CN118804933A

CN118804933A - Method for culturing ROR1 binding protein-expressing cells

Info

Publication number: CN118804933A
Application number: CN202280084762.7A
Authority: CN
Inventors: S·K·沃德纳拉; V·克里什纳莫尔蒂; S·帕克; Q·冯; B·萨特
Original assignee: Lyle Immune Pharmaceutical Co ltd
Current assignee: Lyle Immune Pharmaceutical Co ltd
Priority date: 2021-10-28
Filing date: 2022-10-27
Publication date: 2024-10-18

Abstract

Disclosed herein are methods of culturing immune cells in a medium comprising at least about 5mM potassium ions, wherein the medium is capable of increasing the stem properties of the immune cells. In some aspects, immune cells cultured using the methods provided herein are modified to express ROR1 binding proteins and have increased levels of c-Jun protein. In some aspects, the immune cells are administered to a subject in need thereof.

Description

Method for culturing ROR1 binding protein-expressing cells

Cross Reference to Related Applications

The PCT application claims U.S. provisional application Ser. No. 63/263,230, filed on 10/28 of 2021; U.S. provisional application No. 63/309,400, filed on 11, 2, 2022; and U.S. provisional application No. 63/339,347, filed 5/6 of 2022; each of the U.S. provisional applications is incorporated by reference herein in its entirety.

Reference to a sequence Listing submitted electronically via EFS-WEB

The contents of the electronically submitted sequence listing submitted in the present application (name 4385_082503_seqlipping_ST26. Xml, size 123,699 bytes, and creation date: 10 month 26 of 2022) are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to methods of culturing cells (e.g., sub-totipotent (pluripotent), pluripotent (multipotent) and/or immune cells (e.g., T cells and/or NK cells)) that have been modified to have increased levels of c-Jun protein, e.g., as compared to an unmodified corresponding cell, and that comprise an exogenous polynucleotide encoding a ROR1 binding protein. In some aspects, the methods disclosed herein promote enrichment of poorly differentiated cells (less-DIFFERENTIATED CELL) and/or undifferentiated cells in culture while retaining their effector activity. In some aspects, the culture methods provided herein can also help increase expression of a protein of interest (e.g., c-Jun) in a cell. Cells cultured using the methods disclosed herein can be used in a variety of cell therapies, including, but not limited to, chimeric Antigen Receptor (CAR) T cell therapies, TCR T cell therapies (including neoantigen-directed T cell therapies), and TIL therapies.

Background

Cancer immunotherapy relies on the use of T cells (the primary killer of the immune system against infected and diseased cells) to attack and kill tumor cells. However, the ability of immune cells to target and kill tumor cells is inhibited by the presence of various immune response inhibitors present in the tumor microenvironment. Thus, while CAR T cells have met with various success in treating certain cancers (e.g., KYMRIAH ^TM (se Li Fuming (tisagenlecleucel), novartis) and YESCARTA ^TM (albolnescen (axicabtagene ciloleucel), kit/Gilead) have been FDA approved), challenges remain. For example, the success of CAR T cell immunotherapy is often limited by the extent of CAR T expansion in the recipient, which typically requires a large cell infusion. Furthermore, depletion and persistence of transferred CAR T cells have been observed, leading to loss of clinical efficacy and potential recurrence.

One way to overcome T cell depletion is to selectively administer T cells with a low differentiation state. For example, T memory stem cells (T _SCM) persist in patients longer than highly differentiated T central memory (T _CM) or T effector memory (T _EM) cells after administration, and the effect of T _SCM on tumor size is more pronounced and longer than highly differentiated cells. However, a variety of Adoptive Cell Therapy (ACT) cell preparations contain an ambiguous mixture of immune cells in various differentiation states that are ineffective in eradicating solid tumors. For healing, T cell products with enhanced self-renewing stem cell/effector properties are needed. Thus, there remains a need in the art for methods of efficiently enriching for poorly differentiated and/or naive T cells from a mixed population of isolated T cells.

Disclosure of Invention

Provided herein is a method of increasing the stem performance of an immune cell (e.g., a human immune cell) during in vitro or in vitro culture, the method comprising culturing the immune cell (e.g., a human immune cell) in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have increased levels of a c-Jun polypeptide. Provided herein is a method of increasing the yield of immune cells (e.g., human immune cells) during in vitro or in vitro culture, the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of a c-Jun polypeptide. Provided herein is a method of preparing a population of immune cells (e.g., human immune cells) for immunotherapy, the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of a c-Jun polypeptide. Provided herein is a method of increasing the stem properties of immune cells (e.g., human immune cells) for immunotherapy, while increasing the yield of immune cells (e.g., human immune cells), during in vitro or in vitro culture, the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration above 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of a c-Jun polypeptide. Provided herein is a method of expanding a population of stem cell-like immune cells ex vivo or in vitro, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of a c-Jun polypeptide.

Also provided herein is a method of increasing cytokine production by an immune cell in response to antigen stimulation, wherein the method comprises culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of a c-Jun polypeptide.

In some aspects, the cytokine comprises IL-2. In some aspects, the cytokine produced in response to antigen stimulation is increased at least about 1-fold, 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, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-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 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more after culturing, as compared to a reference immune cell.

The present disclosure also provides a method of increasing effector function of an immune cell in response to a persistent antigen stimulus, the method comprising culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have an increased level of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have an increased level of a c-Jun polypeptide.

In some aspects, the immune cells retain effector function in at least one, at least two, or at least three additional rounds of antigen stimulation analysis as compared to a reference immune cell. In some aspects, effector functions include the following capabilities: (i) killing tumor cells, (ii) producing cytokines upon further antigen stimulation, or (iii) both (i) and (ii). In some aspects, the cytokine comprises IFN- γ.

In some aspects, after culturing, the immune cells have an increase in effector function in response to persistent antigen stimulation of at least about 1-fold, 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, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-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 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more, as compared to a reference immune cell.

Provided herein is a method of altering the phenotypic expression of one or more markers on an immune cell, the method comprising culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have increased levels of a c-Jun polypeptide.

In some aspects, the one or more markers comprise CD39, LAG3, PD1, TIGIT, TIM3, or a combination thereof. In some aspects, the expression of the one or more markers is reduced 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 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to a reference immune cell. In some aspects, the expression of the one or more markers is reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least about 5-fold as compared to a reference immune cell.

In any of the above methods, in some aspects, the reference immune cell comprises the following corresponding immune cell: (i) A c-Jun polypeptide that has been modified to have increased levels and is cultured in a medium that does not contain potassium ions at a concentration above 5 mM; (ii) Unmodified to have increased levels of c-Jun polypeptide and cultured in a medium comprising potassium ions at a concentration above 5 mM; (iii) Unmodified to have increased levels of c-Jun polypeptide and cultured in a medium that does not contain potassium ions at a concentration above 5 mM; or (iv) any combination of (i) to (iii).

In any of the above methods, in some aspects, the immune cells have been modified by an exogenous polynucleotide encoding a chimeric polypeptide, a c-Jun polypeptide, or both, such that upon modification, the immune cells have increased levels of the c-Jun polypeptide, as compared to an unmodified corresponding immune cell. In some aspects, the c-Jun polypeptide is endogenous to an immune cell, and wherein the immune cell has been modified by a transcriptional activator capable of increasing expression of the endogenous c-Jun polypeptide. In some aspects, the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity.

Also provided herein is a method of preparing an immune cell for immunotherapy ex vivo or in vitro, the method comprising modifying an immune cell with an exogenous polynucleotide encoding (i) a c-Jun polypeptide and (ii) a chimeric polypeptide comprising a ROR1 binding protein in a medium comprising potassium ions at a concentration above 5 mM. The present disclosure also provides a method of preparing immune cells for immunotherapy ex vivo or in vitro, the method comprising using, in a medium comprising potassium ions at a concentration of greater than 5 mM: (i) A transcriptional activator capable of increasing expression of an endogenous c-Jun polypeptide; and (ii) an exogenous polynucleotide encoding a chimeric polypeptide comprising a ROR1 binding protein. In some aspects, the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity.

In some aspects, the chimeric polypeptide comprises a Chimeric Antigen Receptor (CAR). In some aspects, the c-Jun polypeptide is capable of preventing or reducing the depletion of immune cells.

Provided herein is a method of increasing the stem performance of an immune cell (e.g., a human immune cell) during in vitro and in vitro culture, the method comprising culturing an immune cell (e.g., a human immune cell) in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to express a chimeric polypeptide comprising: (i) a ROR1 binding protein, (ii) a spacer comprising the amino acid sequence set forth in SEQ ID NO:51, and (iii) a transmembrane domain. Provided herein is a method of increasing the yield of immune cells (e.g., human immune cells) during in vitro or in vitro culture, the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising: (i) a ROR1 binding protein, (ii) a spacer comprising the amino acid sequence set forth in SEQ ID NO:51, and (iii) a transmembrane domain. Provided herein is a method of preparing a population of immune cells (e.g., human immune cells) for immunotherapy, the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising: (i) a ROR1 binding protein, (ii) a spacer comprising the amino acid sequence set forth in SEQ ID NO:51, and (iii) a transmembrane domain. Provided herein is a method of increasing the stem properties of immune cells (e.g., human immune cells) for immunotherapy during in vitro or in vitro culture while increasing the yield of immune cells (e.g., human immune cells), the method comprising culturing immune cells (e.g., human immune cells) in a medium comprising potassium ions at a concentration above 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising: (i) a ROR1 binding protein, (ii) a spacer comprising the amino acid sequence set forth in SEQ ID NO:51, and (iii) a transmembrane domain. Provided herein is a method of expanding a population of stem cell-like immune cells ex vivo or in vitro, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising: (i) a ROR1 binding protein, (ii) a spacer comprising the amino acid sequence set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

In any of the above methods, in some aspects, the immune cell is further modified by an exogenous polynucleotide encoding a chimeric polypeptide.

Provided herein is a method of preparing an immune cell for immunotherapy ex vivo or in vitro, the method comprising modifying the immune cell with an exogenous polynucleotide encoding a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain in a medium comprising potassium ions at a concentration of greater than 5 mM.

In some aspects, the chimeric polypeptide of any of the methods provided above comprises a Chimeric Antigen Receptor (CAR).

In some aspects, the immune cells of any of the methods provided above further overexpress a c-Jun polypeptide. In some aspects, the immune cells have been modified by an exogenous polynucleotide encoding a chimeric polypeptide, a c-Jun polypeptide, or both, such that upon modification, the immune cells have increased levels of the c-Jun polypeptide, as compared to a corresponding immune cell not modified to have increased levels of the c-Jun polypeptide. In some aspects, the c-Jun polypeptide is endogenous to the immune cell and wherein the immune cell has been modified by a transcriptional activator capable of increasing expression of the endogenous c-Jun polypeptide. In some aspects, the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity. In some aspects, the c-Jun polypeptide is capable of preventing or reducing the depletion of immune cells.

In some aspects, the c-Jun polypeptide comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID NO. 13. In some aspects, the c-Jun polypeptide is encoded by an exogenous polynucleotide comprising a nucleotide sequence having at least about 60%, at least about 70%, at least about 80%, 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 a nucleotide sequence as set forth in any one of SEQ ID NO:12、SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9 or SEQ ID NO. 10.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 12.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 1. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 1.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having 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 a nucleic acid sequence as set forth in SEQ ID NO. 2. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 2.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having 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%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a nucleic acid sequence as set forth in SEQ ID NO. 4. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 4.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having 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 about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a nucleic acid sequence as set forth in SEQ ID NO. 5. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 5.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having at least 88%, at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 6. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 6.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having at least 82%, at least 83%, at least 84%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 7. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 7.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having 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 a nucleic acid sequence as set forth in SEQ ID NO. 8. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 8.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having at least 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 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 a nucleic acid sequence as set forth in SEQ ID NO 9. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 9.

In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 10. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 10.

In some aspects, the c-Jun polypeptide and the chimeric polypeptide are on the same sequence (i.e., vector). In some aspects, the c-Jun polypeptide and the chimeric polypeptide are on different sequences (i.e., vectors).

In some aspects, the ROR1 binding protein of any one of the methods provided above comprises an antibody or antigen binding portion thereof that specifically binds to ROR 1. In some aspects, the ROR1 binding protein specifically binds to the same epitope as the R12 antibody. In some aspects, the ROR1 binding protein comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 of an R12 antibody and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 of an R12 antibody. In some aspects, VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:57, VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:58, and VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 59. In some aspects, VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:61, VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:62, and VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63. In some aspects, the VH of the ROR1 binding protein comprises the amino acid sequence set forth in SEQ ID NO:56 and the VL of the ROR1 binding protein comprises the amino acid sequence set forth in SEQ ID NO: 60. In some aspects, the ROR1 binding protein comprises an amino acid sequence that has at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence as set forth in SEQ ID NO. 83.

In any of the above methods, in some aspects, the chimeric polypeptide further comprises a transmembrane (^T M) domain. In some aspects, the TM domain is derived from CD8a, CD2, CD4, CD28, CD45, PD1, CD152, or any combination thereof. In some aspects, the TM domain is derived from CD28. In some aspects, the TM domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID NO. 75.

In some aspects, the chimeric polypeptide further comprises a spacer between the ROR1 binding protein and the TM domain. In some aspects, the spacer is derived from an immunoglobulin hinge region or CD8. In some aspects, the spacer comprises the amino acid sequence set forth in SEQ ID NO. 51. In some aspects, the spacer further comprises a linker. In some aspects, the linker comprises GGGSG (SEQ ID NO: 40).

In some aspects, the chimeric polypeptide further comprises an intracellular signaling domain. In some aspects, the intracellular signaling domain comprises a cd3ζ activation domain, a cd3δ activation domain, a cd3ε activation domain, a cd3η activation domain, a CD79A activation domain, a DAP 12 activation domain, a FCER1G activation domain, a DAP10/CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a cd3ζ activation domain. In some aspects, the cd3ζ activating domain comprises an amino acid sequence having 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%, at least about 99%, or at least about 100% sequence identity to an amino acid sequence as set forth in SEQ ID No. 84.

In some aspects, the chimeric polypeptide further comprises an intracellular co-stimulatory domain. In some aspects, the intracellular co-stimulatory domain comprises a co-stimulatory domain of: interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, ligand that binds specifically to CD83, CD28 (ICA) that lacks Lck binding, OX40, BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, fc receptor gamma chain, fc receptor epsilon chain, ligand that binds specifically to CD83, or any combination thereof.

In some aspects, the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence having 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%, at least about 99%, or at least about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 76.

In some aspects, the c-Jun polypeptide and the ROR1 binding protein are linked by a linker. In some aspects, the linker is a cleavable linker. In some aspects, the cleavable linker comprises a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof.

In some aspects, the chimeric polypeptide further comprises a truncated EGF receptor (EGFRt). In some aspects, EGFRt comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID NO. 24.

In some aspects, EGFRt is linked to the c-Jun polypeptide and/or the ROR1 binding protein by a linker. In some aspects, the linker is a cleavable linker. In some aspects, the cleavable linker comprises a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof.

In any of the above methods, in some aspects, the chimeric polypeptide further comprises a signal peptide. In some aspects, the signal peptide is derived from hIgK. In some aspects, hIgK signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 54. In some aspects, the chimeric polypeptide comprises 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 the amino acid sequence set forth in SEQ ID NO. 54.

In some aspects, the chimeric polypeptide is encoded by an exogenous polynucleotide comprising a nucleotide sequence having 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%, at least about 99%, or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 86.

In some aspects, the exogenous polynucleotide of any of the methods provided above comprises a vector. In some aspects, the exogenous polynucleotide further comprises a myeloproliferative sarcoma virus enhancer, a deleted negative control region, a dl587rev primer binding site substitution (MND) promoter, an EF1a promoter, an ubiquitin promoter, or any combination thereof. In some aspects, the MND promoter comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID NO. 92.

In any of the above methods, in some aspects, the potassium ion is at a concentration of greater than about 10mM, greater than about 15mM, greater than about 20mM, greater than about 25mM, greater than about 30mM, greater than about 35mM, greater than about 40mM, greater than about 45mM, greater than about 50mM, greater than about 55mM, greater than about 60mM, greater than about 65mM, greater than about 70mM, greater than about 75mM, greater than about 80mM, greater than about 85mM, or greater than about 90mM. In some aspects, the concentration of potassium ions is selected from the group consisting of about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, and about 80 mM. In some aspects, the concentration of potassium ions is between about 30mM and about 80mM, between about 40mM and about 80mM, between about 50mM and 80mM, between about 60mM and about 80mM, between about 70mM and about 80mM, between about 40mM and about 70mM, between about 50mM and about 70mM, between about 60mM and about 70mM, between about 40mM and about 60mM, between about 50mM and about 60mM, or between about 40mM and about 50 mM. In some aspects, the concentration of potassium ions is about 50mM, about 60mM, or about 70mM.

In some aspects, the medium further comprises sodium ions. In some aspects, the medium further comprises NaCl. In some aspects, the medium comprises less than about 140mM, less than about 130mM, less than about 120mM, less than about 110mM, less than about 100mM, less than about 90mM, less than about 80mM, less than about 70mM, less than about 60mM, less than about 50mM, or less than about 40mM NaCl.

In some aspects, the culture medium is hypotonic or isotonic.

In some aspects, the medium is hypotonic, and wherein the sum of potassium ion concentration and sodium ion concentration multiplied by 2 is less than 280mM. In some aspects, the medium is hypotonic, and wherein the sum of potassium ion concentration and sodium ion concentration multiplied by 2 is greater than 240mM and less than 280mM. In some aspects, the medium is isotonic, and wherein the sum of potassium ion concentration and sodium ion concentration multiplied by 2 is greater than or equal to 280mM and less than 300mM.

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

In some aspects, the immune cells of the methods provided above comprise T cells, B cells, regulatory T cells (tregs), tumor Infiltrating Lymphocytes (TILs), natural Killer (NK) cells, natural Killer T (NKT) cells, or any combination thereof. In some aspects, the immune cells have been engineered in vitro or ex vivo.

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), interleukin-21 (IL-21), interleukin-15 (IL-15), or any combination thereof. In some aspects, the one or more cytokines comprise IL-2, IL-7, and IL-15.

In some aspects, the medium comprises IL-2 at a concentration of about 50IU/mL to about 500IU/mL. In some aspects, the concentration of IL-2 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL. In some aspects, the concentration of IL-2 is between about 100IU/mL and about 300 IU/mL. In some aspects, the concentration of IL-2 is about 200IU/mL.

In some aspects, the medium comprises IL-21 at a concentration of about 50IU/mL to about 500IU/mL. In some aspects, the concentration of IL-21 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL. In some aspects, the concentration of IL-21 is between about 100IU/mL and about 300 IU/mL. In some aspects, the concentration of IL-21 is about 200IU/mL.

In some aspects, the medium comprises IL-7 at a concentration of about 500IU/mL to about 1,500IU/mL. In some aspects, the concentration of IL-7 is about 500IU/mL, about 550IU/mL, about 600IU/mL, about 650IU/mL, about 700IU/mL, about 750IU/mL, about 800IU/mL, about 850IU/mL, about 900IU/mL, about 950IU/mL, about 1,000IU/mL, about 1,050IU/mL, about 1,100IU/mL, about 1,150IU/mL, about 1,200IU/mL, about 1,250IU/mL, about 1,300IU/mL, about 1,350IU/mL, about 1,400IU/mL, about 1,450IU/mL, or about 1,500IU/mL. In some aspects, the concentration of IL-7 is from about 1,000IU/mL to about 1,400IU/mL. In some aspects, the concentration of IL-7 is about 1,200IU/mL.

In some aspects, the medium comprises IL-15 at a concentration of about 50IU/mL to about 500IU/mL. In some aspects, the concentration of IL-15 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL. In some aspects, the concentration of IL-15 is between about 100IU/mL and about 300 IU/mL. In some aspects, the concentration of IL-15 is about 200IU/mL.

In some aspects, the medium further comprises a cell expansion agent. In some aspects, the cell expansion agent comprises a GSK3B inhibitor, ACLY inhibitor, PI3K inhibitor, AKT inhibitor, or any combination thereof. In some aspects, the PI3K inhibitor is selected from hydroxycitrate, LY294002, pitelist (pictilisib), CAL101, IC87114, or any combination thereof. In some aspects, the AKT inhibitor is selected from MK2206, A443654, AKTi-VIII, or any combination thereof.

In some aspects, the media of the methods provided above are capable of being in the final cell product as compared to immune cells cultured in media that do not contain high concentrations of potassium ions and/or immune cells that do not contain the c-Jun polypeptide, as compared to starting immune cells: a) Increasing the number and/or percentage of poorly differentiated and/or undifferentiated cells; b) Increasing transduction efficiency; c) Increasing stem cell-like immune cells; d) Increasing the activity in vivo; e) Increasing cellular potency; f) Preventing cell depletion; or g) any combination thereof.

In some aspects, the medium further comprises calcium ions, glucose, or both.

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

In some aspects, the medium further comprises calcium ions, and wherein the concentration of calcium ions is greater than about 0.4mM. In some aspects of the present invention, the concentration of calcium ions is about 0.4mM to about 2.8mM, about 0.4mM to about 2.5mM, about 0.5mM to about 2.0mM, about 1.0mM to about 2.0mM, about 1.1mM to about 2.0mM, about 1.2mM to about 2.0mM, about 1.3mM to about 2.0mM, about 1.4mM to about 2.0mM, about 1.5mM to about 2.0mM, about 1.6mM to about 2.8mM, about 1.7mM to about 2.0mM, about 1.8mM to about 2.0mM, about 1.2 to about 1.3mM, about 1.2 to about 1.4mM, about 1.2 to about 1.5mM about 1.2 to about 1.6mM, about 1.2 to about 1.7mM, about 1.2 to about 1.8mM, about 1.3 to about 1.4mM, about 1.3 to about 1.5mM, about 1.3 to about 1.6mM, about 1.3 to about 1.7mM, about 1.3 to about 1.8mM, about 1.4 to about 1.5mM, about 1.4 to about 1.6mM, about 1.4 to about 1.7mM, about 1.4 to about 1.8mM, about 1.5 to about 1.6mM, about 1.5 to about 1.7mM, about 1.5 to about 1.8mM, about 1.6 to about 1.7mM, about 1.6 to about 1.8mM, or about 1.7 to about 1.8mM. In some aspects, the concentration of calcium ions is about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, about 2.0mM, about 2.1mM, about 2.2.mM, about 2.3mM, about 2.4mM, about 2.5mM, about 2.6mM, about 2.7mM, about 2.8mM, about 2.9mM, or about 3.0mM.

In some aspects, the immune cells are cd3+, CD45RO-, ccr7+, cd45ra+, cd62l+, cd27+, cd28+, or tcf7+, or any combination thereof, after culturing. In some aspects, the immune cells are formulated in a pharmaceutical composition and a pharmaceutically acceptable carrier.

Provided herein are populations of immune cells (e.g., human immune cells) prepared by any of the methods described herein.

Provided herein is a population of immune cells capable of achieving long-term persistence in vivo, wherein the immune cells have: (i) Culturing in a medium comprising potassium ions at a concentration of greater than 5mM, and (ii) modifying to express a chimeric polypeptide comprising a ROR1 binding protein and having an increased level of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have an increased level of a c-Jun polypeptide. In some aspects, the population of immune cells described herein is capable of lasting at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months in a subject in need thereof when administered to the subject.

Also provided herein is a pharmaceutical composition comprising a population of immune cells (e.g., human immune cells) described herein and a pharmaceutically acceptable carrier.

The present disclosure also provides a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject any of the population of immune cells (e.g., human immune cells) or the pharmaceutical composition provided herein. In some aspects, the tumor is derived from a cancer comprising breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some aspects, the tumor is a solid tumor.

In some aspects, after administration, the tumor size (tumor size) is reduced compared to a reference tumor size. In some aspects, referencing the tumor size comprises: (i) tumor size prior to administration, (ii) tumor size of a corresponding subject not receiving administration (e.g., receiving administration of a corresponding immune cell transduced and cultured in a medium that does not comprise potassium ions at a concentration greater than 5 mM), or (iii) both (i) and (ii). In some aspects, the tumor size is reduced 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 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to a reference tumor size.

In some aspects, the subject's survival duration increases after administration as compared to a reference survival duration. In some aspects, the reference survival duration comprises the survival duration of a corresponding subject that did not receive administration (e.g., received administration of a corresponding immune cell transduced and cultured in a medium that did not comprise potassium ions at a concentration above 5 mM). In some aspects, the survival duration is increased by at least about one week, at least about two weeks, at least about three weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about 10 months, at least about 11 months, or at least about one year as compared to the reference survival duration.

In some aspects, the methods of treating a tumor provided herein further comprise administering at least one additional therapeutic agent to the subject. In some aspects, the at least one additional therapeutic agent comprises a chemotherapeutic drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, a co-stimulatory molecule activator, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. In some aspects, the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, or any combination thereof.

Also provided herein is a composition comprising a population of cd4+ T cells and cd8+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of a c-Jun polypeptide, wherein: (i) At least about 20% of the modified cd4+ T cells are surface positive for CCR7 and CD45 RA; (ii) At least about 20% of the modified cd8+ T cells are surface positive for CCR7 and CD45 RA; or (iii) both (i) and (ii).

Provided herein is a composition comprising a population of cd4+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of c-Jun polypeptide, wherein at least about 20% of the modified cd4+ T cells are surface positive for CCR7 and CD45 RA. Provided herein is a composition comprising a population of cd8+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of c-Jun polypeptide as compared to corresponding immune cells that have not been modified to have increased levels of c-Jun polypeptide, wherein at least about 20% of the modified cd8+ T cells are surface positive for CCR7 and CD45 RA.

Also provided herein is a composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of c-Jun polypeptide as compared to corresponding immune cells that have not been modified to have increased levels of c-Jun polypeptide, wherein at least about 4% of the cells are progenitor depleted T cells. Some aspects of the disclosure relate to a composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of c-Jun polypeptide as compared to corresponding immune cells that have not been modified to have increased levels of c-Jun polypeptide, wherein between about 4% and about 6% of cells are progenitor depleted T cells. Also provided herein is a composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of a c-Jun polypeptide, wherein at least about 4% of the cells are progenitor depleted T cells and at least about 4% of the cells are stem cell-like T cells.

