatory cytokines such as IL-6 and TNF-alpha, highlighting a key pathway through which HRH2 signaling impacts immune responses (Shi et al., 2019) (Esbenshade et al., 2003). Importantly, HRH2 activation is known to increase intracellular cAMP levels through G protein-coupled receptor activation of adenylate cyclase, which in turn activates PKA (Protein Kinase A), thereby modulating numerous downstream signaling pathways, including those that suppress NF-KB activity (Zhong et al., 1997). These mechanisms collectively contribute to the immunosuppressive effects observed upon HRH2 activation, including the suppression of cytokine production crucial for T cell function. Knockout of HRH2, may decrease cAMP levels, leading to reduced PKA activity and less CREB-mediated competition, thereby facilitating NF-KB translocation to the nucleus and increasing IL-2 transcription.

[0125] OR13D1

[0126] 0R13D1 is one of the many olfactory receptors (ORs) encoded in the human genome. Olfactory receptors are a large family of G-protein-coupled receptors (GPCRs) predominantly known for their role in detecting odors in the olfactory epithelium.

[0127] Existing literature on G protein-coupled receptors (GPCRs), which include olfactory receptors, demonstrates that these receptors can significantly impact immune cell function by modulating key signaling cascades (Dorsam and Gutkind, 2007; Neuhaus et al., 2009). Specifically, the involvement of cAMP and PKA in regulating gene expression within T cells-23-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 has been well-documented, particularly their role in controlling the transcriptional activity of factors like NF AT and NF-Kb which directly influence IL-2 production (Gerlo et al., 2011).

[0128] Applicant’s observations suggest that knockout of 0R13D1 leads to increased IL-2 secretion. This observation aligns with the known effects of cAMP elevation, which generally promotes T-cell activation and cytokine production through PKA activation (Bielenberg et al., 2024). Under normal conditions, 0R13D1 may act to maintain a suppressive influence on IL-2 secretion via modulation of cAMP levels. The deactivation or inhibition of 0R13D1, as observed in the knockout models, removes this suppressive signal, thereby leading to increased cAMP accumulation, enhanced PKA activity, and ultimately elevated IL-2 production. It is noteworthy that one specific isoform of the OR13 family is enriched in the screen rather than other members.[01291 Each gene was targeted by four distinct sgRNAs, as shown in FIGURE 5B. Gene Ontology (GO) enrichment analysis of significantly enriched sgRNAs highlighted the top immune regulatory pathways, as shown in FIGURE 5C.

[0130] Functional validation of IL-2 regulatory gene knockouts revealed a CD8+helper-like phenotype and transcriptional reprogramming. Enzyme-linked immunosorbent assay (ELISA) quantification of IL-2, IFN-y, and TNF-a demonstrated significantly enhanced cytokine secretion in primary human CD8+T cells following targeted knockout of IL-2 regulatory genes, including LOXL2, MC1R, HRH2, OR13D1, IL1RL1, PTGER1, TBKBP1, RASA4, ACKR4, and ITGB1, as shown in FIGURE 6A. Long-term survival analysis of knockout and control T cell populations cultured in the absence of exogenous IL-2 revealed improved persistence in knockout cells, as shown in FIGURE 6B. Flow cytometric profiling of canonical exhaustion markers under IL-2-deprived conditions shown in FIGURE 6C indicated reduced exhaustion phenotypes in knockout T cells relative to controls. Prolonged co-culture cytotoxicity assays using A375 melanoma cells demonstrated sustained antitumor activity of knockout T cells compared to control populations, as shown in FIGURE 6D. Flow cytometric profiling of degranulation markers and cytotoxic markers under IL-2- deprived conditions indicated improved degranulation activity and cytotoxicity in knockout T cells relative to controls, as shown in FIGURE 6E. Differential gene expression analysis via-24-4870-3559-0385.3Atty. Dkt. No.: 121384-0289RNA sequencing of L0XL2-deficient CD8+T cells revealed significant transcriptional changes, with upregulated and downregulated genes visualized in a volcano plot, as shown in FIGURE 6F. Gene set enrichment analysis (GSEA) highlighted key Hallmark pathways that were significantly upregulated or downregulated in LOXL2-deficient CD8+T cells, as shown in FIGURE 6G

