WO2022183076A1

WO2022183076A1 - Enhanced immune cell therapy targeting ny-eso-1

Info

Publication number: WO2022183076A1
Application number: PCT/US2022/018034
Authority: WO
Inventors: Helle Groothaert JENSEN; Blythe D. SATHER; Rachel C. LYNN; Bijan A. BOLDAJIPOUR; Hajime Hiraragi; Shobha POTLURI; Rachel A. FUKUDA; Megan L. MURT; Spencer PARK; Queenie M. VONG; Ying Wang; Nicole Christ; Oliver Jon MENDES
Original assignee: Lyell Immunopharma, Inc.; Glaxosmithkline Intellectual Property Development Limited
Priority date: 2021-02-25
Filing date: 2022-02-25
Publication date: 2022-09-01
Also published as: TW202246511A

Abstract

The present application concerns engineered human cells T cells expressing a NY-ESO-1 tumor antigen-targeted TCR, which are enhanced by co-expression with c-jun in the vectors used to transform the cells; these cells are for use in cancer therapy. Also provided are expression constructs for making such engineered cells, in particular an expression construct comprising one or more expression cassettes for expressing: (a) a T cell receptor (TCR) that specifically binds to a peptide from a human NY-ESO-1 protein complexed with an HLA-A molecule; and (b) a human c-Jun polypeptide. The c-Jun polypeptide can be the human wild type c-Jun or a mutant human c-Jun.

Description

ENHANCED IMMUNE CELL THERAPY TARGETING NY-ESO-1

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Applications 63/153,939, filed February 25, 2021, and 63/236,789, filed August 25, 2021, the disclosures of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The electronic copy of the Sequence Listing, created on February 11, 2022, is named 026225_WO017_SL.txt and is 39,562 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Adoptive T cell therapy has been studied intensively in recent years as a potential venue for cancer treatment. In many studies, therapeutic T cells are engineered to express an antigen receptor specific for a tumor antigen. However, one challenge facing T cell therapy is the lack of persistence of T cells in vivo due to a phenomenon known as T cell exhaustion. (See, e.g., Fraietta et ak, Nat Med. (2018) 24(5):563-71; Long et ah, Nat Med. (2015) 21(6):581-90; and Eyquem et ah, Nature (2017) 543(7643): 113-7). T cell exhaustion is characterized by marked changes in metabolic function, transcriptional reprogramming, loss of effector functions (e.g., reduced cytokine secretion and cytotoxicity), increased expression of multiple surface inhibitory receptors, and apoptosis. T cell exhaustion has been attributed to constant antigen exposure, leading to continuous TCR signaling, or to tonic antigen- independent signaling through an engineered antigen receptor on T cells (see, e.g., Long, supra). Prevention or reversal of T cell exhaustion has been sought as a means to enhance T cell effectiveness, e.g., in patients with cancer or chronic infections and in T cell therapy.

See, e.g., WO 2019/118902, the disclosure of which is incorporated by reference herein in its entirety.

[0004] Thus, there remains a need for improved T cell therapy in which the engineered T cells have high as well as sustained tumor-killing potency. SUMMARY OF THE INVENTION

[0005] The present disclosure provides compositions and methods for enhancing immune cell therapy targeting NY-ES O-l -expression in cancer. In one aspect, the present disclosure provides an expression construct comprising one or more expression cassettes for expressing: a) a T cell receptor (TCR) that specifically binds to a peptide from a human NY-ESO-1 protein complexed with an HLA-A molecule; and b) a human c-Jun polypeptide.

[0006] In one aspect, the present disclosure provides a method of reducing dysfunction (e.g., exhaustion) of an engineered immune cell. In a related aspect, the present disclosure provides a method of inhibiting or reducing exhaustion of an engineered immune cell (e.g., as indicated by reduced expression of T cell exhaustion markers as further described below), and a method of increasing function or activity of an engineered immune cells (e.g., as indicated by increased antigen-induced cytokine production, cytotoxicity, and proliferation). These methods comprise introducing into the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises one or more expression constructs comprising one or more expression cassettes for expressing: a) a T cell receptor (TCR) that specifically binds to a peptide from a NY-ESO-1 protein complexed with an MHC class I molecule; and b) a human c-Jun polypeptide. In some embodiments, the immune cell is a T cell, e.g., a human T cell.

[0007] In some embodiments, the c-Jun is a wildtype human c-Jun, optionally comprising SEQ ID NO: 13 or 16, or an amino acid sequence at least 90% (e.g., at least 95, 96, 97, 98 or 99%) identical thereto. In other embodiments, the c-Jun is a mutant human c-Jun, optionally comprising an inactivating mutation in its transactivation domain or delta domain. In particular embodiments, the c-Jun comprises (i) S63A and S73A mutations or (ii) a deletion between residues 2 and 102 or between residues 30 and 50 as compared to wildtype c-Jun. [0008] In some embodiments, the NY-ESO-1 peptide is derived from a human NY-ESO-1 protein and is human NY-ESO-1157-165 (SEQ ID NO: 19), and the HLA-A molecule is HLA- A*02.

[0009] In some embodiments, the TCR comprises an a chain and a b chain, wherein the a chain comprises the CDRl-3 in SEQ ID NO:5 and the b chain comprises the CDRl-3 in SEQ ID NO:6. In other embodiments, the TCR a CDRl-3 comprise SEQ ID NOs:7-9, respectively, and the TCR b CDRl-3 comprise SEQ ID NOs: 10-12, respectively. In other embodiments, the TCR a chain comprises a variable domain comprising SEQ ID NO: 5 or an amino acid sequence at least 90% (e.g., at least 95, 96, 97, 98 or 99%) identical thereto and the TCR b chain comprises a variable domain comprising SEQ ID NO:6 or an amino acid sequence at least 90% (e.g., at least 95, 96, 97, 98 or 99%) identical thereto embodiments, the TCR a and b chains comprise SEQ ID NOs:3 and 4, respectively, or SEQ ID NOs:17 and 18, respectively, or amino acid sequences sequence at least 90% (e.g., at least 95, 96, 97, 98 or 99%) identical thereto.

[0010] In some embodiments, the expression constructs herein are viral vectors, e.g., lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.

[0011] In some embodiments, the expression construct herein comprises a tri-cistronic expression cassette for expressing c-Jun, TCR a chain, and TCR b chain. In some embodiments, the tri-cistronic expression cassette is for expressing: a) an ab T cell receptor (TCR) that specifically binds to human NY-ESO-1157-165 peptide complexed with HLA-A*02; and b) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 13 and coding sequences for SEQ ID NOs:3 and 4, optionally wherein the coding sequences are separated in frame by a sequence selected from a 2A-coding sequence and a furin cleavage consensus sequence. In some embodiments, the coding sequence for SEQ ID NO: 13 comprises SEQ ID NO:21, the coding sequence for SEQ ID NO:3 comprises SEQ ID NO:l, the coding sequence for SEQ ID NO:4 comprises SEQ ID NO:2, and/or the expression construct comprises SEQ ID NO: 14, or a nucleotide sequence at least 80% identical thereto.

[0012] In some embodiments, the expression cassette comprises a constitutive or inducible promoter, optionally an EF-la promoter, optionally wherein the expression construct is a lentiviral vector.

[0013] In another aspect, the present disclosure also provides a recombinant virus comprising the tri-cistronic expression construct disclosed herein, optionally wherein the expression construct is a lentiviral vector.

[0014] In another aspect, the present disclosure provides a method of engineering immune cells, comprising: (a) providing a starting cell population, (b) introducing the expression construct(s) or the recombinant virus disclosed herein into the starting cell population, (c) optionally selecting cells that express the TCR and the c-Jun, and (d) deriving engineered immune cells from the cells of step (b) or (c), optionally wherein the immune cells are human cells. In some embodiments, the starting cell population comprises immune cells, optionally autologous or allogeneic T cells. In other embodiments, the starting cell population comprises pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells, optionally T cells. [0015] In one aspect, the present disclosure provides a population of hui comprising the expression construct(s) disclosed herein or the recombinant virus disclosed herein, optionally wherein the human cells are immune cells. The present disclosure also provides a population of immune cells obtained by methods disclosed herein, optionally wherein the immune cells are human cells. In some embodiments, the cells are T cells, optionally CD8⁺ T cells. In some embodiments, the cells: a) express a lower level of an exhaustion marker (e.g., CD39, PD-1, TIGIT, TIM-3, or LAG-3), and/or b) express a higher level of IL-2 and/or IFN-g, as compared to corresponding cells that do not overexpress c-Jun. In some embodiments, no more than about 5%-15% of the T cells are TIGIT positive after 14 days of persistent antigen stimulation. In some embodiments, no more than about 2%-5% of the T cells are PD-1 positive after 14 days of persistent antigen stimulation. In some embodiments, no more than about 20%-45% of the T cells are CD39 positive after 14 days of persistent antigen stimulation. In some embodiments, the T cells secrete at least about 2-fold more IL-2, INF-g, and/or TNF-a at day 0 and/or day 14 of persistent antigen stimulation at a 1:1, 1:5, 1:10, or 1:20 ratio of effector (e.g., T) cells to target cells, as compared to a control population of engineered T cells that do not overexpress c-Jun. In some embodiments, the T cells proliferate at least about 2-fold more in response to antigen as compared to a control population of engineered T cells that do not overexpress c-Jun.

[0016] The present disclosure also provides pharmaceutical compositions comprising the expression constructs, viruses, or engineered cells herein, and a pharmaceutically acceptable carrier.

[0017] In one aspect, the present disclosure provides a method of killing target cells, comprising contacting the target cells with the engineered immune cells (e.g., T cells such as CD8⁺ T cells) or pharmaceutical composition herein under conditions that allow killing of the target cells by the immune cells, wherein the target cells are cancer cells expressing NY- ESO-1, optionally wherein the immune cells express a lower level of an exhaustion marker (e.g., CD39, PD-1, TIGIT, TIM-3, or LAG-3) when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression.

[0018] In another aspect, the present disclosure provides a method of treating a human patient in need thereof, comprising administering the human cells or pharmaceutical composition disclosed herein to the patient. The patient may have, e.g., NY-ESO-1- expressing cancer (e.g., metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, myxoid/round cell liposarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prosta bladder cancer).

[0019] Also provided in the present disclosure are the use of the expression constructs, viruses, or engineered cells herein for the manufacture of a medicament for treating a patient in need thereof. Further provided are expression constructs, viruses, cells, or pharmaceutical compositions for use in treating a patient in need thereof in a treatment method as described herein.

[0020] In another aspect, the present disclosure provides an ab T cell receptor (TCR) specific for NY-ESO-I157-165 peptide complexed with HLA-A*02, wherein the a chain of the TCR comprises CDRl-3 comprising SEQ ID NOs:7-9, respectively, and the b chain of the TCR comprises CDRl-3 comprising SEQ ID NOs: 10-12, respectively. In some embodiments, the a and b chains of the TCR comprise SEQ ID NOs:5 and 6, respectively; SEQ ID NOs:3 and 4, respectively; or SEQ ID NOs:17 and 18, respectively.

[0021] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESRIPTION OF THE DRAWINGS [0022] FIG. 1 is a schematic showing polycistronic c-Jun NY-ESO-1 TCR constructs used in the experiments described below. Two different promoters (EF-la promoter and synthetic MND promoter) were evaluated for their ability to drive high levels of c-Jun expression. Both wildtype (“WT”) and mutated versions (S63A/S73A; “AA”) of human c- Jun were evaluated to assess the potential safety risk for oncogenic transformation. P2A: P2A self-cleaving peptide. Furin: furin cleavage site. NY-ESO-1 TCR: a TCR specific for a NY-ESO-1 peptide presented by a MHC class I molecule.

[0023] FIGs. 2A and 2B are graphs showing c-Jun expression from constructs shown in FIG. 1. FIG. 2A shows intracellular (“IC”) c-Jun expression levels as measured by flow cytometry in CD8⁺ T cell transduced with an MND promoter or an EF-la promoter constructs shown in FIG 1. FIG. 2B shows c-Jun expression levels as measured by using Western blot analysis. MFI: mean fluorescence intensity. The MND promoter is derived from the myeloproliferative sarcoma virus enhancer with the negative control region deleted and the dl587rev primer-binding site substituted. [0024] FIG. 3 is a panel of flow cytometry dot plots showing that EF-lc constructs displayed more stable expression of c-Jun after stimulation as compared to MND promoter constructs. Control or transduced cells were unstimulated (top panels), stimulated with CD3/CD8 TransAct™ beads (Miltenyi Biotec) (middle panels), or stimulated with NY- ESO-l⁺ target cells (bottom panels).

[0025] FIG. 4 is a diagram showing an overview of the IncuCyte® Immune Cell Killing and the MSD (Meso Scale Discovery) cytokine functional assays.

[0026] FIGs. 5A and 5B are panels of graphs showing IncuCyte® Immune Cell Killing data for T cells transduced with mock, NY-ESO-1, c-JunWT_NY-ESO-l, and c-JunAA_NY- ESO-1 constructs with either the EF-la promoter (FIG. 5A) or the MND promoter (FIG. 5B). The killing efficiency was determined by tracking the kinetics of A375 (high Ag), H1703 (medium Ag), and TCCSup (low Ag) cancer cell clearance at an E:T ratio of 1: 10 or 1:20 (A375 and H1703) and 1:40 or 1:80 (TCCSup) over a 162 hr time course using the IncuCyte® S3 Live-Cell Analysis System. Killing data are representative of five donors. Lower E:T ratios showed similar findings and are not shown on the graphs. Ag: antigen.

E:T: ratio of effector cells to target cells. The target cells were stably transduced to express NucLight™ Red (NLR) to allow quantification using IncuCyte® or similar instrument.

[0027] FIGs. 6A and 6B are bar graphs depicting IFN-g (FIG. 6A) and IL-2 (FIG. 6B) levels in supernatants after 24 hours of co-culture of A375 NLR target cells with T cells transduced with mock, NY-ESO-1 TCR, c-Jun WT NY-ESO-l TCR, and c-JunAA_NY- ESO-1 TCR constructs either with the EFla promoter or the MND promoter. The E:T ratio was 1:1.

[0028] FIGs. 7A-7C are bar graphs showing the fold change in cell number (proliferation) of stimulated T cells transduced with NY-ESO-1 TCR, c-JunWT_NY-ESO-l TCR, and c- JunAA_NY-ESO-l constructs either with the EF-la promoter or the MND promoter, from three healthy human donors (FIGs. 7A, 7B, and 7C, respectively).

[0029] FIG. 8 is a schematic of the serial re-stimulation assay that models persistent antigen stimulation. Co-culture plates were coated with poly-L-omithine one day prior to each co-culture setup; irradiated parental (pA375) target cells were plated and rested for about 4 hours prior to addition of T cells; 10 IU/mL (1,000 pg/mL) IL-2 was added at each co-culture reset, but not to IncuCyte® experiments; co-culture was reset at an E:T ratio of 1: 1 based on T cell count and %TCRvpi3.1⁺ on live T cells on each harvest day. [0030] FIGs. 9A-9C are graphs showing c-Jun NY-ESO-1 TCR T cells human donors (FIGs. 9A, 9B, and 9C, respectively) have similar or increased proliferation in response to antigen when compared to control.

[0031] FIGs. 10A-10E are graphs showing that c-Jun overexpression reduces the percentage of NY-ESO-1 TCR⁺ T cells expressing exhaustion markers after serial re stimulation. For FIGs. 10A-10C, the T cells were from Donor 3035680. FIG. 10A: % TIGIT in CD8⁺TCR⁺ cells; FIG. 10B: % PD-1 in CD8⁺TCR⁺ cells; FIG. IOC: % CD39 in CD8⁺TCR⁺ cells. Data are representative and were similar across donors for PD-1 and TIGIT. Reduction in CD39 expression was not as drastic in two of the donors or in CD4⁺ cells. TCR⁺ refers to NY-ESO-1 TCR⁺. For FIGs. 10D and 10E, a multi-marker analysis using Boolean gating was performed based on the % TIGIT⁺, % PD-1⁺, and % CD39⁺ populations obtained for each T cell product from three donors on Day 14 of the serial re stimulation assay. The gray bar (c-Jun) in each statistical analysis comparing the % expression of the indicated markers on EFla_NY-ESO-l TCR and EFla c-JunWT NY- ESO-1 TCR T cell products was performed by a paired t-test and the results were defined as significant at p<0.05.

[0032] FIGs. 11A-11L show results from evaluation of NY-ESO-1 TCR T cell cytotoxicity. The killing efficiency of mock, EFla_NY-ESO-l, EFla_c-JunWT_NY-ESO-l TCR, or EFla_c-JunAA_NY-ESO-l T cells from a representative donor (Donor 3035610) at Day 0 and Day 14 of the serial re-stimulation assay was determined by tracking the kinetics of A375 and H1703 NLR cancer cell clearance at an E:T ratio of 1:1 (FIGs. 11A and 11B), 1:5 (FIGs. 11D and HE), 1:10 (FIGs. 11G and 11H), or 1:20 (FIGs. 11J and 11K) over a 162 hr time course using the IncuCyte™ S3 Live-Cell Analysis System. The figures show the results for A375 target cells from a representative donor (Donor 3035610). Similar data were seen across donors for H1703 target cells (data not shown). FIGs. 11C, 11F, 111, and 11L show the fold change of area under the curve (AUC) at Day 0 and Day 14 of EFla c- JunWT_NY-ESO-l TCR compared to EFla_NY-ESO-l TCR (the lower the value, the more killing).