Drawings

FIGS. 1A, 1B and 1C provide a comparison of anti-ROR 1 (R12) CAR transduction efficiencies of T cells cultured (or grown) in TCM (i.e., control medium) and MRM. The T cells (including cd4+ and cd8+ T cells) are derived from different donors: donor #1 (fig. 1A), donor #2 (fig. 1B), and donor #3 (fig. 1C). The different transduction conditions (or test groups) were as follows: (1) non-transduced T cells cultured in TCM; (2) T cells transduced with control CD19T-R12CAR (i.e., R12CAR without c-Jun) and cultured in TCM; (3) T cells transduced with a c-Jun-R12 CAR (i.e., R12CAR with c-Jun) and cultured in TCM; (4) non-transduced T cells cultured in MRM; (5) T cells transduced with control CD19T-R12CAR and cultured in MRM; and (6) T cells transduced with c-Jun-R12 CAR and cultured in MRM.

Figures 2A, 2B and 2C show the percentages of cd4+ (black bars) and cd8+ (white bars) T cells present in the total transduced T cell populations from the different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. Transduced T cells are derived from three different donors: donor #1 (fig. 2A), donor #2 (fig. 2B), and donor #3 (fig. 2C).

FIGS. 3A, 3B and 3C show the effect of MRM on C-Jun protein expression levels (shown as Median Fluorescence Intensity (MFI)) of transduced T cells from different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. Transduced T cells are derived from three different donors: donor #1 (fig. 3A), donor #2 (fig. 3B), and donor #3 (fig. 3C).

Fig. 4A, 4B, 4C, 4D, 4E and 4F provide a comparison of the percentages of stem cell-like transduced cd4+ T cells (fig. 4A, 4B and 4C-three different donors) and cd8+ T cells (fig. 4D, 4E and 4F-from three different donors) from different test groups. The different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. As explained in example 2, stem cell-like cells were identified as CD45RO ^-CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺TCF7⁺.

Fig. 4G, 4H, 4I, 4J, 4K and 4L show the percentages of cd4+ T cells (fig. 4G, 4H, 4I) and cd8+ T cells (fig. 4J, 4K, 4L) in naive and stem cell memory T cells from the same three donors. Naive and stem cell memory T cells were identified as CCR7 ⁺CD45RA⁺ as described in example 2.

Fig. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, and 5J provide a comparison of the expression of various surface markers on transduced T cells (derived from donor # 1) from different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. Fig. 5A, 5B, 5C, 5D and 5E show the percentage of anti-ROR 1 (R12) CAR transduced cd4+ T cells expressing CD39, LAG3, PD1, TIGIT and TIM3, respectively. Fig. 5F, 5G, 5H, 5I, and 5J show the percentage of anti-ROR 1 CAR transduced cd8+ T cells expressing CD39, LAG3, PD1, TIGIT, and TIM3, respectively.

Fig. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, and 6J provide a comparison of the expression of various surface markers on transduced T cells (derived from donor # 2) from different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. Fig. 6A, 6B, 6C, 6D, and 6E show the percentage of anti-ROR 1 (R12) CAR transduced cd4+ T cells expressing CD39, LAG3, PD1, TIGIT, and TIM3, respectively. Fig. 6F, 6G, 6H, 6I, and 6J show the percentage of anti-ROR 1 CAR transduced cd8+ T cells expressing CD39, LAG3, PD1, TIGIT, and TIM3, respectively.

Fig. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I and 7J provide a comparison of the expression of various surface markers on transduced T cells (derived from donor # 3) from different test groups. The different test groups are identical to the test groups described in fig. 1A-1C. Fig. 7A, 7B, 7C, 7D and 7E show the percentage of anti-ROR 1 (R12) CAR transduced cd4+ T cells expressing CD39, LAG3, PD1, TIGIT and TIM3, respectively. Fig. 7F, 7G, 7H, 7I and 7J show the percentage of anti-ROR 1 (R12) CAR transduced cd8+ T cells expressing CD39, LAG3, PD1, TIGIT and TIM3, respectively.

FIGS. 8A, 8B and 8C provide a comparison of IL-2 production by T cells transduced and cultured in MRM or TCM after primary antigen stimulation. Transduced T cells are derived from three different donors: donor #1 (fig. 8A), donor #2 (fig. 8B), and donor #3 (fig. 8C). The different test groups were as follows: (1) T cells transduced with control CD19T-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in TCM (closed circles); (2) T cells transduced with c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in TCM (closed squares); (3) T cells transduced with control CD19T-R12 CAR and cultured in MRM (open circles); and (4) T cells transduced with c-Jun-R12 CAR and cultured in MRM (open squares). The x-axis provides the effector to target (E: T) ratio (i.e., the ratio of transduced T cells to target tumor cells).

FIGS. 9A, 9B and 9C provide a comparison of IFN- γ production by T cells transduced and cultured in MRM or TCM after multiple rounds of antigen stimulation. As also provided in example 3, when the number of transduced T cells required for the next round of re-seeding is not reached, the continuous stimulation analysis is terminated: (i) four rounds of antigen stimulation against donor #1 (fig. 9A), (ii) three rounds of antigen stimulation against donor #2 (fig. 9B), and (iii) two rounds of antigen stimulation against donor #3 (fig. 9C). The different test groups were as follows: (1) T cells transduced with control CD19T-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in TCM (closed circles); (2) T cells transduced with c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in TCM (closed squares); (3) T cells transduced with control CD19T-R12 CAR and cultured in MRM (open circles); and (3) T cells transduced with c-Jun-R12 CAR and cultured in MRM (open squares). The x-axis provides the effector to target (E: T) ratio (i.e., the ratio of transduced T cells to target tumor cells).

Fig. 10A, 10B, 10C, 10D, 10E and 10F illustrate the ability to transduce cd8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. Transduced T cells are derived from three different donors: donor #1 (fig. 10B and 10E), donor #2 (fig. 10A and 10D), and donor #3 (fig. 10C and 10F). Fig. 10A, 10B and 10C provide results for cd8+ T cells transduced with control CD19T-R12 CAR (i.e., R12 CAR without C-Jun) (black bars) or C-Jun-R12 CAR (i.e., R12 CAR with C-Jun) (white bars) and cultured in control medium (i.e., TCM). FIGS. 10E, 10F and 10G provide the results of CD8+ T cells transduced with control CD19T-R12 CAR (black bars) or c-Jun-R12 CAR (white bars) and cultured in MRM. The x-axis provides the effector to target (E: T) ratio (i.e., the ratio of transduced T cells to target tumor cells).

Fig. 11A, 11B, 11C, 11D, 11E, and 11F provide a comparison of anti-tumor effects after administration of T cells transduced as described herein and cultured in MRM. Mice subcutaneously implanted with human ROR1 positive H1975NSCLC cells were treated intravenously with one of the following: (i) A simulated (non-transduced) T cell cultured in MRM ("simulated-MRM"; round), (ii) a T cell transduced with a control CD19T-R12 CAR (i.e., R12CAR without c-Jun) and cultured in MRM ("control R12 CAR-MRM"; square); and (3) T cells transduced with a c-Jun-R12 CAR (i.e., R12CAR with c-Jun) and cultured in MRM ("c-Jun-R12-CAR-MRM"; triangle). The T cells were administered to mice at one of two doses: (a) 5×10 ⁶ cells (fig. 11A, 11C, and 11E) or (B) 2.5×10 ⁶ cells (fig. 11B, 11D, and 11F). After T cell administration, the animals were assessed for tumor size (fig. 11A and 11B), body weight (fig. 11C and 11D), and survival (fig. 11E and 11F) at various times after administration.

Fig. 12A and 12B show the persistence of T cells transduced as described herein and cultured in MRM and administered to H1975 tumor-bearing NSG mice with MHC class I and II knockouts. As shown, the animals received a single intravenous administration of one of the following: (i) A simulated (non-transduced) T cell cultured in MRM ("simulated-MRM"; round), (ii) a T cell transduced with a control CD19T-R12 CAR (i.e., R12CAR without c-Jun) and cultured in MRM ("control R12 CAR-MRM"; square); and (3) T cells transduced with a c-Jun-R12 CAR (i.e., R12CAR with c-Jun) and cultured in MRM ("c-Jun-R12-CAR-MRM"; triangle). The T cells were administered to mice at one of two doses: (a) 5X 10 ⁶ cells (FIG. 12A) or (B) 2.5X 10 ⁶ cells (FIG. 12B). Next, at various moments after administration, peripheral blood was collected and flow cytometry was used to quantify the number of T cells (mock) or car+ T cells (control and c-Jun R12 group) per mL of blood.

Figures 13A and 13B provide transcriptome maps of anti-ROR 1 CAR T cells following successive antigen stimulation. As further described in example 6, some anti-ROR 1 CAR T cells were modified to overexpress the c-Jun protein and/or cultured in MRM. The different test groups shown are as follows: (1) T cells transduced with control CD19T-R12 CAR (i.e., R12CAR without c-Jun) and cultured in control medium (grey bars); and (2) T cells transduced with c-Jun-R12CAR (i.e., R12CAR with c-Jun) and cultured in MRM (black bars). Fig. 13A shows the proportion of CD8 ⁺ T cells enriched for stem cell-like genes on days 7 and 10 of the continuous antigen stimulation assay. FIG. 13B shows the proportion of CD8 ⁺ T cells enriched for the T cell terminal depletion gene.

Fig. 14A, 14B, 14C, 14D and 14E provide transcriptome maps of anti-ROR 1 CAR T cells (with or without C-Jun overexpression) following adoptive transfer to tumor bearing mice. Tumor bearing mice were treated with c-Jun ROR1 CAR T cells (c-Jun R12 CAR MRM) cultured in MRM or control ROR1 CAR T cells (control R12 CAR MRM) cultured in MRM as further described in example 7. And then, adoptively transferred transduced T cells are isolated from the tumor and single cell RNA-seq analysis is performed. Fig. 14A provides UMAP of CD8 ⁺ T cells from all samples from both treatment groups. FIG. 14B shows the proportion of CD8 ⁺ T cells enriched for the T cell terminal depletion gene. Figure 14C shows the proportion of CD8 ⁺ T cells enriched for the T cell progenitor depletion gene. Fig. 14D shows the proportion of CD8 ⁺ T cells enriched for stem cell-like genes. FIG. 14E shows the proportion of CD8+ T cells enriched for T cell activation related genes.

Fig. 15A, 15B and 15C are bar graphs showing C-Jun expression in T cells transduced with ROR1 CAR and cultured in TCM or MRM containing different concentrations of potassium ions. As further described in example 8, the potassium ion concentration of the different MRMs tested ranged between 40-80mM (i.e., low to high concentration). The different transduction conditions (or test groups) were as follows: (1) T cells transduced with control CD19T-R12 CAR (i.e., R12CAR without c-Jun) and cultured in TCM; (2) T cells transduced with a c-Jun-R12 CAR (i.e., R12CAR with c-Jun) and cultured in TCM; (3) T cells transduced with control CD19T-R12 CAR and cultured in MRM with different potassium concentrations; and (4) T cells transduced with c-Jun-R12 CAR and cultured in MRM with different potassium concentrations. FIGS. 15A, 15B and 15C each provide C-Jun expression from biological replicates of T cells isolated from three independent donors.

Fig. 16A, 16B and 16C provide a comparison of the percentages of stem cell-like cd4+ T cells transduced and cultured in TCM or MRM containing potassium ions at concentrations ranging from 40-80m M (i.e., low to high concentrations). The different test groups are identical to those described in fig. 15A-15C. The stem cell-like cells were identified using the cell surface marker CD45RO ^-CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺TCF7⁺. Fig. 16A, 16B and 16C each provide the results of a biological repeat experiment of T cells isolated from three independent donors.

Fig. 17A, 17B and 17C provide a comparison of the percentages of stem cell-like cd8+ T cells transduced and cultured in TCM or MRM containing potassium ions at concentrations ranging from 40-80m M (i.e., low to high concentrations). The different test groups are identical to those described in fig. 15A-15C. The stem cell-like cells were identified using the cell surface marker CD45RO ^-CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺TCF7⁺. Fig. 17A, 17B and 17C each provide the results of a biological repeat experiment of T cells isolated from three independent donors.

FIGS. 18A, 18B, 18C, 18D, 18E, 18F, 18G, 18H, and 18I show IFN- γ (FIGS. 18A, 18B, and 18C), IL-2 (FIGS. 18D, 18E, and 18F), and TNF- α (FIGS. 18G, 18H, and 18I) production by anti-ROR 1 CAR T cells following primary antigen stimulation at an effector to target (E: T) ratio of 1:1. As further described in example 8, T cells (isolated from three independent donors) were transduced and cultured in TCM or MRM containing potassium ions at concentrations ranging from 40-80mM (i.e., low to high concentrations). T cells were transduced with the following: (1) T cells control CD19T-R12CAR (i.e., R12CAR without c-Jun) (closed circle); or (2) c-Jun-R12 CAR (i.e., R12CAR with c-Jun) (closed square).

FIGS. 19A, 19B, 19C, 19D, 19E, 19F, 19G, 19H and 19I show IFN-gamma (FIGS. 19A, 19B and 19C), IL-2 (FIGS. 19D, 19E and 19F) and TNF-alpha (FIGS. 19G, 19H and 19I) production by anti-ROR 1 CAR T cells following primary antigen stimulation at an effector to target (E: T) ratio of 1:4. As further described in example 8, T cells (isolated from three independent donors) were transduced and cultured in TCM or MRM containing potassium ions at concentrations ranging from 40-80mM (i.e., low to high concentrations). T cells were transduced with the following: (1) T cells control CD19T-R12CAR (i.e., R12CAR without c-Jun) (closed circle); or (2) c-Jun-R12 CAR (i.e., R12CAR with c-Jun) (closed square).

Fig. 20A, 20B, 20C and 20D illustrate the ability of anti-ROR 1 CAR cd8+ T cells (transduced and cultured in TCM or MRM containing different concentrations of potassium ions) to kill target tumor cells after multiple rounds of antigen stimulation. As further described in example 8, transduced T cells were stimulated with antigen at an effector to target (E: T) ratio of 1:1 (fig. 20A and 20B) or 1:4 (fig. 20C and 20D). Fig. 20A and 20C provide the results of cd8+ T cells transduced with control CD19T-R12 CAR (i.e., R12 CAR without C-Jun) and cultured in TCM or MRM containing potassium ions at a concentration ranging from 40-80mM (i.e., low to high concentration). FIGS. 20B and 20D provide the results of CD8+ T cells transduced with c-Jun-R12-CAR (i.e., R12 CAR with c-Jun) and cultured in TCM or MRM containing potassium ions at concentrations ranging from 40-80mM (i.e., low to high concentrations). The area under the curve (AUC) from the IncuCyte killing curve was used to calculate the percent tumor viability (the lower the bar, the higher the cytotoxicity). In each of fig. 20A-20D, only tumor cells and non-transduced ("mock") T cells were used as controls.

Detailed Description

The efficacy of cellular immunotherapy depends on a number of factors, including the persistence, pluripotency, and asymmetric cell division of the cellular product infused into the patient. Culture and/or engineering of cells for cell therapies uses media that can profoundly affect the metabolic, epigenetic and phenotypic properties of these cells, thereby affecting their therapeutic potential.

The present disclosure is directed to methods of culturing cells, cells prepared by the methods, and/or compositions or kits for use in the cell culture methods. In some aspects, the disclosure provides methods of generating a population of immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) for Adoptive Cell Therapy (ACT), wherein the immune cells (e.g., T cells or NK cells) have a low differentiation state and retain proliferation capacity. In some aspects, the immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) have a low differentiation state and maintain the ability to target and kill tumor cells. In some aspects, the immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) have a low differentiation state, retain proliferation capacity, and maintain the ability to target and kill tumor cells. In some aspects, immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured according to the methods disclosed herein have increased efficacy in ACT as compared to cells cultured according to conventional methods (e.g., in media with less than 5mM potassium ions). In some aspects, immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured according to the methods disclosed herein have increased persistence in ACT after administration to a subject as compared to immune cells cultured according to conventional methods (e.g., in a medium having less than 5mM potassium ions). Such increased persistence refers to the ability of immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) to infiltrate and function in the tumor microenvironment, the ability to resist or delay the onset of depletion, and persistence of dryness to ensure persistence of amplification and response. In some aspects, immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured according to the methods disclosed herein are stem cell-like cells. Such cells are capable of self-renewal, proliferation and differentiation. In some aspects, immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured according to the methods disclosed herein are stem cell-like cells that also express effector-like markers. In some aspects, immune cells (e.g., T cells or NK cells) cultured according to the methods disclosed herein are stem cell-like cells that also maintain the ability to target and kill tumor cells.

The cell culture methods of the present disclosure can increase the pluripotency and/or sub-totipotency of the cultured cells, or can increase transduction efficiency when the cells are being transduced with a vector. In some aspects, the culture methods can reduce and/or prevent cell depletion when the cells are cultured and/or used in vivo therapies. In some aspects, the culture methods are also capable of increasing in vivo viability, in vivo persistence, in vivo effector function, or any combination thereof. In some aspects, the culture methods disclosed herein are capable of enriching for oligoclonal or polyclonal tumor-reactive stem cell-like T cells and/or CD8 ⁺ TIL. In some aspects, the culture methods disclosed herein are capable of maintaining clonal diversity of TILs derived from cancer patients.

In some aspects, the disclosure is directed to a method of culturing cells (e.g., immune cells (e.g., T cells or NK cells)) comprising placing the cells in a metabolic reprogramming media comprising potassium at a concentration of at least about 5mM (e.g., greater than 5 mM), wherein the media is not hypertonic, e.g., hypotonic or isotonic. Some aspects of the disclosure are directed to methods of culturing cells (e.g., immune cells (e.g., T cells or NK cells)) comprising placing the cells in a medium comprising potassium at a concentration greater than 40mM (e.g., about 40-80mM, e.g., about 50mM-80 mM). In some aspects, the immune cells comprise T cells, tumor-infiltrating lymphocytes (TILs), natural Killer (NK) cells, regulatory T (T _reg) cells, or any combination thereof.

Some aspects of the disclosure are directed to a method of increasing the yield of immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) while increasing the stem properties of the immune cells during in vitro or in vitro culture, the method comprising culturing the cells in a metabolic reprogramming medium comprising potassium ions at a concentration greater than 5mM (e.g., between 40mM and 80 mM) and NaCl at a concentration between 30mM and 100mM, wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. Some aspects of the disclosure are directed to a method of preparing a population of immune cells (e.g., T cells or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) for use in immunotherapy, the method comprising culturing the cells in a medium comprising potassium ions at a concentration above 5mM (e.g., between 40mM and 80 mM) and NaCl at a concentration between 30mM and 100mM, wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. Some aspects of the disclosure are directed to a method of increasing the stem properties of an immune cell (e.g., a T cell or NK cell (e.g., modified to express a ROR1 binding protein and having an increased level of c-Jun protein)) during in vitro or in vitro culture, the method comprising culturing the immune cell (e.g., a T cell or NK cell (e.g., modified to express a ROR1 binding protein and having an increased level of c-Jun protein)) in a medium comprising potassium ions at a concentration greater than 5mM (e.g., between 40mM and 80 mM) and NaCl at a concentration between 30mM and 100mM, wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the immune cell is a T cell.

In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In certain aspects, the medium further comprises Interleukin (IL) -2, IL-21, IL-7, IL-15, or any combination thereof. In some aspects, the medium comprises IL-2, IL-7 and IL-15. In some aspects, the medium comprises IL-2 and IL-21. In some aspects, the medium further comprises sodium ions, calcium ions, glucose, or any combination thereof.

As described herein, in some aspects, with reference to a method, wherein: (i) The immune cells are modified (e.g., to overexpress c-Jun), but are not cultured in a medium comprising potassium at a concentration above 5 mM; (ii) The immune cells are not modified but are cultured in a medium comprising potassium at a concentration above 5 mM; or (iii) modifying an immune cell (e.g., a T cell or NK cell) in a medium comprising potassium ions at a concentration greater than 5mM compared to (i) express a ROR1 binding protein, and (ii) having increased levels of a c-Jun polypeptide (e.g., with an exogenous nucleotide sequence encoding a c-Jun and/or a transcriptional activator capable of increasing expression of an endogenous c-Jun polypeptide) can also improve one or more characteristics of the immune cell. Additional aspects of such methods are provided throughout this disclosure.

I. terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this disclosure, each of the following terms shall have the meanings set forth below, unless the context clearly provides otherwise. Additional definitions are set forth throughout the present disclosure.

Throughout this disclosure, the term "a/an" entity refers to one or more of the entities; for example, it is understood that "chimeric polypeptide" represents one or more chimeric polypeptides. Thus, the terms "a/an", "one or more", and "at least one" are used interchangeably herein. Furthermore, the use of "or" means an open list of components in the list. For example, "wherein X comprises a or B" means that X comprises a, X comprises B, X comprises a and B, or X comprises a or B and any other components.

Furthermore, "and/or" as used herein should be taken as specifically disclosing each of the two specified features or components together with or without the other. Thus, as used herein in phrases such as "a and/or B," the term "and/or" is intended to include "a and B," "a or B," "a" (alone) and "B" (alone). Also, as used in phrases such as "A, B and/or C," the term "and/or" 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).

It should be understood that in any event the aspects are described herein in the language "comprising," other similar aspects described in terms of "consisting of … …" and/or "consisting essentially of … … are also provided.

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 pertains. For example Concise Dictionary of Biomedicine and Molecular Biology, juo, pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3 rd edition, 1999,Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, revisions, 2000,Oxford University Press provide a general dictionary of many terms to those skilled in the art that are used in the present disclosure.

Units, prefixes, and symbols are expressed in terms of their international system of units (Syst re me International de Unites, SI). Unless explicitly stated otherwise, numerical ranges include numbers defining the ranges.

Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in greater detail in the following subsections.

The term "about" or "substantially comprises" means that the value or composition is within an acceptable error range for the particular value or composition as determined by one of skill in the art, which will depend in part on the manner in which the value or composition is measured, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to practice in the art. Or "about" or "substantially comprising" may mean a range of up to 10% (e.g., a range of values within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater or less) unless otherwise specified or otherwise apparent from the context (unless such numbers would exceed 100% of the possible values). For example, "about 55mM" as used herein includes 49.5mM to 60.5mM. In addition, especially for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in the application and claims, unless otherwise indicated, the meaning of "about" or "consisting essentially of" shall be assumed to be within an acceptable error range for the particular value or composition.

As used herein, the term "about" when applied to one or more related values refers to a value that is similar to the stated reference value. In some aspects, unless specified otherwise or otherwise apparent from the context (unless such numbers would exceed 100% of the possible values), the term "about" (as the term "about") refers to a range of values within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater than or less).

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the stated range, and fractions thereof (such as tenths and hundredths of integers) as appropriate.

As used herein, the term "control medium", "conventional medium" or "reference medium" refers to any medium compared to the Metabolic Reprogramming Media (MRM) disclosed herein. The control medium may comprise the same components as the metabolic reprogramming medium, except for certain ion concentrations, such as potassium ions. In some aspects, the metabolic reprogramming media described herein are prepared from a control medium by adjusting one or more ion concentrations (e.g., potassium ion concentrations) as described herein. In some aspects, the control medium comprises a basal medium, such as CTS ^TMOPTMIZER^TM. In some aspects, the control culture genes thus comprise one or more additional components, including but not limited to amino acids, glucose, glutamine, T cell stimulators, antibodies, substitutions, and the like, that are also added to the metabolic reprogramming media, but the control media has certain ion concentrations that are different from the metabolic reprogramming media. Unless otherwise indicated, the terms "medium" and "medium" are used interchangeably.

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

As used herein, the term "expansion" in reference to immune cell culture refers to the process of stimulating or activating cells and culturing the cells. After the cells are stimulated or activated and cultured, the expansion process may result in an increase in the proportion or total number of cells required, for example, in the population of cultured cells. The number of all cell types in the cell population that do not require culture for expansion increases. More specifically, in some aspects, only a subset of cells in the cultured cell population increases in number during expansion, while the number of other cell types may not change or may decrease.

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

As used herein, the term "metabolic reprogramming media (metabolic reprogramming media/metabolic reprogramming medium)" or "MRM" refers to media of the present disclosure, wherein the media has an increased potassium concentration. In some aspects, the metabolic reprogramming media comprises potassium ions at a concentration greater than 5 mM. In some aspects, the metabolic reprogramming media comprises potassium ions at a concentration greater than 40mM. In some aspects, the MRM comprises potassium ions at a concentration between about 40mM and about 80 mM. In some aspects, the metabolic reprogramming media comprises a potassium ion concentration of at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 55mM, at least about 60mM, at least about 65mM, at least about 70mM, at least about 75mM, at least about 80mM, at least about 85mM, at least about 90mM, at least about 95mM, or at least about 100 mM. In some aspects, the MRM comprises potassium ions at a concentration of about 40mM. In some aspects, the MRM comprises potassium ions at a concentration of about 50 mM. In some aspects, the MRM comprises potassium ions at a concentration of about 60 mM. In some aspects, the MRM comprises potassium ions at a concentration of about 70 mM. In some aspects, the MRM comprises potassium ions at a concentration of about 80 mM. In some aspects, the metabolic reprogramming media comprises about 40mM to about 80mM NaCl, about 40mM to about 90mM KCl, about 0.5mM to about 2.8mM calcium, and about 10mM to about 24mM glucose. In some aspects, the MRM comprises from about 40mM to about 80mM NaCl, from about 40mM to about 80mM potassium ion, from about 0.5mM to about 2.8mM calcium, and from about 10mM to about 24mM glucose. In some aspects, the MRM comprises about 55mM to about 90mM NaCl and about 40mM to about 80mM potassium ions. In some aspects, the metabolic reprogramming media further comprises an osmolality of about 250 to about 300 mOsmol.

As used herein, the term "above" means greater than but not equal to. For example, "above 5mM" means any amount exceeding 5mM but excluding 5mM.