[0131] Further, the results suggested that MC1R and LOXL2 modulated IL-2 secretion in human CD8+T cells through distinct signaling mechanisms. FIGURE 7A depicts the molecular regulation of IL-2 secretion following LOXL2 knockout or without LOXL2 knockout in primary human CD8+T cells. Western blot evaluates protein expression before (WT) and after LOXL2 knockout (LOXL2-KO) indicated the proteins involved in the IL-2 regulation pathway in human primary cytotoxic T cells, as shown in FIGURE 7B. Treatment with a NFKB and HIF-loc inhibitor led to increased IL-2 secretion in both control and LOXL2-deficient T cells, as shown in FIGURE 7C. Edited T cells performed similar cytotoxicity against human primary skin cells compared to un-edited T cells under 1% O2 hypoxic condition, indicating edited T cells were biocompatible, as shown in FIGURE 7D. Cytotoxicity assays against Nalm6 (leukemia), SKOV3, and SKBR3 (breast cancer) cell lines under hypoxic conditions (1% O2) demonstrated that LOXL2 and MC1R knockout T cells retained robust antitumor activity compared to controls, as shown in FIGURE 7E. RNA sequencing of LOXL2-deficient CD8+T cells revealed the significant upregulated CD4 helper-like marker genes expression and with strong alignment with published RNA sequencing profile of human CD4+ T cells, as shown in FIGURE 7F.

[0132] LOXL2- and MClR-deficient CD8+T cells exhibited in vivo therapeutic efficacy in syngeneic KPC pancreatic cancer models. In vivo experiments were performed to assess tumor suppression and survival outcomes following adoptive T cell transfer, as illustrated in FIGURE 8A. FIGURE 8B demonstrates tumor growth kinetics in KPC-bearing immunocompetent mice treated with vehicle control, wild-type (WT) CD8+T cells, LOXL2- knockout T cells (20% ACT dosage), or MClR-knockout T cells (20% ACT dosage). Both LOXL2- and MClR-deficient T cells exhibited superior tumor control compared to WT or control -treated groups. Kaplan-Meier survival analysis revealed significantly prolonged survival in mice receiving LOXL2 or MClR-knockout T cells, indicating enhanced antitumor-25-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 efficacy in vivo, as shown in FIGURE 8C. Flow cytometric analysis of tumor-infiltrating lymphocytes showed altered immune cell composition and reduced exhaustion marker expression in tumors treated with knockout T cells relative to WT and control groups, as shown in FIGURE 8D. Tumor growth kinetics in CT26-bearing immunocompetent mice treated with vehicle control, wild-type (WT) CD8+T cells, or LOXL2-knockout T cells (20% ACT dosage) suggested that the LOXL2 -deficient T cells exhibited superior tumor control compared to WT or control-treated groups, as shown in FIGURE 8E. Kaplan-Meier survival analysis revealed significantly prolonged survival in mice receiving LOXL2-knockout T cells, indicating enhanced antitumor efficacy in vivo, as shown in FIGURE 8F. Flow cytometric analysis of tumor-infiltrating lymphocytes showed altered immune cell composition and reduced exhaustion marker expression in tumors treated with knockout T cells relative to WT and control groups, as shown in FIGURE 8G. LOXL2-deficient T cells demonstrated significantly improved tumor control compared with WT or control groups, while body weight remained stable across all treatment conditions, as shown in FIGURE 8H.

[0133] Embodiments

[0134] 1. A method of immunotherapy comprising enhancing IL-2 production in a subject in need thereof concurrently with administering immunotherapy to the subject in need thereof.

[0135] 2. The method of embodiment 1, wherein at least one regulator of IL-2 production is knocked down or knocked out in a T cell population administered to the subject in need thereof, thereby enabling increased endogenous IL-2 production in the T cell population.

[0136] 3. The method of embodiment 1 or 2, wherein the at least one regulator of IL-2 production is selected from HRH2, IL1R11, PTGER2, OR13D1, MC1R, LOXL2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, TBKBP1, RASA4, ACKR4, ITGB1, and AKR1A1.-26-4870-3559-0385.3Atty. Dkt. No.: 121384-0289[0137| 4. The method of embodiment 1 or 2, wherein the at least one regulator of IL-2 production is selected from HRH2, IL1R11, PTGER2, 0R13D1, MC1R, TBKBP1, RASA4, ACKR4, ITGB1, and L0XL2.

[0138] 5. A T cell comprising at least one modification that enhances IL-2 production.

[0139] 6. The T cell of embodiment 5, wherein the T cell is selected from a CD3+ cell,CD8+ cell, CD4+ cell, regulatory T cell, a gamma delta T cell (ybT), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Th 17 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, or a naive T (TN) cell.

[0140] 7. The T cell of embodiment 6, wherein the T cell is a CD8+ cell.

[0141] 8. The T cell of embodiment 6, wherein the T cell has been engineered to include a T cell receptor (TCR).

[0142] 9. The T cell of embodiment 6, wherein the T cell has been engineered to include a synthetic agonist receptor (SAR).

[0143] 10. The T cell of embodiment 6, wherein the T cell has been engineered to include a chimeric antigen receptor (CAR).

