WO2023077028A1

WO2023077028A1 - Enhanced t cell therapy targeting ny-eso-1

Info

Publication number: WO2023077028A1
Application number: PCT/US2022/078820
Authority: WO
Inventors: Suman Kumar VODNALA; Veena KRISHNAMOORTHY
Original assignee: Lyell Immunopharma, Inc.
Priority date: 2021-10-28
Filing date: 2022-10-27
Publication date: 2023-05-04

Abstract

Some aspects of the present disclosure are directed to methods of culturing T cells in a culture medium comprising at least about 55 mM potassium ion, wherein the T cells express an anti-NY-ESO-1 T cell receptor (TCR) that specifically binds the NY-ESO-1157-165/HLA-A*02 complex. Some aspects of the present disclosure are directed to a population of T cells cultured in a medium comprising at least about 55 mM potassium ion, wherein the T cells express an anti-NY-ESO-1 T cell receptor (TCR) that specifically binds the NY-ESO-1157-165/HLA-A*02 complex. Other aspects of the present disclosure are directed to methods of treating a disease or condition (e.g., a cancer) in a subject in need thereof comprising administering a population of T cells to the subject, wherein the population of T cells are cultured in a medium comprising at least about 55 mM potassium ion, and wherein the T cells express an anti-NY-ESO-1 T cell receptor (TCR) that specifically binds the NY-ESO-1157-165/HLA-A*02 complex.

Description

ENHANCED T CELL THERAPY TARGETING NY-ESO-1

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application Nos. 63/273,139 filed October 28, 2021, and 63/341,862 filed May 13, 2022, each of which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS- WEB

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name: 4385_080PC02_Seqlisting_ST26; Size: 54,531 bytes; and Date of Creation: October 27, 2022) filed with the application is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] The present disclosure is directed to methods of ex vivo or in vitro culture of Tcells, wherein the T cells express a T cell receptor (TCR) that specifically binds to a peptide from a human NY-ESO-1 protein complexed with an HL A- A molecule.

BACKGROUND

[0004] Cancer immunotherapy relies on harnessing T cells — the immune system’ s primary killers of infected and diseased cells — to attack and kill tumor cells. However, there is an important stumbling block for immunotherapy: T cells’ ability to kill can fade, a phenomenon often referred to as exhaustion or terminal differentiation of T cells. Immune checkpoint blockade, ex vivo- expanded Tumor-Infiltrating Lymphocytes (TILs) therapy, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor-engineered (TCR) T cell therapy are treatments that make use of functionally active T cells isolated from patients and require highly functional T cells in order to be effective. These T cells are engineered and expanded ex vivo to recognize antigens on target cancer cells. T cell therapies have not been consistently effective at curing solid cancers, in part because the T cells lose their ability to proliferate or kill over time.

[0005] One means of overcoming T cell exhaustion is to selectively administer T cells having a less-differentiated state. For example, T memory stem cells (TSCM) persist for a greater period in patients following administration than do more differentiated T central memory (TCM) or T effector memory (TEM) cells, and TSCM elicit a more pronounced and prolonged effect on tumor size than more differentiated cells. However, there remains a need in the art for methods of efficiently enriching for less differentiated and/or naive T cells from a mixed population of isolated T cells.

SUMMARY OF THE DISCLOSURE

[0006] Some aspects of the present disclosure are directed to methods of increasing sternness of human T cells ex vivo or in vitro comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds to the NY-ESO-li57-i65/HLA-A*02 complex.

[0007] Some aspects of the present disclosure are directed to methods of increasing the yield of human T cells during ex vivo or in vitro culture comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex. [0008] Some aspects of the present disclosure are directed to methods of preparing a population of human T cells for immunotherapy comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex.

[0009] Some aspects of the present disclosure are directed to methods of increasing sternness of human T cells while increasing the yield of human T cells during ex vivo or in vitro culture for an immunotherapy comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex.

[0010] Some aspects of the present disclosure are directed to methods of expanding a population of stem-like T cells ex vivo or in vitro comprising culturing T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex. In some aspects, the method further comprises transfecting the T cells with a polynucleotide encoding the TCR.

[0011] Some aspects of the present disclosure are directed to methods of preparing T cells ex vivo or in vitro for immunotherapy comprising transfecting T cells with a polynucleotide which encodes a T cell receptor (TCR) that specifically binds the NY-ESO-l i57-i65/HLA-A*02 complex; wherein the T cells are transfected in a medium comprising potassium ion at a concentration higher than 55 mM [0012] In some aspects, the TCR comprises an a chain and a P chain, wherein the a chain comprises the CDR1-3 in SEQ ID NO: 5 and the P chain comprises the CDR1-3 in SEQ ID NO: 6, wherein the TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex. In some aspects, the TCR a chain CDR1-3 comprise SEQ ID NOs: 7-9, respectively, and the TCR P chain CDR1-3 comprise SEQ ID NOs: 10-12, respectively. In some aspects, 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 P chain comprises a variable domain comprising SEQ ID NO: 6 or an amino acid sequence at least 90% identical thereto. In some aspects, the TCR a and P chains comprise SEQ ID NOs: 3 and 4, respectively, or SEQ ID NOs: 17 and 18, respectively.

[0013] In some aspects, the TCR that specifically binds the NY-ESO-l i57-i65/HLA-A*02 complex is encoded by an expression construct, 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. In some aspects, the expression construct comprises a bi-cistronic expression cassette for expressing a TCR a chain and a TCR P chain.

[0014] In some aspects, the concentration of potassium ion is higher than about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM. In some aspects, the concentration of potassium ion is selected from the group consisting of about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, and about 80 mM. In some aspects, the concentration of potassium ion is between about 30 mM and about 80 mM, about 40 mM and about 80 mM, about 50 mM and 80 mM, about 60 mM and about 80 mM, about 70 mM and about 80 mM, about 40 mM and about 70 mM, about 50 mM and about 70 mM, about 60 mM and about 70 mM, about 40 mM and about 60 mM, about 50 mM and about 60 mM, or about 40 mM and about 50 mM. In some aspects, the concentration of potassium ion is about 50 mM, about 60 mM, or about 70 mM.

[0015] In some aspects, the medium further comprises sodium ion. In some aspects, the medium further comprises NaCl. In some aspects, the medium comprises less than about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 90 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, or about 40 mM NaCl. [0016] In some aspects, the medium is hypotonic or isotonic. In some aspects, the medium is is less than 280. In some aspects, the medium is more than 240 and less than 280. In some aspects, the medium is more than or equal to 280 and less than 300.

[0017] In some aspects, the concentration of potassium ion is about 60 mM, and the concentration of NaCl is less than 80 mM, less than 75 mM, less than 70 mM, less than 65 mM, or less than 60mM. In some aspects, the concentration of potassium ion is about 55 mM, and the concentration of NaCl is less than 85 mM, less than 80 mM, less than 75 mM, less than 70 mM, or less than 65mM. In some aspects, the concentration of potassium ion is about 50 mM, and the concentration of NaCl is less than 90 mM, less than 85 mM, less than 80 mM, less than 75 mM, or less than 70 mM.

[0018] In some aspects, the medium further comprises one or more cytokines. In some aspects, the one or more cytokines comprise Interleukin-2 (IL-2), Interleukin-7 (IL-7), or Interleukin- 15 (IL- 15), or any combination thereof. In some aspects, the one or more cytokines comprise IL-2, IL-7, and IL-15.

[0019] In some aspects, the medium further comprises calcium ion, glucose, or any combination thereof.

[0020] In some aspects, the medium is capable of: (i) increasing the number and/or percentage of less differentiated cells (e.g., stem-like T cells); (ii) increasing the number and/or percentage of more differentiated cells, (iii) increasing the number and/or percentage of both less differentiated cells (e.g., stem-like T cells) and more differentiated cells, (v) increasing in vivo viability; (iv) increasing cell potency; (v) preventing cell exhaustion; or (vi) any combination thereof; in the final cell product as compared to the starting T cells and/or the T cells cultured in cell medium different from MRM described herein.

[0021] In some aspects, the medium further comprises glucose. In some aspects, the concentration of glucose is more than about 10 mM. In some aspects, the concentration of glucose is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM. In some aspects, the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In some aspects, the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

[0022] In some aspects, the medium further comprises calcium ion. In some aspects, the concentration of calcium ion is more than about 0.4 mM. In some aspects, the concentration of calcium ion is from about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 mM to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about

1.6 mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 mM to about 1.6 mM, about 1.3 mM to about 1.7 mM, about 1.3 mM to about 1.8 mM, about 1.4 mM to about 1.5 mM, about 1.4 mM to about 1.6 mM, about 1.4 mM to about 1.7 mM, about 1.4 mM to about 1.8 mM, about 1.5 mM to about 1.6 mM, about 1.5 mM to about 1.7 mM, about 1.5 mM to about 1.8 mM, about 1.6 mM to about 1.7 mM, about 1.6 mM to about 1.8 mM, or about 1.7 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

[0023] In some aspects, the T cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, TCF7+, or any combination thereof, following the culturing.

[0024] In some aspects, the medium comprises IL-2 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL. In some aspects, the concentration of IL-2 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL. In some aspects, the concentration of IL-2 is about 1.0 ng/mL. In some aspects, the concentration of IL-2 is about 10 ng/mL.

[0025] In some aspects, the MRM comprises between about 50 lU/mL to about 500 lU/mL of IL-2. In some aspects, the culture medium comprises about 50 lU/mL, about 60 lU/mL, about 70 lU/mL, about 80 lU/mL, about 90 lU/mL, about 100 lU/mL, about 125 lU/mL, about 150 lU/mL, about 175 lU/mL, about 200 lU/mL, about 225 lU/mL, about 250 lU/mL, about 275 lU/mL, about 300 lU/mL, about 350 lU/mL, about 400 lU/mL, about 450 lU/mL, or about 500 IU/mL of IL-2.

[0026] In some aspects, the medium comprises IL-7 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL. In some aspects, the concentration of IL-7 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL. In some aspects, the concentration of IL-7 is about 1.0 ng/mL. In some aspects, the concentration of IL-7 is about 10 ng/mL.

[0027] In some aspects, the MRM comprises between about 500 lU/mL to about 1,500 lU/mL of IL-7. In some aspects, the culture medium comprises about 500 lU/mL, about 550 lU/mL, about 600 lU/mL, about 650 lU/mL, about 700 lU/mL, about 750 lU/mL, about 800 lU/mL, about 850 lU/mL, about 900 lU/mL, about 950 lU/mL, about 1,000 lU/mL, about 1,050 lU/mL, about 1,100 lU/mL, about 1,150 lU/mL, about 1,200 lU/mL, about 1,250 lU/mL, about 1,300 lU/mL, about 1,350 lU/mL, about 1,400 lU/mL, about 1,450 lU/mL, or about 1,500 lU/mL of IL-7. [0028] In some aspects, the medium comprises IL- 15 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL. In some aspects, the concentration of IL- 15 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL. In some aspects, the concentration of IL- 15 is about 1.0 ng/mL. In some aspects, the concentration of IL- 15 is about 10 ng/mL.

[0029] In some aspects, the MRM comprises between about 50 lU/mL to about 500 lU/mL of IL-15. In some aspects, the culture medium comprises about 50 lU/mL, about 60 lU/mL, about 70 lU/mL, about 80 lU/mL, about 90 lU/mL, about 100 lU/mL, about 125 lU/mL, about 150 lU/mL, about 175 lU/mL, about 200 lU/mL, about 225 lU/mL, about 250 lU/mL, about 275 lU/mL, about 300 lU/mL, about 350 lU/mL, about 400 lU/mL, about 450 lU/mL, or about 500 lU/mL of IL-15.

[0030] Some aspects of the present disclosure are directed to a population of human T cells prepared by a method disclosed herein. In some aspects, the cells are T cells, optionally CD8⁺ T cells. In some aspects the cells are a mixture of CD4+ and CD8+ T cells.

[0031 ] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising cells disclosed herein, and a pharmaceutically acceptable carrier.

[0032] Some aspects of the present disclosure are directed to methods of killing target cells, comprising contacting the target cells with T cells disclosed herein or a pharmaceutical composition disclosed herein under conditions that allow killing of the target cells by the T cells, wherein the target cells express NY-ESO-1. In some aspects, the target cells are cancer cells expressing NY-ESO-1. In some aspects, the targets cells are cancer cells expressing NY-ESO-1157- 165 complexed with HLA-A*02. [0033] Some aspects of the present disclosure are directed to methods of treating a patient in need thereof, comprising administering human cells disclosed herein or the pharmaceutical composition disclosed herein to the patient. In some aspects, the patient has a NY-ESO-1- expressing cancer. In some aspects, the patient has a cancer expressing NY-ESO-1157-165 complexed with HLA-A*02. In some aspects, the NY-ESO-1 -expressing cancer is selected from the group consisting of 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, and bladder cancer.

[0034] Some aspects of the present disclosure are directed to the use of human cells disclosed herein for the manufacture of a medicament for treating a patient in need thereof in a method disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGs. 1A-1B provide graphical representations of the proportions of NY-ESO-1 TCR T cells that express stem-like markers CCR7 and CD45RA following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2"). The naive and memory T-cell subsets were defined as follows: Naive T-cells/T stem cell memory (TN/TSCM) (CCR7+/CD45RA+/CD45RO-; also referred to as "T stem-like"), T central memory (TCM) (CCR7+/CD45RA-/CD45RO+), T effector memory (TEM) (CCR7-/ CD45RA-/CD45RO+), and T effector cells (CCR7-/CD45RA+/CD45RO- ; also referred to as "Temra"). Experiments were repeated using five healthy donors: D10744, D7179, D14174, D14294, and D18878 (FIG. 1A); and four synovial sarcoma patient samples: ESO 2, ESO 3, ESO 4, and ESO5 (FIG IB).

[0036] FIGs. 2A-2B are graphical representations (density flow plots) of the frequency of TCR7+ and/or CD39- T cells following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2"), using T cells obtained from five healthy donors (FIG. 2A) and four synovial sarcoma patient samples (FIG. 2B).

[0037] FIGs. 3A-3D are line graphs showing the percentage of less differentiated (e.g., stem-like) T cells (FIGs. 3A and 3C) and the percentage of more-differentiated (e.g., effector-like) T cells (FIGs. 3B and 3D) following culture in MRM (FIGs. 3A-3D), TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"; FIGs. 3A-3D), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2"; FIGs. 3A-3B), using T cells obtained from six healthy donors (FIGs. 3A-3B) and two synovial sarcoma patient samples (FIGs. 3C-3D).

[0038] FIGs. 4A-4B are graphical representations of the relative number of stem-like T cells (per 10⁶ CD3+/TCRv[3+ cells) following culture in MRM (FIGs. 4A-4B), TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"; FIGs. 4A-4B), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL- 7, and 200 lU/mL IL-15 ("Control 2"; FIGs. 4A), using T cells obtained from five healthy donors (FIG. 4A) and four synovial sarcoma patient samples (FIG. 4B).

[0039] FIGs. 5A-5T illustrate the differentiation and metabolic profile of T cells cultured in MRM or control media. FIGs. 5A-5E provide graphical representations (as shown by UMAP) of gene set enrichment analyses of CD8+ T cells. FIG. 5F is a UMAP of single-cell RNA-Seq data for CD8+ T cells from NY-ESO-1 TCR T-cell products produced at large-scale from 2 donors, comparing LYL132 (orange; 1) and the first-generation product (teal; 2). Each dot represents a single cell projected onto a 2-dimensional space. FIG. 5G shows clusters identified in product CD8+ T cells. Less-differentiated clusters are marked as "1" and "8", and the most-differentiated clusters are marked as "5" and "7". FIGs. 5H-5I are UMAP visualizations of TCF7 RNA expression (FIG. 5H) and CD39 protein expression (FIG. 51) measured by CITE-Seq. FIGs. 5J-5K are graphical representations of percentages of CD8+ T cells comprising less-differentiated and most- differentiated clusters, respectively, in CD8+ NY-ESO-1 TCR T-cell products. FIGs. 5L-5O are images of flow cytometry density plots showing CD39 and TCF7 protein expression in NY-ESO- 1 TCR T-cell products produced at large-scale from 2 donors, comparing MRM (FIGs. 5L and 5N) to controls (FIGs. 5M and 50), with analysis gated on live CD3+/TCRvpi3.1+ T cells. The frequency of the least-differentiated population (TCF7+/CD39-) is shown in the top left comer of each figure. FIGs. 5P-5T provide graphical representations (as shown by UMAP) of metabolic gene set enrichment analyses of CD4+ T cells. Bulk RNA-Seq analysis was performed on CD8+ or CD4+ T cells sorted from CD45+TCRv[313.1+ NY-ESO-1 TCR T cells expanded in MRM or control media.

[0040] FIGs. 6A-6B are western blot images showing the level of proteins associated with the OXPHOS pathway (Complex I-V; FIGs. 6A-6B), proteins associated with oxidative stress (Complex I, II and III; FIGs. 6A-6B), and proteins in autophagy pathway (LC3b I and LC3b II; FIG. 6A) in T cells following culture in MRM (FIGs. 6A-6B), TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"; FIG. 6B), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2"; FIG. 6A), using T cells obtained from two healthy donors (FIG. 6A) and three synovial sarcoma patient samples (FIG. 6B).

[0041] FIGs. 7A-7G are graphical representations (as shown by UMAP) of gene set enrichment analyses (single-cell RNA-seq data) of CD8+ NY-ESO-1 TCR T-cells expanded in MRM compared to cells expanded in control media from six healthy donors (FIGs. 7A-7E) and patient samples (FIGs. 7F-7G). Projection of the unfolded protein response gene set is shown on the same UMAP. CXCR3 and ITGA4 gene expression values measured by CITE-Seq are shown on the same UMAP represention. FIGs. 7A-7E show UMAP of single-cell RNA-Seq data for CD8+ T cells from NY-ESO-1 TCR T-cell products from 6 healthy donors produced at researchscale, comparing MRM and two control media. Each dot represents a single cell projected onto a 2-dimensional space. FIGs. 7F-7G show UMAP of single-cell RNA-Seq data for CD8+ T cells from NY-ESO-1 TCR T-cell products derived from 2 patients with synovial sarcoma, produced at research-scale, comparing MRM and control.

[0042] FIG. 8 provides graphical representations illustrating the proportion of T-cells producing multiple cytokines following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2"), using T cells from five healthy donors.

[0043] FIG. 9 provides graphical representations illustrating the proportion of T-cells producing multiple cytokines following culture in MRM or TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), using T cells from four synovial sarcoma patient samples.

[0044] FIGs. 10A-10B provides graphical representations of the frequency of divided cells after exposure for 5 days to H1703 without IL-2 (FIG. 10A, first panel), H1703 with IL-2 (FIG. 10 A, second panel) or A375 without IL-2 (FIG. 10 A, third panel) and the absolute count of CCR7+/CD62L+ T cells (FIG. 10B) following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL- 15 ("Control 2"). [0045] FIGs. 11A-11D are graphical representations of gene set enrichment analyses (single-cell RNA-seq data) of CD45+/TCRvpi3.1+ T cells expanded in MRM compared to cells expanded in control media. Clusters identified in single-cell RNA-Seq analysis of CD8+ NY-ESO- 1 TCR T-cells collected on Dayl4 of serial stimulation, shown by UMAP (FIG. 11 A). Each dot represents a single-cell projected onto a 2-dimensional space. Cluster Cl exhibited enrichment of both Gattinoni naive-associated gene signature score and CD39-CD69- double negative gene signature score. Clusters CO and C5 exhibited enrichment of Gattinoni effector-associated gene signature score (FIG. 11 A). Projection of refined CD39-CD69-DN gene signatures (left), as well as the naive-associated (middle) and effector-associated (right) gene sets described in Gattinoni et al., (Nature Medicine 77(70): 1290-1297 (2011)) (FIG. 1 IB). For each gene set, a normalized average expression level of the gene set was calculated on a single-cell level. CD39, CD69, CD27 and CD28 protein expression values measured by CITE-Seq, visualized in the same UMAP space (FIG. 11C). Proportions of the stem-like cluster Cl and the more-differentiated clusters (C0+C5) in the 2 donors (FIG. 1 ID).

[0046] FIG. 12 provides bar graphs illustrating cytotoxicity of NY-ESO-1 TCR T-cell products against A375 and H1703 cell lines at varying E:T ratios, following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL- 15 ("Control 2").

[0047] FIG. 13 provides graphs showing mean cytokine levels with ratio paired t-test analysis for NY-ESO-1 TCR T-cell products against A375, H1703, and COL0205 cells; following culture in MRM, TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 ("Control 1"), or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7, and 200 lU/mL IL-15 ("Control 2").

[0048] FIGs. 14A-14G present data illustrating the proliferative capacity, cytotoxicity and cytokine secretion of T cells cultuted in MRM as compared to control media. FIGs. 14A-14B are scatter plots illustrating T-cell expansion during serial restimulation with NY-ESO-1 antigen in NY-ESO-1 TCR T-cell products produced at large-scale from 2 donors. Shown is theoretical fold expansion of T cells from the control media (1) and MRM (2) on Days 0, 3, 7, 10, and 14 of the serial restimulation assay. FIGs. 14C-14D are scatter plots illustrating cytotoxicity at Day 14 of the serial restimulation assay (E:T ratio, 1 :10) for NY-ESO-1 TCR T-cell products derived from 2 donors. The mean fold change of NLR count over time zero was used to track target cell death for the control media (1) and MRM (2). Gray line (3) depicts NLR count of tumor only. FIGs. 14E- 14J are bar graphs illustrating cytokine secretion during serial restimulation by NY-ESO-1 TCR T cells. IFNy (FIGs. 14E-14F), IL-2 (FIGs. 14G-14H), and TNFa (FIGs. 14I-14J) cytokine secretion by cell cultured in control media or MRM was assessed on Day 14 post serial restimulation after overnight co-culture with A375 NLR cancer cell lines at an E:T ratio of 1 : 1.

[0049] FIGs. 15A-15G present data illustrating phenotypes of T cells evaluated after serial restimulation using transcriptional profiling by single-cell CITE-Seq. FIG. 15A is a UMAP of CD8+ T-cell clusters identified by single-cell RNA-Seq analysis of NY-ESO-1 TCR T cells produced at large-scale from 2 donors and collected on Day 14 of serial restimulation. Each dot represents a single cell projected onto a 2-dimensional space. The cluster exhibiting enrichment of both Gattinoni naive-associated gene signature score and CD39-/CD69- double-negative (DN) gene signature score is labelled as "1". FIG. 15B is a projection of the previously described naive- associated gene setl (left; Gattinoni L et al. Nat Med. 2011; 17(10): 1290-1297) and refined CD39- /CD69- DN gene signatures2 (right; Krishna S et al. Science. 2020;370(6522): 1328-1334). For each gene set, normalized average expression was calculated on a single-cell level. FIGs. 15C-15F are illustrations of CD39 (FIG. 15C), CD69 (FIG. 15D), CD27 (FIG. 15E), and CD28 (FIG. 15F) protein expression measured by CITE-Seq, visualized in the same UMAP space. FIG. 15G is a graphical representation of the proportions of Cl cells on Day 14 in T cells cultured in MRM or control media, derived from 2 donors.