As used herein, the term "tonicity" refers to the calculated effective osmolality gradient across the cell membrane, expressed by the sum of the potassium ion concentration and the sodium chloride (NaCl) concentration multiplied by 2. Tonicity can be expressed in terms of osmolality (mOsm/kg) or osmolality (mOsm/L) of a solution (e.g., medium). Osmolality and osmolarity are measures of solute osmolarity per mass (osmolality) and per volume (osmolality) of solvent. As used herein, isotonic medium has a tonicity of about 280mOsm/L (e.g., ([ k+ ] + [ NaCl ]) x2 = 280).

As used herein, hypotonic solutions have a tonicity of less than 280mOsm/L (e.g., ([ k+ ] + [ NaCl ]) x 2< 280). In some aspects, the hypotonic medium has a tonicity of at least about 210mOsm/L to less than about 280 mOsm/L. In some aspects, the hypotonic medium has a tonicity of at least about 220mOsm/L to less than about 280 mOsm/L. In some aspects, the hypotonic medium has a tonicity of at least about 230mOsm/L to less than about 280 mOsm/L. In some aspects, the hypotonic medium has a tonicity of at least about 240mOsm/L to less than about 280 mOsm/L. In some aspects, the hypotonic culture medium described herein has a tonicity of about 250 mOsm/L.

As used herein, hypertonic solutions have a tonicity of greater than 300mOsm/L (e.g., ([ k+ ] + [ NaCl ]) x 2> 300). In some aspects, the hypertonic medium described herein has a tonicity of about 320 mOsm/L. In some aspects, the tonicity of a solution (e.g., a culture medium) is adjusted by increasing or decreasing the concentration of potassium ions and NaCl. In some aspects, the tension of the medium may be maintained by counteracting the increase in one solute with the decrease in the other solute. For example, increasing the potassium ion concentration in the medium without changing the sodium ion concentration can increase the tonicity of the medium. However, if the potassium ion concentration is increased and the sodium ion concentration is decreased, the tension of the original medium can be maintained.

As used herein, the terms "potassium", "potassium ion", "potassium cation" and "k+" are used interchangeably to refer to elemental potassium. Elemental potassium is present in the solution as a cation. However, it should be apparent to one of ordinary skill in the art that a standard manner of preparing a solution comprising potassium ions includes diluting a potassium-containing salt (e.g., KCl) into the solution. Thus, a solution (e.g., medium) comprising a molar (M) concentration of potassium ions can be described as comprising an equimolar (M) concentration of potassium-comprising salt.

As used herein, the terms "sodium ion" and "sodium cation" are used interchangeably to refer to elemental sodium. Elemental sodium is present in the solution as a monovalent cation. However, it should be apparent to one of ordinary skill in the art that a standard manner of preparing a solution containing sodium ions includes diluting a sodium-containing salt (e.g., naCl) into the solution. Thus, a solution (e.g., medium) comprising a molar (M) concentration of sodium ions can be described as comprising an equimolar (M) concentration of sodium-comprising salts.

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 should be apparent to one of ordinary skill in the art that a standard manner of preparing a solution comprising calcium ions includes diluting a calcium salt (e.g., caCl ₂) into the solution. Thus, a solution (e.g., medium) comprising a molar (M) concentration of calcium ions can be described as comprising an equimolar (M) concentration of a salt comprising calcium.

As used herein, the term "immune cell" refers to a cell of the immune system. In some aspects, the immune cell is selected from T lymphocytes ("T cells"), B lymphocytes ("B cells"), natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils. As used herein, a "population" of cells refers to a collection of more than one cell (e.g., a plurality of cells). In some aspects, the cell population comprises more than one immune cell, e.g., a plurality of immune cells. In some aspects, the cell population is a heterogeneous mixture comprising cells, the mixture comprising a plurality of types of cells, e.g., a heterogeneous mixture of immune cells and non-immune cells. In some aspects, the cell population comprises a plurality of T cells.

As used herein, the term "reference immune cell" (or "reference cell") refers to a cell that is unmodified and/or cultured using the methods provided herein. For example, in some aspects, the reference cells comprise cells (e.g., corresponding immune cells) that have not been modified as described herein (e.g., with any of the c-Jun nucleotide sequences and/or transcriptional activators provided herein). In some aspects, the reference cells comprise such cells (which are not modified as described herein) that are cultured in a medium of the disclosure (e.g., comprising potassium ions at a concentration greater than 5 mM). In some aspects, the reference cells comprise such cells (which are not modified as described herein) that are cultured in a medium that does not comprise potassium ions at a concentration above 5mM (i.e., the reference medium). In some aspects, the reference cells comprise cells that have been modified as described herein (e.g., with any of the c-Jun nucleotide sequences and/or transcriptional activators provided herein), but are cultured in a reference medium. Thus, unless otherwise indicated, a reference cell may comprise any of the following: (1) Cells that are not modified as described herein (e.g., corresponding immune cells); (2) Cells (e.g., corresponding immune cells) that are neither modified as described herein nor cultured in the media of the disclosure; (3) Cells that are not modified as described herein, but are cultured in the media of the invention (e.g., corresponding immune cells); (4) Cells (e.g., corresponding immune cells) that have been modified as described herein, but cultured in a reference medium; or (5) any combination of (1) to (4). Based at least on the present disclosure, it should be apparent to those skilled in the art that the scope of the term "reference cell" as used herein.

As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. Non-limiting classes of T cells include effector T cells and T helper (Th) cells (such as CD4 ⁺ or CD8 ⁺ T cells). In some aspects, the T cell is a Th1 cell. In some aspects, the T cell is a Th2 cell. In some aspects, the T cell is a Tc17 cell. In some aspects, the T cell is a Th17 cell. In some aspects, the T cell is a T _reg cell. In some aspects, the T cell is a tumor infiltrating cell (TIL).

As used herein, the term "memory" T cell refers to a T cell that has previously encountered and responded to its cognate antigen (e.g., in vivo, in vitro, or ex vivo) or has been stimulated (e.g., in vitro or ex vivo) with, for example, an anti-CD 3 antibody. Immune cells have a "memory-like" phenotype after secondary exposure, and such memory T cells can proliferate to produce a faster and more intense immune response than during initial exposure. In some aspects, the memory T cells comprise central memory T cells (T _CM cells), effector memory T cells (T _EM cells), tissue resident memory T cells (T _RM cells), stem cell-like memory T cells (T _SCM cells), or any combination thereof.

As used herein, the term "stem cell-like memory T cells", "T memory stem cells" or "T _SCM cells" refers to memory T cells that express CD95, CD45RA, CCR7 and CD62L and are endowed with the ability to self-renew and reconstruct the full range of memory and effector T cell subsets.

As used herein, the term "central memory T cell" or "T _CM cell" refers to memory T cells that express CD45RO, CCR7 and CD 62L. Central memory T cells are commonly found in the intra-lymph node and in the peripheral circulation.

As used herein, the term "effector memory T cell" or "T _EM cell" refers to a memory T cell that expresses CD45RO but lacks CCR7 and CD62L expression. Because effector memory T cells lack lymph node homing receptors (e.g., CCR7 and CD 62L), these cells are commonly found in peripheral circulation and in non-lymphoid tissues.

As used herein, the term "tissue resident memory T cells" or "T _RM cells" refers to memory T cells that do not circulate and remain resident in peripheral tissues such as skin, lung, and gastrointestinal tract. In some aspects, the tissue resident memory T cells are also effector memory T cells.

As used herein, the term "naive T cellsT cell) "or" T _N cell "refers to a T cell that expresses CD45RA, CCR7, and CD62L but does not express CD 95. T _N cells represent the most highly undifferentiated cells in the T cell lineage. The interaction between T _N cells and Antigen Presenting Cells (APC) induces differentiation of T _N cells into activated T _EFF cells and an immune response.

As used herein, the term "stem-like" or "poorly differentiated" refers to immune cells (e.g., T cells, NK cells, or TIL) that express a marker consistent with a more naive phenotype. For example, poorly differentiated T cells may express one or more markers specific for T _N or T _SCM cells. In some aspects, "poorly differentiated" or "stem cell-like" T cells express CD45RA, CCR7, and CD62L. In some aspects, "poorly differentiated" or "stem cell-like" T cells express CD45RA, CCR7, CD62L, and TCF7. In some aspects, the "poorly differentiated" or "stem cell-like" T cells do not express CD45RO, or CD45RO ^{Low and low}. In some aspects, the methods disclosed herein promote immune cells (e.g., T cells and/or NK cells) having a low differentiation phenotype. Without being bound by any particular mechanism, in some aspects, the methods disclosed herein block, inhibit, or limit differentiation of poorly differentiated immune cells (e.g., T cells and/or NK cells), resulting in an increase in the number of stem cell-like cells in culture. For example, adoptive transfer of poorly differentiated immune cells (e.g., T cells and/or NK cells) having a stem cell-like memory or central memory phenotype is generally considered to be preferred for effective control of tumors. See Gattinoni, L. et al, J.Clin. Invest.115:1616-1626 (2005); gattinoni, L. et al, nat Med 15 (7): 808-814 (2009); lynn, R.C. et al, nature 576 (7786): 293-300 (2019); gattinoni, L. et al, nat Rev 12:671-684 (2012); klebanoff, C.et al, J.Immunother35 (9): 651-670 (2012) and Gattinoni, L.et al, nat Med 17 (10): 1290-1297 (2011).

Dryness is characterized by self-renewing capacity, pluripotency and persistence of proliferative potential. In some aspects, dryness is characterized by a specific gene signature (gene signature), such as a combined expression pattern of multiple genes. In some aspects, stem cell-like cells can be identified by transcriptome analysis, for example, using the dry genetic imprinting disclosed herein (see, e.g., examples 6 and 7). In some aspects, the genetic imprinting comprises one or more genes ：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、OCM2、GCET2、LRRN1、IL6ST、LRRC16A、IGSF9B、EFHA2、LOC129293、APP、PKIA、ZC3H12D、CHMP7、KIAA0748、SLC22A17、FLJ13197、NRCAM、C5orf13、GIPC3、WNT7A、FAM117B、BEND5、LGMN、FAM63A、FAM153B、ARHGEF11、RBM11、RIC3、LDLRAP1、PELI1、PTK2、KCTD12、LMO7、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 selected from the group consisting of, and any combination thereof (see, e.g., gattinoni et al, nature Medicine 17 (10): 1290-97 (2011)). In some aspects, the genetic imprinting comprises one or more genes ：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、C7orf10、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、PROS1、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 selected from the group consisting of and any combination thereof (see, e.g., gattinoni, l. Et al, nat Med 17 (10): 1290-1297 (2011)). In some aspects, the genetic imprinting comprises one or more genes ：SELL、CCR7、S1PR1、KLF3、TCF7、GPR183、SC5D、FAAH2、LTB、SESN3、MAL、TSHZ2、LEF1、AP3M2、SLC2A3、ICAM2、PLAC8、SCML1、IL7R、ABLIM1、RASGRP2、TRABD2A、SATB1、ALG13、ARID5A、BACH2、PABPC1、GPCPD1、NELL2、TAF4B、FCMR、ARRDC2、C1orf162、FAM177A1、ANKRD12、TXK、SORL1、AQP3、ADTRP、FXYD7、CD28、P2RY8、CRYBG1、TNFSF8、BEX2、PGAP1、PTGER4、MAML2、BEX3、PCSK1N、INPP4B、AC119396.1、CXCR5、LINC00402、CCR4、IL6R、ZBTB10、ITGA6、ARMH1、RILPL2、FOXP1、TESPA1、YPEL5、LPAR6、CMSS1、RIPOR2、ZNF331、EMP3、GIMAP7、WDR74、RIC3、CYSLTR1、ITGB1、CD5、SAMHD1、SERINC5 selected from the group consisting of, and any combination thereof (see, e.g., caushi et al, nature 596:126-132 (2021)).

As used herein, the term "effector-like" or "effector cell-like" refers to tumor cell killing ability and cytokine versatility, e.g., the ability of a cell to produce inflammatory cytokines and/or cytotoxic molecules. In some aspects, effector-like cells are characterized by a particular marker expressed by the cells. In some aspects, those effector-like markers comprise one or more of pstat5+, stat5+, pstat3+, and stat3+. In some aspects, the effector-like marker comprises STAT targets ：AKT1、AKT2、AKT3、BCL2L1、CBL、CBLB、CBLC、CCND1、CCND2、CCND3、CISH、CLCF1、CNTF、CNTFR、CREBBP、CRLF2、CSF2、CSF2RA、CSF2RB、CSF3、CSF3R、CSH1、CTF1、EP300、EPO、EPOR、GH1、GH2、GHR、GRB2、IFNA1、IFNA10、IFNA13、IFNA14、IFNA16、IFNA17、IFNA2、IFNA21、IFNA4、IFNA5、IFNA6、IFNA7、IFNA8、IFNAR1、IFNAR2、IFNB1、IFNE、IFNG、IFNGR1、IFNGR2、IFNK、IFNL1、IFNL2、IFNL3、IFNLR1、IFNW1、IL10、IL10RA、IL10RB、IL11、IL11RA、IL12A、IL12B、IL12RB1、IL12RB2、IL13、IL13RA1、IL13RA2、IL15、IL15RA、IL19、IL2、IL20、IL20RA、IL20RB、IL21、IL21R、IL22、IL22RA1、IL22RA2、IL23A、IL23R、IL24、IL26、IL2RA、IL2RB、IL2RG、IL3、IL3RA、IL4、IL4R、IL5、IL5RA、IL6、IL6R、IL6ST、IL7、IL7R、IL9、IL9R、IRF9、JAK1、JAK2、JAK3、LEP、LEPR、LIF、LIFR、MPL、MYC、OSM、OSMR、PIAS1、PIAS2、PIAS3、PIAS4、PIK3CA、PIK3CB、PIK3CD、PIK3CG、PIK3R1、PIK3R2、PIK3R3、PIK3R5、PIM1、PRL、PRLR、PTPN11、PTPN6、SOCS1、SOCS2、SOCS3、SOCS4、SOCS5、SOCS7、SOS1、SOS2、SPRED1、SPRED2、SPRY1、SPRY2、SPRY3、SPRY4、STAM、STAM2、STAT1、STAT2、STAT3、STAT4、STAT5A、STAT5B、STAT6、TPO、TSLP、TYK2 selected from the group consisting of and any combination thereof. In some aspects, the effector-like cells are characterized by transcriptome analysis. In some aspects, the effector-like markers comprise Kaech et al, cell 111:837-51 (2002); tripathi et al, J.immunology 185:2116-24 (2010); and/or Johnnidis et al, science Immunology 6:eabe3702 (2021, 1, 15 days), each of which is incorporated herein by reference in its entirety.

In some aspects, effector-like cells are characterized using the effector-related gene set described in Gattinoni, L. Et al, nat Med 17 (10): 1290-97 (2011). In some aspects, the genetic imprinting of the effector-like cells comprises one or more genes ：MTCH2、RAB6C、KIAA0195、SETD2、C2orf24、NRD1、GNA13、COPA、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、C1orf58、C11orf63、C6orf129、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、MYO5A、KIR2DL3、DLG3、MXD4、RALGDS、S1PR5、WSB2、CCR3、TIPARP、SP140、CD151、SOX13、KRTAP5-2、NF1、PEA15、PARP8、RNF166、UEVLD、LIMK1、CACNB1、TMX4、SLC6A6、LBA1、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、ABI3、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、FOSL2、RGS2、TGFBR3、PRF1、MYO1F、GAB3、C17orf66、MICAL2、CYTH3、TOX、HLA-DRA、SYNE1、WEE1、PYHIN1、F2R、PLD1、THBS1、CD58、FAS、NETO2、CXCR6、ST6GALNAC2、DUSP4、AUTS2、C1orf21、KLRG1、TNIP3、GZMA、PRR5L、PRDM1、ST8SIA6、PLXND1、PTPRM、GFPT2、MYBL1、SLAMF7、FLJ16686、GNLY、ZEB2、CST7、IL18RAP、CCL5、KLRD1、KLRB1 selected from the group consisting of, and any combination thereof (see, e.g., gattinoni, L. et al, nat Med 17 (10): 1290-97 (2011)).

As further described herein (see, e.g., example 7), in some aspects, transcriptome analysis can be used to assess characteristics of cells (e.g., T cells and/or NK cells) by comparing up-and/or down-regulation of different sets of genes associated with T cell activation (also referred to herein as "TACT genes"), T cell progenitor depletion (also referred to herein as "TPE genes"), T cell terminal depletion (also referred to herein as "TTE genes").

In some aspects, the TTE-related gene set described in Oliveira et al, nature 596:119-125 (2021) is used to characterize terminal depleted T cells. In some aspects, the genetic imprinting of TTE cells comprises one or more or all of the genes ：KRT86、RDH10、ACP5、CXCR6、HMOX1、LAYN、CLIC3、HAVCR2、AC243829.4、PRF1、SLC2A8、CHST12、GALNT2、ENTPD1、LAG3、GZMB、PDCD1、CARD16、CTLA4、SLA2、CD27、RALA、VCAM1、SYNGR2、NKG7、LSP1、CCL5、RARRES3、CD7、CTSW、MTSS1、PTMS、BATF、KIR2DL4、AKAP5、CD38、RAB27A、GZMH、IGFLR1、ATP8B4、CD63、HOPX、TNFRSF18、ADGRG1、PLPP1、CSF1、TNFSF10、SNAP47、LINC01871、MYO1E、ZBED2、AHI1、ABI3、FASLG、TYMP、ZBTB38、CTSB、PLSCR1、AFAP1L2、ITGAE、TNS3、DUSP16、CASP1、CARS、DUSP5、IFIT1、SLC1A4、GOLIM4、RSAD2、DNPH1、NBL1、ACOT9、ABHD6、OAS1、SLC27A2、ZBP1、CD200R1、OAS3、CMPK2、TNFSF4、POLR1E、CADM1、HELZ2、SYTL2、AGPAT2、UBE2F、GIMAP6、ZBTB32、RIN3、PLEKHF1、CHPF、PACSIN2、ABCB1、SPATS2L、USP18、TMEM9、KLRC1、MPST. selected from the group consisting of, in some aspects, characterizing progenitor cell depleted T cells (TPE) using the TPE-related gene set described in Oliveira et al, nature 596:119-125 (2021). In some aspects, the genetic imprinting of TPE cells comprises one or more or all of the genes ：FXYD6、CAV1、GNG4、XCL1、CRTAM、CXCL13、GEM、XCL2、FXYD2、HLA-DRA、LANCL2、RASSF4、BAG3、HSPA1B、HLA-DQA1、HSPB1、FABP5、MS4A6A、SERPINH1、HLA-DPA1、HLA-DRB1、HSPA1A、RGS2、DRAIC、CD74、HSPD1、HSPA6、HSPE1、CD82、TOX、CD200、HLA-DPB1、NR4A2、VCAM1、BEX3、AIF1、DNAJA1、HSPH1、DNAJB1、HIPK2、LHFPL6、HLA-DMA、GK、TSHZ2、LPL、C16orf45、ZFAND2A、CD80、ETV1、NMB、DEDD2、CMC1、PON2、SEMA4A、ENC1、GRAMD1A、MYL6B、BCAT1、ARMH1、TIAM1、PIKFYVE、MRPL18、INPP5F、LMCD1、SESN3、CCDC6、KIAA1324、CHN1、ANKRD10、CD70、PRRG4、TNFSF4、CORO1B、DNAJB4、MAGEH1、ICAM1、GGT1、NINJ2、BLVRA、FAAH2、TOX2、SLK、CCDC141、ATF3、INPP1、FAM3C、GADD45G、APP、MAL、SIT1、DRAM1、CLECL1、MDFIC、PMCH、HLA-DMB、PHF6、AFAP1L2、BTN2A2、CCL4L2. selected from the group consisting of, in some aspects, characterizing activated T cells (TACT) using the TACT-related gene set described in Oliveira et al, nature 596:119-125 (2021). In some aspects, the genetic signature of the activated T cells comprises one or more or all of the genes selected from the group consisting of ：EGR1、HSPA6、FOS、HSPA1B、GADD45B、NR4A1、FOSB、ATF3、DNAJB1、DUSP1、JUNB、CD69、NR4A2、NFKBIA、PPP1R15A、KLF6、DNAJA1、JUN、SRSF7、SLC2A3、ZFP36L1、IER2、HSPA1A、EIF4A2、ID1、IFRD1、CCNL1、RSRP1、SERTAD1、DEDD2、KLF10、AL118516.1、KLF2、ZFAND2A、CLK1、RSRC2、IER3、BTG2、MYLIP、MAFF、CSRNP1、ID2、ZC3H12A、BAG3、SNHG12、TNF、DDIT4、SGK1、SNHG15、DNAJB4、NR4A3、NFKBID、SCML1、RASD1、ATF4、AREG、RASGEF1B、AC020916.1、DDIT3、SNHG8、CITED2、TXNIP、TOB1、PIM2、SOCS3、GADD45G、RGS16、TIPARP、NFKBIZ、CCL4、CD83、PPP1R10、CCL4L2、SESN2、CHMP1B、LEF1、CSKMT、HEXIM1、HSPA2、MRPL18、RBKS、CD55、ARRDC2、SC5D、FAM53C、ATP2B1-AS1、IFNG、MYC、TSC22D2、SERPINH1、LRIF1、ARRDC3、ILF3-DT、INTS6、ZNF10、PRMT9、ATM、SELL、AC243960.1.

As used herein, the term "basal" medium refers to any starting medium supplemented with one or more additional elements disclosed herein, such as potassium, sodium, calcium, glucose, IL-2, IL-7, IL-15, IL-21, or any combination thereof. The basal medium can be any medium used to culture immune cells (e.g., T cells and/or NK cells). In some aspects, the basal Medium comprises balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), dulbecco's Modified Eagle's Medium, DMEM, kelvin Medium (Click's Medium), minimal Essential Medium (MEM), eaglian basal Medium (Basal Medium Eagle, BME), F-10, F-12, RPMI 1640, glasgo minimal essential Medium (Glasgow MINIMAL ESSENTIAL Medium, GMEM), alpha minimal essential Medium (alpha MEM), iscove Modified Dulvin Medium (IMDM), M199, OPTMIZER ^TM Pro、OPTMIZER^TMCTS^TM T cell expansion basal Medium (ThermoFisher)、OPTMIZER^TM、OPTMIZER^TMComplete、IMMUNOCULT^TMXF(STEMCELL^TMTechnologies)、AIM V^TM、TEXMACS^TM Medium,T cell CDM, X-VIVO ^TM15(Lonza)、TRANSACT^TM TIL expansion medium, or any combination thereof. In some aspects, the basal medium is serum-free. In some aspects, the basal medium comprisesT cell CDM. In some aspects, the basal medium comprises OPTMIZER ^TM. In some aspects, the basal medium comprises OPTMIZER ^TM Pro. In some aspects, the basal medium further comprises an Immune Cell Serum Replacement (ICSR). For example, in some aspects, the basal medium comprises OPTMIZER ^TM Complete supplemented with ICSR, AIM V ^TM supplemented with ICSR, IMMUNOCULT ^TM XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS ^TM supplemented with ICSR, or any combination thereof. In some aspects, suitable basal media include Kjeldahl medium, OPTMIZER ^TM(CTS^TM) medium,T cell expansion Medium (Sigma-Aldrich), AIM V ^TM Medium (CTS ^TM)、TEXMACS^TM Medium (Miltenyi Biotech), IMMUNOCULT ^TM Medium (Stem Cell Technologies),T cell expansion XSFM (Irvine Scientific), iscoves medium, and/or RPMI-1640 medium. In some aspects, the basal medium comprises CTS ^TMOPTMIZER^TM free of NaCl. In some aspects, the basal medium comprises one or more sodium salts in addition to NaCl.

As used herein, the term "cytokine" refers to small secreted proteins released by cells that have a specific effect on interactions and communication between cells. Non-limiting examples of cytokines include interleukins (e.g., interleukins (IL) -1, 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- α, IFN- β and IFN- γ), 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 immune cells (e.g., T cells and/or NK cells or one or more of the engineered immune cells 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, IL-21, or any combination thereof. IL-2 (UniProtKB-P60568) is produced by T cells in response to antigen or mitotic stimuli. IL-2 is known to stimulate T cell proliferation and other activities critical for the regulation of immune responses. IL-7 (UniProtKB-P13232) is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitor cells. IL-7 is believed to play a role in proliferation during certain phases of B cell maturation. Like IL-2, IL-15 (UniProtKB-P40933) is a cytokine that stimulates T lymphocyte proliferation. IL-21 (UniProtKB-Q9 HBE 4) is a cytokine with immunomodulatory activity. IL-21 is thought to promote a switch between innate and adaptive immunity and induce the production of IgG1 and IgG3 in B cells. IL-21 may also play a role in Natural Killer (NK) cell proliferation and maturation in conjunction with IL-15, and IL-21 may regulate proliferation of mature B cells and T cells in response to activating stimuli. In conjunction with IL-15 and IL-18, IL-15 also stimulates interferon gamma production in T cells and NK cells, and IL-21 can also inhibit dendritic cell activation and maturation during T cell mediated immune responses.

As used herein, "administering" refers to physically introducing a therapeutic agent or composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems. Different routes of administration of the therapeutic agents described herein (e.g., immune cells or populations of immune cells modified to express ROR1 binding protein and having increased levels of c-Jun polypeptide and cultured as described herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion.

As used herein, the phrase "parenteral administration (PARENTERAL ADMINISTRATION)" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intratumoral, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural and intrasternal injection and infusion, and in vivo electroporation.

Alternatively, the therapeutic agents described herein (e.g., immune cells modified to express ROR1 binding protein and having increased levels of c-Jun polypeptide and cultured as described herein) can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

As used herein, "cell engineering" or "cell modification" (including derivatives thereof) refers to targeted modification of a cell (e.g., an immune cell as disclosed herein). In some aspects, cellular engineering includes viral genetic engineering, non-viral genetic engineering, introduction of a receptor to allow tumor specific targeting (e.g., ROR1 binding protein), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell (e.g., T cell) function (e.g., polynucleotides encoding c-Jun polypeptides such that the immune cells exhibit increased c-Jun expression compared to an unmodified corresponding cell), or any combination thereof. As further described elsewhere in this disclosure, in some aspects, the cells can be engineered or modified by a transcriptional activator (e.g., based on a CRISPR/Cas system), wherein the transcriptional activator is capable of inducing and/or increasing endogenous expression of a protein of interest (e.g., c-Jun).