[0033] FIGs. 12A-12F are graphs showing IFN-g (FIGs. 12A and 12D), IL-2 (FIGs.l2B and 12E) and TNF-a (FIGs.l2C and 12F) secretion by mock, EFla_NY-ESO-l TCR,

EF 1 a_c-JunWT_NY -ESO- 1 TCR, or EFla_c-JunAA_NY-ESO-l T cells from Day 0 (FIGs. 12A-C) and Day 14 (FIGs. 12D-F) of the serial re-stimulation assay determined after 24 hr co-culture with A375 NLR cancer cells at an E:T ratio of 1:1, 1:5, 1:10 or 1:20. The figures show the results for A375 target cells from Donor 3035680. Similar data w donors for H1703 target cells (data not shown).

[0034] FIG. 13 is a schematic of the T2 dose-response assay.

[0035] FIGs. 14A-14C are graphs showing that c-Jun overexpression in transduced T cells from three donors (FIGs. 14A, 14B, and 14C, respectively) increases the IL-2 production after co-culture with T2 cells pulsed with NY-ESO-1157-165, whereas no increase was observed after co-culture with T2 cells pulsed with HPV16 E786-93 (irrelevant peptide).

T cells tested were mock or transduced with EFla_NY-ESO-l TCR, EFla c-JunWT NY- ESO-1 TCR, or EFla_c-JunAA_NY-ESO-l construct.

[0036] FIGs. 15A-15C are graphs showing that c-Jun overexpression in transduced T cells from three donors (FIGs. 15A, 15B, and 15C, respectively) increases the IFN-g production after co-culture with T2 cells pulsed withNY-ESO-li57-i65, whereas no increase was observed after co-culture with T2 cells pulsed with HPV16 E786-93. T cells tested were mock or transduced with EFla_NY-ESO-l TCR, EFla_c-JunWT_NY-ESO-l TCR or EFla_c-JunAA_NY-ESO-l construct.

[0037] FIGs. 16A-16C are graphs showing the results of an antigen-independent growth assay demonstrating that c-Jun overexpression did not drive uncontrolled cell growth with or without cytokine support. Data are shown for Donor 3035610, which was representative (data consistent across donors).

[0038] FIGs. 17A-17F are graphs showing the impact of c-Jun overexpression in transduced T cells from Donor 1 (3035610). The cells were stained for exhaustion and differentiation markers to determine the percentages of CD4⁺ and CD8⁺ populations (FIG. 17A); the percentage of TCR⁺ cells (FIG. 17B); the cell exhaustion profile of CD8⁺ T cells (FIG. 17C); the cell exhaustion profiles of CD4⁺ T cells (FIG. 17D); and the relative ratios of naive T cells (“Tnaive”; CD45RA⁺CCR7⁺CD95 ), T memory stem cells (“Tscm”; CD45RA⁺CCR7⁺CD95⁺), central memory T cells (“Tcm”; CD45RA CCR7⁺), effector memory T cells (“Tern”; CD45RA CCR7 ). and effector T cells (“Teff”; CD45RA CCR7 ) among CD8⁺ (FIG. 17E) or CD4⁺ (FIG. 17F) T cells.

[0039] FIGs. 18A-18B are graphs showing the cytotoxicity of transduced T cells from Donor 1 (3035610; FIG. 18A) and Donor 3 (3035702; FIG. 18B). The T cells were co cultured with A375-NucLight cells at a 1:1 or 1:5 effector-to-target ratio and cytotoxicity was assessed in an IncuCyte®-based assay for 120 hours. [0040] FIGs. 19A-19D are graphs showing the characterization of trans Donor 1 and Donor 3. FIG. 19A shows the percentages of viable, apoptotic, and dead cell subpopulations within thawed T cells that were untransduced, expressing NY-ESO-1 TCR, or expressing c-JunWT-NY-ESO-1 TCR, as determined by flow cytometry. FIG. 19B shows the total number of TCR⁺ T cells per mL of blood as measured by flow cytometry at 24 hours post-T cell infusion. FIGs. 19C and 19D show the total number of TCR⁺ T cells per mL of blood as measured by flow cytometry at different time-points of the study.

[0041] FIGs. 20A-20D are graphs showing tumor growths in NSG mice (n = 10 or 15 per group) implanted subcutaneously with 5xl0⁶ A-375 (human melanoma) cells. At day 7 post implant, the animals were treated with EFla_NY-ESO-l, MND_NY-ESO-l, EFla c- JunWT NY -ESO- 1 , or MND_c-JunWT_NY-ESO-l TCR T cells at a total dose of 5xl0⁶ TCR⁺ T cells. Untransduced T cells were used as control. FIGs. 20A and 20B show fold change in tumor volume. Significant differences in tumor growth were noted at D21 for EF 1 a_c-JunWT_NY -ESO-1 versus EFla_NY-ESO-l groups (p = 0.033). FIGs. 20C and 20D show the estimated time (days) to reach target tumor volume (1000 mm³) for each of the groups and the ratio of time to target volume analyses, which show significant differences between EFla_c-JunWT_NY-ESO-l versus EFla_NY-ESO-l treatment groups.

[0042] FIGs. 21A-21C are graphs showing a histopathological quantitative analysis of NY-ESO-1 antigen expression and T cell infiltration in A-375 subcutaneous tumors treated with EFla_NY-ESO-l) or EFla_c-JunWT_NY-ESO-l TCR+ T cells from two donors. Untransduced T cells were used as control. FIG. 21A shows a histology H-score quantification of NY-ESO-1 across the intratumoral (tumor and tumor stroma) areas in the treatment groups. FIGs. 21B and 21C show quantification of CD3⁺ and CD3⁺TCR⁺ T cell infiltration across the intratumoral (tumor and tumor stroma) areas in the treatment groups. [0043] FIG. 22 is a panel of graphs showing single cell RNA sequencing data for exhaustion markers (PDCD1, CTLA4, and TOX) on tumor-infiltrating lymphocytes isolated from a CDX mouse model comparing NY-ESO-1 TCR and c-JunWT_NY-ESO-l TCR. [0044] FIGS. 23A-23B are graphs showing the total number of TCR⁺ T cells per mL of blood as measured by flow cytometry at different time-points of the study for two T cell doses. FIG. 23A: T cells from for Donor 1. FIG. 23B: T cells from Donor 3.

[0045] FIG. 24 is pair of graphs showing the tumor volumes over time of a CDX mouse model with subcutaneous injection of A-375 cells, comparing two T cell doses and untransduced cells. The T cells were from two donors. NTD: non-transduced (untransduced). [0046] FIG.25 is pair of graphs showing Kaplan Meier survival curves for the time to reach the arbitrary tumor volume of 1000 mm³ in a CDX mouse model with subcutaneous injection of A-375 cells, comparing two T cell doses and untransduced cells. The T cells were from two donors. [0047] FIG.26 is a panel of graphs showing the level of serum IFN-γ in a CDX mouse model with subcutaneous injection of A-375 cells, comparing two T cell doses and untransduced cells. The T cells were from two donors. [0048] FIG.27 is a graph showing IFN-γ secretion by tumor-infiltrating lymphocytes after they were isolated from mice and cultured with A-375 tumor cells. The percentage of TCR⁺ T cells is indicated in brackets behind the identification number of each mouse. [0049] FIG.28 is graph showing the tumor volumes over time of a CDX mouse model with intravenous injection of A-375 cells, comparing two T cell doses and untransduced cells. The T cells were from two donors. [0050] FIG.29 is a graph showing the level of serum IFN-γ in a CDX mouse model with intravenous injection of A-375 cells, comparing two T cell doses and untransduced cells. The T cells were from two donors. [0051] FIGs.30A-30D are graphs showing FOXP3 and CD25 expression in NY-ESO-1 TCR T cell products after stimulation. FOXP3 and CD25 expression was determined on mock T cells (untransduced) and NY-ESO-1 TCR T cell products after 7 days of stimulation with A-375 target cells with and without TGF-β. Shown are the CD25^highFOXP3⁺ quadrant gates for donors 3048935 (FIG.30A), 3048947 (FIG.30B), and 3048957 (FIG.30C) of TCRvβ13.1⁺ CD4⁺ and CD8⁺ T cell subsets from the EF1α_NY-ESO-1 TCR and EF1α_c- JunWT_NY-ESO-1 TCR T cell products or TCRvβ13.1- CD4⁺ and CD8⁺ T cell subsets from the mock T cell samples. FIG.30D shows the % CD25^highFOXP3⁺ values (i.e., the Q10 quadrant gates shown in FIGs.30A-30C of the TCRvβ13.1⁺ CD4⁺ and CD8⁺ T cell subsets from the EF1α_NY-ESO-1 TCR and EF1α_c-JunWT_NY-ESO-1 TCR T cell products, or TCRvβ13.1- CD4⁺ and CD8⁺ T cell subsets from the mock T cell samples). The graphs show mean ± SD from 3 donors. Statistical analysis comparing the EF1α_NY-ESO-1 TCR and EF1α_c-JunWT_NY-ESO-1 TCR T cell products was performed using a paired t-test and only p values ^0.05 are shown (*p= 0.0174, CD8⁺, plus TGF-β). DETAILED DESCRIPTION OF THE INVENTION [0052] The present disclosure provides engineered human cells (e.g., immune cells such as T cells) comprising expression constructs for co-expressing a recombinant T cell receptor (TCR) and a c-Jun protein. The recombinant TCR binds aNY-ESO-1 pept with an HLA-A molecule. The NY-ESO-1 protein is expressed by a range of tumors (Chen et al., PNAS (1997) 94: 1914-8). Peptides derived from this protein are presented by Class I HLA molecules of the tumor cells on the tumor cells’ surface. Thus, the NY-ESO-1 peptide/HLA complex provides a cancer marker that therapeutic T cells can target.

[0053] Overexpression of c-Jun in these therapeutic (e.g., T) cells helps sustain the active state of the cells by, e.g., alleviating, reducing, or preventing T cell dysfunction (e.g., T cell exhaustion). The present engineered immune cells such as T cells exhibit sustained, potent cytotoxicity against NY-ESO-1 -bearing tumor cells. As compared to T cells that do not overexpress c-Jun (e.g., through an exogenously introduced c-Jun gene sequence), the present engineered T cells display fewer signs of T cell exhaustion. The engineered cells may have one or more of the following characteristics: (i) they do not have increased expression of exhaustion markers PD-1, TIGIT, and/or CD39 over time, (ii)they have reduced rates of apoptosis, (iii) they maintain an active biological state including secretion of cytokines including IL-2 and INF-g, (iv) they have enhanced cytotoxicity; (v) they display increased recognition of tumor targets with low surface antigen; (vi) they have enhanced proliferation in response to antigen; and (vii) maintain survival and functionality after repeated antigen stimulation.

I. Immune Cell Sources

[0054] The source of the engineered immune cells of the present disclosure may be a patient to be treated (i.e., autologous cells) or from a donor who is not the patient to be treated (e.g., allogeneic cells). In some embodiments, the engineered immune cells are engineered T cells. The engineered T cells herein may be CD4⁺CD8 (i.e., CD4 single positive) T cells, CD4 CD8⁺ (i.e., CD8 single positive) T cells, or CD4⁺CD8⁺ (double positive) T cells. Functionally, the T cells may be cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells, or a mixture thereof. The T cells to be engineered may be autologous or allogeneic.

[0055] Primary immune cells, including primary T cells, can be obtained from a number of tissue sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and/or tumor tissue. Leukocytes, including PBMCs, may be isolated from other blood cells by well-known techniques, e.g., FICOLL™ separation and leukapheresis. Leukapheresis products typically contain lymphocytes (including T and B cells), monocytes, granulocytes, and other nucleated white blood cells. T c isolated from other leukocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3⁺, CD25⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, GITR⁺, and CD45RO⁺ T cells, can be further isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, T cells may be isolated by incubation with any of a variety of commercially available antibody-conjugated beads, such as Dynabeads®, CELLection™, DETACHaBEAD™ (Thermo Fisher) or MACS® cell separation products (Miltenyi Biotec), for a time period sufficient for positive selection of the desired T cells or negative selection for removal of unwanted cells.

[0056] In some instances, autologous T cells are obtained from a cancer patient directly following cancer treatment. It has been observed that following certain cancer treatments, in particular those that impair the immune system, the quality of T cells collected shortly after treatment may have an improved ability to expand ex vivo and/or to engraft after being engineered ex vivo.

[0057] Whether prior to or after genetic modification, T cells can be activated and expanded generally using methods as described, for example, in U.S. Pats. 5,858,358; 5,883,223; 6,352,694; 6,534,055; 6,797,514; 6,867,041; 6,692,964; 6,887,466; 6,905,680; 6,905,681; 6,905,874; 7,067,318; 7,144,575; 7,172,869; 7,175,843; 7,232,566; 7,572,631; and 10,786,533. Generally, T cells may be expanded in vitro or ex vivo by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatins) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule may be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody may be employed.

[0058] The cell culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any ot designed to activate the cells. In some embodiments, the culture conditions include addition ofIL-2, IL-7 and/or IL-15.

[0059] In some embodiments, the cells to be engineered may be pluripotent or multipotent cells that are differentiated into mature T cells after engineering. These non-T cells may be allogeneic and may be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem or progenitor cells. For ease of description, pluripotent and multipotent cells are collectively called “progenitor cells” herein.

[0060] In certain embodiments, where allogeneic cells are used, they are engineered to reduce graft-versus-host rejection (e.g., by knocking out the endogenous B2M and/or TRAC genes).

II. Engineering of Immune or Progenitor cells

[0061] As used herein, the term “cell engineering” or “cell modification” (including derivatives thereol) refers to the targeted modification of a cell, e.g., an immune cell disclosed herein. In some aspects, the cell engineering comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., aNY-ESO-1 peptide complexed with HLA-A), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell function (e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression compared to a corresponding cell that has not been modified), or any combination thereof. As further described elsewhere in the present disclosure, in some aspects, a cell can be engineered or modified with a transcription activator (e.g., CRISPR/Cas system-based transcription activator), wherein the transcription activator is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun).

[0062] In some aspects, a cell described herein has been modified with a transcriptional activator, which is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As used herein, the term “transcriptional activator” refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to enhancers or promoter-proximal elements of a nucleic acid sequence and thereby, inducing its transcription). Non-limiting examples of such transcriptional activators that can be used with the present disclosure include: Transcription Activator-like Effector (TALE)-based transcriptional activator, zinc finger protein (ZFP)-based transcriptional activator, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-assoc (Cas) system-based transcriptional activator, or a combination thereof. See, e.g., Kabadi et al., Methods (2014) 69(2): 188-97, which is incorporated herein by reference in its entirety. [0063] In some aspects, a cell described herein has been modified with a CRISPR/Cas- sy stem-based transcriptional activator, such as CRISPR activation (CRISPRa). See, e.g., Nissim et al., Molecular Cell (2014) 54(4):698-710; Perez-Pinera et al., Nat. Methods (2013) 10(10):973-76; Maeder et al., Nat. Methods (2013) 10(10):977-79; Cheng et al., Cell Res. (2013) 23(10): 1163-71 ; Farzadfard et al ., ACS Synth. Biol. (2013) 2(10):604-13; all of which are incorporated herein by reference in their entirety. CRISPRa is a type of CRISPR tool that comprises the use of modified Cas proteins that lacks endonuclease activity but retains the ability to bind to its guide RNA and the target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins which can be used with the present disclosure are known in the art. See, e.g., Pandelakis et al., Cell Systems (2020) 10(1): 1-14, which is incorporated herein by reference in its entirety. In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as “dCas9”). In some aspects, the modified Cas protein comprises a modified Cas 12a protein. In some aspects, a modified Cas protein that is useful for the present disclosure is bound to a guide polynucleotide (e.g., small guide RNA) (“modified Cas-guide complex”), wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In certain aspects, the guide polynucleotide comprises a recognition sequence that is complementary to the promoter region of an endogenous nucleic acid sequence encoding a protein of interest. In some aspects, one or more transcriptional activators are attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when the modified Cas-guide complex is introduced into a cell, the one or more transcription activators can bind to a regulatory element (e.g., a promoter region) of an endogenous gene and thereby induce and/or increase the expression of the encoded protein (e.g., c-Jun). Illustrative examples of common general activators that can be used include the omega subunit of RNAP, VP16, VP64 and p65 (see, e.g., Kabadi and Gersbach , Methods (2014) 69(2): 188-97). [0064] In some aspects, one or more transcriptional repressors (e.g., Kruppel-associated box domain (KRAB)) can be attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when introduced into a cell, the one or more transcriptional repressors can repress or reduce the transcription of a gene, e.g., such as those that can interfere with the expression of c-Jun (e.g., Bach2). See, e.g., US20200030379A1 and Yang et al., J TranslMed. (2021) 19:459, each of ^. incorporated herein by reference in its entirety. In some aspects, a modified Cas protein useful for the present disclosure can be attached to both one or more transcriptional activators and one or more transcriptional repressors.

[0065] Not to be bound by any one theory, in some aspects, the use of such modified Cas proteins can allow for the conditional transcription and expression of a gene of interest. For example, in some aspects, a cell (e.g., T cells) is modified to comprise a recombinant antigen receptor (e.g., an anti-NY-ESO-l/HLA-A TCR), which is linked to a protease (e.g., tobacco etch virus (TEV)) and a single guide RNA (sgRNA) targeting the promoter region of c-Jun.

In some aspects, the cell is modified to further comprise a linker for activation of T cells (LAT), complexed to the modified Cas protein attached to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., TEV-cleavable linker). Upon activation of the antigen receptor, the modified Cas protein is released for nuclear localization and conditionally and reversibly induces the expression of c-Jun. See, e.g., Yang et al., J Immunother Cancer (2021) 9(Suppl2):A164, which is herein incorporated by reference in its entirety.