DETAILED DESCRIPTION

[0050] T-cell therapies have shown curative potential for treatment of recurrent or high- risk tumors. Engineered TCR T-cell therapies targeting the cancer testis antigen, NY-ESO-1, have shown clinical activity with some patients having complete responses (Dudley et al., Science 298(5594)'.850-854 (2002); Robbins et al., J Clin Onco 29:917-24 (2011); Robbins et al., Clin Cancer Res 27(5/1019-27 (2015); Stadtmauer et al., Blood Advances 5(75 2022-34 (2019); D’Angelo et al., Cancer Discov 5(5/944-57 (2018)). NY-ESO-1 is expressed in -70% to 80% of patients with synovial cell sarcoma, -25% of patients with melanoma and common epithelial tumors, and -30% to 50% of patients with multiple myeloma. Not all patients benefit, however, from engineered T-cell therapies, and proposed improvements of these T-cell therapies include strategies to enhance T-cell sternness, metabolic fitness, product activity and persistence.

[0051] Traditional T-cell expansion conditions result in progressive differentiation of T- cells that is defined by an initial gain of effector functions and deterioration of long-term antitumor potential. The methods of the invention described herein, e.g., MRM, reprograms metabolic demands and preserves less differentiated, e.g., naive (TN) and/or stem cell memory (TSCM) cells, and reduces the progressive differentiation of the other T cell subsets to terminal effector T cells. Maintenance of such less differentiated cells in adoptive cell therapy products has been correlated with improved clinical efficacy in several studies (Rosenberg, CCR 2010, Fraietta et al; Krishna et al).

[0052] A growing body of evidence suggests that the performance of aerobic glycolysis, with rapid glucose consumption actively promotes effector differentiation, while enhancing the capacity for lipid consumption skews T-cell development towards memory formation and improved persistence (Sukumar et al., Journal of Clinical Investigation 723(70/4479-4488 (2013); O’Sullivan et al., Immunity 41(1) :75-88 (2014)). The methods of the invention described herein, e.g., MRM, promotes a state of nutrient conservation that limits the acquisition of exhaustion programs and augments phenotypic marker expression that correlates with sternness and persistence. Expansion of T cells in MRM induces functional caloric restriction which reprograms cells to stave off exhaustion and maintain persistence, self-renewal and thereby T-cell “sternness” - identifying a dynamic link between local ion concentrations, subcellular metabolite abundance, and T-cell anti-tumor capacity.

[0053] Some aspects of the present disclosure are directed to methods of preparing a population of T cells for immunotherapy comprising culturing T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the Tcells express a TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex. Some aspects of the present disclosure are directed to methods of increasing sternness, increasing the population of more- differentiated cells (e.g., effector-like cells), or increasing both sternness and the population of more-differentiated cells (e.g., effector-like cells), ex vivo or in vitro comprising culturing the Tcells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor TCR that specifically binds the NY-ESO-l i57-i65/HLA-A*02 complex.

[0054] Other aspects of the present disclosure are directed to methods of treating a patient in need thereof comprising administering a population of T cells prepared according to the methods disclosed herein to the patient. In some aspects, the patient has an NY-ESO-1 -expressing cancer.

[0055] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0056] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

I. Terms

[0057] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0058] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a chimeric polypeptide," is understood to represent one or more chimeric polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. In addition, "or" is used mean an open list of the components in the list. For example, “wherein X comprises A or B” means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

[0059] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0060] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of' are also provided.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0062] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, unless otherwise explicitly stated.

[0063] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0064] The terms "about" or "comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, z.e., the limitations of the measurement system. For example, "about" or "comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of' can mean a range of up to 10% (e.g., 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 (except where such number would exceed 100% of a possible value)). For example, "about 55 mM," as ued herein, includes 49.5 mM to 60.5 mM. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of' should be assumed to be within an acceptable error range for that particular value or composition.

[0065] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately," like the term "about," 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 (except where such number would exceed 100% of a possible value).

[0066] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0067] The term "control media" as used herein refers to any media in comparison to a metabolic reprogramming media (MRM) disclosed herein. Control media can comprise the same components as the metabolic reprogramming media except certain ion concentrations, e.g., potassium ion. In some aspects, metabolic reprogramming media described herein are prepared from control media by adjusting one or more ion concentrations, e.g., potassium ion concentration, as described herein. In some aspects, control media comprise basal media. In some aspects, control media thus comprises one or more additional components, including, but not limited to, amino acids, glucose, glutamine, T cell stimulators, antibodies, substituents, etc. that are also being added in the metabolic reprogramming media, but control media have certain ion concentrations different from the metabolic reprogramming media. The term "conventional methods" as used herein refers to culturing conditions that comprise a medium having less than 5 mM potassium, e.g., with a control media.

[0068] The term "culturing" as used herein refers to the controlled growth of cells ex vivo and/or in vitro. As used herein, "culturing" includes the growth of cells, e.g., T cells, e.g., one or more engineered T cell disclosed herein, during cell expansion, or cell engineering (e.g., transduction with a construct for expressing a TCR). In some aspects, the cultured cells are obtained from a subject, e.g., a human subject/patient. In some aspects, the cultured cells comprise T cells obtained from a human subject/patient. In some aspects, the cultured cells comprise one or more engineered T cell disclosed herein. In some aspects, the T cells are purified prior to the culture.

[0069] The term "expand" or "expansion," as used herein in reference to T cell culture refers to the process of stimulating or activating the cells and culturing the cells. The expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of less differentiated T cells, in a population of cultured cells, after the cells are stimulated or activated and cultured. Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease.

[0070] As used herein, the term "yield" refers to the total number of cells following a culture method or a portion thereof. In some aspects, the term "yield" refers to a particular population of cells, e.g., stem-like T cells in a population of T cells. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample.

[0071] As used herein, the term "metabolic reprogramming media," "metabolic reprogramming medium," or "MRM," as used herein, refers to a medium of the present disclosure, wherein the medium comprises potassium ion at a concentration higher than 55 mM. In some aspects, the MRM comprises a concentration of potassium ion of at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In certain aspects, the metabolic reprogramming media comprises about 40 mM to about 80 mM NaCl, about 55 mM to about 90 mM KC1, about 0.5 mM to about 3 mM calcium, and about 10 mM to about 25 mM glucose. In some aspects, the MRM further comprises an osmolality of about 250 to about 340 mOsmol.

[0072] As used herein, the term "higher than" means greater than but not equal to. For example, "higher than 4 mM" means any amount that is more than 4 mM, but which does not include 4 mM.

[0073] As used herein, the term "tonicity" refers to the calculated effective osmotic pressure gradient across a cell membrane, represented by the sum of the concentration of potassium ion and the concentration of sodium chloride (NaCl), multiplied by two. Tonicity can be expressed in terms of the osmolality (mOsm/kg) or osmolarity (mOsm/L) of the solution, e.g., the media. Osmolality and osmolarity are measurements of the solute osmotic concentration of a solvent per mass (osmolality) and per volume (osmolarity). As used herein, a solution, e.g., medium, is considered "isotonic" when the concentration of solutes in the media is equivalent to the concentration of solutes inside the cell. As used herein, an isotonic medium has a tonicity of about 280 mOsm/L (e.g., ([K+] + [NaCl]) X 2 = 280).

[0074] As used herein, a solution, e.g., a medium, is considered "hypotonic" if the concentration of solutes in the solution is lower than the concentration of solutes in the cell. As used herein, a hypotonic solution has a tonicity of less than 280 mOsm/L (e.g., ([K+] + [NaCl]) X 2 < 280). In some aspects, a hypotonic medium has a tonicity from at least about 210 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic medium has a tonicity from at least about 220 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic medium has a tonicity from at least about 230 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic medium has a tonicity from at least about 240 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic medium described herein has a tonicity of about 250 mOsm/L.

[0075] As used herein, a solution, e.g., a medium, is considered "hypertonic" if the concentration of solutes in the solution is higher than the concentration of solutes in the cell. As used herein, a hypertonic solution has an osmolality of greater than 300 mOsm/L (e.g., ([K+] + [NaCl]) X 2 > 300). In some aspects, a hypertonic medium described herein has a tonicity of about 320 mOsm/L.

[0076] In some aspects, the tonicity of the solution, e.g., medium is adjusted by increasing or decreasing the concentration of potassium ions and/or NaCl. In some aspects, the tonicity of a medium can be maintained by offsetting the increase of one solute with a decrease in a second solute. For example, increasing the concentration of potassium ion in a medium without changing the concentration of sodium ions can increase the tonicity of the medium. However, if the concentration of potassium ions is increased and the concentration of sodium ions is decreased, the tonicity of the original medium can be maintained.

[0077] As used herein, the terms "potassium," "potassium ion," "potassium cation," and "K+" are used interchangeably to refer to elemental potassium. Elemental potassium exists in solution as a positive ion. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising potassium ion include diluting a potassium-containing salt (e.g., KC1) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of potassium ion, can be described as comprising an equal molar (M) concentration of a salt comprising potassium.

[0078] As used herein, the terms "calcium ion" and "calcium cation" are used interchangeably to refer to elemental calcium. Elemental calcium exists in solution as a divalent cation. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising calcium ion include diluting a calcium-containing salt (e.g., CaCh) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of calcium ion, can be described as comprising an equal molar (M) concentration of a salt comprising calcium.

[0079] As used herein, the terms "sodium ion" and "sodium cation" are used interchangeably to refer to elemental sodium. Elemental sodium exists in solution as a monovalent cation. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising sodium ion include diluting a sodium-containing salt (e.g., NaCl) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of sodium ion, can be described as comprising an equal molar (M) concentration of a salt comprising sodium.

[0080] As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocytes produced or processed by the thymus gland. Non-limiting classes of T cells include effector T cells and helper T (Th) cells (such as CD4⁺ or CD8⁺ T cells).

[0081] As used herein, the term "memory" T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or which have been stimulated with, e.g., an anti-CD3 antibody (e.g., in vitro or ex vivo). T cells having a "memory-like" phenotype upon secondary exposure can reproduce to mount a faster and stronger immune response than during the primary exposure. In some aspects, memory T cells comprise central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), or any combination thereof.

[0082] As used herein, the term "stem cell-like memory T cells," "T memory stem cells," or "TSCM cells" refer to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.

[0083] As used herein, the term "central memory T cells" or "TCM cells" refer to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found within the lymph nodes and in peripheral circulation.

[0084] As used herein, the term "effector memory T cells" or "TEM cells" refer to memory T cells that express CD45RO but lack expression of CCR7 and CD62L. Because effector memory T cells lack lymph node-homing receptors (e.g., CCR7 and CD62L), these cells are typically found in peripheral circulation and in non-lymphoid tissues. In the presence of prolonged antigen exposure, such as in many cancers, more differentiated T cells, e.g., effector and effector memory T cells, often become exhausted and lose their anti-tumor function.

[0085] As used herein, the term "tissue resident memory T cells" or "TRM cells" refer to memory T cells that do not circulate and remain resident in peripheral tissues, such as the skin, lung, and the gastrointestinal tract. In certain aspects, tissue resident memory T cells are also effector memory T cells.

[0086] As used herein, the term "naive T cells" or "TN cells" refers to T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95. TN cells represent the most undifferentiated cell in the T cell lineage. The interaction between a TN cell and an antigen- presenting cell (APC) induces differentiation of the TN cell towards an activated TEFF cell and an immune response.

[0087] As used herein, the term "sternness," "stem cell-like," "stem -like," or "less- differentiated" refers to a T cell that expresses markers consistent with a more naive phenotype. For example, a less differentiated T cell can express one or more marker characteristic of a TN or a TSCM cell. In some aspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, and CD62L, and does not express CD45RO. In some aspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, CD62L, TCF7, CD27, and CD28, and does not express CD45RO. In some aspects, the methods disclosed herein promote T cells having a less- differentiated phenotype. Without being bound by any particular mechanism, in some aspects, the methods disclosed herein block, inhibit, or limit differentiation of less-differentiated T cells, resulting in an increased number of stem-like cells in culture. For example, it is generally thought that to effectively control tumors, adoptive transfer of less-differentiated T cells with a stem celllike memory or central memory phenotype are preferred. See Lynn, R.C., et al., Nature 576(7786):293-300 (2019); and Gattinoni, L., et a!., Nat Med 17(10/ 1290-1297 (2011).

[0088] Sternness is characterized by the capacity to self-renew, the multipotency, and the persistence of proliferative potential. In some aspects, sternness is characterized by a particular gene signature, e.g., a combined pattern of expression across a multitude of genes. In some aspects, the gene signature comprises one or more genes selected from ACTN1, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, 0CM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orfl3, GIPC3, WNT7A, FAM117B, BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LM07, CEP68, SDK2, MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, SORBS3, IL4R, SERPINF1, C16orf45, SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB, C16orf74, AEBP1, SNORD115-11, SNORD115-11, GRAP, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10) .1290-1297 (2011)). In some aspects, the gene signature comprises one or more gene selected from NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR1, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orfl0, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SNORA5C, UBASH3A, FLJ43390, RP6- 213H19.1, INPP5A, PIM2, TNFRSF10D, SNRK, LOC100128288, PIGV, LOC100129858, SPTBN1, PR0S1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD, TARBP1, STRADB, MT1F, TMEM41B, PDHX, KDM6B, LOC100288322, UXS1, LGMN, NANOS2, PYGB, RASGRP2, C14orf80, XPO6, SLC24A6, FAM113A, MRM1, FBXW8, NDUFS2, KCTD12, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10)'. 1290-1297(2011)). In some aspects, the gene signature comprises CD45RA, CD62L, CD27, CD28 and TCF7. Biomarkers, e.g., T cell markers, can be measured using any methods. In some aspects, T cells are identified using antibody-staining following by gated flow cytometry.

[0089] As used herein, the term "basal" media refers to any starting media that is supplemented with one or more of the additional elements disclosed herein, e.g., potassium, sodium, calcium, glucose, IL-2, IL-7, IL- 15, or any combination thereof. The basal media can be any media for culturing T cells. In some aspects, the basal media comprises a balanced salt solution e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Click’s medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM), alpha Minimal Essential Medium (alpha MEM), Iscove’s Modified Dulbecco's Medium (IMDM), M199, OPTMIZER™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, OPTMIZER™ Pro, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, PRIME-XV T cell CDM, X-VIVO™ 15 (LONZA) or any combination thereof. In some aspects, the basal medium further comprises immune cell serum replacement (ICSR).

[0090] As used herein, the term "cytokine" refers to small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells. Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL)-l, IL-2, IL-4, IL-7, IL-9, IL- 13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21 and IL-23), interferons (IFN; e.g., IFN-a, IFN-P, and IFN-y), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members. Some aspects of the present disclosure are directed to methods of culturing and/or expanding a population of T cells, e.g., one or more engineered T cell disclosed herein, in a medium comprising a cytokine. In some aspects, the cytokine is an interleukin. In some aspects, the cytokine comprises IL-2, IL-7, IL- 15, or any combination thereof. IL-2 (UniProtKB - P60568) is produced by T cells in response to antigenic or mitogenic stimulation. IL-2 is known to stimulate T cell proliferation and other activities crucial to regulation of the immune response. IL-7 (UniProtKB - P13232) is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. IL-7 is believed to play a role in proliferation during certain stages of B-cell maturation. IL-15 (UniProtKB - P40933), like IL-2, is a cytokine that stimulates the proliferation of T-lymphocytes.

[0091] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent to a subject or patient, using any of the various methods and delivery systems. The different routes of administration for a therapeutic agent described herein (e.g., a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex cultured as described herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.

The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0092] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "cognate antigen" refers to an antigen which a T cell recognizes and thereby, induces the activation of the T cell (e.g., triggering intracellular signals that induce effector functions, such as cytokine production, and/or proliferation of the cell). In some aspects, the antigen comprises the NY-ESO-l i57-i65 peptide. [0093] A "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" as used herein refers to primary, metastatic and recurrent cancers.

[0094] The term "hematological malignancy" or "hematological cancer" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues. Non-limiting examples of hematological malignancies include those affecting tissues of the blood, bone marrow, lymph nodes, and lymphatic system, including acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. Hematological malignancies are also referred to as "liquid tumors." Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies.

[0095] A "solid tumor," as used herein, refers to an abnormal mass of tissue. Solid tumors may be benign or malignant. Nonlimiting examples of solid tumors include sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of a solid tumor includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed, and which may provide a supporting microenvironment.

[0096] In some aspects, the cancer is selected from adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment. In some aspects, the cancer is selected from chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, myxoid/round cell liposarcoma, or telangiectaltic sarcoma. In some aspects, the cancer is selected from acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma. In some aspects, the cancer is selected from acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidernoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum. In some aspects, the cancer is selected from Leukemia, Hodgkin's Disease, Non- Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Mullerian cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma. In some aspects, the cancer is a NY-ESO-1 -expressing cancer. In some aspects, the NY-ESO-1- expression cancer is selected from 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.

[0097] As used herein, the term "immune response" refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of a cell of the immune system (e.g., a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4⁺ or CD8⁺ T cell, or the inhibition of a Treg cell.

[0098] As used herein, the term "anti-tumor immune response" refers to an immune response against a tumor antigen.

[0099] A "subject" or "patient" includes any human or nonhuman animal. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein. As used herein, the phrase "subject in need thereof includes subjects, such as mammalian subjects, that would benefit, e.g., from administration of T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- 1157-165/HLA-A*O2 complex, cultured as described herein, to control tumor growth. In some aspects, the patient is a human patient.

[0100] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex, cultured as described herein) that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to solid tumors, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations.

[0101] The effective amount of the composition (e.g., a T cell expressing an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex cultured as described herein) can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent and can stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and can stop tumor metastasis); (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0102] In some aspects, a "therapeutically effective amount" is the amount of a composition disclosed herein (e.g., a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex cultured as described herein), which is clinically proven to effect a significant decrease in cancer or slowing of progression (regression) of cancer, such as an advanced solid tumor. The ability of a therapeutic agent of the present disclosure (e.g., a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157-165/HLA- A*02 complex cultured as described herein) to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0103] The terms "effective" and "effectiveness" with regard to a treatment can include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a composition disclosed herein (e.g., cells cultured as described herein) to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ, and/or organism level (adverse effects) resulting from administration of a composition disclosed herein (e.g., cells cultured as described herein).

[0104] As used herein, the term "T cell receptor" or "TCR" refers to a heterodimer composed of 2 different transmembrane polypeptide chains: an a chain and a P chain, each consisting of a constant region, which anchors the chain inside the T-cell surface membrane, and a variable region, which recognizes and binds to the antigen presented by a major histocompatibility complex (MHC). The TCR complex is associated with 6 polypeptides forming 2 heterodimers, CD3ys and CD36s, and 1 homodimer CD3

which together forms the CD3 complex. T-cell receptor-engineered T-cell therapy utilizes the modification of T cells that retain these complexes to specifically target the antigens expressed by particular tumor cells. As used herein, the term "TCR" includes naturally occurring TCRs and engineered TCRs.

[0105] As used herein, an "engineered TCR" or "engineered T-cell receptor" refers to a T- cell receptor (TCR) engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC)/peptide target antigen that is selected, cloned, and/or subsequently introduced into a population of T cells. [0106] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0107] Various aspects described herein are described in further detail in the following subsections.

II. Metabolic Reprogramming Media

[0108] Some aspects of the present disclosure are directed to methods of culturing a T cell (e.g, a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- i65/HLA-A*02 complex disclosed herein or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnlGF-betaRII polypeptide disclosed herein) in a culture condition, wherein the culture condition (e.g., certain ion concentrations, tonicity of the medium, cytokines, and/or any combination thereof) is capable of reducing, limiting, or preventing the differentiation of the cells, thereby affecting or improving their use in a cell therapy. In some aspects, the T cells (e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) are cultured in MRM.

[0109] In some aspects, the present disclosure provides T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i6s/HLA-A*02 complex (or an anti- NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) of the present disclosure, grown in a medium having a high concentration of potassium ion (e.g., higher than about 55 mM, e.g., between 55 mM and 70 mM), are capable of preserving a stem-like phenotype (e.g., minimal differentiation) of the cultured cells. In some aspects, the cultured cells have more stem-like phenotypes (e.g., less differentiated) than cells grown in a medium having a lower potassium concentration. In some aspects, the medium further comprises interleukin (IL)-2, IL-7, IL- 15, or any combination thereof. In some aspects, the medium further comprises sodium ion, calcium ion, glucose, or any combination thereof.

[0110] In some aspects, a population of T cells, e.g, T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), cultured using the methods disclosed herein exhibits an increased number of stem-like cells relative to a population of cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects, the T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A 02 complex and a c-J n, ( D8 lpha co leceptor, and/or dnTGF-betaE.II polypeptide disclosed herein) of the present disclosure, exhibit increased expression of markers characteristic of stem-like cells relative to the starting population of T cells (i.e., prior to the culturing in a medium having potassium ion at a concentration higher than 55 mM).

[0111] In some aspects, the starting population of T cells comprises T cells obtained from a human subject. In some aspects, the starting population of T cells comprises TN cells, TSCM cells, TCM cells, TEM cells, or any combination thereof. In some aspects, the starting population of T cells comprises T cells prior to transfection with a construct encoding an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex.

[0112] Increased cell multipotency can be measured using any methods known in the art. In some aspects, cell sternness is measured by antibody staining followed by gated flow cytometry. In some aspects, the cell sternness is measured by autophagy flux. In some aspects, the cell sternness is measured by glucose uptake. In some aspects, the cell sternness is measured by fatty acid uptake. In some aspects, the cell sternness is measured by mitochondrial biomass. In some aspects, the cell sternness is measured by RNA quantification/expression analysis (e.g., microarray, qPCR (taqman), RNA-Seq., single-cell RNA-Seq., or any combinations thereof). In some aspects, the cell sternness is measured by transcripts that are linked to a metabolism assay (e.g., a Seahorse metabolism assay, analysis of extracellular acidification rate (ECAR); analysis of oxygen consumption rate (OCR); analysis of spare respiratory capacity; and/or analysis of mitochondrial membrane potential. In some aspects, sternness is measured using one or more in vivo or in vitro functional assays (e.g., assaying cell persistence, antitumor capacity, antitumor clearance, multipotency, cytokine relase assay, cell killing or any combination thereof).

[0113] In some aspects, the differentiation status of the T cells, e.g., engineered T cells of the present disclosure, is characterized by increased numbers of cells expressing markers typical of less differentiated cells. In some aspects, an increase in the number of stem-like cells is characterized by increased numbers of T cells, e.g., engineered T cells of the present disclosure, expressing markers typical of TN and/or TSCM cells. In some aspects, an increase in the number of stem-like T cells, e.g., engineered T cells of the present disclosure, is characterized by increased numbers of cells expressing markers typical of TSCM cells. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that express CD45RA. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that express CCR7. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that express CD62L. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that express CD28. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that express CD95. In some aspects, the cells do not express CD45RO. In some aspects, the population of cells cultured according to the methods disclosed herein exhibits an increased number of cells that are CD95⁺, CD45RA⁺, CCR7⁺, and CD62L⁺. In some aspects, the cells exhibit an increased number of cells that express TCF7. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that are CD95⁺, CD45RA⁺, CCR7⁺, CD62L⁺, and TCF7⁺. In some aspects, the cells express CD3. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that are CD3⁺, CD95⁺, CD45RA⁺, CCR7⁺, CD62L⁺, and/or TCF7⁺. In some aspects, the cells express CD27. In some aspects, the population of T cells, e.g., engineered T cells of the present disclosure, cultured according to the methods disclosed herein exhibits an increased number of cells that are CD27⁺, CD3⁺, CD95⁺, CD45RA⁺, CCR7⁺, CD62L⁺, and/or TCF7⁺. In some aspects, a population of T cells cultured according to the methods disclosed herein exhibits an increased number of TSCM cells. In some aspects, a population of T cells cultured according to the methods disclosed herein exhibits an increased number of TN cells. In some aspects, a population of T cells cultured according to the methods disclosed herein exhibits an increased number of TSCM and TN cells.