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 that is recognized by an immune cell (e.g., a T cell) and thereby induces immune cell activation (e.g., triggers an intracellular signal that induces effector functions, such as cytokine production, and/or for cell proliferation). In some aspects, the antigen comprises a tumor antigen. In some aspects, the antigen comprises a neoantigen.

"Cancer" refers to a large group of various diseases characterized by uncontrolled growth of abnormal cells in the body. Deregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distal parts of the body via the lymphatic system or blood flow. As used herein, "cancer" includes primary, metastatic, and recurrent cancers. The terms "cancer" and "tumor" are used interchangeably unless indicated otherwise.

The term "hematological malignancy" or "hematological cancer" refers to cancers and tumors of mammalian hematopoietic and lymphoid tissues. Non-limiting examples of hematological malignancies include those affecting the blood, bone marrow, lymph nodes, and tissues of the 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 lymphoma. Hematological malignancies are also referred to as "liquid tumors". Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies.

As used herein, "solid tumor" refers to an abnormal tissue mass. Solid tumors may be benign or malignant. Non-limiting 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 parenchyma (cancer cells) and supporting stromal cells in which the cancer cells are dispersed and which can provide a supporting microenvironment.

In some aspects, the cancer is selected from adrenocortical cancer, advanced cancer, anal cancer, aplastic anemia, cholangiocarcinoma, bladder cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, primary unknown cancer, castleman's disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, ewing family of tumors (EWING FAMILY of tumors), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hodgkin's disease, kaposi's sarcoma (Kaposi's sarcoma), renal cell carcinoma, laryngeal and hypopharynx cancer, and, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, lung carcinoid tumor, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, adult soft tissue sarcoma, basal cell and squamous cell skin cancer, melanoma, Carcinoma of the small intestine, gastric cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, fahrenheit macroglobulinemia (Waldenstrom macroglobulinemia), wilms tumor (Wilms tumor), and secondary cancers caused by cancer therapy. In some aspects, the cancer is selected from the group consisting of chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, myxoid/round cell liposarcoma, osteosarcoma, ebole's sarcoma (Abemethy's sarcoma), liposarcoma (adipose sarcoma/liposarcoma), acinar soft tissue sarcoma, ameloblastic sarcoma, grape sarcoma, green pulp sarcoma, choriocarcinoma, embryonal sarcoma, wilms tumor sarcoma, endometrial sarcoma, interstitial sarcoma, ewing's sarcoma, fascia sarcoma, blastoma, giant cell sarcoma, granulomatous sarcoma, Hodgkin's sarcoma, idiopathic multiple pigment hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, zhan Senshi sarcoma (Jensen's sarcomas), kaposi's sarcoma (Kupffer cell sarcoma), angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, osteo-parasarcoma, reticulocyte sarcoma, rous sarcoma (Rous sarcomas), serous sarcoma, synovial sarcoma, or telangiectasia sarcoma. In some aspects, the cancer is selected from acromelanic melanoma, non-melanoma, benign juvenile melanoma, claudenman melanoma (Cloudman' S melanoma), S91 melanoma, hao-pandi melanoma (Harding-Passey melanoma), juvenile melanoma, lentigo malignancies, malignant melanoma, metastatic melanoma, nodular melanoma, subungual melanoma, or superficial diffuse melanoma. In some aspects, the cancer is selected from the group consisting of acinar cancer (acinar carcinoma), acinar cancer (acinous carcinoma), adenocystic cancer (adenocystic carcinoma), adenoid cystic cancer (adenoid cystic carcinoma), adenomatous cancer, adrenocortical cancer, follicular cell cancer, basal cell cancer (basal cell carcinoma/carcinoma basocellulare), basal cell-like cancer, basal squamous cell cancer, bronchioloalveolar carcinoma, medullary carcinoma, cholangiocellular carcinoma, choriocarcinoma, glioblastoma, acne carcinoma, uterine body carcinoma, ethmoid carcinoma, armor carcinoma, sores, cylindrical carcinoma (CYLINDRICAL CARCINOMA), cylindrical cell carcinoma (CYLINDRICAL CELL carpinoma), ductal carcinoma, hard carcinoma, embryonal carcinoma, brain-like carcinoma, epidermoid carcinoma, adenoid carcinoma, ectogenic carcinoma, ulcerative carcinoma, fibrocarcinoma, glioblastoma (gelatiniform carcinoma), glioblastoma (gelatinous carcinoma), Giant cell carcinoma (GIANT CELL carcinoma/carcinoma gigantocellulare), adenocarcinoma, granulosa cell carcinoma, kerogen cancer (hair-matrix carcinoma), blood sample carcinoma, hepatocellular carcinoma, xu Teer cell carcinoma (Hurthle cell carcinoma), vitreous carcinoma, adrenal gland-like carcinoma, naive embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, kluken's cell carcinoma (Krompecher's carcinoma), coulter-matrix carcinoma (Kulchitzky-cell carcinoma), Large cell carcinoma, lenticular carcinoma (lenticular carcinoma/carcinoma lenticulare), lipomatoid carcinoma, lymphatic epithelial carcinoma, medullary carcinoma (carcinoma medullare/medullary carcinoma), melanoma, soft carcinoma (carcinoma molle), mucous carcinoma (mucinous carcinoma), mucous carcinoma (carcinoma muciparum), mucous cell carcinoma, mucous epidermoid carcinoma, mucous carcinoma (carcinoma mucosum/mucous carcinoma), and, Myxomatoid cancer, nasopharyngeal cancer, oat cell cancer, ossified cancer, bone-like cancer, papillary cancer, periportal cancer, non-invasive cancer, acanthocellular cancer, erosive cancer, renal cell carcinoma, stock cell carcinoma (RESERVE CELL carpinoma), sarcoid cancer, schneider's cancer (SCHNEIDERIAN CARCINOMA), hard cancer (scirrhous carcinoma), scrotal cancer, ring cell carcinoma, simple cancer, small cell carcinoma, potato-like cancer, globular cell carcinoma, spindle cell carcinoma, spongiform cancer, squamous cell carcinoma, string-binding cancer (string carpinoma), vasodilatory cancer (carcinoma telangiectaticum/carcinoma telangiectodes), transitional cell carcinoma, tubular cancer (carcinoma tuberosum), nodular cancer, wart cancer (verrucous carcinoma), or villous cancer. 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 thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical cancer, prostate cancer, mu LLERIAN CANCER, ovarian, peritoneal, fallopian tube or papillary serous carcinoma of the uterus. in some aspects, the cancer is selected from metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, bladder cancer, or any combination thereof.

As used herein, the term "immune response" refers to a biological response within a vertebrate against exogenous agents that protects the organism against these agents and diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural Killer (NK) cells, NKT cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either of these cells or the liver, which results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate body of invasive pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal human cells or tissues. Immune responses include, for example, activation or suppression of T cells, e.g., effector T cells or Th cells, such as CD4 ⁺ or CD8 ⁺ T cells, or suppression of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some aspects, the T cell is a cd4+ T cell. In some aspects, the T cell is a cd8+ T cell. In some aspects, the T cell is a NKT cell.

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

"Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (such as mice, rats, and guinea pigs). In some aspects, the subject is a human. The terms "subject," "patient," "individual," and "host" are used interchangeably herein. As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from administration of immune cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun polypeptide and cultured using the methods provided herein) as described, for example, to control tumor growth.

The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent that provides a desired biological, therapeutic, and/or prophylactic result (e.g., an immune cell that has been modified to express a ROR1 binding protein and has increased levels of a c-Jun polypeptide and is cultured as described herein). The result may be a reduction, improvement, alleviation, diminishment, delay and/or diminishment of one or more signs, symptoms or causes of the disease, or any other desired alteration of the biological system. With respect to solid tumors, an effective amount comprises an amount sufficient to cause tumor shrinkage and/or to reduce the tumor growth rate (e.g., inhibit tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor progression. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations.

An effective amount of a composition (e.g., an immune cell as described herein, e.g., modified to express a ROR1 binding protein and having an increased level of a c-Jun polypeptide and cultured as described herein) can, e.g., (i) reduce the number of cancer cells; (ii) reducing tumor size; (iii) Inhibit, delay, slow down and can stop cancer cell infiltration into surrounding organs to some extent; (iv) Inhibition (i.e., slowing down to some extent and may stop tumor metastasis); (v) inhibiting tumor growth; (vi) preventing or delaying the onset and/or recurrence of a tumor; and/or (vii) alleviating to some extent one or more symptoms associated with cancer.

In some aspects, a "therapeutically effective amount" is an amount of a composition disclosed herein (e.g., an immune cell modified to express ROR1 binding protein and having increased levels of c-Jun polypeptide and cultured as described herein) that clinically demonstrates significantly reduced cancer or slowed progression of cancer (regression), such as advanced solid tumors. The ability of a therapeutic agent of the present disclosure (e.g., immune cells modified and cultured as described herein) to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, such as in a human subject during a clinical trial, in an animal model system that predicts human efficacy, or by analyzing the activity of the agent in an in vitro assay.

The terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a composition disclosed herein (e.g., immune cells modified and cultured as described herein) to promote regression of cancer in a patient. Physiological safety refers to toxic levels or other adverse physiological effects (side effects) at the cellular, organ and/or organism level that result from administration of a composition disclosed herein (e.g., immune cells modified and cultured as described herein).

As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a set of polypeptides, typically two polypeptides in their simplest form, that when in an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and produce an intracellular signal. In some aspects, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), comprising a functional signaling domain derived from a stimulatory molecule and/or co-stimulatory molecule as defined below. In some aspects, the set of polypeptides are in the same polypeptide chain, e.g., constitute a chimeric fusion protein. In some aspects, the set of polypeptides are discontinuous with each other, e.g., in different polypeptide chains. In some aspects, the set of polypeptides includes a dimerization switch that can couple the polypeptides to each other when a dimerization molecule is present, e.g., can couple an antigen binding domain to an intracellular signaling domain. In some aspects, the stimulatory molecule of the CAR is a zeta chain (e.g., cd3ζ) associated with the T cell receptor complex. In some aspects, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3- ζ). In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some aspects, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD 137), CD27, and/or CD28.

In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen binding domain and the transmembrane domain are linked by a CAR spacer. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising one functional signaling domain derived from a co-stimulatory molecule and one functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR. In some aspects, the CAR further comprises a leader sequence at the N-terminus of the antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

The antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, typically the surface of a malignant tumor expresses an antigen. The antigen-specific extracellular domain specifically binds an antigen, for example, when it binds an antigen with an affinity constant or interaction affinity (K _D) of between about 0.1pM to about 10 μm (e.g., about 0.1pM to about 1 μm or about 0.1pM to about 100 nM). Methods for determining interaction affinities are known in the art. The antigen-specific extracellular domain suitable for use in the CARs of the present disclosure can be any antigen-binding polypeptide, a variety of which are known in the art. In some aspects, the antigen binding domain is a single chain Fv (scFv). Other antibody-based recognition domains, such as cAb VHH (camelid antibody variable domain) and humanized versions thereof, igNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domain are also suitable for use in the CARs of the present disclosure. In some aspects, recognition domains based on T Cell Receptors (TCRs), such as single chain TCRs (scTv, i.e., single chain double domain TCRs containing vαvβ), are also suitable for use in ROR1 binding proteins of the present disclosure.

As used herein, the term "T cell receptor" or "TCR" refers to a heterodimer consisting of 2 distinct transmembrane polypeptide chains: alpha and beta chains, each consisting of a constant region that anchors the chain inside the T cell surface membrane and a variable region that recognizes and binds to antigen presented by MHC. The TCR complex associates with 6 polypeptides to form 2 heterodimers, cd3γepsilon and cd3δepsilon, and 1 homodimer, cd3ζ, which combine to form a CD3 complex. T cell receptor engineered T cell therapies utilize modifications of T cells that retain these complexes to specifically target antigens expressed by specific tumor cells. As used herein, the term "TCR" includes naturally occurring TCRs and engineered TCRs.

As used herein, "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 (e.g., MHC/ROR1 peptide) in a population of selected, cloned, and/or subsequently introduced immune cells (e.g., T cells and/or NK cells).

"TCR mimetic" or "TCRm" refers to an engineered chimeric TCR type comprising an antigen binding domain (e.g., derived from an antibody) that recognizes an epitope comprising both a peptide and an MHC-I molecule, similar to the recognition of such complexes by TCRs on T cells. TCR mimics also comprise a T Cell Receptor Module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. An exemplary TCR mimetic is described, for example, in U.S. patent No. 10,822,413, incorporated by reference herein in its entirety.

The terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence" and "polynucleotide" are used interchangeably and refer to the phosphate polymer form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecules"), or any phosphate analog thereof (such as phosphorothioates and thioesters), in single-stranded form or in double-stranded helices. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule and in particular DNA or RNA molecule refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, this term includes double stranded DNA found in particular in linear or circular DNA molecules (e.g. restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein according to common practice, i.e., sequences are only presented in the 5 'to 3' direction along the non-transcribed DNA strand (i.e., the strand having a sequence homologous to mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semisynthetic DNA. The "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein. As described herein, in some aspects, a polynucleotide of the present disclosure may comprise a single nucleotide sequence encoding a single protein (e.g., a codon-optimized c-Jun nucleotide sequence) ("monocistronic"). In some aspects, the polynucleotides of the present disclosure are polycistronic (i.e., comprise two or more cistrons). In some aspects, each cistron of the polycistronic polynucleotide can encode a protein disclosed herein (e.g., a c-Jun protein, ROR1 binding protein, or EGFRt). In some aspects, each cistron can be translated independently of the other.

As used herein, the term "polypeptide" encompasses peptides and proteins unless otherwise indicated. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants and analogs of the foregoing. The polypeptides may be single polypeptides or may be a multi-molecular complex, such as a dimer, trimer or tetramer. They may also comprise single-or multi-chain polypeptides. Most commonly disulfide linkages are found in multi-chain polypeptides. The term polypeptide may also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid. In some aspects, a "peptide" may be less than or equal to 50 amino acids long, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

As used herein, the term "fragment" of a polypeptide (e.g., a C-Jun polypeptide) refers to an amino acid sequence of a polypeptide that is shorter than a naturally occurring sequence, lacks the N-terminal and/or C-terminal ends, or lacks any portion of the polypeptide as compared to the naturally occurring polypeptide. Thus, fragments need not necessarily have only N-terminal and/or C-terminal amino acids deleted. Polypeptides in which internal amino acids have been deleted relative to the naturally occurring sequence are also considered fragments.

As used herein, the term "functional fragment" refers to a polypeptide fragment that retains the function of the polypeptide. Thus, in some aspects, the functional fragment of an Ig hinge retains the ability to localize an antigen binding domain (e.g., scFv) in the ROR1 binding protein at a distance from a target epitope (e.g., a tumor antigen) such that the antigen binding domain (e.g., scFv) can effectively interact with the target epitope (e.g., a tumor antigen). Also, in some aspects, a c-Jun functional fragment is a fragment that, when expressed in an immune cell (e.g., CAR T cell), results in an immune cell having, for example, 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 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 99%, or about 100% of the activity of a reference immune cell that expresses the corresponding full-length c-Jun. Non-limiting examples of such activities are further described elsewhere in this disclosure.

"Recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. For the purposes of this disclosure, recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique. Polypeptides encoded by the polynucleotides disclosed herein (e.g., ROR1 binding protein, c-Jun, and/or EGFRt) can be recombinantly produced using methods known in the art. In some aspects, polypeptides (e.g., ROR1 binding protein, c-Jun, and/or EGFRt) encoded by polynucleotides of the present disclosure are produced by a cell (e.g., a T cell) after transfection with at least one polynucleotide or vector encoding a polypeptide described herein.

As used herein, a "coding region," "coding sequence," or "translatable sequence" is a portion of a polynucleotide that consists of codons that can be translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is typically not translated into an amino acid, it can be considered as part of the coding region, while any flanking sequences (e.g., promoter, ribosome binding site, transcription terminator, intron, etc.) are not part of the coding region. The boundaries of the coding region are typically determined by a start codon at the 5 'end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxy terminus of the resulting polypeptide).

The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each comprising a nucleobase sequence) or an oligomer and a target gene being related to each other by Watson-Crick base pairing rules. Ext> forext> exampleext>,ext> theext> nucleobaseext> sequenceext> "ext> Text> -ext> Gext> -ext> Aext> (ext> 5ext> 'ext> toext> 3ext>'ext>)ext>"ext> isext> complementaryext> toext> theext> nucleobaseext> sequenceext> "ext> Aext> -ext> Cext> -ext> Text> (ext> 3ext> 'ext> toext> 5ext>'ext>)ext>"ext>.ext> Complementarity may be "partial" in which less than all of a given nucleobase sequence matches another nucleobase sequence according to the base pairing rules. For example, in some aspects, the complementarity between a given nucleobase sequence and another nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Thus, in some aspects, the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% match or complement to a target nucleic acid sequence (e.g., a nucleotide sequence encoding a c-Jun). Or there may be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence to allow the embodiment to continue. In some aspects, the degree of complementarity between nucleobase sequences has a significant impact on the efficiency and intensity of hybridization between sequences.

As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product (e.g., a c-Jun polypeptide). It includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a "gene product". As used herein, a gene product may be a nucleic acid, such as a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein also include nucleic acids having post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides having post-translational modifications (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

As used herein, the term "identity" refers to the conservation of overall monomers between polymer molecules (e.g., between polynucleotide molecules). The term "identical" (e.g., polynucleotide a is identical to polynucleotide B) without any additional qualifiers means that the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity".

For example, calculation of the percent identity of two sequences may be performed by aligning the two polypeptide or polynucleotide sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second polypeptide or polynucleotide sequences to achieve optimal alignment and non-identical sequences may be ignored for comparison purposes). In some aspects, the length of the sequences aligned for comparison purposes is 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%, at least about 95%, or about 100% of the length of the reference sequence. The amino acids at the corresponding amino acid positions are then compared, or in the case of polynucleotides, the bases.

When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences varies with the number of identical positions shared by the sequences, with the length of each gap being introduced to achieve optimal alignment of the two sequences taking into account the number of gaps and the length of the gap. A mathematical algorithm may be used to complete the comparison of sequences and the determination of the percent identity between two sequences.

Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) can be obtained from a variety of sources. A suitable program for determining the percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information (National Center for Biotechnology Information) BLAST website (BLAST. Ncbi. Lm. Nih. Gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to perform a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example Needle, stretcher, water or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from European Bioinformatics Institute (EBI) at world WideWeb.ebi.ac.uk/Tools/psa.

Sequence alignment may be performed using methods known in the art, such as MAFFT, clustal (ClustalW, clustal X, or Clustal Omega), MUSCLE, and the like.

Different regions within a single polynucleotide or polypeptide target sequence that are aligned with a polynucleotide or polypeptide reference sequence may each have their own percent sequence identity. It should be noted that the value of the percent sequence identity is rounded to the decimal point and then to the next digit. For example, 80.11, 80.12, 80.13 and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18 and 80.19 are rounded up to 80.2. It should also be noted that the length value will always be an integer.

In some aspects, the percent identity (ID%) of a first amino acid sequence (or nucleic acid sequence) relative to a second amino acid sequence (or nucleic acid sequence) is calculated as ID% = 100x (Y/Z), where Y is the number of amino acid residues (or nucleobases) that are the same match score in an alignment of the first and second sequences (as aligned by visual inspection or a specific sequence alignment procedure) and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percent identity of the first sequence relative to the second sequence will be higher than the percent identity of the second sequence relative to the first sequence.

It will be appreciated by those skilled in the art that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons driven by only primary sequence material. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., the location of mutations), or phylogenetic data. A suitable procedure for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available at the world Widewebtcoffe.org, and alternatively available, for example, from EBI. It should also be appreciated that the final alignment used to calculate the percent sequence identity may be performed automatically or manually.

As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to the state of a formulation of a desired composition of the present disclosure that has undergone one or more purification processes. In some aspects, isolation or purification as used herein is a process of removing, including partially removing (e.g., a portion of), a composition of the present disclosure (e.g., a modified immune cell expressing a ROR1 binding protein and having increased levels of c-Jun protein) from a sample containing contaminants.

In some aspects, the isolated composition does not have a detectable undesired activity, or alternatively, the level or amount of undesired activity is an acceptable level or amount or less. In some aspects, the isolated composition has an amount and/or concentration of the desired composition of the present disclosure that is an acceptable amount and/or concentration and/or activity, or is greater than an acceptable amount and/or concentration and/or activity. In some aspects, the isolated composition is enriched compared to the starting material from which the composition was obtained. Such enrichment may be at least about 10%, at least about 20%, 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%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.

In some aspects, the isolated preparation is substantially free of residual biological products. In some aspects, the isolated formulation is 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological substance. Residual biological products may include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

As used herein, the term "linked" refers to the covalent or non-covalent attachment of a first amino acid sequence or polynucleotide sequence to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence may be directly joined or juxtaposed to the second amino acid or polynucleotide sequence, or alternatively, the insertion sequence may covalently join the first sequence to the second sequence. The term "linked" means not only that the first polynucleotide sequence is fused to the second polynucleotide sequence at the 5 'end or the 3' end, but also that the entire first polynucleotide sequence (or second polynucleotide sequence) is inserted into any two nucleotides in the second polynucleotide sequence (or first polynucleotide sequence, respectively). The first polynucleotide sequence may be linked to the second polynucleotide sequence by a phosphodiester bond or linker. The linker may be, for example, a polynucleotide.

"Treatment" or "therapy" of a subject (including any grammatical derivations thereof) refers to any type of intervention or procedure performed on the subject or administration of an active agent to the subject with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of symptoms, complications, disorders, or biochemical markers associated with a disease. In some aspects, the term refers to inducing an immune response against an antigen in a subject.

As used herein, the term "prevent/preventing" and variations thereof refers to partially or completely delaying the onset of a disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or clinical expressions of a particular disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or expressions of a particular disease, disorder, and/or condition; partially or completely delay progression of a particular disease, disorder and/or condition; and/or reduce the risk of developing a pathology associated with a disease, disorder, and/or condition. In some aspects, the prophylactic result is achieved via prophylactic treatment.

As used herein, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. Promoters may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that most often result in expression of a gene in most cell types are commonly referred to as "constitutive promoters". Promoters that cause expression of a gene in a particular cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters. Promoters that cause expression of a gene at a particular developmental or cellular differentiation stage are commonly referred to as "developmental specific promoters" or "cellular differentiation specific promoters. Promoters that are induced and result in gene expression after exposure or treatment of cells with agents that induce promoters, biomolecules, chemicals, ligands, light, etc., are commonly referred to as "inducible promoters" or "regulatable promoters. It is further recognized that DNA fragments of different lengths may have the same promoter activity, since in most cases the exact boundaries of regulatory sequences have not been fully defined.

As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively.

Various aspects of the disclosure are described in greater detail in the following subsections.

Methods of the present disclosure

II.A. Metabolic reprogramming Medium

Some aspects of the present disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) under culture conditions (e.g., in a culture medium) wherein the culture conditions (e.g., certain ion concentrations, medium tonicity, cytokines, and/or any combination thereof) are capable of reducing, limiting, or preventing differentiation of immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), thereby affecting or improving their use in cell therapies (e.g., adoptive cell therapies). In some aspects, immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) are cultured in the Metabolic Reprogramming Media (MRMs) disclosed herein. In some aspects, immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured in MRM have a higher proportion of stem cell-like cells as compared to cells cultured using conventional methods, e.g., in media with less than 5mM potassium ions. In some aspects, immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured in MRM have a higher proportion of effector-like cells as compared to cells cultured using conventional methods, e.g., in media with less than 5mM potassium ions. In some aspects, immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured in MRM have a higher proportion of stem cell-like and effector-like cells as compared to cells cultured using conventional methods, e.g., in media having less than 5mM potassium ions. In some aspects, immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured in MRM have a higher proliferative potential as compared to cells cultured using conventional methods, e.g., in media with less than 5mM potassium ions.

Some aspects of the disclosure are directed to methods of preparing a population of immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) comprising culturing the cells in a medium comprising potassium ions at a concentration above 5mM (e.g., metabolic reprogramming media disclosed herein). Some aspects of the disclosure are directed to methods of preparing a population of T cells comprising culturing T cells (e.g., modified to express a ROR1 binding protein and having increased levels of c-Jun protein) in a medium comprising potassium ions at a concentration greater than 5mM (e.g., metabolic reprogramming media disclosed herein). In some aspects, the disclosure provides methods of preparing immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) comprising culturing the cells in a medium comprising potassium ions at a concentration above 5mM (e.g., above 40mM, e.g., between 40mM and 80mM, e.g., between 55mM and 70 mM), the methods being capable of maintaining a stem cell-like phenotype (e.g., minimal differentiation) of the cultured cells. In some aspects, the disclosure provides methods of preparing T cells comprising culturing T cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein) in a medium comprising potassium ions at a concentration above 5mM (e.g., above 40mM, e.g., between 40mM and 80mM, e.g., between 55mM and 70 mM), the methods being capable of maintaining a stem cell-like phenotype (e.g., minimal differentiation) of the cultured T cells. In some aspects, the cultured cells have a more stem cell-like phenotype (e.g., low differentiation) than cells grown in a medium having a lower potassium concentration. In some aspects, the medium further comprises Interleukin (IL) -2, IL-21, IL-7, IL-15, or any combination thereof. In some aspects, the medium further comprises sodium ions (e.g., naCl), calcium ions, glucose, or any combination thereof.