[0066] As will be apparent to those skilled in the art, in some aspects, a cell described herein has been modified using a combination of multiple approaches. For instance, in some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., an antiNY-ESO-1 TCR and a truncated EGFR (EGFRt)) and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun). In some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding a first protein (e.g., an anti-NY-ESO-1 TCR) and (ii) an exogenous nucleotide sequence encoding a second protein (e.g., a c-Jun protein). In some aspects, the modified cell can further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first, second, and third proteins can be part of a single polycistronic vector.

[0067] Unless indicated otherwise, the one or more exogenous nucleotide sequences and/or transcriptional activators can be introduced into a cell using any suitable methods known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to a cell include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof. [0068] In some aspects, a cell has been modified with a transcriptional activator (e.g., CRISPR/Cas-system-based transcription activator, e.g., CRISPRa), such that the expression of the endogenous c-Jun protein is increased compared to a corresponding cell that has not been modified with the transcriptional activator. [0069] While certain disclosures provided herein generally relate to modifying an immune cell to comprise an exogenous nucleotide sequence encoding a c-Jun protein (wild-type c-Jun or a variant thereof), it will be apparent to those skilled in the art that other suitable methods can be used to induce and/or increase c-Jun protein expression (either wild-type or a variant thereof) in a cell. For instance, as described herein, in some aspects, the endogenous c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa). Unless indicated otherwise, disclosures provided herein using exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa). [0070] The immune cells (e.g., T cells) or progenitor cells herein may be engineered to express an exogenous (i.e., recombinant) TCR and overexpress c-Jun (e.g., a human c-Jun). The recombinant TCR may bind specifically to a ligand on a tumor cell (e.g., a tumor antigen peptide complexed with HLA). As used herein, a receptor (e.g., TCR) is said to specifically bind to a ligand (e.g., an antigen peptide/HLA complex) when the binding has a KD less than or equal to l μM and/or has an off-rate (k_off) of 1x10^-3 S^-1 or slower, as measured by surface plasmon resonance (using, e.g., a Biacore™ or Octet™ system). A. Recombinant TCR [0071] In some embodiments, the recombinant TCR expressed by the engineered immune cells (e.g., T cells) is an αβ TCR, i.e., a heterodimeric dimer comprising a TCR α chain and a TCR β chain. In particular embodiments, the recombinant TCR binds a human NY-ESO-1 peptide presented by (i.e., complexed with) with an MHC class I molecule, such as an HLA- A molecule. By “recombinant,” it is meant that the TCR is not endogenously expressed by the immune cells, but is expressed from exogenous nucleotide sequences (e.g., expression construct(s)) that have been introduced to the immune cells. [0072] In certain embodiments, the human NY-ESO-1 peptide is NY-ESO-1_157-165, having the sequence of SLLMWITQC (SEQ ID NO:19). The NY-ESO-1157-165 peptide is derived from the NY-ESO-1 protein, which is expressed by a range of tumors (Chen et al., PNAS (1997) 94:1914-8). The HLA Class I molecules of these cancerous cells present peptides from this protein, including NY-ESO-1_157-165 peptide. Therefore, this peptide complexed with an HLA class I molecule provides a cancer marker that therapeutic T c through their recombinant TCR.

[0073] In some embodiments, the NY-ESO-1 peptide is complexed with HLA-A*02. In further embodiments, the HLA-A molecule may be any one of HLA-A*02:01-555, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA- A*02:06, HLA-A* 02: 07, HLA-A*02:08, HLA-A*02:09, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:15, HLA-A*02:16, HLA- A*02:17, HLA-A* 02: 18, HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. In some embodiments, the recombinant TCR specifically recognizes NY- ESO-1157-165 presented by (i.e., complexed with) HLA-A*02:01. See, e.g., WO 2005/113595. [0074] In some embodiments, the present TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex comprises the following TCRa sequence (with or without the signal peptide (in box)), or an amino acid sequence at least 85, 90, 95, 96, 97, 98, or 99% identical thereto; and/or the following TCR b sequence (with or without the signal peptide (in box)), or an amino acid sequence at least 85, 90, 95, 96, 97, 98, or 99% identical thereto:

In the above sequences, the variable domains (not counting the signal peptides, which are cleaved after processing) are italicized (SEQ ID NOs:5 and 6 for a and b, respectively), and the CDRs are underlined (SEQ ID NOs:7-9 and SEQ ID NOs: 10-12 for a and b, respectively).

[0075] The boundaries of the variable domains and CDRs may vary based on different TCR structure analysis systems. The present disclosure encompasses TCRs comprising the variable domains or the six CDRs, as defined by any one of the systems, in chains set forth above.

[0076] In particular embodiments, the present recombinant TCR is a heterodimer of an a chain and a b chain comprising SEQ ID NO:3, without the signal peptide (amino acids 1-19), and SEQ ID NO:4, without the signal peptide (amino acids 1-22), respectively. In certain embodiments, the present recombinant TCR is a heterodimer of an a chain and a b chain consisting of SEQ ID NO:3, without the signal peptide (amino acids 1-19), and SEQ ID NO:4, without the signal peptide (SEQ ID NO: 1-22), respectively.

[0077] In certain embodiments, the TCR a sequence comprises the variable domain amino acid sequence provided in SEQ ID NO:5 and the TCR b sequence comprises the variable domain amino acid sequence provided in SEQ ID NO:6. Illustrative TCR a and b constant domain sequences are identified herein and other useful constant domain sequences may be identified for use with the recombinant TCR a/b variable domains, for example at IMGT database (Lefranc et al., Nucleic Acids Res. (2015) 43(Database issue):D413-22. Epub 2014 Nov 5).

[0078] The full-length TCR a chain polypeptide, including the signal peptide, may be encoded by, for example, SEQ ID NO: 1, or a degenerate variant or codon-optimized version thereof. The full-length TCR b chain polypeptide, including the signal peptide, may be encoded by, for example, SEQ ID NO:2, or a degenerate variant or codon-optimized version thereof.

[0079] In some embodiments, the variable domain of the TCR a chain comprises SEQ ID NO:5, or an amino acid sequence at least 90, 95, 96, 97, 98, or 99% thereto; and/or the variable domain of the TCR b chain comprises SEQ ID NO:6, or an amino acid sequence at least 90, 95, 96, 97, 98, or 99% thereto.

[0080] In some embodiments, the present TCR comprises TCR a CDRl-3 comprising SEQ ID NOs:7-9, respectively and TCR b CDRl-3 comprising SEQ ID NOs: 10-12, respectively.

[0081] The present recombinant TCR can form a TCR-CD3 complex by recruiting TCR- associated signaling molecules include CD3ys, CD35s, and zz (also known as CD3z or CD3zz) to help mediate T cell activation. B. c-Jun

[0082] In some embodiments, the c-Jun is a human c-Jun, such as wildtype human c-Jun

(c-Jun WT) having the following sequence (available at GenBank under accession number

AAA59197.1 or at UniProtKB under accession number P05412.2):

See also Hattori et al., PNAS (1988) 85:9148-52. Alternatively, the c-Jun is a mutant human c-Jun so long as the mutant c-Jun does not impact the mutant’s ability to rescue dysfunctional (exhausted) T cells. In some embodiments, a mutant c-Jun comprises at least 70% (e.g., at least 75, 80, 85, 90, 95, or 99%) sequence identity with the C-terminal amino acid residues (e.g., C-terminal 50, 75, 100, 150, 200, or 250 or more residues), the C-terminal portion (e.g., quarter, third, or hall) or C-terminal domains (e.g., epsilon, bZIP, and amino acids C-terminal thereol) of a wildtype c-Jun. In some embodiments, the N-terminal amino acid residues (e.g., N-terminal 50, 75, 100, or 150 or more), the N-terminal portion (e.g., quarter, third, or hall) or N-terminal domains (e.g., delta, transactivation domain, and amino acids N-terminal thereol) of a wildtype c-Jun are deleted, mutated, or otherwise inactivated.

[0083] In some embodiments, the c-Jun comprises an inactivating mutation (e.g., substitutions, deletions, or insertions) in its transactivation domain and/or its delta domain. In some embodiments, the c-Jun comprises one or both of S63A and S73A mutations (the positions are boxed above). In some embodiments, the c-Jun has a deletion between residues 2 and 102 or between residues 30 and 50 as compared to wildtype human c-Jun.

[0084] Due to introduction of an exogenously introduced c-Jun coding sequence, the engineered T cells overexpress, i.e., express a higher level (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least 2-, 3-, 4-, 5-, or 10-fold more) of, c-Jun than T cells without such a sequence. In certain embodiments, the engineered T cells express at least about 2-100 fold more, about 5-50 fold more, about 5-40 fold more, about 5-30 fold more, about 5-20 fold more, about 8-20 fold more, or about 10-20 fold more c-Jun than T cells without such a sequence.

[0085] In some embodiments, the immune cells herein are engineered to overexpress c- Jun through activation of the endogenous c-Jun gene in the cells, as described above. C. Nucleic Acids

[0086] The TCR and the c-Jun may be introduced to the T cells or progenitor cells through one or more nucleic acid molecules (e.g., DNA or RNA such as mRNA). In some embodiments, the nucleic acid molecules may be placed on one or more DNA or RNA vectors for introduction into the host cells.

[0087] The nucleic acid molecules (e.g., DNA or RNA vectors containing them) may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in- water emulsions, micelles, mixed micelles, and liposomes). Alternatively, the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules. Examples of viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus. In certain embodiments, the recombinant virus is pseudotyped with a heterologous envelope protein. In one embodiment, the recombinant virus is a lentivirus pseudotyped with an envelope glycoprotein derived from vesicular stomatitis virus (VSV), measles virus, or another virus (see e.g., Cronin et al., Curr Gene Ther. (2005) 5(4):387-98; Gutierrez-Guerrero et al., Viruses (2020) 12(9): 1016).

[0088] In some embodiments, the coding sequences for the TCR polypeptide chains and the c-Jun may be placed on separate expression constructs. In some embodiments, the coding sequences for the two polypeptide chains of the ab TCR and the c-Jun may be placed on a single expression construct. The three coding sequences may be placed into one or more expression cassettes on the construct, each cassette being its own transcription unit (e.g., with its own promoter and polyadenylation site and other transcription control elements). In particular embodiments, the three coding sequences may be placed into a single expression cassette (e.g., a tri-cistronic expression cassette), with the three coding sequences being transcribed under a common promoter. In a polycistronic arrangement, the coding sequences are in-frame and separated from each other by the coding sequence of a self-cleaving peptide (e.g., a 2A self-cleaving peptide such as a T2A, P2A, E2A, or F2A peptide) and/or a consensus recognition sequence for a Furin protease (see, e.g., Limstra et al., J Virol. (1999) 73(8):6299-6306 and Thomas, G, Nat Rev Mol Cell Biol. (2002) 3(10):753-66).

Alternatively, the coding sequences may be separated from each other by a ribosomal internal entry site (IRES). Thus, the polycistronic (e.g., tri-cistronic) expression cassette is transcribed into a single RNA but ultimately the single RNA is processed a separate polypeptides.

[0089] In particular embodiments of a tri-cistronic expression cassette, the coding sequence for c-Jun is separated from the coding sequence of the TCR a chain by a 2A- encoding sequence; the coding sequence of the TCR a chain is separated from the coding sequence of the TCR b chain by a coding sequence for a furin cleavage consensus sequence and a 2A-coding sequence. See, e.g., FIG. 1 and SEQ ID NO: 14. In some embodiments, the c-Jun coding sequence precedes the TCR coding sequences in the tri-cistronic expression cassette. In some embodiments, unlike the construct structure shown in FIG. 1, the TCR b chain coding sequence precedes the TCR a chain coding sequence.

[0090] The expression cassettes (polycistronic or monocistronic) may contain a promoter that is constitutively active in mammalian (e.g., human or human T) cells. Such promoters include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor- la (EF-la) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Core or minimal promoters derived from the aforementioned promoters also are contemplated. Alternatively, the expression cassettes may comprise an inducible promoter system. Exemplary inducible promoter systems include, without limitation, hormone-regulated elements, synthetic ligand- regulated elements, ionizing radiation-regulated elements, tetracycline (Tet) systems (e.g., “Tet-Off” and “Tet-On” systems), andNFAT systems (see, e.g., Kallunki et ak, Cells (2019) 8(8):796; Uchibori et al. ,Mol Ther Oncolytics. (2018) 12:16-25). In some embodiments, the expression cassette contains an elongation factor-la (EF-la) promoter.

[0091] In some embodiments, the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and/or translation of the coding sequences. For example, a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3’ untranslated region of the expression cassette.

[0092] In the expression cassettes, the transcription/translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts. [0093] In certain embodiments, the present disclosure provides a single- (e.g., a lentiviral vector) comprising a tri-cistronic expression cassette, comprising a mammalian promoter, a c-Jun coding sequence, coding sequences for the two TCR chains (a/b), and a polyadenylation signal sequence. The coding sequences are linked by one or more nucleotide linkers selected from a coding sequence for a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A, or functional equivalents thereol) and a furin cleavage consensus sequence. By way of example, FIG. 1 illustrates such an expression cassette, where the promoter is an EF- la promoter.

[0094] In particular embodiments, the expression cassette encodes a c-Jun comprising SEQ ID NO: 13 or a functional analog thereof, and a TCR comprising two polypeptide chains comprising SEQ ID NOs:3 and 4 (or variants thereol), respectively. The construct may be a recombinant lentiviral vector and may further comprise a central polypurine tract (cPPT) upstream of the EF- la promoter, and an SV40 polyadenylation signal, or other sequences for efficient transduction and expression in mammalian cells.

[0095] The coding sequences in the expression cassettes may be codon-optimized for optimal expression levels in a host cell of interest (e.g., human cells).

[0096] The nucleic acid molecules encoding the TCR and the c-Jun may be integrated into the genome of the engineered cells, or remain episomal. The integration may be targeted integration occurring through gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases, and meganucleases).

[0097] The engineered cells can be enriched for by positive selection techniques. For example, the cells can be selected for their ability to bind to the target antigen (NY-ESO-1 or NY-ESO-1157-165 /HLA-A2) in, e.g., flow cytometry assays. To confirm c-Jun expression, RT-PCR may be performed on the engineered immune (e.g., T) cells. The positive selection may lead to enrichment of TCR⁺c-Jun⁺ cells in a cell population, where the double positive T cells constitute more than 30, 35, 40, 45 ,50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the total cell population. The engineered cells may be cryopreserved until use.

D. T Cell Exhaustion

[0098] Overexpression of c-Jun in T cells helps sustain the active state of the cells by, e.g., alleviating or preventing T cell dysfunction (e.g., T cell exhaustion). The present engineered immune cells, such as T cells, exhibit sustained, potent cytotoxicity against NY-ESO-1 - bearing tumor cells. As compared to T cells that do not overexpress c-Jun, the present engineered T cells display fewer signs of T cell exhaustion and increased signs of persistent effector cells. [0099] In certain embodiments, the cells engineered to express NY-ESC have reduced expression of one or more exhaustion markers, including but not limited to, TIGIT, PD-1, TIM-3, LAG-3, and CD39. Expression of exhaustion markers can be measured in bulk populations by flow cytometry, using bulk RNA-Seq transcriptome analysis. Alternatively, individual cell transcriptome analysis may be carried out using single cell RNA-Seq. In certain embodiments, expression of one or more exhaustion markers in NY- ESO-1 TCR engineered T cells overexpressing c-Jun is reduced by at least about 1.5, 2, 2.5, 3.0, 3.5, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100-fold. In certain embodiments, expression of TIGIT inNYESOl TCR engineered T cells overexpressing c- Jun is reduced by at least about 1.5, 2, 2.5, 3.0, 3.5, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100-fold. In certain embodiments, expression of PD-1 inNY-ESO-1 TCR engineered T cells overexpressing c-Jun is reduced by at least about 1.5, 2, 2.5, 3.0, 3.5, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100-fold. In certain embodiments, expression of CD39 in NY-ESO-1 TCR engineered T cells overexpressing c-Jun is reduced by at least about 1.5, 2, 2.5, 3.0, 3.5, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100-fold.

[0100] In certain embodiments, a population of the present NY-ESO-1 TCR⁺_c-Jun cells has no more than about 5%, 6%, 7%, 8%, 9%, or 10% TIGIT⁺ cells after 14 days of persistent antigen stimulation. In some embodiments, a population of NY-ESO-1 TCR⁺_c-Jun T cells as described herein has no more than about 5%-10%, 5%-15%, 8%-12%, or 8%-15% TIGIT⁺ cells after 14 days of persistent antigen stimulation. In this regard, %TIGIT⁺ cells within a population of NY-ESO-1 TCR⁺_c-Jun T cells such as CD4⁺ or CD8⁺ T cells can be measured by methods known in the art such as flow cytometry.

[0101] In certain embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells has no more than about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% PD- 1⁺ cells after about 14 days of persistent stimulation. In some embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells has no more than about 2%-5% PD-1 positive cells after 14 days of persistent antigen stimulation. In this regard, % PD-1 positive cells within a population of CD4+ and/or CD8+ TCR⁺ c-Jun⁺ T cells can be measured using methods known in the art such as by flow cytometry.

[0102] In certain embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells has no more than about 20%-60% CD39⁺ cells after 14 days of persistent stimulation. In some embodiments, a population of the present engineered NYESOl TCR⁺ c-Jun T cells has no more than about 20%-40% or 25%-45% or 30%-40% CD39⁺ cells after 14 days of persistent stimulation. The percentage of CD39⁺ cells with T cells can be measured by, e.g., flow cytometry.