[0114] In some aspects, the number of stem-like cells in the culture is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, relative to the number of stem-like cells prior to culture. In some aspects, the number of stem-like cells in the culture is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5- fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the number of stem-like cells prior to culture.

[0115] In some aspects, following culture of T cells according to the methods disclosed herein, stem-like T cells constitute at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, or at least about 15% of the total number of CD8⁺ T cells in the culture. In some aspects, following culture of T cells according to the methods disclosed herein, stem-like T cells constitute at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, or at least about 15% of the total number of CD4⁺ T cells in the culture.

[0116] In certain aspects, the T cells are transduced using a viral vector. In some aspects, the vector comprises a lentiviral vector, retrovirus vector, adenoviral vector, adeno-associated viral vector, vaccinia vector, herpes simplex viral vector, and Epstein-Barr viral vector. In some aspects, the viral vector comprises a retrovirus. In some aspects, the viral vector comprises a lentivirus. In some aspects, the viral vector comprises an AAV.

[0117] In some aspects, the T cells are transduced before culturing according to the methods disclosed herein. In some aspects, the T cells are transduced after culturing according to the methods disclosed herein. In some aspects, the T cells are cultured according to the methods disclosed herein, e.g., in a medium comprising at least 55 mM potassium ion, prior to, during, and after transduction.

[0118] In some aspects, upon adoptive transfer of the T cells expressing an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex, cultured according to the methods disclosed herein, the transferred cells exhibit decreased cell exhaustion, as compared to cells cultured using a control culture medium. In some aspects, upon adoptive transfer of the cultured cells, the transferred cells persist for a longer period of time in vivo, as compared to cells cultured using a control medium. In some aspects, the transferred cells, e.g., engineered T cells of the disclosure, have a greater in vivo efficacy, e.g., tumor-killing activity, as compared to cells cultured using a control medium. In some aspects, a lower dose of the cells cultured according to the methods disclosed herein is needed to elicit a response, e.g., decreased tumor volume, in a subject as compared to cells cultured using a control medium. [0119] In some aspects, the T cells are cultured in a medium disclosed herein, immediately upon isolation from a subject. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein during expansion of the cells. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein during engineering of the cells, e.g., during transduction with a construct encoding an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein following engineering of the cells, e.g., following transduction with a construct encoding an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein throughout expansion and engineering. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein throughout viral genetic engineering. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein throughout non-viral genetic engineering. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein during introduction of receptors to allow for tumor specific targeting (e.g., a TCR). In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein throughout introduction of one or more endogenous genes that improve T cell function. In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in a medium disclosed herein throughout introduction of one or more synthetic genes that improve T cell function.

[0120] In some aspects, the T cells, e.g., engineered T cells disclosed herein, are cultured in the medium disclosed herein for the entirety of ex vivo culture, e.g., from the time the T cells are isolated from a subject, through growing, expansion, engineering, and until administration. In some aspects, the T cells, e.g, engineered T cells disclosed herein, are cultured in the medium disclosed herein for the duration of expansion. In some aspects, the T cells, e.g, engineered T cells disclosed herein, are cultured in the medium disclosed herein until the total number of viable T cells, e.g., engineered T cells disclosed herein, is at least about 10⁴, 5 x 10⁴, 10⁵, 5 x 10⁵, 10⁶, or 5 x 10⁶ total cells. ILA. Potassium

[0121] Some aspects of the disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) disclosed herein, in a medium comprising an increased concentration of potassium ion (e.g., greater than about 55 mM, greater than about 60 mM, greater than about 65 mM, or greater than about 70 mM), relative to a control medium. In some aspects, the medium comprises at least about 55 mM to at least about 90 mM potassium ion, at least about 55 mM to at least about 80 mM potassium ion, at least about 55 mM to at least about 75 mM potassium ion, at least about 55 mM to at least about 70 mM potassium ion, at least about 55 mM to at least about 65 mM potassium ion, at least about 55 mM to at least about 60 mM potassium ion.

[0122] In some aspects, the medium comprises at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, or at least about 80 mM potassium ion. In some aspects, the medium comprises at least about 55 mM potassium ion. In some aspects, the medium comprises at least about 60 mM potassium ion. In some aspects, the medium comprises at least about 65 mM potassium ion. In some aspects, the medium comprises at least about 70 mM potassium ion. In some aspects, the medium comprises at least about 75 mM potassium ion. In some aspects, the medium comprises at least about 80 mM potassium ion.

[0123] In some aspects, the medium comprises an increased concentration of potassium ion, e.g., at least about 55 mM potassium ion, and the medium is hypotonic. In some aspects, the medium comprises potassium ion at a concentration between about 55 mM and about 80 mM and NaCl at a concentration between about 30 mM and about 100 mM, wherein the total concentration of potassium ion and NaCl is between about 110 mM and about 140 mM.

[0124] In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 90 mM, about 55 mM to about 80 mM, about 55 mM to about 70 mM, or about 55 mM to about 60 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 90 mM, about 55 mM to about 80 mM, about 55 mM to about 70 mM, about 55 mM to about 60 mM, or about 55 mM to about 50 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, or about 55 mM to about 60 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, or about 55 mM to about 60 mM, and wherein the medium is optionally hypotonic.

[0125] In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 90 mM.

[0126] In some aspects, the medium comprises at least about 55 mM potassium ion and less than about 90 mM NaCl. In some aspects, the medium comprises at least about 60 mM to about 120 mM potassium ion and less than about 80 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 65 mM to about 120 mM potassium ion and less than about 75 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 70 mM to about 120 mM potassium ion and less than about 70 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 75 mM to about 120 mM potassium ion and less than about 65 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 80 mM to about 120 mM potassium ion and less than about 60 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 85 mM to about 120 mM potassium ion and less than about 65 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 90 mM to about 120 mM potassium ion and less than about 50 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 95 mM to about 120 mM potassium ion and less than about 55 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 100 mM to about 120 mM potassium ion and less than about 50 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 105 mM to about 120 mM potassium ion and less than about 35 mM to about 20 mM NaCl. In some aspects, the medium comprises at least about 110 mM to about 120 mM potassium ion and less than about 30 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0127] In some aspects, the concentration of potassium ion in a medium of the present disclosure is about 55 mM to about 90 mM, and the concentration of NaCl is less than about 90 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 80 mM, and the concentration of NaCl is less than about 90 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM, and the concentration of NaCl is less than about 90 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM, and the concentration of NaCl is less than about 80 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 90 mM, and the concentration of NaCl is less than about 70 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 80 mM, and the concentration of NaCl is less than about 70 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 90 mM, and the concentration of NaCl is less than about 60 mM to about 50 mM.

[0128] In some aspects, the concentration of potassium ion is about 60 mM to about 65 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 65 mM, and the concentration of NaCl is less than about 80 mM to about 75 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM, and the concentration of NaCl is less than about 75 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 75 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 75 mM, and the concentration of NaCl is less than about 70 mM to about 65 mM. In some aspects, the concentration of potassium ion is about 75 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 75 mM to about 80 mM, and the concentration of NaCl is less than about 65 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 85 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 85 mM, and the concentration of NaCl is less than about 60 mM to about 55 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 90 mM, and the concentration of NaCl is less than about 55 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 95 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 95 mM, and the concentration of NaCl is less than about 50 to about 45. In some aspects, the concentration of potassium ion is about 95 mM to about 100 mM. In some aspects, the concentration of potassium ion is about 95 mM to about 100 mM, and the concentration of NaCl is less than about 45 mM to about 40 mM. In some aspects, the concentration of potassium ion is about 100 mM to about 105 mM. In some aspects, the concentration of potassium ion is about 100 mM to about 105 mM, and the concentration of NaCl is less than about 40 mM to about 35 mM. In some aspects, the concentration of potassium ion is about 105 mM to about 110 mM. In some aspects, the concentration of potassium ion is about 105 mM to about 110 mM, and the concentration of NaCl is less than about 35 to about 30. In some aspects, the concentration of potassium ion is about 110 mM to about 115 mM. In some aspects, the concentration of potassium ion is about 110 mM to about 115 mM, and the concentration of NaCl is less than about 30 mM to about 25 mM. In some aspects, the concentration of potassium ion is about 115 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 115 mM to about 120 mM, and the concentration of NaCl is less than about 25 mM to about 20 mM.

[0129] In some aspects, the concentration of potassium ion is about 55 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM, wherein the medium is hypotonic or isotonic.

[0130] In some aspects, the medium comprising a high concentration of potassium ion can be prepared by adding a sufficient amount of a potassium salt in a medium. In some aspects, nonlimiting examples of potassium salt include potassium aminetrichloroplatinate, potassium aquapentachlororuthenate, potassium bis(oxalato)platinate(II) dihydrate, potassium bisulfate, potassium borohydride, potassium bromide, potassium carbonate, potassium chloride, potassium chromate, potassium dichromate, potassium dicyanoargentate, potassium dicyanoaurate, potassium fluoride, potassium fluorosulfate, potassium hexachloroiridate, potassium hexachloroosmate, potassium hexachloropalladate, potassium hexachloroplatinate, potassium hexachlororhenate, potassium hexacyanochromate, potassium hexacyanoferrate, potassium hexacyanoruthenate(II) hydrate, potassium hexafluoroantimonate, potassium hexafluoronickel ate, potassium hexafluorophosphate, potassium hexafluorotitanate, potassium hexafluorozirconate, potassium hexahydroxoantimonate, potassium hexaiodoplatinate, potassium hexaiodorhenate, potassium hydroxide, potassium iodate, potassium iodide, potassium manganate, potassium metavanadate, potassium molybdate, potassium nitrate, potassium nitrosodi sulfonate, potassium osmate(VI) dihydrate, potassium pentachloronitrosylruthenate, potassium perchlorate, potassium perrhenate, potassium perruthenate, potassium persulfate, potassium phosphate dibasic, potassium phosphate monobasic, potassium pyrophosphate, potassium selenocyanate, potassium selenocyanate, potassium stannate trihydrate, potassium sulfate, potassium tellurate hydrate, potassium tellurite, potassium tetraborate tetrahydrate, potassium tetrabromoaurate, potassium tetrabromopalladate, potassium tetrachloropalladate, potassium tetrachloroplatinate, potassium tetracyanopalladate, potassium tetracyanoplatinate, potassium tetrafluoroborate, potassium tetranitroplatinate, potassium tetrathionate, potassium p-toluenethiosulfonate, potassium hydroxycitrate tribasic monohydrate, or any combination thereof. In certain aspects, the potassium salt comprises potassium chloride (KC1). In certain aspects, the potassium salt comprises potassium gluconate. In certain aspects, the potassium salt comprises potassium citrate. In certain aspects, the potassium salt comprises potassium hydroxy citrate.

II.B. Sodium

[0131] Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1 i57-i6₅/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), comprising placing the cells in a medium comprising (i) potassium ion at a concentration of at least about 55 mM and (ii) sodium ion at a concentration of less than about 115 mM. In some aspects, the medium is hypotonic or isotonic. In some aspects, the target concentration of sodium is reached by starting with a basal medium comprising a higher concentration of sodium ion, and diluting the solution to reach the target concentration of sodium ion. In some aspects, the target concentration of sodium ion is reached by adding one or more sodium salts. Non-limiting examples of sodium salts include sodium (meta)periodate, sodium arsenyl tartrate hydrate, sodium azide, sodium benzyloxide, sodium bromide, sodium carbonate, sodium chloride, sodium chromate, sodium cyclohexanebutyrate, sodium ethanethiolate, sodium fluoride, sodium fluorophosphate, sodium formate, sodium hexachloroiridate(III) hydrate, sodium hexachloroiridate(IV) hexahydrate, sodium hexachloroplatinate(IV) hexahydrate, sodium hexachlororhodate(III), sodium hexafluoroaluminate, sodium hexafluoroantimonate(V), sodium hexafluoroarsenate(V), sodium hexafluoroferrate(III), sodium hexafluorophosphate, sodium hexafluorosilicate, sodium hexahydroxyplatinate(IV), sodium hexametaphosphate, sodium hydrogen difluoride, sodium hydrogen sulfate, sodium hydrogencyanamide, sodium hydroxide, sodium iodide, sodium metaborate tetrahydrate, sodium metasilicate nonahydrate, sodium metavanadate, sodium molybdate, sodium nitrate, sodium nitrite, sodium oxalate, sodium perborate monohydrate, sodium percarbonate, sodium perchlorate, sodium periodate, sodium permanganate, sodium perrhenate, sodium phosphate, sodium pyrophosphate, sodium selenate, sodium selenite, sodium stannate, sodium sulfate, sodium tellurite, sodium tetraborate, sodium tetrachloroaluminate, sodium tetrachloroaurate(III), sodium tetrachloropalladate(II), sodium tetrachloroplatinate(II), sodium thiophosphate tribasic, sodium thiosulfate, sodium thiosulfate pentahydrate, sodium yttrium oxyfluoride, Trisodium trimetaphosphate, or any combination thereof. In some aspects, the sodium salt comprises sodium chloride (NaCl). In some aspects, the sodium salt comprises sodium gluconate. In some aspects, the sodium salt comprises sodium bicarbonate. In some aspects, the sodium salt comprises sodium hydroxycitrate. In some aspects, the sodium salt comprises sodium phosphate.

[0132] In some aspects, the concentration of the sodium ion in a medium of the present disclosure is less than that of the basal medium. In some aspects, the concentration of the sodium ion is reduced as the concentration of potassium ion is increased. In some aspects, the concentration of the sodium ion is from about 25 mM to about 115 mM. In some aspects, the concentration of the sodium ion is from about 25 mM to about 100 mM, about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 40 mM to about 80 mM, about 50 mM to about 55 mM, about 50 mM to about 60 mM, about 50 mM to about 65 mM, about 50 mM to about 70 mM, about 50 mM to about 75 mM, about 50 mM to about 80 mM, about 55 mM to about 60 mM, about 55 mM to about 65 mM, about 55 mM to about 70 mM, about 55 mM to about 75 mM, about 55 mM to about 80 mM, about 60 mM to about 65 mM, about 60 mM to about 70 mM, about 60 mM to about 75 mM, about 60 mM to about 80 mM, about 70 mM to about 75 mM, about 70 mM to about 80 mM, or about 75 mM to about 80 mM. In some aspects, the concentration of the sodium ion is from about 40 mM to about 80 mM. In some aspects, the concentration of the sodium ion is from about 50 mM to about 85 mM. In some aspects, the concentration of the sodium ion is from about 55 mM to about 80 mM. In some aspects, the concentration of the sodium ion is from about 30 mM to about 35 mM. In some aspects, the concentration of the sodium ion is from about 35 mM to about 40 mM.

In some aspects, the concentration of the sodium ion is from about 40 mM to about 45 mM. In some aspects, the concentration of the sodium ion is from about 45 mM to about 50 mM. In some aspects, the concentration of the sodium ion is from about 50 mM to about 55 mM. In some aspects, the concentration of the sodium ion is from about 55 mM to about 60 mM. In some aspects, the concentration of the sodium ion is from about 60 mM to about 65 mM. In some aspects, the concentration of the sodium ion is from about 65 mM to about 70 mM. In some aspects, the concentration of the sodium ion is from about 70 mM to about 75 mM. In some aspects, the concentration of the sodium ion is from about 75 mM to about 80 mM. In some aspects, the concentration of the sodium ion is from about 80 mM to about 85 mM.

[0133] In some aspects, the concentration of the sodium ion is about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM. In certain aspects, the concentration of sodium ion is about 40 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 45 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 50 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 55 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 60 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 65 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 70 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 75 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 80 mM. In some aspects, the medium comprises about 55 mM to about 90 mM potassium ion and about 40 mM to about 80 mM sodium ion (e.g., NaCl).

[0134] In some aspects, the medium comprises about 55 mM to about 75 mM potassium ion and about 80 mM to about 90 mM sodium ion. (e.g., NaCl) In some aspects, the medium comprises about 60 mM to about 75 mM potassium ion and about 80 mM to about 90 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 65 mM to about 75 mM potassium ion and about 80 mM to about 85 mM sodium ion e.g., NaCl). In some aspects, the medium comprises about 65 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 66 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 67 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 68 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 69 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 70 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 71 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 72 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 73 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 74 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 75 mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 65 mM potassium ion and about 80 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 65 mM potassium ion and about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 65 mM potassium ion and about 90 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 70 mM potassium ion and about 80 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 70 mM potassium ion and about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 70 mM potassium ion and about 90 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 75 mM potassium ion and about 80 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 75 mM potassium ion and about 85 mM sodium ion (e.g., NaCl). In some aspects, the medium comprises about 75 mM potassium ion and about 90 mM sodium ion (e.g., NaCl).

[0135] In some aspects, the medium comprises about 55 mM potassium ion and less than or equal to about 80 mM NaCl (e.g., about 80 mM, about 79 mM, about 78 mM, about 77 mM, about 76 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM NaCl). In some aspects, the medium comprises about 60 mM potassium ion and less than or equal to about 75 mM NaCl (e.g., about 75 mM, about 74 mM, about 73 mM, about 72 mM, about 71 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM NaCl). In some aspects, the medium comprises about 65 mM potassium ion and less than or equal to about 70 mM NaCl (e.g., about 70 mM, about 69 mM, about 68 mM, about 67 mM, about 66 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM NaCl). In some aspects, the medium comprises about 70 mM potassium ion and less than or equal to about 70 mM NaCl (e.g., about 65 mM, about 64 mM, about 63 mM, about 62 mM, about 61 mM, about 60 mM, about 55 mM, or about 50 mM NaCl). In some aspects, the medium comprises about 75 mM potassium ion and less than or equal to about 60 mM NaCl (e.g., about 60 mM, about 59 mM, about 58 mM, about 57 mM, about 56 mM, about 55 mM, about 50 mM, about 45 mM, or about 40 mM NaCl). In some aspects, the medium comprises about 80 mM potassium ion and less than or equal to about 55 mM NaCl (e.g., about 55 mM, about 54 mM, about 53 mM, about 52 mM, about 51 mM, about 50 mM, about 45 mM, about 40 mM, or about 35 mM NaCl). In some aspects, the medium comprises about 85 mM potassium ion and less than or equal to about 50 mM NaCl (e.g., about 50 mM, about 49 mM, about 48 mM, about 47 mM, about 46 mM, about 45 mM, about 40 mM, about 35 mM, or about 30 mM NaCl). In some aspects, the medium comprises about 90 mM potassium ion and less than or equal to about 45 mM NaCl (e.g., about 45 mM, about 44 mM, about 43 mM, about 42 mM, about 41 mM, about 40 mM, about 35 mM, about 30 mM, or about 25 mM NaCl). In some aspects, the medium comprises about 70 mM potassium ion and about 60 mM NaCl. In some aspects, the medium comprises about 70 mM potassium ion and about 61 mM NaCl. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic.

[0136] Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1 i57-i6₅/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) disclosed herein, comprising placing the T cells in a medium comprising (i) potassium ion at a concentration higher than 55 mM and (ii) NaCl at a concentration of less than about 135 mM. Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF- betaRII polypeptide) disclosed herein, comprising placing the T cells in a medium comprising (i) potassium ion at a concentration higher than 55 mM and (ii) NaCl at a concentration of less than about 100 mM. Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1 i57-i6₅/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CDSalpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein, comprising placing the T cells in a medium comprising (i) potassium ion at a concentration higher than 55 mM and (ii) NaCl at a concentration of less than about 90 mM.

II.C. Tonicity

[0137] The tonicity of the medium useful for the present application (e.g., (concentration of potassium ion and concentration of NaCl) X 2) is adjusted based on the concentration of potassium ion and concentration of NaCl. In some aspects, the tonicity of the medium is lower than that of the basal medium. In some aspects, the tonicity of the medium is higher than that of the basal medium. In some aspect, the tonicity of the medium is the same as that of the basal medium. The tonicity of the medium can be affected by modifying the concentration of potassium ion and/or NaCl in the media. In some aspects, increased potassium ion concentration is paired with an increase or a decrease in the concentration of NaCl. In some aspects, this pairing affects the tonicity of the medium. In some aspects, the concentration of potassium ion is increased while the concentration of NaCl is decreased.

[0138] In some aspects, the medium useful for the present media can be prepared based on the function of potassium ion and tonicity. For example, in some aspects, if the medium useful for the present disclosure is hypotonic (e.g., less than 280 mOsm) and comprises at least about 55 mM of potassium ion, a concentration of NaCl that is sufficient to maintain the medium as hypotonic can be determined based on the following formula: NaCl concentration = (desired tonicity (280)/2)

- potassium ion concentration, (i.e., the concentration of NaCl (mM) is equal to or lower than (140

- potassium ion concentration)). In some aspects, the hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 110 mM and 140 mM. Therefore, for hypotonic medium, the concentration of potassium ion can be set at a concentration between 50 mM and 90 mM, and the NaCl concentration can be between 90 mM and 50 mM, or lower, so long as the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 115 mM and 140 mM. In some aspects, the hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 120 mM and 140 mM. [0139] In some aspects, the medium is isotonic (between 280 mOsm and 300 mOsm) and comprises a concentration of potassium ion between about 55 mM and 70 mM. The corresponding concentration of NaCl can be again calculated based on the formula: NaCl concentration = (desired tonicity/2) - potassium ion concentration. For example, if the concentration of potassium is 55 mM and the desired tonicity is 300 mOsm, the NaCl concentration can be 100 mM.

[0140] In some aspects, the medium is isotonic. In some aspects, the medium has a tonicity of about 280 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 1 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 2 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 3 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 4 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 5 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 6 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 7 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 8 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 9 mOsm/L. In some aspects, the medium has a tonicity of 280 mOsm/L ± 10 mOsm/L. In some aspects, the medium has a tonicity of about 280 mOsm/L to about 285 mOsm/L, about 280 mOsm/L to about 290 mOsm/L, about 280 mOsm/L to about 295 mOsm/L, about 280 mOsm/L to about 300 mOsm/L, about 280 mOsm/L to about 305 mOsm/L, about 280 mOsm/L to about 310 mOsm/L, about 280 mOsm/L to about 315 mOsm/L, or about 280 mOsm/L to less than 320 mOsm/L. In some aspects, the medium has a tonicity of about 285 mOsm/L, about 290 mOsm/L, about 295 mOsm/L, about 300 mOsm/L, about 305 mOsm/L, about 310 mOsm/L, or about 315 mOsm/L.

[0141] In some aspects, the medium is hypotonic. In some aspects, the medium has a tonicity lower than about 280 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 280 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 275 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 270 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 265 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 260 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 265 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 260 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 255 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 250 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 245 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 240 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 235 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 230 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 225 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the tonicity is higher than about 220 mOsm/L; as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity from about 230 mOsm/L to about 280 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity from about 240 mOsm/L to about 280 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two.

[0142] In some aspects, the medium has an osmolality lower than about 220 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has an osmolality lower than about 215 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has an osmolality lower than about 210 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has an osmolality lower than about 205 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has an osmolality lower than about 200 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two.