In some aspects, a population of immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) cultured using the methods disclosed herein exhibits an increased number of stem cell-like cells relative to a population of cells cultured using conventional methods, e.g., in a medium having less than 5mM potassium ions. In some aspects, a population of T cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein) cultured using the methods disclosed herein exhibits an increased number of stem cell-like T cells relative to a population of T cells cultured using conventional methods, e.g., in a medium having less than 5mM potassium ions. In some aspects, the immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) exhibit increased expression of a marker characteristic of stem cell-like cells relative to the starting immune cell population (i.e., prior to culturing). In some aspects, the T cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein) exhibit increased expression of a marker characteristic of stem cell-like cells relative to the starting T cell population (i.e., prior to culturing). In some aspects, the starting immune cell population comprises immune cells (e.g., T cells and/or NK cells) obtained from a human subject. In some aspects, the starting immune cell population comprises T cells obtained from a human subject. In some aspects, the starting immune T cell population comprises T _N cells, T _SCM cells, T _CM cells, T _EM cells, or any combination thereof. In some aspects, the starting immune cell population comprises T cells prior to modification as described herein (e.g., transfection with a polynucleotide encoding a ROR1 binding protein, c-Jun protein, and/or EGFRt).

Any method known in the art may be used to measure increased cellular pluripotency. In some aspects, cell stem properties are measured sequentially by antibody staining, gated flow cytometry. In some aspects, cell stem properties are measured by autophagy flux. In some aspects, cell stem performance is measured by glucose uptake. In some aspects, cell stem performance is measured by fatty acid uptake. In some aspects, cell stem properties are measured by mitochondrial biomass. In some aspects, cell stem performance is measured by RNA quantification/expression analysis (e.g., microarray, qPCR (taqman), RNA-seq, single cell RNA-seq, or any combination thereof). In some aspects, cell stem performance is measured by transcripts associated with metabolic analysis (e.g., seahorse metabolic analysis, extracellular acidification rate analysis (ECAR), oxygen consumption rate analysis (OCR), backup respiration rate analysis, and/or mitochondrial membrane potential analysis). In some aspects, the dryness is measured using one or more in vivo or in vitro functional assays (e.g., assaying for cell persistence, anti-tumor capability, anti-tumor clearance, viral clearance, pluripotency, cytokine release, cell killing, or any combination thereof).

In some aspects, the differentiation status of immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) is characterized by an increased number of cells expressing markers characteristic of poorly differentiated cells. In some aspects, the differentiation state of a T cell is characterized by an increased number of cells expressing a marker characteristic of a poorly differentiated T cell. In some aspects, the increased number of stem cell-like cells is characterized by an increased number of T cells expressing markers characteristic of T _N and/or T _SCM cells. In some aspects, the increase in the number of stem cell-like T cells is characterized by an increase in the number of cells expressing a marker unique to the T _SCM cells. In some aspects, the T cell population exhibits an increase in the number of cells expressing CD45 RA. In some aspects, the population of T cells exhibits an increase in the number of CCR7 expressing cells. In some aspects, the T cell population exhibits an increase in the number of cells expressing CD 62L. In some aspects, the T cell population exhibits an increase in the number of cells expressing CD 28. In some aspects, the population of T cells exhibits an increase in the number of cells expressing CD 95. In some aspects, the cell is CD45RO ^{Low and low}. In some aspects, the cell does not express CD45RO. In some aspects, the cell population exhibits an increase in the number of CD45RA ⁺ and CCR7 ⁺ cells (e.g., cd4+ and/or cd8+ T cells). In some aspects, the cell population exhibits an increase in the number of CD45RA ⁺、CCR7⁺ and CD62L ⁺ cells. In some aspects, the cell population exhibits an increase in the number of CD95 ⁺、CD45RA⁺、CCR7⁺ and CD62L ⁺ cells. In some aspects, the cell population exhibits an increase in the number of cells expressing TCF 7. In some aspects, the T cell population exhibits an increase in the number of CD45RA ⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. In some aspects, the T cell population exhibits an increase in the number of CD95 ⁺、CD45RA⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. in some aspects, the T cell population exhibits an increased number of CD3 ⁺、CD45RA⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. In some aspects, the T cell population exhibits an increased number of CD3 ⁺、CD95⁺、CD45RA⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. In some aspects, the cell expresses CD27. In some aspects, the T cell population exhibits an increase in the number of CD27 ⁺、CD3⁺、CD45RA⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. In some aspects, the T cell population exhibits an increase in the number of CD27 ⁺、CD3⁺、CD95⁺、CD45RA⁺、CCR7⁺、CD62L⁺ and TCF7 ⁺ cells. In some aspects, the T cell population exhibits an increase in the number of CD39 ^- and CD69 ^- cells. In some aspects, the T cell population exhibits an increased number of TCF7 ⁺ and CD39 ^- cells. In some aspects, the population of cells exhibits an increased number of T _SCM cells. In some aspects, the population of cells exhibits an increased number of T _N cells. In some aspects, the cell population exhibits an increased number of T _SCM and T _N cells. In some aspects, the population of cells exhibits an increased number of stem cell-like T cells. In some aspects, the T cell is a cd4+ cell; in some aspects, the T cells are cd8+ cells. In some aspects, the T cells comprise cd4+ T cells and cd8+ T cells.

In some aspects, the number of stem cell-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 cell-like cells prior to culturing with the MRM. In some aspects, the number of stem cell-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 cell-like cells prior to culturing with the MRM.

In some aspects, after culturing T cells according to the methods disclosed herein, the stem cell-like T cells comprise 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, stem cell-like T cells (e.g., CD45RA ⁺ and CCR7 ⁺) constitute at least about 20% of CD8 ⁺ T cells. In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), the stem cell-like T cells comprise 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. In some aspects, stem cell-like T cells (e.g., CD45RA ⁺ and CCR7 ⁺) constitute at least about 20% of CD4 ⁺ T cells.

As described herein, in some aspects, the methods of the present disclosure can be used to modify T cells (e.g., CD8 ⁺ T cells and/or CD4 ⁺ T cells) to (a) express ROR1 binding proteins (e.g., anti-ROR 1 CARs) and (b) have increased levels of c-Jun protein. Thus, in some aspects, after culturing, CD8 ⁺ T cells express ROR1 binding protein and have increased levels of c-Jun protein and at least about 20% of the modified CD8 ⁺ T cells are stem cell-like T cells (e.g., CD45RA ⁺ and CCR7 ⁺). In some aspects, after culturing, the CD4 ⁺ T cells express ROR1 binding protein and have increased levels of c-Jun protein and at least about 20% of the modified CD4 ⁺ T cells are stem cell-like T cells (e.g., CD45RA ⁺ and CCR7 ⁺).

In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), the stem cell-like T cells comprise at least about 10% to at least about 70% of the total number of T cells in the culture. In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and increased levels of c-Jun protein), the stem cell-like T cells comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD8 ⁺ T cells in the culture. In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), the stem cell-like T cells comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD4 ⁺ T cells in the culture.

In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), at least about 10% to at least about 40% of the total number of T cells in the culture are CD39 ^-/CD69^- T cells. In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 ^-/CD69^- T cells.

In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), at least about 10% to at least about 70% of the total number of T cells in the culture are CD39 ^-/TCF7⁺ T cells. In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 ^-/TCF7⁺ T cells. In some aspects, the T cell is a CD4 ⁺ T cell. In some aspects, the T cell is a CD8 ⁺ T cell.

In some aspects, at least about 10% to at least about 70% of the total number of T cells are CD45RA ⁺ and CCR7 ⁺ T cells after culturing the T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein). In some aspects, after culturing T cells according to the methods disclosed herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein), at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD45RA ⁺ and CCR7 ⁺ T cells. In some aspects, the T cell is a CD4 ⁺ T cell. In some aspects, the T cell is a CD8 ⁺ T cell. In some aspects, the T cells comprise CD4 ⁺ T cells and CD8 ⁺ T cells.

In some aspects, immune cells of the disclosure (e.g., engineered immune cells (e.g., T cells and/or NK cells, modified to express ROR1 binding protein and increased levels of c-Jun protein)) cultured according to methods disclosed herein exhibit increased transduction efficiency.

In some aspects, a greater percentage of cells express, e.g., a target transgene encoding a ligand binding protein after transduction, as compared to cells transduced and cultured similarly using conventional methods (e.g., in a medium containing less than 5mM K ⁺), wherein the cells are cultured according to the methods disclosed herein. In certain aspects, a greater percentage of cells cultured according to the methods disclosed herein express the ligand binding protein after lentiviral transduction of the cells, as compared to similarly transduced cells cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺). In some aspects, transduction efficiency is increased by at least about 1.5-fold relative to a similarly transduced cell cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺). In some aspects, transduction efficiency is increased by at least about 2-fold relative to a similarly transduced cell cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺). As used herein, the term "transduction efficiency" refers to: (i) An amount of material (e.g., an exogenous polynucleotide) that can be physically introduced into the cell over a defined period of time; (ii) The amount of time it takes to physically introduce a given amount of material into a cell; (iii) The cell population absorbs the level of the target material (e.g., exogenous polynucleotide, i.e., transgene) (e.g., percentage of cells expressing the transgene); or (iv) any combination of (i) - (iii). In some aspects, by increasing transduction efficiency, the culture methods provided herein can allow for the introduction of larger amounts of exogenous nucleotide sequences into cells and/or reduce the amount of time required to introduce a given amount of exogenous nucleotide sequences. Without being bound by any theory, in some aspects, such effects may increase expression of the encoded protein (e.g., c-Jun polypeptide) in modified immune cells.

In some aspects, immune cells (e.g., T cells and/or NK cells) are transduced prior to culturing according to the methods disclosed herein. In some aspects, immune cells (e.g., T cells and/or NK cells) are transduced after culturing according to the methods disclosed herein. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured prior to, during, and after transduction according to the methods disclosed herein, e.g., by contacting the immune cells with APC-MS in a medium comprising at least 5mM potassium ions (e.g., above 5mM, e.g., between about 40mM and about 80 mM).

In certain aspects, viral vectors are used to transduce immune cells. In some aspects, the vector comprises a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia vector, a herpes simplex virus vector, and an Epstein-Barr virus 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 AAV.

In some aspects, non-viral methods are used to transduce immune cells. In some aspects, the non-viral method comprises using a transposon. In some aspects, use of a non-viral delivery method allows reprogramming of immune cells (e.g., T cells and/or NK cells), and infusion of cells directly into a subject. In some aspects, polynucleotides can be inserted into the genome of a target cell (e.g., T cell) or host cell (e.g., cell for recombinant expression of the encoded protein) by using CRISPR/Cas systems and genome editing alternatives, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (meganucleases, MN).

In some aspects, after adoptive transfer of immune cells (e.g., T cells and/or NK cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein)) optionally expressing ligand binding proteins cultured according to the methods disclosed herein, the transferred cells exhibit reduced cell depletion as compared to cells cultured using conventional methods (e.g., in medium containing less than 5mM K ⁺). In some aspects, following adoptive transfer of T cells (e.g., modified to express increased levels of c-Jun protein) optionally expressing ligand binding proteins cultured according to the methods disclosed herein, the transferred T cells exhibit reduced cell depletion as compared to T cells cultured using conventional methods (e.g., in medium containing less than 5mM K ⁺).

In some aspects, after adoptive transfer of cells cultured according to the methods disclosed herein, the transferred cells persist in vivo for a longer period of time as compared to cells cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺). In some aspects, the transferred cells (e.g., T cells and/or NK cells) have greater in vivo efficacy, e.g., tumor killing activity, as compared to cells cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺). In some aspects, lower doses of cells cultured according to the methods disclosed herein are required to elicit a response, e.g., reduced tumor volume, in a subject, as compared to cells cultured using conventional methods (e.g., in a medium containing less than 5mM K ⁺).

In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured immediately after isolation from a subject according to the methods disclosed herein, e.g., in a medium comprising at least 5mM potassium ions (e.g., above 5mM, e.g., between about 40mM and about 80 mM). In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein during cell expansion. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein during engineering of the cells, e.g., during transduction with constructs encoding transgenes (e.g., ligand binding proteins). In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein after engineering of the cells, e.g., after transduction with constructs encoding transgenes (e.g., ligand binding proteins). In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein throughout the amplification and engineering process. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein throughout the viral genetic engineering process. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein throughout the non-viral genetic engineering process. In some aspects, during the introduction of a ligand binding protein into an immune cell (e.g., a T cell and/or NK cell) to allow for tumor-specific targeting (e.g., CAR, TCR, or TCR mimetic), the immune cell (e.g., T cell and/or NK cell) is cultured according to the methods disclosed herein. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein throughout the introduction of one or more endogenous genes (e.g., c-Jun) that improve T cell function. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein throughout the introduction of one or more synthetic genes that improve T cell function (e.g., an exogenous polynucleotide encoding a c-Jun protein, or an exogenous polynucleotide encoding CAR, TCR, caTCR, CSR or a TCR mimetic).

In some aspects, according to the methods disclosed herein, immune cells (e.g., T cells and/or NK cells) are cultured, e.g., in a medium comprising at least 5mM potassium ions (e.g., greater than 5mM, e.g., between about 40mM and about 80 mM), for whole ex vivo culture, e.g., upon isolation of immune cells (e.g., T cells and/or NK cells) from a subject, by growth, expansion, engineering, and until administration into a subject in need of adoptive cell therapy. In some aspects, T cells are cultured, e.g., according to the methods disclosed herein, in a medium comprising at least 5mM potassium ions (e.g., greater than 5mM, e.g., between about 40mM and about 80 mM), for whole ex vivo culture, e.g., by growth, expansion, engineering, and until administration to a subject in need of adoptive cell therapy, e.g., upon isolation of T cells from the subject. in some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein for the duration of expansion. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured according to the methods disclosed herein until the total number of viable immune cells (e.g., T cells and/or NK cells) is at least about 10 ⁴, at least about 5 x 10 ⁴, at least about 10 ⁵, At least about 5X 10 ⁵, at least about 10 ⁶, at least about 5X 10 ⁶, at least about 1X 10 ⁷, At least about 5X10 ⁷, at least about 1X 10 ⁸, at least about 5X10 ⁸, at least about 1X 10 ⁹, at least about 5X10 ⁹, at least about 1X 10 ¹⁰, at least about 5X10 ¹⁰, at least about 1X 10 ¹¹, At least about 5 x 10 ¹¹, at least about 1 x 10 ¹², or at least about 5 x 10 ¹² total cells. In some aspects, T cells are cultured according to the methods disclosed herein until the total number of viable T cells is at least about 10 ⁴, at least about 5 x 10 ⁴, at least about 10 ⁵, at least about 5 x 10 ⁵, At least about 10 ⁶, at least about 5X 10 ⁶, at least about 1X 10 ⁷, at least about 5X 10 ⁷, at least about 1X 10 ⁸, at least about 5X10 ⁸, at least about 1X 10 ⁹, at least about 5X10 ⁹, At least about 1X 10 ¹⁰, at least about 5X10 ¹⁰, at least about 1X 10 ¹¹, at least about 5X10 ¹¹, At least about 1x 10 ¹² or at least about 5 x10 ¹² total T cells.

In some aspects, the medium further comprises a cell expansion agent. As used herein, a "cell expansion agent" refers to an agent, such as a small molecule, polypeptide, or any combination thereof, that promotes the growth and proliferation of cultured cells, such as immune cells (e.g., T cells and/or NK cells), in vitro and/or ex vivo. In some aspects, the cell expansion agent comprises a PI3K inhibitor. In some aspects, the medium further comprises an AKT inhibitor. In some aspects, the medium further comprises a PI3K inhibitor and an AKT inhibitor. In some aspects, the PI3K inhibitor comprises LY294002. In some aspects, the PI3K inhibitor comprises IC87114. In some aspects, the PI3K inhibitor comprises Italy (idelalisib) (see, e.g., peterson et al, blood adv.2 (3): 210-23 (2018)). In some aspects, the medium further comprises a GSK3B inhibitor. In some aspects, the GSK3B inhibitor comprises TWS119. In some aspects, the culture medium further comprises ACLY inhibitors. In some aspects, the ACLY inhibitor comprises tripotassium hydroxycitrate monohydrate. In some aspects, the PI3K inhibitor comprises hydroxycitrate. In some aspects, the PI3K inhibitor comprises pitiriprist (pictilisib). In some aspects, the PI3K inhibitor comprises CAL-101. In some aspects, the AKT inhibitor comprises MK2206, A443654, or AKTi-VIII (CAS 612847-09-3).

In some aspects, the metabolic reprogramming media comprises mitochondrial fuel. In some aspects, the metabolic reprogramming media comprises O-acetyl-L-carnitine hydrochloride. In some aspects, the metabolic reprogramming media comprises at least about 0.1mM, at least about 0.5mM, at least about 1.0mM, at least about 5mM, or at least about 10mM O-acetyl-L-carnitine hydrochloride. In some aspects, the metabolic reprogramming media comprises at least about 1.0mM O-acetyl-L-carnitine hydrochloride.

In some aspects, the metabolic reprogramming media further comprises one or more of the following: (i) one or more cell expansion agents, (ii) sodium ions (e.g., naCl), (iii) one or more sugars, (iv) calcium ions, and (v) one or more cytokines.

II.A.1. Potassium

Some aspects of the present disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium (i.e., a metabolic reprogramming medium as disclosed herein) comprising an increased potassium ion concentration (e.g., greater than about 5mM, greater than about 40mM, greater than about 45mM, greater than about 50mM, greater than about 55mM, greater than about 60mM, greater than about 65mM, or greater than about 70 mM) relative to a control medium. in some aspects, the metabolic reprogramming media comprises at least about 5mM to at least about 100mM potassium ion, at least about 5mM to at least about 90mM potassium ion, at least about 5mM to at least about 80mM potassium ion, at least about 5mM to at least about 75mM potassium ion, at least about 5mM to at least about 70mM potassium ion, at least about 5mM to at least about 65mM potassium ion, at least about 5mM to at least about 60mM potassium ion, at least about 5mM to at least about 55mM potassium ion, at least about 5mM to at least about 50mM potassium ion, at least about 5mM to at least about 45mM potassium ion, at least about 5mM to at least about 40mM potassium ion, At least about 10mM to at least about 80mM potassium ion, at least about 10mM to at least about 75mM potassium ion, at least about 10mM to at least about 70mM potassium ion, at least about 10mM to at least about 65mM potassium ion, at least about 10mM to at least about 60mM potassium ion, at least about 10mM to at least about 55mM potassium ion, at least about 10mM to at least about 50mM potassium ion, at least about 10mM to at least about 45mM potassium ion, at least about 10mM to at least about 40mM potassium ion, at least about 20mM to at least about 80mM potassium ion, at least about 20mM to at least about 75mM potassium ion, At least about 20mM to at least about 70mM potassium ion, at least about 20mM to at least about 65mM potassium ion, at least about 20mM to at least about 60mM potassium ion, at least about 20mM to at least about 55mM potassium ion, at least about 20mM to at least about 50mM potassium ion, at least about 20mM to at least about 45mM potassium ion, at least about 20mM to at least about 40mM potassium ion, at least about 30mM to at least about 80mM potassium ion, at least about 30mM to at least about 75mM potassium ion, at least about 30mM to at least about 70mM potassium ion, at least about 30mM to at least about 65mM potassium ion, at least about 30mM to at least about 60mM potassium ion, at least about 30mM to at least about 55mM potassium ion, at least about 30mM to at least about 50mM potassium ion, at least about 30mM to at least about 45mM potassium ion, at least about 30mM to at least about 40mM potassium ion, at least about 40mM to at least about 80mM potassium ion, at least about 40mM to at least about 75mM potassium ion, at least about 40mM to at least about 70mM potassium ion, at least about 40mM to at least about 65mM potassium ion, at least about 40mM to at least about 60mM potassium ion, at least about 40mM to at least about 55mM potassium ion, at least about 40mM to at least about 50mM potassium ion, at least about 40mM to at least about 45mM potassium ion, at least about 45mM to at least about 80mM potassium ion, at least about 45mM to at least about 75mM potassium ion, at least about 45mM to at least about 70mM potassium ion, at least about 45mM to at least about 65mM potassium ion, at least about 45mM to at least about 60mM potassium ion, at least about 45mM to at least about 55mM potassium ion, at least about 45mM to at least about 50mM potassium ion, at least about 50mM to at least about 80mM potassium ion, at least about 50mM to at least about 75mM potassium ion, at least, At least about 50mM to at least about 70mM potassium ion, at least about 50mM to at least about 65mM potassium ion, at least about 50mM to at least about 60mM potassium ion, or at least about 50mM to at least about 55mM potassium ion.

In some aspects, the metabolic reprogramming media comprises at least about 5mM, at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 55mM, at least about 60mM, at least about 65mM, at least about 70mM, at least about 75mM, or at least about 80mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 5mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 10mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 15mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 20mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 25mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 30mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 35mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 40mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 45mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 50mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 55mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 60mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 65mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 70mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 75mM potassium ions. In some aspects, the metabolic reprogramming media comprises at least about 80mM potassium ions. In some aspects, the MRM comprises between about 40mM to about 80mM potassium ions (e.g., between 40-80 mM).