[0103] In certain embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells (e.g., CD8⁺ T cells) have reduced FOXP3 and CD25 expression after 7 days of persistent stimulation, as compared engineered NY-ESO-1 TCR⁺ T cells that do not overexpress c-Jun. The reduced FOXP3 or CD25 expression can be, e.g., 10, 20, 30, 40, 50, or more percent. The expression of FOXP3 expression can be measured by intracellular staining, and the expression of CD25 can be measured by, e.g., flow cytometry.

[0104] In certain embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells secretes at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, or 150-fold more of IL-2, INF-g, and/or TNF-a as compared to a control population of engineered T cells that do not overexpress c-Jun. In particular embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells express at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 10, or 15-fold more of IL-2, INF-g, and/or TNF-a at day 0 and/or day 14 of persistent antigen stimulation at a 1:1, 1:5, 1:10 or 1:20 E:T ratio, as compared to a control population of engineered T cells that do not overexpress c-Jun. Cytokine secretion can be measured by methods known in the art such as ELISA and Meso Scale Discovery (MSD) analysis.

[0105] In certain embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c-Jun T cells demonstrates at least about 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 75 or 100-fold higher enhanced cytotoxicity efficiency as compared to a control population of engineered CD8⁺ T cells that do not overexpress c-Jun, for example as quantified by area under curve (AUC).

[0106] In some embodiments, a population of the present engineered NY-ESO-1 TCR⁺_c- Jun T cells demonstrate about the same, or at least about 1.5, 2, 2.5, 3, 3.5, 4, 5, 8, 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 200, 225, 250, 300, 400, or 500-fold more enhanced proliferation in response to antigen, as compared to a control population of engineered T cells that do not overexpress c-Jun. Antigen-induced proliferation can be tested by proliferation assays known in the art, such as those described herein.

[0107] Assays useful for measuring exhaustion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, cytokine release, and gene expression profiles are known in the art and include, for example flow cytometry, intracellular cytokine staining (ICS), IncuCyte® immune cell killing analysis, MSD or similar assay, persistent antigen stimulation assay, sequential antigen stimulation assay (similar to persistent antigen stimulatic without resetting E:T cell ratio with each round of restimulation), bulk and single cell RNA- seq, cytotoxicity assays, ELISA, Western blot, and other standard molecular and cell biology methods. See, e.g., Geraci et al., Fron Genet. (2020) 11:220; Sturm et al., Bioinformatics (2019) 35(14):i436-45; Van den Berge et al Ann Rev Biomed. (2019) 2:139-73); Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021).

III. Pharmaceutical Compositions and Uses

[0108] The present disclosure provides pharmaceutical compositions comprising the engineered T cells using the expression constructs described herein. The pharmaceutical compositions may comprise a pharmaceutically acceptable carrier that is suitable to maintain the health of the cells before introduction into the patient.

[0109] In some embodiments, engineered cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline (e.g., phosphate buffered saline), physiological saline, water, Hanks' solution, Ringer’s solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. It is preferred that the carrier is isotonic. In some embodiments, the carrier can be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol may also be included.

[0110] The pharmaceutical T cell compositions may be administered in a therapeutically effective amount to a cancer patient systemically (e.g., through intravenous or portal vein injection) or locally (e.g., through intratumoral injection). In some embodiments, the compositions such as those targeting NY-ESO-1 are used to treat a patient with metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, myxoid round cell liposarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, and bladder cancer. As used herein, the term “treatment” or “treating” refers to an approach for obtaining beneficial or desired results in the treated subject. Such results include, but are not limited to: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease (e.g., reducing tumor volumes), stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, delaying the recurrence or relapse of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, improving the quality of life, restoring body weight, and/or extension of survival (e.g., overall survival or progression-free survival).

[0111] A therapeutically effective amount of the composition refers to the number of engineered T cells sufficient to achieve a desired clinical endpoint. In some embodiments, a therapeutically effective amount contains more than 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ of the engineered cells. In certain embodiments, a subject is administered with a range of about 10⁶-10ⁿ engineered cells.

[0112] The pharmaceutical composition in some embodiments comprises the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0113] The cells and compositions in some embodiments are administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition containing a genetically modified cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0114] In one aspect, the present disclosure provides pharmaceutical compositions comprising the nucleic acid molecules for expressing the TCR and c-Jun. The nucleic acid molecules may be as described above, such as the viral vectors (e.g., lentiviral vectors) described above. The pharmaceutical compositions are used ex vivo to eng progenitor cells, which are then introduced to the patient. The pharmaceutical compositions comprise the nucleic acid molecules or the recombinant viruses whose genome comprise the expression cassettes for the TCR and c-Jun and a pharmaceutically acceptable carrier such as a buffered solution that optionally comprises other agents such as preservatives, stabilizing agents, and the like.

[0115] The pharmaceutical compositions may be provided as articles of manufacture, such as kits, that include vials (e.g., single-dose vials) comprising the biological materials (the cells or the nucleic acid molecules or recombinant viruses) and optionally instructions for use.

[0116] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of immunology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0117] In order that this invention may be beher understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLES

[0118] The materials and methods for experiments described in the present working examples are first described.

Materials and Methods

Lentiviral Vector

[0119] Lentiviral vector (LVV) constructs were produced for delivery of the expression cassettes shown in FIG. 1. The cytomegalovirus (CMV) promoter/enhancer drives constitutive Tat-independent transcription of the vector genomic RNA. The partial Gag sequence encompasses the packaging signal (y) and Rev response element and is followed by a region containing the central polypurine tract. These elements are required for efficient manufacture of functional LVV. The U3 region of the long terminal repeat (LTR) is deleted (ALB) to abolish its promoter/enhancer activity, as required for a self-inactivating vector. An SV40 polyadenylation signal is included downstream of the 3’ LTR to improve transcriptional termination of vector genomic RNA during manufacture. An SV40 origin of replication is thought to enable plasmid amplification in transfected HEK293T cells, potentially increasing vector titer. A high copy (pUC) origin of replication and a kanamycin resistance cassette are basic features contained in the plasmid backbone to allow amplification and selection, respectively, of the plasmid in E. coli.

T Cell Production

[0120] The LVV vectors were used to transduce human T cells using standard LVV transduction protocols. Generally, T cells were transduced with a MOI of 1 to 4 of the LVV preparations.

[0121] T cell products were generated using CD4⁺ and CD8⁺ cells isolated from HLA- A*02⁺ healthy donors (less than 50 years of age) and frozen at the vendor (e.g., AllCells, Alameda, CA, USA). The vendor collected samples via apheresis, from which CD4⁺ and CD8⁺ cells were isolated separately in order of CD8⁺ cells positively selected first, followed by positive selection for CD4⁺ cells of the flow-through from the CD8 selection. The CD4⁺ and CD8⁺ cells were isolated using Miltenyi CliniMACS® beads on CliniMACS® machines. The isolated CD4⁺ or CD8⁺ cells were frozen at about 30E+06 cells per vial in vendors’ proprietary freeze medium containing IMDM, FBS, dimethyl sulfoxide (DMSO), and hetastarch.

Six-well G-Rex T Cell Production

[0122] On Day 0, CD4⁺ and CD8⁺ cells were thawed in TexMACS™ medium and combined at a CD4:CD8 ratio of 50:50. For all donors, cells were plated at a concentration of 2E+06 cells/mL in 4 mL (i.e., 8E+06 cells in total) in 6-well G-Rex plate for 24 hours with CD3/CD28 TransAct™ beads (Miltenyi) at a final dilution of 1:100 in TexMACS™ medium supplemented with 100 IU/mL IL-2 (Sigma). On Day 1, T cells were transduced with LVVs encoding the test or control transgene at an MOI of 1 to 4, based on the Day 1 cell count. On Day 3, cells were diluted with fresh media supplemented with IL-2 to equal a final concentration of 100 IU/mL in 35 mL. On Day 6, fresh IL-2 was added to each well to equal a final concentration of 100 IU/mL in 35 mL per well. On Day 8, a medium change was performed where 30 mL of the culture medium in the sample was removed and replaced with 30 mL fresh medium containing enough IL-2 to bring the total concentration to 100 IU/mL in 35 mL. On Day 10, fresh IL-2 was added to each well to equal a final concentration of 100 IU/mL in 35 mL per well. On Day 13, cells were harvested, counted and frozen using standard protocols.

100M G-Rex T Cell Production Protocol

[0123] On Day 0, CD4⁺ and CD8⁺ cells were thawed in TexMACS™ medium and combined at a CD4:CD8 ratio of 50:50. Cells were plated at a concentration of 2E+06 cells/mL in 50 mL (i.e., 100E+06 cells in total) in 100M G-Rex bottles and activated for 24 hours with CD3/CD28 TransAct™ beads (Miltenyi) at a final dilution of 1:100 in TexMACS™ medium supplemented with 100 IU/mL IL-2 (Sigma). On Day 1, T cells were transduced with LVVs at an MOI of 1 to 4, based on the Day 1 cell count. On Day 3, cells were diluted with 950 mL fresh TexMACS™ medium supplemented with IL-2 to a final concentration of 100 IU/mL in 1 L total volume per 100M G-Rex bottle. On Days 6, 8, and 10, fresh IL 2 was added to each 100M G-Rex bottle to equal a final concentration of 100 IU/mL in 1 L. On Day 13, cells were harvested, counted and frozen in CS10. In some cases, T cell products were normalized within each donor, based on the transduction efficiency, by adding mock cells prior to freeze-down to account for variability between the T cell products.

Assessment of NY-ESO-1 TCR and c-Jun Expression by Flow Cytometry [0124] Surface NY-ESO-1 TCR expression was detected using either PE-labelled NY- ESO-1 specific peptide - MHC Class I dextramer complex or PE-labelled anti-TCRvP 13. 1 antibody (Ab) by gating on mock T cells. Intracellular transgenic c-Jun expression was detected in fixed and permeabilized cells using AF647 labelled anti-c-Jun Ab. All Abs and staining reagents were commercially available, for example from BD Bioscience (San Jose, CA), Thermo Fisher Scientific (Waltham, MA), Beckman Coulter (Indianapolis, IN), BioLegend (San Diego, CA ) and Cell Signaling Technology (Danvers, MA). Assessment of NY-ESO-1 TCR Surface Expression for Norn

Evaluation of Transduction Efficiency

[0125] For surface staining with PE - TCRvpi3.1, -2E+05 cells were incubated in the dark at RT for 25 minutes in CSB FACS buffer (Biolegend®) and washed with CSB FACS buffer. For surface staining with PE - NY-ESO-1 dextramer, -2E+05 cells were incubated in the dark at RT for 25 minutes in FBS FACS buffer (BD Biosciences) and washed with FBS FACS buffer. Dead cells were detected using the live/dead staining reagent eBioscience™ 7- aminoactinomycin D (7AAD). Following TCRvP 13.1 or dextramer staining, the cells were stained with 7AAD in CSB FACS buffer in the dark at RT for 5 minutes before data acquisition using a Bio-Rad ZE5™ cell analyzer.

Assessment of NY-ESO-1 TCR Plus c-Jun Expression for Evaluation of

Transduction Efficiency and Expression Level [0126] Generally, dead cells were detected using a live/dead fixable dye. Approximately 2E+05 cells were stained with live-dead fixable dye in the dark at RT for 10 minutes and subsequently washed with FACS buffer. Cells were then stained with Abs against surface markers in the dark at RT for 25 minutes and subsequently washed with FACS buffer. For surface staining with PE - NY-ESO-1 dextramer, cells were stained in the dark at RT for 25 minutes and washed with FACS buffer prior to staining with additional surface markers as described above. After surface staining, cells were fixed and permeabilized with True- Nuclear™ fix/permeabilization buffer (BioLegend®) in the dark at RT for 30 minutes and washed with True-Nuclear permeabilization wash buffer (BioLegend®). Cells were then blocked with rabbit and mouse serum in the dark at RT for 10 to 15 minutes before stained with Abs against intracellular markers in the dark at RT for 30 minutes in True-Nuclear™ permeabilization wash buffer. Cells were washed with True-Nuclear™ permeabilization wash buffer followed by a wash in FACS buffer. Samples were re suspended in FACS buffer and acquired using a BioRad ZE5™ Cell analyzer or Cytek ™ Aurora Spectral Flow Cytometer.

Phenotype Assessment of T Cell Products

[0127] Phenotype assessment was carried out using commercially available antibodies. Dead cells were detected using live/dead fixable eFluor™ 780. Cells were stained with live/dead fixable eFluor™ 780 either in the dark at RT for 10 minutes prior to surface staining or included in the surface Ab mix. Approximately 2E+05 cells were washed with FACS buffer and blocked with mouse serum and human IgG in the dark at 37°C for 10 minutes before staining with anti-CCR7 Ab in the dark at 37°C for 15 minutes. The cells were then washed with FACS buffer, stained with Ab mix containing the re markers in the dark at RT for 25 minutes. After surface staining, cells were washed with FACS buffer and fixed and permeabilized with Foxp3 fixation/permeabilization Buffer (Thermo Fisher Scientific) in the dark at RT for 30 minutes and washed with Foxp3 permeabilization wash buffer (Thermo Fisher Scientific). Cells were then blocked with rabbit and mouse serum in the dark at RT for 10 to 15 minutes before stained with Abs against intracellular markers in the dark at RT for 30 minutes in Foxp3 permeabilization wash buffer. Cells were washed with Foxp3 permeabilization wash buffer followed by a wash in FACS buffer. Samples were re suspended in FACS buffer and acquired with a Cytek™ Aurora Spectral Flow Cytometer.

Flow Cytometry Analysis

[0128] All samples were acquired using a BioRad ZE5™ Cell analyzer or Cytek™ Aurora Spectral Flow Cytometer and analyzed using FlowJo™ version 10.6.1 software. For all samples, the TE was determined as % dextramer positive cells based on the corresponding mock T cell sample gates. The expression level of NY-ESO-1 TCR and c-Jun was reported as Median Fluorescence Intensity (MFI). The c-Jun MFI was determined for the NY-ESO-1 TCR⁺ population based on endogenous c-Jun gate in the control samples.

[0129] For assessment of T cell phenotype, live NY-ESO-1 TCR⁺ CD4⁺ or CD8⁺ T cells were gated as live-dead fixable eFluor™ 780 low, CD3⁺, CD45⁺, and CD4⁺ or CD8⁺, and TCRvpi3. 1 '. Additional phenotype markers were gated based on negative populations when possible or using Fluorescence Minus One (FMO) controls. When applicable, the expression level of phenotype markers was reported as Median Fluorescence Intensity (MFI).

[0130] For assessment of FOXP3 and CD25 expression before (Day 0) and after target cell stimulation (Day 7), T cells were gated as live (i.e., cPARP and live-dead fixable eFluor780 low) singlet cells, followed by CD3⁺CD45⁺ and then CD4⁺ or CD8⁺ gating. TCRvpi3.1⁺ cells were gated based on the mock T cell samples. FOXP3⁺ cells were gated based on an FMO control. The CD25^hlgh gate was gated based on the Day 0 EFla_NY-ESO- 1 T cell samples.

Assessment of c-Jun Expression by Western Blotting [0131] 1E+06 total T cells from each sample were lysed in RIPA lysis buffer supplemented with lx EDTA-free Halt proteinase inhibitor cocktail using a -80°C freeze- thaw cycle. During thaw, the lysates were incubated on ice with vortexing every 5 to 10 minutes and subsequently cleared for cell debris by centrifugation. The total protein concentration from the cleared lysates was determined using a Pierce BSA following the manufacturer’s guidelines. Equivalent concentration of total protein from each lysate was combined with lx EZ standard master mix (Protein Simple), denatured at 95°C for 5 minutes, and rested on ice according to the manufacturer’s guidelines. The denatured lysates were loaded onto a 12-230 kDa Jess Separation Module (Protein Simple), together with an anti-c-Jun antibody (CST, clone #60A8, 1:20 final dilution) and the reagents from an anti-rabbit detection module for Jess (Protein Simple) following the manufacturers guidelines. The c-Jun protein band was visualized at ~50 kDA with the automated Jess Protein Simple Western system using the standard manufacturer setting. The c-Jun protein band intensities were obtained using Compass analysis software (version 4.0.0) and corresponds to the peak area intensity of the c-Jun protein band identified at ~50 kDA.

IncuCyte® Killing Assay

[0132] A schematic of the IncuCyte® Assay is shown in FIG. 4. The IncuCyte® killing assay was set-up in 96 well flat-bottom moat assay plates 3 different NucLight™ Red (NLR) target cell lines at a T cell effector: NLR target cell (E:T) ratio of 1:1, 1:5, 1:10 or 1:20. The NLR target cell lines, A375 (highNY-ESO-1 antigen level), H1703 (medium NY-ES O-l antigen level), and Colo205 (NY-ESO-1 antigen negative), were generated using NucLight™ Red (NLR) Lentivirus reagent (Essen Bioscience) according to the manufacturer's guidelines. The NLR target cells were thawed and maintained in R10 medium for at least two passages before used in the assay. For the semi-adherent Colo205 NLR cell line, the assay plates were coated with 50 pL of poly-L-omithine. On the day of assay set-up, the NLR target cell lines were harvested using accutase, washed with fresh R10 medium and counted. 5E+04 target cells were plated onto the assay plates and allowed to adhere for ~6 hr at 37°C and 5% CO2 prior to adding the T cells at the indicated E:T ratios (e.g., 1:1, 1:5, 1:10, and 1 :20). The number of NY-ESO-1 TCR⁺ T cells was calculated based on the total T cell count obtained and the % NY-ESO-1 TCR⁺ T cells that was determined prior to freeze down or at the time ofharvest. Forthe E:T ratio of 1:1, 5E+04 NY-ESO-1 TCR⁺ T cells were added to 5E+04 NLR target cells. For the E:T ratio of 1:5, 1:10 or 1:20, the T cells were diluted further in fresh R10 medium and 1E+04, 0.5E+04, or 0.25E+04 NY-ESO-1 TCR⁺ T cells were added to 5E+04 NLR target cells, respectively. Each sample was set-up in duplicates. The assay plates were incubated at 37°C and 5% CO2 in a humidified IncuCyte® S3 Live-Cell Analysis System and scheduled for image acquisition at 6-hour intervals for seven days (-162 hr). After 24 hr co-culture, an aliquot of supernatant was collected from each sample and stored at -80°C for subsequent cytokine analysis, and the I plates were returned to the IncuCyte® S3.

