Interleukin-9 receptor variants for in VIVO enhancement of adoptive transferred t cells

Abstract

The present disclosure relates generally to compositions and methods for robustly improving therapeutic properties of recombinant cells (e.g., recombinant T cells) for adoptive cell therapy. More particularly, some embodiments of the disclosure relate to recombinant cytokine receptors that include an intracellular signaling domain (ICD) of an interleukin 9 receptor (IL-9R), wherein the ICD includes a proline-to-alanine (P‒>A) substitution in its STAT binding site, recombinant nucleic acids encoding the recombinant cytokine receptors, recombinant cells (e.g., recombinant T cells) comprising the recombinant cytokine receptors and / or nucleic acids encoding the recombinant cytokine receptors. Also provided herein are methods of generating the engineered T cells, methods of administering the engineered T cells, and methods of treating individuals of relevant health conditions

Description

Attorney Docket No.: 078430-543001 WOINTERLEUKIN-9 RECEPTOR VARIANTS FORTY VIVO ENHANCEMENT OF ADOPTIVE TRANSFERRED T CELLSSTATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with Government support under contracts CA244711 and CA273074 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 745,258, filed on January 14, 2025, and U.S. Provisional Patent Application Serial No. 63 / 768,766, filed on March 7, 2025. The disclosures of the above-referenced applications are herein expressly incorporated by reference it their entireties, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “078430_543001WO_Sequence Listing_ST26.XML,” was created on January 8, 2026 and is 18,982 bytes in size.BACKGROUND

[0004] Adoptive transfer of genetically modified immune cells, e.g., T cells, has emerged as a potent therapy for various malignancies. For example, current modalities of adoptive T cell therapy include cells modified to express receptors specific for cancer antigens, such as chimeric antigen receptors (CARs) and high-affinity T cell receptors (TCRs). Upon exposure to the cancer antigen, the modified T cells exhibit cytolytic activity and / or send signals to initiate an immune response against the cancer.

[0005] In adoptive T cell therapies, modified T cells are generally activated by exposure to the cognate antigen in vitro or ex vivo, expanded, and then administered to the subject, where they proliferate and have anticancer activity. Recent clinical trials using CAR-modified T cells (CAR-T cells) specific for the CD19 molecule on B-cell malignancies demonstrated marked disease regression in a subset of patients with advanced cancers. However, despite clinical success33'34, T cell therapies for solid tumors remain constrained by the need for large-scale manufacturing andAttorney Docket No.: 078430-543001 WOtoxic, suboptimal trafficking, chronic antigen exposure and hostile tumor microenvironments. Cytokines offer a regulatable approach to modulate engineered T cells in vivo but dose-limiting toxicities have limited their therapeutic potential35'38.

[0006] Thus, new compositions, methods, and strategies are needed for generating improved therapeutic cells, e.g., T cells, for adoptive cell therapy. The presently disclosed aspects and embodiments address these needs and provide other related advantages.SUMMARY

[0007] The present disclosure relates generally to, mter alia, novel compositions and methods for the prevention and / or treatment of various health conditions. In particular, some embodiments of the disclosure relate to recombinant cytokine receptors that include an intracellular signaling domain (ICD) of an interleukin 9 receptor (IL-9R), wherein the ICD includes a proline-to-alanine (P->A) substitution in its STAT binding site. Some embodiments of the disclosure provide recombinant nucleic acids encoding a recombinant cytokine receptor as disclosed herein. Also provided, in some embodiments, are recombinant cells, e.g., T cells that have been engineered to express a recombinant cytokine receptor as disclosed herein. Further provided are methods for generating a population of recombinant cells, e.g, engineered T cells with improved therapeutic properties for adoptive cell therapy, and pharmaceutical compositions containing such a population of engineered T cells with enhanced therapeutic properties, as well as methods and kits for the prevention and / or treatment of a health condition in subjects in need thereof.

[0008] In one aspect, provided herein are recombinant cytokine receptors including: (a) an extracellular domain (ECD) and an intracellular signaling domain (ICD) of a IL-9 receptor (IL-9R), wherein the ICD includes a proline-to-alanine (P->A) substitution in the STAT binding site; and (b) a transmembrane domain (TMD) operably inserted between the ECD the ICD.

[0009] Non-limiting exemplary embodiments of the disclosed recombinant cytokine receptors can include one or more of the following features. In some embodiments, the IL-9R is a murine IL-9R. In some embodiments, the IL-9R is a human IL-9R. In some embodiments, the TMD is a transmembrane domain derived from the same IL-9R (e.g., the IL-9R from which the ECD and / or the ICD is derived). In some embodiments, the TMD is a heterologous transmembrane domain. In some embodiments, the heterologous TMD is derived from a type-1 transmembraneAttorney Docket No.: 078430-543001 WOspanning protein. In some embodiments, the type-1 transmembrane spanning protein is CD3 , CD4, CD8, CD28, B7-H3, IL-2RB, IL-4R, IL-7R, IL-9R, or IL-21R. In some embodiments, the heterologous TMD is derived from a surface receptor subunit. In some embodiments, the surface receptor subunit is selected from the group consisting of IL3Ra, IL4Ra, IL5Ra, IL6Ra, IL7Ra, ILlORa, ILlORb, IL12Rpl, IL12Rp2, IL12p40, IL13RA1, IL15Ra, IL20R, IL21Ra, IL22R, IL23R, IL28R, IL31 Ra, GMCSFRa, LIFR, CNTFR, CLF1, OSMR, GCSFR, EPOR, TPOR, GHR, PRLR, LEPR, IFNAR2, IFNAR1, IFNGR1, and IFNGR2.

[0010] In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2.

[0011] In some embodiments of the disclosure, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced (e.g., decreased) by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, confers an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in promoting less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the reference T cell includes a wild-type IL-9 receptor.

[0012] In one aspect, provided herein are recombinant nucleic acid molecules including a nucleic acid sequence encoding a recombinant cytokine receptor as disclosed herein. Nonlimiting exemplary embodiments of the disclosed nucleic acid molecules can include one or more of the following features. In some embodiments, the recombinant nucleic acid moleculeAttorney Docket No.: 078430-543001 WOincludes a nucleic acid sequence having at least 80% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 3-4.

[0013] In some embodiments of the disclosure, the recombinant nucleic acid molecule is operably linked to one or more heterologous nucleic acid sequences. In some embodiments, the one or more heterologous nucleic acid sequences includes one or more expression control elements. In some embodiments, the one or more expression control elements is independently selected from the group consisting of ribosomal binding sites, promoters, translational start sequences, translational termination sequences, transcriptional start sequences, transcriptional termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, enhancer elements, activator elements, replication elements, RNA processing and export elements, insulator sequences, internal ribosome entry sites (IRES), 5’-UTRs, 3’-UTRs, mRNA 3 ’ end processing sequences, or any combination thereof. In some embodiments, the one or more heterologous nucleic acid sequences includes a promoter. In some embodiments, the promoter is a CD4 cell-specific promoter or a CD 8 cell-specific promoter. In some embodiments, the promoter is selected from the group consisting of murine stem cell virus (MSCV) promoter, EFla promoter, CMV promoter, C AG promoter, CD4 promoter, CD 8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cellAttorney Docket No.: 078430-543001 WOimmunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GAT A3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, and NR4A3 promoter.

[0014] In some embodiments of the disclosure, the recombinant nucleic acid molecule is incorporated into an expression cassette or a vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a RNP-packaging virus-like particle. In some embodiments, the viral vector is a lentivirus vector, a baculovirus vector, a retrovirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some embodiments, the recombinant nucleic acid molecule is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a protein nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM).

[0015] In another aspect, some embodiments of the disclosure relate to recombinant cells including: (a) a recombinant cytokine receptor as disclosed herein; and / or (b) a recombinant nucleic acid as disclosed herein. Non-limiting exemplary embodiments of the disclosed recombinant cells can include one or more of the following features. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0016] In some embodiments, the recombinant cell is T cell. In some embodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell. In some embodiments, the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, theAttorney Docket No.: 078430-543001 WOCD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells. In some embodiments, the T cell is an exhausted T cell or a non-exhausted T cell. In some embodiments, the T cell is obtained leukapheresis of a sample obtained from a subject.

[0017] In some embodiments, the recombinant T cell of the disclosure further includes: (a) an engineered immune receptor; and / or (b) a recombinant nucleic acid encoding the engineered immune receptor of (a). In some embodiments, the engineered immune receptor is a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the engineered immune receptor is a chimeric antigen receptor (CAR) having specificity for a target ligand.

[0018] In some embodiments, the target ligand is a tumor antigen. Non-limiting examples of tumor antigens include alpha feto-protein (AFP) / HLA-A2, ADAM12, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD 171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican- 3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-Al, MAGE- A3, MAGE-A4, Melan A, mesothehn, MG7 (glycosylated CEA), MMP, MRC2, MSX1, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC- A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PRAME, PSCA, PSMA, R0R1, ROR2, SSX1, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof. In some embodiments, the target ligand is expressed on or associated with a cancer cell. In some embodiments, the cancer is a B-cell malignancy, lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, or renal cancer. In some embodiments, the B-cell malignancy is a B-cell lymphomas or leukemia.Attorney Docket No.: 078430-543001 WO

[0019] In some embodiments, the recombinant T cell of the disclosure includes a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced (e.g., decreased) by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure includes an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure includes an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor.

[0020] In another aspect, some embodiments of the disclosure relate to methods for producing a recombinant T cell, the methods including introducing into a T cell a recombinant cytokine receptor as disclosure herein, and / or a recombinant nucleic acid molecule as disclosure herein.

[0021] Non-limiting exemplary embodiments of the disclosed methods for producing a recombinant T cell can include one or more of the following features. In some embodiments, the recombinant T cell includes a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced (e.g., decreased) by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell includes an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell includes an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In someAttorney Docket No.: 078430-543001 WOembodiments, the recombinant T cell promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the methods further include introducing into the T cell at least one engineered immune receptor. In some embodiments, the at least one engineered immune receptor includes an engineered T cell receptor (TCR) and / or an engineered chimeric antigen receptor (CAR). Accordingly, recombinant T cells produced by the methods disclosed herein are also encompassed within the present disclosure.

[0022] In a related aspect, some embodiments of the disclosure relate to cell cultures including at least one recombinant T cell of the disclosure and a culture medium.

[0023] In one aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable excipient and one or more of the following: (a) a recombinant cytokine receptor as disclosed herein; (b) a recombinant nucleic acid as disclosed herein; and (c) a recombinant cell as disclosed herein. In some embodiments, the pharmaceutical compositions of the disclosure include a recombinant cell as disclosed herein and a pharmaceutically acceptable carrier.

[0024] In another aspect, provided herein are methods for preventing and / or treating a health condition in a subject in need thereof, the methods include administering to the subject a composition including a pharmaceutically effective amount of one or more of the following: (a) a recombinant cytokine receptor as disclosed herein or a signaling component thereof; (b) a recombinant nucleic acid as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) pharmaceutical composition as disclosed herein.

[0025] Non-limiting exemplary embodiments of the methods for preventing and / or treating a health condition described herein can include one or more of the following features. In some embodiments, the recombinant cells are allogeneic relative to the subject. In some embodiments, the recombinant cells are autologous relative to the subject. In some embodiments, the health condition is a proliferative disorder (e.g., cancers), an infectious disease, an autoimmune disease, and / or inflammatory disease. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is a B-cell malignancy, lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer,Attorney Docket No.: 078430-543001 WOesophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, or renal cancer. In some embodiments, the B-cell malignancy is a B-cell lymphomas or leukemia.

[0026] In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor a signaling component thereof. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced (e.g., decreased) by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor or a signaling component thereof. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor or a signaling component thereof. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increase in circulating recombinant T cells in the blood of the subject. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increased accumulation of the recombinant T cells in lymphoid and non-lymphoid organs. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor or a signaling component thereof.

[0027] In another aspect, some embodiments of the disclosure relate to kits for the prevention and / or treatment of a condition in a subject in need thereof, the kits include one or more of the following: (a) a recombinant cytokine receptor as disclosed herein or a signaling component thereof; (b) a recombinant nucleic acid as disclosed herein; (c) a recombinant cell as disclosed herein; and (d) pharmaceutical composition as disclosed herein.

[0028] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, furtherAttorney Docket No.: 078430-543001 WOaspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A-1G summarize the results of experiments performed to illustrate the nomination of IL-9 as a naturally orthogonal cytokine for T cell therapy.

[0030] FIG. 1A: scRNA-seq of PBMCs from healthy donors. Expression of common yc cytokines receptors in single cell RNA sequencing data of PBMC from normal healthy donors. tSNE plots with cell-type annotations are shown in the top panel as a reference. For each cytokine yc receptor, a feature plot colored by scaled expression level (left panel) and a scatter plot summarizing expression for each cell type (right panel) are shown.

[0031] FIG. IB: As in FIG. 1A, but for lamina propria lymphocytes from the uninflamed ileum of patients with Crohn’s disease.

[0032] FIG. 1C: Expression scatterplot of normalized expression of receptors for yc cytokines in publicly available RNA sequencing data of 37 normal tissues from the GTEx database.Dashed line marks the nTPM threshold of 0.7, below which expression is not detected or negligible.

[0033] FIG. ID: Dot plot of intratumoral expression of a panel of cytokine receptors across malignant and non-malignant cell types, based on single cell RNA-sequencing data from soft tissue sarcoma (scRNA-seq; n=65,945 cells from 15 patients).

[0034] FIG. IE: Differential expression of genes (number of genes, magnitude of response) across lymph node cell types from mice treated with individual cytokines versus PBS control.

[0035] FIG. IF: Reported median Shet values for ycand non-yc cytokines. Shet quantifies (e.g., estimates) the impact of predicted heterozygous loss-of-function variants on evolutionary fitness, with higher scores associated with greater impact on evolutionary fitness. Error bars represent 95% highest posterior density. The dashed line at 0.02 represents the median Shet value across canonical transcripts.

[0036] FIG. 1G: Dot plot of yc cytokine receptor expression in sorted CD 19 CAR T cells (n=66,042) from 15 pediatric patients at various timepoints after infusion. Data presented separately for CD4 (left) and CD8 (right).

[0037] PBMC: peripheral blood mononuclear cells; TPM: transcripts per million; GTEX: Genotype-Tissue Expression; CAR: chimeric antigen receptor.Attorney Docket No.: 078430-543001 WO

[0038] FIGS. 2A-2J summarize the results of experiments performed to illustrate that exogenous IL-9 is well-tolerated and enhances tissue infiltration, sternness, and anti-tumor activity of IL-9R-engineered antigen-specific T cells.

[0039] FIG. 2A: Body weight (percentage change from baseline) of mice (n=8 mice / group) treated with PBS, IL-9 (50 pg or 100 pg), or IL-2 (50 pg) i.p. every other day.

[0040] FIG. 2B: Survival of mice from FIG.2A.

[0041] FIG. 2C: Sum of distance traveled over 30 second period in mice from FIG.2 A, normalized to the PBS group, e.g., plotted relative to the average distance traveled by mice in the PBS group.

[0042] FIG. 2D: Serum IFNy levels measured by ELISA six days after initiating treatment (n=3 mice / group) as described in FIGS. 2A-2C.

[0043] FIG. 2E: B16-F10 tumor growth (mean ± SEM, n=5 mice / group) after ACT with pmel T cells engineered with o9R or IL-9R (0.4 106transduced cells, i.v.), and corresponding cytokine treatment with oIL-2 or IL-9, respectively (5 / 104IU i.p., daily for 5 days starting with ACT). Data are representative of three independent experiments.

[0044] FIG. 2F: Survival of mice from FIG. 2E.

[0045] FIG. 2G: Quantification of transduced pmel T cells in the blood five days after ACT (n=5 mice / group).

[0046] FIG. 2H: Quantification of transduced pmel T cells (Thyl.l+YFP+) across different tissues five days after ACT (n=5 mice / group).

[0047] FIG. 21: Transduced (YFP+) pmel T cells as percentage of pmel T cells within the tumor 14 days after ACT (left panel) and total number of CD8+T cells per gram of tumor 14 days after ACT (right panel) (n=5 mice / group).

[0048] FIG. 2J: Proportion of TSCM cells for o9R or IL9R transduced C57BL / 6 T cells stimulated for 24h with IL-2 (lOnM), IL-9 (lOnM), oIL-2 (lOpM), or a combination as noted. Statistical comparisons refer to the differences in TSCM frequency across conditions. Data from two independent experiments, with 4 technical replicates per condition.

[0049] For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.0005, ****p < 0.0001 (one-way ANOVA for FIGS. 2A-2G; unpaired t test for FIGS.2H-2I; two-way ANOVA for FIG. J).Attorney Docket No.: 078430-543001 WO

[0050] FIGS. 3A-3I summarize the results of experiments performed to illustrate that IL-9R signaling results in phosphorylation of STAT4, in addition to STAT1, STAT3, and STAT5.

[0051] FIG. 3A: Dose-response curves of STAT1, STAT3, and STAT5 phosphorylation in IL-9R or o9R transduced (YFP+) pmel T cells stimulated with either oIL-2 or IL-9 for 20 minutes (shown are technical duplicates; representative of at least three independent experiments).

[0052] FIG. 3B: Volcano plots depicting differential gene expression by RNA-seq in IL-9R or o9R transduced C57BL / 6 T cells and treated with IL-9 (10 nM), IL-2 (10 nM), or oIL2 (10 pM) for 48 hours. Comparisons for each volcano plot are shown below the x-axis. Significance (red) indicates adjusted p < 10'5and absolute fold change >2.

[0053] FIG. 3C: Differentially phosphorylated proteins between IL-9R transduced C57BL / 6 T cells stimulated for 20 minutes with either (i) IL-2 (lOnM) or no cytokine (left), or (ii) IL-9 (lOnM) versus no cytokine (right). Significance (red) indicates adjusted p<0.05 and log2(fold change)> 0.5.

[0054] FIG. 3D: Dose-response curve for STAT4 phosphorylation in IL-9R transduced (YFP+) pmel T cells treated for 20 minutes with IL-9 or IL- 12, or o9R transduced T cells treated with oIL-2 (shown are technical duplicates; representative of two independent experiments).

[0055] FIG. 3E: Waterfall plot of inferred transcription factor enrichment scores based on RNA-seq data of IL-9R T cells treated with IL-9 or IL-2 (top 15 for each). Enrichment score is inferred by fitting a linear model that predicts observed gene expression based on prior knowledge of a curated set of transcription factors and their target genes.

[0056] FIG. 3F: Heat map of the expression of the Biocarta IL-12 Pathway gene set based on RNA-seq from FIG.3B. Samples and genes clustered hierarchically without supervision.

[0057] FIG. 3G: In vitro expansion of IL-9R (YFP+) pmel T cells treated with 1 OnM cytokine starting on day 3 after activation (n=3 technical replicates / group). Data are representative of three biological replicates and three independent experiments.

[0058] FIG. 3H: Representative contour plots of CD44 and CD62L expression of T cells from FIG. 3G after 24h cytokine exposure.

[0059] FIG. 31: Quantification of naive CD62L+CD44‘ (left) and stem-like CD62L+CD44 Sca" 1+(right) T cells from FIG. 3H. Data from two independent experiments.

[0060] *P < 0.05, **P < 0.005, ***P < Oj.001, ****P < 0.0001 (one-way ANOVA for FIG. 3G; two-way ANOVA for FIGS.3G, 31).Attorney Docket No.: 078430-543001 WO

[0061] FIGS. 4A-4G summarize the results of experiments performed to illustrate that structure-based attenuation or amplification of IL-9 / IL-9R signaling diminishes the anti -tumor qualities of T cells signaling through the native receptor complex.

[0062] FIG. 4A: Structural prediction of the interleukin-9 (IL-9) receptor complex based on AlphaFold2. The complex consists of the IL-9 receptor (IL-9R, green), IL-9 (blue), and the yc(pink). The inset demonstrates the interaction of the glutamine at amino acid position 115 of IL-9 (QI 15) with the yc, which was subsequently mutated (IL-9Q115T) to generate an attenuated cytokine (e.g., to attenuate signaling).

[0063] FIG. 4B: Dose-response curves of each pSTAT in IL-9R pmel T cells stimulated with IL-9WT(blue) or IL-9Q115T(light gray) for 20 minutes. Shown are technical duplicates; representative of two independent experiments. ECso and Emax values are provided in the accompanying table.

[0064] FIG. 4C: Tumor growth after ACT with IL-9R pmel T cells seven days after B16-F10 tumor inoculation. Mice were treated with either IL-9WT(n=7 mice) or IL-9Q115T(n=9 mice) (10 doses, every other day). Data are representative of two independent experiments.

[0065] FIG. 4D: Peripheral blood IL-9R transduced pmel T cell counts over time in mice treated as in FIG.4C with ACT and either IL-9WTor IL-9Q115Tcytokine (each cytokine dose indicated by a vertical dashed line). In the IL-9Q115Tgroup, cells were undetectable by day 41 or after three additional cytokine doses on days 44-48, only the IL-9WTgroup received doses beyond day 48. Data are representative of two independent experiments.

[0066] FIG. 4E: Schematic of IL- 9R variants with either three (IL-9R3x) or five (IL-9R5x) repeated phospho-tyrosine (pY) elements within the intracellular domain of the IL-9 receptor (created with Biorender.com). Sequence of the phosphotyrosine element shown in the legend (SEQ ID NO: 5).

[0067] FIG. 4F: Phosphorylation of indicated STATs at Emax(1 OOnM) for IL-9RWT, IL-9R3x, and IL-9R5Xtransduced pmel T cells stimulated with IL-2 or IL-9 for 20 minutes. Data are representative of three biological experiments (mean ± SEM).

[0068] FIG. 4G: B16-F10 tumor growth after ACT with either IL-9RWT(n=7 mice) or IL-9R3xtransduced pmel T cells (n=8 mice). Mice were also treated with IL-9 (5 104IU i.p. , every other day for 5 doses starting with ACT). Shown are individual tumor growth curves (left and middle) and group mean ± SEM (right). Data are representative of two independent experiments.Attorney Docket No.: 078430-543001 WO

[0069] *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. (two-way ANOVA for C; Welch's t-test for FIGS.4D and 4G; one-way ANOVA for FIG. 4F) (see also FIGS. 11A-11F).

[0070] FIGS. 5A-5I summarize the results of experiments performed to illustrate that IL-9R intracellular domain mutants skew STAT phosphorylation and alter in vivo proliferative capacity and anti-tumor efficacy.

[0071] FIG. 5A: Schematic of the IL-9 receptor complex, highlighting the phosphotyrosine (pY) site within the IL-9R intracellular domain (ICD) and three adjacent residues. A panel of ten single amino acid substitutions of the proline / glutamine residues adjacent to the pY site were generated (created with Biorender.com). See also FIG. 5B.

[0072] FIG. 5B: Heat map of pSTAT MFI (log-scaled and normalized to IL-9RWT[YLPQ]) at Emax for C57BL / 6 T cells transduced with wild-type IL-9R or one of ten IL-9R mutants described in FIG. 5A. Transduced pmel T cells (technical duplicates) were stimulated with IL-9 for 20 minutes. Data are representative of two independent experiments. Raw MFI values are shown in each cell.