[0144] 11. The T cell of embodiment 6, wherein the T cell is a tumor infiltrating lymphocyte (TIL).

[0145] 12. The T cell of embodiment 11, wherein the TIL has been engineered to have enhanced tumor homing ability, longevity, cytotoxicity, and / or trafficking into the tumor.

[0146] 13. The T cell of embodiment 11, wherein the TIL has been engineered to have an insertion of the Neo JI TN Fa, TRAIL, IL2, NF AT, IL-12, CXCR2, or CXCR1 gene.-27-4870-3559-0385.3Atty. Dkt. No.: 121384-0289

[0147] 14. A T cell population, comprising a T cell of any of embodiments 5-13.

[0148] 15. A composition, comprising the T cell of any of embodiments 5-13 or T cell population of embodiment 14 and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.

[0149] 16. The composition of embodiment 15, further comprising an additional therapeutic agent.

[0150] 17. The composition of embodiment 16, further comprising a bispecific antibody, wherein the bispecific antibody, wherein the bispecific antibody can recognize both the T-cell and a tumor.[0151 [ 18. A method to promote longevity, growth, or expansion of a T cell population grown in vitro in culture medium, the method comprising growing a T cell population of embodiment 14 in culture medium, wherein exogenous IL-2 is not included in the culture medium.

[0152] 19. A method to promote longevity, growth, or expansion of a T cell population in a tumor microenvironment or tumor draining lymph node of a subject in need, the method comprising administering to the subject in need the T cell of any of embodiments 5-13, T cell population of embodiment 14, or composition of any of embodiments 15-17 wherein exogenous IL-2 is not administered concurrently or consecutively to the T cell or T cell population in the subject in need.

[0153] 20. A method to inhibit the growth of a cancer and / or increase the survival rate of a subject in need, comprising administering to the subject in need the T cell of any of embodiments 5-13, T cell population of embodiment 14, or composition of any of embodiments 15-17 wherein exogenous IL-2 is not administered concurrently or consecutively to the T cell or T cell population in the subject in need.

[0154] 21. The method of embodiment 20, wherein the cancer is a solid tumor.

[0155] 22. The method of embodiment 20 or 21, wherein the cancer is a small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, -28-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.[0156| 23. The method of any of embodiments 19-22, wherein the administration is parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intramedullary and intrathecal), intraperitoneal, or intratumoral.

[0157] 24. The method of any of embodiments 19-22, further comprising administering an additional therapeutic agent to the subject in need.

[0158] 25. The method of any of embodiments 19-24, wherein the subject in need is a mammal, optionally a human.

[0159] 26. A kit comprising the composition of any of embodiments 15-17 and instructions for use.* * *[016(>| Equivalents[01611 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0162] Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.[01631 The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be-29-4870-3559-0385.3Atty. Dkt. No.: 121384-0289 read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.[0164| Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0165] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0166] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0167] All publications, patent applications, patents, GenBank citations, ATCC citations, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the specification, including definitions, will control.ReferencesTakahashi, N., et al. (2002). Inhibition of the NF-kappaB transcriptional activity by protein kinase A. European journal of biochemistry, 269(18), 4559-4565. DOI: 10.1046 / j .1432- 1033.2002.03157.x.-30-4870-3559-0385.3Atty. Dkt. No.: 121384-0289Barry-Hamilton, V., et al. (2010). Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment. Nature medicine, 16(9), 1009-1017. DOI: 10.1038 / nm.2208.Das, L., & Levine, A. D. (2008). TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector / memory T cells in the presence of sustained TCR signal transduction. Journal of immunology (Baltimore, Md. : 1950), 180(3), 1490-1498. DOI: 10.4049 / jimmunol.l80.3.1490.Shi, Z., et al. (2019). Distinct roles of histamine Hl- and H2-receptor signaling pathways in inflammation-associated colonic tumorigenesis. American journal of physiology.Gastrointestinal and liver physiology, 316(1), G205-G216. DOI: 10.1152 / ajpgi.00212.2018.Esbenshade, T. A., et al. (2003). Differential activation of dual signaling responses by human Hl and H2 histamine receptors. Journal of receptor and signal transduction research, 23(1), 17-31. DOI: 10.1081 / rrs-120018758.Zhong, H., et al. (1997). The transcriptional activity of NF-kappaB is regulated by the IkappaB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell, 89(3), 413-424. DOI: 10.1016 / s0092-8674(00)80222-6.Dorsam, R. T., & Gutkind, J. S. (2007). G-protein-coupled receptors and cancer. Nature reviews. Cancer, 7(2), 79-94. DOI: 10.1038 / nrc2069.Neuhaus, E. M., et al. (2009). Activation of an olfactory receptor inhibits proliferation of prostate cancer cells. The Journal of biological chemistry, 284(24), 16218-16225. DOI: 10.1074 / jbc.M109.012096.Gerlo, S., et al. (2011). Cyclic AMP: a selective modulator ofNF-xB action. Cellular and molecular life sciences : CMLS, 68(23), 3823-3841. DOI: 10.1007 / s00018-011-0757-8.Bielenberg, M., et al. (2024). A mini-review: phosphodiesterases in charge to balance intracellular cAMP during T-cell activation. Frontiers in immunology, 15, 1365484. DOI: 10.3389 / fimmu.2024.1365484.-31-4870-3559-0385.3