[0143] In some aspects, the medium has a tonicity from about 100 mOsm/L to about 280 mOsm/L, about 125 mOsm/L to about 280 mOsm/L, about 150 mOsm/L to about 280 mOsm/L, about 175 mOsm/L to about 280 mOsm/L, about 200 mOsm/L to about 280 mOsm/L, about 210 mOsm/L to about 280 mOsm/L, about 220 mOsm/L to about 280 mOsm/L, about 225 mOsm/L to about 280 mOsm/L, about 230 mOsm/L to about 280 mOsm/L, about 235 mOsm/L to about 280 mOsm/L, about 240 mOsm/L to about 280 mOsm/L, about 245 mOsm/L to about 280 mOsm/L, about 250 mOsm/L to about 280 mOsm/L, about 255 mOsm/L to about 280 mOsm/L, about 260 mOsm/L to about 280 mOsm/L, about 265 mOsm/L to about 280 mOsm/L, about 270 mOsm/L to about 280 mOsm/L, or about 275 mOsm/L to about 280 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity from about 250 mOsm/L to about 270 mOsm/L. In some aspects, the medium has a tonicity from about 250 mOsm/L to about 255 mOsm/L, about 250 mOsm/L to about 260 mOsm/L, about 250 mOsm/L to about 265 mOsm/L, about 255 mOsm/L to about 260 mOsm/L, about 255 mOsm/L to about 265 mOsm/L, about 255 mOsm/L to about 265 mOsm/L, about 260 mOsm/L to about 265 mOsm/L, or about 254 mOsm/L to about 263 mOsm/L. In some aspects, the medium has a tonicity from about 254 mOsm/L to about 255 mOsm/L. In some aspects, the medium has a tonicity from about 255 mOsm/L to about 256 mOsm/L. In some aspects, the medium has a tonicity from about 256 mOsm/L to about 257 mOsm/L. In some aspects, the medium has a tonicity from about 257 mOsm/L to about 258 mOsm/L. In some aspects, the medium has a tonicity from about 258 mOsm/L to about 259 mOsm/L. In some aspects, the medium has a tonicity from about 260 mOsm/L to about 261 mOsm/L. In some aspects, the medium has a tonicity from about 261 mOsm/L to about 262 mOsm/L. In some aspects, the medium has a tonicity from about 262 mOsm/L to about 263 mOsm/L. In some aspects, the medium has a tonicity from about 263 mOsm/L to about 264 mOsm/L. In some aspects, the medium has a tonicity from about 264 mOsm/L to about 265 mOsm/L. In some aspects, the medium has a tonicity from about 220 mOsm/L to about 280 mOsm/L; as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two.

[0144] In some aspects, the medium has a tonicity of about 100 mOsm/L, about 125 mOsm/L, about 150 mOsm/L, about 175 mOsm/L, about 200 mOsm/L, about 210 mOsm/L, about 220 mOsm/L, about 225 mOsm/L, about 230 mOsrn/L, about 235 mOsrn/L, about 240 mOsrn/L, about 245 mOsm/L, about 250 mOsm/L, about 255 mOsm/L, about 260 mOsm/L, about 265 mOsm/L, about 270 mOsm/L, or about 275 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two.

[0145] In some aspects, the medium has a tonicity of about 250 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 262.26 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 260 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 259.7 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 257.5 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 257.2 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 255.2 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 254.7, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 255 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity of about 260 mOsm/L, as calculated by adding the potassium ion concentration and the NaCl concentration, and multiplying by two.

[0146] In some aspects, the medium comprises about 50 mM potassium ion and (i) about

80.5 mM NaCl; (ii) about 17.7 mM glucose; and (iii) about 1.8 mM calcium ion.

[0147] In some aspects, the medium comprises about 55 mM potassium ion and (i) about 76 mM NaCl; (ii) about 17.2 mM glucose; and (iii) about 1.7 mM calcium ion.

[0148] In some aspects, the medium comprises about 60 mM potassium ion and (i) about 72.2 mM NaCl; (ii) about 16.8 mM glucose; and (iii) about 1.6 mM calcium ion.

[0149] In some aspects, the medium comprises about 65 mM potassium ion and (i) about

67.6 mM NaCl; (ii) about 16.3 mM glucose; and (iii) about 1.5 mM calcium ion. [0150] In some aspects, the medium comprises about 70 mM potassium ion and (i) about 63.9 mM NaCl; (ii) about 15.9 mM glucose; and (iii) about 1.4 mM calcium ion.

[0151] In some aspects, the medium comprises about 75 mM potassium ion and (i) about 59.3 mM NaCl; (ii) about 15.4 mM glucose; and (iii) about 1.3 mM calcium ion.

[0152] In some aspects, the medium comprises about 80 mM potassium ion and (i) about 55.6 mM NaCl; (ii) about 15 mM glucose; and (iii) about 1.2 mM calcium ion.

[0153] The tonicity of the medium can be adjusted, e.g., to an isotonic or hypotonic state disclosed herein, at any point. In some aspects, the tonicity of the medium can be adjusted, e.g., to an isotonic or hypotonic state disclosed herein, before the cells are added to the medium. In some aspects, the cells are cultured in the hypotonic or isotonic medium prior to cell engineering, e.g., prior to transduction with a construct expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A 02 complex and a c-Jun, CD8alpha co leceptor, and/or dnTGF-betalFII polypeptide) disclosed herein. In some aspects, the cells are cultured in the hypotonic or isotonic medium during cell engineering, e.g., during transduction with a construct expressing an anti-NY- ESO-1 TCR that specifically binds the NY-ESO-1157-165/HL A- A*02 complex (or an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein. In some aspects the cells are cultured in the hypotonic or isotonic medium after cell engineering, e.g., after transduction with a construct expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA- A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CDSalpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein. In some aspects, the cells are cultured in the hypotonic or isotonic medium throughout cell expansion.

II.D. Saccharides

[0154] Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1157-165/HL A- A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CDSalpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein, comprising placing the cells in a medium comprising (i) potassium ion at a concentration of at least about 55 mM and (ii) a saccharide. In some aspects, the medium is hypotonic or isotonic. [0155] In some aspects, the target concentration of the saccharide is reached by starting with a basal medium comprising a higher concentration of the saccharide, and diluting the solution to reach the target concentration of the saccharide. In some aspects, the target concentration of the saccharide is reached by raising the concentration of the saccharide by adding the saccharide until the desired concentration is reached. In some aspects, the saccharide is a monosaccharide, a disaccharide, or a polysaccharide. In some aspects, the saccharide is selected from glucose, fructose, galactose, mannose, maltose, sucrose, lactose, trehalose, or any combination thereof. In certain aspects, the saccharide is glucose. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM and (ii) glucose. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM and (ii) mannose. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) glucose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM and (ii) mannose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0156] In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM to at least about 100 mM and (ii) glucose. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM to at least about 100 mM and (ii) mannose. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM to at least about 100 mM and (ii) glucose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the medium comprises (i) potassium ion at a concentration of at least about 55 mM to at least about 100 mM and (ii) mannose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0157] In some aspects, the concentration of the saccharide, e.g., glucose, is about 10 mM to about 24 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is less than about 4.29 g/L. In some aspects, the concentration of the saccharide, e.g., glucose, is less than about 24 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is more than about 5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 5 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 5 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 5 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 15 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 14 mM to about 14.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 14.5 mM to about 15 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 15 mM to about 15.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 15.5 mM to about 16 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 16 mM to about 16.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 16.5 mM to about 17 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 17 mM to about 17.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 17.5 mM to about 18 mM.

[0158] In some aspects, the concentration of the saccharide, e.g., glucose, is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, is about 10.5 mM, about 11 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 14.5 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about 17 mM, about 17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, about 20 mM, about 20.5 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

II.E. Calcium

[0159] Some aspects of the present disclosure are directed to methods of culturing T cells, e.g., one or more T cells that expresses an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1 i57-i6₅/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein, comprising placing the cells in a medium comprising (i) potassium ion at a concentration of at least about 55 mM and (ii) calcium ion. In some aspects, the medium is hypotonic or isotonic.

[0160] In some aspects, the target concentration of calcium is reached by starting with a basal medium comprising a higher concentration of calcium ion, and diluting the solution to reach the target concentration of calcium ion. In some aspects, the target concentration of calcium is reached by raising the concentration of calcium ion by adding one or more calcium salts. Nonlimiting examples of calcium salts include calcium bromide, calcium carbonate, calcium chloride, calcium cyanamide, calcium fluoride, calcium hydride, calcium hydroxide, calcium iodate, calcium iodide, calcium nitrate, calcium nitrite, calcium oxalate, calcium perchlorate tetrahydrate, calcium phosphate monobasic, calcium phosphate tribasic, calcium sulfate, calcium thiocyanate tetrahydrate, hydroxyapatite, or any combination thereof. In some aspects, the calcium salt comprises calcium chloride (CaCh). In some aspects, the calcium salt comprises calcium gluconate.

[0161] In some aspects, the concentration of the calcium ion is less than that of the basal medium. In some aspects, the concentration of the calcium ion is greater than that of the basal medium. In some aspects, the concentration of calcium ion is more than about 0.4 mM. In some aspects, the concentration of calcium ion is less than about 2.8 mM. In some aspects, the concentration of calcium ion is less than about 2.5 mM. In some aspects, the concentration of calcium ion is less than about 2.0 mM. In some aspects, the concentration of calcium ion is less than about 1.9 mM. In some aspects, the concentration of calcium ion is less than about 1.8 mM. In some aspects, the concentration of calcium ion is less than about 1.7 mM. In some aspects, the concentration of calcium ion is less than about 1.6 mM. In some aspects, the concentration of calcium ion is less than about 1.5 mM. In some aspects, the concentration of calcium ion is less than about 1.4 mM. In some aspects, the concentration of calcium ion is less than about 1.3 mM. In some aspects, the concentration of calcium ion is less than about 1.2 mM. In some aspects, the concentration of calcium ion is less than about 1.1 mM. In some aspects, the concentration of calcium ion is less than about 1.0 mM.

[0162] In some aspects, the concentration of calcium ion is from about 0.4 mM to about 2.8 mM, about 0.4 mM to about 2.7 mM, about 0.4 mM to about 2.5 mM, about 0.5 mM to about

2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about

2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about

2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 0.8 to about 0.9 mM, about 0.8 to about 1.0 mM, about 0.8 to about 1.1 mM, about 0.8 to about 1.2 mM, about 0.8 to about 1.3 mM, about 0.8 to about 1.4 mM, about 0.8 to about 1.5 mM, about 0.8 to about 1.6 mM, about 0.8 to about 1.7 mM, about 0.8 to about 1.8 mM, about

0.9 to about 1.0 mM, about 0.9 to about 1.1 mM, about 0.9 to about 1.2 mM, about 0.9 to about

1.3 mM, about 0.9 to about 1.4 mM, about 0.9 to about 1.5 mM, about 0.9 to about 1.6 mM, about 0.9 to about 1.7 mM, about 0.9 to about 1.8 mM, about 1.0 to about 1.1 mM, about 1.0 to about 1.2 mM, about 1.0 to about 1.3 mM, about 1.0 to about 1.4 mM, about 1.0 to about 1.5 mM, about

1.0 to about 1.6 mM, about 1.0 to about 1.7 mM, about 1.0 to about 1.8 mM, about 1.1 to about

1.2 mM, about 1.1 to about 1.3 mM, about 1.1 to about 1.4 mM, about 1.1 to about 1.5 mM, about

1.1 to about 1.6 mM, about 1.1 to about 1.7 mM, about 1.1 to about 1.8 mM, about 1.2 to about

1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about

1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about

1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about

1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about

1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about

1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM.

[0163] In some aspects, the concentration of calcium ion is from about 0.8 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about 0.9 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about 1.0 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about 1.1 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about 1.2 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about 1.3 mM. In some aspects, the concentration of calcium ion is from about 1.3 mM to about 1.4 mM. In some aspects, the concentration of calcium ion is from about 1.4 mM to about 1.5 mM. In some aspects, the concentration of calcium ion is from about 1.5 mM to about 1.6 mM. In some aspects, the concentration of calcium ion is from about 1.7 mM to about 1.8 mM. [0164] In some aspects, the concentration of calcium ion is about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM. In some aspects, the concentration of calcium ion is about 0.6 mM. In some aspects, the concentration of calcium ion is about 0.7 mM. In some aspects, the concentration of calcium ion is about 0.8 mM. In some aspects, the concentration of calcium ion is about 0.9 mM. In some aspects, the concentration of calcium ion is about 1.0 mM. In some aspects, the concentration of calcium ion is about 1.1 mM. In some aspects, the concentration of calcium ion is about 1.2 mM. In some aspects, the concentration of calcium ion is about 1.3 mM. In some aspects, the concentration of calcium ion is about 1.4 mM. In some aspects, the concentration of calcium ion is about 1.5 mM. In some aspects, the concentration of calcium ion is about 1.6 mM. In some aspects, the concentration of calcium ion is about 1.7 mM. In some aspects, the concentration of calcium ion is about 1.8 mM.

ILF. Cytokines

[0165] In some aspects, the medium comprises a cytokine. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In some aspects, the medium is hypertonic. In some aspects, the cytokine is selected from IL-2, IL-7, IL-15, and any combination thereof. In some aspects, the medium does not comprise IL-2. In some aspects, the medium comprises IL-2 and IL7. In some aspects, the medium comprises IL-2 and IL-15.

[0166] The cytokine can be added to the medium at any point. In some aspects, the cytokine is added to the medium before the T cells, e.g., one or more T cells that expresses an anti-NY- ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide) disclosed herein, are added to the medium. In some aspects, the T cells are cultured in the medium comprising (i) potassium at a concentration disclosed herein, and (ii) a cytokine prior to cell engineering, e.g., prior to transduction with a construct encoding an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA- A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the T cells are cultured in the medium comprising (i) potassium at a concentration disclosed herein, and (ii) a cytokine during cell engineering, e.g., during transduction with a construct encoding an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA- A*02 complex (or an anti-NY-ESO-1 TCRthat specifically binds theNY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jim, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the T cells are cultured in the medium comprising (i) potassium at a concentration disclosed herein, and (ii) a cytokine after cell engineering, e.g., after transduction with a construct encoding an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the T cells are cultured in the medium comprising (i) potassium at a concentration disclosed herein, and (ii) a cytokine throughout cell expansion.

[0167] In some aspects, the MRM described herein (e.g., comprising potassium ion at a concentration greater than 55 mM) comprises between about 50 lU/mL to about 500 lU/mL of IL- 2. In some aspects, the culture medium comprises about 50 lU/mL, about 60 lU/mL, about 70 lU/mL, about 80 lU/mL, about 90 lU/mL, about 100 lU/mL, about 125 lU/mL, about 150 lU/mL, about 175 lU/mL, about 200 lU/mL, about 225 lU/mL, about 250 lU/mL, about 275 lU/mL, about 300 lU/mL, about 350 lU/mL, about 400 lU/mL, about 450 lU/mL, or about 500 lU/mL of IL-2.

[0168] Therefore, in some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 50 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 60 lU/mL of IL- 2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 70 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 80 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 90 lU/mL of IL- 2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 100 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 125 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 150 lU/mL of IL- 2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 175 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 200 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 225 lU/mL of IL- 2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 250 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 275 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 300 lU/mL of IL- 2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 350 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 400 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 555 mM and (ii) about 450 lU/mL of IL-2. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 500 lU/mL of IL-2. In some aspects, the medium comprising potassium ion and IL-2 further comprises NaCl at a concentration less than about 115 nM.

[0169] In some aspects, the medium comprises at least about 0.1 ng/mL IL-2. In some aspects, the medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-2. [0170] In some aspects, the medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-2. In some aspects, the medium comprises at least about 1.0 ng/mL IL-2. In some aspects, the medium comprises at least about 2.0 ng/mL IL-2. In some aspects, the medium comprises at least about 3.0 ng/mL IL-2. In some aspects, the medium comprises at least about 4.0 ng/mL IL-2. In some aspects, the medium comprises at least about 5.0 ng/mL IL-2. In some aspects, the medium comprises at least about 6.0 ng/mL IL-2. In some aspects, the medium comprises at least about 7.0 ng/mL IL-2. In some aspects, the medium comprises at least about 8.0 ng/mL IL-2. In some aspects, the medium comprises at least about 9.0 ng/mL IL-2. In some aspects, the medium comprises at least about 10 ng/mL IL-2. [0171] In some aspects, the medium comprises at least about 0.1 ng/mL IL-2. In some aspects, the medium comprises from about 50 ng/mL to about 600 ng/mL, about 50 ng/mL to about 500 ng/mL, about 50 ng/mL to about 450 ng/mL, about 50 ng/mL to about 400 ng/mL, about 50 ng/mL to about 350 ng/mL, about 50 ng/mL to about 300 ng/mL, about 100 ng/mL to about 600 ng/mL, about 100 ng/mL to about 500 ng/mL, about 100 ng/mL to about 450 ng/mL, about 100 ng/mL to about 400 ng/mL, about 100 ng/mL to about 350 ng/mL, about 100 ng/mL to about 300 ng/mL, about 200 ng/mL to about 500 ng/mL, about 200 ng/mL to about 450 ng/mL, about 200 ng/mL to about 400 ng/mL, about 200 ng/mL to about 350 ng/mL, about 200 ng/mL to about 300 ng/mL, about 250 ng/mL to about 350 ng/mL, about 300 ng/mL to about 600 ng/mL, about 300 ng/mL to about 500 ng/mL, about 300 ng/mL to about 450 ng/mL, about 300 ng/mL to about 400 ng/mL, about 300 ng/mL to about 350 ng/mL, about 250 ng/mL to about 300 ng/mL, or about 275 ng/mL to about 325 ng/mL IL-2.

[0172] In some aspects, the medium comprises at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90 ng/mL, at least about 100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about 130 ng/mL, at least about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least about 170 ng/mL, at least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at least about 210 ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240 ng/mL, at least about 250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about 280 ng/mL, at least about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least about 320 ng/mL, at least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at least about 360 ng/mL, at least about 370 ng/mL, at least about 380 ng/mL, at least about 390 ng/mL, at least about 400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about 430 ng/mL, at least about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least about 470 ng/mL, at least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at least about 510 ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540 ng/mL, at least about 550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about 580 ng/mL, at least about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the medium comprises at least about 50 ng/mL IL-2. In some aspects, the medium comprises at least about 60 ng/mL IL-2. In some aspects, the medium comprises at least about 70 ng/mL IL-2. In some aspects, the medium comprises at least about 73.6 ng/mL IL-2. In some aspects, the medium comprises at least about 75 ng/mL IL-2. In some aspects, the medium comprises at least about 80 ng/mL IL-2. In some aspects, the medium comprises at least about 90 ng/mL IL-2. In some aspects, the medium comprises at least about 100 ng/mL IL-2. In some aspects, the medium comprises at least about 200 ng/mL IL-2. In some aspects, the medium comprises at least about 300 ng/mL IL-2. In some aspects, the medium comprises at least about 400 ng/mL IL-2. In some aspects, the medium comprises at least about 500 ng/mL IL-2. In some aspects, the medium comprises at least about 600 ng/mL IL-2.

[0173] In some aspects, the MRM described herein (e.g., comprising potassium ion at a concentration greater than 55 mM) comprises between about 500 lU/mL to about 1,500 lU/mL of IL-7. In some aspects, the culture medium comprises about 500 lU/mL, about 550 lU/mL, about 600 lU/mL, about 650 lU/mL, about 700 lU/mL, about 750 lU/mL, about 800 lU/mL, about 850 lU/mL, about 900 lU/mL, about 950 lU/mL, about 1,000 lU/mL, about 1,050 lU/mL, about 1,100 lU/mL, about 1,150 lU/mL, about 1,200 lU/mL, about 1,250 lU/mL, about 1,300 lU/mL, about 1,350 lU/mL, about 1,400 lU/mL, about 1,450 lU/mL, or about 1,500 lU/mL of IL-7.

[0174] In some aspects, the medium useful for the present disclosure comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 500 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 550 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 600 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 650 lU/mL of IL- 7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 700 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 750 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 800 lU/mL of IL- 7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 850 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 900 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 950 lU/mL of IL- 7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,000 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,050 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,100 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,150 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,200 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,250 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,300 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,350 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,400 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,450 lU/mL of IL-7. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 1,500 lU/mL of IL-7. In some aspects, the medium comprising potassium ion and IL-7 further comprises NaCl at a concentration less than about 115 nM.

[0175] In some aspects, the medium comprises at least about 0.1 ng/mL IL-7. In some aspects, the medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-7. [0176] In some aspects, the medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-7. In some aspects, the medium comprises at least about 1.0 ng/mL IL-7. In some aspects, the medium comprises at least about 2.0 ng/mL IL-7. In some aspects, the medium comprises at least about 3.0 ng/mL IL-7. In some aspects, the medium comprises at least about 4.0 ng/mL IL-7. In some aspects, the medium comprises at least about 5.0 ng/mL IL-7. In some aspects, the medium comprises at least about 6.0 ng/mL IL-7. In some aspects, the medium comprises at least about 7.0 ng/mL IL-7. In some aspects, the medium comprises at least about 8.0 ng/mL IL-7. In some aspects, the medium comprises at least about 9.0 ng/mL IL-7. In some aspects, the medium comprises at least about 10 ng/mL IL-7.

[0177] In some aspects, the MRM described herein (e.g., comprising potassium ion at a concentration greater than 55 mM) comprises between about 50 lU/mL to about 500 lU/mL of IL- 15. In some aspects, the culture medium comprises about 50 lU/mL, about 60 lU/mL, about 70 lU/mL, about 80 lU/mL, about 90 lU/mL, about 100 lU/mL, about 125 lU/mL, about 150 lU/mL, about 175 lU/mL, about 200 lU/mL, about 225 lU/mL, about 250 lU/mL, about 275 lU/mL, about 300 lU/mL, about 350 lU/mL, about 400 lU/mL, about 450 lU/mL, or about 500 lU/mL of IL-15. [0178] Therefore, in some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 50 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 60 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 70 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 80 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 90 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 100 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 125 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 150 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 175 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 200 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 225 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 250 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 275 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 300 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 350 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 400 lU/mL of IL-15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 450 lU/mL of IL- 15. In some aspects, the medium comprises (i) potassium ion at a concentration higher than 55 mM and (ii) about 500 lU/mL of IL-15. In some aspects, the medium comprising potassium ion and IL- 15 further comprises NaCl at a concentration less than about 115 nM.

[0179] In some aspects, the medium comprises at least about 0.1 ng/mL IL-15. In some aspects, the medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-15. [0180] In some aspects, the medium comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL- 15. In some aspects, the medium comprises at least about 1.0 ng/mL IL-15. In some aspects, the medium comprises at least about 2.0 ng/mL IL-15. In some aspects, the medium comprises at least about 3.0 ng/mL IL-15. In some aspects, the medium comprises at least about 4.0 ng/mL IL-15. In some aspects, the medium comprises at least about 5.0 ng/mL IL-15. In some aspects, the medium comprises at least about 6.0 ng/mL IL-15. In some aspects, the medium comprises at least about 7.0 ng/mL IL-15. In some aspects, the medium comprises at least about 8.0 ng/mL IL-15. In some aspects, the medium comprises at least about 9.0 ng/mL IL-15. In some aspects, the medium comprises at least about 10 ng/mL IL-15. In some aspects, the medium further comprises NaCl, wherein the total concentration of potassium ion and NaCl is from 110 mM to 140 mM.

[0181] In some aspects, the medium comprises at least about 55 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 60 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 65 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 70 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 75 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 80 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 85 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the medium comprises at least about 90 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15.

II.G. Basal Media

[0182] In some aspects, the basal medium comprises a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM), alpha Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium (IMDM), M199, OPTMIZER™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, OPTMIZER™ Pro, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS,™ PRIME-XV T cell CDM, OR X-VIVO™ 15 (LONZA) medium, or any combination thereof. In some aspects, the basal medium is serum free. In some aspects, the basal medium further comprises immune cell serum replacement (ICSR). For example, in some aspects, the basal medium comprises OPTMIZER™ Complete supplemented with ICSR, AIM V supplemented with ICSR, IMMUNOCULT™ XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS™ supplemented with ICSR, or any combination thereof.