IFN-g, IL-2, and TNF-a Detection in Co-Culture Supernatants by MSD [0133] A schematic of the MSD Assay set up is shown in FIG. 4. The IFN-g, IL-2, and TNF-a cytokine analysis was conducted using a modified 3-plex version from the V-plex® Proinflammatory Panel 1 Human Kit (Meso Scale Diagnostics) according to the manufacturer’s guidelines and the plates were read using an MSD Sector® S 600 Imager. Cytokine concentrations were calculated using the MSD Workbench software based on the dilution factor of the supernatants and the concentration of the calibrators.

Co-Culture Set-Up for Serial Restimulation Assay [0134] A schematic of the serial (persistent) restimulation assay is shown in FIG. 8.

Mock T cells and NY-ESO-1 TCR T cells were serially re-stimulated in 24-well tissue culture plates every 3 or 4 days with irradiated A375 target cells at aNY-ESO-1 TCR+ T cell Effector: Target cell (E:T) ratio of 1 : 1 for a total of 4 rounds of stimulation. One day prior to each round of assay set-up, the assay plates were coated with 300 pL poly-L-omithine per well. On the day of assay set-up, parental A375 target cells were detached using accutase, resuspended in PBS, and irradiated with 10 Gy using a RAD Source Quastar® RS 1800 Q irradiator according to the manufacturer’s guidelines. Immediately after irradiation the cells were washed and plated at 1.5E+05 cells per well into the 24-well plates and allowed to adhere for ~4 hours in a humidified 37°C and 5% CO2 incubator before addition of T cells at a E:T ratio of 1 : 1. For Day 0 of the assay, the T cells were thawed and rested for ~30 minutes, washed and resuspended in fresh R10 medium containing IL-2 for a final concentration of 10 IU/mL. The number of NY-ESO-1 TCR⁺ T cells was calculated based on the total T cell count and the % NY-ESO-1 TCR⁺ T cells that was determined prior to freeze down after completed production. For Day 3, 7, and 10 of the assay, the T cells were harvested from the 24-well plates of the previous round of stimulation, counted, and resuspended in fresh R10 medium containing IL-2 for a final concentration of 10 IU/mL.

The number of NY-ESO-1 TCR⁺ T cells was calculated based on the total T cell count and the % TCRvpi3. 1 ¹ determined on live T cells. IncuCyte® killing assessment and IFN-g, IL- 2, and TNF-a cytokine detection was performed as previously described.

Co-Culture Set-up for T2 Dose-Response Assay [0135] A schematic of the T2 dose-response assay is shown in FIG. 13. The T2 dose- response assay was set-up in 96 well flat-bottom plates. The T2 cell line was thawed and maintained in RIO medium for at least two passages before used in the T2 c assay. Two HLA- A* 02 -restricted peptides were purchased from Cambridge Research Biochemicals, Discovery® Peptides (Billingham, England). The NY-ESO-I157-165 SLLMWITQC (SEQ ID NO: 19) peptide was used as the target peptide and the HPV16 E7s6- 93 TLGIVCPI (SEQ ID NO:20) peptide was used as the negative control peptide (Table 7). The peptides were diluted in DMSO immediately prior to the experiment to a stock concentration of 10 mM, incorporating differences in “net peptide content” listed in the peptide data sheet provided with each lot. The peptide stocks were further diluted 1 : 1000 in serum free RPMI 1640 medium containing GlutaMax™ to a starting concentration of 10 mM (i.e., 10^'5 M) for the peptide pulse of T2 cells. The peptides were then serial diluted 10-fold for a total of eight times in serum free RPMI 1640 medium. The T2 cells were washed and pulsed with the diluted peptide preparations in serum free RPMI 1640 medium for 2 hr at 37°C and 5% CO2, with vortexing every 30 min. After pulsing, the T2 cells were washed with fresh R10 medium, counted, and plated into a 96-well flat-bottom plates at 5E+04 peptide-pulsed T2 cells per well. The T cells were added to the plates immediately after at an E:T ratio of 1:1 or 1:5. The number ofNY-ESO-1 TCR⁺ T cell effector cells was calculated based on the total T cell count and the % NY-ESO-1 dextramer+ on total live T cells (i.e., transduction efficiency on total live T cells), which was determined for the T cell products prior to freeze down after completed production. For the E:T ratio of 1 : 1, 5E+04 NY-ESO-1 TCR⁺ T cells were added to 5E+04 peptide-pulsed T2 cells. For E:T ratio of 1:5, the NY- ESO-1 TCR T cell products were diluted 1:5 in R10 medium before 1E+04 NY-ESO-1 TCR⁺ T cells were added to 5E+04 peptide-pulsed T2 cells. The co-culture plates were incubated for 24 hr at 37°C and 5% CO2 and the supernatants were collected into 96-well U-bottom plates and stored at -80°C for subsequent cytokine analysis as previously described.

Antigen-Independent Growth Assay

[0136] The antigen-independent growth assay was conducted in TexMACS™ medium, using 3 different culture conditions: 1) TexMACS™ without cytokines, 2) TexMACS™ supplemented with 100 IU/mL IL-2, or 3) TexMACS™ supplemented with 1200 IU/mL IL-7 and 200 IU/mL IL-15. The workflow of the antigen-independent growth assay included counting every 3 or 4 days and reseeding into fresh medium every 7 days until the cell counts had diminished and there were not enough cells to continue the assay.

[0137] On Day 0, the T cell products were thawed and counted, and lxl 0⁶ cells were transferred to each well in 24-well G-Rex plates for assay setup. For the “no cytokine” culture condition, 7 mL of TexMACS™ medium without cytokines was ad volume of 8 mL per well. For the IL-2 culture condition, 7 mL of TexMACS™ medium was added containing IL-2 at a final concentration of 100 IU/mL IL-2 for the total 8 mL sample. For the IL-7+IL-15 culture condition, 7 mL of TexMACS™ media was added containing IL- 7 and IL-15 at a final concentration of 1200 IU/mL IL-7 and 200 IU/mL IL-15 for the total 8 mL sample size. Each T cell sample was set up in duplicate. The 24-well G-Rex plates were incubated in a humidified 37°C and 5% CCh incubator for 4 days. On Day 4, 4 mL of culture media from each T cell sample was removed and replaced with 4 mL of pre-warmed TexMACS™ medium with or without cytokines. IL-2 was added at a final concentration of 100 IU/mL and IL-7+IL-15 was added at final concentrations of 1200 IU/mL IL-7 and 200 IU/mL IL-15 for a total 8 mL sample size. The T cell samples were gently mixed and counted using the Attune™ NxT cell counter. On Day 7, the T cell samples were gently resuspended and counted using the Attune™ NxT cell counter. Due to low cell count, the “no cytokine” culture condition was discontinued and the IL-2 and IL-7+IL-15 duplicate samples were combined into one 50 mL tube, centrifuged at 300 ^c g for 5 minutes at RT, and resuspended in TexMACS™ medium without cytokines for a concentration of lxl 0⁶ cells/mL (donor 3035610) or 0.5xl0⁶ cells/mL (donor 3035680 and 3035702). lxlO⁶ cells (donor 3035610) or 0.5xl0⁶ cells (donor 3035680 and 3035702) from each sample were transferred into new 24-well G-Rex® plates, and 7 mL of the fresh TexMACS™ medium with IL-2 or IL-7+IL-15 cytokines were added for a total volume of 8 mL per well as described above. Due to low cell counts there were not enough cells to setup the assay in duplicate on Day 7. On Day 11, a media change was performed, as had been performed on Day 4, as described above. On Day 14, the T cell samples were gently mixed and counted.

On Day 14, there were not enough cells from the IL-2 and IL 7+IL 15 culture conditions to continue the assay.

Example 1: Production of Human T Cells Expressing High Affinity NY-ESO-1 TCR +/- c-Jun

[0138] The effect of c-Jun overexpression on human T cells expressing a recombinant high affinity NY-ESO-1 TCR was investigated. In particular, two different promoters were tested to identify a lead candidate for the study of c-Jun overexpression in the setting of a recombinant TCR.

[0139] Constructs were made having an affinity-enhanced NY-ESO-1 TCR recognizing the SLLMWITQC (SEQ ID NO:19)/HLA-A*02 peptide complex (Robbins et al., J Immunol. (2008) 180(9):6116-31) along with a codon optimized human wildtype c-Ji JunAA, which contains two serine to alanine mutations at position 63 and 73 in the N- terminal region of human c-Jun. The SLLMWITQC (SEQ ID NO: 19) peptide (NY-ESO- 1157-164) is derived from the NY-ESO-1 and LAGE- la family of cancer/testis antigens and is expressed in complex with HLA-A*02 on multiple malignancies, including non-small cell lung cancer (NSCLC), synovial cell sarcoma, melanoma, and multiple myeloma (D’Angelo et al., Cancer Discov. (2018) 8(8):944-57; Mackall et al ,, J Clin Oncol. (2016) 34:TPS3101- TPS3101; Robbins et al ,, J Clin Onco. (2011) 29:917-24; Robbins et al. (2015) Clin Cancer Res. 21(5): 1019-27; Stadtmauer et al., Blood Advances (2019) 3(13):2022-34). [0140] Initial experiments were carried out to identify optimal codon usage to increase c-

Jun transgene expression. These experiments were carried out in a model system using a multi-cistronic construct having an anti -ROR1 -specific CAR with a truncated EGFR (EGFRt). The structured Domain III of human EGFR is targeted by cetuximab (Erbitux®), a FDA-licensed biologic. Separating the cetuximab-binding ability of EGFR from its biological activity by selective truncation of the receptor offers the potential for an inert, fully human cell surface marker (Li et al., Cancer Cell (2005) 7(4):301-11; Wang et al., Blood (2011) 118(5): 1255-63). As such, the EGFRt functions as a safety switch to eliminate modified cells with cetuximab.

[0141] Multiple algorithms were used to generate codon optimized variants which were then tested for c-Jun expression measured by flow cytometry. The results from 2 screens are shown in Table 1 below (MFI: mean fluorescence intensity).

Table 1: Codon Optimization of c-Jun

[0142] As shown in Table 1, the codon-optimized c-Jun sequence in construct LP 2071 maintained c-Jun, anti-RORl CAR, and EGFRt transgene expression as measured by MFI in flow cytometry. The nucleotide sequence for the codon-optimized c-Jun coding sequence in the LP 2071 construct is show in SEQ ID NO:21. This codon optimized c-Jun sequence was used in the constructs described below (see, e.g.. SEQ ID NO: 14). A single T to C nucleotide substitution was made at position 798 of SEQ ID NO:21 for cloning purposes to remove a restriction site.

[0143] Several expression constructs were made as shown in the schematic of FIG. 1. In particular, two different promoters, elongation factor-la (EF-la) promoter and MND promoter (MND), were shown to drive high level expression of c-Jun. Wildtype and mutated versions of c-Jun were evaluated to mitigate the potential safety risk for oncogenic transformation. The c-JunAA variant contains inactivating serine-to-alanine mutations at position 63 and 73 (i.e., S63A and S73A) in the N-terminal region of c-Jun.

[0144] The design of constructs placed the c-Jun open reading frame (codon-optimized sequence of cJun gen huml P05412 - either wildtype (WT) or containing inactivating mutations (AA)) - upstream of the NY-ESO-l^c259 TCR a and b chains with all 3 coding sequences being under the control of either the constitutive human EF-la promoter or MND promoter. Although transcribed into a single mRNA, the presence of P2A ribosomal skipping sequences encoded between each component leads to production of 3 separate polypeptides. In addition, a furin cleavage site was placed adjacent to the P2A sequence located at the C-terminus of TCR a to remove parts of the ribosomal skipping protein sequence from the TCR a subunit.

Example 2: High, Stable Expression of Both c-Jun and NY-ESO-1 TCR in Transduced T cells

[0145] Human T cells were transduced with LVV containing the expression constructs shown in FIG. 1 and expression levels of the recombinant transgenes were assessed by standard Western blot and by flow cytometry as described above in Materials and Methods. Although MND resulted in higher levels of c-Jun as determined by flow cytometry (FIG. 2A) or by Western blotting analysis (FIG. 2B), EF-la constructs showed more stable expression of c-Jun following stimulation with either Trans Act™ or with NY-ESO-1 ⁺ target cells (FIG. 3). Example 3: Overexpression of c-Jun Enhances T Cell Killing and Cyt<

[0146] Human T cells transduced with LVV expressing the constructs shown in FIG. 1 were stimulated and tested in an IncuCyte® assay for killing and cytokine function above as described in Materials and Methods. An overview of the functional assay is shown in the schematic of FIG. 4.

[0147] Primary stimulation of NY-ESO-1 TCR +/- c-Jun T cells at a variety of E:T showed better killing (FIGs. 5A and B) and cytokine production (FIGs. 6A and B) by c-Jun constructs driven by the EF-la promoter. The results also showed that the c-Jun WT and AA variants functioned similarly in the killing assay.

Example 4: Cells Overexpressing c-Jun Proliferate More in Response to Target Cells [0148] Proliferation of T cells transduced with the NY-ESO-1 TCR constructs with and without c-Jun driven either by the EF-la or MND promoter was evaluated 7 days after target cell stimulation and reported as the fold change from starting culture compared to EF-la NY- ESO-1 control TCR. As shown in FIGs. 7A-7C, T cells transduced with EF-la NY-ESO-1 TCR + c-Jun constructs expanded the most across donors, and results obtained with c-Jun WT and AA variants were similar.

[0149] The above studies indicated that c-Jun provided benefit to NY-ESO-1 TCR transduced T cells in particular when expressed under the control of the EF-la promoter.

Example 5: c-Jun NY-ESO-1 TCR Cells Demonstrate Improved Functions in Response to Serial Antigen Stimulation

[0150] In view of the results shown in Examples 1-4, additional focused studies were carried out with EF-la NY-ESO-1 TCR constructs with and without c-Jun. In this Example, T-cell exhaustion was modelled in vitro by using a serial re-stimulation assay in which the NY-ESO-1 TCR T cell products were stimulated every 3 or 4 days with irradiated A375 target cells for a total of 4 rounds of stimulation (see schematic in FIG. 8). This assay provides a model for persistent antigen stimulation. Hallmarks of T cell exhaustion include increased co-expression of exhaustion markers such as TIGIT, PD-1, and CD39, as well as progressive loss of T-cell effector functions, such as cytotoxicity and cytokine secretion (McLane et al, Ann Rev Immunol. (2019) 37:457-95). Thus, phenotypic evaluation of the NY-ESO-1 TCR T cells was conducted at the time of production, assay start, after 2 rounds of stimulation, and after 4 rounds of stimulation (i.e., Pre-freeze, Day 0, Day 7, and Day 14, respectively) and included assessment of T cell differentiation and expression of exhaustion markers. Furthermore, functional evaluation of the NY-ESO-1 TCR T cell: the time of assay start and after the 4 rounds of stimulation (i.e., Day 0 and Day 14, respectively) and included measurement of cytotoxicity and cytokine secretion in response to target cell lines expressing various levels of NY-ESO-1 antigen.

[0151] Throughout the 4 rounds of stimulation, a high level of c-Jun expression was maintained by the EF-la promoter in the EFla_c-JunWT_NY-ESO-l TCR T cells (data not shown). c-Jun NY-ESO-1 TCR T cells have similar or increased proliferation in response to antigen when compared to control (FIGs. 9A-9C). The EFla_NY-ESO-l TCR T cells displayed characteristics of exhaustion after 4 rounds of NY-ESO-1 antigen stimulation in that an increase in co-expression of the exhaustion markers TIGIT, PD-1, and CD39 was observed (FIGs. 10A-10E). Note that data were similar across donors and within CD8⁺ and CD4⁺ populations.

[0152] One of the hallmarks of dysfunctional T cells is the co-expression of multiple exhaustion markers (McLane et ak, ibid). A multi-marker analysis using Boolean gating was performed with the TIGIT⁺, PD-1⁺, and CD39⁺ gates obtained within the NY-ESO-1 TCR⁺ CD8⁺and CD4⁺ T cell population on day 14 (i.e., after 4 rounds of stimulation). About 17- 29% of the CD8⁺ T cell population and about 20-71% of the CD4⁺ T cell population from the EFla_NY-ESO-l TCR T cell products co-expressed two or more exhaustion markers (FIGs. 10D and 10E) (‘Total multiple marker^1"). The majority of the CD8⁺ T cells co-expressing multiple exhaustion markers were CD39⁺TIGIT⁺, whereas the majority of the CD4⁺ T cells were CD39⁺PD-1⁺ or CD39⁺PD-1⁺TIGIT⁺ (FIG. 10D and E). A significant decrease in the proportion of CD8+ (i.e., ranging from -2-7%) and CD4⁺ (i.e., ranging from -5-38%) T cell populations co-expressing multiple exhaustion markers was observed in the EFla c- JunWT_NY-ESO-l TCR T cell products. Together these results indicate that overexpression of c-Jun reduces the proportion of both CD8⁺ and CD4⁺ NY-ESO-1 TCR T cells expressing exhaustion markers, TIGIT, PD-1 and CD39, caused by persistent antigen stimulation.