[0073] FIG. 5C: Dose-response curves of pSTATs among transduced IL-9RWT, IL-9RAQ, and IL-9RPRpmel T cells (YFP+) stimulated with recombinant IL-9 for 20 minutes (technical duplicates). Data are representative of three biological replicates. EmaXand ECso are shown in the table.

[0074] FIG. 5D: Relative in vitro expansion of YFP+IL-9RWT, IL-9RAQ, IL-9RPRpmel T cells cultured with IL-9 on day 3 post-activation (10 nM; n=3 technical replicates / group). Data are representative of 3 biological replicates.

[0075] FIG. 5E: Proliferation index of C57BL / 6 T cells engineered with IL-9RWT, IL-9RAQ, and IL-9RPRover the course of nine days post-activation (transduced on day 1), as measured by dilution of CellTrace Violet dye (n=6 replicates / group). Cells received recombinant IL-2 or IL-9 (10 nM) on day 3 after T cell activation.

[0076] FIG. 5F: Peripheral blood quantification of IL-9RWT, IL-9RAQ, or IL-9RPRtransduced (Thyl.l+YFP+) pmel T cells in B16-F10 tumor-bearing mice (n=6-7 mice / group) on the indicated days after ACT. IL-9 treatment (5 104IU i.p., every other day) was given with ACT and continued for 5 doses. Data are representative of at least three independent experiments.Attorney Docket No.: 078430-543001 WO

[0077] FIG. 5G: Quantification of IL-9RWT, IL-9RAQ, or IL-9RPRtransduced (YFP+) pmel T cells within the tumors (B16-F10) of mice (n=6-7 mice / group) seven days post ACT. Data are representative of at least three independent experiments.

[0078] FIG. 5H: B16-F10 tumor growth in mice treated with IL-9RWT(n=7 mice) or IL-9RPR(n=6 mice) pmel T cells. Data are representative of at least three independent experiments.

[0079] FIG. 51: B16-F10 tumor growth in mice treated with IL-9RWTor IL-9RAQpmel T cells (n=10 mice / group). Data are representative of at least 4 independent experiments.

[0080] *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA for FIGS.5D-5F; Welch's t-test for FIGS. 5G-5I).

[0081] FIGS. 6A-6O summarize the results of experiments in which IL-9R variants revealed STAT1 as a rheostat skewing T cells from a stem and memory state toward a terminal effector state.

[0082] FIG. 6A: Schematic for in vivo experiment performed to generate single cell scRNA-seq data. IL-9RWT, IL-9RAQ, or IL-9RPRpmel T cells are adoptively transferred into B16-F10 tumor-bearing mice on Day 7 after tumor inoculation. Mice (n=7-8 mice / group) are treated with IL-9 (5X104I.U. every other day) starting on the day of ACT (Day 7) until tumors are harvested on day 15. Transduced pmel T cells (Thyl.l+YFP+) were FACS-sorted prior to library preparation and scRNA-seq. See also related FIGS. 13A-13B.

[0083] FIG. 6B: UMAP plots based on scRNA-seq of n=6,706 cells pmel T cells from FIG.6A showing ten major annotated clusters. See also related FIGS. 13C-13E.

[0084] FIG. 6C: Propeller composition plots demonstrating the relative proportion of each cluster from FIG.6A, split by treatment group (IL-9RWT, IL-9RAQ, or IL-9RPR).

[0085] FIG. 6D: Violin plots summarizing single cell expression of a gene set differentially expressed between KLRGlhleffector versus naive mouse T cell gene sets (GSE10239). Gene sets are summarized as Seurat module scores and plotted by treatment group.

[0086] FIG. 6E: Violin plots summarizing single cell expression of a gene set differentially expressed between mouse T cells eight days (D8) after encounter with acute infection versus malignancy (GSE60501). Gene sets are summarized as a module score in Seurat and plotted by treatment group.

[0087] FIG. 6F: Ridgeplot of pseudotime scores by treatment group.Attorney Docket No.: 078430-543001 WO

[0088] FIG. 6G: UMAP plot of scRNA-sequencing data from FIG. 6A annotated with pseudotime trajectories. The Tscm-like (Tcf7) cluster was selected as the root for the pseudotime analysis (white circles with black outlines). Black circles with white outlines represent nodes of the differentiation trajectory.

[0089] FIG. 6H: Schematic for linking phosphoflow data with scRNA-seq data. o9R, IL-9RWT, I L-9RAQ, IL-9RPR, or IL-9R5xpmel T cells were treated with cytokines for either 20 mins (phosphoflow) or 48h (RNA-seq). MFIs at Emax for each of pSTATs were merged with transcriptomes to identify genes most correlated with each STAT protein.

[0090] FIG. 61: Histograms of phosphorylation of each pSTAT for transduced (YFP+) T cells for each condition after 20 minutes of cytokine exposure at Emax: IL-2 (lOnM), IL-9 (lOnM) and oIL-2 (10 .M).

[0091] FIG. 6J: Venn diagram of the top 100 genes most correlated with the phosphorylation of each STAT.

[0092] FIG. 6K: Scatterplot relating pSTATl module score (y-axis; built from the top 100 pSTATl -correlated genes) to pSTATl phosphorylation levels (x-axis) in vitro. Module score was calculated from the expression of 100 genes most strongly correlated with STAT1 phosphorylation in vitro. Shown are biological triplicates (RNA-seq) colored by sample condition. For STAT phosphorylation data, shown are technical replicate means. The gray line indicates the linear regression fit.

[0093] FIG. 6L: Violin plot depicting projection of pSTATl module score from FIGS. 6J-6K onto scRNA-seq data from FIG.6A, organized by treatment group.

[0094] FIG. 6M: Overlay of the pSTATl module score from FIG. 8K onto UMAP from FIG.6B

[0095] FIG. 6N: Waterfall plot summarizing transcription factor (TF) enrichment from in vitro RNAseq data comparing IL-9RWTvs IL-9RAQgroups (left) and IL-9RPRVS IL-9RWIgroups (right). Gene expression changes across IL-9RAQIL-9RWTIL-9RPRare captured by increasing activity of STAT1 and related TFs (shown in red).

[0096] FIG. 60: Ridgeplots summarizing STAT1, STAT3, STAT4, and STAT5a regulon activity (AUC) inferred with SCENIC, shown by group with pairwise two-side Wilcoxon rank-Ssm tests).Attorney Docket No.: 078430-543001 WO

[0097] FIGS. 7A-7L summarize the results of experiments performed to illustrate that the balance of JAK / STAT signaling through IL-9R and IL-9R variants, and their impact on antitumor activity, is conserved in human CAR T cells.

[0098] FIG. 7A: Dose-response curves of each STATs in human T cells transduced with human IL-9R or human o9R (YFP+) and stimulated with either human IL-9 or human oIL-2 for 20 minutes. Data are representative of two independent donors.

[0099] FIG. 7B: Schematic of Nalm6 human leukemia orthotopic model in NSG mice treated with second generation human CAR T cells targeting CD 19 (CD19-BBz) and co-transduced with human IL-9R. Mice were also treated with either human IL-2 or IL-9 (or no cytokine).

[0100] FIG. 7C: Weight of NSG mice (n=5 mice / group) in response to treatment schema shown in FIG. 7B.

[0101] FIG. 7D: Bioluminescence (photons / second) of tumors in mice treated as described in FIG. 7B

[0102] FIG. 7E: Survival curves of mice from FIG. 7D.

[0103] FIG. 7F: Schematic of 143B human osteosarcoma orthotopic solid tumor model in NSG mice treated with second generation human CAR T cells targeting Her2 (Her2-BBz) and co-expressing IL-9R (or not) and treated with human IL-9 (or not).

[0104] FIG. 7G: Tumor growth as measured by leg volume of mice (n=5 mice / group) as treated per FIG. 7F.

[0105] FIG. 7H: Survival curves of mice from FIG. 7G.

[0106] FIG. 71: Dose-response curves of each pSTAT in human T cells transduced with human IL-9R, IL-9RPRor IL-9RAQand stimulated with IL-9 for 20 minutes. Shown are technical duplicates and representative of two independent donors. ECso and Emax are shown in the table.

[0107] FIG. 7J: Absolute number of (YFP+) CAR T cells in blood twelve days after tumor inoculation (8 days after ACT).

[0108] FIG. 7K: Tumor growth as measured by leg volume of mice (n=5 mice / group) treated as per schematic in FIG. 7F. Tumor volume was measured three times per week.

[0109] FIG. 7L: Survival curves of mice from FIG. 7K.

[0110] *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 (two-way ANOVA for FIGS.7D, 7G, 71, 7 J; Mantel-Cox test for FIGS. 7E, 7H, 7K; one-way ANOVA for FIG. 7L).Attorney Docket No.: 078430-543001 WO

[0111] FIGS. 8A-8F summarize the results of experiments performed to illustrate the nomination of IL-9 as a naturally orthogonal cytokine (related to FIGS. 1A-1G).FIG. 8A: Relative transcript expression of cytokine receptors in the common gamma chain family in human CD4 and CD8 T cells and unstimulated PBMC as measured by quantitative PCR. Values were normalized to expression of GAPDH in unstimulated PBMC.

[0112] FIG. 8B: Expression [log(FPKM+l)] of receptors for yc cytokines from publicly available RNA sequencing data from sorted populations from normal bone marrow and thymus of human donors representing different stages of hematopoietic and T cell development.

[0113] FIG. 8C: Dose-response curves of STAT1, STAT3, and STAT5 phosphorylation in isolated healthy donor human T cells (n=4 healthy donors) stimulated on Day 6 post activation with either recombinant human IL-2 or IL-9 for 20 minutes..

[0114] FIG. 8D: Expression of receptors for yc cytokines from publicly available RNA sequencing data of 22 stages of fetal and human development spanning 4 weeks post conception (4wpc) through 63 years of age. Plot is colored by tissue type. Vertical dashed lines indicate newborn stage for each tissue type. Horizontal dashed lines indicate threshold RPKM of 1.

[0115] FIG. 8E: Expression dot plots illustrating the percentage of cells expressing various cytokine receptor transcripts and average expression across various T cell subsets obtained from single cell RNA-sequencing data from three patients with non-small cell lung cancer treated with immune checkpoint blockade.

[0116] FIG. 8F: Same as FIG. 8E, except showing expression of the same cytokine receptor transcripts across tissue site, including lymph node (LN), normal tissue, tissue without viable tumor, viable tumor, and adrenal gland.

[0117] FIGS. 9A-9L summarize the results of experiments performed to illustrate the antitumor and phenotypic effects of IL-9 (related to FIGS.2A-2L).

[0118] FIG. 9A: Graphical representation of mouse movement (n=8 mice / group) over a period of 30 seconds at baseline (top panel) and six days after starting treatment (bottom panel).

[0119] FIG. 9B: Flow cytometry contour plots of YFP and surface IL-9R expression in mouse T cells transduced with the IL-9R construct. Right panel shows correlation of IL-9R and YFP expression across three viral titer dilutions (n=3 biological replicates for each titer) .

[0120] FIG. 9C: Representative gating strategy for major immune subsets (left panel) along with expression of IL-9R across each subset in lymphoid (lymph node, spleen, Peyer’s patches)Attorney Docket No.: 078430-543001 WOand non-lymphoid (lung, gut epithelium) tissues in C57BL / 6 mice (n=3-5 mice per group). Frequency of IL-9R+ mouse T cells after transduction with IL-9R is shown as a horizontal dashed line (60%).

[0121] FIG. 9D: B16-F10 tumor growth (from FIG.2F) with data shown for individual mice.

[0122] FIG. 9E: B16-F10 tumor growth (mean ± SEM, n=5 mice / group) in mice treated with either PBS or IL-9 (5 / 104IU, i.p / dose) starting five days after tumor inoculation and continuing every other day for five doses.

[0123] FIG. 9F: Growth of B16 tumors in C57BL / 6 mice treated with untransduced pmel T cells or IL-9R pmel T cells (i.v. on day 5 after tumor inoculation), along with MSA-IL9 (50,000 units i.p.) or PBS every other day for five doses starting with ACT (n=5-6 mice per group).

[0124] FIG. 9G: Same experimental setup as FIG. 9F. Data is representative of three independent experiments.

[0125] FIG. 9H: KP-gplOO tumor growth (mean ± SEM and individual mice, n=6 mice / group) after ACT with pmel T cells engineered with IL-9R (1.6 / 106transduced cells, i.v.), and cytokine treatment with PBS or IL-9, respectively (5x104 IU i.p., every other day for 5 days starting with ACT). Data is representative of two independent experiments.

[0126] FIG. 91: Representative gating strategy of CD44 and CD62L phenotyping for IL-9R or o9R-engineered T cells 24h after stimulation with IL-2 (lOnM), IL-9 (lOnM), or MSA-oIL2 (10pM). See also FIG. 2J.

[0127] FIG. 9J: Related to FIGS.2J and 91. The percentage of naive (CD62L+CD44-), central memory (CD62L+CD44+), effector (CD44+CD62L-) or double negative (CD62L-CD44-) T cells as bar plots.

[0128] FIG. 9K: Related to FIGS.2J and 91. Proportion of TSCM cells (CD44-, CD62L+, Sca-1+) presented as bar plots.

[0129] FIG. 9L: Phenotype (TN, TCM, TEFF) of IL-9R transduced (YFP+) or untransduced (YFP-) T cells in culture with IL-2 or IL-9 (lOnM) for 24 hours.

[0130] For all in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.0005, ****p < 0.0001 (two-way ANOVA for FIG. 9B; Welch's T-test for FIG. 9C; one-way ANOVA for FIG.9D; two-way ANOVA for FIGS.9F-9G.Attorney Docket No.: 078430-543001 WO

[0131] FIGS. 10A-10G summarize the results of experiments performed to illustrate the signaling and transcriptomic effects of IL-9R engagement (related to FIGS. 3A-3I).

[0132] FIG. 10A: Dose-response curves for STAT phosphorylation of IL- 9R transduced T cells treated with MSA-IL9, considering both transduced (YFP+) and untransduced (YFP-) cells within a culture.

[0133] FIG. 10B: Principal component analysis (PCI vs PC2) of RNA sequencing of C57BL / 6 T cells transduced for 48h with IL-9R and treated with IL-9 (10 nM) or IL-2 (10 nM), or with o9R and treated with oIL2 (10 pM). Samples cluster by treatment.

[0134] FIG. 10C: Volcano plots depicting differential gene expression based on RNA-sequencing of C57BL / 6 T cells transduced with IL-9R or o9R and treated with IL-9 (10 nM), IL-2(10 nM) or oIL2 (10 pM) for 48 hours. Shown in this figure is the comparisons between o9R T cells treated with oIL-2 and IL-9R T cells treated with IL-2. Significance (red) indicates adjusted p- value < IxlO'5.

[0135] FIG. 10D: Heatmap of top 100 differentially upregulated (left) and downregulated (right) transcripts in T cell expressing IL-9R treated with IL-9 versus IL-2 for 48h. Shown also in this figure are the o9R samples treated with oIL-2, which mimic the expression of the IL-9R + IL-9 samples.

[0136] FIG. 10E: Differentially phosphorylated proteins between C57BL / 6 T cells transduced with IL-9R and stimulated with IL-2 (lOnM) or IL-9 for 20 minutes. Significance (red) indicates adjusted p < 0.05 and log2(fold change) >0.5.

[0137] FIG. 10F: Representative gating strategy for flow cytometry data for intracellular staining of phosphoproteins in engineered (YFP+) T cells.

[0138] FIG. 10G: Dose-response curves of ERK or AKT phosphorylation in IL-9R or o9R transduced pmel T cells (YFP+) and stimulated with either oIL-2, IL-2, or IL-9 for 20 minutes. Error bars represent min / max of technical duplicates. Data representative of two independent experiments.

[0139] FIGS. 11A-11F summarize the results of experiments performed to illustrate the structure-based attenuation or amplification of IL-9 / IL-9R (related to FIGS. 4A-4G).

[0140] FIG. 11A: Sequence alignment between mouse and human IL-2, IL-9, IL-15, and IL-21 around the conserved glutamine in Helix D. Human IL-21: SEQ ID NO: 6; human IL-15:Attorney Docket No.: 078430-543001 WOSEQ ID NO: 7; mouse IL-2: SEQ ID NO: 8; human IL-2: SEQ ID NO: 9; mouse IL-9: SEQ ID NO: 10; human IL-9: SEQ ID NO: 11.

[0141] FIG. 11B: Frequency of TSCM (CD62L+CD44-Sca-1+) among mouse T cells transduced with IL-9R (CD8+YFP+) and treated with IL-2, IL-9 and IL-9Q115T (lOnM) for 24 hours.

[0142] FIG. 11C: Waterfall plot of maximal tumor size reduction for experiment from FIG.4C, in which B16-F10 tumors in mice (n=7-9 mice / group) treated with IL-9R transduced pmel T cells and either IL-9WTor IL-9Q115T.

[0143] FIG. 11D: Peripheral blood enrichment of IL- 9R transduced pmel T cells over time in mice treated with ACT and either IL-9WTor IL-9Q115Tcytokine (each cytokine dose indicated by a vertical dashed line). Enrichment was calculated as percentage of transduced (YFP+) pmel T cells as a proportion of all pmel T cells. See also FIG.4D.

[0144] FIG. HE: Dose-response curves of phosphorylation of indicated STAT proteins in pmel T cells transduced with IL-9RWT, IL-9R3x, and IL-9R5xand stimulated with IL-2 or IL-9 for 20 minutes. Error bars represent min / max of three biological replicates.

[0145] FIG. 11F: Frequency of TSCM (CD62L+CD44-Sca-1+) among mouse T cells transduced with IL-9RWTor IL-9R3x(CD8+YFP+) and treated with IL-2, or IL-9 (lOnM) for 24 hours. For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. ***P < 0.001 (Welch's t-test for FIG. 11C, two-way ANOVA for FIG. 11D).

[0146] FIGS. 12A-12G summarize the results of experiments performed to illustrate the proliferation of IL-9R and IL-9R intracellular domain mutants (related to FIGS. 5A-5I).

[0147] FIG. 12A: Phosphorylation of indicated STATs at Ema(1 OnM) for IL-9RWT, IL-9RFLPQtransduced C57BL / 6 T cells stimulated with IL-2 or IL-9 for 20 minutes. Bars represent mean ± SEM of technical replicates. Data are representative of two independent experiments.

[0148] FIG. 12B: Representative histograms of CellTrace Violet staining for the groups from FIG 5E

[0149] FIG. 12C: Percentage of YFP+cells undergoing apoptosis (Annexin V+, Zombie Violet") or dead (Annexin V+, Zombie Violet ) shown as bar plots (each point represents the average of technical replicates for each biological replicate).Attorney Docket No.: 078430-543001 WO

[0150] FIG. 12D: Related to FIG. 5D. Percentage of YFP+cells undergoing apoptosis (Annexin V+, L / D ) or dead (Annexin V+, L / D+) shown as bar plots (each point represents the average of technical replicates for each biological replicate).

[0151] FIG. 12E: Frequency of TCM (CD62L+CD44+), TSCM (CD62L+CD44-Sca-1+) and TEFF (CD62L-CD44+), among mouse T cells transduced with IL-9RWT, IL-9RAQ, or IL-9RPR(CD8+YFP+) and treated with IL-2, or IL-9 (lOnM) for 24 hours.

[0152] FIG. 12F: Survival curves of mice from FIG. 5G.

[0153] FIG. 12G: Survival curves of mice from FIG. 5H.

[0154] *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (one-way ANOVA for FIGS.12A-12B, two-way ANOVA for FIG. 5E, Mantel-Cox test for FIGS. 12F-12G).

[0155] FIGS. 13A-13O summarize the results of experiments in which scRNA-seq of tumorinfiltrating pmel T cells engineered with IL-9R or IL-9R variants revealed STAT1 as a rheostat of T cell differentiation (related to FIGS. 6A-6O).

[0156] FIG. 13A: Tumor growth (mean ± SEM) for the experiment described in FIGS. 6A-60. Data are representative of at least two independent experiments.

[0157] FIG. 13B: Transduced (Thyl.l+YFP+) pmel T cells as a percentage of total CD8 T cells in the tumor by treatment group for scRNA-seq experiment from FIGS. 13A-13B.

[0158] FIG. 13C: Merged UMAP plot based on scRNA-seq of n=6,706 cells pmel T cells transduced with IL-9RWT, IL-9RAQ, or IL-9RPR. Ten major clusters colored according to annotation. See also related FIG.6A.

[0159] FIG. 13D: Violin plots summarizing single cell expression of stem and memory markers, Tcf7 (top) and Il7r (bottom), by cluster.

[0160] FIG. 13E: Feature plot of module score defined by the Gene Ontology Myeloid Differentiation gene set demonstrating high expression exclusively in the Tmyeloid-like cluster.

[0161] FIG. 13F: Violin plots summarizing single cell expression of effector molecules, Prfl (top) and Gzmb (bottom), by treatment group.

[0162] FIG. 13G: Ridgeplot of pseudotime scores for single cell data from FIG. 13A organized by annotated cluster.

[0163] FIG. 13H: Frequency of TN and TCM / TEM phenotypes among adoptively transferred IL-9RWT, IL-9RAQand IL-9R3xpmel T cells in the tumor, dLN and spleen seven days after adoptive transfer into C57BL / 6 mice bearing B16-F10 tumors multiple comparison testing.Attorney Docket No.: 078430-543001 WOCD62L+CD44+ cells are considered TCM in the spleen and dLN, and TEM in the tumor.Statistical comparisons performed by unpaired t test (for two groups) or by one-way ANOVA (for three groups).

[0164] FIG. 131: Frequency of TEFFphenotypes among adoptively transferred IL-9RWT, IL-9RAQand IL-9R3xpmel T cells in the tumor (left panel) and dLN (right panel) seven days after adoptive transfer into C57BL / 6 mice bearing B16-F10 tumors. Statistical comparisons performed by unpaired t test (for two groups) or one-way ANOVA (for three groups).

[0165] FIG. 13J: Frequency of Granzyme B+ (left) and TNF-a+ (right) among adoptively transferred IL-9RWT, IL-9RAQand IL-9R3xpmel T cells in the tumor (top panel) or dLN (bottom panel) seven days after adoptive transfer into C57BL / 6 mice bearing B16-F10 tumors. Statistical comparisons performed by unpaired t test (top panel) or one-way ANOVA (bottom panel).

[0166] FIG. 13K: Scatterplot depicting the relationship between the pSTAT module scores (y-axis) and pSTAT phosphorylation levels (x-axis) in vitro. Module scores were calculated from the expression of 100 genes most strongly correlated with STAT phosphorylation in vitro.Shown are biological triplicates for the gene expression data, colored by sample condition. For STAT phosphorylation data, shown are the averages of technical duplicates. See also FIG.6K.

[0167] FIG. 13L: Projection of pSTAT module scores from FIG. 13H onto scRNA-seq data from FIG.6A.