Claims

1. Atty. Dkt. No.: 121384-0289WHAT IS CLAIMED IS:

1. A method of immunotherapy comprising enhancing IL-2 production in a subject in need thereof concurrently with administering immunotherapy to the subject in need thereof.

2. The method of claim 1, wherein at least one regulator of IL-2 production is knocked down or knocked out in a T cell population administered to the subject in need thereof.

3. The method of claim 1 or 2, wherein the at least one regulator of IL-2 production is selected from HRH2, IL1R11, PTGER2, 0R13D1, MC1R, L0XL2, PHC2, KRTAP5-5, HGC6.3, LVRN, ABHD14B, TBKBP1, RASA4, ACKR4, ITGB1, and AKR1A1.

4. The method of claim 1 or 2, wherein the at least one regulator of IL-2 production is selected from HRH2, IL1R11, PTGER2, 0R13D1, MC1R, TBKBP1, RASA4, ACKR4, ITGB1, and L0XL2.

5. A T cell comprising at least one modification that enhances IL-2 production.

6. The T cell of claim 5, wherein the T cell is selected from a CD3+ cell, CD8+ cell, CD4+ cell, regulatory T cell, a gamma delta T cell (ybT), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-celllike memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Th 17 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, or a naive T (TN) cell.

7. The T cell of claim 6, wherein the T cell is a CD8+ cell.

8. The T cell of claim 6, wherein the T cell has been engineered to include a T cell receptor (TCR).

9. The T cell of claim 6, wherein the T cell has been engineered to include a synthetic agonist receptor (SAR).-32-4870-3559-0385.3Atty. Dkt. No.: 121384-028910. The T cell of claim 6, wherein the T cell has been engineered to include a chimeric antigen receptor (CAR).

11. The T cell of claim 6, wherein the T cell is a tumor infiltrating lymphocyte (TIL).

12. The T cell of claim 11, wherein the TIL has been engineered to have enhanced tumor homing ability, longevity, cytotoxicity, and / or trafficking into the tumor.

13. The T cell of claim 11, wherein the TIL has been engineered to have an insertion of the NeoR, TNFa, TRAIL, IL2, IL-12, CXCR2, or CXCR1 gene.

14. A T cell population, comprising a T cell of any of claims 5-13.

15. A composition, comprising the T cell of any of claims 5-13 or T cell population of claim 14 and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.

16. The composition of claim 15, further comprising an additional therapeutic agent.

17. The composition of claim 16, further comprising a bispecific antibody, wherein the bispecific antibody, wherein the bispecific antibody can recognize both the T-cell and a tumor.

18. A method to promote longevity, growth, or expansion of a T cell population grown in vitro in culture medium, the method comprising growing a T cell population of claim 14 in culture medium, wherein exogenous IL-2 is not included in the culture medium.

19. A method to promote longevity, growth, or expansion of a T cell population in a tumor microenvironment or tumor draining lymph node of a subject in need, the method comprising administering to the subject in need the T cell of any of claims 5-13, T cell population of claim 14, or composition of any of claims 15-17 wherein exogenous IL-2 is not administered concurrently or consecutively to the T cell or T cell population in the subject in need.

20. A method to inhibit the growth of a cancer and / or increase the survival rate of a subject in need, comprising administering to the subject in need the T cell of any of claims 5--33-4870-3559-0385.3Atty. Dkt. No.: 121384-028913, T cell population claim 14, or composition of any of claims 15-17 wherein exogenous IL- 2 is not administered concurrently or consecutively to the T cell or T cell population in the subject in need.

21. The method of claim 20, wherein the cancer is a solid tumor.

22. The method of claim 20 or 21, wherein the cancer is a small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

23. The method of any of claims 19-22, wherein the administration is parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intramedullary and intrathecal), intraperitoneal, or intratumoral.

24. The method of any of claims 19-22, further comprising administering an additional therapeutic agent to the subject in need.

25. The method of any of claims 19-24, wherein the subject in need is a mammal, optionally a human.

26. A kit comprising the composition of any of claims 15-17 and instructions for use.-34-4870-3559-0385.3

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