In some aspects, the metabolic reprogramming media comprises an increased concentration of potassium ions, for example at least about 5mM potassium ions, and the media is hypotonic. In some aspects, the metabolic reprogramming media comprises potassium ions at a concentration of between about 40mM and about 80mM and NaCl at a concentration of between about 30mM and about 100mM, wherein the total concentration of potassium ions and NaCl is between about 110mM and about 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 5mM to about 100mM. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 5mM to about 100mM, wherein the media is hypotonic. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 5mM to about 90mM, about 5mM to about 80mM, about 5mM to about 70mM, about 5mM to about 60mM, or about 5mM to about 50mM. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 5mM to about 90mM, about 5mM to about 80mM, about 5mM to about 70mM, about 5mM to about 60mM, or about 5mM to about 50mM, wherein the media is hypotonic. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 25mM to about 100mM. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 25mM to about 100mM, wherein the media is hypotonic. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 25mM to about 90mM, about 25mM to about 80mM, about 25mM to about 70mM, about 25mM to about 60mM, or about 25mM to about 50mM. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 25mM to about 90mM, about 25mM to about 80mM, about 25mM to about 70mM, about 25mM to about 60mM, or about 25mM to about 50mM, wherein the media is hypotonic. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 40mM to about 100mM. In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 40mM to about 100mM, wherein the media is hypotonic. In some aspects, the concentration of potassium ions is about 40mM to about 90mM, about 40mM to about 85mM, about 40mM to about 80mM, about 40mM to about 75mM, about 40mM to about 70mM, about 40mM to about 65mM, about 40mM to about 60mM, about 40mM to about 55mM, or about 40mM to about 50mM. In some aspects, the concentration of potassium ions is about 40mM to about 90mM, about 40mM to about 85mM, about 40mM to about 80mM, about 40mM to about 75mM, about 40mM to about 70mM, about 40mM to about 65mM, about 40mM to about 60mM, about 40mM to about 55mM, or about 40mM to about 50mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is between about 40mM to about 80mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is about 50mM to about 90mM, about 50mM to about 85mM, about 50mM to about 80mM, about 50mM to about 75mM, about 50mM to about 70mM, about 50mM to about 65mM, about 50mM to about 60mM, or about 50mM to about 55mM. In some aspects, the concentration of potassium ions is about 50mM to about 90mM, about 50mM to about 85mM, about 50mM to about 80mM, about 50mM to about 75mM, about 50mM to about 70mM, about 50mM to about 65mM, about 50mM to about 60mM, or about 50mM to about 55mM, and wherein the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 50mM potassium ions and less than about 90mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 50mM to about 120mM. In some aspects, the concentration of potassium ions is about 50mM to about 115mM, about 50mM to about 110mM, about 50mM to about 105mM, about 50mM to about 100mM, about 50mM to about 95mM, about 50mM to about 90mM, about 50mM to about 85mM, about 50mM to about 80mM, about 50mM to about 75mM, about 50mM to about 70mM, about 50mM to about 65mM, about 50mM to about 60mM, or about 50mM to about 55mM. In some aspects, the medium is hypotonic. In some aspects, the medium comprises at least about 50mM to about 120mM potassium ion and less than about 90mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 55mM to about 120mM. In some aspects, the concentration of potassium ions is about 55mM to about 115mM, about 55mM to about 110mM, about 55mM to about 105mM, about 55mM to about 100mM, about 55mM to about 95mM, about 55mM to about 90mM, about 55mM to about 85mM, about 55mM to about 80mM, about 55mM to about 75mM, about 55mM to about 70mM, about 55mM to about 65mM, or about 55mM to about 60mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 55mM to about 120mM potassium ions and less than about 85mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl in the metabolic reprogramming media of the present disclosure is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 60mM to about 120mM. In some aspects, the concentration of potassium ions is about 60mM to about 115mM, about 60mM to about 110mM, about 60mM to about 105mM, about 60mM to about 100mM, about 60mM to about 95mM, about 60mM to about 90mM, about 60mM to about 85mM, about 60mM to about 80mM, about 60mM to about 75mM, about 60mM to about 70mM, or about 60mM to about 65mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 60mM to about 120mM potassium ions and less than about 80mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 65mM to about 120mM. In some aspects, the concentration of potassium ions is about 65mM to about 115mM, about 65mM to about 110mM, about 65mM to about 105mM, about 65mM to about 100mM, about 65mM to about 95mM, about 65mM to about 90mM, about 65mM to about 85mM, about 65mM to about 80mM, about 65mM to about 75mM, or about 65mM to about 70mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 65mM to about 120mM potassium ions and less than about 75mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 70mM to about 120mM. In some aspects, the concentration of potassium ions is about 70mM to about 115mM, about 70mM to about 110mM, about 70mM to about 105mM, about 70mM to about 100mM, about 70mM to about 95mM, about 70mM to about 90mM, about 70mM to about 85mM, about 70mM to about 80mM, or about 70mM to about 75mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 70mM to about 120mM potassium ions and less than about 70mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 75mM to about 120mM. In some aspects, the concentration of potassium ions is about 75mM to about 115mM, about 75mM to about 110mM, about 75mM to about 105mM, about 75mM to about 100mM, about 75mM to about 95mM, about 75mM to about 90mM, about 75mM to about 85mM, or about 75mM to about 80mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 75mM to about 120mM potassium ions and less than about 65mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 80mM to about 120mM. In some aspects, the concentration of potassium ions is about 80mM to about 115mM, about 80mM to about 110mM, about 80mM to about 105mM, about 80mM to about 100mM, about 80mM to about 95mM, about 80mM to about 90mM, or about 80mM to about 85mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 80mM to about 120mM potassium ions and less than about 60mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 85mM to about 120mM. In some aspects, the concentration of potassium ions is about 85mM to about 115mM, about 85mM to about 110mM, about 85mM to about 105mM, about 85mM to about 100mM, about 85mM to about 95mM, or about 85mM to about 90mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 85mM to about 120mM potassium ions and less than about 65mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 90mM to about 120mM. In some aspects, the concentration of potassium ions is about 90mM to about 115mM, about 90mM to about 110mM, about 90mM to about 105mM, about 90mM to about 100mM, or about 90mM to about 95mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 90mM to about 120mM potassium ions and less than about 50mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 95mM to about 120mM. In some aspects, the concentration of potassium ions is about 95mM to about 115mM, about 95mM to about 110mM, about 95mM to about 105mM, or about 95mM to about 100mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 95mM to about 120mM potassium ions and less than about 55mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 100mM to about 120mM. In some aspects, the concentration of potassium ions is about 100mM to about 115mM, about 100mM to about 110mM, or about 100mM to about 105mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 100mM to about 120mM potassium ions and less than about 50mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 105mM to about 120mM. In some aspects, the concentration of potassium ions is about 105mM to about 115mM or about 105mM to about 110mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 105mM to about 120mM potassium ions and less than about 35mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 110mM to about 120mM. In some aspects, the concentration of potassium ions is about 110mM to about 115mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming media comprises at least about 110mM to about 120mM potassium ions and less than about 30mM to about 20mM NaCl. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 50mM to about 90mM. In some aspects, the concentration of potassium ions is about 50mM to about 80mM. In some aspects, the concentration of potassium ions is about 60mM to about 90mM. In some aspects, the concentration of potassium ions is about 60mM to about 80mM. In some aspects, the concentration of potassium ions is about 70mM to about 90mM. In some aspects, the concentration of potassium ions is about 70mM to about 80mM. In some aspects, the concentration of potassium ions is about 80mM to about 90mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 50mM to about 90mM, and the concentration of NaCl is less than about 90mM to about 50mM. In some aspects, the concentration of potassium ions is from about 50mM to about 80mM, and the concentration of NaCl is less than about 90mM to about 60mM. In some aspects, the concentration of potassium ions is about 60mM to about 90mM, and the concentration of NaCl is less than about 90mM to about 60mM. In some aspects, the concentration of potassium ions is about 60mM to about 80mM, and the concentration of NaCl is less than about 80mM to about 60mM. In some aspects, the concentration of potassium ions is about 70mM to about 90mM, and the concentration of NaCl is less than about 70mM to about 50mM. In some aspects, the concentration of potassium ions is about 70mM to about 80mM, and the concentration of NaCl is less than about 70mM to about 60mM. In some aspects, the concentration of potassium ions is about 80mM to about 90mM, and the concentration of NaCl is less than about 60mM to about 50mM. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions in the metabolic reprogramming media of the present disclosure is about 50mM to about 55mM. In some aspects, the concentration of potassium ions is about 50mM to about 55mM, and the concentration of NaCl is less than about 90mM to about 85mM. In some aspects, the concentration of potassium ions is about 55mM to about 60mM. In some aspects, the concentration of potassium ions is about 55mM to about 60mM, and the concentration of NaCl is less than about 85mM to about 80mM. In some aspects, the concentration of potassium ions is about 60mM to about 65mM. In some aspects, the concentration of potassium ions is from about 60mM to about 65mM, and the concentration of NaCl is less than about 80mM to about 75mM. In some aspects, the concentration of potassium ions is about 65mM to about 70mM. In some aspects, the concentration of potassium ions is about 65mM to about 70mM, and the concentration of NaCl is less than about 75mM to about 70mM. In some aspects, the concentration of potassium ions is about 70mM to about 75mM. In some aspects, the concentration of potassium ions is about 70mM to about 75mM, and the concentration of NaCl is less than about 70mM to about 65mM. In some aspects, the concentration of potassium ions is about 75mM to about 80mM. In some aspects, the concentration of potassium ions is about 75mM to about 80mM, and the concentration of NaCl is less than about 65mM to about 60mM. In some aspects, the concentration of potassium ions is about 80mM to about 85mM. In some aspects, the concentration of potassium ions is from about 80mM to about 85mM, and the concentration of NaCl is from less than about 60mM to about 55mM. In some aspects, the concentration of potassium ions is about 85mM to about 90mM. In some aspects, the concentration of potassium ions is about 85mM to about 90mM, and the concentration of NaCl is less than about 55mM to about 50mM. In some aspects, the concentration of potassium ions is about 90mM to about 95mM. In some aspects, the concentration of potassium ions is about 90mM to about 95mM, and the concentration of NaCl is less than about 50mM to about 45mM. In some aspects, the concentration of potassium ions is about 95mM to about 100mM. In some aspects, the concentration of potassium ions is about 95mM to about 100mM, and the concentration of NaCl is less than about 45mM to about 40mM. In some aspects, the concentration of potassium ions is about 100mM to about 105mM. In some aspects, the concentration of potassium ions is about 100mM to about 105mM, and the concentration of NaCl is less than about 40mM to about 35mM. In some aspects, the concentration of potassium ions is about 105mM to about 110mM. In some aspects, the concentration of potassium ions is about 105mM to about 110mM, and the concentration of NaCl is less than about 35mM to about 30mM. In some aspects, the concentration of potassium ions is about 110mM to about 115mM. In some aspects, the concentration of potassium ions is about 110mM to about 115mM, and the concentration of NaCl is less than about 30mM to about 25mM. In some aspects, the concentration of potassium ions is about 115mM to about 120mM. In some aspects, the concentration of potassium ions is about 115mM to about 120mM, and the concentration of NaCl is less than about 25mM to about 20mM. In some aspects, the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of potassium ions is about 40mM to about 90mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 40mM to about 80mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 40mM to about 70mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 50mM to about 90mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 50mM to about 80mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 50mM to about 70mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 55mM to about 90mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 55mM to about 80mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 55mM to about 70mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 60mM to about 90mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 60mM to about 80mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 60mM to about 70mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 65mM to about 90mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 65mM to about 80mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 65mM to about 70mM, wherein the medium is hypotonic or isotonic.

In some aspects, the concentration of potassium ions is greater than about 4mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 4mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 5mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 5mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 6mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 6mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 7mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 7mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 8mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 8mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 9mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 9mM, wherein the medium is hypotonic or isotonic.

In some aspects, the concentration of potassium ions is greater than about 10mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 10mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 11mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 11mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 12mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 12mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 13mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 13mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is greater than about 14mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 14mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 15mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 15mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 16mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 16mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 17mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 17mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 18mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 18mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 19mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 19mM, wherein the medium is hypotonic or isotonic.

In some aspects, the concentration of potassium ions is greater than about 20mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 20mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 21mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 21mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 22mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 22mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 23mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 23mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is greater than about 24mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 24mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 25mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 25mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 26mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 26mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 27mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 27mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 28mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 28mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 29mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 29mM, wherein the medium is hypotonic or isotonic.

In some aspects, the concentration of potassium ions is greater than about 30mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 30mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 31mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 31mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 32mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 32mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 33mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 33mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is greater than about 34mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 34mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 35mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 35mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 36mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 36mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 37mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 37mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 38mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 38mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 39mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 39mM, wherein the medium is hypotonic or isotonic.

In some aspects, the concentration of potassium ions is greater than about 40mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 40mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 41mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 41mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 42mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 42mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 43mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 43mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ions is greater than about 44mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 44mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 45mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 45mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 46mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 46mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 47mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 47mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 48mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 48mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is greater than about 49mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ions is about 49mM, wherein the medium is hypotonic or isotonic.

In some aspects, a metabolic reprogramming media comprising a high concentration of potassium ions is prepared by adding a sufficient amount of potassium salt to the media. In some aspects of the present invention, non-limiting examples of potassium salts include potassium monochloro-amine platinate, potassium pentachlororuthenate hydrate, potassium bis (oxalate) platinate (II) dihydrate, potassium bisulfate, potassium borohydride, potassium bromide, potassium carbonate, potassium chloride, potassium chromate, potassium dichromate, silver potassium cyanide, potassium aurous cyanide, potassium fluoride, potassium fluorosulfate, potassium hexachloroiridium, potassium hexachloroosmium, potassium hexachloropalladium, potassium hexachloroplatinate, potassium hexachlororhenate, potassium hexacyanochromate, potassium hexacyanoferrate, potassium hexacyanoruthenium (II) acid hydrate, potassium hexafluoroantimonate, potassium hexafluoronickelate, potassium hexafluorophosphate, potassium hexafluorotitanate, potassium hexafluorozirconate, potassium hexahydroxyantimonate, potassium hexaiodoplatinate, potassium hexaiodorhenate, potassium hydroxide, potassium iodide potassium manganate, potassium metavanadate, potassium molybdate, potassium nitrate, potassium nitrosodisulfonate, potassium osmium (VI) acid dihydrate, potassium pentachloronitrosylruthenate, potassium perchlorate, potassium perrhenate, potassium perruthenate, potassium persulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium pyrophosphate, potassium selenocyanate, potassium stannate trihydrate, potassium sulfate, potassium tellurate hydrate, potassium tellurite, potassium tetraborate tetrahydrate, potassium tetrabromo-gold acid, potassium tetrabromo-palladium, potassium tetrachloropalladium, potassium tetrachloroplatinate, potassium tetracyanopyrrolate, potassium tetracyanopylatinate, potassium tetrafluoroborate, potassium tetranitroplatinate, potassium tetrathionate, potassium p-toluene-sulfosulfonate, potassium hydroxycitrate tripotassium monohydrate, or any combination thereof. In certain aspects, the potassium salt comprises potassium chloride (KCl). 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 hydroxycitrate.

II.A.2. sodium

Some aspects of the disclosure are directed to methods of culturing immune cells in a medium comprising (i) potassium ions at a concentration of at least about 5mM (e.g., greater than 5mM, e.g., between about 40mM and about 80 mM) and (ii) sodium ions (e.g., naCl) at a concentration of less than about 115 mM. In some aspects, the culture medium is hypotonic or isotonic. In some aspects, the target concentration of sodium (e.g., naCl) is achieved by starting with a basal medium that contains a higher concentration of sodium ions (e.g., naCl), and diluting the solution to achieve the target concentration of sodium ions (e.g., naCl). In some aspects, the target concentration of sodium ions (e.g., naCl) is achieved by adding one or more sodium salts (e.g., more NaCl). Non-limiting examples of sodium salts include sodium (meta) periodate, disodium methyl arsenate hydrate, sodium azide, sodium benzyloxy, sodium bromide, sodium carbonate, sodium chloride, sodium chromate, sodium cyclohexane butyrate, sodium ethanethiol, sodium fluoride, sodium fluorophosphate, sodium formate, sodium hexachloroiridium (III) acid hydrate, sodium hexachloroiridium (IV) acid hexahydrate, sodium hexachloroplatinate (IV) acid hexahydrate, sodium rhodium (III) hexachloride, sodium hexafluoroaluminate, sodium hexafluoroantimonate (V) acid, sodium hexafluoroarsenate (V), sodium hexafluoroferric (III) acid, sodium hexafluorophosphate, sodium hexafluorosilicate, sodium hexahydroxyplatinate (IV) acid, sodium hexametaphosphate, sodium bifluoride, sodium bisulfate, sodium hexa-platinate (IV) hexahydrate, sodium hexa-platinate (IV) sodium hexafluorophosphate, sodium hexafluoroantimonate (III) sodium hexafluoroantimonate, sodium hexafluoroantimonate (IV) sodium salt, sodium hexafluorophosphate, sodium hexafluoroantimonate (IV) sodium salt, sodium salt sodium cyanamide, 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 tetrachlorogold (III) acid, sodium tetrachloropalladium (II) acid, sodium tetrachloroplatinum (II) acid, sodium thiophosphate, sodium thiosulfate pentahydrate, yttrium sodium oxybluoride, sodium 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 hydroxy citrate. In some aspects, the sodium salt comprises sodium phosphate.

In some aspects, the concentration of sodium ions (e.g., naCl) in the metabolic reprogramming media of the present disclosure is lower than the concentration of basal media. In some aspects, the concentration of sodium ions (e.g., naCl) decreases as the concentration of potassium ions increases. In some aspects, the concentration of sodium ions (e.g., naCl) is about 25mM to about 115mM. In some aspects, the concentration of sodium (e.g., naCl) ions is from about 25mM to about 100mM, from about 30mM to about 40mM, from about 30mM to about 50mM, from about 30mM to about 60mM, from about 30mM to about 70mM, from about 30mM to about 80mM, from about 40mM to about 50mM, from about 40mM to about 60mM, from about 40mM to about 70mM, from about 40mM to about 80mM, from about 50mM to about 55mM, from about 50mM to about 60mM, from about 50mM to about 65mM, from about 50mM to about 70mM, from about 50mM to about 75mM, from about 50mM to about 80mM, from about 55mM to about 60mM, from about 55mM to about 65mM, from about 55mM to about 70mM, from about 55mM to about 75mM, from about 55mM to about 80mM, from about 60mM to about 65mM, from about 60mM to about 75mM, from about 70mM to about 80mM, from about 70mM, or from about 80mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 40mM to about 80mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 50mM to about 85mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 55mM to about 80mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 30mM to about 35mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 35mM to about 40mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 40mM to about 45mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 45mM to about 50mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 50mM to about 55mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 55mM to about 60mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 60mM to about 65mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 65mM to about 70mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 70mM to about 75mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 75mM to about 80mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 80mM to about 85mM.

In some aspects, the concentration of sodium ions (e.g., naCl) is about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, or about 90mM. In certain aspects, the concentration of sodium ions (e.g., naCl) is about 40mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 45mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 50mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 55mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 55.6mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 59.3mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 60mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 63.9mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 65mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 67.6mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 70mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 72.2mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 75mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 76mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 80mM. In some aspects, the concentration of sodium ions (e.g., naCl) is about 80.5mM. In some aspects, the metabolic reprogramming media comprises about 40mM to about 90mM potassium ions and about 40mM to about 80mM sodium ions (e.g., naCl).

In some aspects, the metabolic reprogramming media comprises about 50mM to about 75mM potassium ions and about 80mM to about 90mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 55mM to about 75mM potassium ions and about 80mM to about 90mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 60mM to about 75mM potassium ions and about 80mM to about 90mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 65mM to about 75mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 66mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 67mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 68mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 69mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 71mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 72mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 73mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 74mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and about 80mM to about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and about 80mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and about 90mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 80mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 90mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and about 80mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and about 85mM sodium ions (e.g., naCl). In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and about 90mM sodium ions (e.g., naCl).

In some aspects, the metabolic reprogramming media comprises about 40mM to about 90mM potassium ion and about 30mM to about 109mM NaCl, wherein the NaCl concentration (mM) is equal to or lower than (135-potassium ion concentration, meaning 135 minus potassium ion concentration). In some aspects, the metabolic reprogramming media comprises about 40mM potassium ions and less than or equal to about 95mM NaCl (e.g., about 95mM, about 94mM, about 93mM, about 92mM, about 91mM, about 90mM, about 85mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 45mM potassium ions and less than or equal to about 90mM NaCl (e.g., about 90mM, about 89mM, about 88mM, about 87mM, about 86mM, about 85mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 50mM potassium ions and less than or equal to about 85mM NaCl (e.g., about 85mM, about 84mM, about 83mM, about 82mM, about 81mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 55mM potassium ions and less than or equal to about 80mM NaCl (e.g., about 80mM, about 79mM, about 78mM, about 77mM, about 76mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 60mM potassium ions and less than or equal to about 75mM NaCl (e.g., about 75mM, about 74mM, about 73mM, about 72mM, about 71mM, about 70mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and less than or equal to about 70mM NaCl (e.g., about 70mM, about 69mM, about 68mM, about 67mM, about 66mM, about 65mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and less than or equal to about 70mM NaCl (e.g., about 65mM, about 64mM, about 63mM, about 62mM, about 61mM, about 60mM, about 55mM, or about 50mM NaCl). In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and less than or equal to about 60mM NaCl (e.g., about 60mM, about 59mM, about 58mM, about 57mM, about 56mM, about 55mM, about 50mM, about 45mM, or about 40mM NaCl). In some aspects, the metabolic reprogramming media comprises about 80mM potassium ions and less than or equal to about 55mM NaCl (e.g., about 55mM, about 54mM, about 53mM, about 52mM, about 51mM, about 50mM, about 45mM, about 40mM, or about 35mM NaCl). In some aspects, the metabolic reprogramming media comprises about 85mM potassium ions and less than or equal to about 50mM NaCl (e.g., about 50mM, about 49mM, about 48mM, about 47mM, about 46mM, about 45mM, about 40mM, about 35mM, or about 30mM NaCl). In some aspects, the metabolic reprogramming media comprises about 90mM potassium ions and less than or equal to about 45mM NaCl (e.g., about 45mM, about 44mM, about 43mM, about 42mM, about 41mM, about 40mM, about 35mM, about 30mM, or about 25mM NaCl). In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 60mM NaCl. In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 61mM NaCl. In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and about 62mM NaCl.

In some aspects, the medium comprises about 50mM potassium ions and about 75mM NaCl. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic.

Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration greater than 5mM and (ii) NaCl at a concentration less than about 135 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration greater than 40mM and (ii) NaCl at a concentration less than about 100 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration greater than 50mM and (ii) NaCl at a concentration less than about 90 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration greater than 55mM and (ii) NaCl at a concentration less than about 70 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration greater than 60mM and (ii) NaCl at a concentration less than about 70 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration of between about 40mM to about 80mM and (ii) NaCl at a concentration of between about 40mM to about 80 mM. Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration of between about 40mM to about 80mM and (ii) NaCl at a concentration of between about 55mM to about 90 mM.

II.A.3. tension

In some aspects of the disclosure, the tonicity of the metabolic reprogramming media is adjusted based on the concentration of potassium ions and/or NaCl (e.g., (potassium ion concentration plus NaCl concentration) ×2). In some aspects, the tension of the metabolic reprogramming media is lower than the tension of the basal media. In some aspects, the tension of the metabolic reprogramming media is higher than the tension of the basal media. In some aspects, the tension of the medium is the same as the tension of the basal medium. The tonicity of the metabolic reprogramming media can be affected by modifying the concentration of potassium ions and/or NaCl in the media. In some aspects, the increased potassium ion concentration is paired with an increase or decrease in NaCl concentration. In some aspects, this pairing affects the tension of the metabolic reprogramming media. In some aspects, the potassium ion concentration increases and the NaCl concentration decreases.

In some aspects, media useful in the media of the present disclosure are prepared based on the function of potassium ions and tonicity. For example, in some aspects, if the media useful in the present disclosure is hypotonic (e.g., less than 280 mOsm) and comprises at least about 50mM potassium ions, the NaCl concentration sufficient to maintain the hypotonic media can be determined based on the following formula: naCl concentration= (desired tonicity (280)/2) -potassium ion concentration. (i.e., naCl concentration (mM) is equal to or lower than (140-potassium ion concentration)). In some aspects, the hypotonic culture medium disclosed herein comprises potassium ions and NaCl at a total concentration of between 110mM and 140 mM. Thus, for hypotonic media, the potassium ion concentration may be set at a concentration between 50mM and 90mM, and the NaCl concentration may be between 90mM and 50mM, or less, as long as the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the hypotonic culture medium disclosed herein comprises potassium ions and NaCl at a total concentration of between 115mM and 140 mM. In some aspects, the hypotonic culture medium disclosed herein comprises potassium ions and NaCl at a total concentration of between 120mM and 140 mM.

In some aspects, the metabolic reprogramming media is isotonic (between 280 and 300 mOsm) and comprises potassium ions at a concentration between about 50mM and 70 mM. The corresponding NaCl concentration can be calculated again based on the following formula: naCl concentration= (desired tonicity/2) -potassium ion concentration. For example, if the potassium concentration is 50mM and the desired tonicity is 300mOsm, the NaCl concentration may be 100mM.

In some aspects, the metabolic reprogramming media is isotonic. In some aspects, the metabolic reprogramming media has a tonicity of about 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -1 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -2 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L.+ -.3 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -4 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -5 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -6 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -7 mOsm/L. In some aspects, the MRM has a tonicity of 280 mOsm/L.+ -. 8 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L+ -9 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of 280 mOsm/L.+ -.10 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 280 to about 285, about 280 to about 290, about 280 to about 295, about 280 to about 300, about 280 to about 305, about 280 to about 310, about 280 to about 315, or about 280 to less than 320 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 285mOsm/L, about 290mOsm/L, about 295mOsm/L, about 300mOsm/L, about 305mOsm/L, about 310mOsm/L, or about 315 mOsm/L.

In some aspects, the metabolic reprogramming media is hypotonic. In some aspects, the metabolic reprogramming media has a tonicity of less than about 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 280 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 280 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 275 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 275 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 270 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 270 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 265 mOsm/L. in some aspects, the metabolic reprogramming media has a tonicity of less than 265 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 260 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 260 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 265 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 265 mOsm/L; As measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 260 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 260 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than 255 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than 255 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 250 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 250 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 245 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 245 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 240 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 240 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 235 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 235 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 230 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 230 mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of less than about 225 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of less than about 225 mOsm/L. In some aspects, the tonicity is greater than about 220mOsm/L; as measured by adding the potassium ion concentration and the NaCl concentration and multiplying by 2. In some aspects, the metabolic reprogramming media has a tonicity of about 230mOsm/L to about 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 240mOsm/L to about 280 mOsm/L.

In some aspects, the metabolic reprogramming media has an osmolality of less than about 220 mOsm/L. In some aspects, the metabolic reprogramming media has an osmolality of less than about 215 mOsm/L. In some aspects, the metabolic reprogramming media has an osmolality of less than about 210 mOsm/L. In some aspects, the metabolic reprogramming media has an osmolality of less than about 205 mOsm/L. In some aspects, the metabolic reprogramming media has an osmolality of less than about 200 mOsm/L.

In some aspects, the metabolic reprogramming media has about 100mOsm/L to about 280mOsm/L, about 125mOsm/L to about 280mOsm/L, about 150mOsm/L to about 280mOsm/L, about 175mOsm/L to about 280mOsm/L, about 200mOsm/L to about 280mOsm/L, about 210mOsm/L to about 280mOsm/L, about 220mOsm/L to about 280mOsm/L, about 225mOsm/L to about 280mOsm/L, about 230mOsm/L to about 280mOsm/L, A tonicity of about 235mOsm/L to about 280mOsm/L, about 240mOsm/L to about 280mOsm/L, about 245mOsm/L to about 280mOsm/L, about 250mOsm/L to about 280mOsm/L, about 255mOsm/L to about 280mOsm/L, about 260mOsm/L to about 280mOsm/L, about 265mOsm/L to about 280mOsm/L, about 270mOsm/L to about 280mOsm/L, or about 275mOsm/L to about 280 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 250mOsm/L to about 270 mOsm/L. In some aspects of the present invention, the metabolic reprogramming media has a composition of about 250mOsm/L to about 255mOsm/L, about 250mOsm/L to about 260mOsm/L, about 250mOsm/L to about 265mOsm/L, about 255mOsm/L to about 260mOsm/L a tonicity of about 255mOsm/L to about 265mOsm/L, about 260mOsm/L to about 265mOsm/L, or about 254mOsm/L to about 263 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 254mOsm/L to about 255 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 255mOsm/L to about 256 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 256 to about 257 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 257mOsm/L to about 258 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 258 to about 259 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 260 to about 261 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 261 to about 262 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 262 to about 263 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 263 to about 264 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 264mOsm/L to about 265 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 220mOsm/L to about 280 mOsm/L.

In some aspects, the metabolic reprogramming media has a tonicity of about 100mOsm/L, about 125mOsm/L, about 150mOsm/L, about 175mOsm/L, about 200mOsm/L, about 210mOsm/L, about 220mOsm/L, about 225mOsm/L, about 230mOsm/L, about 235mOsm/L, about 240mOsm/L, about 245mOsm/L, about 250mOsm/L, about 255mOsm/L, about 260mOsm/L, about 265mOsm/L, about 270mOsm/L, or about 275 mOsm/L.

In some aspects, the metabolic reprogramming media has a tonicity of about 250 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 262.26 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 260 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 259.7 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 257.5 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 257.2 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 255.2 mOsm/L. In some aspects, the metabolic reprogramming media has a tension of about 254.7. In some aspects, the metabolic reprogramming media has a tonicity of about 255 mOsm/L. In some aspects, the metabolic reprogramming media has a tonicity of about 260 mOsm/L. In some aspects, the MRM comprises (i) potassium ions at a concentration greater than 5mM, (ii) NaCl at a concentration between about 40mM to about 80mM, and (iii) a tonicity of about 250-260 mOsm/L. In some aspects, the MRM comprises (i) potassium ions at a concentration of between about 40mM to about 80mM, (ii) NaCl at a concentration of between about 40mM to about 80mM, and (iii) a tonicity of about 250-260 mOsm/L. In some aspects, the MRM comprises (i) potassium ions at a concentration of between about 40mM to about 80mM, (ii) NaCl at a concentration of between about 55mM to about 90mM, and (iii) a tonicity of about 250-260 mOsm/L.

In some aspects, the metabolic reprogramming media comprises about 50mM potassium ions and (i) about 80.5mM NaCl; (ii) about 24mM glucose; and (iii) about 2.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 40mM potassium ions and (i) about 88.9mM NaCl; (ii) about 24mM glucose; and (iii) about 2.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 60mM potassium ions and (i) about 72.2mM NaCl; (ii) about 24mM glucose; and (iii) about 2.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and (i) about 63.9mM NaCl; (ii) about 24mM glucose; and (iii) about 2.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 80mM potassium ions and (i) about 55.6mM NaCl; (ii) about 24mM glucose; and (iii) about 2.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 50mM potassium ions and (i) about 80.5mM NaCl; (ii) about 17.7mM glucose; and (iii) about 1.9mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 50mM potassium ions and (i) about 80.5mM NaCl; (ii) about 17.7mM glucose; and (iii) about 1.8mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 55mM potassium ions and (i) about 76mM NaCl; (ii) about 17.2mM glucose; and (iii) about 1.7mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 60mM potassium ions and (i) about 72.2mM NaCl; (ii) about 16.8mM glucose; and (iii) about 1.6mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 65mM potassium ions and (i) about 67.6mM NaCl; (ii) about 16.3mM glucose; and (iii) about 1.5mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 70mM potassium ions and (i) about 63.9mM NaCl; (ii) about 15.9mM glucose; and (iii) about 1.4mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 75mM potassium ions and (i) about 59.3mM NaCl; (ii) about 15.4mM glucose; and (iii) about 1.3mM calcium ion.