[0168] FIG. 13M: STAT1 phosphorylation of mouse T cells transduced with IL-9RWT, IL-9RAQand IL-9RPRcultured with IL-9 (lOnM) and increasing doses of IFN-P for 20 minutes. Dashed line indicates STAT1 phosphorylation level of IL-9RPRT cells cultured in IL-9 without IFN-p.

[0169] FIG. 13N: Phenotypic analysis of CD62L and CD44 expression by mouse T cells transduced with IL-9RWT, IL-9RAQor IL-9RPRand cultured with IL-9 (lOnM) with or without IFN-P (lOng / ml) for 24 hours. Shown are pooled results of two independent experiments.Statistical comparisons performed by one-way ANOVA.

[0170] FIG. 130: Hallmark gene set enrichment analysis based on genes differentially expressed among tumor infiltrating IL-9RAQpmel T cells from FIG. 6A.

[0171] FIGS. 14A-14J summarize the results of experiments performed to illustrate phenotypic and functional impact of CAR T cells signaling through IL-9R and IL-9R variants (related to FIGS. 7A-7L).Attorney Docket No.: 078430-543001 WO

[0172] FIG. 14A: Repetitive killing of M407mCheriyhuman melanoma cell lines by NY-ESO-1 TCR T cells co-transduced with ho9R or hIL-9R in the presence of IL-9 (lOnM) or oIL-2 (l M), respectively. Shown is the mean cytotoxicity index as measured on the Incucyte SX5 based on tumor fluorescence (mCherry). Statistical comparisons were performed by two-way ANOVA. Data is representative of two independent experiments.

[0173] FIG. 14B: Expression of second generation CD19 CAR (CD19-BBz) and human IL-9R after co-transduction as measured by idiotype staining and YFP expression, respectively.

[0174] FIG. 14C: In vitro cytotoxicity of second generation CD 19 CAR (CD19-BBz) cotransduced with human IL-9R in a repetitive tumor challenge with NALM6 leukemia at a 2: 1 E:T ratio. Cocultures were supplemented with IL-2, IL-9, or no cytokine. Shown is the mean cytotoxicity index as measured on the Incucyte SX5 based on tumor fluorescence (GFP).Statistical analysis performed by two-way ANOVA.

[0175] FIG. 14D: Schematic demonstrating the conserved amino acids following the phosphotyrosine site of the intracellular domain of the human and mouse IL-9R.

[0176] FIG. 14E: Expression of second-generation Her2 CAR (Her2-BBz) and human IL-9R after co-transduction as measured by idiotype staining and YFP expression, respectively.

[0177] FIG. 14F: In vitro proliferation of human IL-9R transduced T cells grown in complete media supplemented with IL-9 (lOnM).

[0178] FIG. 14G: Percentage of IL-9R+(YFP+) CAR T cells as a percentage of total human T cells twelve days after tumor inoculation (8 days after ACT) from FIG. 14K

[0179] FIG. 14H: Repetitive killing of M407mCheriyhuman melanoma cell line by NY-ESO-1 TCR transduced T cells cotransduced with IL-9RWT, IL-9RAQor IL-9RPRin the presence of IL-9 or no cytokine. Shown is the mean cytotoxicity index based on tumor fluorescence (mCherry). Statistical comparisons performed using two-way ANOVA.

[0180] FIG. 141: Quantification of NY-ESO-1 TCR T cells co-transduced with IL-9RWT, IL-9RAQor IL-9RPRby flow cytometry after three rounds of repetitive tumor killing in the presence of IL-9 or no cytokine. T cells were plated at equal quantity at the start of round three.

[0181] FIG. 14J: Frequency of intracellular Granzyme B expressing NY-ESO-1 TCR T cells co-transduced with IL-9RWT, I L-9RAQor IL-9RPRafter three rounds of repetitive killing in the presence of IL-9 or no cytokine. Statistical comparisons performed by one-way ANOVA with multiple comparison testing.Attorney Docket No.: 078430-543001 WO

[0182] *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.DETAILED DESCRIPTION OF THE DISCLOSURE

[0183] The present disclosure relates generally to, mter alia, novel methods and compositions for the prevention and / or treatment of various health conditions. In particular, some embodiments of the disclosure recombinant cytokine receptors that include an intracellular signaling domain (ICD) of an interleukin 9 receptor (IL-9R), wherein the ICD includes a proline-to-alanine (P->A) substitution in its STAT binding site.

[0184] As described in greater detail below, the recombinant cytokine receptors disclosed herein possess signaling properties that are advantageous for the adoptive transfer of T cells, in particular in vivo engraftment, expansion, and effector function. In adoptive T cell therapies, engineered T cells are typically activated by exposure to the cognate antigen in vitro or ex vivo, expanded, and then administered to the individual, where they proliferate and exhibit cytolytic activity and / or send signals to initiate an immune response against the target cancer.

[0185] The improved in vivo expansion, tissue infiltration and effector function of T cells signaling through the recombinant cytokine receptors disclosed herein has particular relevance for use in the treatment of cancer. Engineered T cells are FDA approved for the treatment of hematologic malignancies, and there are several additional commercial products in the pipeline for the treatment of solid tumors, as well as autoimmune conditions. The signaling properties of the recombinant cytokine receptors disclosed herein would enhance the in vivo engraftment, expansion, trafficking and effector function of these commercial products.

[0186] Some embodiments of the disclosure provide recombinant nucleic acids encoding a recombinant cytokine receptor as disclosed herein. Also provided, in some embodiments, are recombinant cells, e.g., T cells that have been engineered to express a recombinant cytokine receptor as disclosed herein. Further provided are methods for generating a population of recombinant cells, e.g, engineered T cells with improved therapeutic properties for adoptive cell therapy, and pharmaceutical compositions containing such a population of engineered T cells with enhanced therapeutic properties, as well as methods and kits for the prevention and / or treatment of a health condition in subjects in need thereof.Attorney Docket No.: 078430-543001 WODEFINITION

[0187] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0188] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0189] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B”.

[0190] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0191] The term “administration” and its grammatical variations, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0192] The term “cancer” refers to the presence of cells possessing several characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells can aggregate into a mass, such as a tumor, or can exist alone within a subject. A tumor can be a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term “cancer” also encompasses other types of non-tumor cancers. Non-limiting examples include blood cancers orAttorney Docket No.: 078430-543001 WOhematological cancers, such as leukemia. Cancer can include premalignant, as well as malignant cancers.

[0193] The terms “cell,” “cell culture,” and “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications can occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0194] As used herein, the term “functional variant thereof’ refers to a molecule having quantitative and / or qualitative biological activity in common with the wild-type molecule from which the fragment or variant was derived.

[0195] The term “operably linked,” as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is functional link that allows for expression of the polynucleotide of interest. It should be understood that, operably linked elements may be contiguous or non-contiguous. In the context of a polypeptide, “operably linked” refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide. In the present disclosure, various domains of the recombinant polypeptides of the disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the recombinant polypeptides in the cell. Operably linked domains of the recombinant polypeptides of the disclosure may be contiguous or non-contiguous (e.g., linked to one another through a linker).

[0196] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,Attorney Docket No.: 078430-543001 WOor higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity typically is calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul etal., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0197] The term “recombinant” or “engineered” nucleic acid molecule, polypeptide, or cell as used herein, refers to a nucleic acid molecule, polypeptide, or cell that has been altered through human intervention.

[0198] As used herein, and unless otherwise specified, a “therapeutically effective amount” or a “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease, e.g, cancer, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount or number of a compound means an amount or number of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the disease. The term “therapeutically effective amount” can encompass an amount or number that improves overall therapy of the disease, reduces or avoids symptoms or causes of the disease, or enhances therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of theAttorney Docket No.: 078430-543001 WOseverity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0199] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subject) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or a subject who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be a subject who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g, mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0200] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0201] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0202] Headings, e.g, (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.Attorney Docket No.: 078430-543001 WO

[0203] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0204] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0205] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, isAttorney Docket No.: 078430-543001 WOunderstood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps. Furthermore, the use of the term “including” as well as other forms, such as “include,” “includes” and “included,” is not limiting.

[0206] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.INTERLEUKIN 9 RECEPTOR (IL-9R)

[0207] Interleukin 9 receptor (IL9R) also known as CD 129 (Cluster of Differentiation 129) is a type I cytokine receptor. This cytokine receptor specifically mediates the biological effects of interleukin 9 (IL-9). Interleukin 9 (IL-9) has a role in the growth and promotion of T cells and mast cells, so it was initially named as T cell growth factor III (TCGFIII) and / or mast cell growth- enhancing activity (MEA).

[0208] The IL-9 receptor is a heterodimer complex composed of IL-9Ra and common gamma subunit, i.e., y chain (yC). IL-9Ra is a specific receptor chain for IL-9, whereas the yC chain is also observed in many other cytokine receptors, including IL-2, IL-4, IL-7, IL-15, IL-21 and thymic stromal lymphopoietin receptors. The ligand binding of this receptor leads to the activation of various JAK kinases and STAT proteins, which connect to different biologic response.

[0209] In both human and mice, IL-9R is expressed by on T cell lineages and effector T cells, with the exception of naive T cells. Among TH cell subsets, IL-9R is predominantly expressed on TH2 and TR1 cells. Several studies have indicated that multiple cell types can also express 1L-9R including; macrophages, mast cells, dendritic cells, microglia, immature neurons, NK cells, NKT cells, Tregs, B cells (specially on germinal center B cells) and TH9 cells. Although the requirement of IL-9 function in non-hematopoietic cells is not yet known, IL-9Ra expression onAttorney Docket No.: 078430-543001 WOcells including airway and intestinal epithelial cells, smooth muscle cells, and keratinocytes has been identified.

[0210] Cytokines and their receptors enable precise tuning of T cell function. Leveraging this biology holds tremendous promise for optimizing antitumor immunity. Arming T cells with a synthetically orthogonal IL-9 receptor (o9R) for instance, permits facile engraftment and potent anti-tumor functions. A number of experiments described herein have been performed to investigate whether the paucity of wild-type IL-9R expression could be exploited for T cell immunotherapy given that, in mice, and the safety of high doses of IL-9 were well-tolerated without discernible immune modulation. As described in greater detail below, compared to o9R, T cells engineered with wild-type IL-9R exhibited superior tissue infiltration, sternness, and antitumor activity. These qualities were consistent with a stronger JAK / STAT signal, which included canonically IL-12-driven STAT4 in addition to STAT1 / 3 / 5. IL-9R T cells were exquisitely sensitive to perturbations of proximal signaling, including structure-guided attenuation, amplification, and rebalancing of JAK / STAT signals. In addition, biased IL-9R mutants showed STAT1 acts as a rheostat between stem-like and effector states. In summary, the experimental data described herein have identified IL-9 / IL-9R as a naturally orthogonal cytokine-receptor pair with a superior and / or desired JAK / STAT signaling profile for engineered T cell therapy.

[0211] Common y-chain (yc) cytokines are important regulators of T cell differentiation and function, exerting their effects through JAK / STAT signaling pathways1. Their potential to enhance adoptive T cell therapy for cancer has long been recognized2, yet clinical translation of ycand other cytokines has been impeded by safety concerns stemming from their pleiotropic effects3'7. To overcome this challenge, several approaches have been developed to selectively deliver cytokine signals to engineered T cells. In one approach, mutations in IL-2 and its receptor, IL-2RB, yielded orthogonal IL-2 (oIL-2) and orthogonal IL-2RB (o2R), a synthetic and mutually exclusive pair8. This system enables targeted IL-2 signaling in engineered T cells while avoiding activation of other IL-2-responsive populations that contribute to systemic toxicity9.

[0212] To explore which of the yc cytokines induce the desired and / or superior JAK / STAT signals for successful T cell immunotherapy, experiments have been carried out to test an iteration of the orthogonal IL-2 system in which the intracellular domain (ICD) of o2R was replaced with ICDs of different yc cytokine receptors (IL-4R, IL-7R, IL-9R, IL-21R)10. These chimeras (o4R, o7R, o9R, o21R) mimic the unique JAK / STAT signaling pattern of their naturalAttorney Docket No.: 078430-543001 WOreceptor counterpart and induce different phenotypic and functional profiles in T cells. It was observed that o9R, which induces a distinct JAK / STAT signaling profile consisting of STAT1, STAT3, and STAT5 activation, results in T cells that acquire stem-like characteristics, efficiently infiltrate tumors, and substantially improve the anti-tumor activity of TCR- and CAR-T cells in immunocompetent hosts even without conditioning lymphodepletion.

[0213] Building on an orthogonal IL-2 system in which intracellular domains (ICD) of yc cytokine receptors had been swapped, it was previously found that the chimera incorporating the IL-9RICD activates STAT1, STAT3 and STAT5, enriches stem-like T cells, improves infiltration into solid tumors, and enhances antitumor activity in immunocompetent hosts without lymphodepletion.

[0214] Given the relative anonymity of IL-9R signaling among the ycfamily, this surprising result prompted the inventors to explore anti-tumor effects of T cells signaling through the wildtype IL-9 receptor. In particular, the reported low nanomolar affinity of IL-9 for the wild-type IL-9 receptor was found intriguing11. However, it was not clear whether there is sufficient IL-9R expression on T cells to permit a response to IL-9. And furthermore, as the only yc cytokine not tested in humans, potential toxicities of systemic IL-9 administration are not well understood.

[0215] To address these issues, publicly available and in-house genomic, transcriptomic and functional datasets were integrated and examined to characterize the expression and function of IL-9R in normal tissues. Compared to receptors for other common yc cytokines, IL-9R is rarely expressed across immune and non-immune tissues, including throughout fetal and thymic development and in the tumor microenvironment. In fact, population-based genetics suggests limited evolutionary constraint on IL-9.

[0216] Based on these observations, the wild-type IL-9 and IL-9R was nominated as a naturally orthogonal cytokine-receptor pair. In mice, IL-9 was well-tolerated even at very high doses. Tumor-specific T cells engineered with the wild-type IL-9 receptor, and compared to T cells with o9R. It was found that these engineered T cells have superior engraftment, tumor infiltration, sternness and anti-tumor activity compared to o9R. These findings were linked to more potent phosphorylation of STAT1, STAT3 and STAT5, as well as phosphorylation of STAT4, which is not classically associated with common yc signaling.

[0217] As discussed in greater detail below, by attenuatin, amplifying, or biasing IL-9R JAK / STAT signaling of IL-9R, it was found that anti-tumor efficacy is exquisitely sensitive toAttorney Docket No.: 078430-543001 WOboth signal strength and STAT stoichiometry. Mutant receptors revealed a T cell intrinsic STAT1 rheostat that skews T cells from a proliferative stem- and memory-like state toward terminally differentiated effectors. These results were extended to human CAR and TCR T cells, positioning IL-9 / I1-9R as a natural alternative to synthetic orthogonal systems.

[0218] Experimental data described herein indicate that IL-9 stands out among yc cytokines because its private receptor (IL-9R) shows remarkably restricted normal tissue expression across tissues. This likely explains why high doses of half-life extended IL-9 were well-tolerated and induced negligible node transcriptional perturbation.

[0219] The above finding is not to preclude important roles for rare IL-9R expressing cells39. IL-9 participates in type 2 immunity and helminth defense40'43and can amplify airway inflammation42, 44, 45. Even so, IL-9 deficient mice are broadly healthy withnormal immune functions46,47. And based on populationgenetics, IL-9 scored lowest among canonical cytokines with respect to its role in evolutionary fitness.

[0220] As described in greater detail in the Examples, leveraging the paucity of IL-9R expression , coadministration of IL-9 with small numbers of IL-9R engineered tumor-specific T cells generated robust anti-tumor responses without preconditioning lymphodepletion, outperforming the benchmark o9R system. These findings were consistent across multiple mouse and human models, as well as data from Castelli, et al48. IL-9R T cells demonstrate superior signaling, transcriptomic, and biological outputs compared to the synthetic o9R system. The native, high affinity IL-9 / IL-9R interaction likely contributes to stronger proximal signaling than intentionally low-affinity synthetic pairs (o9R and oIL-2)4’49.

[0221] The stronger IL-9 / IL-9R signal revealed the unexpected phosphorylation of STAT4 , providing a route to deliver STAT4 effector programs without the toxicities that have limited IL-1217’37. At the same time, IL-9 preserves STAT5 -associated proliferation and effector capacity21,50and STAT3 -associated sternness22.

[0222] The perturbation studies described herein uncovered a “goldilockswindow” both attenuation and amplification of iL-9R signaling impaired anti-tumor efficacy. Likewise, T cells were exquisitely sensitive to changes in pSTAT stoichiometry51. Leveraging two IL-9R single amino acid variants with divergent pSTATl signals, the role of T cell intrinsic STAT1 in antitumor functions was examined. STAT1 has anti-proliferative effects in T cells26’27, but has also been linked to clonal expansion, memory formation52, and effector function. Amplification ofAttorney Docket No.: 078430-543001 WOpSTATl (IL-9RPR) diminished expansion and reduced anti-tumor efficacy, while attenuation of pSTATl (IL-9RAQ) resulted in superior expansion, particularly in vivo, accompanied with better tumor infiltration.

[0223] Subtle shifts in pSTAT stoichiometry have potent effects on cell fate, even in the presence of an active TCR stimulus. Transcriptomic differences driven by IL9R variants were captured along a pSTATl axis, with high pSTATl driving the development of a terminally differentiated effector state (IL-9RPR), low pSTATl yielding a proliferative, stem- and memorylike state (IL-9RAQ), and IL-9RWToccupying an intermediate state. This is consistent with work demonstrating that interferon signaling, through IRF2, promotes CD8+ T cell exhaustion53, and also provides a plausible explanation for why JAK / STAT inhibition in the context of chronic interferon signaling may restore T cell effector functions54. However, pSTATl may not be entirely detrimental to anti-tumor immunity: lower pSTATl signals (IL-9RAQ) restrained effector differentiation, and IL-9RAQpmel T cells did not improve anti-tumor efficacy despite superior T cell expansion.

[0224] The reduced pSTATl signal produced by IL-9RAQwas also associated with an enrichment in STAT3 and STAT5 activity inferred from single cell transcriptomic data, consistent with their stem, memory22,31and proliferative qualities21,32of IL9RAQpmel T cells. However, this was partly unexpected because IL-9RAQproduces a weaker pSTAT3 signal than IL-9RWTby phosphoflow (and similar pSTAT5 signal). Without being bound to any particular theory, it is hypothesized that the discrepancy between STAT activity assessed by phosphorylation and STAT activity inferred from scRNA-seq data may be related to downstream competition between simultaneously active STAT proteins, a phenomenon described in the fate of Thl7 cells55. The weaker pSTATl signal of IL-9RAQmay result in less competition with pSTAT3, such that even a relatively weak pSTAT3 signal generates a stronger downstream biologic effect.

[0225] In summary, the experimental results described herein uncover and leverage the uniquely restricted normal tissue expression of IL-9R. The IL-9 / IL-9R is a natural cytokinereceptor pair with orthogonal qualities, and in models of engineered T cell therapy, outperforms its synthetic counterpart (o9R). The activity of IL-9R can be linked to its higher affinity and more potent signaling, which unexpectedly includes STAT4, not previously considered a primary target of ycsignaling. The highly potent anti-tumor functions of T cells signaling throughAttorney Docket No.: 078430-543001 WOIL-9R - validated in multiple mouse and human CAR T cell models against hematologic and solid tumors - can be traced to a superior strength and balance of IL-9R signals, with pSTATl tuning cells between a stem- and memory-like state and a terminal effector state.

[0226] As discussed in greater detail below, human primary T cells expressing a recombinant cytokine receptor of the disclosure can be transformed with a chimeric antigen receptor (CAR) or native T cell receptor (TCR) to create CAR T cells that can be subsequently used to treat relevant health conditions and diseases, such as cancers.COMPOSITIONS OF THE DISCLOSURE

[0227] As described in greater details below, one aspect of the present disclosure relates to novel recombinant cytokine receptors for in vivo enhancement of adoptive transferred T cells. Another aspect of the disclosure relates to recombinant nucleic acids encoding a recombinant cytokine receptor as disclosed herein. Also provided, in some embodiments, are recombinant cells, e.g., recombinant T cells that have been engineered to express a recombinant cytokine receptor as disclosed herein. Further provided are pharmaceutical compositions containing such a population of recombinant T cells with enhanced therapeutic properties, as well as kits for the prevention and / or treatment of a health condition in subjects in need thereof.Recombinant cytokine receptors

[0228] As outlined above, one aspect of the present disclosure relates to novel recombinant cytokine receptors for in vivo enhancement of adoptive transferred T cells. In particular, some embodiments of the disclosure relate to recombinant cytokine receptors including: (a) an ECD and an ICD of an IL-9R, wherein the ICD includes a proline-to-alanine (P->A) substitution in the STAT binding site; and (b) a transmembrane domain (TMD) operably inserted between the ECD the ICD.

[0229] Non-limiting exemplary embodiments of the disclosed recombinant cytokine receptors can include one or more of the following features. In some embodiments, the IL-9R is a murine IL-9R or a function variant thereof. In some embodiments, the IL-9R is a human IL-9R or a functional variant thereof.

[0230] Generally, the TMD suitable for the recombinant cytokine receptors disclosed herein can be any transmembrane domain of a Type 1 transmembrane protein. In some embodiments, the TMD is a transmembrane domain derived from the same IL-9R. In some embodiments, theAttorney Docket No.: 078430-543001 WOTMD is a heterologous transmembrane domain relative to the IL-9R. In some embodiments, the heterologous TMD is derived from a type-1 transmembrane spanning protein. Non-limiting suitable TMDs from Type 1 transmembrane proteins include those from CD3 , CD4, CD8, CD28, B7-H3, IL-2RB, IL-4R, IL-7R, IL-9R, or IL-21R. In some embodiments, the heterologous TMD is derived from a surface receptor subunit. Non-limiting examples of TMDs from surface receptor subunits suitable for the compositions and methods of the disclosure include those from IL3Ra, IL4Ra, IL5Ra, IL6Ra, IL7Ra, ILlORa, ILlORb, IL12Rpl, IL12Rp2, IL12p40, IL13RA1, IL15Ra, IL20R, IL21Ra, IL22R, IL23R, IL28R, and IL31 Ra. Additional TMDs from surface receptor subunits suitable for the compositions and methods of the disclosure include, but are not limited to, GMCSFRa, LIFR, CNTFR, CLF1, OSMR, GCSFR, EPOR, TPOR, GHR, PRLR, LEPR, IFNAR2, IFNAR1, IFNGR1, and IFNGR2.

[0231] In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2. In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments of the disclosure, the recombinant cytokine receptor includes the amino acid sequence of SEQ ID NO: 1, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 1 is / are substituted by a different amino acid residue. In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments of the disclosure, the recombinant cytokine receptor includes the amino acid sequence of SEQ ID NO: 2, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 2 is / are substituted by a different amino acid residue. In some embodiments of the disclosure, the recombinant cytokine receptor includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0232] In some embodiments of the disclosure, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in a decreased STAT1 signaling while substantiallyAttorney Docket No.: 078430-543001 WOretains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by at least 10%, e.g., at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor.

[0233] In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, confers an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, in vitro and / or in vivo proliferation / expansion of the recombinant T cell is enhanced by at least 10%, e.g., at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, in vitro and / or in vivo proliferation / expansion of the recombinant T cell is enhanced by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor.