[0183] In some aspects, the medium, e.g., the MRM, further comprises about 2.5% serum supplement (CTS™ Immune Cell SR, Thermo Fisher), 2 mM L-glutamine, 2 mM L-glutamax, MEM Non-Essential Amino Acids Solution, Pen-strep, 20pg/ml fungin™, sodium pyruvate, or any combination thereof.

[0184] In some aspects, the medium further comprises a CD3 agonist. In some aspects, the CD3 agonist is an anti-CD3 antibody. In some aspects, the anti-CD3 antibody comprises OKT-3. [0185] In some aspects, the medium further comprises a CD28 agonist. In some aspects, the CD28 agonist is an anti-CD28 antibody. [0186] In some aspects, the present disclosure includes a cell culture comprising the medium disclosed herein, a cell bag comprising the medium disclosed herein, or a bioreactor comprising the medium disclosed herein.

III. Compositions of the Disclosure

[0187] Some aspects of the present disclosure are directed to methods of culturing a T cell (e.g., a T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- i65/HLA-A*02 complex or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) in a culture medium comprising an increased concentration of potassium ion. Other aspects are directed to a cell composition comprising one or more T cell e.g., T cell expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-l i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein) cultured according to the methods disclosed herein.

III.A. Cells

[0188] In some aspects, a population of cells (e.g., T cells) cultured according to the methods and/or in the medium disclosed herein has an increased number of less-differentiated e.g. , stem-like) cells as compared to comparable cells cultured according to conventional methods. In some aspects, a population of cells cultured according to the methods disclosed herein exhibits increased expression of one or more marker typical of a stem-like phenotype. In some aspects, a population of cells cultured according to the methods and/or in the medium disclosed herein has an increased number of more-differentiated cells (e.g., effector-like cells) as compared to comparable cells cultured according to conventional methods. In some aspects, a population of cells cultured according to the methods disclosed herein exhibits increased expression of one or more marker typical of a more differentiated phenotype. In some aspects, a population of cells cultured according to the methods and/or in the medium disclosed herein has an increased number of less-differentiated (e.g., stem-like) cells and an increased number of more-differentiated (e.g., effector-like cells) cells as compared to comparable cells cultured according to conventional methods.

[0189] In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo viability upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased cell potency. In some aspects, the cells cultured according to the methods disclosed herein exhibit decreased cell exhaustion. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo persistence upon transfusion in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo activity upon transfusion in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit a more durable in vivo response upon transplantation in a subject. In some aspects, the subject is a human.

[0190] In some aspects, at least about 5% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 10% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 15% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 20% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 25% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 30% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 35% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 40% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 45% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 50% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 60% of the cells in the cell composition have a stemlike phenotype. In some aspects, at least about 70% of the cells in the cell composition have a stemlike phenotype.

[0191] In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 1.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 2.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 2.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 3.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 3.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 4.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 4.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 5.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 5.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 6.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 6.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 7.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 7.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 8.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 9.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 10-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 15-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 20-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 30-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 40-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 50-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 75-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 100-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 500-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 1000-fold as compared to the number of cells in the cell composition prior to the culture.

[0192] In some aspects, the cell composition comprises T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti- NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein). In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY- ESO-1 TCRthat specifically binds the NY-ESO-1157-165/HL A- A*02 complex (or an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), and which also express CD95. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CDSalpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which do not express CD45R0. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY- ESO-1157-165/HL A- A*02 complex (or an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO- li57-i6₅/HLA-A*02 complex and a c-Jun, CDSalpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD45RA. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CCR7. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY- ESO-1 TCR that specifically binds the NY-ESO-1157-165/HL A- A*02 complex (or an anti-NY-ESO- 1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD62L. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express TCF7. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-I157- 16S/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157- i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD3. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD27. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD95 and CD45RA. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD95, CD45RA, and CCR7. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF- betaRII polypeptide disclosed herein), which also express CD95, CD45RA, CCR7, and CD62L. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD95, CD45RA, CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO-li57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27, and which do not express CD45R0. In some aspects, the cell composition comprises an increased percent of T cells, e.g., T cells expressing an anti-NY-ESO-1 TCRthat specifically binds the NY-ESO-1 i57-i65/HLA-A*02 complex (or an anti-NY-ESO-1 TCR that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex and a c-Jun, CD8alpha co receptor, and/or dnTGF-betaRII polypeptide disclosed herein), which also express CD45RA, CCR7, CD62L, TCF7, CD27, and CD28, and which do not express CD45RO.

[0193] In some aspects, the cell composition, obtained by any method described herein (e.g., the yield of the final cell product for use as a therapy), comprises at least about 1 x 10⁵, 5 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, or 5 x 10⁹ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 10³, 5 x 10³, 1 x 10⁴, 5 x 10⁴, 1 x 10⁵, 5 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, or 5 x 10⁹ stem-like cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 5 x 10⁹, 6 x 10⁹, 7 x 10⁹, 8 x 10⁹, 9 x 10⁹, 1 x IO¹⁰, 2 x IO¹⁰, 3 x IO¹⁰, 4 x IO¹⁰, 5 x IO¹⁰, 6 x IO¹⁰, 7 x IO¹⁰, 8 x IO¹⁰, 9 x IO¹⁰, 10 x 10¹⁰, 11 x IO¹⁰, 12 x IO¹⁰, 13 x IO¹⁰, 14 x IO¹⁰, or 15 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 10⁶ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 10⁶ stem-like cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 2 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 3 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 4 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 5 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 6 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 7 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 8 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 9 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 10 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 11 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 12 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 13 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 14 x IO¹⁰ cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 15 x IO¹⁰ cells. In some aspects, cell yield represents the total number of CD3+ cells.

[0194] In some aspects, the cell composition, obtained by any method described herein, comprises an increased proportion of T cells that are TCF7+, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM).

[0195] In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having decreased expression of one or more proteins associated with oxidative stress, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM). In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having decreased expression of one or more of Complex I, Complex II, and Complex III, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM).

[0196] In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having enhanced metabolic fitness, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM). In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having increased expression of one or more respiratory electron transport gene sets, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM). In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having increased expression of one or more proteins associated with mitochondrial oxidative phosphorylation, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM). In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having increased expression of autophagy pathway-related proteins, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM).

[0197] In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells having enhanced trafficking capability (e.g., T-cell tumor trafficking), as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM).

[0198] In some aspects, the cell composition, obtained by any method described herein, comprises an increased proportion of T cells that produce multiple cytokines, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM). In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells that produce higher levels of IFNy, IL-2, and/or TNFa levels following antigen-stimulation, as compared to a composition of T cells cultured in a medium that does not have increased potassium ion (e.g., a potassium ion concentration of less than about 5 mM).

[0199] In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells that exhibit high TCF7 expression after antigen encounter. In some aspects, the cell composition obtained by any method described herein comprises a population of T cells that exhibit a higher proportion of CD397CD69" cells. In some aspects, the cell composition, obtained by any method described herein, comprises a population of T cells that exhibit high proliferation after antigen encounter.

III.B. Anti-NY-ESO-1 TCR

[0200] The anti-NY-ESO-1 TCR described herein is an affinity-enhanced TCR capable of recognizing the HLA-A*02:01, *02:05, *02:06-SLLMWITQC antigen complex. The SLLMWITQC (SEQ ID NO: 19) peptide is derived from the NY-ESO-1 and LAGE-la family of cancer-testes antigens (NY-ESO-1157-164) (Robbins et al., J Immunol 750(9/6116-31 (2008); Mackall et al., J Clin Oncol 34:TPS3101-TPS3101 (2016)). In some aspects, the NY-ESO-1 TCR T-cells described herein comprise autologous, CD4+ and CD8+ lentiviral vector transduced T-cells engineered to express an affinity enhanced NY-ESO-1 TCR recognizing the SLLMWITQC/HLA- A*02+ peptide complex. As such, in some aspects, NY-ESO-1 TCR is designed to target NY- ESO-1 and/or LAGE-la positive tissue.

[0201] In some aspects, the present disclosure provides modified T cells engineered to express an exogenous (e.g., recombinant) T Cell Receptor (TCR) that specifically binds the NY- ESO-1157-165/HLA-A*O2 complex. As used herein, a receptor (e.g., TCR) is capable of specifically binding to a ligand (e.g., an antigen peptide/HLA complex) when the binding has a KD less than or equal to 1 pM and/or has an off-rate (k_Off) of IxlO'³ S'¹ or slower, as measured by surface plasmon resonance (using, e.g., a BIACORE™ or OCTET™ system).

[0202] In some aspects, the recombinant TCR expressed by the T cells is an aP TCR, i.e., a heterodimeric dimer comprising a TCR a chain and a TCR P chain. In some aspects, the recombinant TCR binds a human NY-ESO-1 peptide presented by (i.e., complexed 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 T cells, but is expressed from exogenous nucleotide sequences (e.g., expression construct(s)) that have been introduced to the T cells. In some aspects, the recombinant TCR expressed by the T cells is expressed at a higher number compared to a TCR non-recombinantly (e.g., naturally) expressed by T cells. In some aspects, the TCR a chain and a TCR P chain are expressed from an expression construct comprising a bi-cistronic expression cassette for expressing a TCR a chain and a TCR P chain In some aspects, the recombinant TCR is expressed by the T cells at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 fold, at least about 5.0 fold, at least about 5.5 fold, at least about 6.0 fold, at least about 6.5 fold, or at least about 7.0 fold higher than a TCR non-recombinantly (e.g., naturally) expressed by T cells.

[0203] In some aspects, the modified T cells described herein comprises a recombinant TCR capable of binding a human NY-ESO-1 peptide. In some aspects, the human NY-ESO-1 peptide is NY-ESO-1157-165, 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 94: 1914-8 (1997)). The HLA Class I molecules of these cancerous cells present peptides from this protein, including NY-ESO-1157-165 peptide. Therefore, this peptide complexed with an HLA class I molecule provides a cancer marker that therapeutic T cells can target through their recombinant TCR.

[0204] In some aspects, the NY-ESO-1 peptide (e.g., SLLMWITQC (SEQ ID NO: 19) is complexed with HLA-A*02. In some aspects, 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:l l, 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 aspects, 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.

[0205] In some aspects, the recombinant TCR expressed by the T cells is an aP TCR, i.e., a heterodimeric dimer comprising a TCR a chain and a TCR P chain, wherein the TCR a chain comprises a variable region, and wherein the TCR P chain comprises a variable region: wherein the TCR a chain variable region comprises a complementarity determining region (CDR) 1 (a CDR1), an a CDR2, and an a CDR3, and the TCR P chain variable region comprises a PCDR1, a PCDR2, and a PCDR3. In some aspects, the TCR comprises an a CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 9. In some aspects, the TCR comprises an a CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 8. In some aspects, the TCR comprises an a CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7. In some aspects, the TCR comprises a PCDR1 comprising an amino acid sequence with at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 10. In some aspects, the TCR comprises a PCDR2 comprising an amino acid sequence with at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 11. In some aspects, the TCR comprises a PCDR3 comprising an amino acid sequence with at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 12. In some aspects, the CDRs for the TCRs useful for the present disclosure comprise the sequences shown in Table 1 below.

[0206] In some aspects, the TCR a variable region comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 5. In some aspects, the TCR P variable region comprising an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 6. In some aspects, the TCR a variable region comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 5 and the TCR P variable region comprising an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to an amino acid sequence of SEQ ID NO: 6. [0207] In some aspects, the TCR that specifically targets the NY-ESO-1 i57-i65/HLA-A*02 complex comprises a TCR a chain sequence (with or without the signal peptide (in box)) of SEQ ID NO: 3, or comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3. In some aspects, the TCR that specifically targets the NY-ESO-1 i57-i65/HLA-A*02 complex comprises a TCR P chain sequence (with or without the signal peptide (in box)) of SEQ ID NO: 4, or comprises an amino acid sequence having at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4. In some aspects, the TCR that specifically targets the ESO-li57-i65/HLA-A*02 complex comprises a TCR a chain sequence of SEQ ID NO: 3 (with or without the signal peptide (in box)), or comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3 and a TCR P chain sequence of SEQ ID NO: 4 (with or without the signal peptide (in box)), or comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4.

NY- ESO- 1 TCR a chain amino acid sequence

METLLGLLIL WLQLQWVSS|K QEVTQIPAAL SVPEGENLVL NCSFTDSAIY NLQWFRQDPG KGLTSLLLIQ SSQREQTSGR LNASLDKSSG RSTLYIAASQ PGDSATYLCA VRPLYGGSYI PTFGRGTSLI VHPYIQNPDP AVYQLRDSKS SDKSVCLFTD FDSQTNVSQS KDSDVYITDK TVLDMRSMDF KSNSAVAWSN KSDFACANAF NNSI I PEDTF FPSPESSCDV KLVEKSFETD TNLNFQNLSV IGFRILLLKV AGFNLLMTLR LWSS ( SEQ ID NO : 3 )

NY- ESO- 1 TCR p chain amino acid sequence

RMSIGLLCCA ALSLLWAGPV N^GVTQTPKF QVLKTGQSMT LQCAQDMNHE YMSWYRQDPG MGLRLIHYSV GAGITDQGEV PNGYNVSRST TEDFPLRLLS AAPSQTSVYF CASSYVGNTG ELFFGEGSRL TVLEDLKNVF PPEVAVFEPS EAEI SHTQKA TLVCLATGFY PDHVELSWWV NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV LMAMVKRKDS RG ( SEQ ID NO : 4 )

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 P, respectively), and the CDRs are underlined (SEQ ID NOs: 7-9 and SEQ ID NOs: 10-12 for a and P, respectively).

[0208] In some aspects, the boundaries of the variable domains and CDRs can vary based on different TCR structure analysis systems. In some aspects, the present disclosure encompasses TCRs comprising the variable domains or the six CDRs, as defined by any one of the systems, in the TCR a and P chains set forth above.

[0209] In some aspects, the present disclosure provides a recombinant TCR as a heterodimer of an a chain and a P chain comprising SEQ ID NO: 3 or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3, without the signal peptide (amino acids 1-19), and SEQ ID NO: 4, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, without the signal peptide (amino acids 1-22), respectively. In some aspects, the recombinant TCR is a heterodimer of an a chain and a P chain consisting of SEQ ID NO: 3, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3 without the signal peptide (amino acids 1-19), and SEQ ID NO: 4, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, without the signal peptide (SEQ ID NO: 1-22), respectively.

[0210] In some aspects, the TCR a sequence comprises the variable domain amino acid sequence provided in SEQ ID NO: 5 or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% sequence identity to SEQ ID NO: 5, and the TCR P sequence comprises the variable domain amino acid sequence provided in SEQ ID NO: 6, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 6. Illustrative TCR a and P constant domain sequences are identified herein and other useful constant domain sequences can be identified for use with the recombinant TCR a/p variable domains, for example at IMGT database (Lefranc et al., Nucleic Acids Res. 43(Database /.S.S /Q:D4 I 3-22 (2015) Epub 2014 Nov 5).

[0211] In some aspects, the full-length TCR a chain polypeptide, including the signal peptide, can be encoded by, for example, a polynucleotide of SEQ ID NO: 1, or a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 1, or a degenerate variant or codon-optimized version thereof. In some aspects, the full-length TCR P chain polypeptide, including the signal peptide, can be encoded by, for example, SEQ ID NO: 2, or a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2, or a degenerate variant or codon-optimized version thereof. In some aspects, the TCR disclosed herein is encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 1, or a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 1, and a nucleic acid sequence of SEQ ID NO: 2, or a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about

100% sequence identity to SEQ ID NO: 2.

[0212] In some aspects, the variable domain of the TCR a chain comprises SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or the variable domain of the TCR P chain comprises SEQ ID NO: 6, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% thereto.

[0213] In some aspects, the present disclosure provides a TCR comprising TCR a CDR1- 3 comprising SEQ ID NOs: 7-9, respectively and TCR P CDR1-3 comprising SEQ ID NOs: 10- 12, respectively.

Table 1. NY-ESO TCR Nucleotide and Amino Acid Sequences

[0214] In some aspects, the present disclosure provides a TCR (e.g., a recombinant TCR) as a heterodimer of an a chain and a P chain, wherein the a chain comprises a variable domain comprising an a chain CDR1, CDR2, and CDR3; and wherein the P chain comprises a variable domain comprising an P chain CDR1, CDR2, and CDR3. In some aspects, the a chain comprises a variable domain comprising an a chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, an a chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an a chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. In some aspects, the P chain comprises a variable domain comprising a P chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a P chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a P chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. In some aspects, the TCR comprises (i) an a chain comprising an a chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, an a chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an a chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (ii) a P chain comprising a P chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a P chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a P chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0215] In some aspects, the present disclosure provides a TCR (e.g., a recombinant TCR) as a heterodimer of an a chain and a P chain, wherein the a chain comprises a variable domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 20, wherein the TCR is capable of binding an antigen that is expressed on a tumor cell. In some aspects, the a chain of the TCR comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0216] In some aspects, the present disclosure provides a TCR (e.g., a recombinant TCR) as a heterodimer of an a chain and a P chain, wherein the P chain comprises a variable domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some aspects, the P chain of the TCR comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0217] In some aspects, the present disclosure provides a TCR (e.g., a recombinant TCR) as a heterodimer of an a chain and a P chain, wherein (i) the a chain comprises a variable domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 20; and (ii) the P chain comprises a variable domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some aspects, (i) the a chain of the TCR comprises the amino acid sequence set forth in SEQ ID NO: 20, and (ii) the P chain of the TCR comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0218] In some aspects, the present disclosure provides T cells comprising a recombinant TCR that can form a TCR-CD3 complex by recruiting TCR-associated signaling molecules include CD3ys, CD36s, and ( (also known as CD3(^ or CD3(^Q to help mediate T cell activation.

[0219] In some aspects, the TCR can be introduced into the modified T cells through one or more nucleic acid molecules (e.g., DNA or RNA such as mRNA). In some aspects, the nucleic acid molecules may be placed on one or more DNA or RNA vectors for introduction into the host cells.

[0220] The nucleic acid molecules (e.g., DNA or RNA vectors containing them) can 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 can 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 some aspects, the recombinant virus is pseudotyped with a heterologous envelope protein. In some aspects, 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. 56Z/387-98 (2005); Gutierrez-Guerrero et al., Viruses 12(9):1016 (2020)). [0221] In some aspects, T cells useful in the compositions and methods provided herein are modified to a co-express an exogenous CD8 co-receptor polypeptide or fragment thereof in addition to a TCR e.g., an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- 165/HLA-A*02 complex). CD8 co-receptors suitable for use in the compositions and methods of the disclosure are described in, e.g., WO 2020/049496, which is incorporated by reference herein in its entirety. In some aspects, the CD8 co-receptor is a CD8a homodimer. In some aspects, the CD8a co-receptor polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22. In another embodiment, the CD8a co-receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 22. In another embodiment, the CD8a co-receptor polypeptide comprises residues 22 to 235 of the amino acid sequence of SEQ ID NO: 22.

Table 2A: CD8a co-receptor polypeptide (signal peptide is in bold)

[0222] In some aspects, T cell useful in the compositions and methods provided herein are modified to co-express an exogeneous dominant-negative TGF-PRII (dnTGF-PRII) polypeptide or fragment thereof in addition to a TCR (e.g., an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*02 complex). In some aspects, the dnTGF-PRII polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. In some aspects, the dnTGF-PRII polypeptide comprises the amino acid sequence of SEQ ID NO: 23. In some aspects, the dnTGF-PRII polypeptide comprises residues 24 to 199 of the amino acid sequence of SEQ ID NO: 23.

Table 2B: Dominant-negative TGF-PRII (dnTGF-PRII) (signal peptide is in bold)

III.C. c-Jun Polypeptides

[0223] Some aspects of the present disclosure are directed to methods of preparing a population of human T cells for immunotherapy comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express (i) a TCR that specifically targets the NY-ESO-l i57-i65/HLA-A*02 complex and (ii) a c-Jun polypeptide. Some aspects of the present disclosure are directed to methods of increasing sternness and/or yield of human T cells ex vivo or in vitro comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express (i) a TCR that specifically targets the NY-ESO-l i57-i65/HLA-A*02 complex and (ii) a c-Jun polypeptide.

[0224] In some aspects, the cell does not naturally express a c-Jun protein, but has been modified to express the c-Jun protein. In some aspects, the T cell is naturally capable of expressing a c-Jun protein, but has been modified to increase the expression of the endogenous c-Jun protein. Unless indicated otherwise, "c-Jun overexpression" (or derivatives thereof) comprises both such cells. As described herein, any suitable methods known in the art can be used to modify the cells described herein.

[0225] In some aspects, a cell (e.g., a T cell) described herein has been modified with a transcriptional activator, which is capable of inducing and/or increasing the endogenous expression of an endogenous 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-associated protein (Cas) system-based transcriptional activator, or a combination thereof. See, e.g., Kabadi et al., Methods 69(2): 188-197 (Sep. 2014), which is incorporated herein by reference in its entirety.

[0226] In some aspects, a cell (e.g, a T cell) described herein has been modified with a CRISPR/Cas-system-based transcriptional activator, such as CRISPR activation (CRISPRa). See, e.g., Nissim et al., Molecular Cell 54: 1-13 (May 2014), which is incorporated herein by reference in its entirety. CRISPRa is a type of CRISPR tool that comprises the use of modified Cas proteins that lack endonuclease activity but retain the ability to bind to its guide RNA and the target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins that can be used with the present disclosure are described, e.g., by Pandelakis etal., Cell Systems 10(1): 1-14 (Jan. 2020), 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 Casl2a 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 c-Jun. 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 of a nucleic acid sequence, and thereby induce and/or increase the expression of the encoded c-Jun.

[0227] 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 an anti-NY-ESO-1 TCR disclosed herein and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of endogenous protein c-Jun.

[0228] Any c-Jun polypeptide can be used in the methods and compositions disclosed herein. In some aspects, the c-Jun polypeptide is exogenously added. In some aspects, the c-Jun polypeptide is recombinantly expressed in the cell. For instance, in some aspects, a cell described herein has been modified or engineered (e.g., genetically) to comprise a polynucleotide which comprises a nucleotide sequence encoding a c-Jun polypeptide (also referred to herein as "c-Jun nucleotide sequence"). In some aspects, due to introduction of the exogenously introduced c-Jun nucleotide sequence, the engineered cells overexpress, i.e., express a higher level of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least 1.5-, 2-, 3-, 4-, 5-, or 10-fold more), c- Jun polypeptide than corresponding cells without such a nucleotide sequence ("reference cell"). In some aspects, the engineered 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 polypeptide than the reference cell. In some aspects, the expression of the c-Jun polypeptide is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, by at least about 50-fold, by at least about 60-fold, by at least about 70-fold, by at least about 80-fold, by at least about 90- fold, or by at least about 100-fold compared to the expression of the c-Jun polypeptide in the reference cell (i.e., not comprising the c-Jun nucleotide sequence described herein).

[0229] As is apparent from the present disclosure, in some aspects, T cells described herein (e.g., cultured using the methods provided herein) have been modified to express one or more additional transgenes in combination with an increased amount of c-Jun polypeptide. For instance, in some aspects, a T cell useful for the present disclosure has been modified to comprise a first nucleotide sequence encoding an anti-NY-ESO-1 TCR, e.g., as disclosed herein, and a second nucleotide sequence encoding a c-Jun polypeptide. In some aspects, the first and second nucleotide sequences are part of a single polynucleotide (referred to herein as a "polycistronic polynucleotide"). Non-limiting examples of nucleotide sequences encoding a c-Jun polypeptide are provided further below.