[0153] Furthermore, the EFla_NY-ESO-l TCR T cells showed reduced killing capacity (FIGs. 11A-11L) and cytokine production (FIGs. 12A-12F) in response to the NY-ESO-l⁺ target cell lines A375 and H1703. In contrast, the EFla_c-JunWT_NY-ESO-l TCR T cells had reduced expression of exhaustion markers (FIGs. 10A-10C), with the reduction in CD39 expression not being as drastic in two of the donors or in CD4 cells, and maintained the ability to kill (FIGs. 11A-11F) and produce cytokines (FIGs. 12A-12F), even after 4 rounds of NY-ESO-1 antigen stimulation. FIGs. 11A-11L and FIGs. 12A-12F show the results for A375 target cells from a representative donor. Similar data were seen aero:

HI 703 target cells (data not shown).

[0154] Taken together, these data show that c-Jun overexpression can prevent dysfunctional states of NY-ESO-1 TCR T cell products after persistent antigen exposure.

Example 6: c-Jun Overexpression Increases Antigen Sensitivity of NY-ESO-1 TCR Cell Products

[0155] The goal of this experiment was to evaluate whether c-Jun overexpression changes the antigen sensitivity of NY-ESO-1 TCR in comparison to the NY-ESO-1 TCR product without c-Jun. This study was carried out with the EFla_NY-ESO-l TCR constructs with or without c-Jun.

[0156] A schematic of the T2 dose-response assay used for this experiment is shown in FIG. 13. T2 cells express HLA-A*02 but are deficient in transporter associated with antigen processing (TAP) and therefore cannot present endogenous (processed) peptides in complex with HLA-A*02. When pulsed with peptides, the T2 cells will present the pulsed peptide in complex with HLA-A*02. As such, these cells can be used to evaluate T cell responses to different amounts of an exogenous antigen of interest in a non-competitive environment. In particular, for this assay, NY-ESO-1 peptides were diluted fresh, the negative control peptide used was HPV16 E7 (AA 86-93). T2 cells were pulsed at 37°C in serum free RPMI medium, washed in R10 medium before being plated into a 96 well flat-bottom plates. NY-ESO-1 TCR+ T cells were added to the T2 cells directly after plating. About 24 hours after coculture, supernatant was removed from plates for cytokine analysis.

[0157] The results show that c-Jun overexpression increased the antigen sensitivity of the NY-ESO-1 TCR T cell products. In particular, c-Jun overexpression increases the IL-2 production after co-culture with NY-ESO-1 pulsed T2 cells, whereas no increase was observed after coculture with HPV16 E7 pulsed T2 cells (FIGs. 14A-14C). The E:T ratio is 1:5. Calculation of the half maximal effective concentration (EC 50) as summarized in Table 2 below shows that c-Jun overexpression increases the antigen sensitivity of NY-ESO-1 TCR products.

Table 2: EC₅₀ Values of Engineered T Cells

[0158] c-Jun overexpression also increased the IFN-g production after co-culture with NY-ESO-1 pulsed T2 cells while no increase was observed after coculture with HPV16 E7 pulsed T2 cells (FIGs. 15A-15C).

[0159] In summary, overexpression of c-Jun consistently increased the level and threshold for IL-2 and IFN-g production of NY-ESO-1 TCR T cells in response to antigen when compared to NY-ESO-1 TCR controls. Changes to TNF-a expression in response to antigen were not changed in a consistent manner when c-Jun was overexpressed by NY-ESO-1 TCR T cells.

Example 7: Antigen Independent Growth Assay

[0160] The aim of this study was to evaluate the antigen-independent cell growth of the EFla_c-JunWT_NY-ESO-l TCR over time with or without cytokine support as part of the pre-clinical safety assessment.

[0161] EF 1 a_NY -ESO- 1 and EFla_c-JunWT_NY-ESO-l TCR T cell products generated from 3 healthy donors were evaluated in an antigen-independent growth assay as described in the Materials and Methods using 3 different culture conditions: 1) TexMACS™ without cytokines, 2) TexMACS™ supplemented with 100 IU/mL IL-2, or 3) TexMACS™ supplemented with 1200 IU/mL IL-7 and 200 IU/mL IL-15. T cells from each culture condition were counted every 3 or 4 days and the cultures were reseeded into fresh medium every 7 days until the cell counts had diminished and there were not enough cells to continue the assay. The experiment without cytokines was discontinued on Day 7, and the experiments with cytokines (IL-2 or IL-7+IL-15) were discontinued on Day 14 due to insufficient cell numbers.

[0162] From the data acquired across 3 donors as shown in FIGs. 16A-16C, there was no evidence of c-Jun overexpression driving uncontrolled antigen-independent cell growth of NY-ESO-1 TCR T cell products. The T cells persisted longer with IL-2 or IL-7+IL-15 cytokine support and the EFla c JunWT_NY-ESO-l and EFla_NY-ESO-l TCR T cell products exhibited similar kinetics of cell numbers overall. Taken together, these data demonstrate that overexpression of c-Jun does not promote antigen-independent growth of NY-ESO-1 TCR T cell products with or without cytokine support. [0163] In summary, the above Examples present data indicating that NY-ESO-1 TCR T cells overexpressing c-Jun can successfully be generated, that c-Jun overexpression can prevent functional exhaustion of NY-ESO-1 TCR T cells, and that overexpression of c-Jun does not promote antigen-independent growth of NY-ESO-1 TCR T cell products with or without cytokine support. The functional impact of c-Jun overexpression on NY-ESO-1 TCR engineered T cells appears to be greater than what has been observed on CAR T cells. Overall, the NY-ESO-1 TCR T cells overexpressing c-Jun provide a new clinical candidate for improving adoptive T cell therapy for the treatment of solid tumors. Example 8: Comparison of EF1α _NY-ESO-1 and EF1α_ c-JunWT_NY-ESO-1 TCR⁺ T cells on Tumor Growth in A-375 CDX Tumor-Bearing Mice [0164] This example describes the study that evaluated the efficacy of EF1α_c- JunWT_NY-ESO-1 versus EF1α_NY-ESO-1 TCR+ T cells, and the efficacy of MND_c- JunWT_NY-ESO-1 versus MND_NY-ESO-1 TCR+ T cells, against the NY-ESO-1-positive cancer cells in a mouse model. The in vitro studies described above showed that although MND resulted in higher levels of c-Jun as determined by flow cytometry (FIG.2A) or by Western blotting analysis (FIG.2B), EF-1 ^ constructs showed more stable expression of c- Jun following stimulation with either TransAct^TM or with NY-ESO-1⁺ target cells (FIG.3). [0165] To assess the in vivo efficacy and function of the NY-ESO-1 TCR T cell products in solid tumor models, an NY-ESO-1-expressing tumor cell line, A-375, was used to establish a subcutaneous tumor xenograft model. Once subcutaneous A-375 derived tumors had been established, mice were treated intravenously with 5x10⁶ TCR⁺ T cell product (EF1α_c- JunWT_NY-ESO-1; MND_c-JunWT_NY-ESO-1; EF1α_NY-ESO-1; or MND_NY-ESO-1) or non-transduced (NTD; also referred to as untransduced) T cells. Phenotypic characterization of the T cell products was carried out prior to in vivo infusion and histopathological analysis of target antigen (NY-ESO-1) and T cell infiltration was conducted at study endpoint on fixed tumors. 8.1: Materials and Methods: [0166] To assess efficacy of the EF1α/MND_NY-ESO-1 versus EF1α/MND_c- JunWT_NY-ESO-1 TCR-T cells against tumor cells in vivo, we used the NY-ESO-1 antigen- expressing melanoma cell line A-375. [0167] The primary readout of the efficacy study was impact on tumor growth and mouse survival. Secondary readouts were (a) Blood PK assay to investigate T cell persistence over time, (b) assessment of TCR⁺ T cell presence in tumors and murine tissues by CD3 IHC and NY-ESO-1 TCR RNAscope®, (c) evaluation of NY-ESO-1 antigen expression in tumors by IHC, and (d) single cell RNA sequencing to investigate gene expression. T cell and A-375 Tumor Cell Preparation for Inoculation [0168] The TCR⁺ T cells were prepared as above. Tumor cells were upscaled before inoculation into mice. Before inoculation tumor cells were harvested and supernatant was collected for human and murine pathogen testing to confirm pathogen-free status of the cells. Harvested tumor cells were counted and resuspended on ice in pre-chilled PBS: Matrigel (1:1) to a final concentration of 5x10⁶ cells in 100 µL per mouse. Cells were kept on ice and later used for subcutaneous inoculation of cells into the right flank of each NSG female mouse. Preparation for Dosing (Tumor Randomization and T cell Preparation) [0169] Tumor volumes in each mouse were measured using a caliper before the mice were randomized to T cell treatment. From these measurements, mean tumor volume was calculated for each cage and classified as low volume (< median mean tumor volume) or high volume (≥ median mean tumor volume). [0170] TCR⁺ T and non-transduced T cells were thawed and transferred to 50mL tubes containing prewarmed RPMI media and pipetted up and down gently to continue the thawing process. Cells were washed with PBS and counted. Based on TCR transduction efficiencies, cell suspensions were adjusted to 5x10⁶ TCR⁺ T cells/100 µL in sterile PBS for intravenous injection into tail vein of each NSG female mouse. [0171] On the day of thawing, transduction efficiency and phenotype of the NY-ESO-1 TCR T cells was assessed by flow cytometry (method described in the section “T cell Phenotyping” below). Tumor Cell Inoculation [0172] Subcutaneous (s/c) tumor implantations were carried out in a class II sterile cabinet. All equipment used was sterilized prior to use. Animals were briefly anaesthetized in a chamber by isoflurane-oxygen mix and moved to face cone. Right flank was shaved then wiped with alcohol wipe. A total volume of 100 µL of Matrigel and PBS solution with cells were injected s/c per mouse. Animals were moved to recovery area to be monitored until fully recovered before placed back in-home cage and monitored. T cell Dosing

[0173] When tumors were palpable (about 100 mm³) on study day 7, TCR⁺ T cells were dosed via tail vein injection at a dose of 5xl0⁶ TCR⁺ T cells per mouse. The % NY-ESO-1 TCR⁺ on total live T cells that was previously determined prior to freeze down as described in the previous examples. For both donors, the % NY-ESO-1 dextramer⁺ on total live T cells was used for the calculation. Intravenous (i.v.) dose of therapy was carried out in a class II sterile cabinet. Table 3 below shows the details for each TCR⁺ T cell construct tested in vivo and treatment groups.

Table 3: Dosing Protocol

Tumor Measurements, Study Plan, and Endpoints

[0174] Tumor size in all mice was measured by caliper and recorded three times a week to be followed by body weight recording twice a week. Tumor volume was calculated as indicated below: Tumor volume = Tumor length * (Tumor Width^A2) * 0.5

[0175] Mice were culled and tissues harvested at individual endpoints based on predetermined criteria, including maximum tumor volume. Additionally, for selected TCR⁺

T cell treatment groups, tumors were harvested at study days 20-21 for dissociation and further T cell characterization ( n=5 animals/group). For all groups tumors and spleen were collected and either dissociated for T cell characterization (n=5) or fixed for histopathological examination (n=5) as described below.

[0176] Blood samples from all mice on study were collected at 24 hours post-T cell inoculation and weekly thereafter. About 100 pL blood per mouse with an additional 5 pL of blood for wastage per sample was collected. Blood was also collected via cardiac puncture as a part of terminal sampling. T Cell Phenotyping [0177] To determine T cell phenotype before infusion into mice, flow cytometry-based analysis was performed, using differentiation and exhaustion immune panels (exhaustion: CD45, CD4, CD8, TCRvβ13.1, CD223, CD279, CD336 and differentiation: CD45, CD4, CD8, TCRvβ13.1, CD45RA, CD197, CD95). T cells (2x10⁵) were transferred to a 96-well V-bottom plate, centrifuged, resuspended in a blocking solution and incubated for 15 min at 4^oC. Ten μL of antibodies diluted in FACS buffer were added to each well. Cells were incubated with antibodies at 4^oC for 30 min in the dark. After 30 min of incubation, cells were washed once, and 120 μL of diluted 7AAD Live/Dead dye added to each well for 5-10 min in the dark at RT. FMO controls were also prepared for each antibody and plate run on CytoFLEX S Flow Cytometer. [0178] For each immune panel, compensations were performed. Each antibody was mixed with one drop of REA or UltraComp eBeads^TM compensation beads, in a 96-well V- bottom plate, and incubated for 15 min on ice in the dark. For the live/dead stain, an aliquot of cells was incubated for 5 min at 95^oC to induce cell death, mixed with live T cells at 1:1 ratio and further stained with 7AAD. Unstained beads and cells were used as negative controls. The wells were washed with 100 μL, centrifuged and then resuspended in 100 μL of PBS and run on the CytoFLEX Flow Cytometer. Annexin V/Helix NP Staining [0179] Cells were incubated with Annexin V-APC diluted in Annexin V binding buffer in the dark at RT for 15 minutes. Cells were washed in Annexin V binding buffer and stained with Helix NP^TM Blue dead cell dye prior to data acquisition. Gating was performed on the total cell population and a quadrant plot was used to gate the “Viable cell population” (Annexin V-/Helix NP -), “Apoptotic cell population” (Annexin V+/Helix NP -), and “Dead cell population” (Annexin V+ & - /Helix NP +). Data was acquired using a BD Biosciences Fortessa^TM X-20 and analyzed using Flowlogic^TM Flow Cytometry Analysis software. Cytotoxicity Assay [0180] A375-NucLight^TM target cells (4x10⁴/well) were plated in a 96-well plate and allowed to attach for 3-4 hrs in 50 μL of co-culture media (Phenol red free RPMI supplemented with 10% FBS, 1% NEAA, 1% GlutaMAX^TM and 1% NaPyruvate). Donor 1 (3035610) and Donor 3 (3035702) T cells (4x10⁴/well) from each group were added to target cells in 50 μL of co-culture media. For a 1:1 effector-to-target ratio, 4x10⁴ T cells were added per well. For a 1:5 effector-to-target ratio, 8x10³ T cells were plated per well. A375- NucLight target cells only were used as control. Once T cell plating was completed, the 96 well plate was loaded to IncuCyte® S3, where image acquisition, with red fluorescence and phase channel, was scheduled at 2-hour intervals. T cell PK Flow Cytometry [0181] Blood was transferred to V-bottomed 96-well plate. Red blood lysis was performed twice using 150 µL of Red Blood Cell lysis buffer and cells washed with 200 µL cell staining buffer. After lysis, cells were resuspended in 50 μL cell staining buffer supplemented with 10% mouse serum and 100 µg/mL human IgG and incubated for 10 minutes at room temperature. 50 µL of antibody master mix (Live/dead DRAQ7, CD3 FITC, mCD45 BV421, CD4 BV786, CD8 BUV395, TCRvb PE and Dextramer APC) or fluorescent minus one (FMO; for TCR or Dextramer) mix was added to cells and incubated for 25 minutes at room temperature. Cells were washed twice with cell staining buffer. 50 µL of 123count™ eBeads^TM master mix was added to each sample. All samples were acquired using Fortessa^TM X-20 Flow Cytometer. FCS files were exported from FACSDiva^TM and analyzed in FlowJo^TM, primary metrics were exported as csv. and visualized in GraphPad Prism. Histopathology [0182] The spleen and tumor tissues were harvested from all animals, bisected and one half was snap frozen and the other half fixed in formalin for 24 hours, processed overnight and embedded in a paraffin block. All formalin fixed paraffin embedded (FFPE) tumor tissues were sectioned at 4 μm and stained with haematoxylin and eosin for tissue morphology assessment. Immunohistochemistry (IHC) for CD3 and NY-ESO-1 [0183] FFPE tumor tissues serially sectioned at 4 μm were stained by IHC for CD3 using anti-CD3 (clone 2GV6) rabbit monoclonal antibody and for NY-ESO-1 using anti-NY-ESO- 1 (clone E978) mouse monoclonal antibody. Digital histopathology quantification of human CD3 T cell infiltration and NY-ESO-1 expression in the tumor and tumor stroma areas was performed using HALO™ software (v3.2.1851.229) from Indica Labs (Albuquerque, NM, USA). HALO^TM software annotations and IHC quantification was reviewed by an investigator and two pathologists. Dual CD3 IHC and RNAscope in situ Hybridisation (ISH) Assay for NY-ESO-1 TCR [0184] FFPE tumor tissues serially sectioned at 4 μm were used for dual CD3 IHC and RNAscope® Ventana (VS) Universal Red ISH assay. The IHC part of the assay used the anti-CD3 (clone 2GV6) rabbit monoclonal antibody and the pre-treatment conditions used for the RNAscope® ISH part of the assay include antigen retrieval for 16 minu for 16 minutes. Tissue RNA quality was first assessed using human cyclophilin B (PPIB) housekeeping gene probe as a positive control, while a bacterial gene probe (dapB) was used in a smaller set of samples to assess possible background staining. Semi-quantitative scores (0-4) were obtained for all samples to determine QC pass/fail. Samples with good RNA quality were subsequently stained for the NY-ESO-1 TCR and quantification performed using HALO software (v3.2.1851.229) from Indica Labs (Albuquerque, NM, USA).