[0234] In some embodiments, the recombinant cytokine receptor, when expressed in a recombinant T cell, results in promoting less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the reference T cell includes a wild-type IL-9 receptor.Nucleic acids

[0235] In an aspect, provided herein is nucleic acid molecules encoding the recombinant cytokine receptors described herein, expression cassettes encoding the recombinant cytokine receptors described herein, and vectors (e.g., expression vectors) containing the nucleic acidsAttorney Docket No.: 078430-543001 WOencoding the recombinant cytokine receptors described herein. In some embodiments, nucleic acid molecules may be operably linked to expression control elements which facilitate expression of the recombinant cytokine receptors in a host cell.

[0236] The terms “nucleic acid” and “polynucleotide” can be used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acids comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid can contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0237] The nucleic acids of the present disclosure can be nucleic acids of any length, including nucleic acids that are generally between about generally between about 0.5 Kb and about 20 Kb, for example between about 0.5 Kb and about 20 Kb, between about 1 Kb and about 15 Kb, between about 2 Kb and about 10 Kb, or between about 5 Kb and about 25 Kb, for example between about 10 Kb to 15 Kb, between about 15 Kb and about 20 Kb, between about 5 Kb and about 20 Kb, about 5 Kb and about 10 Kb, or about 10 Kb and about 25 Kb.

[0238] In some embodiments, the nucleic acids of the disclosure include a nucleotide sequence encoding a recombinant cytokine receptor as disclosed herein. In some embodiments, the recombinant cytokine receptor includes: (a) an ECD and an ICD of an IL-9R, wherein the ICD includes a proline-to-alanine (P->A) substitution in the STAT binding site; and (b) a TMD operably inserted between the ECD the ICD.

[0239] In some embodiments, the nucleic acids include a nucleotide sequence encoding an recombinant cytokine receptor that includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the recombinant cytokine receptor as disclosed herein or a functional fragment thereof. In some embodiments, the nucleic acids include a nucleotide sequence encoding an recombinant cytokine receptor that includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acids include a nucleotide sequence encoding anAttorney Docket No.: 078430-543001 WOrecombinant cytokine receptor that includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0240] Nucleic acid sequences having a high degree of sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to an amino acid sequence of a recombinant cytokine receptor of interest can be identified and / or isolated by using the sequences identified herein (e.g., SEQ ID NOs: 1-2) or any others as they are known in the art, by genome sequence analysis, hybridization, and / or PCR with degenerate primers or gene-specific primers from sequences identified in the genome.

[0241] In some embodiments, the recombinant nucleic acid molecule includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 3-4. In some embodiments, the recombinant nucleic acid molecule includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 3. In some embodiments, the recombinant nucleic acid molecule includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 4.

[0242] In some embodiments of the disclosure, the recombinant nucleic acid molecule is operably linked to one or more heterologous nucleic acid sequences. In some embodiments, the one or more heterologous nucleic acid sequences includes one or more expression control elements. Non-limiting examples of expression control elements suitable for the compositions and methods disclosed herein include ribosomal binding sites, promoters, translational start sequences, translational termination sequences, transcriptional start sequences, transcriptional termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, and 325 bp poly(A) tracts. Additional expression control elements suitable for the compositions and methods disclosed herein include, but are not limited to, enhancer elements, activator elements, replication elements, RNA processing and export elements, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences. In someAttorney Docket No.: 078430-543001 WOembodiments, the one or more heterologous nucleic acid sequences operably linked to the recombinant nucleic acid molecule includes a promoter sequence. In some embodiments of the disclosure, the promoter is a CD4 cell-specific promoter or a CD8 cell-specific promoter. In some embodiments, the promoter is a CD4 cell-specific promoter. In some embodiments, the promoter is a CD8 cell-specific promoter. Non-limiting examples of promoters suitable for the compositions and methods disclosed herein include murine stem cell virus (MSCV) promoter, EFla promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD 57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, and CD 132 promoter. Additional promoters suitable for the compositions and methods disclosed herein include, but are not limited to, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GAT A3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, and NR4A3 promoter. In some embodiments, the one or more heterologous nucleic acid sequences operably linked to the recombinant nucleic acidAttorney Docket No.: 078430-543001 WOmolecule includes or is a synthetic promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a regulatable promoter, e.g., an inducible promoter or a repressible promoter.

[0243] In some embodiments, the nucleic acids as disclosed herein can be incorporated into an expression cassette or vector, such as an expression vector. Accordingly, some embodiments disclosed herein relate to vectors or expression cassettes including the nucleic acids as disclosed herein. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette can be incorporated into a vector for targeting to a desired host cell and / or into an individual. As such, in some embodiments, an expression cassette of the disclosure includes a coding sequence for a recombinant cytokine receptor as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence. Non-limiting examples of vectors suitable for the compositions and methods of the disclosure include plasmids, synthetic DNA vectors, linear DNA vectors, closed linear DNA vectors, RNA vectors, phagemid vectors, viral vectors, self-replicating RNA viruses, mRNA-packaging virus-like particles, and RNP-packaging virus-like particles. In some embodiments, the recombinant nucleic acid molecule is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a protein nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM).

[0244] In some embodiments, the nucleic acids of the disclosure can be incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector. Accordingly, also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acids encoding any of the recombinant cytokine receptors disclosed herein. The nucleicAttorney Docket No.: 078430-543001 WOacid described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., etal., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al. , “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989).

[0245] It should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art can make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector’s copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the multivalent polypeptides and multivalent antibodies of the present disclosure to be amplified in copy number. Such amplifiable vectors are known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No.4,470,461) or glutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No. 5,122,464 and European published application EP 338,841).

[0246] Accordingly, in some embodiments, the recombinant cytokine receptors of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.

[0247] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.Attorney Docket No.: 078430-543001 WO

[0248] Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0249] The nucleic acid sequences encoding the recombinant cytokine receptors of the present disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. Codon usages within the coding sequence of the recombinant cytokine receptors disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a host cell. In these instances, the expression of the codon optimized polypeptides may be enhanced by at least about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to a reference polypeptide, e.g., the original polypeptide that has not been codon optimized.

[0250] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject recombinant cytokine receptors, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0251] Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). In some embodiments of the disclosure, the viral vector is a lentivirus vector, a baculovirus vector, a retrovirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a retrovirus vector. In some embodiments, the viral vector is a lentivirus vector. In some embodiments, the viral vector is an AAV vector.Attorney Docket No.: 078430-543001 WO

[0252] The nucleic acids of the disclosure are not limited to sequences that encode the recombinant cytokine receptors; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription. Recombinant cells and cell cultures

[0253] The nucleic acid molecule(s) encoding the recombinant cytokine receptors of the present disclosure can be introduced into a host cell, such as, for example, a human T lymphocyte, to produce a recombinant cell (e.g., a recombinant T cell) containing the nucleic acid(s). Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0254] Accordingly, in some embodiments of the disclosure, host cells (e.g., T cells) can be genetically engineered e.g., transduced or transformed or transfected) with, for example, a nucleic acid molecule of the disclosure, such as an expression cassette or a vector construct, to produce a recombinant cell (e.g., recombinant T cell). For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using zinc-finger proteins (ZNF), guide RNA directed CRISPR / Cas9, DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases).Attorney Docket No.: 078430-543001 WO

[0255] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, baculoviral virus or adeno-associated virus (AAV) can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0256] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as genedelivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0257] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the recombinant cytokine receptor of interest.

[0258] Host cells (e.g., T cells) of this disclosure can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0259] In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a nonhuman primate cell.

[0260] In some embodiments, the recombinant cell is a T lymphocyte. In some embodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell. In someAttorney Docket No.: 078430-543001 WOembodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell. Suitable CD8+ T cytotoxic lymphocyte cells include, but are not limited to, naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T cell is a CD4+ T helper lymphocyte cell. Nonlimiting examples of CD4+ T helper lymphocyte cells suitable for the compositions and methods of the disclosure include naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells. In some embodiments, the T cell is an exhausted T cell. In some embodiments, the T cell is a nonexhausted T cell.

[0261] In some embodiments, the cell can be obtained by leukapheresis performed on a sample obtained from a subject. In some embodiments, the subject is a human patient or a subject who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease.

[0262] In some embodiments, the recombinant T cells of the disclosure further include: (a) an engineered immune receptor, such as, a chimeric antigen receptor (CAR) or and T cell receptor (TCR); and / or (b) a recombinant nucleic acid encoding the same. For example, the recombinant T cells can include and / or express an engineered immune receptor, e.g, a receptor that can immunologically recognize and / or specifically bind to target ligand (e.g., an antigen), or an epitope thereof, such that binding of the antigen-specific receptor to the target ligand, or the epitope thereof, elicits an immune response. In some embodiments, the engineered immune receptor has antigenic specificity for a cancer antigen, such as a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0263] In some embodiments, the target ligand is a tumor antigen. Non-limiting examples of tumor antigens include alpha feto-protein (AFP) / HLA-A2, ADAM12, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD 171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, and FAP. Additional tumor antigens suitable for the compositions and methods of the disclosure include, but are not limited to, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican- 3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7Attorney Docket No.: 078430-543001 WO(glycosylated CEA), MMP, MRC2, MSX1, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC- A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PRAME, PSCA, PSMA, R0R1, R0R2, SSX1, TIM-3, TM4SF1, TnMucl, and VEGFR2.

[0264] In some embodiments, the target ligand is expressed on or associated with a cancer cell. In some embodiments, the cancer is a B-cell malignancy (such as a B-cell lymphomas or leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma. Additional suitable cancers include, but are not limited to, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

[0265] In some embodiments, the engineered immune receptor is a T-cell receptor (TCR). A TCR generally includes two polypeptides (e.g., polypeptide chains), such as an a-chain of a TCR, a P-chain of a TCR, a y-chain of a TCR, a 8-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The antigen-specific TCR can include any amino acid sequence, provided that the TCR can specifically bind to and / or immunologically recognize a target ligand (e.g., an antigen), such as a cancer antigen or epitope thereof. In some embodiments, the TCR is an endogenous TCR, e.g., a TCR that is endogenous or native to (e.g., naturally-occurring) the T cell. In such a case, the T cell expressing the endogenous TCR can be a T cell that was isolated from a mammal which is known to express the particular cancer antigen. For example, in some embodiments, the T cell is a primary T cell isolated from a mammal having a cancer. In some embodiments, the T cell is a tumor infiltrating lymphocyte (TIL) or a T cell isolated from a human cancer patient.

[0266] In some embodiments, the recombinant T cells of the disclosure include and / or express a chimeric antigen receptor (CAR). Generally, a CAR includes an antigen binding domain, e.g., a single-chain variable fragment (scFv) of an antibody, fused to a transmembrane domain and an intracellular domain. In this case, the antigenic specificity of a CAR can be encoded by a scFv which specifically binds to the antigen, or an epitope thereof. CARs, and methods of making them, are known in the art.Attorney Docket No.: 078430-543001 WO

[0267] In some embodiments, the immune cells include one or more nucleic acids encoding an exogenous (e.g., recombinant) antigen-specific receptor. In some embodiments, such exogenous antigen-specific receptors, e.g., exogenous TCRs and CARs can confer specificity for additional antigens to the T cell beyond the antigens for which the endogenous TCR is naturally specific.

[0268] In some embodiments, the recombinant T cell of the disclosure has a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced (e.g., decreased) by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure has an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure has an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell of the disclosure promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor.

[0269] In another aspect, provided herein are cell cultures including at least one recombinant cell, e.g., recombinant T cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one engineered cell as disclosed herein and a culture medium are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Pharmaceutical compositions

[0270] The recombinant cytokine receptors, nucleic acids encoding the recombinant cytokine receptors, and recombinant cells expressing the recombinant cytokine receptors of the disclosure can be incorporated into compositions, including pharmaceutical compositions. SuchAttorney Docket No.: 078430-543001 WOcompositions generally can include one or more recombinant cytokine receptors, nucleic acids, and recombinant cells of the disclosure; and a pharmaceutically acceptable excipient, e.g., a carrier. Accordingly, in one aspect, some embodiments of the disclosure relate to pharmaceutical compositions including a pharmaceutically acceptable excipient and one or more of the following: (a) recombinant cytokine receptor of the disclosure; (b) a recombinant nucleic acid of the disclosure; and (c) a recombinant cell of the disclosure. In some embodiments, the pharmaceutical compositions of the disclosure are formulated for the treating, preventing, ameliorating a health condition, e.g., a proliferative disease such as cancer, or for reducing or delaying the onset of the health condition (e.g., disease).

[0271] Non-limiting exemplary embodiments of the pharmaceutical compositions described herein can include one or more of the following features. In some embodiments, the composition includes a nucleic acid encoding one or more recombinant cytokine receptors of the disclosure, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions of the disclosure include at least one recombinant cell as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions of the disclosure include at least one recombinant nucleic acid molecule as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the recombinant nucleic acid is incorporated into an expression cassette or an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus (AAV) vector, or a retroviral vector. In some embodiments, the viral vector an AAV vector. In some embodiments, the viral vector is a retroviral vector.

[0272] In some embodiments, the pharmaceutical composition includes at least one recombinant T cell of the disclosure and a pharmaceutically acceptable excipient. In some embodiments, the at least one recombinant T cell exhibits an enhanced effector function when introduced into a subject, as compared to the effector function of control T cells under similar conditions, e.g., T cells that have not been engineered. In some embodiments, the recombinant T cell includes a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cellAttorney Docket No.: 078430-543001 WOincludes an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell includes an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the recombinant T cell promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the methods further include introducing into the T cell at least one engineered immune receptor. In some embodiments, the at least one engineered immune receptor includes an engineered T cell receptor (TCR) and / or an engineered chimeric antigen receptor (CAR).

[0273] In certain embodiments, the pharmaceutical compositions in accordance with some embodiments of the disclosure include cultures of recombinant T cells disclosed herein that can be washed, treated, combined, supplemented, or otherwise altered prior to administration to an individual in need thereof. Furthermore, administration can be at varied doses, time intervals or in multiple administrations.

[0274] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual. In some embodiments, the individual is a human. One of ordinary skilled in the art will appreciate that the formulation should suit the mode ofAttorney Docket No.: 078430-543001 WOadministration.

[0275] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition can be formulated for intravenous, oral, intraperitoneal, intratracheal, subcutaneous, intramuscular, topical, or intratumoral administration.

[0276] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0277] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparationAttorney Docket No.: 078430-543001 WOare vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0278] In some embodiments, the recombinant T cells of the disclosure can be formulated for administration to a subject using techniques known to the skilled artisan. For example, formulations comprising populations of recombinant T cells can include pharmaceutically acceptable excipient(s). Excipients included in the formulations will have different purposes depending, for example, on the recombinant T cells used and the mode of administration.Examples of generally used excipients included, without limitation: saline, buffered saline, dextrose, water-for-inj ection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising recombinant T cells can have been prepared and cultured in the absence of non-human components, e.g., in the absence of animal serum. A formulation can include one population of recombinant T cells, or more than one, such as two, three, four, five, six or more populations of recombinant T cells.

[0279] Formulations comprising population(s) of recombinant T cells can be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, without limitation, intratumoral, intradermal, subcutaneous (S.C., s.q., sub-Q, Hypo), intramuscular (i.m.), intraperitoneal (i.p.), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Devices useful for parenteral injection of infusion of the formulations can be used to effect such administration.METHODS OF THE DISCLOSURE

[0280] As described in greater details below, one aspect of the present disclosure relates to methods of generating the presently described recombinant cells (e.g., recombinant T cells), methods of administering the recombinant cells (e.g., recombinant T cells), and methods of treating individuals of relevant health conditions, such as proliferative diseases (e.g., cancers) , infectious diseases, autoimmune diseases, and inflammatory diseases.Methods of seneratins recombinant cells

[0281] The recombinant nucleic acids encoding the recombinant cytokine receptors of the present disclosure can be introduced into a host cell, such as, for example, a human TAttorney Docket No.: 078430-543001 WOlymphocyte, to produce a recombinant T cell containing the nucleic acids. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0282] The nucleic acids can be delivered by, for example, viral or non-viral delivery vehicles known in the art. In some embodiments, the nucleic acids can be maintained and replicated in the host cell (e.g., T cell) as an episomal unit. In some embodiments, the nucleic acids can be stably integrated into the genome of the host cell (e.g., T cell). Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acids can present in the host cell (e.g., T cell) as a mini-circle expression vector for transient expression.

[0283] Accordingly, in some embodiments, provided herein is a method for generating (e.g., producing) an recombinant T cell with enhanced effector function, the method comprising introducing into a T cell any one of the recombinant cytokine receptors the disclosure and / or a recombinant nucleic acid encoding the recombinant cytokine receptor. In some embodiments, the introduced recombinant cytokine receptor and / or recombinant nucleic acid results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell under similar condition, e.g., a T cell that does not include the recombinant cytokine receptor and / or recombinant nucleic acid. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by at least 10%, e.g, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor.Attorney Docket No.: 078430-543001 WO

[0284] In some embodiments, the introduced recombinant cytokine receptor and / or recombinant nucleic acid results in an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell under similar condition, e.g., a T cell that does not include the recombinant cytokine receptor and / or recombinant nucleic acid.

[0285] In some embodiments, the introduced recombinant cytokine receptor and / or recombinant nucleic acid results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell under similar condition, e.g., a T cell that does not include the recombinant cytokine receptor and / or recombinant nucleic acid. In some embodiments, the recombinant T cell promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell under similar condition, e.g., a T cell that does not include the recombinant cytokine receptor and / or recombinant nucleic acid.

[0286] In some embodiments, the method further comprises introducing into the T cell at least one recombinant antigen-specific receptor. In some embodiments, the at least one recombinant antigen-specific receptor comprises an engineered T cell receptor (TCR) and / or an engineered chimeric antigen receptor (CAR).

[0287] Accordingly, in a related aspect, recombinant T cells produced by the methods of generating recombinant T cells as disclosed herein are also encompassed within the present disclosure.Methods of treatment

[0288] Administration of any one of the therapeutic compositions described herein, e.g., recombinant cytokine receptors, nucleic acids, recombinant T cells, and pharmaceutical compositions, can be used to treat individuals in the treatment of relevant health conditions, such as proliferative diseases (e.g., cancers) , infectious diseases, autoimmune diseases, and inflammatory diseases. In some embodiments, one or more recombinant cytokine receptors, nucleic acids, recombinant T cells, and pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating a subject who has, who is suspected of having, or who can be at high risk for developing one or more health conditions, such as proliferative diseases (e.g., cancers) , infectious diseases, autoimmune diseases, and inflammatory diseases. In some embodiments, the subject is a mammalian subject. In someAttorney Docket No.: 078430-543001 WOembodiments, the subject has or is suspected of having a proliferative diseases (e.g., cancer an infectious disease, an autoimmune disease, or an inflammatory disease. In some embodiments, the proliferative disease is a cancer. In some embodiments, the subject is a patient under the care of a physician.

[0289] Accordingly, in an aspect, provided herein is a method for preventing and / or treating a health condition in a subject in need thereof, the method comprising administering to the subject a composition comprising: a pharmaceutically effective amount of one or more of the following: (a) a recombinant cytokine receptor as disclosed herein or a signaling component thereof; (b) a recombinant nucleic acid as disclosed herein; (c) a recombinant T cell as disclosed herein; and (d) pharmaceutical composition as disclosed herein.

[0290] In some embodiments, the health condition is a proliferative disorder (e.g., cancers), an infectious disease, an autoimmune disease, and / or inflammatory disease. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is a B-cell malignancy, such as a B-cell lymphomas or leukemia. In some embodiments, the cancer is a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a Merkel cell carcinoma, a melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, or urothelial carcinoma. Additional cancers that can be suitably treated by the methods disclosed herein include, but are not limited to gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

[0291] In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor a signaling component thereof. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by at least 10%, e.g., at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the STAT1 signaling in the recombinant T cell is reduced by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or fromAttorney Docket No.: 078430-543001 WO60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor or a signaling component thereof. In some embodiments, in vitro and / or in vivo proliferation / expansion of the recombinant T cell is enhanced by at least 10%, e.g., at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, in vitro and / or in vivo proliferation / expansion of the recombinant T cell is enhanced by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increase in circulating recombinant T cells in the blood of the subject. In some embodiments, circulating recombinant T cells in the blood of the subject is increased by at least 10%, e.g., at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, circulating recombinant T cells in the blood of the subject is increased by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increased accumulation of the recombinant T cells in lymphoid and non-lymphoid organs. In some embodiments, accumulation of the recombinant T cells in lymphoid and non-lymphoid organs is increased by at least 10%, e.g., at least 15%, at least 20%, at least 25%, or at least 30%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, accumulation of the recombinant T cells in lymphoid and non-lymphoid organs by 10% to 90%, e.g., from 10% to 50%, from 20% to 70%, from 30% to 80%, from 40% to 90%, from 25% to 75%, from 50% to 75%, or from 60% to 90%, as compared to a reference T cell that does not include the recombinant cytokine receptor. In some embodiments, the administered composition or recombinant cytokine receptor (or a signaling component thereof) promotes less effector differentiation while enhancingAttorney Docket No.: 078430-543001 WOproliferation / expansion of the recombinant T cell, as compared to a subject that does not include the recombinant cytokine receptor or a signaling component thereof.

[0292] An effective amount of the compositions described herein, e.g., recombinant cytokine receptors, nucleic acids, recombinant T cells, and / or pharmaceutical compositions, can be determined based on the intended goal, for example cancer regression. For example, where existing cancer is being treated, the amount of a composition disclosed herein to be administered can be greater than where administration of the composition is for prevention of cancer. One of ordinary skill in the art would be able to determine the amount of a composition to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each subject. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0293] Determination of the amount of compositions to be administered will be made by one of skill in the art, and will in part be dependent on the extent and severity of cancer, and whether the recombinant cells, e.g., T cells, are being administered for treatment of existing cancer or prevention of cancer. For example, longer intervals between administration and lower amounts of compositions can be employed where the goal is prevention. For instance, amounts of compositions administered per dose can be 50% of the dose administered in treatment of active disease, and administration can be at weekly intervals. One of ordinary skill in the art, in light of this disclosure, would be able to determine an effective amount of compositions and frequency of administration. This determination would, in part, be dependent on the particular clinical circumstances that are present (e.g., type of cancer, severity of cancer).

[0294] In some embodiments, it can be desirable to provide a continuous supply of a composition disclosed herein to the subject to be treated, e.g., a patient. In some embodiments, continuous perfusion of the region of interest (such as a tumor) can be suitable. The time period for perfusion would be selected by the clinician for the particular subject and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, the dose of the composition viaAttorney Docket No.: 078430-543001 WOcontinuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.