In some aspects, the metabolic reprogramming media comprises about 80mM potassium ions and (i) about 55.6mM NaCl; (ii) about 15mM glucose; and (iii) about 1.2mM calcium ion.

The tonicity of the metabolic reprogramming media can be adjusted at any time to, for example, the isotonic or hypotonic state disclosed herein. In some aspects, the tonicity of the metabolic reprogramming media may be adjusted to an isotonic or hypotonic state such as disclosed herein prior to adding the cells to the metabolic reprogramming media. In some aspects, the cells are cultured in hypotonic or isotonic medium prior to cell engineering, e.g., prior to transduction with a construct that expresses the CAR, TCR, or TCR mimetic. In some aspects, the cells are cultured in hypotonic or isotonic medium during cell engineering, e.g., during transduction with a construct that expresses the CAR, TCR, or TCR mimetic. In some aspects, the cells are cultured in hypotonic or isotonic media after cell engineering, e.g., after transduction with a construct that expresses the CAR, TCR, or TCR mimetic. In some aspects, the cells are cultured in a hypotonic or isotonic medium during whole cell expansion.

II.A.4. sugar

Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration of at least about 5mM (e.g., greater than 5mM, e.g., between about 40mM and about 80 mM) and (ii) a sugar. In some aspects, the culture medium is hypotonic or isotonic.

In some aspects, the target sugar concentration is achieved by starting with a basal medium comprising a higher concentration of sugar, and diluting the solution to achieve the target concentration of sugar. In some aspects, the target concentration of sugar is achieved by adding sugar to raise the concentration of sugar until the desired concentration is achieved. In some aspects, the sugar is a monosaccharide, disaccharide, or polysaccharide. In some aspects, the sugar is selected from glucose, fructose, galactose, mannose, maltose, sucrose, lactose, trehalose, or any combination thereof. In certain aspects, the sugar is glucose. In some aspects, the medium comprises (i) potassium ions at a concentration of at least about 5mM and (ii) glucose. In some aspects, the medium comprises (i) potassium ions at a concentration greater than 40mM and (ii) glucose. In some aspects, the medium comprises (i) potassium ions at a concentration of at least about 5mM and (ii) mannose. In some aspects, the medium comprises (i) potassium ions at a concentration of at least about 50mM 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 ions at a concentration greater than 40mM and (ii) glucose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the medium comprises (i) potassium ions at a concentration greater than 50mM and (ii) glucose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the medium comprises (i) potassium ions at a concentration of at least about 40mM and (ii) mannose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the medium comprises (i) potassium ions at a concentration of at least about 50mM and (ii) mannose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) glucose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration of at least about 30mM to at least about 100mM and (ii) glucose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 40mM and (ii) glucose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) mannose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration of at least about 30mM to at least about 100mM and (ii) mannose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 40mM and (ii) mannose. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration of at least about 50mM and (ii) mannose. In some aspects, the metabolic reprogramming media is hypotonic. In some aspects, the medium is isotonic. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 40mM and (ii) glucose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 50mM and (ii) glucose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration of at least about 40mM and (ii) mannose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration of at least about 50mM and (ii) mannose; wherein the total concentration of potassium ions and NaCl is between 110mM and 140 mM.

In some aspects, the concentration of sugar (e.g., glucose) is about 10mM to about 24mM. In some aspects, the concentration of sugar (e.g., glucose) is less than about 4.29g/L. In some aspects, the concentration of sugar (e.g., glucose) is less than about 24mM. In some aspects, the concentration of sugar (e.g., glucose) is greater than about 5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 5mM to about 20mM. In some aspects, the concentration of sugar (e.g., glucose) is about 10mM to about 20mM. In some aspects, the concentration of the sugar (e.g., glucose) is about 10mM to about 25mM, about 10mM to about 20mM, about 10mM to about 5mM, about 15mM to about 25mM, about 15mM to about 20mM, about 15mM to about 19mM, about 15mM to about 18mM, about 15mM to about 17mM, about 15mM to about 16mM, about 16mM to about 20mM, about 16mM to about 19mM, about 16mM to about 18mM, about 16mM to about 17mM, about 17mM to about 20mM, about 17mM to about 19mM, or about 17mM to about 18mM. In some aspects, the concentration of sugar (e.g., glucose) is about 5mM to about 20mM. In some aspects, the concentration of sugar (e.g., glucose) is about 10mM to about 20mM. In some aspects, the concentration of sugar (e.g., glucose) is about 10mM to about 15mM. In some aspects, the concentration of sugar (e.g., glucose) is about 14mM to about 14.5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 14.5mM to about 15mM. In some aspects, the concentration of sugar (e.g., glucose) is about 15mM to about 15.5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 15.5mM to about 16mM. In some aspects, the concentration of sugar (e.g., glucose) is about 16mM to about 16.5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 16.5mM to about 17mM. In some aspects, the concentration of sugar (e.g., glucose) is about 17mM to about 17.5mM. In some aspects, the concentration of sugar (e.g., glucose) is about 17.5mM to about 18mM.

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

In some aspects, media useful in the present disclosure comprise (i) potassium ions at a concentration greater than 5mM (e.g., between about 40mM and about 80 mM), (ii) NaCl at a concentration between about 40mM and about 80mM, and (iii) glucose. In some aspects, media useful in the present disclosure comprise (i) potassium ions at a concentration above 5mM (e.g., between about 40mM and about 80 mM), (ii) NaCl at a concentration between about 40mM and about 80mM, (iii) glucose, and (iv) a tonicity of about 250-260 mOsm/L. In some aspects, media useful in the present disclosure comprise (i) potassium ions at a concentration above 5mM (e.g., between about 40mM and about 80 mM), (ii) NaCl at a concentration between about 40mM and about 80mM, (iii) glucose at a concentration between about 10mM and about 24mM, and (iv) a tonicity of about 250-260 mOsm/L.

II.A.5. calcium

Some aspects of the disclosure are directed to methods of culturing immune cells (e.g., T cells and/or NK cells) in a medium comprising (i) potassium ions at a concentration of at least about 5mM (e.g., greater than 5mM, e.g., between about 40mM and about 80 mM) and (ii) calcium ions. In some aspects, the culture medium is hypotonic or isotonic.

In some aspects, the target calcium concentration is achieved by starting with a basal medium comprising a higher concentration of calcium ions, and diluting the solution to achieve the target concentration of calcium ions. In some aspects, the target calcium concentration is achieved by increasing the concentration of calcium ions by adding one or more calcium salts. Non-limiting 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 dihydrogen phosphate, tricalcium phosphate, calcium sulfate, calcium thiocyanate tetrahydrate, hydroxyapatite, or any combination thereof. In some aspects, the calcium salt comprises calcium chloride (CaCl ₂). In some aspects, the calcium salt comprises calcium gluconate.

In some aspects, the concentration of calcium ions is lower than the concentration of basal medium. In some aspects, the concentration of calcium ions is higher than the concentration of basal medium. In some aspects, the concentration of calcium ions exceeds about 0.4mM. In some aspects, the concentration of calcium ions is less than about 2.8mM. In some aspects, the concentration of calcium ions is less than about 2.5mM. In some aspects, the concentration of calcium ions is less than about 2.0mM. In some aspects, the concentration of calcium ions is less than about 1.9mM. In some aspects, the concentration of calcium ions is less than about 1.8mM. In some aspects, the concentration of calcium ions is less than about 1.7mM. In some aspects, the concentration of calcium ions is less than about 1.6mM. In some aspects, the concentration of calcium ions is less than about 1.5mM. In some aspects, the concentration of calcium ions is less than about 1.4mM. In some aspects, the concentration of calcium ions is less than about 1.3mM. In some aspects, the concentration of calcium ions is less than about 1.2mM. In some aspects, the concentration of calcium ions is less than about 1.1mM. In some aspects, the concentration of calcium ions is less than about 1.0mM.

In some aspects of the present invention, the concentration of the calcium ion is about 0.4mM to about 2.8mM, about 0.4mM to about 2.7mM, about 0.4mM to about 2.5mM, about 0.5mM to about 2.0mM, about 1.0mM to about 2.0mM, about 1.1mM to about 2.0mM, about 1.3mM to about 2.0mM, about 1.4mM to about 2.0mM, about 1.5mM to about 2.0mM, about 1.6mM to about 2.0mM, about 1.7mM to about 2.0mM, about 1.8mM to about 2.0mM, about 0.8 mM to about 1.0mM, about 0.8 to about 1.1mM, about 0.8 to about 1.2mM, about 0.8 mM to about 1.3mM, about 0.8 to about 1.4mM, about 1.8mM, about 1.5mM, about 1.4mM to about 2.0mM, about 1.7mM to about 2.0mM, about 1.8mM, about 1.9mM, about 1.8mM to about 1.9mM, about 1.8mM, about 1.9mM, about 1.0mM, about 1.8mM to about 1.0mM, about 1.9mM, about 1.0mM, about 1.8 to about 1.9 mM; about 1.0 to about 1.5mM, about 1.0 to about 1.6mM, about 1.0 to about 1.7mM, about 1.0 to about 1.8mM, about 1.0 to about 1.9mM, about 1.1 to about 1.2mM, about 1.1 to about 1.3mM, about 1.1 to about 1.4mM, about 1.1 to about 1.5mM, about 1.1 to about 1.6mM, about 1.1 to about 1.7mM, about 1.1 to about 1.8mM, about 1.1 to about 1.9mM, about 1.2 to about 1.3mM, about 1.2 to about 1.4mM, about 1.2 to about 1.5mM, about 1.2 to about 1.6mM, about 1.2 to about 1.3mM, about 1.3 to about 1.4mM, about 1.3 to about 1.3mM, about 1.3 to about 1.5mM, about 1.1.1 to about 1.5mM, about 1.3mM, about 1.1 to about 1.7mM, about 1.3mM, about 1.2 to about 1.5mM, about 1.3mM, about 1.2 to about 1.5mM, about 1.3mM, about 1.1 to about 1.7mM, about 1.6mM, about 1.3mM, about 1.1 to about 1.3 mM.

In some aspects, the concentration of calcium ions is about 0.8mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 0.9mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 1.0mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 1.1mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 1.2mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 0.8mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 0.8mM to about 0.9mM. In some aspects, the concentration of calcium ions is about 0.9mM to about 1.0mM. In some aspects, the concentration of calcium ions is about 1.0mM to about 1.1mM. In some aspects, the concentration of calcium ions is about 1.1mM to about 1.2mM. In some aspects, the concentration of calcium ions is about 1.2mM to about 1.3mM. In some aspects, the concentration of calcium ions is about 1.3mM to about 1.4mM. In some aspects, the concentration of calcium ions is about 1.4mM to about 1.5mM. In some aspects, the concentration of calcium ions is about 1.5mM to about 1.6mM. In some aspects, the concentration of calcium ions is about 1.7mM to about 1.8mM. In some aspects, the concentration of calcium ions is about 1.8mM to about 1.9mM.

In some aspects, the concentration of calcium ions is about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, or about 2.0mM. In some aspects, the concentration of calcium ions is about 0.6mM. In some aspects, the concentration of calcium ions is about 0.7mM. In some aspects, the concentration of calcium ions is about 0.8mM. In some aspects, the concentration of calcium ions is about 0.9mM. In some aspects, the concentration of calcium ions is about 1.0mM. In some aspects, the concentration of calcium ions is about 1.1mM. In some aspects, the concentration of calcium ions is about 1.2mM. In some aspects, the concentration of calcium ions is about 1.3mM. In some aspects, the concentration of calcium ions is about 1.4mM. In some aspects, the concentration of calcium ions is about 1.5mM. In some aspects, the concentration of calcium ions is about 1.6mM. In some aspects, the concentration of calcium ions is about 1.7mM. In some aspects, the concentration of calcium ions is about 1.8mM. In some aspects, the concentration of calcium ions is about 1.9mM.

In some aspects, the media useful in the present disclosure comprise: (i) Potassium ions at a concentration of between about 40mM and about 80mM and (ii) calcium at a concentration of between about 0.5mM and about 2.8 mM. In some aspects, the medium comprises: (i) potassium ions at a concentration of between about 40mM and about 80mM, (ii) NaCl at a concentration of between about 40mM and about 80mM, and (iii) calcium at a concentration of between about 0.5mM and about 2.8 mM. In some aspects, the medium comprises: (i) potassium ions at a concentration of between about 40mM and about 80mM, (ii) NaCl at a concentration of between about 40mM and about 80mM, (iii) glucose at a concentration of between about 10mM and about 24mM, and (iv) calcium at a concentration of between about 0.5mM and about 2.8 mM. In some aspects, the medium comprises: (i) potassium ions at a concentration of between about 40mM and about 80mM, (ii) NaCl at a concentration of between about 40mM and about 80mM, (iii) glucose at a concentration of between about 10mM and about 24mM, (iv) calcium at a concentration of between about 0.5mM and about 2.8mM, and (v) a tonicity of about 250-260 mOsm/L.

II.A.6. cytokines

In some aspects, the metabolic reprogramming media comprises cytokines. 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 the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof. In some aspects, the metabolic reprogramming media does not comprise IL-2. In some aspects, the metabolic reprogramming media comprises IL-2 and IL-21. In some aspects, the metabolic reprogramming media comprises IL-2, IL-21, and IL-15.

Cytokines can be added to the culture medium at any time. In some aspects, the cytokine is added to the medium prior to the addition of immune cells (e.g., T cells and/or NK cells) to the medium. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured in a medium comprising (i) potassium at a concentration disclosed herein (e.g., above 5mM, e.g., between about 40mM and about 80 mM) and (ii) a cytokine, prior to cell engineering, e.g., prior to transduction with a construct encoding a ligand binding protein. In some aspects, immune cells (e.g., T cells and/or NK cells) are cultured during cell engineering, e.g., during transduction with a ligand binding protein, in a medium comprising (i) potassium at a concentration disclosed herein (e.g., above 5mM, e.g., between about 40mM and about 80 mM) and (ii) a cytokine. In some aspects, after cell engineering, e.g., after transduction with a construct encoding a polypeptide ligand binding protein, immune cells (e.g., T cells and/or NK cells) are cultured in a medium comprising (i) potassium at a concentration disclosed herein (e.g., above 5mM, e.g., between about 40mM and about 80 mM) and (ii) a cytokine. In some aspects, in whole cell expansion, immune cells (e.g., T cells and/or NK cells) are cultured in a medium comprising (i) potassium at a concentration disclosed herein (e.g., above 5mM, e.g., between about 40mM and about 80 mM) and (ii) a cytokine.

In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-2. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-2. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-2. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-7. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-7. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-7. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-15. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-21. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-15. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7 and IL-15. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7 and IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-2, and the metabolic reprogramming media does not comprise IL-7 and IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-2 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-2 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-2 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-7 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-7 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-7 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 5mM potassium ion and (ii) IL-15 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) greater than 40mM potassium ion and (ii) IL-15 and IL-21. In some aspects, the metabolic reprogramming media comprises (i) at least about 50mM potassium ion and (ii) IL-15 and IL-21. In some aspects, the metabolic reprogramming media is hypotonic. In some aspects, the metabolic reprogramming media is isotonic. In some aspects, the metabolic reprogramming media further comprises NaCl, wherein the total concentration of potassium ions and NaCl is 110mM to 140mM.

In some aspects, the metabolic reprogramming media described herein (e.g., comprising potassium ions at a concentration greater than 5 mM) comprises between about 50IU/mL to about 500IU/mL of IL-2. In some aspects, the metabolic reprogramming media comprises about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL IL-2.

Thus, in some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 50IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 60IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 70IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 80IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 90IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 100IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 125IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 150IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 175IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 200IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 225IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 250IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 275IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 300IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 350IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 400IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 450IU/mL IL-2. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 500IU/mL IL-2. In some aspects, the metabolic reprogramming media comprising potassium ions and IL-2 further comprises NaCl at a concentration of less than about 115 nM.

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL IL-2. In some aspects, the metabolic reprogramming media comprises about 0.1 to about 20ng/mL, about 1 to about 15ng/mL, about 1 to about 14ng/mL, about 1 to about 13ng/mL, about 1 to about 12ng/mL, about 1 to about 11ng/mL, about 1 to about 10ng/mL, about 1 to about 9ng/mL, about 1 to about 8ng/mL, about 1 to about 7ng/mL, about 1 to about 6ng/mL, about 1 to about 5ng/mL, about 1 to about 4ng/mL, about 1 to about 3ng/mL, about 1 to about 2ng/mL, about 5 to about 15ng/mL, about 5 to about 10ng/mL, about 10 to about 20ng to about 15ng/mL, or about 15 to about 15 ng/mL.

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

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL IL-2. In some aspects, the metabolic reprogramming media comprises about 50 to about 600ng/mL, about 50 to about 500ng/mL, about 50 to about 450ng/mL, about 50 to about 400ng/mL, about 50 to about 350ng/mL, about 50 to about 300ng/mL, about 100 to about 600ng/mL, about 100 to about 500ng/mL, about 100 to about 450ng/mL, about 100 to about 400ng/mL, about 100 to about 350ng/mL, about 100 to about 300ng/mL, about 200 to about 500ng/mL, about 200 to about 450ng/mL, about 200 to about 400ng/mL, about 200 to about 350ng/mL, about 200 to about 300ng/mL, about 250 to about 350ng/mL, about 300 to about 600ng/mL, about 300 to about 300ng/mL, about 300 to about 300ng/mL, about 500ng to about 500 ng/mL.

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

In some aspects, the metabolic reprogramming media described herein (e.g., comprising potassium ions at a concentration greater than 5 mM) comprises between about 50IU/mL to about 500IU/mL of IL-21. In some aspects, the medium comprises about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL IL-21.

In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 50IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 60IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 70IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 80IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 90IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 100IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 125IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 150IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 175IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 200IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 225IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 250IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 275IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 300IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 350IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 400IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 450IU/mL IL-21. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 500IU/mL IL-21. In some aspects, the metabolic reprogramming media comprising potassium ions and IL-21 further comprises NaCl at a concentration of less than about 115 nM.

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises about 0.1 to about 20ng/mL, about 1 to about 15ng/mL, about 1 to about 14ng/mL, about 1 to about 13ng/mL, about 1 to about 12ng/mL, about 1 to about 11ng/mL, about 1 to about 10ng/mL, about 1 to about 9ng/mL, about 1 to about 8ng/mL, about 1 to about 7ng/mL, about 1 to about 6ng/mL, about 1 to about 5ng/mL, about 1 to about 4ng/mL, about 1 to about 3ng/mL, about 1 to about 2ng/mL, about 5 to about 15ng/mL, about 5 to about 10ng/mL, about 10 to about 20ng/mL, about 21 to about 15ng/mL, or about 15 to about 15 ng/mL.

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL, at least about 0.5ng/mL, at least about 1ng/mL, at least about 2ng/mL, at least about 3ng/mL, at least about 4ng/mL, at least about 5ng/mL, at least about 6ng/mL, at least about 7ng/mL, at least about 8ng/mL, at least about 9ng/mL, at least about 10ng/mL, at least about 11ng/mL, at least about 12ng/mL, at least about 13ng/mL, at least about 14ng/mL, at least about 15ng/mL, at least about 16ng/mL, at least about 17ng/mL, at least about 18ng/mL, at least about 19ng/mL, or at least about 20ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 1.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 2.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 3.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 4.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 5.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 6.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 7.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 8.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 9.0ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 10ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 10ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 15ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 20ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 25ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 30ng/mL IL-21. In some aspects, the metabolic reprogramming media comprises at least about 35ng/mL IL-21.

In some aspects, the metabolic reprogramming media described herein (e.g., comprising potassium ions at a concentration greater than 5 mM) comprises between about 500IU/mL to about 1,500IU/mL of IL-7. In some aspects, the medium comprises about 500IU/mL, about 550IU/mL, about 600IU/mL, about 650IU/mL, about 700IU/mL, about 750IU/mL, about 800IU/mL, about 850IU/mL, about 900IU/mL, about 950IU/mL, about 1,000IU/mL, about 1,050IU/mL, about 1,100IU/mL, about 1,150IU/mL, about 1,200IU/mL, about 1,250IU/mL, about 1,300IU/mL, about 1,350IU/mL, about 1,400IU/mL, about 1,450IU/mL, or about 1,500IU/mL IL-7.

In some aspects, metabolic reprogramming media useful in the present disclosure comprise (i) potassium ions at a concentration greater than 5mM and (ii) about 500IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 550IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 600IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 650IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 700IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 750IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 800IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 850IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 900IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 950IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,000IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,050IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,100IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,150IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,200IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,250IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,300IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,350IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,400IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,450IU/mL IL-7. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 1,500IU/mL IL-7. In some aspects, the metabolic reprogramming media comprising potassium ions and IL-7 further comprises NaCl at a concentration of less than about 115 nM.

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL IL-7. In some aspects, the metabolic reprogramming media comprises about 0.1 to about 20ng/mL, about 1 to about 15ng/mL, about 1 to about 14ng/mL, about 1 to about 13ng/mL, about 1 to about 12ng/mL, about 1 to about 11ng/mL, about 1 to about 10ng/mL, about 1 to about 9ng/mL, about 1 to about 8ng/mL, about 1 to about 7ng/mL, about 1 to about 6ng/mL, about 1 to about 5ng/mL, about 1 to about 4ng/mL, about 1 to about 3ng/mL, about 1 to about 2ng/mL, about 5 to about 15ng/mL, about 5 to about 10ng/mL, about 10 to about 20ng/mL, about 10 to about 15ng/mL, or about 15 to about 15 ng/mL.

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

In some aspects, the metabolic reprogramming media described herein (e.g., comprising potassium ions at a concentration greater than 5 mM) comprises between about 50IU/mL to about 500IU/mL of IL-15. In some aspects, the medium comprises about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL IL-15.

Thus, in some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 50IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 60IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 70IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 80IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 90IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 100IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 125IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 150IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 175IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 200IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 225IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 250IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 275IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 300IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 350IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 400IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 450IU/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) potassium ions at a concentration greater than 5mM and (ii) about 500IU/mL IL-15. In some aspects, the metabolic reprogramming media comprising potassium ions and IL-15 further comprises NaCl at a concentration of less than about 115 nM.

In some aspects, the metabolic reprogramming media comprises at least about 0.1ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises about 0.1 to about 20ng/mL, about 1 to about 15ng/mL, about 1 to about 14ng/mL, about 1 to about 13ng/mL, about 1 to about 12ng/mL, about 1 to about 11ng/mL, about 1 to about 10ng/mL, about 1 to about 9ng/mL, about 1 to about 8ng/mL, about 1 to about 7ng/mL, about 1 to about 6ng/mL, about 1 to about 5ng/mL, about 1 to about 4ng/mL, about 1 to about 3ng/mL, about 1 to about 2ng/mL, about 5 to about 15ng/mL, about 5 to about 10ng/mL, about 10 to about 20ng to about 15ng/mL, or about 15 to about 15 ng/mL.

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

In some aspects, the metabolic reprogramming media comprises at least about 30mM to at least about 100mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises greater than 40mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 45mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 50mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 55mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 60mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 65mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 70mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 75mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 80mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 85mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises at least about 90mM potassium ion, about 300ng/mL IL-2, and about 0.4ng/mL IL-15. In some aspects, the metabolic reprogramming media comprises (i) at least about 70mM potassium ion, (ii) about 60mM NaCl, (iii) about 1.4mM calcium, (iv) about 16mM glucose, (v) about 300ng/mL IL-2, and (vi) about 0.4ng/mL IL-15.

II.A.7. Basal medium

In some aspects, the basal medium comprises a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), darbeck Modified Eagle Medium (DMEM), minimal Essential Medium (MEM), eagle Basal Medium (BME), F-10, F-12, RPMI1640, glasgang Minimal Essential Medium (GMEM), alpha minimal essential medium (alpha MEM), iscove Modified Duchenne Medium (IMDM), M199, OPTMIZER ^TMCTS^TM T cell expansion basal medium (ThermoFisher)、OPTMIZER^TMComplete、IMMUNOCULT^TMXF(STEMCELL^TMTechnologies)、IMMUNOCULT^TM、AIM V、TEXMACS^TM medium,T cell CDM, X-VIVO ^TM 15(Lonza)、TRANSACT^TM TIL expansion medium, or any combination thereof. In some aspects, the basal medium comprises PRIME-XV T cell CDM. In some aspects, the basal medium comprises OPTMIZER ^TM. In some aspects, the basal medium comprises OPTMIZER ^TM Pro. In some aspects, the basal medium is serum-free. In some aspects, the basal medium further comprises an Immune Cell Serum Replacement (ICSR). For example, in some aspects, the basal medium comprises OPTMIZER ^TM Complete supplemented with ICSR, AIM V supplemented with ICSR, IMMUNOCULT ^TM XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS ^TM supplemented with ICSR, or any combination thereof. In a particular aspect, the basal medium comprises OPTMIZER ^TM complete.

In some aspects, the medium (e.g., MRM) further comprises about 2.5% serum supplement (CTS ^TM Immune Cell SR, thermo Fisher), 2mM L-glutamine, 2mM L-glutamax, MEM nonessential amino acid solution, pen-strep, 20 μg/ml fungin ^TM, sodium pyruvate, or any combination thereof. In some aspects, the culture medium further comprises O-acetyl-L-carnitine hydrochloride. In some aspects, the medium further comprises a kinase inhibitor.

In some aspects, the medium further comprises a CD3 agonist. In some aspects, the CD3 agonist is an anti-CD 3 antibody. In some aspects, the anti-CD 3 antibody comprises OKT-3.