Tumor Growth Curves

[0185] A linear mixed model is fit to tumor volumes, with fixed effects for construct, time, and mouse tumor baseline volume. Random effects are used for cage, donor, and mouse. Tumor volumes are logio transformed to ensure homoskedasticity. Marginal mean tumor volumes are calculated for each construct group, at each timepoint. Linear contrasts are constructed to compare tumor volumes at arbitrary timepoints. These are fold changes as tumor volumes are logio transformed.

Survival Analysis

[0186] Data was reformatted to “time to 1000 mm³ tumor volume,” with mice considered “censored” if they dropped out (died) or reached the end of the study first. Survival analysis (parametric survival regression) was fit to this data, with effects of construct and donor. Marginal means (as median time to arbitrary tumor volume) was reported in each construct and compared between groups as linear contrasts (converted back to fold changes). Separately, this data can also be visualized as Kaplan-Meier curves.

Tumor -Infiltrating Lymphocyte Isolation

[0187] Tumors were washed with RPMI and any extra connective tissue or skin removed with the use of clean forceps. Each tumor was cut into 2-4 mm pieces with clean forceps. The tumor pieces were placed into a gentleMACS™ C tube containing 4.85 mL of RPMI- 1640 media and 100 pL of Enzyme H, 50 pL of Enzyme R and 12.5 pL of Enzyme A were added. The sealed C tube was placed on the gentleMACS™ Dissociator and the appropriate program was run for the specific tumor type as per the manufacturer’s protocol. Once the incubation completed, the cell solution was passed through a 70 pm filter and the filter was washed twice with 35 mL of RPMI- 1640 and centrifuged. The pellets were resuspended in 30 mL of RPMI. Cell suspensions were further separated using 13 mL Ficoll-Pague which was carefully layered with 30 mL of cell suspension. Two distinct layers were visible, and any mixing was avoided. Cells were centrifuged with slow acceleration and brake off. The layer containing immune cells was collected and washed with RPMI. Pellets were resuspended in 1-5 mL of FACS buffer (lxPBS + 2% FBS + 1 mM EDTA) pellet size and counted. Immune cells were further isolated with CD45⁺ selection using magnetic beads following manufacturer’s protocol.

Single Cell RNA Sequencing

[0188] Tumor-infiltrating lymphocytes were stained with hashing antibodies in Curiox Laminar plates for 30 minutes on ice to allow multiplexing of samples. Cells were then washed using the Curiox system using cell staining buffer. Cells were then pooled, resuspended in 500 pL of 0.5% BSA in PBS and filtered through a 40 pm Flowmi® cell strainer. 20,000 or 25,000 cells were loaded for each emulsion. Emulsions, cDNA processing, library preparation and sequencing on NovaSeq 6000 was done according to Chromium Single Cell V(D)J Reagent Kits with Feature Barcode technology for Cell Surface Protein.

Single Cell RNA Sequence Analysis

[0189] The following QC metrics and (global) thresholds were used for cell QC: a) number of detected genes per cell > 800, b) number of total counts per cell > 2000, and c) percent of counts in mitochondrial genes < 10%. Cells were demultiplexed using a threshold- based approach with expressed/not expressed antibodies defined as follows: expressed antibodies: > 200 counts; not expressed: < 50 counts. The data were normalized using the SCTransform method in the Seurat package v.3.1.3. The principal component analysis results were confirmed by other dimensionality reduction methods (UMAP). The FindClusters function (Seurat v3.1.3) with default options (resolution = 0.5) and 20 PCs was used for clustering. Clusters that did not express T cell markers and expressed epithelial, myeloid and stromal cells markers were removed along with TRAB/TRBV genes. The remaining data were re-processed following the same steps described above.

[0190] Differential expression analysis was performed on pseudo-bulk samples (sum of counts of cells). Lowly expressed genes (<10 counts) were not considered in the analysis.

The data were normalized for library size following guidelines for differential expression analysis with DESeq2 and accounting for the donor effect in the data.

Single Cell RNA Sequencing for Exhaustion Marker Expression [0191] Single Cell RNA sequencing was performed on T cells isolated from the tumor samples to analyze RNA expression such as exhaustion markers. Gene expression was determined for T cells from mice at the end of the in vivo study and shown combined for both donors (FIG. 22). Key exhaustion markers are PD1 (PDCD1 gene) and CTLA4, both of which were significantly downregulated in EFla_c-JunWT_NY-ESO-l T cells as compared to EF1α_NY-ESO-1 T cells. In addition, the transcription factor Tox, a key gene driving exhaustion, was less expressed in EF1α_c-JunWT_NY-ESO-1 T cells. 8.2: T cell Characterization and Blood PK Profile [0192] Prior to in vivo administration, flow cytometry analysis was performed to determine the exhaustion profile and cellular subset composition of T cell products. For both donors and cell conditions, most transduced T cells were CD8⁺ (FIG.17A), with CD4⁺ cells ranging from 8-24%. The % transduction efficiency (TE) of the NY-ESO-1 TCR products was determined in all donor samples and was an average of 57% across the conditions (FIG. 17B). Although a low percentage of TCR⁺CD8⁺ T cells expressed exhaustion markers LAG3 and PD-1 (<10%), most cells expressed TIM-3 (FIG.17C). High levels of TCR⁺CD4⁺ T cells also expressed the exhaustion marker TIM-3, with very low percentages of cells expressing LAG-3 (<5%) for all conditions (FIG.17D). A higher percentage of TCR⁺CD4⁺ within the EF1α_NY-ESO-1 (46.2%) and NTD (43.2%) T cell groups expressed PD-1, with significantly lower levels of EF1α_c-JunWT_NY-ESO-1 TCR⁺CD4⁺PD-1⁺ T cells (20.3%) (FIG.17D). Similar results were also observed with the MND constructs. Another key difference observed was a significantly decreased TIM-3⁺ population in MND_NY-ESO-1 versus EF1α _NY-ESO-1 TCR+ T cell group. [0193] To gain insight into the composition of T cell products and distinguish memory populations, cell surface markers, such as CD45RA, CCR7 and CD95 were used. Both TCR⁺CD8⁺ and CD4⁺ cells had a predominantly stem cell memory, central memory, and effector phenotype. Both MND_c-JunWT_NY-ESO-1 and EF1α_c-JunWT_NY-ESO-1 TCR⁺ CD8⁺ T cells presented a more differentiated phenotype with higher percentage of Tcm and reduction in Tscm, as compared to NTD, MND_NY-ESO-1 and EF1α_NY-ESO-1 TCR+ T cells (FIG.17E). Significant differences in CD8⁺ T cell subpopulations were observed between the EF1α and MND c-JunWT NY-ESO-1 groups (FIG.17E). In particular, the MND_c-JunWT_NY-ESO-1 group had a significantly increased percentage of Teff and a decreased percentage of Tscm populations as compared to the EF1α_c-JunWT_NY-ESO-1 group (FIG.17E). The differences in the CD4⁺ T cell subpopulations percentages were not significantly (FIG.17F). [0194] To evaluate EF1α_NY-ESO-1 and EF1α_c-JunWT_NY-ESO -1 T cell mediated killing potential, an IncuCyte®-based cytotoxicity assay was set up, using freshly thawed T cells, originating from the same ex vivo expansion process. In more detail, A375-NucLight^TM cells and T cells were co-cultured at a 1:1 or 1:5 effector-to-target ratio with images taken at 2-hour intervals. Both EF1α_NY-ESO-1 and EF1α_c-JunWT__NY-ESO-1 T demonstrated significant killing potential with no differences observed between constructs or donors (FIGs. 18A and 18B). [0195] In addition to T cell phenotyping of the cells pre-dosing, cellular viability, determined by Annexin V/Helix NP staining, was measured. The viability status showed that the percentage of viable cells obtained via cell counter and flow cytometer were of similar levels, with the apoptotic fraction accounting for <2% of the total population (FIG.19A). Additionally, a blood PK flow cytometry analysis of the T cells in the blood at 24 hours post- infusion showed equivalence in the TCR⁺ T cell number between the groups, with no significant difference observed between treatment groups for each of the donors (FIG.19B). These data provided confidence in the accuracy of the total cell doses delivered to the mice in the in vivo efficacy study. [0196] The PK profile of the TCR⁺ T cells was monitored over the in vivo study duration and showed persistence of the cells in the blood, with an increase in the total TCR⁺ T cell number for the treatment groups at study endpoint (FIGs.19C and 19D). Multiple t test comparison was performed for each time-point where there was data. No significant difference was seen between groups. 8.3: Impact of c-Jun Overexpression on Anti-tumor Efficacy of NY-ESO-1 TCR T Cell Treatment of A-375 Tumor-Bearing Mice [0197] The in vivo study demonstrated that EF1α_c-JunWT_NY-ESO-1 did not significantly differ from EF1α_NY-ESO-1 treatment in terms of the mean tumor volume at D14 or D28, although there was a decrease in tumor volume with EF1α_c-JunWT_NY-ESO- 1 treatment at the later time-point. EF1α_c-JunWT_NY-ESO-1 treatment resulted in significantly smaller tumors at an intermediate time-point, D21 (FIG.20A). In the MND treatment groups, however, c-Jun expression resulted in significantly larger tumors at D14 and a nominal increase in tumor volumes at both intermediate- and end- points (D21 & D28) (FIG.20B). [0198] An analysis of the time taken to reach an arbitrary tumor volume (1000mm³), illustrated via time versus target volume graphs for both EF1α (FIG.20C) and MND (FIG. 20D) groups, showed that the EF1α_c-JunWT_NY-ESO-1 group had increased mouse survival and time to target volume, as compared to EF1α_NY-ESO-1 treatment, indicating slower tumor growth and therefore enhanced efficacy in this group (FIG.20C). A significant difference was observed in the ratio of time to 1000mm³ in EF1α_c-JunWT_NY-ESO-1 compared to EFla_NY-ESO-l treatment (p= 0.014). The converse effect \ the MND_c-JunWT_NY-ESO-l group, where a significantly faster tumor growth was observed as compared to MND_NY-ESO-l treatment, with a reduced time to target volume, indicating a lack of efficacy in this group (p= 0.002) (FIG. 20D).

8.4: Histopathological Examination ofNY-ESO-1 Target Antigen Expression and TCR⁺ T Cell Infiltration of Fixed Tumors Taken fromA-375 Tumor-Bearing Mice [0199] H&E and IHC was performed to confirm the presence of tumor and NY-ESO-1 antigen expression in the A-375 subcutaneous tumors in the different treatment groups. Histopathological examination confirmed the presence of tumor and moderate to strong expression (H-score between 140-240) ofNY-ESO-1 antigen in the A-375 subcut tumors across different treatment groups (FIG. 21A). A significant decrease in NY-ESO-1 expression was observed between NTD and both TCR T cell-treated groups.

[0200] CD3 IHC and RNAscope™ for NY-ESO-1 TCR was performed to determine the extent of human CD3⁺TCR⁺ T cells infiltration in the tumors. The presence of human T cells was low in the NTD groups, and significantly higher levels of T cell infiltration were observed in the majority of both TCR T cell treated groups (FIG. 21B and FIG. 21C). No significant differences were found in CD3⁺TCR⁺ T cell tumor infiltration between EFla c- JunWT_NY-ESO-l and EFla_NY-ESO-l treated groups.

8.5: Summary

[0201] The above data show that there was a statistically significant reduction in PD- 1⁺CD4⁺ cells within the MND_c-JunWT_NY-ESO-l TCR¹ and EFla_c-JunWT_NY-ESO-l TCR+ T cell groups as compared to both NTD and MND_NY-ESO-l or EFla_NY-ESO-l T cells.

[0202] Further, within the CD8⁺ subpopulation of T cells, an overall decrease in %Tscm and increase in %Tcm was observed in MND_c-JunWT_NY-ESO-l and EFla c- JunWT_NY-ESO-l TCR T cells, as compared to MND_NY-ESO-l and EFlo_NY-ESO-l TCR T cells. The MND_c-JunWT_NY-ESO-l TCR T cell group demonstrated the highest levels of differentiated cell subpopulations.

[0203] Observations from the IncuCyte®-based cytotoxicity assay provided confidence in cytotoxic potential of the T cells, with no differences observed between donors or EFlaJMY- ESO-1 and EFla_c-JunWT_NY-ESO-l TCR T cells. [0204] The pre-infusion T cell viability status showed that there was no increased apoptotic cell fraction upon thaw and a blood PK flow cytometry analysis of the T cells in the blood at 24 hours post-infusion showed equivalence in the TCR⁺ T cell number between the groups, giving overall confidence in the TCR⁺ T cell dose administered for each of the treatment groups. [0205] The in vivo study demonstrates that the NY-ESO-1 TCR products showed enhanced anti-tumor efficacy and increased T cell persistence in the blood as compared with NTD control cells. Furthermore, within the fixed tumors taken at study endpoint, histological analysis showed the presence of human T cells was low to negligible in the NTD groups, and significantly higher levels of T cell infiltration were observed in the TCR⁺ T cell treated groups. Additionally, a significant decrease in NY-ESO-1 antigen expression was observed in the TCR⁺ T cell-treated groups as compared to the control. [0206] A comparison of the treatment groups showed that EF1α_c-JunWT_NY-ESO-1 had smaller tumors at D21 and D28 time-points as compared to EF1α_NY-ESO-1 treatment, with statistical significance reached at D21. An analysis of the time taken to reach an arbitrary tumor volume of 1000 mm³ showed that the EF1α_c-JunWT_NY-ESO-1 group had significantly increased time to target volume as compared to EF1α_NY-ESO-1 treatment, indicating slower tumor growth and therefore enhanced efficacy in this group. This enhanced efficacy was not due to increased levels of T cells in the tumor as no significant differences were found in a histological analysis of CD3⁺TCR⁺ T cell tumor infiltration between EF1α_c- JunWT_NY-ESO-1 and EF1α_NY-ESO-1 treated groups. The converse effect was observed for the MND treatment, where c-Jun expression resulted in significantly larger tumors at D14 and a nominal increase in tumor volumes at both intermediate- and end- points (D21 & D28). Additionally, the MND_c-JunWT_NY-ESO-1 group showed a significantly faster tumor growth as compared to MND_NY-ESO-1 treatment, with a reduced time to target volume, indicating a lack of efficacy in this group. [0207] Single cell RNA sequencing on tumor-infiltrating lymphocytes suggest that EF1α_c-JunWT_NY-ESO-1 T cells had reduced expression of exhaustion markers compared to EF1α_NY-ESO-1 at the end of the in vivo study. [0208] Taken together, these data support the hypothesis that c-Jun overexpression in the context of the EF1α promoter can prevent dysfunctional states of NY-ESO-1 TCR T cell products and lead to enhanced efficacy against NY-ESO-1-expressing tumor models. Example 9: Comparison of EFla NY-ESO-l and EFla_ c-JunWT_N T Cells on Tumor Growth and Ex Vivo Readouts in A-375 CDX Tumor-Bearing Mice [0209] The experiments described in Example 8 were repeated with two doses of T cells and additional ex vivo readouts to measure whether the T cell response was preserved when removing T cells from the tumor at the end of the study.

9.1: Materials and Methods

Tumor and T cell Preparations and Inoculation [0210] The in vivo study was conducted as described in Example 8 with the following exceptions:

- T cells were as isolated as previously described with the following changes: CD4⁺ and CD8⁺ T cells were isolated together (rather than separately) using a Miltenyi CliniMACS Prodigy®; and CD4:CD8 ratio was not adjusted to 50:50, but instead left at the ratio isolated initially and varied between donors; - Only a comparison between EFla_NY-ESO-l and EFla_c-JunWT_NY-ESO-l

TCR-T cells was made;

- The primary readout of the efficacy study was impact on tumor growth and mouse survival. Secondary readouts were (a) Blood PK assay to investigate T cell persistence over time, (b) assessment of TCR⁺ T cell presence in tumors and murine tissues by CD3 IHC and NY-ESO-l TCR RNAscope®, (c) evaluation of

NY-ESO-l antigen expression in tumors by IHC, and (d) assessment of ex vivo activity of tumor-infiltrating lymphocytes.

- A dose of 3xl0⁶ T cells was assessed in addition to the dose of 5xl0⁶ cells assessed in Example 8; and - No harvest occurred on study days 20-21.

[0211] The following donors, constructs and number of mice were used and untransduced cells were injected as top dose per donor (Table 4):

Table 4: Construct and Dosing Protocol

Ex vivo Stimulation and Cytokine Measurements [0212] T cell:tumor cell cocultures were performed as described above in Example 8, except a 3:1 ratio of T cell: tumor cell was used, instead of a 1:1 ratio.

[0213] MSD was performed as described above in Example 8, except that a U-Plex CAR- T ceil combo 1 (hu) kit was used instead of a V-plex® Proinflammatory Panel 1 Human Kit (Meso Scale Diagnostics).

9.2: T cell Characterization and Blood PK Profile

[0214] Prior to in vivo administration, flow cytometry and a cytotoxicity assay were performed and resulted in similar data as described above in Example 8.

[0215] A blood PK flow cytometry analysis of the T cells in the blood at 24 hours post- infusion showed a reduction in the TCR⁺ T cell number, particularly for donor 1 EFla c- JunWT_NY-ESO-l compared to the higher dose of EFla_NY-ESO-l (FIGs. 23A-23B). These differences could potentially be due to technical problems at time of infusion or different in vivo fate. An increase in the total TCR⁺ T cell number for the treatment groups at study endpoint was observed, similar to what was observed in Example 8.