[0295] In some embodiments, administration is by intravenous infusion. An effective amount of the recombinant cytokine receptors, nucleic acids, recombinant T cells, and / or pharmaceutical compositions disclosed herein can be determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the number of cells to be administered can be greater than where administration of the engineered immune cells, e.g., T cells, disclosed herein is for prevention of cancer. One of ordinary skill in the art would be able to determine the number of cells to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.Administration of recombinant cells

[0296] In some embodiments, the methods of the disclosure involve administering an effective amount or number of the recombinant cells, e.g., recombinant T cells, provided here to a subject in need thereof. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells, e.g, recombinant T cells, can be infused directly in the subject’s bloodstream or otherwise administered to the subject.

[0297] In some embodiments, the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant T cells into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant T cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a fewAttorney Docket No.: 078430-543001 WOdays, to as long as several years, or even the lifetime of the individual, e.g., long-term engraftment.

[0298] When provided prophylactically, the recombinant T cells described herein can be administered to a subject in advance of any symptom of a disease or health condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant T cell population prevents the occurrence of symptoms of the disease or health condition.

[0299] When provided therapeutically in some embodiments, recombinant T cells are provided at (or after) the onset of a symptom or indication of a disease or health condition, e.g., upon the onset of disease or health condition.

[0300] For use in the various embodiments described herein, an effective amount of recombinant T cells as disclosed herein, can be at least 102cells, at least 5 102cells, at least 103cells, at least 5 103cells, at least 104cells, at least 5 104cells, at least 105cells, at least 2 x 105cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 105cells, at least 6 x 105cells, at least 7 x io5cells, at least 8 x 105cells, at least 9 x 105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3 x 106cells, at least 4 x 106cells, at least 5 x 106cells, at least 6 x 106cells, at least 7 x io6cells, at least 8 x 106cells, at least 9 x 106cells, or multiples thereof.

[0301] In some embodiments, the recombinant T cells are non-autologous to the subject in need of treatment. In some embodiments, the adoptive cell therapy is an allogeneic adoptive cell therapy. For example, in some embodiments, the recombinant T cells are allogeneic to the subject in need of treatment. In an allogeneic adoptive cell therapy, the recombinant T cells are not derived from the individual receiving the adoptive cell therapy. Allogeneic cell therapy generally refers to a therapy whereby the individual (donor) who provides the T cells is a different individual (of the same species) than the individual receiving the cell therapy. For example, a population of recombinant T cells being administered to an individual is derived from one more unrelated donors, or from one or more non-identical siblings. Accordingly, the recombinant T cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the recombinant T cells are expanded in culture prior to administration to a subject in need thereof.

[0302] In some embodiments, the delivery of a cell composition (e.g., a composition including a plurality of recombinant T cells according to any of the recombinant cells described herein) into a subject by a method or route results in at least partial localization of the cell composition atAttorney Docket No.: 078430-543001 WOa desired site. A composition including recombinant T cells can be administered by any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 * 104cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, delivery by injection or infusion is often considered a standard mode of administration.

[0303] In some embodiments, the recombinant T cells are administered systemically, e.g., via infusion or injection. For example, a population of recombinant T cells as described herein are administered other than directly into a target site, tissue, or organ, such that it enters, the subject’s circulatory system and, thus, is subject to metabolism and other similar biological processes.

[0304] The efficacy of a treatment including any of the compositions provided herein for the prevention or treatment of a disease or health condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a prevention or treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g, arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0305] Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced. In general, a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solidAttorney Docket No.: 078430-543001 WOtumor or cancer, suitable parameters can include reduction in the number and / or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and / or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and / or disease severity and / or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.

[0306] As discussed above, a therapeutically effective amount of a pharmaceutical composition can be an amount of the pharmaceutical composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease or health condition. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or health condition, alter the course of a symptom of the disease or health condition (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease or health condition. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation. Additional therapies

[0307] As discussed above, any one of the compositions as disclosed herein, e.g., recombinant cytokine receptors, recombinant nucleic acids, recombinant T cells, and pharmaceutical compositions, can be administered to a subject in need thereof as a single therapy (e.g., monotherapy). In addition or alternatively, in some embodiments of the disclosure, one or more of the engineered immune cells and pharmaceutical compositions described herein can be administered to the subject in combination with one or more additional therapies, e.g., at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include therapeutic agents such as chemotherapeutics, anticancer agents, and anti-cancer therapies.Attorney Docket No.: 078430-543001 WO

[0308] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. The term chemotherapy as used herein encompasses anti-cancer agents. Various classes of anti-cancer agents can be suitably used for the methods disclosed herein. Non-limiting examples of anticancer agents include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.

[0309] Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a single therapy (e.g., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.Kits

[0310] Also provided herein are kits for the practice of a method described herein. A kit can include one or more of the recombinant cytokine receptors, nucleic acids encoding the recombinant cytokine receptors, recombinant cells expressing the recombinant cytokine receptors, and / or pharmaceutical compositions as described and provided herein. For examples, provided herein, in some embodiments, are kits that include one or more recombinant cytokine receptors of the disclosure. In some embodiments, provided herein are kits that include one or more recombinant nucleic acids of the disclosure. In some embodiments, provided herein are kits that include one or more recombinant cells (e.g., T cells) of the disclosure. In someAttorney Docket No.: 078430-543001 WOembodiments, provided herein are kits that include one or more pharmaceutical compositions of the disclosure. In some embodiments, the kits of disclosure further include written instructions for making the recombinant cells (e.g., T cells), recombinant cytokine receptors, nucleic acids encoding the recombinant cytokine receptors, recombinant cells expressing the recombinant cytokine receptors, and / or pharmaceutical compositions of the disclosure and using the same.

[0311] In some embodiments, the kits of the disclosure further include one or more delivery vehicles or systems, such as syringes (including pre-filled syringes) and / or catheters (including pre- filled syringes) used to administer one any of the provided compositions, e.g, recombinant cytokine receptors, recombinant nucleic acids, recombinant cells (e.g., recombinant T cells), and pharmaceutical compositions to a subject in need thereof. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g, for modulating an activity of a cell, inhibiting a target cell (e.g., cancer cell), or treating a health condition or diseases in a subject in need thereof.

[0312] For example, any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control cell populations, positive control cell populations, reagents for ex vivo production of the cell populations.

[0313] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. For example, in some embodiments of the disclosure, the kit includes one or more of the provided recombinant cytokine receptors, nucleic acids, recombinant T cells, and / or pharmaceutical compositions as described herein in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0314] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure can be supplied in the insert:Attorney Docket No.: 078430-543001 WOpharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and intellectual property information.

[0315] In some embodiments, a kit can include further instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0316] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0317] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0318] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.Attorney Docket No.: 078430-543001 WO

[0319] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.EXAMPLES

[0320] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press;Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0321] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0322] More information regarding the experiments described in the following Examples can be found in Jiang H. et al., Immunity 59, 1-18, January 13, 2026, which is herein expressly incorporated by reference.Attorney Docket No.: 078430-543001 WOEXAMPLE 1General materials and methodsProtein production

[0323] DNA encoding mouse and human IL-2 and IL-9, and mouse IL-9Q115Twere cloned into the mammalian expression vector pD649, which includes a C-terminal 8xHis tag for affinity purification. DNA encoding mouse serum albumin (MSA) was purchased from Integrated DNA Technologies (IDT) and cloned into pD649 as an N-terminal fusion. Mammalian expression DNA constructs were transfected into Expi293E cells using the Expi293 Expression System (Thermo Fisher Scientific) for secretion and purified for the clarified supernatant by nickel affinity resin (Ni-IMAC, Thermo Fisher Scientific) followed by size-exclusion chromatography with a Superdex-200 column (Cytiva) and formulated in sterile phosphate-buffered saline (PBS) for injection. Endotoxin was removed using the Proteus NoEndo HC Spin column kit following the manufacture’s recommendations (VivaProducts) and endotoxin removal was confirmed using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Fisher Scientific). Proteins were concentrated and stored at -80°C until use.Mammalian Expression Vectors

[0324] cDNA encoding mouse orthogonal IL-2R|3 extracellular domain (ECD) and gene block cDNA encoding mouse ICDs of IL-9R (IDT) were cloned into the retroviral vector pMSCV-MCS-IRES-YFP by PCR and isothermal assembly (ITA). Human orthogonal IL2R|3-ECD-IL9R-ICD (ho9R) were similarly cloned into the pMSCV vector. cDNA encoding the native mouse and human IL9R-P2A-YFP were synthesized (VectorBuilder) and cloned into the pMSCV vector. CD19-4-lBB^ and HER2-4-lBB^ constructs were generous gifts from Dr. Crystal Mackall.57NY-ESO-1 TCR (1G4) was acquired from Dr. Antoni Ribas as part of a material transfer agreement.

[0325] Single amino acid mutations of the IL-9R, including those at and near Y405 (mouse) and Y407 (human), and the single amino acid mutation of IL-9 (QI 15T), were made through Q5 site-directed mutagenesis (New England Biolabs). To generate IL-9R receptors with repetitive phosphotyrosine motifs (IL-9R3xand IL-9R5x), gene blocks encoding amino acids from position 386 to position 425 were repeated three times or five times in tandem, followed by the remainder of the IL-9R intracellular domain.Retrovirus productionAttorney Docket No.: 078430-543001 WO

[0326] For production of retrovirus to engineer mouse T cells, HEK293T (ATCC) cells were seeded at 4.5 x 105cells per well in a 6-well tissue culture treated plate. 6-well tissue culture treated plates were coated with 0.01% poly-l-lysine (Sigma-Aldrich) prior to seeding. For each well 2.5 pg of plasmid (1.25 pg of pMSCV retroviral vector plus 1.25 pg of pCL-Eco packaging vector) was added to 250 pl of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific), followed by 5.5 pl of Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) and 5 pl of P3000™ enhancer reagent. After 18 to 20 h, the medium was replaced with fresh DMEM-c with 20 mM HEPES and cultured for another 24 hours before viral collection at 48 hours posttransfection.

[0327] For production of retrovirus to engineer human T cells, HEK293GP (ATCC) cells were cultured in DMEM-c. Prior to plating of HEK293GP cells, 10cm tissue-culture treated dishes were coated with 5 mL of 0.01% poly-L-lysine. Cells were subsequently plated at a density of 6.5 x 106ml’1per dish in 10 mL total DMEM-c and incubated overnight at 37°C. After 24 hours, cells were transfected with 9 pg vector plasmid, 1.5 mL of Mix A (1.5 mL Opti-Mem, 4.5 pg RD114), and 1.5 mL of Mix B (1.5 mL Opti-MEM, 33uL Lipofectamine 3000, and 2uL P3000 per pg vector plasmid) per plate. After 24 hours, fresh media was replenished. Virus was collected at 48- and 72-hours post-transfection. Viral supernatants were filtered through a 0.22 pM Whatman filter (Cytiva). If not used immediately, the virus was frozen for storage in -80°C.Activation and retroviral transduction of primary mouse T cells

[0328] For retroviral transduction of mouse T cells, splenocytes from the five- to ten- week-old mice were mechanically digested and filtered through a 70 pM strainer (Fisher Scientific). Red blood cells were lysed with eBioscience RBC Lysis Buffer (Thermo Fisher Scientific) for 5 minutes at 4°C. Splenocytes were resuspended in PBS with 2% FBS and 1 mM Ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific), and enriched for CD3+cells by magnetic bead separation, according to the manufacturer's protocol (StemCell). C57BL / 6-derived mouse T cells were activated with a 1 : 1 bead: cell ratio of mouse CD3 / 28 Dynabeads™ (Thermo Fisher Scientific) in fresh T cell medium with 100 U ml’1rmIL-2 overnight. On day of transduction, CD3 / 28 Dynabeads™ were removed prior to transduction by magnetic bead separation. Isolated pmel T cells were activated with 100 U ml’1recombinant mouse IL-2 (rmlL-2) (P eprotech) and 1 pg ml’1human gplOO peptide (Anaspec) the day before transduction. One day before transduction, six- well non-tissue culture treated plates (Thermo Fisher Scientific)Attorney Docket No.: 078430-543001 WOwere coated with 37.5 pg ml’1retronectin (Takara Bio) in PBS per well and placed at 4°C overnight. The following day, retronectin-coated plates were blocked with 0.5% BSA (Sigma-Aldrich) in PBS for 30 minutes at 25°C and washed out with PBS. Viral supernatant (2 ml) was added to each well and spun at 2000g for two hours at 32°C without brake. Viral supernatant was carefully removed, 3 ml of activated T cells (1 x 106ml’1) were added to each well with 100 U ml’1rmIL-2, and spun at 2000g for 10 minutes at 32°C without brake. Cells were then cultured for 18-22 hours at 37°C. Cells were collected via gentle pipetting and resuspended at 1 x 106ml’1in fresh mouse T cell medium with 100 U ml’1rmIL-2 and expanded overnight before further downstream cellular assays. For in vivo tumor assays, transduced cells were used either immediately after collection or one day post-transduction. YFP served as a surrogate marker for the expression of all mouse constructs used and was checked on day of use.Activation and retroviral transduction of primary human T cells

[0329] Healthy donor Leukocyte Reduction System (LRS) chambers were purchased from the Stanford Blood Center according to an institutional review board (IRB)-exempt protocol.Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (Cytiva) density gradient centrifugation and frozen in a Mr. Frosty™ Freezing Container in aliquots of 10 x 106PBMCs mL’1of CELLBANKER 2 serum-free cell cryopreservation medium (AMSBIO). Aliquots were stored frozen at -80°C.

[0330] Human T cells were isolated from cryopreserved PBMCs using the EasySep™ Human T Cell Isolation Kit (StemCell) according to the manufacturer’s protocol. T cells were activated using Dynabeads™ Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) for T Cell expansion and activation at a 3: 1 ratio of beads: cells and cells were incubated at 37°C for three days at a concentration of 1 x 106cells ml’1. Transduction was performed on days 3 and 4 after activation. Twelve-well, non-tissue culture treated plates (Thermo Fisher Scientific) were coated with 18.75 pg mL’1retronectin (Takara Bio) in PBS per well and left at 4°C overnight or for two hours at 25 °C. On the day of transduction, the retronectin was transferred to a new 12- well nontissue culture treated plate, which was subsequently stored at 4°C overnight. The original retronectin-treated plate was then blocked with 1 mL of 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS per well for 10-20 minutes. Afterward the BSA was removed and 1 mL of 48 hour retroviral supernatant was added per well. For co-transductions, 1 mL of supernatant per construct was added per well (for a total of 2 mL per well). Following the addition of viralAttorney Docket No.: 078430-543001 WOsupernatant, the plate was spun at 2000 x g for 2 hours at 32°C. Subsequently, viral supernatant was aspirated, and the activated Day 3 T cells were added at a concentration of 0.5 x 106cells ml'1in 1 mL per well. The plate was then incubated at 37°C overnight. The transduction was repeated the following day using 72 hour supernatant. On Day 5 post activation, the DynaBeads were magnetically removed from T cells and cells were resuspended in fresh media at a concentration of 0.5 x 106cells ml’1. On Day 7 post activation, T cells were passaged and resuspended in fresh media at a concentration of 0.5 x 106cells ml'1. On Day 10, transduction efficiency was assessed by flow cytometry and T cells were used in subsequent assays.Quantitative Reverse-Transcriptase PCR (pRT-PCR)

[0331] Total RNA was extracted from human cells with the RNeasy Mini Kit (Qiagen) as directed by the manufacturer. 0.6 pg of total RNA was reversed transcribed to synthesize cDNA, using High Capacity cDNA Reverse Transcription Kit (ThermoFisher). Real-time quantitative PCR was run in duplicate using iTaq SYBRGreen (Bio-Rad), following manufacturer’s instructions. PCR amplification was monitored using QuantStudio5 detection system (AppliedBiosystems). Data were expressed as relative mRNA abundance normalized to GAPDH expression level in each sample. The primer sequences are listed in the key resources table. Phospho flow signaling assays

[0332] Actively growing mouse or human primary T cells were rested at a concentration of 2 x 106cells mL'1in T cell mediumwithout IL-2 for 12-18 hours before signaling assays. Cells were plated in a 96- well round bottom plate in T cell medium with reduced FBS (2%) for two hours prior to the assay. Cells were stimulated by addition of recombinant cytokines for 20 minutes at 37°C, and the reaction was terminated by fixation with 2.1% paraformaldehyde (BD Cytofix) for 30 minutes at 37°C. Cells were washed and permeabilized with ice-cold methanol (BD Phosflow Perm Buffer III) for 30 minutes on ice or stored at -20°C overnight. Cells were washed with staining buffer (eBioscience) before staining with pSTAT antibodies for 30 min to 1 hour at 4°C in the dark. Cells were washed and analyzed on a CytoFlex (Beckman Coulter) or Novocyte Quanteon / Penteon (Agilent Technologies). Data represent the median fluorescence intensity (MFI), and points were fit to a log(agonist) versus dose-response model using Prism 10.2.3 (GraphPad). For the gating strategy, see FIG. 10E. Summary of all ECso and Emax values are provided in Table S6 of Jiang H. et al., 2026, supra, which is herein expressly incorporated by reference.Attorney Docket No.: 078430-543001 WOTandem mass tag (TMT)-based global phospho-proteomics

[0333] C57BL / 6-derived mouse T cells were activated and transduced as previously described. Transduced T cells were sorted (2.5 x 107per replicate) based on expression of YFP+using an Aria II cell sorter (BD Biosciences) and stimulated for 20 minutes at 37°C using the respective cytokines: rmIL-2 and rmIL-9 in T cell medium. Stimulated T cells were washed twice with PBS. Supernatant were removed, pellet was snap-frozen in liquid nitrogen and stored at -80°C before downstream analyses at the IDeA National Resource for Quantitative Proteomics.

[0334] Total protein from each sample was reduced, alkylated, and purified by chloroform / methanol extraction prior to digestion with sequencing grade trypsin and LysC (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo), combined into two multiplex sample groups, then enriched using High-Select TiO2 and Fe-NTA phosphopeptide enrichment kits (Thermo) following the manufacturer’s instructions. Both enriched and un-enriched labeled peptides were separated into 46 fractions on a 100 x 1.0 mm Acquity BEH Cl 8 column (Waters) using an UltiMate 3000 UHPLC system (Thermo) with a 50 min gradient from 99: 1 to 60:40 buffer A:B ratio under basic pH conditions, then consolidated into 18 super-fractions for the enriched and un-enriched sample sets (Buffer A = 0.1% formic acid, 0.5% acetonitrile; Buffer B = 0.1% formic acid, 99.9% acetonitrile). Both buffers were adjusted to pH 10 with ammonium hydroxide for offline separation. Each super- fraction was then further separated by reverse phase XSelect CSH Cl 8 2.5 um resin (Waters) on an in-line 150 x 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 75 min gradient from 98:2 to 60:40 buffer A:B ratio. Eluted peptides were ionized by electrospray (2.4 kV) followed by mass spectrometric analysis on an Orbitrap Eclipse Tribrid mass spectrometer (Thermo) using multi-notch MS 3 parameters. MS data were acquired using the FTMS analyzer in top-speed profile mode at a resolution of 120,000 over a range of 375 to 1500 m / z. Following CID activation with normalized collision energy of 31.0, MS / MS data were acquired using the ion trap analyzer in centroid mode and normal mass range. Using synchronous precursor selection, up to 10 MS / MS precursors were selected for HCD activation with normalized collision energy of 55.0, followed by acquisition of MS3 reporter ion data using the FTMS analyzer in profile mode at a resolution of 50,000 over a range of 100-500 m / z.Attorney Docket No.: 078430-543001 WO

[0335] Proteins were identified and reporter ions quantified by searching the UniprotKB database using MaxQuant (Max Planck Institute) with a parent ion tolerance of 3 ppm, a fragment ion tolerance of 0.5 Da, a reporter ion tolerance of 0.001 Da, trypsin / P enzyme with 2 missed cleavages, variable modifications including oxidation on M, Acetyl on Protein N-term, and phosphorylation on STY, and fixed modification of Carbamidomethyl on C. Protein identifications were accepted if they could be established with less than 1.0% false discovery. Proteins identified only by modified peptides were removed. Protein probabilities were assigned by the Protein Prophet algorithm [Anal. Chem. 75: 4646-58 (2003)]. TMT MS3 reporter ion intensity values are analyzed for changes in total protein using the unenriched lysate sample. Phospho(STY) modifications were identified using the samples enriched for phosphorylated peptides. The enriched and un-enriched samples are multiplexed using two TMTIO-plex batches, one for the enriched and one for the un-enriched samples.

[0336] Following data acquisition and database search, the MS3 reporter ion intensities were normalized using ProteiNorm (Graw et al). The data was normalized using VSN (Huber et al) and analyzed using ProteoViz to perform statistical analysis using Linear Models for Microarray Data (limma) with empirical Bayes (eBayes) smoothing to the standard errors (Storey et al, Ritchie et al). A similar approach is used for differential analysis of the phosphopeptides, with the addition of a few steps. The phosphosites were filtered to retain only peptides with a localization probability > 75%, filter peptides with zero values, and log2 transformed. Limma was also used for differential analysis. Proteins and phosphopeptides with an FDR-adjusted p-value < 0.05 and an absolute fold change > 2 were considered significant.Mouse T cell in vitro expansion, proliferation (CellTrace) and apoptosis cell death assays

[0337] Actively growing mouse T cells were rested for 24 hours post-transduction in T cell medium with 100 U ml’1rmIL-2. On day of assay, T cells were washed with T cell medium lacking cytokine. IL-9R transduced T cell proliferation was assessed by seeding 100,000 cells per well in a round bottom 96-well plate in the presence of equipotent doses of recombinant murine IL-2 (rmIL-2), rmIL-9, or rmIL-12. T cells were fed with fresh media and cytokine 48 hours after seeding, and every subsequent 48 hours until the end of the assay. Counts were also acquired on the day of replenishment by staining an aliquot of cells with Acridine Orange / Propidium Iodide (AO / PI, The DeNovix Company) and analyzing on a cell counter (The DeNovix Company). An aliquot of cells were also stained with Zombie Violet Live DeadAttorney Docket No.: 078430-543001 WO(BioLegend) and analyzed for YFP+ on the NovoCyte Penteon (Agilent Technologies). For CellTrace assays, resuspension of dye was followed as recommended by the manufacturer. Proliferation index was calculated in FlowJo by modeling dye dilution peaks to determine the number of cells in each generation, then dividing the total number of divisions (sum of cells in each generation multiplied by their division number) by the number of cells that underwent at least one division. This yields the average number of divisions per responding (divided) cell, excluding undivided cells from the denominator. To measure apoptosis, cells were removed every 48 hours post cytokine treatment. Cells were subsequently resuspended at 1 x 106 cells / mL and stained with Zombiet Violet (BioLegend) and Annexin V (Thermo Fisher) in Annexin Binding buffer (BioLegend) at 25°C for 15 minutes. After the incubation, cells were diluted with 2x Annexin Binding buffer, placed on ice, and immediately analyzed on the Novocyte Quanteon (Agilent Technologies).ELISA

[0338] Serum IFNy ELISA was conducted using the Mouse IFNy ELISA MAX kit (Biolegend) using the manufacturer's recommendations. In short, the day prior to running the ELISA, the Capture Antibody was diluted in Coating Buffer and 100 pL of this capture solution to all relevant wells of a 96- well plate. The plate was sealed and incubated overnight at 4°C. On the day of running the ELISA, the plate was washed 4 times with 300 pL of Wash Buffer per well to remove capture solution. After the wash was complete, the plate was then blocked with 200 pL of Assay Diluent per well, and then incubated at 25°C for 1 hour on a plate shaker (500 rpm). Following the blocking, the plate was washed 4 times with Wash Buffer, and then 100 pL of standard dilutions and samples were added in duplicates. The plate was then incubated at 25°C for 2 hours with shaking. After the incubation, the plate was washed 4 times as previously described and 100 pL of diluted Detection Antibody solution was added to each well and incubated at 25°C for 1 hour with shaking. Following the detection incubation, the plate was washed 4 times and add 100 pL of diluted Avidin-HRP solution was added and incubated at 25°C for 30 minutes with shaking. After the incubation, the plate was washed 5 times with Wash Buffer, with the buffer soaking in each well for 30 seconds per wash to minimize background. After the washes were complete, 100 pL of TMB Substrate Solution was added and incubated at 25°C in the dark until the wells turn blue. The reaction was stopped by adding 100 pL of StopAttorney Docket No.: 078430-543001 WOSolution to each well and then immediately read on a fluorescent microplate reader (Thermo Fisher Scientific) (absorbance read at 450 nm).Coculture, cytotoxicity and proliferation assays for human T cells

[0339] Human T cells transduced with a CAR or NY-ESO-1 TCR, and co-transduced with IL-9R or an IL-9R variant, were resuspended at 1 x 106cells ml’1in RPMI-c with either no cytokine, 1 nM recombinant human IL-2, or 10 nM recombinant human IL-9 (Peprotech). T cells were serially diluted to achieve specified effector: target ratio and triplicates were cocultured with 5 x 104cells ml’1(50pL) NALM6-FFluc or 5 x 103M407mCherrycells in a 96 well plate. 50 pL of media was added to bring the total volume of each well to 200 pL. The plate was imaged in the Incucyte Live-Cell Analysis System (Sartorius) for 72 hours, and killing was quantified by measuring the Total Green or Red Object Integrated Intensity metric of GFP+or mCherry+tumor cells. Cytotoxicity index was calculated by normalizing to the fluorescence intensity at the first time point for each triplicate. Data were analyzed in GraphPad Prism.