[0230] c-Jun is an oncogenic transcription factor belonging to the activator protein- 1 (AP- 1) family. It interacts with various proteins (e.g., c-Fos) to form dimeric complexes that modulate a diverse range of cellular signaling pathways, including cell proliferation and tumor progression. Accordingly, increased c-Jun expression has been observed in certain cancers, and there has been much interest in developing c-Jun antagonists to treat such cancer. See, e.g., Brennan, A., et al., J Exp Clin Cancer Res 39(1): 184 (Sep. 2020).

[0231] In humans, the c-Jun polypeptide is encoded by the JUN gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047 of GenBank Accession No. NC_000001.11, minus strand orientation). Synonyms of the JUN gene, and the encoded protein thereof, are known and include "Jun proto-oncogene, AP-1 transcription factor subunit," "v-Jun avian sarcoma virus 17 oncogene homolog," "transcription factor AP-1," "Jun oncogene," "AP-1," "Jun activation domain binding protein," “p39” and "enhancer-binding protein API." The wild-type human c-Jun polypeptide sequence is 331 amino acids in length. The amino acid and nucleic acid sequences of the wild-type human c-Jun are provided in Tables 1 and 2, respectively.

[0232] The wild type human c-Jun (UniProt identifier: P05412-1) protein sequence is 331 amino acids in length (SEQ ID NO: 24). The amino acid and nucleic acid sequences are shown in Tables 3A and 3B, respectively.

Table 3A. c-Jun polypeptide sequence

[0233] In some aspects, the T cells disclosed herein have been modified to comprise an exogenous nucleotide sequence encoding a wild-type c-Jun polypeptide, such as the wild-type nucleotide sequence set forth in SEQ ID NO: 24. Alternatively, in some aspects, the T cells described herein are modified to comprise an exogenous nucleotide sequence encoding a mutant c-Jun polypeptide, which retains the ability to prevent and/or reduce exhaustion in the T cells. In some aspects, a mutant c-Jun polypeptide, which can be expressed on the T cells disclosed herein, comprises at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 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 half) or C-terminal domains (e.g., epsilon, bZIP, and amino acids C-terminal thereof) of a wildtype c-Jun (i.e., SEQ ID NO: 24). In some aspects, 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 half) or N-terminal domains (e.g., delta, transactivation domain, and amino acids N-terminal thereof) of a wildtype c-Jun (i.e., SEQ ID NO: 24) are deleted, mutated, or otherwise inactivated. Non-limiting examples of mutant c-Jun polypeptides that are useful for the present disclosure are provided in US 2019/0183932 Al and US 2017/0037376 Al, each of which is incorporated herein by reference in its entirety.

[0234] In some aspects, a T cell described herein has been modified to comprise an exogenous nucleotide sequence encoding a c-Jun polypeptide, wherein the exogenous nucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 25 to 35. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 25 to 35.

[0235] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 26. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 26. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 26.

[0236] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 27. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 27. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 27.

[0237] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 28. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 28. In some aspects, the exogeous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 28.

[0238] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 29. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% to the nucleic acid sequence set forth in SEQ ID NO: 29. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 29.

[0239] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 30. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 30. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 30.

[0240] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 31. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 31. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 31.

[0241] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 32. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 32. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 32.

[0242] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 33. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 33. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 33.

[0243] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 34. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 34. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 34.

[0244] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 35. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 35. In some aspects, the exogenous comprises the nucleotide sequence set forth in SEQ ID NO: 35.

[0245] Exemplary c-Jun nucleotide sequences are provided in Table XX (below).

Table XX. c-Jun Nucleotide Sequences

[0246] The c-Jun nucleotide sequence disclosed herein can be codon-optimized using any methods known in the art. For instance, in some aspects, the codons of a c-Jun nucleotide sequence disclosed herein has been optimized to modify (e.g., increase or decrease) one or more of the following parameters compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 25): (i) codon adaptation index (z.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and unstable motifs; (iv) repeat sequences (e.g., direct repeats, inverted repeats, dyad repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

[0247] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide provided herein is capable of increasing the expression of the encoded c-Jun polypeptide when transfected, transduced or otherwise introduced into a T cell (e.g., human T cell), as compared to a corresponding expression in a cell transfected with the wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 25). In some aspects, the expression of the c-Jun polypeptide in the T cell modified to comprise the exogenous polynucleotide is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3 -fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, or by at least about 50-fold, compared to the corresponding expression in the cell transfected, transduced, or otherwise genetically modified to express with the wild-type c-Jun nucleotide sequence (e.g, SEQ ID NO: 25).

[0248] While certain disclosures provided above 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 above 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).

[0249] In some aspects, the increased expression of the c-Jun polypeptide can improve and/or enhance one or more properties of the modified T cells (e.g., T cells, such as CD4+ and/or CD8+ T cells). Non-limiting examples of such properties include: resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers, such as PD-1, CD39, TIM-3, and/or LAG-3; increased persistence/survival; delay of the onset of dysfunctional states; and/or increased cytokine production), increased expansion/proliferation, increased antigen sensitivity, improved effector function, in particular, improved effector function following repeated antigen stimulation (e.g, cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or combinations thereof.

[0250] Assays useful for measuring exhaustion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, cytokine production/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, Meso Scale Discovery (MSD) or similar assay, persistent antigen stimulation assays, bulk and single cell RNAseq (see, e.g., Fron Genet. 77:220 (2020);; Bioinformatics 354436-445 (2019); Annual Review of Biomed. Data Sci. 2: 139-173 (2019)), cytotoxicity /killing assays, ELISA, western blot and other standard molecular and cell biology methods such as described herein or as described, for example, in Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021) or elsewhere.

[0251] In some aspects, the increased expression of the c-Jun polypeptide increases the resistance of the T cell to exhaustion. In some aspects, the resistance to exhaustion is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20- fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, or by at least about 50-fold, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun polypeptide expression).

[0252] In some aspects, the increased c-Jun polypeptide expression can decrease exhaustion in an exhausted cell. In some aspects, the increased expression of the c-Jun polypeptide can decrease exhaustion by at least about 5%, at least about 10%, at least about 15%, at least about

20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about

45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about

70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about

95%, or about 100%, compared to a reference cell (e.g., corresponding exhausted cell that was not modified to have increased c-Jun polypeptide expression), as measured, for example, using one or more assays as described herein.

[0253] In some aspects, the increased c-Jun protein expression can delay the onset of exhaustion in a cell. In some aspects, the increased expression of the c-Jun protein can delay the onset of exhaustion by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about

95%, or about 100%, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun protein expression), as measured, for example, using one or more assays as described herein. [0254] Accordingly, in some aspects, the expression of one or more exhaustion markers (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3) in a cell described herein is decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun protein expression).

[0255] In some aspects, the expression of one or more exhaustion markers (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3) in a cell described herein is decreased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4-fold, at least 4.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold or more, compared to a reference cell (e.g., corresponding cell that has not been engineered to overexpress c-Jun).

[0256] In some aspects, the exhaustion state of a population of T cells (e.g., modified and cultured using the methods provided herein) can be determined by quantifying the amount (e.g., number and/or percentage) of cells within the population of T cells that express a given exhaustion marker (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3). For instance, when a population of T cells is modified to express an increased level of a c-Jun polypeptide (e.g., modified and cultured using the methods provided herein), the amount (e.g., number and/or percentage) of cells that express a given exhaustion marker is reduced, compared to the amount in a corresponding population of T cells that was not modified as described herein. Accordingly, in some aspects, the amount of cells that express a given exhaustion marker in a population of modified T cells described herein is decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the amount in a corresponding population of T cells that was not modified as described herein.

[0257] In some aspects, the increased expression of the c-Jun polypeptide can increase the persistence/survival of the T cell, e.g., when administered to a subject in vivo. In some aspects, the persistence/survival of the cell is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30- fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, or by at least about 50-fold, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun polypeptide expression).

[0258] In some aspects, increased expression of the c-Jun protein can increase the persistence/survival of the immune cell described herein is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the amount in a corresponding population of immune cells that was not modified as described herein.

[0259] In some aspects, the increased expression of the c-Jun polypeptide can increase the expansion/proliferation of the T cell, e.g., upon antigen stimulation. In some aspects, the expansion/proliferation of the cell is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16- fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, or by at least about 50-fold, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun polypeptide expression).

[0260] In some aspects, increased expression of the c-Jun protein can increase expansion/proliferation of the immune cell, e.g., upon antigen stimulation, by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to the amount in a corresponding population of immune cells that was not modified as described herein.

[0261] In some aspects, the increased expression of the c-Jun polypeptide can increase the effector function of the cell, e.g., increased cytokine (e.g., IFN-y, TNF-a, and/or IL-2) production, granzyme release, and/or cytotoxicity. In some aspects, the effector function of the cell is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20- fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45-fold, or by at least about 50-fold, compared to a reference cell (e.g., corresponding cell that was not modified to have increased c-Jun polypeptide expression).

[0262] In some aspects, the increased expression of the c-Jun protein can increase the effector function of the cell by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, compared to a reference cell.

[0263] In some aspects, a cell modified to express an increased level of c-Jun (e.g., as described herein) retains effector function, e.g., increased cytokine (e.g., IFN-y, TNF-a, and/or IL- 2) production, granzyme release, and/or cytotoxicity (e.g., ability to kill relevant target cells) for at least one, at least two, at least three, or more, additional rounds of a serial, chronic or sequential stimulation assay (see, e.g., Zhao et al., 2015 Cancer Cell 28(4):415-428; Kunkele et al., 2015 Cancer Immunology Research 3(4):368-379; each of which is incorporated herein by reference in its entirety) as compared to control cells (e.g., cells not overexpressing c-Jun).

[0264] In some aspects, as compared to the corresponding immune cells that were cultured in the reference culture medium, immune cells cultured in metabolic reprogramming media of the present disclosure (e.g., comprising potassium ion at a concentration higher than 5 mM, e.g., MRM) are able to produce higher amounts of cytokines (e.g., IFN-y and/or IL-2) after at least two rounds of antigen stimulation, after at least three rounds of antigen stimulation, after at least four rounds of antigen stimulation, after at least five rounds of antigen stimulation, after at least six rounds of antigen stimulation.

[0265] Increased expression of c-Jun in T cells can help sustain the active state of the cells by, e.g., alleviating or preventing T cell dysfunction (e.g., T cell exhaustion). Accordingly, the different approaches to increasing c-Jun protein expression in a cell provided herein (e.g., modifying the cell with an exogenous polynucleotide encoding a c-Jun polypeptide and/or a transcriptional activator that is capable of increasing the expression of endogenous c-Jun) can be used to engineer T cells, such as T cells expressing an anti-NY-ESO-1 TCR, as disclosed herein, which then exhibit sustained, potent cytotoxicity against desired target cells (e.g., target cells expressing NY-ESO-1). As compared to T cells that do not overexpress c-Jun, engineered T cells disclosed herein (which have increased expression of the c-Jun polypeptide) display fewer signs of T cell exhaustion as described above.

[0266] Additionally, as is apparent from the present disclosure, in some aspects, when T cells comprising an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i6s/HLA-A*02 complex and a c-Jun polypeptide are cultured using the methods provided herein (e.g., in MRM comprising potassium ion at a concentration higher than 55 mM), one or more of the abovedescribed properties are further enhanced. For instance, in some aspects, compared to a reference cell (e.g., modified to express an increased level of a c-Jun polypeptide but not cultured in MRM and/or cultured in a MRM but not modified to express a c-Jun polypeptide), the T cell comprising an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i65/HLA-A*02 complex and a c-Jun polypeptide, wherein the T cell is cultured in MRM is capable of exhibiting one or more of the following: (i) increased resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers, such as PD-1, CD39, TIM-3, and/or LAG-3; increased persistence/survival; delay of the onset of dysfunctional states; and/or increased cytokine production), (ii) increased expansion/proliferation, (iii) increased antigen sensitivity, (iv) increased effector function (particularly following repeated antigen stimulation) (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or (v) any combination thereof.

III.D. Delivery Vectors

[0267] In some aspects, provided herein are vectors (e.g., expression vectors) that can be used to modify a T cell described herein (e.g., cultured using the methods provided herein). In some aspects, a vector described herein comprises multiple (e.g., 2, 3, or 4 or more) polynucleotides, wherein the multiple polynucleotides each encode a protein described herein (e.g., an anti-NYESO-1 TCR a chain and an anti-NY-ESO-1 TCR P wherein the anti-NY-ESO-1 TCR specifically targets the NY-ESO-li57-i65/HLA-A*02 complex). Accordingly, in some aspects, a vector comprises a polycistronic vector (e.g., bicistronic vector or tricistronic vector). In some aspects, the polynucleotides described herein are comprised on the same vector (e.g., on a multi ci str onic expression vector). In some aspects, the polynucleotides encoding the proteins described herein (e.g., an anti-NYESO-1 TCR a chain and an anti-NY-ESO-1 TCR P wherein the anti-NY-ESO-1 TCR specifically targets the NY-ESO-li57-i65/HLA-A*02 complex) are provided on one or more separate vectors.

[0268] As described herein, such vectors are useful for recombinant expression in host cells and cells targeted for therapeutic intervention. The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked; or an entity comprising such a nucleic acid molecule capable of transporting another nucleic acid. In some aspects, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In some aspects, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors, or polynucleotides that are part of vectors, are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, "plasmid" and "vector" can sometimes be used interchangeably, depending on the context, as the plasmid is the most commonly used form of vector. However, also disclosed herein are other forms of expression vectors, such as viral vectors (e.g., lentiviruses, replication defective retroviruses, poxviruses, herpesviruses, baculoviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0269] In some aspects, a vector comprises a polynucleotide described herein (e.g., encoding an anti-NYESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex) and a regulatory element. For instance, in some aspects, a vector comprises a polynucleotide described herein (e.g, encoding an anti-NYESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex), operatively linked to a promoter. In some aspects, the vector can comprise multiple promoters (e.g., at least two, at least three, at least four, at least five or more). For instance, in some aspects, the nucleotide sequence encoding the anti-NYESO-1 TCR a chain can be under the control of a first promoter, and the nucleotide sequence encoding the anti- NY-ESO-1 TCR P can be under the control of a second promoter, wherein the TCR specifically targets the NY-ESO-1157-165/HLA-A*O2 complex. In some aspects, each of the multiple promoters are the same. In some aspects, one or more of the multiple promoters are different.

[0270] Any suitable promoter known in the art can be used with the present disclosure. In some aspects, the promoters useful for the present disclosure comprises a mammalian or viral promoter, such as a constitutive or inducible promoter. In some aspects, the promoters for the present disclosure comprises at least one constitutive promoter and at least one inducible promoter, e.g., tissue specific promoter.

[0271] Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. As described herein, in some aspects, promoters that can be used with the present disclosure are inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. When multiple inducible promoters are present, they can be induced by the same inducer molecule or a different inducer.

[0272] In some aspects, the promoter comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter, EFla promoter, or both.

[0273] In some aspects, a vector useful for the present disclosure (e.g., comprising one or more nucleotide sequences encoding an anti -NY-ESO-1 TCR that specifically targets the NY- ESO-1157-165/HLA-A*O2 complex) further comprises one or more additional regulatory elements. Non-limiting examples of regulatory elements include a translation enhancer element (TEE), a translation initiation sequence, a microRNA binding site or seed thereof, a 3’ tailing region of linked nucleosides, an AU rich element (ARE), a post transcription control modulator, a 5' UTR, a 3' UTR, a localization sequence (e.g., membrane-localization sequences, nuclear localization sequences, nuclear exclusion sequences, or proteasomal targeting sequences), post-translational modification sequences (e.g., ubiquitination, phosphorylation, or dephosphorylation), or combinations thereof.

[0274] In some aspects, the vector can additionally comprise a transposable element. Accordingly, in some aspects, the vector comprises a polynucleotide described herein (e.g., encoding an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex), which is flanked by at least two transposon-specific inverted terminal repeats (ITRs). In some aspects, the transposon-specific ITRs are recognized by a DNA transposon. In some aspects, the transposon-specific ITRs are recognized by a retrotransposon. Any transposon system known in the art can be used to introduce the nucleic acid molecules into the genome of a host cell, e.g., a T cell. In some aspects, the transposon is selected from hAT-like Tol2, Sleeping Beauty (SB), Frog Prince, piggyBac (PB), and any combination thereof. In some aspects, the transposon comprises Sleeping Beauty. In some aspects, the transposon comprises piggyBac. See, e.g., Zhao et al., Transl. Lung Cancer Res. 5(1)'.120-25 (2016), which is incorporated by reference herein in its entirety.

[0275] In some aspects, the vector is a transfer vector. The term "transfer vector" refers to a composition of matter which comprises an isolated nucleic acid (e.g, a polynucleotide described herein) and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0276] In some aspects, the vector is an expression vector. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0277] In some aspects, the vector is a viral vector, a mammalian vector, or bacterial vector. In some aspects, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, a hybrid vector, and an adeno associated virus (AAV) vector.

[0278] In some aspects, the adenoviral vector is a third generation adenoviral vector. ADEASY™ is by far the most popular method for creating adenoviral vector constructs. The system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. The transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme Pmel. This construct is then transformed into ADEASIER-1 cells, which are BJ5183 E. coli cells containing PADEASY™. PADEASY™ is a ~33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production. The shuttle vector and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination of the transgene into the adenoviral plasmid. One can also co-transform standard BJ5183 with supercoiled PADEASY™ and the shuttle vector, but this method results in a higher background of non-recombinant adenoviral plasmids. Recombinant adenoviral plasmids are then verified for size and proper restriction digest patterns to determine that the transgene has been inserted into the adenoviral plasmid, and that other patterns of recombination have not occurred. Once verified, the recombinant plasmid is linearized with PacI to create a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7- 10 days later. In addition to this method, other methods for creating adenoviral vector constructs known in the art at the time the present application was filed can be used to practice the methods disclosed herein.

[0279] In some aspects, the viral vector is a retroviral vector, e.g, a lentiviral vector (e.g., a third or fourth generation lentiviral vector). The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect nondividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8)'.1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0280] Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (env) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. The virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system. Current third generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-mediated generation of a replication-competent virus. In fourth generation lentiviral vectors, the retroviral genome has been further reduced (see, e.g., TAKARA® LENTI-X™ fourth-generation packaging systems).

[0281] In some aspects, non-viral methods can be used to deliver a polynucleotide described herein (e.g., encoding an anti-NY-ESO-1 TCR that specifically targets the NY-ESO- 1157-165/HLA-A*O2 complex) into a T cell. In some aspects, the non-viral method includes the use of a transposon. In some aspects, use of a non-viral method of delivery permits reprogramming of cells, e.g., T cells, and direct infusion of the cells into the subject. In some aspects, the polynucleotide can be inserted into the genome of a target cell (e.g., a T cell) or a host cell (e.g., a cell for recombinant expression of the encoded proteins) by using CRISPR/Cas systems and genome edition alternatives such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).

[0282] In some aspects, the polynucleotides disclosed herein (e.g., encoding an anti-NY- ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex) are DNA (e.g., a DNA molecule or a combination thereof), RNA (e.g., a RNA molecule or a combination thereof), or any combination thereof. In some aspects, the polynucleotides are single stranded or double stranded RNA or DNA (e.g. , ssDNA or dsDNA) in genomic or cDNA form, or DNA-RNA hybrids, each of which can include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. As described herein, such nucleic acid sequences can comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleotide sequences will encode the different polypeptides described herein (e.g., an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i65/HLA-A*02 complex).

IV. Methods of Treatment

[0283] Some aspects of the present disclosure are directed to methods of administering a T cell described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-l i57-i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein). Some aspects of the present disclosure are directed to methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a T cell described herein. For instance, in some aspects, disclosed herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a T cell that has been modified to express an anti-NY- ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex (or modified to express an anti-NY-ESO-1 TCR that specifically targets theNY-ESO-li57-i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide). In some aspects, the disease or condition comprises a tumor, /.<?., a cancer. In some aspects, the method comprises stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, comprising administering a T cell described herein. In some aspects, the target cell population comprises a tumor. In some aspects, the tumor is a solid tumor.

[0284] In some aspects, administering a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) reduces a tumor volume in the subject compared to a reference tumor volume. In some aspects, the reference tumor volume is the tumor volume in the subject prior to the administration. In some aspects, the reference tumor volume is the tumor volume in a corresponding subject that did not receive the administration. In some aspects, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to the reference tumor volume.

[0285] In some aspects, treating a tumor comprises reducing a tumor weight in the subject. In some aspects, administering a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) reduces the tumor weight in a subject when administered to the subject. In some aspects, the tumor weight is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to a reference tumor weight. In some aspects, the reference tumor weight is the tumor weight in the subject prior to the administration. In some aspects, the reference tumor weight is the tumor weight in a corresponding subject that did not receive the administration.

[0286] In some aspects, administering a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) to a subject, e.g., suffering from a tumor, increases the duration of a T cell response in a subject relative to the duration of a T response in a corresponding subject that did not receive the administration (e.g., treated with a corresponding cell not cultured according to the methods disclosed herein). In some aspects, the duration of the immune response is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% or more compared to a reference (e.g., corresponding subject that did not receive the administration). In some aspects, the duration of the immune response is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more compared to a reference (e.g., corresponding subject that did not receive the administration). [0287] A T cell or a population of T cells described herein e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA- A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) can be used to treat variety of cancers. Non-limiting examples of cancers that can be treated include adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, secondary cancers caused by cancer treatment, and combinations thereof. In some aspects, the cancer is associated with a solid tumor. In some aspects, the cancer comprises one or more tumor cells that express NY-ESO-1 and/or LAGE-la. In some aspects, the cancer is selected from melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, myxoid/round cell liposarcoma (MRCLS), gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, or bladder cancer. In some aspects, the cancer is metastatic. In some aspects, the cancer is recurrent. In some aspects, the cancer is relapsed. In some aspects the cancer is advanced, e.g., locally advanced. In some aspects, the cancer is metastatic melanoma.

[0288] In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i6s/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) is used in combination with other therapeutic agents (e.g., anti-cancer agents and/or immunomodulating agents). Accordingly, in some aspects, a method of treating a disease or disorder (e.g., tumor) disclosed herein comprises administering a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex, and cultured using the methods provided herein) in combination with one or more additional therapeutic agents. Such agents can include, for example, chemotherapeutic drug, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgery, radiotherapy, activator of a costimulatory molecule, immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. [0289] In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i6s/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) is administered to the subject prior to or after the administration of the additional therapeutic agent. In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) is administered to the subject concurrently with the additional therapeutic agent. In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157-165/HLA-A*O2 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) and the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti- NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i6s/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) and the additional therapeutic agent are administered concurrently as separate compositions. [0290] In some aspects, a T cell or a population of T cells described herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-li57-i65/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) is used in combination with a standard of care treatment (e.g., surgery, radiation, and chemotherapy). Methods described herein can also be used as a maintenance therapy, e.g., a therapy that is intended to prevent the occurrence or recurrence of tumors.

[0291] In some aspects, a T cell or a population of T cells provided herein (e.g., modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1 i57-i6s/HLA-A*02 complex or modified to express an anti-NY-ESO-1 TCR that specifically targets the NY-ESO-1157- i65/HLA-A*02 complex and an increased level of a c-Jun polypeptide, and cultured using the methods provided herein) is used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway can be targeted. Non-limiting examples of such combinations include: a therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immune suppressing cells (e.g., myeloid- derived suppressor cells); a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40, and/or CD40 or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapy that impacts the function of suppressor myeloid cells in the tumor; a therapy that enhances immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell transfer including genetically engineered cells, e.g., cells engineered to express a chimeric antigen receptor (CAR-T therapy); a therapy that inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase; a therapy that reverses/prevents T cell anergy or exhaustion; a therapy that triggers an innate immune activation and/or inflammation at a tumor site; administration of immune stimulatory cytokines; blocking of immuno repressive cytokines; or any combination thereof.