9.3: Impact of c-Jun Overexpression on Anti-Tumor Efficacy of NY-ESO-1 TCR T Cell Treatment of A-375 Tumor-Bearing Mice

[0216] The in vivo study showed low tumor growth control for Donor 1 for all constructs, indicating technical problems for this donor (FIG. 24). For Donor 3, efficacy of all constructs was visible without significant differences between constructs. Similar observations were made when assessing Kaplan Meier survival curves for the time to reach the arbitrary tumor volume of 1000 mm³ (FIG. 25).

9.4: Impact of c-Jun Overexpression on Serum IFN-g of NY-ESO-1 TCR T Cell Treatment of A-375 Tumor-Bearing Mice

[0217] IFN-g in the serum of mice was measured as an indication of an ongoing immune response. At both T cell doses, an initial peak of IFN-g was observed with EFla_c- JunWT_NY-ESO-l compared to EFla_NY-ESO-l and untransduced (NTE first 2 weeks of the study (FIG. 26). At later time points, all constructs and untransduced T cells had an increase in IFN-g, potentially indicative of an emerging graft-versus-host response.

9.5: Ex Vivo Stimulation of Tumor-Infiltrating Lymphocytes to Assess Cytokine Production

[0218] An additional ex vivo stimulation of tumor-infiltrating lymphocytes at the end point of the in vivo study was conducted to assess whether T cell function was preserved. Despite the lower percentage of TCR⁺ T cells for EFla_c-JunWT_NY-ESO-l (numbers in brackets, FIG. 27), cytokine secretion was increased, suggesting increased functionality of EF 1 a_c-JunWT_NY -ESO- 1 compared to EFla_NY-ESO-l.

9.6: Summary [0219] In contrast to the results of the in vivo study in Example 8, no significant difference in efficacy was observed for EFla_c-JunWT_NY-ESO-l compared to EFla_NY-ESO-l. However, the lack of increased efficacy was likely due to the fact that these mouse models developed graft-versus-host disease.

[0220] Serum IFN-g observed at early timepoints suggests an increased response of EFla_c-JunWT_NY-ESO-l compared to EFla_NY-ESO-l which was masked by elevated IFN-g at the end of the study, indicative of a graft-versus-host response.

[0221] IFN-g secretion upon stimulation of tumor-infiltrating T cells ex vivo suggest an increased response of EFla_c-JunWT_NY-ESO-l compared to EFla_NY-ESO-l and thus preserved functionality by c-Jun overexpression. [0222] Taken together, the observed secondary endpoints of enhanced T cell function and preserved T cell activity indicate increased functionality of EFla_c-JunWT_NY-ESO-l compared to EFla_NY-ESO-l which may result in increased effector functions in more suitable mouse models that do not develop graft-versus-host disease. Example 10: Comparison of EF1α _NY-ESO-1 and EF1α_ c-JunWT_NY-ESO-1 TCR⁺ T cells on Tumor Growth in A-375 CDX Tumor-Bearing Mice After Intravenous Injection of Tumor Cells [0223] The same mouse model described in Examples 8 and 9 was used, except that tumor cells were injected intravenously instead of subcutaneously to allow the establishment of tumors in internal organs. 10.1: Materials and Methods: Tumor and T cell Preparations and Inoculation [0224] The in vivo study was conducted as described in Example 8 with the following exceptions: - Instead of A-375 tumor cells, luciferase⁺ A-375 was used; - The primary readout of the efficacy study was impact on tumor growth. Secondary readouts were (a) assessment of TCR⁺ T cell presence in tumors and murine tissues by CD3 IHC and NY-ESO-1 TCR RNAscope®, and (b) assessment of ex vivo activity of tumor-infiltrating lymphocytes; - Tumor burden was measured by bioluminescence using the Bruker In Vivo Xtreme imaging system and randomized by signal strength; - Transduction efficiency and phenotype on the day of thawing were not assessed; and - Cells were injected intravenously to establish tumors in internal organs and the following constructs and number of mice were used (Table 5): Table 5: Construct and Dosing Protocol Treatment – T cells Dose (TCR+ T cells) Mice/ group Donor 1: 3035610 EF1a NY-ESO-1 1x10⁶ 10

0. : mpact of c-Jun Overexpression on nti-tumor fficacy of N - SO- C Cell Treatment of Mice Bearing Tumors After Intravenous Injection of A-375 [0225] Intravenous injection of tumor cells results in tumor development in internal organs. This model was used to assess efficacy of EF1a_c-JunWT_NY-ESO-1 compared to EF1a_NY-ESO-1 and untransduced (NTD) T cells. Both constructs showed significantly increased tumor control compared to untransduced T cells (FIG.28). However, there was no difference in tumor growth control between EF1a_c-JunWT_NY-ESO-1 compared to EF1a_NY-ESO-1. [0226] CD3 IHC and RNAscope^TM were performed and similarly to Example 8, tumor cells expressed the antigen and there was infiltration of transduced T cells into the tumor, but without difference between the constructs. [0227] The presence of IFN-γ was measured in the serum as readout for an active immune response. Both constructs showed higher serum cytokine levels compared to untransduced cells and EF1a_c-JunWT_NY-ESO-1 had higher levels of IFN-γ compared to EF1a_NY- ESO-1, particularly towards the end of the in vivo study (FIG.29). 10.3: Summary [0228] In contrast to the results of the in vivo study in Example 8, no significant difference in efficacy was observed for EF1α_c-JunWT_NY-ESO-1 compared to EF1α_NY-ESO-1. [0229] Serum IFN-γ suggests an increased response of EF1α_c-JunWT_NY-ESO-1 compared to EF1α_NY-ESO-1 which was further elevated at the end of the study. [0230] Taken together, the secondary endpoint of enhanced T cell function as assessed by serum IFN-γ indicates increased functionality of EF1α_c-JunWT_NY-ESO-1 compared to EF1α_NY-ESO-1 which may result in increased effector functions in more suitable mouse models that do not have graft-versus host response. Example 11: Assessment of FOXP3 and CD25 Expression of T Cell Products After Stimulation by Flow Cytometry [0231] This example describes the study that evaluated the expression of FOXP3 in EF1 ^_NY-ESO-1 TCR versus EF1 ^_c-JunWT_NY-ESO-1 TCR T cells after by stimulation by A-375 target cells in the absence or presence of TGF-β. [0232] Expression of FOXP3, a transcription factor known to control the differentiation and function of regulatory T cells (Treg), has been shown to be induced in CD8⁺ T cells after TCR stimulation in vitro and in vivo and can be increased in the presence of TGF-β (Lozano et al., Cancer Letters (2022) 528:45–58). Increased FOXP3 expression by CD8⁺ T cells has been associated with impairment of the proliferation, cytokine production, lytic activity, and antitumor efficacy, as well as immunosuppressive functions (Kiniwa et al., Clin Cancer Res. (2007) 13(23):6947-58; Bisikirska et al., J Clin Invest. (2005) 115:2904-13). Transcriptional profiling of EF1 ^_NY-ESO-1 TCR and EF1 ^_c-JunWT_NY-ESO-1 TCR T cell products that had been exposed to persistent antigen stimulation in vitro showed that c-Jun overexpression reduced the proportion of CD8⁺ T cells expressing FOXP3. [0233] On Day 0 before stimulation, a low level of FOXP3 was expressed by the CD4⁺ and CD8⁺ T cells from the mock and the EF1 ^_NY-ESO-1 TCR and EF1 ^_c-JunWT_NY- ESO-1 TCR T cell products (i.e., between 2-5% FOXP3⁺CD4⁺ T cells and between 1-4% FOXP3⁺CD8⁺ T cells was observed) (Table 6). Table 6: Day 0 FOXP3⁺ Values CD8⁺ T Cells (%FOXP3⁺)

[0 3 ] owever, a ter 7 ays o -375 target ce st mu at on, t e O 3 express on was increased in both the CD4⁺ and CD8⁺ T cells from the EF1 ^_NY-ESO-1 TCR and EF1 ^_c- JunWT_NY-ESO-1 TCR T cell products (FIGs.30A-30C). The majority of the CD4⁺ and CD8⁺ T cells from the EF1 ^_NY-ESO-1 TCR and EF1 ^_c-JunWT_NY-ESO-1 TCR T cell products expressing FOXP3 after 7 days of target cell stimulation was found to also express high levels of CD25 (FIGs.30A-30C). A decrease in the proportion of CD25^highFOXP3⁺CD8⁺ T cells was observed from the EF1α_c-JunWT_NY-ESO-1 TCR T cell products compared to the EF1α_NY-ESO-1 TCR T cell products after 7 days of target cell stimulation in the absence or presence of TGF- ^ (i.e., 2-8% compared to 14-30%, respectively, in the absence of TGF- ^; and 5-19% compared to 20-35%, respectively, in the presence of TGF- ^), with a significant decrease observed in the presence of TGF- ^ (p= 0.0174 from paired t-test) (FIGs.30A-30D and Table 7). Table 7: Day 7 CD25^highFOXP3⁺ Values CD8⁺ T Cells (%CD25^highFOXP3⁺) Donor Mock EF1α NY-ESO-1 EF1α c-JunWT NY-ESO-1

[0 35] n contrast, tt e erence was o serve n t e percentage o C 5 O 3⁺ CD4⁺ T cells from the EF1α_c-JunWT_NY-ESO-1 TCR T cell products compared to the EF1α_NY-ESO-1 TCR T cell products after 7 days of target cell stimulation in the absence or presence of TGF- ^ (i.e., 5-15% compared to 5-17%, respectively, in the absence of TGF- ^ and 3-8% compared to 4-12%, respectively, in the presence of TGF- ^) (FIGs.30A-30D and Table 7). In the mock T cells, which did not receive TCR-mediated stimulation after co- culture with the A-375 target cells, the percentage of CD25^highFOXP3⁺ cells was below 2% within both the CD4⁺ and CD8⁺ T cell populations (Table 7). [0236] Together these results show that stimulation of NY-ESO-1 TCR T cell products by A-375 target cells in the absence or presence of TGF- ^ can increase the FOXP3 expression by CD4⁺ and CD8⁺ T cells and that c-Jun overexpression can reduce the proportion of CD8⁺ T cells from the NY-ESO-1 TCR T cell products that become FOXP3 positive after stimulation.

Claims

1. An expression construct comprising one or more expression cassettes for expressing: a) a T cell receptor (TCR) that specifically binds to a peptide from a human NY- ESO-1 protein complexed with an HLA-A molecule; and b) a human c-Jun polypeptide.

2. A method of reducing dysfunction of an engineered immune cell, comprising introducing into the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises one or more expression constructs comprising one or more expression cassettes for expressing: a) a T cell receptor (TCR) that specifically binds to a peptide from aNY-ESO-1 protein complexed with an MHC class I molecule; and b) a human c-Jun polypeptide.

3. The expression construct of claim 1 or the method of claim 2, wherein the c-Jun is a wildtype human c-Jun, optionally comprising SEQ ID NO: 13 or 16, or an amino acid sequence at least 90% identical thereto.

4. The expression construct of claim 1 or the method of claim 2, wherein the c-Jun is a mutant human c-Jun, optionally comprising an inactivating mutation in its transactivation domain or delta domain.

5. The expression construct or method of claim 4, wherein the c-Jun comprises (i) S63A and S73A mutations or (ii) a deletion between residues 2 and 102 or between residues 30 and 50 as compared to wildtype c-Jun.

6. The expression construct or method of any one of the preceding claims, wherein the peptide is human NY-ESO-1157-165 (SEQ ID NO: 19) and the HLA-A molecule is HLA-A*02.

7. The expression construct or method of any one of the preceding claims, wherein the TCR comprises an a chain and a b chain, wherein the a chain comprises the CDRl-3 in SEQ ID NO:5 and the b chain comprises the CDRl-3 in SEQ ID NO:6.

8. The expression construct or method of claim 7, wherein the TCR a comprise SEQ ID NOs:7-9, respectively, and the TCR b chain CDRl-3 comprise SEQ ID NOs: 10-12, respectively.

9. The expression construct or method of claim 7, wherein the TCR a chain comprises a variable domain comprising SEQ ID NO:5 or an amino acid sequence at least 90% identical thereto, and the TCR b chain comprises a variable domain comprising SEQ ID NO:6 or an amino acid sequence at least 90% identical thereto.

10. The expression construct or method of claim 9, wherein the TCR a and b chains comprise SEQ ID NOs:3 and 4, respectively, or SEQ ID NOs: 17 and 18, respectively.

11. The expression construct or method of any one of the preceding claims, wherein the expression construct is a viral vector, optionally selected from a lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia vector, herpes simplex viral vector, and Epstein-Barr viral vector.

12. The expression construct or method of any one of the preceding claims, wherein the expression construct comprises a tri-cistronic expression cassette for expressing c-Jun, a TCR a chain, and a TCR b chain.

13. A tri-cistronic expression construct comprising an expression cassette for expressing: a) an ab T cell receptor (TCR) that specifically binds to human NY-ESO-I157-165 peptide complexed with HLA-A*02; and b) a human c-Jun polypeptide.

14. The expression construct or method of the preceding claims, wherein the expression cassette is a tri-cistronic expression cassette comprising a coding sequence for SEQ ID NO: 13 and coding sequences for SEQ ID NOs:3 and 4, optionally wherein the coding sequences are separated in frame by a sequence selected from a 2A-coding sequence and a furin cleavage consensus sequence.

15. The expression construct or method of claim 14, wherein the coding sequence for SEQ ID NO: 13 comprises SEQ ID NO:21, the coding sequence for SEQ ID NO: 3 comprises SEQ ID NO: 1 the coding sequence for SEQ ID NO:4 comprises SEQ ID NO:2, and/or the expression construct comprises SEQ ID NO: 14, or a nucleotide sequence at least 80% identical thereto.

16. The expression construct or method of any one of the preceding claims, wherein the expression cassette comprises a constitutive or inducible promoter, optionally an EF-la promoter, optionally wherein the expression construct is a lentiviral vector.

17. A recombinant virus comprising the tri-cistronic expression construct of claims 13-16, optionally wherein the expression construct is a lentiviral vector.

18. A method of engineering immune cells, comprising: a) introducing the expression construct(s) of any one of claims 1 and 3-16 or the recombinant virus of claim 17 into a starting cell population, b) optionally selecting cells that express the TCR and the c-Jun, and c) deriving engineered immune cells from the cells of step a) or b), optionally wherein the immune cells are human cells.

19. The method of claim 18, wherein the starting cell population comprises immune cells, optionally autologous or allogeneic T cells.

20. The method of claim 18, wherein the starting cell population comprises pluripotent or multipotent cells, and step c) comprises differentiating the cells of step a) or b) into immune cells, optionally T cells.

21. A population of human cells comprising the expression construct(s) of any one of claims 1 and 3-16 or the recombinant virus of claim 17, optionally wherein the human cells are immune cells.

22. A population of immune cells obtained by the method of any one of claims 2-16 and 18-20, optionally wherein the immune cells are human cells.

23. The cells of claim 21 or 22, wherein the cells are T cells, optionally CD8⁺ T cells.

24. The cells of any one of claims 21-23, wherein the cells i) express a lower level of an exhaustion marker, optionally wherein the exhaustion marker is CD39, PD-1, TIGIT, TIM-3, or LAG-3, and/or ii) express a higher level of IL-2 and/or IFN-g, as compared to corresponding cells that do not overexpress c-Jun.

25. The cells of claim 24, wherein the cells are T cells and wherein i) no more than about 5%-15% of the T cells are TIGIT positive after 14 days of persistent antigen stimulation, ii) no more than about 2%-5% of the T cells are PD-1 positive after 14 days of persistent antigen stimulation, iii) no more than about 20%-45% of the T cells are CD39 positive after 14 days of persistent antigen stimulation, iv) the T cells secrete at least about 2-fold more IL-2, INF-g, and/or TNF-a at day 0 and/or day 14 of persistent antigen stimulation at a 1:1, 1:5, 1:10, or 1:20 ratio of T cells to target cells, as compared to a control population of engineered T cells that do not overexpress c-Jun, and/or v) the T cells proliferate at least about 2-fold more in response to antigen as compared to a control population of engineered T cells that do not overexpress c-Jun.

26. A pharmaceutical composition comprising the expression construct of any one of claims 1 and 3-16, the recombinant virus of claim 17, or the cells of any one of claims 21-25, and a pharmaceutically acceptable carrier.

27. A method of killing target cells, comprising contacting the target cells with the immune cells of any one of claims 21-25 or the pharmaceutical composition of claim 26 under conditions that allow killing of the target cells by the immune cells, wherein the target cells are cancer cells expressing NY-ESO-1, optionally wherein the immune cells express a lower level of an exhaustion marker when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression, optionally wherein the exhaustion marker is CD39, PD-1, TIGII

3, and optionally wherein the immune cells comprise T cells, optionally CD8⁺ T cells.

28. A method of treating a patient in need thereof, comprising administering the human cells of any one of claims 21-25 or the pharmaceutical composition of claim 26 to the patient, optionally wherein the patient is a human.

29. The method of claim 28, wherein the patient has a NY-ESO-1 -expressing cancer, optionally metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, bladder cancer, or myxoid round cell liposarcoma (MRCLS).

30. Use of the expression construct of any one of claims 1 and 3-16, the recombinant virus of claim 17, or the human cells of any one of claims 21-25 for the manufacture of a medicament for treating a patient, optionally a human patient, in need thereof in the method of any one of claims 27-29.

31. The expression construct(s) of any one of claims 1 and 3-16, the recombinant virus of claim 17, the human cells of any one of claims 21-25, or the pharmaceutical composition of claim 26, for use in treating a patient, optionally a human patient, in need thereof in the method of any one of claims 27-29.