[0340] For repetitive killing assays using non-adherent tumor cells, additional tumor cells were added in the same effectortarget ratio as originally plated once tumor killing plateaued, while maintaining cytokine concentrations. For repetitive killing assays using non-adherent tumor cells, T cells were harvested from each well after tumor killing plateaued, counted, and replated at the same E:T ratio in a new 96 well plate with fresh tumor cells.

[0341] In vitro proliferation assays were performed in triplicate in 96-well round-bottom plates. T cells were counted and percent live YFP+cells were assessed by flow cytometry every 48 hours to calculate fold expansion, then re-plated in fresh media with cytokine at their original concentration. Flow cytometry data were analyzed using FlowJo and graphs were generated in GraphPad Prism.In vivo mouse tumor studies

[0342] For in vivo tumor growth experiments using immunocompetent mice, early-passage cell lines were used (fewer than 10 passages). B16-F10 or KP-gplOO tumor cells (5 x 105cells) were resuspended in 100 pl of PBS and injected subcutaneously in the right flank of 6-10-week-old C57BL / 6J mice. Prior to adoptive cell transfer, mice were randomized based on average tumor size. 2 x 106- 5 x 106non-sorted, transduced T cells (derived from pmel mice) were adoptively transferred five or six days after tumor inoculation unless otherwise indicated. Specifically, T cells were resuspended in 50 pl of PBS per mouse and administered by retroorbital intravenousAttorney Docket No.: 078430-543001 WOinjection. Where indicated, mice received treatment with cytokines: mouse serum albumin (MSA)-bound mouse IL-2 (MSA-IL2), MSA-IL9, MSA-IL9Q115T, MSA-oIL2 (5 x 104units per dose, intraperitoneal) for five doses (or longer, where indicated), administered every other day starting on the day of adoptive cell transfer (unless otherwise indicated). Tumor size (length x width) was measured with calipers three times a week and volume was calculated as (length x width2) / 2). Peripheral blood (10 pl) was collected at indicated time points from the tail vein for quantification of adoptively transferred pmel T cells by flow cytometry. Mice were euthanized when the total tumor volume exceeded 2,000 mm3, as per IACUC guidelines.

[0343] We also performed xenograft studies of human T cells and human tumors in immunodeficient NSG mice. For the leukemia model, NALM6-FFluc cells (1 x 106ml’1in 200 pL sterile PBS) were injected via tail vein into each mouse. On Day 3 post tumor inoculation, luciferin-based imaging was performed to confirm tumor engraftment. Upon confirmation, T cells were injected via tail vein at a dose of 1 x 106cells or 3 x 106cells in 200 pL per mouse. Beginning on Day 3 as well, mice received a dose of 25,0001.U. of MSA-hIL-9 in 100 pL of sterile PBS (i.p.). Mice were injected with cytokine every other day for a total of ten doses. Luciferase-based imaging was performed twice per week for at least three weeks to assess tumor growth. Specifically, mice were anaesthetized with isoflurane and 200 uL of 13mg mL’1D-luciferin was injected i.p. per mouse, incubated for 4 minutes, and imaged for 30 seconds with heavy binning or automatic exposure detection. Mice were closely monitored and sacrificed upon reaching any one of the morbidity criteria, as outlined under IACUC guidelines (tumor flux exceeding 1 x 1011p / s, rear leg paralysis, or other conditions that necessitated euthanasia).

[0344] For the osteosarcoma model, the right leg of each mouse was shaved, and width and depth were measured prior to tumor inoculation. 1 x 106143B-FFluc cells in 100 pL sterile PBS were injected intramuscularly into the right leg of each mouse. On Day 4 post tumor inoculation, luciferin-based imaging was performed to confirm tumor engraftment. Upon confirmation, T cells were injected via tail vein at a dose of 7 x 106cells in 200 pL per mouse. Beginning on Day 4 as well, mice received a dose of 25,000 I.U. of MSA-hIL-9 intraperitoneally. Mice were injected with cytokine every over day for a total of 10 doses. Their right leg was measured three times a week. Mice were closely monitored and sacrificed upon reaching any one of the morbidity criteria, as outlined under IACUC guidelines (tumor size, inability to move their right leg, or other conditions that necessitated euthanasia).Attorney Docket No.: 078430-543001 WOIn vivo cytokine dose calculations

[0345] For in vivo experiments, cytokines were dosed based on activity (international units; LU.). For MSA-mIL2, MSA-hIL2 or MSA-oIL2 (3A10), 1 pg of protein was considered equal to 32801.U. For MSA-mIL9, 1 pg was considered equal to 1118 I.U. For MSA-hIL9, 1 pg MSA-hIL9 was considered equal to 1428 I.U.In vivo toxicology studies

[0346] For measuring the toxicity of cytokines in vivo, mice were weighed and video recorded individually for 30 second intervals at baseline and every 48 hours as indicated. Videos were processed using EthoVision Software to quantify mobility.Analysis ofT cell biodistribution and phenotype

[0347] Spleen, tumor-draining lymph nodes, tumor, liver, and lung were harvested and dissociated into single-cell suspensions at the indicated timepoints post adoptive cell transfer. Spleens and lymph nodes were weighed, dissociated mechanically, and filtered through a 70- pM cell strainer. Tumors were minced into 2-4 mm pieces and lungs were perfused with PBS and separated by lobe. Tumor, lung and liver tissues were enzymatically and mechanically dissociated in a gentleMACS Octo Dissociator (Miltenyi Biotec) for 40, 30 and 30 minutes, respectively, using enzymes included in the tissue-specific dissociation kit (Miltenyi Biotec). All single-cell suspensions of dissociated tissues were strained in 70 pM-filters. Dissociated tumors were resuspend in 5 ml Percoll 40% (sigma- Aldrich) and then laid on 3 ml Percoll 67% in a 15ml polypropylene tube. Tubes were spun at 2000 rpm for 20 min at room temperature.Mononuclear cells were collected from the interface of the 40:67% Percoll gradient and washed with complete RPMI prior to antibody staining. Peripheral blood (10 pl) was obtained through tail-vein sampling prior to collection of other organs. Red blood cells were lysed with eBioscience RBC Lysis Buffer (1 mL per spleen; Thermo Fisher Scientific) for 5 minutes at 4°C and washed with PBS. After red blood cell lysis, cells were washed with PBS.

[0348] T cells collected from in vitro or in vivo experiments were initially stained with viability dye in PBS for 15 minutes at 4°C. After washing, cells were surface stained with antibody panel diluted in staining buffer (Flow Cytometry Staining Buffer, Ebioscience) for 30 mins at 4°C, then washed. In some cases, cells were fixed in 1.6% paraformaldehyde (PF A) for 15 minutes at 25°C and washed and stored prior to analysis. For the intracellular staining, after fixation, cells were permeabilized (Fixation and Permeabilization Kit, eBiosciences) and stainedAttorney Docket No.: 078430-543001 WOwith antibody panel diluted in staining buffer for 2h at 4°C according to the manufacturer’s protocol. Cells were washed and resuspended in staining buffer. Analysis was performed on an LSRII (BD Biosciences), Symphony A5, (BD Biosciences), Fortessa (BD Biosciences), Aurora (Cytek Biosciences), or NovoCyte Penteon (Agilent Technologies). For analysis of transduced T cells from tissues of mice, 350 cells were required for immunophenotypic analysis; groups with less than three biological replicates meeting this criteria were excluded.

[0349] Single cell RNA-seq of sorted live thyl .1+YFP+ pmel T cells was performed using the 10X Genomics platform using lOx Genomics reagents according to the manufacturer’s instructions. The 3' Gene Expression libraries were sequenced using a NovaSeq 6000 with a sequencing depth of 120M paired reads (240M total reads) per sample to achieve >20K reads per cell.

[0350] FASTQ files were processed using the CellRanger count pipeline [CellRanger version 7.0.1 (lOx Genomics)] using the mmlO mouse reference genome. Raw gene expression matrices were constructed into a Seurat object and imported into R software using Seurat (version 5). To filter out low-quality cells, cells that had either a low or high number of detected genes and cells that had more than 10-15% of mitochondrial UMI counts in the scRNA-seq data were removed and remaining 6706 cells were used for downstream analysis. To normalize the gene expression levels, the LogNormalize method implemented in Seurat was utilized. Principal component analysis (PCA) was performed to reduce the dimensionality of scRNA-seq data.

[0351] Cell clustering and cell type annotation: The top 30 principal components (PCs) were selected to construct the UMAP embeddings. The FindClusters function was used to identify cell clusters. Total of 10 major cell clusters were obtained, and cluster specific markers were identified using "Find AllMarkers" or "FindMarkers" functions in Seurat. Only positive markers expressed in at least 1% of cells were considered. The nonparametric Wilcoxon rank-sum test was used to obtain the p value for comparisons, and the adjusted p value based on Bonferroni correction was calculated. Genes with adjusted p < 0.05 were considered as enriched in a particular cluster.

[0352] The “AddModuleScore” function in Seurat with default parameters was used to score sets of genes in the single cell data. We calculated the average expression levels of each gene set on a single cell level, subtracted by the aggregated expression of control gene sets. All analyzedAttorney Docket No.: 078430-543001 WOfeatures are binned based on averaged expression, and the control features are randomly selected from each bin. The scored expression was used for plotting.

[0353] Trajectory analysis of scRNA-seq data: To simulate and understand the differentiation trajectories between cells, “monocle3” package (version 1.3.7) was used. The T cell clusters and differential gene expression analysis with Seurat were extracted from the scRNA-seq data and used to construct the monocle object. Pseudotime trajectories were constructed with default parameters and visualized with the plot_cells() function. Cells from the “TscM-like (Tcf7)” cluster were used as the reference starting point (root nodes) from which potential differentiation pathways resonate. The identified paths were mapped on to the UMAP projection for visualization. Ridge plots show the pseudotime scores across treatment groups or clusters.

[0354] Transcription factor acitivitv inference from scRNA-seq data: Top regulons (gene sets regulated by the same transcription factor) and their activity were inferred and evaluated using the SCENIC pipeline (version 1.3.1), thereby defining the top regulon activity in every cell. The input files consisted of the expression matrix and metadata. Then, the co-expression network was calculated by GRNBoost2, and the regulons were identified by RcisTarget. Next, the regulon activity for each cell was scored by A UCell. Scaled expression of regulons were used for the plotting.Analysis of in-house bulk RNA-seq data

[0355] Libraries were prepared using KAPA mRNA HyperPrep Kit from total RNA. Paired-end sequencing was performed on a Novaseq 6000 / X Plus (40M reads per sample).

[0356] Read alignment was performed using STAR aligner on GRCm38 mouse reference transcriptome and transcript level quantification using Salmon. Quality control of the paired-end bulk RNA-seq data was performed using the FastQC program. Adapter trimming was carried out by Cutadapt method. Initial quality control of the count data was carried out by PCA and samplesample distance after variance stabilizing normalization. DESeq2 was used to perform the differential gene expression analysis on samples.

[0357] Transcription factor activity inference from bulk RNA-seq data: To infer TF activity in bulk RNA-seq data, the Univariate Linear Model (ulm) method using the decoupleR package was ran. For each sample in our dataset (mat) and each TF in our network (net), the inventors fit a linear model that predicts the observed gene expression based solely on TF-Gene interactionAttorney Docket No.: 078430-543001 WOweights. Once fitted, the obtained t-value of the slope is the score. If it is positive, the TF is interpreted being as active and if it is negative the TF is interpreted being as inactive.

[0358] Gene set scoring for bulk RNA-seq data: The gene set score of the bulk RNA-seq data was calculated based on the average normalized expression of the genes. From this, the enrichment score of randomly selected control genes with similar expression levels are subtracted. These control gene sets are defined by first binning all genes into bins (n= 24) of aggregate expression level and then, for each gene in the gene set of interest, 100 genes from the same expression bin as that gene are randomly selected. The score is then set to range from zero, meaning no enrichment compared to random sets of genes with similar expression, to one, reflecting the highest average expression of all genes within the gene set of interest. This gene set score is calculated per sample for bulk RNA-seq samples.

[0359] Functional geneset enrichment analysis was performed using fGSEA package in R to infer enriched biological pathways in the treatment groups.

[0360] Linking bulk RNA-seq and phosphoflow data to generate pSTAT gene modules: A set of samples with matched RNA-seq and phosphoflow data was utilized. The RNA-seq data was obtained from in vitro experiments, while the phosphoflow data consisted of MFI measurements at maximum stimulation (Emax) for phosphorylated STAT proteins (pSTATl, pSTAT3, pSTAT4, and pSTAT5). These two datasets were merged to create a combined matrix for subsequent analysis. To investigate the relationship between gene expression and STAT phosphorylation, Pearson correlation coefficients between the RNA-seq counts and each pSTAT measurement were computed. This analysis was performed using the cor. test function from the R stats package. The correlation test was conducted for each gene against each pSTAT, resulting in a comprehensive correlation matrix. For each pSTAT, the top 100 genes exhibiting the highest absolute correlation coefficients were selected. These genes were then visualized using scatter plots and the coefficient of determination (R2) was calculated for each pSTAT-gene set relationship to quantify the proportion of variance in pSTAT levels explained by gene expression. Gene modules were created for each pSTAT using the top 100 correlated genes identified in the previous step and applying the AddModuleScore function in Seurat. These gene modules were then projected onto the scRNA-seq dataset as violin and feature plots.Analysis of publicly availableAttorney Docket No.: 078430-543001 WO

[0361] PBMC and lamina propria scRNA-seq datasets: The raw expression data was obtained from the Single Cell Portal (Broad Institute) for Human PBMC (“Single Cell Comparison:PBMC data”)5and Lamina Propria (“ICA: Ileum Lamina Propria Immunocytes”)6. Raw gene expression matrices were constructed into a Seurat object and imported into R software using Seurat (version 5). Data processing was performed as described in sections above. t-Distributed stochastic neighbor embedding (t-SNE) in Seurat was used for data visualization. Azimuth (version 0.5.0) in Seurat was used to annotate the clusters obtained at resolution 0.2 (PBMC) and 0.5 (lamina propria). To specifically visualize the expression of yc cytokines and their receptors, feature plots and dot plots were plotted to examine the expression across different cell types in PBMC and Lamina Propria.

[0362] Analysis of publicly available bulk scRNA-seq data from Human Protein Atlas: RNA GTEx (genotype tissue expression) tissue gene data was downloaded from The Human Protein Atlas58(vl9.proteinatlas.org / about / download). Transcript expression levels summarized per gene in 36 tissues based on RNA-seq. The tab-separated file includes Ensembl gene identifier ("Gene"), analysed sample ("Tissue"), transcripts per million ("TPM"), protein-transcripts per million ("pTPM") and normalized expression ("NX"). The data was obtained from GTEx and is based on The Human Protein Atlas version 19.3 and Ensembl version 92.38. The expression values for IL2RB, IL4R, IL7R, IL9R, and IL21R across different tissues were used for visualization. A threshold of 0.7 was defined, below which expression is not detected or negligible.

[0363] Analysis of publicly available scRNA-seq data of mouse lymph nodes in response to cytokines: Data was obtained from previously published work on the single cell transcriptomic responses of mouse immune cells (from lymph nodes) in response to a large panel of different cytokines11. Data were downloaded as single cell data objects that were reanalyzed to quantify the overall magnitude of transcriptomic responses of each cytokine across different cell types. A dot plot was created with two metrics: the number of differentially expressed genes (DEGs) and the magnitude of change (based on Euclidean distance) across the entire transcriptome. The number of DEGs was the total number of genes in each cytokine signature. The overall magnitude of cytokine-induced differential expression was computed as the Euclidean distance between the centroid vectors of cytokine-treated cells and PBS-treated cells. A distinct color ramp was used for each cell type to emphasize that cell types have different properties (e.g.,Attorney Docket No.: 078430-543001 WOdifferent numbers of genes expressed on average) and were independently analyzed. Cytokinecell type combinations with five or more cells sampled were included in this analysis.

[0364] Analysis of publicly available scRNA-seq data from human CAR T cells sorted from patients: Publicly available data from a scRNA-seq data set of sorted CAR T cell populations at various timepoints after patient administration14were utilized. Across all patients and time points, the authors sequenced 66,042 post-infusion CAR T cells, with an average of 11,549 cells per patient (SD = 7,335) and 20,532 cells per time point (SD = 37,898). Notably, the month 6 post-infusion time point only consists of 7 CAR T cells. Seurat objects containing different samples at various time points were downloaded, fetched the expression of relevant cytokine receptors across different cell types, and generated dot plots of the data.Analysis of soft tissue sarcoma single-cell RNA-seq samples

[0365] Previously published synovial sarcoma lOx Genomics and SMART-seq2 scRNA-seq data were downloaded from GEO (GSE131309)59. The undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS) samples were obtained GEO (GSE212527)60. Lognormalization was performed independently for each sample using the Seurat function NormalizeData. After this step, the Seurat objects for each independent sample were merged preserving the individual normalization of the data. Sample integration and clustering were performed separately for each histotype, accounting for batch effects and differences in sequencing technologies. The top 2000 highly variable genes of the merged and normalized expression matrices were identified using the function FindVariableFeatures, and then centered and scaled using the ScaleData function. Principal component analysis was then run using the top 2000 highly variable genes previously identified. To integrate multiple samples, the harmony R package^ (version 0.1.0) was employed for batch correction. Harmony was ran using both the sample of origin and the sequencing technology variables as arguments of “group. by. vars,” and based on the first 25 PCA dimensions previously identified. UMAP embeddings were then obtained using the first 25 Harmony dimensions, and clusters were obtained by calculating the k-nearest neighbors (k-NN) and a shared nearest-neighbor graph using the Louvain algorithm implemented in the FindClusters function of the Seurat package with a resolution of 1.0.Endothelial cells, myeloid cells, lymphoid cells, and CAFs were annotated using the differentially expressed genes discovered with the FindMarkers function in Seurat, which was run with the default parameters except for only.pos = T and min.pct = 0.25. Canonical cellAttorney Docket No.: 078430-543001 WOidentity markers obtained from the literature were used for cell type annotations. A small number of cells could not be uniquely assigned to a specific cell identity and were removed from further downstream analysis. UMAP plots were generated with Seurat and scCustomize (4). Density plots were generated with the R package Nebulosa (5).Quantification and statistical analysis

[0366] Statistical comparisons between cell clusters, treatment conditions and regulon activity from the scRNA-seq data were performed using R. The Kruskal- Wallis test was used to compare the medians of groups. P < 0.05 was considered statistically significant.

[0367] Statistical analysis for other experimental data was performed using GraphPad Prism 9 (GraphPad software), except where indicated. All values and error bars are shown as mean ± SEM except as indicated. Comparisons of two groups were performed by using two-tailed unpaired Student’s t test. Comparisons of multiple groups were performed by using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test unless otherwise indicated. In some cases with unequal variance, Kruskal-Wallis with Dunn’s test was performed to compare multiple groups. Experiments that involved repeated measures over a time course, such as tumor growth were performed by using two-way ANOVA with Tukey’s multiplecomparisons post-test. Survival data were analyzed using the Log-rank (Mantel-Cox) test. For analysis of in vivo experiments, outliers were identified across groups using the ROUT method.Data and code availability

[0368] The raw and processed bulk and scRNA-seq data generated in the present disclosure are available at Zenodo (Record #145360225). Relevant code is available at github.com / KalbasiLab. Raw source data from presented experiments (and replicate experiments, when applicable) are available in the source data table (Table S7 of Jiang H. et al., 2026, supra).EXAMPLE 2Expression and activity of IL-9R is restricted in T cells and across normal tissues

[0369] This Example describes experiments perform the illustrate that the expression and activity of IL-9R is restricted in T cells and across normal tissues.

[0370] To investigate the expression of IL-9R in normal T cells, experiments were carried out to examine single cell RNA-sequencing (scRNA-seq) of 28,964 peripheral blood mononuclear cells (PBMCs) from two healthy donors, capturing major hematopoietic lineage cell types (FIG.Attorney Docket No.: 078430-543001 WOIA)5. Compared to receptors for other common yc cytokines (e.g., IL-2 / IL-15 (IL2RB), IL-4 (IL4R), IL-7 (IL7R), and IL-21 (IL21R)), IL9R was expressed at low levels and in very few cells, including rare B cells (0.53%) and CD4 T cells (0.26%). An analysis was extended to tissue resident immune cells in a scRNA-seq dataset of 39,560 lamina propria immunocytes (FIG. IB)6. Again, IL9R was rarely and weakly expressed, in contrast to IL2RB, IL4R, IL7R, and IL21R qRT-PCR of activated human CD4 and CD8 T cells confirmed low IL9R expression (FIG. 8A). Even across thymic development, IL9R transcript levels are nearly absent (FIG. 8B)7Consistent with the low transcript expression, activated human T cells failed to phosphorylate STATs in response to IL-9 (FIG. 8C).