[0292] In some aspects, an anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through the particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors include the following: anti-PDl antibody (e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF- 06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-Ll antibody (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and anti-CTLA-4 antibody (e.g., ipilimumab (YERVOY®), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof).

[0293] In some aspects, an anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through the particular immune checkpoint pathway). In some aspects, immune checkpoint activator comprises 0X40 agonist (e.g., anti-OX40 antibody), LAG-3 agonist (e.g. anti-LAG-3 antibody), 4-1BB (CD137) agonist (e.g., anti-CD137 antibody), GITR agonist (e.g., anti-GITR antibody), TIM3 agonist (e.g., anti-TIM3 antibody), or combinations thereof.

***

[0294] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd Ed. CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.), each of which is incorporated by reference herein in its entirety.

[0295] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Production of Cells

[0296] NY-ESO-1 TCR T-cells that specifically binds the NY-ESO-1 i57-i6₅/HLA-A*02 complex were produced from isolated human CD4+ and CD8+ T-cells or from total CD3+ T-cells collected and frozen from five HL A- A* 02-positive healthy donors (donors 10744, 7179, 14174, 14294 and 18878) or four synovial sarcoma patient samples (ESO2, ESO3, ESO4, ESO5). Cells were activated with CD3/CD28 TRANSACT™ beads in either control media for 24 hours (either TEXMACS™ media supplemented with 100 international units (IU)/mL interleukin (IL)-2 (control 1) or OPTMIZER™ complete media supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7 and 200 lU/mL IL-15 (control 2)) or MRM supplemented with 200 lU/mL IL-2, 1200 lU/mL IL-7 and 200 lU/mL IL-15) for 44 hours. Activated T-cells in control media or MRM were transduced with lentiviral vector (LVV) encoding NY-ESO-1 TCR. Cells were analyzed as described below.

[0297] Media preparation: Basal media was supplemented with immune Cell Serum Replacement (Thermo Fisher), 2 mM L-glutamine (Gibco), 2 mM GLUTAMAX™ (Gibco), MEM Non-Essential Amino Acids Solution (Gibco), sodium pyruvate (Gibco), IL-2, 200 lU/mL; IL-7, 120 lU/ml; and IL-15, 20 lU/ml.

[0298] For hypotonic conditioning medium, basal media with varying concentrations of sodium, potassium, glucose and calcium were adjusted by adding NaCl, glucose, and calcium free RPMI. After adding defined NaCl free RPMI to Basal media, the final concentrations were in the range of: NaCl (40-80 mM), KC1 (40-80 mM), calcium (0.5-2.8mM), glucose (10-24mM) and osmolality (-250-260 mOsmol). See Table 4. Table 4: Media with varying concentrations of potassium, sodium, glucose, and calcium

*Tonicity is calculated based on the following formula: 2 X (concentration of K + concentration of NaCl)

[0299] We also tested the effect of tonicity on T cells by maintaining constant tonicity conditions (250 mOsmol - hypotonic, 280 mOsmol - isotonic, 320 mOsmol - hypertonic) with varying potassium concentrations. Final concentrations in hypotonic conditions, NaCl (35-75mM), KC1 (50-90 mM), final concentrations in isotonic conditions NaCl (50-90 mM), KC1 (50-90 mM), final concentrations in hypertonic conditions NaCl (70-110 mM), KC1 (50-90 mM). See Table 5.

Table 5: Hypotonic, isotonic, hypertonic solutions with varying concentrations of potassium and NaCl

* Tonicity is calculated according to the formula: Tonicity = ([K] + [NaCl]) x 2 wherein "[K]" is the potassium concentration and "[NaCl]" is the sodium chloride concentration of the media.

Example 2: NY-ESO-1 T-cells expanded in MRM exhibit superior in vitro phenotypic and functional properties.

[0300] NY-ESO-1 T-cell products from five HLA-A*02+ healthy donors and four synovial sarcoma patient samples expanded in either MRM orcontrol media (described above) were evaluated for their phenotype and in vitro potency including assessment of the sternness phenotype by flow cytometry, single-cell sequencing, intra-cellular cytokine profile, short term proliferative capacity in response to antigen, INCUCYTE™ killing, and cytokine production by Mesoscale Discovery (MSD) assay. Results presented here demonstrated that NY-ESO-1 TCR T-cells expanded in MRM have a greater than 10-25 % frequency of TCF7+ cells compared to cells expanded in control media. Furthermore, these findings are corroborated by single-cell RNA-Seq analysis. The proportion of less-differentiated cells in CD8+ NY-ESO-1 TCR T-cells expanded in MRM was increased when compared to cells expanded in control media. These data strongly support evidence of sternness in T cell products expanded with MRM.

[0301] Assessment of sternness phenotype:

[0302] Using polychromatic flow cytometry and a stringent gating strategy, CD45RO-/CCR7+/CD45RA+/CD62L+/TCF7+ markers were used to determine the proportion of stem-like T-cells in the T-cell product. Briefly, donor cell products (healthy donors and patient samples) were harvested at the specified harvest times and counted using a Cellaca MX High- Throughput Automated Cell Counter using standard protocols. The cells were then re-suspended at 4xl0⁶ cells/mL, and 800,000 T-cells (200 pL) from each sample was transferred to a 96-well V- bottom plate. Cells were first washed with cell staining buffer (CSB) to remove traces of media, following which they were stained with chemokine (C-C motif) receptor 7 (CCR7) in a final volume of 50 pL and incubated for 15 minutes at 37°C. Cells were then stained with antibodies against specific surface markers in the dark for 30 minutes at 4°C and subsequently washed twice with CSB. To determine the expression of the transcription factor TCF7, the cells were fixed and permeabilized with FoxP3 fix/permeabilization buffer (eBioscience) in the dark at room temperature (RT) for 20 minutes and washed with IX FoxP3 permeabilization wash buffer (EBIO SCIENCE™). Cells were then stained with antibodies against intracellular markers in the dark at 4°C for 30 minutes in IX FoxP3 permeabilization wash buffer. Cells were washed twice with IX FoxP3 permeabilization wash buffer followed by a wash in CSB. Samples were then re-suspended in CSB with 123 COUNT EBEADS™ (Invitrogen) and acquired using a CYTEK® Aurora analyzer.

[0303] NY-ESO-1 TCR T-cells cultured in MRM exhibited higher proportions of stemlike T-cells compared to NY-ESO-1 TCR T-cells cultured in control media (Figure 1 A and IB). In these studies, CCR7, CD45RA, CD62L, CD45RO, and TCF7 expression was determined to define naive and memory CD4+ and CD8+ T-cell subsets within the NY-ESO-1 TCR+ population. The naive and memory T-cell subsets were defined as follows: Naive T-cells/T stem cell memory (TN/TSCM) (CCR7+/CD45RA+/CD45RO-; also referred to as "Tstem like"), T central memory (TCM) (CCR7+/CD45RA-/CD45RO+), T effector memory (TEM) (CCR7-/ CD45RA-/CD45RO+), and T effector cells (CCR7-/CD45RA+/CD45RO-; also referred to as "Temra") (Mahnke et al., Ez/r J Immunol 43(11) :2797-2809 (2013)). Additional markers such as CD62L and CD45RA were also used to identify similar T-cell subsets: Naive T-cells/T stem cell memory (TN/TSCM) (CD62L+/CD45RA+), T central memory (TCM) (CD62L+/CD45RA-), T effector memory (TEM) (CD62L-/CD45RA-), and CD45RA+ T effector memory (TEMRA) (CD62L-/CD45RA+).

[0304] Of the additional phenotypic markers evaluated, the sternness transcription factor TCF7 and the activation marker CD39 stood out as being differentially expressed in T-cells conditioned in MRM media. It is reported that in adoptive T-cell therapy, a higher number of infused CD39-/CD69-/CD8+ tumor-infiltrating lymphocytes (TILs), which were also TCF7-hi, was associated with complete cancer regression in metastatic melanoma patients and improved progression free survival (Krishna et al., Science 370: 1328-1334 (2020)). In flow data, the proportion of TCF7+ cells was increased, and the proportion of CD39+ cells was decreased in NY- ESO-1 TCR T-cells expanded in MRM compared to cells expanded in control media (FIGs. 2A- 2B), suggesting an advantage in anti-tumor efficacy and retention of a stem-like state of NY-ESO- 1 TCR T-cells expanded in MRM.

[0305] To more robustly assess the sternness phenotype in the products, single-cell RNA- Seq was performed on CD45+/TCRvpi3.1+ NY-ESO-1 TCR T-cells. In UMAPs generated after single-cell RNA-Seq analysis, the CD8+ NY-ESO-1 TCR T-cells expanded in MRM exhibited a distinct transcriptional profile compared to that from cells expanded with control media (data not shown). Post-clustering on the single-cells, a set of “less-differentiated clusters” were defined using naive-associated gene set described in Gattinoni, L., et al., Nat Med 77(70): 1290-1297 - I l l -

(2011), as well as the positivity of conventional naive-associated markers such as TCF7. These less-differentiated clusters also exhibited high TCF7 RNA expression and low CD39 protein expression. A set of “most-differentiated clusters” was also defined based on the effector- associated gene set described in Gattinoni, L., et al., Nat Med 17(10) : 1290-1297 (2011). The proportion of the less-differentiated clusters was increased in CD8+ NY-ESO-1 TCR T-cells cultured in MRM as compared to cells expanded in control media (FIGs. 3A-3D), which was consistent with the increase in the proportion of TCF7+ cells and the decrease in the proportion of CD39+ cells observed in NY-ESO-1 TCR T-cells expanded in MRM in the flow data described above.

[0306] A set of “most-differentiated clusters” was also defined based on the effector- associated gene set described in Gattinoni, L., et al., Nat Med 77(70): 1290- 1297 (2011). In some aspects the gene signature for “most-differentiated clusters” comprises one or more genes selected from MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COP A, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, Clorf58, Cl lorf63, C6orfl29, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MY05A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIP ARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LB Al, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMI, NAT13, TGFB1, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3,, ADCY9, CD300A, SCD5, ABB, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A,, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MYO6, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MBOAT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, F0SL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, Clorf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686,, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, KLRB1, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 77 70): 1290-1297 (2011). The proportion of cells in the most-differentiated clusters (a smaller proportion) were also greater in CD8+ NY-ESO-1 TCR T-cells expanded in MRM (FIGs. 3B and 3D). These observations were confirmed using CD45+TCRvpi3.1+ T-cells sorted from NY-ESO- 1 TCR T cells expanded in MRM from two synovial sarcoma patient samples. These cells exhibited an increasing proportion of both less-differentiated clusters (also TCF7-hi CD39-low) and most- differentiated clusters (FIGs. 3A-3D).

[0307] Stem-like T-cell yield in the product was also determined using the following markers: CCR7, CD45RO, CD62L, CD45RA, CD27, CD28 and TCF7. In both healthy donor and synovial sarcoma patient samples, stem-like T-cell yield per million CD3+/TCRvpi3.1+ population was significantly higher in NY-ESO-1 T-cells culture in MRM compared to cells expanded in control media (FIGs. 4A-4B). These results show that expanding cells in MRM enriches for less-differentiated stem-like cells in NY-ESO-1 TCR T-cell products in both healthy and patient samples.

[0308] Assessment of metabolic fitness

[0309] T-cell metabolism and immunological function are closely connected: stem-like T- cells predominantly use oxidative phosphorylation (OXPHOS) for energy production, whereas in effector T-cells glycolysis is preferentially utilized (Pearce et al., Immunity 41 ( 7):75-88 (2013); Bourgeois et al., Frontiers in Oncology 8.231 (2018)). Therefore, OXPHOS is considered more favorable in T-cell products used in adoptive cell therapy. In addition, Vardhana et al., Nature Immunology 27:1022-33 (2020) demonstrated that constant antigen exposure leads to altered metabolism thereby limiting intratumoral T-cell proliferation and self-renewal. The hallmark of this altered metabolism is a rapid induction of mitochondrial oxidative stress that limits the ability of T-cells to engage in oxidative phosphorylation, resulting in bioenergetic limitations that are sufficient to block T-cell proliferation.

[0310] To assess the metabolic fitness of the T cell products, bulk RNA-Seq analysis was performed on CD8+ or CD4+ T cells sorted from CD45+TCRv[313.1+ NY-ESO-1 TCR T cells expanded in MRM or control media. Gene set enrichment analysis revealed that genes upregulated in NY-ESO-1 TCR T cells expanded in MRM were significantly enriched for the OXPHOS and respiratory electron transport gene sets, and genes downregulated in these cell products were significantly enriched for the glycolysis and hypoxia gene sets compared to cells expanded in control media in both CD8+ and CD4+ cells (Table 6). The enrichment/depletion of the 4 pathways was also confirmed in single cell RNA-Seq data by projecting differential genes contributing to pathway enrichment (FIGs. 5A-5T; Table 7). T cells obtained from 2 healthy donors were cultured in MRM at large-scale. The differentiation profiles of T cells (Tstem-iike vs more-differentiated subsets) in MRM-cultured T-cells were compared to control-cultured T cell products (FIGs. 5F-5O). Metabolic fitness of the products was assessed by examining genetic signatures associated with oxidative phosphorylation, fatty acid oxidation, hypoxia, and glycolysis, and maturation of protein complexes associated with the autophagy pathway (LC3b I and LC3b II) was examined by immunoblot. These results suggest that the T cell products expanded in MRM have better metabolic fitness, as MRM was observed to enhance the frequency of Tstem-iike cells and improves metabolic fitness of T-cells manufactured at large-scale.

[0311] Results of flow cytometry and RNA-Seq showed an increased proportion of less- differentiated CD8+ and CD4+ T cells in MRM-cultured product as compared to control media- cultured products and/or controls expanded from most donors at large-and research-scale. Genetic signatures of oxidative phosphorylation were upregulated in MRM-cultured TCR T cell products compared to control media-cultured TCR T cell products at both large- and research-scale. RNA- Seq analysis demonstrated that genetic signatures of oxidative phosphorylation are upregulated in MRM-cultured T cell product compared to the contro-cultured product at both large- and researchscale. Proportions of autophagy flux proteins were higher in MRM-cultured T cell productrelative to the control media-cultured product. Table 6: Fgsea normalized enrichment score (NES) and adjusted p-values of selected metabolic pathways in bulk RNA-Seq analysis

[0312] Gene set enrichment analysis was performed using bulk RNA-seq data from NY- ESO-1 TCR T cells expanded in MRM or control from two donors. Normalized enrichment score (NES) and adjusted p-values (Padj) of selected T cell metabolism and T cell infiltration associated pathways generated using fgsea are shown (see also Table 7, below). Positive NES values indicate upregulation of corresponding pathways in the T cell product expanded in MRM. Negative NES values indicate downregulation of corresponding pathways in T cell product expanded in MRM. Table 7: Leading edges projected on single cell CITE-Seq data

Shown above are the genes used for projecting the significantly enriched gene set identified by fgsea on single cell CITE-Seq data. For each gene set, leadingEdges (i.e. genes contributing the most to the enrichment signal of the gene set) reported by fgsea were filtered by their log2 fold changes (log2FC) and p values (pval) reported by DESeq2 in bulk RNA-Seq differential analysis. Genes with log2FC > 0.25 and pval < 0.05 were used for projection.

[0313] Metabolic fitness of the T-cells was also assessed by looking at the proteins associated with the proteins within the OXPHOS pathway (Complex I-V) that are associated with oxidative stress (Complex I, II and III) and the proteins in autophagy pathway (*LC3bI and LC3b II) by western analysis. The relative abundance of the proteins associated with Oxidative stress (Complex I, II and III) was examined by immunoblot. The levels of all three complexes were lower in NY-ESO-1 TCR T-cells expanded in MRM compared to NY-ESO-1 TCR T-cells expanded in control media (FIGs. 6A-6B).

[0314] It is reported that rapid induction of mitochondrial oxidative stress limits the ability of T-cells to engage in oxidative phosphorylation, resulting in bioenergetic limitations that are sufficient to block T-cell proliferation (Vardhana et al., Nature Immunology 21 : 1022-33 2020). Oxidative stress is interlinked with elevation of reactive oxygen species and T-cell exhaustion (Redman et al., Cell Metabolism 27(4/805-815 (2018)). Oxidative stress related complexes that were associated with ROS production were expressed at lower levels in NY-ESO-1 TCR T-cells expanded in MRM. In addition, transcriptomic analysis revealed that genes associated with OXPHOS and respiratory electron transport genes were significantly upregulated in these cells. These results suggest that the majority of the cellular energy in this cell product is supported by mitochondrial oxidative phosphorylation (OXPHOS). Autophagy, another means to determine metabolic fitness of cells, has also been shown to be essential for memory formation and for preventing senescence in cells (Xu et al., 2014). Autophagy pathway-related proteins were higher in NY-ESO-1 TCR T-cells expanded in MRM. These results indicate that expansion of T-cells in MRM generates products that confer metabolic fitness partly by reduced oxidative stress and improved autophagy that is needed for a sternness phenotype.

[0315] Collectively these data suggest that T-cells expanded in MRM acquire a metabolic state defined by functional caloric restriction, an ongoing autophagy and reduced oxidative stress to maintain a state of metabolic fitness.

[0316] Assessment of T-cell trafficking capability:

[0317] A successful anti-tumor immune response relies on the trafficking of T-cells to the tumor site (Slaney et al., Cancer Research 74(24)'. 7168-74 (2014)), and T-cells high in unfolded protein response have been reported to have reduced infiltration to the tumor (Song et al., Nature Letters 562:423-428 (2018)). Bulk RNA-Seq gene set enrichment analysis revealed that the unfolded protein response gene set was downregulated in CD8+ NY-ESO-1 TCR T-cells expanded in MRM compared to cells expanded in control media for T cells from five healthy donors (FIGs. 7A-7E). Single-cell RNA-Seq analysis demonstrated that the expressions of the chemokine receptor gene CXCR3 and the integrin gene ITGA4 are increased in CD8+ NY-ESO-1 TCR T- cells expanded in MRM when compared to control 2 but not when compared to control 1 (FIGs. 7C-7D). CXCR3 is crucial to T-cell trafficking and function (Groom and Luster, Experimental Cell Research 377(5/620-31 2011), and CXCR3 -mediated T-cell tumor trafficking has been shown to be required for anti-PD-1 therapy (Han et al., EBioMedicine 48: 169-177 (2019)). ITGA4 encodes an integrin subunit reported to be indispensable for the homing of CD4+ Thl cells in central nervous system (Glatigny et al., Journal of Immunology 187 (12) :6176-9 (2012)).

[0318] In addition, T cells were isolated from patients with synovial sarcoma and cultured at research scale in MRM or control media. The differentiation profiles of T cells (Tstem-iike vs more- differentiated subsets) were then analyzed by UMAP in T cells cultured in MRM compared to T cells cultuted in control media (FIGs. 7F-7G).

[0319] Together these results suggest that NY-ESO-1 TCR T-cells expanded in MRM have increased trafficking capability.

[0320] Assessment of the intra-cellular cytokine profile:

[0321] IL-2, IFNy, TNFOC, and CCR7 expression in the NY-ESO-1 TCR cells was determined to assess for the presence of functionally less differentiated CD3+ or CD4+/CD8+/TCRvpi3.1+ T-cell subsets that were capable of producing multiple cytokines indicating polyfunctionality of T-cells. The following cytokine producing subsets were assessed: single cytokine producing subsets (the homeostatic cytokine IL-2+ or the effector cytokine IFNy+), polyfunctional cytokine producing T-cells (IL-2+, IFNy+) and /or CCR7+ less differentiated T- cells either producing IL-2 or IFNy.

[0322] Briefly, donor cell products (healthy donors and patient samples) were harvested at specified harvest times and counted. They were then re-suspended at 4xl0⁶ cells/mL, and 400,000 T-cells (100 .L of T-cell suspension) was plated in a LT- or V-bottom plate for ICS stimulation. 50 pL of a 1 : 125 dilution of phorbol myristate acetate (PMA)-ionomycin was added to the cells and incubated at 37°C for 1 hour. After 1 hour, 50 pL of a 1 :375 Golgi-stop and 1 :250 Golgi-plug dilution was added to the cells with PMA-ionomycin and incubated for 4 hours at 37°C. The cells were then stained with the sternness and cytokine panel as described above.

[0323] The proportion of T-cells producing multiple cytokines was significantly higher in NY-ESO-1 TCR T-cells expanded in MRM (FIGs. 8 and 9). In addition, the proportion of T-cells that did not produce IL-2 or IFNy in response to the PMA-ionomycin stimulation was lower in these cells. Of the less differentiated T-cells producing either IL-2 or IFNy, donors cells expanded with MRM had higher frequencies of CCR7+ IL-2+ T-cells (FIGs. 8-9). Overall, these data suggest that despite donor dependent variabilities, T-cells expanded in MRM have a higher proportion of less differentiated T-cells that produce multiple cytokines.

[0324] Evaluation of in-vitro potency of NY-ESO-1 TCR T-cell products against target cell lines expressing various levels NY-ESO-l/LAGE-la antigen

[0325] The in vitro potency of NY-ESO-1 TCR T-cell products against three Nuclight Red

(NLR) target cell lines expressing various mRNA levels of NY-ESO-1 and/or LAGE-la was evaluated in a proliferative capacity assay that included assessment of cytotoxic activity over a 7- day co-culture, assessment of cytokine secretion 24 hours after co-culture, and assessment of T- cell proliferation after 5 days of co-culture. Of the three NLR target cell lines used, A375 was identified as being NY-ESO-l/LAGE-la antigen (Ag) high, H1703 was Ag medium, and Colo205 was Ag negative. T-cell proliferation was determined after 5 days of co-culture with low-to-high Ag target cell lines at an effector to target (E:T) ratio of 1 : 1. The proliferative capacity of the T- cell products was calculated based on the dilution of the proliferative dye cell trace violet (CTV). Results showed that NY-ESO-1 TCR T-cells expanded in MRM exhibit high TCF7 expression and have increased proliferative potential after antigen encounter (FIGs. 10A-10B).

[0326] The cytotoxic activity of NY-ESO-1 TCR T-cell products was determined using the INCUCYTE™ S3 Live-Cell Analysis System in which the kinetics of NLR target cell clearance were tracked over a 162-hour time course. Overall, a lower tumor cell viability was observed when using NY-ESO-1 TCR T-cells expanded in MRM as compared to control 2 (data not shown). Importantly, no killing of the Ag-negative cell line Colo205 was observed by any of the NY-ESO- 1 TCR T-cell products.

[0327] IFNy, IL-2, and TNFa cytokine secretion levels were determined in supernatants collected from the E:T ratio of 1 : 1 from the proliferative assay plates 24 hours after co-culture start using MSD analysis. Similar to the cytotoxic activity, variability in cytokine secretion by the NY- ESO-1 TCR T-cell products was observed across all donors and target cell lines. NY-ESO-1 TCR T-cells expanded in MRM produced higher IFNy, IL-2, and TNFa levels against A375 and H1703 (high- and medium-expressing target cells) as compared to cells expanded in control media. The cytokine levels were determined from co-culture supernatants and therefore did not take into account any IL-2 utilized by the T-cells themselves in an autocrine and/or paracrine manner. After a 24-hour co-culture with the Ag-negative cell line, Colo205, the NY-ESO-1 TCR T-cell products secreted ~55 pg/mL of IFNy, ~47 pg/mL of IL-2, and ~41 pg/mL of TNFa (data not shown).