[0371] Even throughout stages of thymic development, IL9R transcript levels are nearly absent (FIG. 8A)14. Consistent with the low transcript expression in the vast majority of developing and mature T cells, activated human T cells from three independent healthy donors did not phosphorylate STAT proteins in response to IL-9 (FIG. 8B).

[0372] The analysis was extended beyond immune cells to 37 human tissue types using publicly available bulk RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project15,16. IL9R expression was broadly restricted: only four of 37 tissues exceeded logio(nTPM)>0.7: lung, small intestine, spleen and urinary bladder (FIG. 1C)8,9. Restricted IL9R expression was also restricted during fetal development (FIG. 8D).

[0373] To evaluate whether IL-9R expression can be induced across T cell differentiation states, additional experiments were performed to analyze scRNA-seq data from tumor, adjacent normal tissues and lymph nodes from three patients with non-small cell lung cancer after immune checkpoint blockade10. IL-9R transcripts were low across twelve distinct T cell subsets and across tissues of origin (FIGS. 8E-8F). Given the inflammatory tumor microenvironment might induce IL9R expression, additional analysis was performed using scRNA-seq from soft tissue sarcoma samples (n=65,945 cells, 15 patients). Again, restricted IL9R expression was observed across major cell types (see, e.g., FIG. ID).

[0374] Subsequently, a functional assessment was used to evaluate the potential toxicity of systemically administered IL-9. For this, scRNA-seq data obtained from lymph nodes of mice treated with one of 86 different cytokines (n=3 mice per cytokine) or PBS (n=14 mice)11were evaluated. For each cell type, differential gene expression in response to each cytokine compared to PBS control treated mice was evaluated to ascertain the magnitude of the globalAttorney Docket No.: 078430-543001 WOtranscriptomic response as well as the total number of significantly upregulated genes. Unlike many yc and non-yc cytokines, IL-9 minimally perturbed gene expression relative to control, with <1 differentially expressed gene (|log2(fold change)|>0.25, FDR<0.05, Wilcoxon) per cluster (FIG. IE).

[0375] It was hypothesized that IL-9 may be less essential for evolutionary fitness than other cytokines. To explore this, an analysis from population-scale exomes (n=983,578; Regeneron Genetics Center Million Exome project) was utilized. IL-2, IL-7, and IL-15 showed high indispensability (Shet), whereas IL-9 ranked most dispensable with mean Shet 0.0019 (95% highest posterior density: [0.0017, 0.0022]), below the canonical transcript mean (0.073)( FIG. IF).

[0376] Given sufficient IL-9R expression on T cells, exploiting the IL-9 / IL-9R axis in T cell therapy would require genetic modification. Consistent with this, scRNA-seq from 66,042 postinfusion CAR T cells from 16 pediatric patients showed absent IL-9R expression, unlike IL2RB / IL2R (primarily CD8) an II.4R II.7R (primarily CD4) (FIG. 1G)14.EXAMPLE 3IL-9 is well-tolerated and drives potent anti-tumor efficacy of T cells engineered with IL-9R

[0377] This Example describes experiments performed to illustrate that IL-9 is well-tolerated and drives potent anti-tumor efficacy of T cells engineered with IL-9R.

[0378] To evaluate the in vivo effects of IL-9, a half-life extended version of mouse IL-9 was engineered by tagging mouse serum albumin to the N-terminus (MSA-IL9), which unlike MSA-IL2, was well-tolerated by weight, survival and mobility, even at 1 OOpg every other day for three weeks (FIGS.2A-2C, 9A). MSA-IL2 increased serum IFNy levels, whereas MSA-IL9 did not, consistent with the low surface expression of IL-9R on major immune subsets across tissues (FIG. 2D and 9B)

[0379] The anti-tumor efficacy of H2-Db / gpl00-specific pmel-1 T cells (hereafter, pmel) engineered was then compared with IL-9R or another benchmark, synthetic o9R, in B16-F10 bearing mice. IL-9R was co-expressed with YFP (P2A), which was a reliable surrogate for IL-9R surface expression (FIG. 9C). With corresponding cytokine treatment (MSA-IL9 or MSA-oIL2), IL-9R pmel T cells achieved tumor control and survival compared to o9R pmel T cells (FIGS. 2F-2F, 9D) .MSA-IL9 alone has no effect (FIG.9E) did not augment untransduced pmel T cells (FIG. 9F), and IL-9R pmel T cells required exogenous MSA-IL9 (FIG. 9G).Attorney Docket No.: 078430-543001 WOEfficacy extended to a sarcoma model derived from KrasG12D / 'P53floxmice and modified to express gplOO (KP-gplOO; FIG. 9H).

[0380] The superior efficacy associated with improved peripheral expansion of IL-9R pmel T cells, which scaled with higher IL-9 dose (FIG.2G). An enhanced early tissue infiltration was also noted (lung, liver and tumor (FIG.2H) and sustained tumor enrichment after cytokine withdrawal (FIG.21), despite no enrichment in normal tissues (FIG. 9H).

[0381] It has been previously demonstrated3that o9R expands a stem cell memory (TSCM, CD62L+CD44'Sca-l+) phenotype. Here, IL-9R signaling more efficiently induced naive (TN, CD62L+CD44‘ T cells; FIGS.9I-9J), including TSCM (FIGS.2L and 9K), with effects confined to IL-9R+ (YFP+) cells, whereas IL-2 affected both YFP+ and YFP- cells (FIG. 9L). In sum, wild-type IL-9R promotes sternness and tumor infiltration, and yields more potent anti-tumor function than o9R.EXAMPLE 4In addition to robust activation of STAT1, STAT3 and STAT5, IL-9R recruits STAT4 signaling

[0382] This Example describes experiments performed to illustrate that in addition to robust activation of STAT1, STAT3 and STAT5, IL-9R recruits STAT4 signaling.

[0383] Experiments were carried out to elucidate the unique signaling characteristics of IL-9R that contribute to its enhanced anti-tumor activity, by first comparing the signaling strength of IL- 9R versus o9R. Dose-response curves of STAT1, STAT3, and STAT5 phosphorylation showed that IL-9R produced higher Emax and lower ECso for STAT1, STAT3, and STAT5 than o9R, indicating both higher affinity and stronger STAT signaling (FIG. 3A) with no cytokineindependent tonic signaling of IL-9R (FIG. 10A). RNA-seq 48h after stimulation showed IL-9R T cells (plus IL-9) and o9R T cells (plus oIL-2) elicited relatively similar transcriptomes when compared with their IL-2-treated controls (FIGS. 3B and 10B-10D). While 7673 differentially expressed genes (DEGs) were observed between IL-9R + IL-9 and IL-9R + IL-2 groups, and 7166 DEGs between o9R + oIL-2 and IL-9R + IL-2 groups, only 3031 DEGs were noted between IL-9R + IL-9 and o9R + oIL-2 groups (FIGS. 3B and 10B-10D). Thus, IL-9R and o9R activate similar gene expression patterns, consistent with their shared ICDs, yet their transcriptomic profiles are distinguishable due to differences in signal strength.

[0384] Phoshphoproteomic analysis (20-minute stimulation) revealed broad changes with IL-2: 228 differentially abundant phosphoproteins, consistent with its known activation of multipleAttorney Docket No.: 078430-543001 WOsignaling pathways including JAK / STAT, ERK and AKT (FIGS. 3C and 10D; and Table SI of Jiang H. et al., 2026, supra). However, the phosphoproteomic footprint of IL-9 was highly restricted, with only four differentially abundant phosphoproteins. The JAK / STAT pathway accounted for three of the four differentially abundant phosphoproteins. With phosphoflow cytometry, it was confirmed that IL-9 did not phosphorylate of ERK or AKT in IL-9R engineered T cells (FIGS. 10E-10F).

[0385] Unexpectedly, the most differentially abundant phosphoprotein upon IL-9 stimulation was pSTAT4 (FIG.3C), which has not been canonically implicated in IL-9 or, more broadly, ycsignaling1. Phosphoflow pSTAT4 activity comparable to IL- 12 signaling (FIG.3D), the canonical activator of STAT4 in T cells17and a well-described promoter of anti-tumor functions in T cells (FIG. 3D)17'20. Transcription factor inference based on RNA-seq data supported STAT1, STAT2, STAT3, and STAT4 engagement with IL-9R versus IL-2 (FIG.3E).Additionally, both IL-9R and o9R signaling increased expression of 15 of 18 Biocarta IL- 12 Pathway genes compared to IL-2 (FIG.3F).

[0386] Unlike IL-12 (STAT4-only), IL-9 activates STAT1, STAT3, STAT4 and STAT5. This distinction was evident in the functional outcomes of IL-9 and IL- 12 signaling. Functionally, IL-9 provided a modest proliferation to IL-9R T cells (less than 11-2, more than IL- 12 (FIG.3G).This was likely attributable to a higher STAT5 signal from IL-221. IL-9 also enriched naive and TSCM cells compared to IL- 12 (FIGS.3H-3I), consistent with its stronger STAT3 signal22.

[0387] In summary, these results demonstrate that IL-9R robustly activates STAT1, STAT3, and STAT5; and also recruites STAT4, producing a transcriptional and phenotypic program blending features of IL- 12 and yc cytokines and contributing to the IL-9R T cell efficacy.EXAMPLE 5Either attenuation or amplification of IL-9R signaling disrupt its anti-tumor properties

[0388] This Example describes experiments performed to illustrate that either attenuation or amplification of IL-9R signaling disrupt its anti-tumor properties.

[0389] Given its unique JAK / STAT signaling profile, experiments were carried out to test how proximal signaling magnitude affect efficacy, we first attenuated IL-9 by mutating the yc-contact glutamine within helix D (QI 15T; FIG. 11 A, predicted complex in FIG.4A). In other yc cytokines, a mutation in this glutamine residue attenuates signaling23. The inventors thus selected to mutate the corresponding glutamine residue at position 115 of IL-9 to threonine (QI 15T). AAttorney Docket No.: 078430-543001 WOpredicted structure of the IL-9R complex also supported the interaction of QI 15 with the yc. Indeed, a reduced Emax and EC50 of STAT1, STAT3, STAT4, and STAT5 signaling of IL-9R engineered T cells was observed in response to IL-9Q115Tcompared to IL-9WT(FIG. 4B) and diminished TSCM induction versus IL-9.

[0390] The attenuation of IL-9 diminished the anti -tumor efficacy of IL-9R pmel T cells against Bl 6 melanoma, both as measured by tumor growth and maximal treatment response (FIGS. 4C and 11C). Blood expansion and persistence were initially similar, but by Day 41, IL-9R pmel T cells were detectable with IL-9Q115T, while IL-9R pmel T cells in the IL-9WTgroup persisted (FIGS. 4D and 11D). In fact, IL-9R pmel T cells were detectable 115 days after initial adoptive transfer, at which point a third cycle of IL-9WTtreatment resulted in a third peak of IL-9R pmel T cells, that exceeded levels of the initial expansion.

[0391] To amplify IL-9R signaling, the fact that IL-9R signaling is driven by a single phosphotyrosine site (Y405) downstream of the Box 1 motif24was leveraged. The inventors generated IL-9R mutants in which a 39 amino acid sequence flanking the phosphotyrosine site Y405 (SEQ ID NO: 5) were repeated three or five times (IL-9R3xor IL-9R5x, respectively) (FIG.4E). These mutants IL-9R3xand IL- 9R5xincreased the Emax of IL-9 for pSTATl, pSTAT3, and pSTAT4 (not pSTAT5) with significance for IL-9R5x(FIGS.4F and HE), and minimally augmented TSCM enrichment in vitro (Fig. 11F).

[0392] Despite subtle amplification of the IL-9R signal, IL-9R3xreduced anti-tumor activity of pmel T cells compared to IL-9RWT, with earlier outgrowth of B16-F10 and fewer complete responses. (1 / 8 versus 6 / 7; FIG. 4G). Thus, both weaker and stronger IL-9R signaling impaired efficacy, suggesting a superior JAK / STAT signaling window.EXAMPLE 6IL-9R intracellular domain mutants skew STAT phosphorylation profiles and alter in vivo proliferation and anti-tumor efficacy

[0393] This Example describes experiments performed to illustrate that IL-9R intracellular domain mutants skew STAT phosphorylation profiles and alter in vivo proliferation and antitumor efficacy.

[0394] Because IL-9R activates multiple STATs, experiments were performed to explore how stoichiometry -not just magnitude- contributes to its superior anti-tumor functions. It was confirmed that Y405 is the primary hub for IL-9R STAT signaling, as a Y405F substitutionAttorney Docket No.: 078430-543001 WOablated STAT1, STAT3 and STAT4 activation and diminished STAT5 activation (FIG. 12A)24.Amino acids immediately adjacent to a phosphotyrosine site can influence STAT binding and activation25, so the inventors generated ten single amino acid mutants of the C-terminal proline or glutamine residue of Y405 (FIG. 5A). T cells engineered with these IL-9R mutants displayed an array of STAT signaling profiles, each yielding distinct pSTAT stoichiometry (FIG. 5B).

[0395] Additional experiments were perform to further study the contribution of STAT1, in which IL-9RPR(higher pSTATl but similar pSTAT3, pSTAT4 and pSTAT5 compared to IL-9RWT) and IL-9RAQ(reduced pSTATl with similar pSTAT3, pSTAT4 and pSTAT5 compared to IL-9RWT) were selected. Two other mutants, IL-9RKQand IL-9RSQ, also produced lower STAT1 phosphorylation than IL-9RWTwhile maintaining phosphorylation levels of the remaining STATs, but for simplicity, additional experiments were conducted with IL-9RAQ. It was confirmed that IL-9RPRincreased pSTATl Emax and IL-9RAQreduced pSTATl Emax in T cells (FIG. 5C). Notably, IL-9RAQand IL-9RPRphosphorylated STAT3, STAT4, and STAT5 at similar levels. Compared to IL-9RWT, IL-9RAQand IL-9RPRproduced a similar pSTAT5, but weaker pSTAT3 and pSTAT4 signals.

[0396] A superior in vitro expansion of mouse T cells engineered with IL-9RAQwas observed, as compared to IL-9RWT(FIG. 5D) (IL-9RAQT cells expanded more than IL-9RWTT cells (Fig.5D), whereas IL-9RPRT cells expanded less). IL-9RAQT cells had a higher proliferative index than compared to IL-9RPRT cells as measured by dilution of CellTrace Violet dye (FIGS. 5E and 12B). An increase in apoptotic or dead cells with either mutant (FIGS. 12C-12D) was not observed, which is consistent with the cytostatic effects of STAT126‘27

[0397] Phenotypically, IL-9RAQT cells favored a central memory (TCM) phenotype (CD62L+CD44+) in vitro, and aligned most with IL-2-treated T cells, while IL-9RPRskewed away from TCM (FIG. 12E). Across TSCM and TEFF(CD62L CD44+) subsets, receptors ordered according to their pSTATl Emax (IL-9RPR> IL-9RWT> IL-9R<)).

[0398] The improved proliferation of IL-9RAQT cells was pronounced in vivo where the cells expanded ~5- 10-fold greater in the blood between five and ten days after adoptive cell transfer (ACT) compared to IL-9RWT(FIG. 5F). More IL-9RAQT cells were also observed in the tumor seven days after ACT (FIG. 5G).

[0399] Compared to IL-9RWTpmel T cells, IL-9RPRpmel T cells resulted in significantly reduced tumor control, fewer complete responses, and worse survival (FIGS. 5G and 12F),Attorney Docket No.: 078430-543001 WOconsistent with their diminished proliferative capacity. IL-9RAQpmel T cells, despite their increased peripheral expansion and superior tumor infiltration, did not surpass the anti-tumor efficacy of IL-9RWTpmel T cells (FIGS. 5H and 12G).EXAMPLE 7T cell intrinsic STAT1 acts as a rheostat between stem and effector fates of tumor-infiltrating T cells

[0400] This Example describes experiments performed to illustrate that T cell intrinsic STAT1 acts as a rheostat between stem and effector fates of tumor-infiltrating T cells.

[0401] To further explore the fate of tumor-specific T cells signaling through IL-9R and the biased IL-9R mutants, scRNA-seq was performed on tumor- infiltrating pmel T cells engineered with IL-9RWT, IL-9RAQor IL-9RPR, eight days after adoptive transfer (FIGS.6A, 13A). As expected, the frequency of engineered (YFP+) pmel T cells among infiltrating CD8+ T cells was highest in the IL-9RAQgroup (17.0%), followed by IL-9RWT(13.4%) and IL-9RPR(0.7%) (FIG.13B)

[0402] Most cells mapped to effector subtype (see, e.g., FIG. 13C and Table S2 of Jiang H. et al., 2026, supra), including early activated effectors (T early activated, expressing Pdcdl, Lag3, Havcr2, and Vsir, along with Gzmb an Prf! expression), proliferative effectors (Teff-l / prolif and Teff-2 / prolif), and effectors with high Statl expression (Teff-5). Two clusters consisted of mixed effector, naive and either precursor exhausted (Teff-3 / pex / naive) or effector memory (Teff-4 / naive / em) T cells. Aside from effectors, a cluster with features of both effector memory and resident memory T cells (Tem / rm), marked by Il7r, Itgal, Itga4, Cxcr3, Ccl5 and Gzma expression (FIG. 13D) was identified. A cluster of stem cell memory-like T cells (TscM-like) had uniquely high expression of Tcf7, as well as Il7r (FIG. 13D). Additionally, clusters of proliferative T cells (Tprolif) and T cells and enriched for a myeloid differentiation gene set (FIG. 13E)28were also identified. Transcription factor and gene regulatory network inference using the Single-Cell rEgulatory Network Inference and Clustering (SCENIC) package aided annotations (Table S2 of Jiang H. et al., 2026, supra).

[0403] Despite the tumor TCR stimulus, T cell fates diverged by cytokine receptor. IL-9RPRpmel T cells (high pSTATl signal) localized to Teff-5 (Statl / Gzmb / Prfl cluster); IL9RAQpmel T cells (lower pSTATl) enriched in Tem / rm and TscM-like clusters (FIGS.6C and 13D) and were less represented among effector clusters; IL-9RWTpmel T cells occupied an intermediateAttorney Docket No.: 078430-543001 WOstate, enriched for both early activated effectors (Teff-l / activated) and TEM / RM and TscM-like clusters. Consistent with this gradient of effector phenotype (IL-9RPR> IL-9RWT> IL-9RAQ), the overall expression of a gene set distinguishing KLRGlhleffector versus naive T cells was lowest in IL-9RAQ, and highest in_IL-9RPRpmel T cells (GSE10239; FIG.6D)29. A similar trend was seen for the expression of Prfl and Gzmb (FIG. 13F).

[0404] The inventors did not find clusters fitting a canonical definition of exhaustion as most cells lacked expression of Tox and Eomes, consistent with the early timepoint. Instead, the inventors examined an acute-infection-versus-malignancy gene set (GSE6O5O1)30, which was most highly expressed among IL-9RAQ, and lowest among IL-9RPRpmel T cells (FIG.6E). Pseudotime analysis anchored on the Tcf7+Tscm-like cluster further supported an earlier differentiation state for IL-9RAQ, intermediate for IL-9RWT, and later for IL-9RPR(FIGS.6G and 13G). In summary, compared to IL-9RWT, IL-9RAQdrives a memory and stem-like state at the expense of effector differentiation, while IL-9RPRdrives a purely effector state with early features of exhaustion.

[0405] Phenotypic analysis of T cells seven days after ACT into mice bearing B16-F10 melanoma confirmed more TN / TCM cells with IL-9RAQacross tumor, spleen, and tumor draining lymph node (tdLN) (FIG. 13H). The phenotypic analysis was limited to IL-9RWTand IL-9RAQT cells in the spleen, draining lymph node (tdLN) and tumor, and IL-9R3xT cells in the tdLN, due to the poor expansion and tumor- infiltration in other groups (Table S3 of Jiang H. et al., 2026, supra). A small increase was observed in frequency of TEFF cells among IL-9RWTversus IL-9RAQcells in the tumor (a similar trend was observed in the tdLN; FIG. 131). Tumor-infiltrating IL-9RWTpmel T cells also demonstrated greater frequency of Granzyme B (p=0.03) and TNF-a (p=0.09) expression than IL-9RAQpmel T cells (FIG. 13 J), explaining their similar anti -tumor function despite fewer cells (FIGS. 5H and 12G).

[0406] To link signaling to transcription, STAT phosphorylation patterns (20 minutes) were correlated with bulk RNA-seq (48 hours) across six conditions with different patterns of STAT phosphorylation (FIGS. 6H-6I). For each condition: IL-9R, IL-9RAQ, IL-9RPRor IL-9R5xplus IL-9, o9R + oIL-2, and IL-9R + IL-2(FIGS.6H-6I ). The top 100 pSTAT correlated genes demonstrated the entirely unique gene programs induced by pSTATl and pSTAT5 (see, e.g., FIG. 6 J and Table S4 of Jiang H. et al., 2026, supra). Sixty-six of 100 genes most correlatedAttorney Docket No.: 078430-543001 WOwith pSTAT3 and pSTAT4 were shared. pSTATl had the strongest gene expression correlation, reflecting the range and even distribution of pSTATl activation across the sample set (FIG.6K).

[0407] Gene expression modules were generated based on the top pSTAT-correlated genes were projected onto the scRNA-seq data from FIG.6A. The pSTATl module distinguished IL-9RPRfrom IL-9RWTand IL-9RAQpmel T cells, validating the fidelity between the in vitro and in vivo transcriptomic effects of IL-9R signaling (FIG.6L). Differences in pSTAT3, pSTAT4, or pSTAT5 gene modules between the groups were not found (FIG. 13L), though these were less sensitive for detecting differences given the lower correlation between gene expression and STAT phosphorylation.

[0408] With exogenous IFN- , pSTATl in IL-9RWTand IL-9RAQT cells to those of IL-9RPRT cells were selectively increased (FIG. 13M). To evaluate the functional impact of STAT1 modulation, additional experiments were focused on the TCM subset, as previous experiments showed that IL-9RAQ, IL-9RWT, and IL-9RPRT cells exhibited graduated TCM phenotypes both in vitro and in vivo (IL-9RAQ> IL-9RWT> IL-9RPR). The increase in pSTATl reduced the TCM phenotype in IL-9RAQand IL-9RWTT cells to levels observed in IL-9RPRT cells (FIG. 13N), supporting the STAT1 -driven differentiation observed in single cell analyses described herein.

[0409] Additional experiments were performed to understand if the low pSTATl signal of IL-9RAQ Jce||scontributed to their less-differentiated state. Indeed, transcription factor inference from the in vitro RNA-seq data identified STAT1 and related TFs (IRF1, IRF2 and IRF9), as the dominant distinguishing feature between IL-9RWTand IL-9RAQT cells (FIG. 6N). SCENIC analysis of the scRNA-seq dataset also demonstrated a modest increase in the STAT1 regulon in tumor-infiltrating IL-9RWTcompared to IL-9RAQpmel T cells (FIG.60). In fact, among STAT1, STAT3, STAT4 and STAT5, STAT1 was the only regulon enriched in IL-9RWTcompared to IL-9RAQpmel T cells.

[0410] It was noted that STAT3, STAT4 and STAT5 regulons were enriched in tumorinfiltrating IL-9R<)pmel T cells compared to both IL-9RWTand IL-9RPRpmel T cells.Furthermore, genes differentially expressed by IL-9RAQT cells were enriched for genes from Hallmark IL2 / STAT5 signaling and IL6 / JAK / STAT3 signaling gene sets (adjusted p-value = 0.001 and 0.07, respectively; FIG. 130).

[0411] The above observation is consistent with the stem, memory22, 31and proliferative qualities21, 32of IL9RAQpmel T cells (FIG.6N), but is also partly counterintuitive consideringAttorney Docket No.: 078430-543001 WOthat IL-9RAQresults in stronger pSTAT3 and pSTAT4 (and similar pSTAT5) compared to IL-9RWT. It is hypothesized that differences between STAT profiles based on proximal signaling versus in vivo transcriptomics may relate to complex downstream interplay and competition between STAT proteins (see discussion).

[0412] Overall, T cell intrinsic STAT1 titrates fate from a less-differentiated, stem-like state (IL-9RAQ, STATl-low) to a terminally differentiated effector (IL-9RPR, STATl-hi), with IL-9RWT(STATl-mid) encompassing both stem-like and effector states.EXAMPLE 8Potency and sensitivity of proximal IL-9R signaling for human CAR T cells

[0413] This Example describes experiments performed to investigate potency and sensitivity of proximal IL-9R signaling for human CAR T cells.

[0414] In human T cells, IL-9R produced stronger phosphorylation of STAT1, STAT3, and STAT5 than human orthogonal chimeric o9R (ho9R), and induced pSTAT4 (FIG. 7A). In repetitive tumor killing assays with NY-ESO-1 TCR (1G4) effectors and human melanoma M407mCheriytumor cell targets, hIL-9R + IL-9 outperformed ho9R + hoIL-2 (FIG. 14A).

[0415] CD19-BBz CAR T cells was also co-engineered with human IL-9R (hIL-9R; FIG. 14B). In a repetitive killing assay with NALM6 tumor cells, IL-9R plus IL-9 outperformed the same cells cultured with no cytokine or IL-2 between five and nine rounds of tumor killing (FIG.14C). In Nalm6 leukemia-bearing NSG mice, IL-2 caused rapid weight loss; IL-9 did not (FIGS.7B-7C). Dual CD19-BBz CAR plus IL-9R T cells resulted in prolonged tumor control and survival (FIGS. 7D-7E), whereas IL-9 did not had no effect in the absence of IL-9R-engineered T cells.

[0416] Against a solid tumor (143B osteosarcoma) expressing Her2 (FIG. 7F; CAR T cells targeting Her2 (H2-BBz) were minimally effective alone (FIGS. 7G-7H), but H2-BBz hIL9R T cells improved tumor control and survival (FIGS. 7G-7H).

[0417] It was also found that the signaling patterns of 11-9 mutant receptors, IL-9RAQand IL-9RPR, were generally conserved between mouse and human systems (FIGS. 14D-14E): IL-9RAQreduced and IL-9RPRincreased pSTATl (FIG. 71); pSTAT5 levels were similar; and pSTAT3 and pSTAT4 were modestly lower than IL-9RWT. Unlike the mouse system, we observed slight differences pSTAT3 and pSTAT4 levels between human IL-9RAQand IL-9RPRmutants (although for STAT3 this was only observed in selected donors). In vitro, IL-9RAQexpanded more , andAttorney Docket No.: 078430-543001 WOIL-9RPRexpanded less than IL-9RWT(FIG. 14F). In vivo, the expansion and enrichment of CAR T cells co-engineered with IL-9RAQwas superior to IL-9RWTor IL-9RPR(FIGS. 7 J and 14G).Treatment with IL-9 improved tumor control in mice treated with CAR T cells co-engineered with IL-9RWTand I L-9RAQ, but not IL-9RPR(FIG. 7K), consistent with the inferior anti -tumor functions of mouse IL-9RPRpmel T cells. Survival with IL-9RWTCAR T cells exceeded IL-9RPR(p=0.078) and IL-9RAQCAR T cells, though survival after IL-9RAQCAR T cells was confounded by toxicity from rigorous CAR expansion. Consistent with the in vivo data, IL-9RWTand IL-9RAQoutperformed IL-9RPRin the repetitive killing of M407mCheriywith NY-ESO-1 TCR effectors (FIG. 14H). IL-9RAQT cells expanded more after each round of killing (Fig. S7I), yet IL-9RWTmaintained similar anti-tumor efficacy (Fig. S7F), consistent with a higher frequency of Granzyme B expression among IL-9RWTco-transduced cells compared to IL-9RAQ(FIG. 14 J) after three rounds of tumor killing. These findings mirrored our results with mouse T cells and further supported the higher effector capacity of IL-9RWTversus IL-9RAQon a per cell basis.

[0418] Altogether, human IL-9R signaling, its single amino acid mutational tuning, and resultant expansion / efficacy mirror the mouse system, further supporting its role as a driver of anti -tumor functions in T cells that is exquisitely sensitive to its superior balance of JAK / STAT signaling.

[0419] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.Attorney Docket No.: 078430-543001 WOINFORMAL SEQUENCE LISTINGAttorney Docket No.: 078430-543001 WOAttorney Docket No.: 078430-543001 WOREFERENCES E Leonard, W.J., Lin, J.X., and O'Shea, J.J. (2019). The y(c) Family of Cytokines: Basic Biology to Therapeutic Ramifications. Immunity 50, 832-850.10.1016 / j.immuni.2019.03.028.2. Dwyer, C.J., Knochelmann, H.M., Smith, A.S., Wyatt, M.M., Rangel Rivera, G.O., Arhontoulis, D.C., Bartee, E., Li, Z., Rubinstein, M.P., and Paulos, C.M. (2019). Fueling Cancer Immunotherapy With Common Gamma Chain Cytokines. Frontiers in Immunology 10. 10.3389 / fimmu.2019.00263.1. 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Expression of Interleukin 9 in the Lungs of Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness. Journal of Experimental Medicine 188, 1307-1320. 10.1084 / jem.188.7.1307.McMillan , S.J., Bishop , B., Townsend , M.J., McKenzie , A.N., and Lloyd , C.M.(2002). The Absence of Interleukin 9 Does Not Affect the Development of Allergen-induced Pulmonary Inflammation nor Airway Hyperreactivity. Journal of Experimental Medicine 195, 51-57. 10.1084 / jem.20011732.Townsend, M. J., Fallon, P.G, Matthews, D. J., Smith, P., Jolin, H.E., and McKenzie, A.N. J. (2000). IL-9-Deficient Mice Establish Fundamental Roles for IL-9 in Pulmonary Mastocytosis and Goblet Cell Hyperplasia but Not T Cell Development. Immunity 13, 573-583. 10.1016 / S1074-7613(00)00056-X.Attorney Docket No.: 078430-543001 WOCastelli, S., Wilson, W.V., Uslu, U., Finck, A., Assenmacher, C.-A., Atoche, S.J., Siurala, M., Young, R.M., and June, C.H. (2025). IL-9 signaling redirects CAR T cell fate toward CD8+ memory and CD4+ cycling states, enhancing anti-tumor efficacy. bioRxiv, 2025.2001.2030.635582. 10.1101 / 2025.01.30.635582.Sockolosky, J.T., Trotta, E., Parisi, G., Picton, L., Su, L.L., Le, A.C., Chhabra, A., Silveria, S.L., George, B.M., King, I.C., et al. (2018). Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes. Science 359, 1037-1042.10.1126 / science. aar3246.Beltra, J.C., Abdel-Hakeem, M.S., Manne, S., Zhang, Z., Huang, H., Kurachi, M., Su, L., Picton, L., Ngiow, S.F., Muroyama, Y., et al. (2023). Stat5 opposes the transcription factor Tox and rewires exhausted CD8(+) T cells toward durable effector-like states during chronic antigen exposure. Immunity 56, 2699-2718. e2611.10.1016 / j . immum.2023.11.005.Demoulin, J.B., Uyttenhove, C., Van Roost, E., DeLestre, B., Donckers, D., Van Snick, J., and Renauld, J.C. (1996). A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9. Mol Cell Biol 16, 4710-4716. 10.1128 / mcb.16.9.4710.Quigley, M., Huang, X., and Yang, Y. (2008). STAT1 Signaling in CD8 T Cells Is Required for Their Clonal Expansion and Memory Formation Following Viral Infection InVivol. The Journal of Immunology 750, 2158-2164. 10.4049 / jimmunol.180.4.2158. Lukhele, S., Rabbo, D.A., Guo, M., Shen, J., Elsaesser, H.J., Quevedo, R., Carew, M., Gadalla, R., Snell, L.M., Mahesh, L., et al. (2022). The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity. Immunity 55, 2369-2385. e2310. / / doi.org / 10.1016 / j.immum.2022.10.020.Mathew, D., Marmarelis, M.E., Foley, C., Bauml, J.M., Ye, D., Ghinnagow, R., Ngiow, S.F., Klapholz, M., Jun, S., Zhang, Z., et al. (2024). Combined JAK inhibition and PD-1 immunotherapy for non-small cell lung cancer patients. Science 384, eadfl329. doi:10.1126 / science.adfl329.Yang, X.P., Ghoreschi, K., Steward-Tharp, S.M., Rodriguez-Canales, J., Zhu, J., Grainger, J.R., Hirahara, K., Sun, H.W., Wei, L., Vahedi, G, et al. (2011). Opposing regulation of the locus encoding IL- 17 through direct, reciprocal actions of STAT3 and STAT5. Nat Immunol 72, 247-254. 10.1038 / m.l995.Radpour, R., Simillion, C., Wang, B., Abbas, H.A., Riether, C., and Ochsenbein, A.F. (2024). IL-9 secreted by leukemia stem cells induces Thl -skewed CD4+ T cells, which promote their expansion. Blood 144, 888-903. 10.1182 / blood.2024024000.Tousley, A.M., Rotiroti, M.C., Labanieh, L., Rysavy, L.W., Kim, W.-J., Lareau, C., Sotillo, E., Weber, E.W., Rietberg, S.P., Dalton, G.N., etal. (2023). Co-opting signalling molecules enables logic-gated control of CAR T cells. Nature 615, 507-516.10.1038 / s41586-023-05778-2.Uhlen, M., Fagerberg, L., Hallstrom, B.M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, A., Kampf, C., Sjostedt, E., Asplund, A., et al. (2015). Tissue-based map of the human proteome. Science 347, 1260419. doi:10.1126 / science.1260419.Jerby-Arnon, L., Neftel, C., Shore, M.E., Weisman, H.R., Mathewson, N.D., McBride, M.J., Haas, B., Izar, B., Volorio, A., Boulay, G., et al. (2021). Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma. Nat Med 27, 289-300. 10.1038 / s41591-020-01212-6.Attorney Docket No.: 078430-543001 WOSubramanian, A., Nemat-Gorgani, N., Ellis-Caleo, T.J., van, I.D.G.P., Sears, T. J., Somani, A., Luca, B.A., Zhou, M.Y., Bradic, M., Torres, I. A., et al. (2024). Sarcoma microenvironment cell states and ecosystems are associated with prognosis and predict response to immunotherapy. Nat Cancer 5, 642-658. 10.1038 / s43018-024-00743-y.

Claims

Attorney Docket No.: 078430-543001 WOCLAIMS WHAT IS CLAIMED IS:

1. A recombinant cytokine receptor comprising:a) an extracellular domain (ECD) and an intracellular signaling domain (ICD) of a IL-9 receptor (IL-9R), wherein the ICD comprises a proline-to-alanine (P->A) substitution in its STAT binding site; andb) a transmembrane domain (TMD) operably inserted between the ECD the ICD.

2. The recombinant cytokine receptor of claim 1 , wherein the IL-9R is a murine IL-9R.

3. The recombinant cytokine receptor of claim 1, wherein the IL-9R is a human IL-9R.

4. The recombinant cytokine receptor of any one of claims 1-3, wherein the TMD is a transmembrane domain derived from the same IL-9R.

5. The recombinant cytokine receptor of any one of claims 1-3, wherein the TMD is a heterologous transmembrane domain.

6. The recombinant cytokine receptor of claim 5, wherein the heterologous TMD is derived from a type-1 transmembrane spanning protein.

7. The recombinant cytokine receptor of claim 6, wherein the type-1 transmembrane spanning protein is selected from the group consisting of CD3 , CD4, CD8, CD28, B7-H3, IL-2RB, IL-4R, IL-7R, IL-9R, and IL-21R.

8. The recombinant cytokine receptor of claim 5, wherein the heterologous TMD is derived from a surface receptor subunit.

9. The recombinant cytokine receptor of claim 8, wherein the surface receptor subunit is selected from the group consisting of IL3Ra, IL4Ra, IL5Ra, IL6Ra, IL7Ra, ILlORa, ILlORb, IL12Rpl, IL12Rp2, IL12p40, IL13RA1, IL15Ra, IL20R, IL21Ra, IL22R, IL23R, IL28R, IL31 Ra, GMCSFRa, LIFR, CNTFR, CLF1, OSMR, GCSFR, EPOR, TPOR, GHR, PRLR, LEPR, IFNAR2, IFNAR1, IFNGR1, and IFNGR2.

10. The recombinant cytokine receptor of any one of claims 1-9, wherein the recombinant cytokine receptor comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2.Attorney Docket No.: 078430-543001 WO11. The recombinant cytokine receptor of any one of claims 1-10, wherein the recombinant cytokine receptor, when expressed in a recombinant T cell, results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

12. The recombinant cytokine receptor of claim 11, wherein the STAT1 signaling in the recombinant T cell is decreased by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

13. The recombinant cytokine receptor of any one of claims 1-12, wherein the recombinant cytokine receptor, when expressed in a recombinant T cell, confers an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

14. The recombinant cytokine receptor of any one of claims 1-13, wherein the recombinant cytokine receptor, when expressed in a recombinant T cell, results in an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

15. The recombinant cytokine receptor of any one of claims 1-14, wherein the recombinant cytokine receptor, when expressed in a recombinant T cell, results in promoting less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

16. The recombinant cytokine receptor of any one of claims 11-15, wherein the reference T cell comprises a wild-type IL-9 receptor.

17. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant cytokine receptor of any one of claims 1-16.

18. The recombinant nucleic acid molecule of claim 17, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence having at least 80% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 3-4.Attorney Docket No.: 078430-543001 WO19. The recombinant nucleic acid molecule of any one of claims 17-18, wherein the recombinant nucleic acid molecule is operably linked to one or more heterologous nucleic acid sequences.

20. The recombinant nucleic acid molecule of claim 19, wherein the one or more heterologous nucleic acid sequences comprises one or more expression control elements.

21. The recombinant nucleic acid molecule of claim 20, wherein the one or more expression control elements is independently selected from the group consisting of ribosomal binding sites, promoters, translational start sequences, translational termination sequences, transcriptional start sequences, transcriptional termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, enhancer elements, activator elements, replication elements, RNA processing and export elements, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, or any combination thereof.

22. The recombinant nucleic acid molecule of any one of claims 19-21, wherein the one or more heterologous nucleic acid sequences comprises a promoter.

23. The recombinant nucleic acid molecule of claim 22, wherein the promoter is a CD4 cellspecific promoter or a CD8 cell-specific promoter.

24. The recombinant nucleic acid molecule of any one of claims 22-23, wherein the promoter is selected from the group consisting of murine stem cell virus (MSCV) promoter, EFla promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter,Attorney Docket No.: 078430-543001 WOCTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B-and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD130 promoter, NR4A1 promoter, NR4A2, and NR4A3 promoter.

25. The recombinant nucleic acid molecule of any one of claims 17-24, wherein the recombinant nucleic acid molecule is incorporated into an expression cassette or a vector.

26. The recombinant nucleic acid molecule of claim 25, wherein the vector is an expression vector.

27. The recombinant nucleic acid molecule of any one of claims 25-26, wherein the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a RNP-packaging virus-like particle.

28. The recombinant nucleic acid molecule of claim 27, wherein the viral vector is a lentivirus vector, a baculovirus vector, a retrovirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.

29. The recombinant nucleic acid molecule of any one of claims 25-28, wherein the recombinant nucleic acid molecule is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a protein nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM).

30. A recombinant cell comprising:Attorney Docket No.: 078430-543001 WO(a) a recombinant cytokine receptor according to any one of claims 1-16; and / or(b) a recombinant nucleic acid according to any one of claims 17-29.

31. The recombinant cell of claim 30, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.

32. The recombinant cell of claim 31, wherein the recombinant cell is a eukaryotic cell.

33. The recombinant cell of claim 32, wherein the eukaryotic cell is a mammalian cell.

34. The recombinant cell of claim 33, wherein the mammalian cell is a human cell.

35. The recombinant cell of any one of claims 33-34, wherein the recombinant cell is T cell.

36. The recombinant cell of claim 35, wherein the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell.

37. The recombinant cell of claim 36, wherein the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells.

38. The recombinant cell of claim 36, wherein the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

39. The recombinant cell of any one of claims 35-38, wherein the T cell is an exhausted T cell or a non-exhausted T cell.

40. The recombinant cell of any one of claims 35-39, wherein the T cell is obtained leukapheresis of a sample obtained from a subject.

41. The recombinant cell of any one of claims 35—40, wherein the T cell further comprises:(a) an engineered immune receptor; and / or(b) a recombinant nucleic acid encoding the engineered immune receptor of (a).

42. The recombinant cell of claim 41, wherein the engineered immune receptor is a chimeric antigen receptor (CAR) or and T cell receptor (TCR).Attorney Docket No.: 078430-543001 WO43. The recombinant cell of claim 42, wherein the engineered immune receptor is a chimeric antigen receptor (CAR) having specificity for a target ligand.

44. The recombinant cell of claim 43, wherein the target ligand is a tumor antigen selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, ADAMI 2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD 171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican- 3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE- A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MRC2, MSX1, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC- A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PRAME, PSCA, PSMA, R0R1, R0R2, SSX1, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

45. The recombinant cell of any one of claims 43-44, wherein the target ligand is expressed on or associated with a cancer cell.

46. The recombinant cell of claim 45, wherein the cancer cell is of a B-cell malignancy (such as a B-cell lymphomas or leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, or renal cancer.

47. The recombinant cell of any one of claims 35-46, wherein the recombinant T cell comprises a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.Attorney Docket No.: 078430-543001 WO48. The recombinant cell of claim 47, wherein the STAT1 signaling in the recombinant T cell is decreased by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

49. The recombinant cell of any one of claims 35-48, wherein the recombinant T cell comprises an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

50. The recombinant cell of any one of claims 35-49, wherein the recombinant T cell comprises an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

51. The recombinant cell of any one of claims 35-50, wherein the recombinant T cell promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

52. A method for producing a recombinant T cell, the method comprising introducing into a T cell a recombinant cytokine receptor according to any one of claims 1-16, or a recombinant nucleic acid molecule according to any one of claims 17-29.

53. The method of claim 52, wherein the recombinant T cell comprises a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

54. The method of claim 53, wherein the STAT1 signaling in the recombinant T cell is decreased by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

55. The method of any one of claims 52-54, wherein the recombinant T cell comprises an enhanced stem cell-like memory T cell (TSCM) phenotype, superior engraftment, enhanced tumor infiltration, and enhanced anti-tumor activity; as compared to a reference T cell that does not comprise the recombinant cytokine receptor.Attorney Docket No.: 078430-543001 WO56. The method of any one of claims 52-55, wherein the recombinant T cell comprises an enhanced in vitro and / or in vivo proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

57. The method of any one of claims 52-56, wherein the recombinant T cell promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a reference T cell that does not comprise the recombinant cytokine receptor.

58. The method of any one of claims 52-57, further comprising introducing into the T cell at least one engineered immune receptor.

59. The method of any one of claims 52-58, wherein the at least one engineered immune receptor comprises an engineered T cell receptor (TCR) and / or an engineered chimeric antigen receptor (CAR).

60. A recombinant T cell produced by the method of any one of claims 52-59.

61. A cell culture comprising at least one recombinant T cell according to any one of claims 35-51 and 60.

62. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following:(a) a recombinant cytokine receptor according to any one of claims 1-16;(b) a recombinant nucleic acid according to any one of claims 17-29; and(c) a recombinant cell according to any one of claims 35-51 and 60.

63. The pharmaceutical composition of claim 62, wherein the composition comprises a recombinant cell according to any one of claims 35-51 and 60, a pharmaceutically acceptable carrier.

64. A method of preventing and / or treating a health condition in a subject in need thereof, comprising administering to the subject a composition comprising a pharmaceutically effective amount of one or more of the following:(a) a recombinant cytokine receptor according to any one of claims 1-16, or a signaling component thereof;(b) a recombinant nucleic acid according to any one of claims 17-29;(c) a recombinant cell according to any one of claims 35-51 and 60; andAttorney Docket No.: 078430-543001 WO(d) pharmaceutical composition according to any one of claims 62-63.

65. The method of claim 64, wherein the recombinant cells are allogeneic relative to the subject.

66. The method of claim 64, wherein the recombinant cells are autologous relative to the subject.

67. The method of any one of claims 64—66, wherein the health condition is a proliferative disorder (e.g., cancers), an infectious disease, an autoimmune disease, and / or inflammatory disease.

68. The method of claim 67, wherein the proliferative disorder is a cancer.

69. The method of claim 68, wherein the cancer cell is of a B-cell malignancy (such as a B-cell lymphomas or leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, sarcoma, neuroendocrine cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, or renal cancer.

70. The method of any one of claims 64—69, wherein the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in a decreased STAT1 signaling while substantially retains STAT3 signaling and / or STAT5 signaling in the recombinant T cell, as compared to a subject that does not comprise the recombinant cytokine receptor a signaling component thereof.

71. The method of claim 70, wherein said STAT1 signaling in the recombinant T cell is decreased by at least 10%, 15%, 20%, 25%, or 30%, as compared to a reference T cell that does not comprise the recombinant cytokine receptor or a signaling component thereof.

72. The method of any one of claims 64—71 , wherein the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an enhanced in vitroAttorney Docket No.: 078430-543001 WOand / or in vivo proliferation / expansion of the recombinant T cell, as compared to a subject that does not comprise the recombinant cytokine receptor or a signaling component thereof .

73. The method of any one of claims 64—72, wherein the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increase in circulating recombinant T cells in the blood of the subject.

74. The method of any one of claims 64—73, wherein the administered composition or recombinant cytokine receptor (or a signaling component thereof) results in an increased accumulation of the recombinant T cells in lymphoid and non-lymphoid organs.

75. The method of any one of claims 64—74, wherein the administered composition or recombinant cytokine receptor (or a signaling component thereof) promotes less effector differentiation while enhancing proliferation / expansion of the recombinant T cell, as compared to a subject that does not comprise the recombinant cytokine receptor or a signaling component thereof.

76. A kit for the prevention and / or treatment a health condition in a subject, the kit comprising one or more of the following:(a) a recombinant cytokine receptor according to any one of claims 1-16, or a signaling component thereof;(b) a recombinant nucleic acid according to any one of claims 17-29;(c) a recombinant cell according to any one of claims 35-51 and 60; and(d) pharmaceutical composition according to any one of claims 62-63.

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