[0328] We observed a reduced proportion of IFNY alone cells in cells expanded in MRM by intracellular staining, but a large fraction of these cells are polyfunctional as assessed by IL- 2+/IFNy+, IL-2+/CCR7+, and IFNy+/CCR7+. This is consistent with the notion that stem-like cells have cytokine secreting abilities similar to that of less differentiated memory subsets. These results suggest that cells expanded in MRM produce cell products that exhibit qualities consistent with stem-like memory subsets with superior polyfunctional cytokine secretion, higher proliferative capacity and also retain effector function proportion of TEM cells associated with cytotoxicity.

[0329] The data above show that culturing NY-ESO-1 T-cell products in MRM prevents rapid progression of T cell differentiation without compromising T cell expansion. Additionally, these NY-ESO-1 T-cells exhibited increased proportions of less differentiated T cells that are metabolically fit and have the properties expected to elicit superior effector functions against tumors expressing NY-ESO-1.

Example 3: NY-ESO-1 TCR T-cells expanded in MRM exhibit superior cytotoxicity, proliferation and preservation of cells with stem-like properties after persistent antigen exposure.

[0330] In order to model T-cell persistent antigen exposure in vitro, NY-ESO-1 TCR T- cells expanded in MRM or control media were serially re-stimulated every 3 or 4 days by coculture with irradiated A375 NY-ESO+ cancer cells for a total of 4 rounds of stimulation. At each day of harvest, NY-ESO-1 TCR expression was determined, as well as the fold expansion of T-cells. Phenotypic evaluation of the NY-ESO-1 TCR T-cell products by flow cytometry was conducted as described above before stimulation, after 2 rounds and after 4 rounds of NY-ESO-1 antigen stimulation (i.e., Day 0, Day 7, and Day 14, respectively) and assessed for T-cell differentiation. T-cell phenotypes were also evaluated using transcriptomic profiling by single-cell CITE-Seq conducted on Day 14. In addition, functional evaluation of the NY-ESO-1 TCR T-cell products was conducted on Day 0, Day 7, and Day 14 of the serial re-stimulation assay, including measurement of cytotoxicity and cytokine secretion in response to target cell lines expressing various levels of NY-ESO-1 antigen.

[0331] Phenotypic evaluation of CD45RA and CCR7 expression using flow cytometry after repeated rounds of NY-ESO-1 antigen stimulation demonstrated that all of the cell products, regardless of expansion media, exhibited differentiation towards an effector memory T-cell (Tem) phenotype (data not shown). To further evaluate phenotypic differences after persistent antigen exposure, single-cell RNA-Seq was performed on CD45+/TCRvpi3.1+ T-cells. To assess the stem-like properties of cells after persistent antigen exposure, gene signatures from published literature were used (Krishna et al. 2020, Gattinoni, L., et al., Nat Med 17(10) : 1290-1297 (2011)). The naive-associated gene set described in Gattinoni, L., et al., Nat Med 17(10) .1290-1297 (2011) and the refined gene signatures of the CD39-/CD69- double negative (DN) subset post-clustering on the single cells (Krishna et al., Science 370:1328-1334 (2020)) were used to identify less- differentiated cells. The analysis demonstrated enrichment of both the Gattinoni naive-associated score and CD39-/CD69- DN gene signature score in cluster 1 (Cl) (FIGs. 11A-11B). Protein expression data measured by CITE-Seq antibody staining confirmed that Cl exhibited high expression of CD27 and CD28 as well as low expression of CD39 and CD69 proteins, as was seen in the CD39-/CD69- DN subset in TILs (FIG. 11C, Krishna et al., Science 370: 1328-1334 (2020)). Cl also showed intermediate TCF7 expression (73% cells in Cl had TCF7 expression > 0.5), lower than C2 and C13 (96% cells in C2 and 97% cells in C13 had TCF7 expression > 0.5. C13 comprised <0.3% of cells) but higher than the rest of the clusters. An increase in the proportion of Cl ranging from 5% to 20% was observed in NY-ESO-1 TCR T-cells expanded in MRM as compared to cells expanded in control media (FIG. 11D). In addition to the stem-like cluster Cl, a set of “more- differentiated clusters” was also defined based on the effector-associated gene set described in Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011) (FIGs. 11A-11B). The proportion of the more-differentiated clusters was higher in NY-ESO-1 TCR T-cells expanded in MRM (FIG. 1 ID). Taken together, these results suggest that NY-ESO-1 TCR T-cell product expanded in MRM has both stem-like T-cells as well as effector-like T-cells after persistent antigen exposure.

[0332] Another hallmark of dysfunctional T-cells is the loss of effector functions, such as cytotoxicity and cytokine secretion (McLane et al., Annu Rev Immunol 26(37) :457-495 (2019)). Thus, the functional activity of NY-ESO-1 TCR T-cell products was evaluated at the beginning of the serial re-stimulation assay and after four rounds of stimulation (i.e., Day 0, Day 7, and 14, respectively), which included measurement of cytotoxicity and cytokine secretion in response to the NY-ESO-1+ target cell lines, A375 and H1703, and the NY-ESO-1 negative control cell line, Colo205.

[0333] The functional activity of NY-ESO-1 TCR T-cell products was determined using the INCUCYTE™ S5 Live-Cell Analysis System in which the kinetics of NLR target cell clearance was tracked over a 144- to 168-hour time-course. The cytotoxic activity of NY-ESO-1 TCR T-cell products was evaluated immediately post-thaw and at the 3^rd and 5^th round of serial stimulation (Day 0, 7, and 14, respectively) against the A375 (high antigen), H1703 (medium antigen), and Colo205 (antigen negative) NLR target cell lines at an E:T ratio of 1 : 1, 1 :5, 1 : 10, and 1 :20. The AUC was calculated from the INCUCYTE™ killing kinetics for each T-cell product at the E:T ratios of 1 : 1, 1 :5, 1 :10, and 1 :20.

[0334] At Day 0 (i.e., post-thaw), all the NY-ESO-1 TCR T-cell products, regardless of expansion media, showed substantial killing capacity against A375 and H1703 cell lines, even at the lower E:T ratios, where fewer NY-ESO-1 TCR+ T-cells per target cell were added to the samples (FIG. 12). After two and four rounds of stimulation with NY-ESO-1 antigen (i.e., Day 7), the NY-ESO-1 TCR T-cells expanded in MRM and control 1 continued to maintain the cytotoxicity effects against both A375 and H1703 cell lines (FIG. 12). No cytotoxic activity was observed from the NY-ESO-1 TCR T-cell products against the antigen-negative cell line Colo205 on Day 0 or 14 (FIG. 12).

[0335] IFNy, IL-2, and TNF-a cytokine levels were assayed in supernatants, which were collected from the INCUCYTE™ killing assay plates 20 to 22 hours after co-culture start, using MSD analysis. FIG. 13 provides graphs showing mean cytokine levels with ratio paired t-test analysis. NY-ESO-1 TCR T-cell products expanded in MRM generally secreted higher levels of IFNy throughout the multiple rounds of serial stimulation (Day 0, 7, and 14) as compared to cells expanded in control 2. At day 0, NY-ESO-1 TCR T-cells expanded in MRM also secreted higher levels of IL-2 and TNFa against A375 and significantly higher levels of IL-2 (p-value of 0.006) and TNFa (p-value of 0.007) against H1703 (FIG. 13). After 3 to 5 rounds of stimulation, similar or higher IL-2 and TNFa production were observed from NY-ESO-1 TCR T-cells expanded in MRM as compared to control 1.

[0336] NY-ESO-1 TCR T-cells expanded in MRM produced higher amount of IFNy against H1703, similar IFNy production against A375 in 2 donors (D10744 and D7179) and less in 1 donor (D 14174) at primary stimulation. In the later rounds of stimulations, cells expanded in control media exhibited similar or better IFNy production compared to NY-ESO-1 TCR T-cells expanded in MRM. NY-ESO-1 TCR T-cells expanded in MRM and cocultured with A375 showed higher levels of secreted IL-2 than cells expanded in control media at primary stimulation. This difference was significant (p-value of 0.011) when the cells were cultured against H1703 at the primary stimulations. After a 20- to 22-hour co-culture with the Ag-negative cell line, Colo205, the NY-ESO-1 TCR T-cells secreted less than 50 pg/mL of IFNy, 6 pg/mL of IL-2, and 5 pg/mL of TNFa (FIG. 13).

[0337] Together these data show that the NY-ESO-1 TCR T-cells expanded in MRM displayed killing capacity and cytokine production after four rounds of antigen stimulation (i.e., on Day 0, Day 7, and Day 14).

[0338] Proliferative Capacity and Cytokine Secretion

[0339] To assess proliferative capacity and cytotoxic function after persistent antigen exposure, T cells cultured in either MRM or control media were serially restimulated every 3-4 days with irradiated NY-ESO-1 antigen-positive A375 target cells at an effector (E):target (T) ratio of 1 : 1 for a total of 4 rounds of stimulation. T-cell expansion and proliferative capacity, cytotoxicity potential, and cytokine secretion were then evaluated. T-cell expansion during serial restimulation with NY-ESO-1 antigen in NY-ESO-1 TCR T-cell products produced at large-scale from 2 donors are shown in FIGs. 14A-14B. Shown is theoretical fold expansion of T cells from the control media-cultured T cell product (1) and MRM-cultured T cell product (2) on Days 0, 3, 7, 10, and 14 of the serial restimulation assay. Cytotoxicity at Day 14 of the serial restimulation assay (E:T ratio, 1 :10) for NY-ESO-1 TCR T-cell products derived from 2 donors is shown in FIGs. 14C-14D. The mean fold change of NLR count over time zero was used to track target cell death for the control media-cultured T cell product (1) and MRM-cultured T cell product (2). Gray line (3) depicts NLR count of tumor only. Cytokine secretion during serial restimulation by NY- ESO-1 TCR T cells was consistently higher in MRM-cultured T cells as compared to control media-cultured T cells for IFNy (FIGs. 14E-14F), IL-2 (FIGs. 14G-14H), and TNFa (FIGs. MI- 14 J), as assessed on Day 14 post serial restimulation after overnight co-culture with A375 NLR cancer cell lines at an E:T ratio of 1 : 1. These data show that MRM-cultured T cells display persistent anti-tumor efficacy, similar-to-improved proliferative capacity, and sustained cytokine production after serial antigen encounter, as compared to the control media-cultured T cells.

[0340] T Cell Phenotypes

[0341] T-cell phenotypes were evaluated after serial restimulation using transcriptional profiling by single-cell CITE-Seq, which captures both mRNA and protein. UMAP of CD8+ T- cell clusters identified by single-cell RNA-Seq analysis of NY-ESO-1 TCR T cells produced at large-scale from 2 donors and collected on Day 14 of serial restimulation is shown in FIG. 15 A. Each dot represents a single cell projected onto a 2-dimensional space. The cluster exhibiting enrichment of both Gattinoni naive-associated gene signature score and CD39-/CD69- double- negative (DN) gene signature score is labeled as "1". Projection of the previously described naive- associated gene setl (left) and refined CD39-/CD69- DN gene signatures2 (right) is shown in FIG. 15B. For each gene set, normalized average expression was calculated on a single-cell level. CD39, CD69, CD27, and CD28 protein expression measured by CITE-Seq is visualized in the same UMAP space and shown in FIGs. 15C-15F. T cell product from one of the two donors showed a higher percentage of Cl cells (exhibiting enrichment of both Gattinoni naive-associated gene signature score and CD39-/CD69- gene signature score) on Day 14 in MRM-cultured cells as compared to control media-cultured T cells (FIG. 15G). These data show that MRM-cultured T cells maintain higher proportions of Tstem-iike cell populations after serial antigen encounter, as compared to T cells cultured in control media.

[0342] MRM-cultured T cells further displayed a proliferative capacity similar to or higher than that of control media-cultured T cells derived from both donors, and MRM-cultured T cells showed persistent cytotoxicity and production of cytokines (IFNy and IL-2) in response to antigenexpressing cells. After persistent antigen exposure, the proportion of CD8+ Tstem-like cells in MRM-cultured T cells was similar to or higher than that of T cells cultured in control media.

[0343] These data suggest that epigenetic reprogramming of NY-ESO-1 TCR T-cell products with MRM improves sternness, metabolic fitness, and functional persistence relative to NY-ESO-1 TCR T-cell products developed without MRM. The durable sternness observed provides evidence that MRM can be used to generate autologous T-cell products with attributes known to correlate with anti-tumor efficacy and response durability. These results also suggest that MRM confers metabolic fitness, potentially by reducing glycolysis and driving metabolic reprogramming that may be required to preserve the sternness phenotype. T cell cultured in MRM demonstrated potent functional activity in vitro, with similar-to-higher functional capacity and greater persistence of Tstem-iike cell populations after serial antigen encounter relative to a control NY-ESO-1 TCR T-cell product. MRM-cultured T cells also maintained improved cytotoxicity and cytokine production throughout four rounds of antigen stimulation.

Example 4: Analysis of the Effect of Metabolic Reprogramming Medium NY-ESO-1 TCR T-cells that Overexpress c-Jun

[0344] To determine whether the improved biological effects observed above in Examples 1-3 can be further enhanced, NY-ESO-1 TCR T-cells will be further modified to overexpress c- Jun. The NY-ESO-1 TCR T-cells are modified and cultured in either a metabolic reprogramming medium described herein (e.g., comprising potassium ion at a concentration higher than 5 mM) or in a control medium that does not comprise potassium ion at a concentration higher than 5 mM). The modified NY-ESO-1 TCR T-cells will then be assessed for various properties, including but not limited to, transduction efficiency, sternness phenotype, effector function (e.g., the ability of the modified NY-ESO-1 TCR T-cells overexpressing c-Jun to recognize and kill NY-ESO-1- expressing target cells, including after repeated stimulation), or resistance to exhaustion.

***

[0345] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0346] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0347] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0348] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0349] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

Claims

What is claimed is:

1. A method of increasing sternness of human T cells ex vivo or in vitro comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-I 157- 16S/HLA-A*02 complex

2. A method of increasing the yield of human T cells during ex vivo or in vitro culture comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex.

3. A method of preparing a population of human T cells for immunotherapy comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-I157- 16S/HLA-A*02 complex.

4. A method of increasing sternness of human T cells while increasing the yield of human T cells during ex vivo or in vitro culture for immunotherapy comprising culturing human T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-li57-i65/HLA-A*02 complex.

5. A method of expanding a population of stem-like T cells ex vivo or in vitro comprising culturing T cells in a medium comprising potassium ion at a concentration higher than 55 mM, wherein the T cells express a T cell receptor (TCR) that specifically binds the NY-ESO-I 157- i65/HLA-A*02 complex.

6. The method of any one of claims 1 to 5, further comprising transfecting the T cells with a polynucleotide encoding the TCR prior to, concurrently with, or after culturing the T cells in the medium comprising potassium ion at a concentration higher than 55 mM.

7. A method of preparing T cells ex vivo or in vitro for immunotherapy comprising transfecting T cells with a polynucleotide which encodes a T cell receptor (TCR) that specifically binds the NY-ESO-1157-165/HLA-A*O2 complex; wherein the T cells are transfected in a medium comprising potassium ion at a concentration higher than 55 mM.

8. The method of any one of claims 1 to 7, wherein the TCR comprises an a chain and a P chain, wherein the a chain comprises the CDR1-3 in SEQ ID NO:5 and the P chain comprises the CDR1-3 in SEQ ID NO:6.

9. The method of claim 8, wherein the TCR a chain CDR1-3 comprise SEQ ID NOs:7-9, respectively, and the TCR P chain CDR1-3 comprise SEQ ID NOs: 10-12, respectively.

10. The method of claim 8, 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 P chain comprises a variable domain comprising SEQ ID NO:6 or an amino acid sequence at least 90% identical thereto.

11. The method of claim 10, wherein the TCR a and P chains comprise SEQ ID NOs:3 and 4, respectively, or SEQ ID NOs:20 and 21, respectively.

12. The method of any one of claims 1 to 11, wherein the TCR is encoded by an expression construct, 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.

13. The method of claim 12, wherein the expression construct comprises a bi-cistronic expression cassette for expressing a TCR a chain and a TCR P chain.

14. The method of any one of claims 1 to 13, wherein the concentration of potassium ion is higher than about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM.

15. The method of any one of claims 1 to 13, wherein the concentration of potassium ion is selected from the group consisting of about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, and about 80 mM.

16. The method of any one of claims 1 to 13, wherein the concentration of potassium ion is between about 55 mM and 80 mM, about 60 mM and about 80 mM, about 70 mM and about 80 mM, about 50 mM and about 70 mM, about 60 mM and about 70 mM, or about 50 mM and about 60 mM.

17. The method of any one of claims 1 to 13, wherein the concentration of potassium ion is about 50 mM, about 60 mM, or about 70 mM.

18. The method of any one of claims 1 to 17, wherein the potassium ion comprises KC1.

19. The method of any one of claims 1 to 18, wherein the medium further comprises sodium ion.

20. The method of any one of claims 1 to 19, wherein the medium further comprises NaCl.

21. The method of any one of claims 1 to 20, wherein the medium comprises 1 mM to less than about 140 mM, 1 mM to less than about 130 mM, 1 mM to less than about 120 mM, 1 mM to less than about 110 mM, 1 mM to less than about 100 mM, 1 mM to less than about 90 mM, 1 mM to less than about 80 mM, 1 mM to less than about 70 mM, 1 mM to less than about 60 mM, 1 mM to less than about 50 mM, or 1 mM to less than about 40 mM NaCl.

22. The method of any one of claims 1 to 21, wherein the medium is hypotonic or isotonic.

23. The method of any one of claims 1 to 22, wherein the medium is hypotonic, and wherein the sum of the potassium concentration and the sodium chloride concentration, multiplied by two is less than 280.

24. The method of any one of claims 1 to 22, wherein the medium is hypotonic, and wherein the sum of the potassium concentration and the sodium chloride concentration, multiplied by two is more than 240 and less than 280.

25. The method of any one of claims 1 to 22, wherein the medium is isotonic, and wherein the sum of the potassium concentration and the sodium chloride concentration, multiplied by two is more than or equal to 280 and less than 300.

26. The method of any one of claims 1 to 22, wherein the concentration of potassium ion is about 60 mM, and the concentration of NaCl is less than 80 mM, less than 75 mM, less than 70 mM, less than 65 mM, or less than 60mM.

27. The method of any one of claims 1 to 22, wherein the concentration of potassium ion is about 55 mM, and the concentration of NaCl is less than 85 mM, less than 80 mM, less than 75 mM, less than 70 mM, or less than 65mM. - 129 -

28. The method of any one of claims 1 to 22, wherein the concentration of potassium ion is about 50 mM, and the concentration of NaCl is less than 90 mM, less than 85 mM, less than 80 mM, less than 75 mM, or less than 70 mM.

29. The method of any one of claims 1 to 28, wherein the medium further comprises one or more cytokines.

30. The method of claim 29, wherein the one or more cytokines comprise Interleukin-2 (IL-2), Interleukin-7 (IL-7) or Interleukin- 15 (IL- 15), or any combination thereof.

31. The method of claim 29, wherein the one or more cytokines comprise IL-2, IL-7, and IL- 15.

32. The method of any one of claims 1 to 31, wherein the medium further comprises calcium ion, glucose, or any combination thereof.

33. The method of any one of claims 1 to 31, wherein the medium further comprises glucose.

34. The method of claim 33, wherein the concentration of glucose is more than about 10 mM.

35. The method of claim 33 or 34, wherein the concentration of glucose is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.

36. The method of any one of claims 32 to 34, wherein the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

37. The method of any one of claims 33 to 36, wherein the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

38. The method of any one of claims 1 to 37, wherein the medium further comprises calcium ion. - 130 -

39. The method of claim 38, wherein the concentration of calcium ion is more than about 0.4 mM.

40. The method of claim 38 or 39, wherein the concentration of calcium ion is from about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about 1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM.

41. The method of any one of claims 38 to 40, wherein the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

42. The method of any one of claims 1 to 41, wherein the T cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, or TCF7+, or any combination thereof, following the culturing.

43. The method of any one of claims 1 to 42, wherein the medium comprises IL-2 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL.

44. The method of claim 43, wherein the concentration of IL-2 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 - 131 - ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL.

45. The method of claim 43 or 44, wherein the concentration of IL-2 is about 1.0 ng/mL.

46. The method of claim 43 or 44, wherein the concentration of IL-2 is about 10 ng/mL.

47. The method of any one of claims 1 to 46, wherein the medium comprises IL-7 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL.

48. The method of claim 47, wherein the concentration of IL-7 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL.

49. The method of claim 47 or 48, wherein the concentration of IL-7 is about 1.0 ng/mL.

50. The method of claim 47 or 48, wherein the concentration of IL-7 is about 10 ng/mL.

51. The method of any one of claims 1 to 50, wherein the medium comprises IL- 15 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL - 132 - to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL.

52. The method of claim 51, wherein the concentration of IL- 15 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL.

53. The method of claim 51 or 52, wherein the concentration of IL-15 is about 1.0 ng/mL.

54. The method of claim 51 or 52, wherein the concentration of IL- 15 is about 10 ng/mL.

55. The method of any one of claims 1 to 54, wherein the T cells further express a c-Jun polypeptide.

56. The method of claim 55, wherein the TCR and the c-Jun polypeptide are on the same sequence.

57. The method of claim 1 to 55, wherein the TCR and the c-Jun polypeptide are on different sequences.

58. A population of human T cells prepared by the method of any one of claims 1 to 57.

59. A population of human T cells, wherein at least about 5% of the T cells in the population of T cells have a stem-like phenotype.

60. The population of human T cells of claim 59, wherein at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of the T cells in the population of T cells have a stem-like phenotype.

61. The population of human T cells of claim 59 or 60, wherein the T cells having a stem-like phenotype are TCF7⁺. - 133 -

62. The population of human T cells of any one of claims 59 to 61, wherein the T cells having a stem-like phenotype are CD3⁺, CD45RO’, CCR7⁺, CD45RA⁺, CD62L⁺, CD27⁺, CD28⁺, and TCF7⁺.

63. The population of human T cells of any one of claims 59 to 62, wherein the T cells having a stem -like phenotype are CD39" and CD69".

64. The population of human T cells of any one of claims 58 o 63, wherein the T cells are CD8⁺ T cells.

65. A pharmaceutical composition comprising the population of human T cells of any one of claims 58 to 64, and a pharmaceutically acceptable carrier.

66. A method of killing target cells, comprising contacting the target cells with the population of human T cells of any one of claims 58 to 64 or the pharmaceutical composition of claim 65 under conditions that allow killing of the target cells by the T cells, wherein the target cells express NY-ESO-1.

67. The method of claim 66, wherein the target cells are cancer cells expressing NY-ESO-1.

68. A method of treating a patient in need thereof, comprising administering the population of human T cells of any one of claims 58 to 64 or the pharmaceutical composition of claim 65 to the patient.

69. A method of inducing an anti-tumor immune response in a patient, comprising administering the population of T cells of any one of claims 58 to 64 or the pharmaceutical composition of claim 65 to the patient.

70. The method of claim 68 or 69, wherein the patient has a NY-ESO-1 -expressing cancer.

71. The method of claim 70, wherein the NY-ESO-1 expressing cancer comprises a solid tumor.

72. The method of claim 70, wherein the NY-ESO-1 -expressing cancer is selected from the group consisting of 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. - 134 -

73. Use of the population of human T cells of any one of claims 58 to 64, or the pharmaceutical composition of claim 65 for the manufacture of a medicament for treating a patient in need thereof in the method of any one of claims 67 to 72.

74. The population of human T cells of any one of claims 58 to 64 or the pharmaceutical composition of claim 65 for use in a method of treating a cancer in a patient in need thereof.