In some aspects, the medium further comprises a CD28 agonist. In some aspects, the CD28 agonist is an anti-CD 28 antibody. In some aspects, the medium further comprises a CD27 ligand (CD 27L). In some aspects, the medium further comprises a 4-1BB ligand (4-1 BBL).

In some aspects, the disclosure includes a cell culture comprising a medium disclosed herein, a cell bag comprising a medium disclosed herein, or a bioreactor comprising a medium disclosed herein.

Sources and activation of b. cells

Immune cells of the present disclosure (which may be modified and cultured using the methods described herein, including primary T cells) may be obtained from a variety of tissue sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and/or tumor tissue. White blood cells, including PBMCs, may be separated from other blood cells by well known techniques, such as FICOLL ^TM separation and white blood cell separation. The leukapheresis product typically contains lymphocytes (including T and B cells), monocytes, pellets and other nucleated leukocytes. T cells may be further isolated from other white blood cells, for example by gradient centrifugation through PERCOLL ^TM or elutriation by countercurrent centrifugation. Specific subsets of T cells, such as CD3 ⁺、CD25⁺、CD28⁺、CD4⁺、CD8⁺、CD45RA⁺、GITR⁺ and/or CD45RO ⁺ T cells, may be further isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, the antibody may be bound by any of a variety of commercially available antibodies, such asCELLECTION ^TM、DETACHaBEAD^TM (Thermo Fisher) orThe cell isolation product (Miltenyi Biotec) is incubated for a period of time sufficient to positively select for the desired T cells or to negatively select for removal of unwanted cells to isolate T cells.

In some cases, autologous T cells are obtained directly from the cancer patient after the cancer treatment. It is observed that after certain cancer treatments, particularly those that damage the immune system, the quality of T cells collected shortly after treatment may have improved ability to expand ex vivo and/or to be implanted after ex vivo engineering.

Whether before or after genetic modification (e.g., using any of the modification methods described herein), T cells can generally be activated and expanded using methods as described, for example, in U.S. patent 5,858,358;5,883,223;6,352,694;6,534,055;6,797,514;6,867,041;6,692,964;6,887,466;6,905,680;6,905,681;6,905,874;7,067,318;7,144,575;7,172,869;7,175,843;7,232,566;7,572,631; and 10,786,533, each of which is expressly incorporated by reference in its entirety. In general, T cells can be expanded in vitro or ex vivo by surface contact with an agent attached to stimulate a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cell. In some aspects, the T cell population can be stimulated, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof or an anti-CD 3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryoid) and a calcium ionophore. For co-stimulation of the accessory molecules on the surface of the T cells, ligands that bind to the accessory molecules may be used. For example, a population of T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate proliferation of CD4 ⁺ T cells or CD8 ⁺ T cells, anti-CD 3 antibodies and anti-CD 28 antibodies may be used. In some aspects, T cells are activated and expanded using, for example, DYNABEADS ^TM or commercial nanoparticles, such as TRANSACT ^TM (Miltenyi Biotech) or other known activators.

In some aspects, the methods described herein comprise contacting a human immune cell (e.g., a T cell and/or NK cell, modified to express an increased level of c-Jun protein) with a programmable cell signaling scaffold (PCS) in a medium (e.g., metabolic reprogramming medium) comprising potassium ions at a concentration of greater than 5mM, as described herein. Non-limiting examples of programmable cell signaling scaffolds (PCS) are described in WO 2018/013977 and Chung et al (Nature Biotechnology (2)) 160-169 (2018), the contents of which are incorporated herein by reference in their entirety, in aspects, the programmable cell signaling scaffolds of the present disclosure comprise a first layer comprising a high surface area mesoporous silica micro-rod (MSR), a second layer comprising a lipid coating the first layer, and a plurality of functional molecules supported on the scaffold, in aspects, including but not limited to a stimulatory molecule (T cell activating molecule) that activates T cells, in aspects, the stimulatory molecule activates T cells by engagement and/or clustering components of a T cell receptor complex, in aspects, the stimulatory molecule comprises an anti-CD 3 antibody or antigen binding portion thereof, in aspects, the functional molecules comprise one or more co-stimulatory molecules that specifically bind to one or more co-stimulatory antigens, representative examples of the co-stimulatory molecules include but are not limited to CD28, OX4, CD 40, CD 37 (CD 40), CD 37, or CD 37, such as is generally contemplated by the present in the scaffolds (e.g., CD 37, or CD 37, and/or CD-effector cell-specific to such as is contemplated by the scaffolds (e.g., the present in the FRCD-35) and/or the target-specific-inducing-effector-cell-binding molecules in aspects, t cells) and the various presented by the scaffold direct or indirect interactions between functional molecules are used to mediate these effects. In some aspects, the scaffold modulates the survival of target cells (e.g., T cells), the growth of target cells (e.g., T cells), and/or the function of target cells (e.g., T cells) via the physical or chemical characteristics of the scaffold itself.

II.C. cells

The disclosure also provides a modified cell that expresses the ROR1 binding protein and has an increased level of the c-Jun polypeptide as compared to a reference cell (e.g., a corresponding cell that is not modified to have an increased level of the c-Jun polypeptide). In some aspects, the cell does not naturally express the c-Jun protein, but has been modified to express the c-Jun protein. In some aspects, the cell is naturally capable of expressing the c-Jun protein, but has been modified to increase expression of the endogenous c-Jun protein. Unless otherwise indicated, "c-Jun overexpression" (or derivatives thereof) comprises two such modified cells. Any suitable method known in the art may be used to modify the cells described herein, as described herein.

In some aspects, cells useful in the present disclosure have been modified to include exogenous nucleotide sequences encoding a protein of interest such that the encoded protein is expressed in the cell. As described herein, in some aspects, after modification, expression of the encoded protein is increased compared to a reference cell (e.g., a corresponding cell that is not modified to comprise the exogenous nucleotide sequence). In some aspects, the cells described herein have been modified to include a plurality of exogenous nucleotide sequences encoding different related proteins (e.g., ROR1 binding proteins, c-Jun polypeptides, and/or EGFRt). Where multiple exogenous nucleotide sequences are involved, in some aspects, the multiple exogenous nucleotide sequences may be part of a single polycistronic polynucleotide.

In some aspects, the cells described herein have been modified by a transcriptional activator capable of inducing and/or increasing endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As described herein, in some aspects, after modification, endogenous expression of the protein is increased as compared to a reference cell (e.g., a corresponding cell that has not been modified by a transcriptional activator). As used herein, the term "transcriptional activator" refers to a protein that increases transcription of a gene or collection of genes (e.g., by binding to an enhancer or promoter proximal element of a nucleic acid sequence and thereby inducing its transcription). Non-limiting examples of such transcriptional activators that may be used with the present disclosure include: transcriptional activators based on transcription activator-like effectors (TALEs), transcriptional activators based on Zinc Finger Proteins (ZFPs), transcriptional activators based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) systems, or combinations thereof. See, e.g., kabadi et al, methods 69 (2): 188-197 (month 9 of 2014), which is incorporated by reference herein in its entirety.

In some aspects, the cells described herein have 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 (month 5 of 2014), which is incorporated by reference herein in its entirety. CRISPRa is a type of CRISPR tool comprising the use of a modified Cas protein lacking endonuclease activity but retaining the ability to bind to its guide RNA and target DNA nucleic acid sequences. Non-limiting examples of the modified Cas proteins that can be used with the present disclosure are known in the art. See, e.g., PANDELAKIS et al, CELL SYSTEMS (1): 1-14 (month 1 of 2020), which is incorporated by reference herein in its entirety. In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as "dCas 9"). In some aspects, the modified Cas protein comprises a modified Cas12a protein. In some aspects, modified Cas proteins useful in the present disclosure bind to a guide polynucleotide (e.g., a small guide RNA) ("modified Cas-guide complex") wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In some aspects, the guide polynucleotide comprises a recognition sequence complementary to a promoter region of an endogenous nucleic acid sequence encoding a protein of interest. In some aspects, one or more transcriptional activators are linked to a modified Cas-directed complex (e.g., the N-terminus and/or the C-terminus of a modified Cas protein) such that when the modified Cas-directed complex is introduced into a cell, the one or more transcriptional activators can bind to a regulatory element (e.g., a promoter region) of a nucleic acid sequence and thereby induce and/or increase expression of the encoded protein (e.g., C-Jun). In some aspects, one or more transcriptional activators may bind to a regulatory element (e.g., a promoter region) of an endogenous gene and thereby induce and/or increase expression of the encoded protein (e.g., c-Jun). Non-limiting illustrative examples of common general activators that may be used include the omega subunit of RNAP, VP16, VP64, and p65. See, e.g., kabadi and Gersbach, methods 69:188-197 (2014), which are incorporated by reference herein in their entirety.

In some aspects, one or more transcription repressors (e.g., kruppel-related cassette domain (KRAB)) can be linked to the modified Cas-directed complex (e.g., the N-terminus and/or C-terminus of the modified Cas protein) such that when introduced into a cell, the one or more transcription repressors can repress or reduce transcription of genes (e.g., those genes that can interfere with C-Jun expression (e.g., bach 2)). See, e.g., US20200030379A1 and Yang et al, J TRANSL MED 19:459 (2021), each of which is incorporated by reference herein in its entirety. In some aspects, modified Cas proteins useful in the present disclosure may be linked to one or more transcriptional activators and one or more transcriptional repressors.

Without wishing to be bound by any theory, in some aspects, the use of such modified Cas proteins may allow conditional transcription and expression of the relevant genes. For example, in some aspects, a cell (e.g., a T cell) is modified to include a ligand binding protein (e.g., an anti-ROR 1 CAR) linked to a protease (e.g., tobacco Etch Virus (TEV)) and a single guide RNA (sgRNA) targeting the c-Jun promoter region. In some aspects, the cells are modified to further comprise a Linker (LAT) for activating T cells, the linker complexed with a modified Cas protein linked to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., TEV-cleavable linker). Upon activation of the ligand-binding protein, the modified Cas protein is released for nuclear localization and conditionally and reversibly induces expression of c-Jun. Yang et al J Immunother Cancer (journal 2) A164 (2021), which is incorporated by reference herein in its entirety.

As should be apparent to one of skill in the art, in some aspects, the cells described herein have been modified using a combination of methods. For example, in some aspects, the cells have been modified to include (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., ROR1 binding protein and EGFRt), and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun). In some aspects, the cell has been modified to include (i) an exogenous nucleotide sequence encoding a first protein (e.g., ROR1 binding protein), and (ii) an exogenous nucleotide sequence encoding a second protein (e.g., c-Jun protein). In some aspects, the modified cell may further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first, second, and third proteins may be part of a single polycistronic vector.

Unless otherwise indicated, one or more exogenous nucleotide sequences and/or transcriptional activators may be introduced into the cell using any suitable method known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to a cell include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.

In some aspects, immune cells of the disclosure (which may be modified and cultured using the methods described herein) are isolated from a human subject, e.g., prior to in vitro or ex vivo culturing. In some aspects, immune cells are isolated from a human subject for allogeneic cell therapy. In some aspects, immune cells are isolated from a human subject for autologous cell therapy. In some aspects, the immune cells are T cells (e.g., cd4+ T cells and/or cd8+ T cells). In some aspects, the immune cell is an NK cell. In some aspects, the immune cells are tregs.

In some aspects, the cells (e.g., T cells and/or NK cells) are engineered prior to culturing according to the methods disclosed herein. In some aspects, the cells (e.g., T cells and/or NK cells) are engineered after culturing according to the methods disclosed herein. In some aspects, the cells (e.g., T cells and/or NK cells) are cultured prior to, during, and after cell engineering according to the methods disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least 5mM potassium ions (e.g., above 5mM, e.g., between about 40mM and about 80 mM). In some aspects, cells (e.g., T cells and/or NK cells) are engineered to express a Chimeric Antigen Receptor (CAR). In some aspects, cells (e.g., T cells and/or NK cells) are engineered to express an engineered T Cell Receptor (TCR). In certain aspects, culturing cells (e.g., T cells and/or NK cells) under the conditions disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least about 5mM potassium ions, results in higher transduction efficiency. In some aspects, transduction efficiency in cells (e.g., T cells and/or NK cells) cultured in a hypotonic or isotonic medium comprising at least about 60mM potassium ions according to the methods disclosed herein is at least about 2-fold higher as compared to cells (e.g., T cells and/or NK cells) cultured in a medium comprising 4mM potassium ions or less. In some aspects, transduction efficiency in cells (e.g., T cells and/or NK cells) cultured in a hypotonic or isotonic medium comprising at least about 65mM potassium ions according to the methods disclosed herein is at least about 2.5-fold higher as compared to cells (e.g., T cells and/or NK cells) cultured in a medium comprising 4mM potassium ions or less.

As is apparent from the present disclosure, in some aspects, immune cells useful in the present disclosure (e.g., modified and cultured using the methods provided herein) comprise any suitable immune cells known in the art. Furthermore, as further described elsewhere in this disclosure, the immune cells of the present disclosure have been modified such that they differ from the corresponding immune cells naturally occurring in nature. For example, the immune cells described herein have been modified to express one or more proteins that help to confer unique properties on the immune cells. In particular, in some aspects, a modified immune cell provided herein expresses increased levels of a c-Jun protein as compared to a reference cell (e.g., a corresponding immune cell that has not been modified as described herein). In some aspects, the modified immune cells described herein also express a chimeric binding protein (e.g., CAR) that is not naturally expressed in the immune cells. As will be apparent from the disclosure, in some aspects, a chimeric binding protein (e.g., ROR1 binding protein) can be expressed in a cell by modifying the cell with an exogenous polynucleotide encoding the chimeric binding protein. Additional proteins that can be encoded by exogenous polynucleotides and thus expressed in immune cells are described elsewhere in this disclosure. Non-limiting disclosure relating to such polynucleotides is provided below.

II.C.1.ROR1 binding proteins

As is apparent from the present disclosure, the cells described herein (e.g., immune cells, e.g., cultured in MRM) are modified to comprise an exogenous nucleotide sequence encoding a ROR1 binding protein. Thus, in some aspects, the cells described herein have been modified to express a ROR1 binding protein (e.g., by transducing the cells to include an exogenous nucleotide sequence encoding the ROR1 binding protein) as compared to reference cells (e.g., corresponding cells that are not modified, such as those found naturally in nature). In some aspects, the expression of the ROR1 binding protein is increased by at least about 0.5 fold, at least about 1 fold, 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, at least about 10 fold, at least about 12 fold, at least about 14 fold, at least about 16 fold, at least about 18 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, or at least about 50 fold as compared to a reference cell.

As used herein, the term "ROR1 binding protein" refers to any protein (e.g., an antibody or antigen binding fragment thereof), or fragment thereof (e.g., expressed on tumor cells or peptide/MHC complexes), that can specifically bind to ROR1 protein. In some aspects, the ROR1 binding protein comprises an antibody, an engineered antibody (such as scFv), a CAR, an engineered TCR, a TCR mimetic (e.g., an antibody-T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), or a Chimeric Signaling Receptor (CSR). In some aspects, the ROR1 binding protein comprises a natural ligand of ROR 1. Additional disclosure regarding such ROR1 binding proteins is also provided below. In some aspects, the ROR1 binding protein comprises an antibody or fragment thereof comprising VH and/or VL sequences of 2A2, R11, and R12 anti-ROR 1 monoclonal antibodies described in: for example Hudecek et al, clin.cancer Res.19 (12): 3153-64 (2013); baskar et al, MAbs 4:349-61 (2012); yang et al, PLoS ONE 6:e21018 (2011); US 9,316,646 B2; and U.S. Pat. No. 9,758,586 B2, which is incorporated by reference herein in its entirety. For example, in some aspects, the ROR1 binding protein is capable of cross-competing with an anti-ROR 1 antibody (e.g., an R12 antibody). The R12 antibody sequences are shown in table 14.

In some aspects, the ROR1 binding protein comprises VH and/or VL sequences (or one or more CDR sequences) of any of the anti-ROR 1 binding proteins disclosed in the following documents (e.g., anti-ROR 1 antibodies): for example, WO2019225992A1 (e.g., AB4, A2F2, A2F3, BA6, CC9, C2E3, DG6, D2B12, A2F 2M 1 and BA 6M 1; e.g., SEQ ID NOS: 43-58); US20210317204A1 (e.g., SEQ ID NO: 45-59); WO2022129622A1 (e.g., ,B1、B1G4、1E2、1E5、1B11、C3CP、2G5、1G12、G5CP、2F4、1G9、1H8、G11CP、D9CP、1B6、1F10、E6CP、F2CP、B6CP、1G1、A10CP、G3CP、G3CP G4、G3CP V15、1H8 G4、1H8 V15、C3CP G4、C3CP V15、P3A1 G1, includes variants; see the sequences in tables 1 and 2); WO2022020388A1 (e.g., SEQ ID NOS: 641-644 and 641-744); US20200338210A1 (e.g., m2A2, h2A2 and Y31; SEQ ID NOS: 1,2, 9, 10, 36 and 37); US20190153092A1 (e.g., 2A2, hu2A2B, rbQ11, rbD4, rbQ12, huR12_4, huR12_7, huR12_11, and huR12_16; SEQ ID NOS 7-14, 21-28 and 35-44); WO2021048564A2 (e.g., 1D4, 3F6, 4E2, 5D2, 8B3, and 9G1; SEQ ID NOs: 7, 8, 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, and 56); WO2022029431A1 (e.g., SEQ ID NO: 5); WO2017156479A1 (e.g., anti- ROR1 CAR24-SEQ ID NO:146;SEQ ID NO:7、8、15、16、23、24、31、32、39、40、47、48、55、56、63、64、71、72、79、80、87、88、95、96、103、104、111、112、119 and 120); US20180147271A1 (e.g. ,SEQ ID NO:7、15、23、31、39、47、55、63、71、79、87、95、103、111、119、127、135、143、151、159、167、175、183、191、199、207、215、223、231、239、247、255、263、271、279、287、295、303、311、319、327、335、343、351、359、8、16、24、32、40、48、56、64、72、80、88、96、104、112、120、128、136、144、152、160、168、176、184、192、200、208、216、224、232、240、248、256、264、272、280、288、296、304、312、320、328、336、344、352 and 360); US10752684B2 (e.g., 2A2, 4A5, D10, G6, G3, H10, 2A4 and 1C11;SEQ ID NO:11, 15, 19, 23, 27, 32, 37, 41, 45, 49, 53, 58, 63, 67, 71 and 75); US10759868B2 (e.g., H8, A1, A2, and A3; SEQ ID NOS: 11-18); WO2021190629A1 (e.g., antibodies #1, 11, 32, 101, 103, 115, 140, and 162); US20200157174A1 (e.g. SEQ ID NOs: 105, 113, 121, 129, 137, 145, 153, 109, 117, 125, 133, 141, 149 and 157); US20200255521A1 (e.g., SEQ ID NO: 2-9); US20170306018A1 (e.g., MAB1, MAB2, MAB3, and MAB4; SEQ ID NOS.2, 6 and 42-47); WO2022042488A1 (e.g., mAb004, including humanized variants; SEQ ID NOS: 8-20); WO2022026759A1 (e.g., SEQ ID NOs: 450, 454, 458 and 462); U.S. Pat. No. 3, 20210155692A1 (e.g., I2A-3, I2A-4, I2A-6, I2-A8, I2-A12, I2-A20, I2-A25, I2-A26, I2-A27, I2-A30, I2-A32, I2-A33, and I2-A37; The sequences provided in table 6); U.S. Pat. No. 3, 11155615,2,2, (e.g., sequences provided in tables 6 and 7; sequences provided in tables 147,601-149,601-28,601-37,601-4,601-5,601-50,601-65,601-66,601-70,601-87 and 601-9); US10968275B2 (e.g., SEQ ID NO: 12-16); US20210145882A1 (e.g., 2A2, R12, R11, Y31, UC-961, D10, and H10; SEQ ID NOS: 1-45); EP4039707A1 (e.g., PR000374; SEQ ID NOS: 84 and 85); WO2021115497A2 (e.g., 1015M2-H4; SEQ ID NOS: 63 and 71); WO2022048581A1 (e.g., SEQ ID NOS: 93-134 and 186-197); US20220195041A1 (e.g., SEQ ID NOS: 156 and 166); WO2021057822A1 (e.g., C3, G3, and G6; SEQ ID NOS 1, 5, 15, 19, 29 and 33); WO2022150831A1 (e.g., SEQ ID NOS: 34-36 and 256-270); US20210177902A1 (e.g., SEQ ID NO: 15-74); US10889652B2 (e.g., clones 83B, 83, 305, 298, 350, 20, 16, 48, 43, 366, 40, 461, 65, 81 and 7; sequences provided in table 3B); US20220168344A1 (e.g., SEQ ID NO: 152-157); US10647768B2 (e.g., SEQ ID NOS: 51 and 52); WO2022152168A1 (e.g., SEQ ID NOs: 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 68, 69, 72, 73, 76, 77, 80, 81, 84, 85, 88, 89, 92 and 93); WO2020026987A1 (e.g., SEQ ID NOS: 17 and 22); US20190153092A1 (e.g., SEQ ID NO: 9-44); US20210139579A1 (e.g., SEQ ID NOs: 20, 21, 4,5, 8,9, 22, 23, 2 and 3); US10758556B2 (e.g., SEQ ID NOs: 4 and 5); US2021379194A1 (e.g., SEQ ID NOs: 1,2, 20 and 21); US10618959B2 (SEQ ID NO: 130-141); WO2022084440A2 (e.g., SEQ ID NOS: 212-214 and 221-223); WO2022167460A1 (e.g., SEQ ID NOS: 7-12 and 20-29); US9228023B2 (e.g., A1-A14; SEQ ID NOS: 1-14, 29-42, 268 and 270); WO2021202863A1 (e.g., SEQ ID NOs: 3 and 4); US2021277109A1 (e.g., 226E12, 323H7, 324C7, 323D10, 324E2, 324C6, 338H4 and 330F11;SEQ ID NO:4、8、12、16、20、24、28、32、44、48、52、56、60、64、68、72、76、80、84、88、92、96、100、104、108、112、116、120、124、128 and 132); US2019276540A1 (e.g., SEQ ID NOS: 65, 69, and 79); US20200405759A1 (e.g., SEQ ID NO: 3-14); US9938350B2 (e.g., SEQ ID NOs: 1 and 2); US11312787B2 (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 27-32); US20190112380A1 (e.g., SEQ ID NOS: 454, 455, 755, and 756); EP3548055A2 (see, e.g., exemplary sequences in tables 6 and 7); US20210137977A1 (e.g., SEQ ID NOS: 9699, 9637, 9638, and 11145-11193); WO2021188599A1 (e.g., SEQ ID NO: 19065-19133); US2021253729A1 (e.g., SEQ ID NO: 24-26); US20220152214A1 (e.g., ab1; SEQ ID NOS: 3 and 4); US20220227866A1 (e.g., ab2, ab3, ab6, ab7; SEQ ID NOS: 72-75, 100 and 103); WO2021159029A1; WO2022011075A1; US20220133901A1; each of which is incorporated by reference herein in its entirety.

Receptor tyrosine kinase-like orphan receptor 1 (ROR 1) is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. It is also known as neurotrophic tyrosinase kinase receptor related 1 (ntrfr 1). Human amino acid and nucleic acid sequences can be found in public databases such as GenBank, uniProt and Swiss-Prot. For example, the amino acid sequences of isoforms 1 and 2 precursors of human ROR1 are found at accession numbers np_005003.2 and np_001077061.1, respectively, and the mRNA sequences encoding the sequences are found at accession numbers nm_005012.3 and nm_001083592.1, respectively. As used herein, "ROR1" includes proteins comprising mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants) of full-length wild-type ROR 1.

ROR1 has been described as being overexpressed in a variety of cancers including Triple Negative Breast Cancer (TNBC) and non-small cell lung cancer (NSCLC) adenocarcinomas. Balakrishnan et al CLIN CANCER RES 23:3061-3071 (2017). Receptor tyrosine kinase-like orphan receptor 1 positive (ROR 1 ⁺) solid tumors can be safely targeted by anti-ROR 1 CAR T cells (Specht et al, 2019Cancer Res (79) (4 journals) P2-09-13); however, efficacy has been limited in part because CAR T cells exhibit depletion or dysfunction following infusion in patients with solid tumor malignancies. In addition, solid tumors have immunosuppressive disorders that limit the anti-tumor activity of immunotherapy (such as CAR T cells) (Newick 2016,Srivastava2018,Martinez 2019).

Chimeric Antigen Receptor (CAR)

As described herein, in some aspects, ROR1 binding proteins useful in the present disclosure comprise a CAR. Thus, in some aspects, immune cells that can be cultured using the methods provided herein have been modified to express a CAR and increased levels of c-Jun protein. In some aspects, the immune cells are cd8+ T cells and express the CAR and increased levels of c-Jun protein. In some aspects, the immune cells are cd4+ T cells and express a CAR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd8+ T cells and cd4+ T cells, wherein the cd8+ T cells and cd4+ T cells each express a CAR and increased levels of c-Jun protein. In some aspects, the CAR-expressing cells disclosed herein are CAR T cells, e.g., single CAR T cells, genome-edited CAR T cells, dual CAR T cells, or tandem CAR T cells. Examples of such CAR T cells are provided in international publication No. WO2020028400, which is incorporated herein by reference in its entirety.

In some aspects, the CAR (e.g., which can be expressed in combination with the c-Jun protein) is designed as a standard CAR. In a "standard CAR," the different components (e.g., extracellular targeting domain, transmembrane domain, and intracellular signaling/activation domain) are linearly constructed as a single fusion protein. In some aspects, the CAR is designed as a first generation CAR. A "first generation" CAR is composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains. All first generation CARs contained the cd3ζ chain domain as an intracellular signaling domain. In some aspects, the CAR is designed as a second generation CAR. The "second generation" CAR additionally contains a co-stimulatory domain (e.g., CD28 or 4-1 BB). In some aspects, the CAR is designed as a third generation CAR. "third generation" CARs are similar to second generation CARs except that they contain multiple co-stimulatory domains (e.g., CD28-4-1BB or CD28-OX 40). In some aspects, the CAR is designed as a fourth generation CAR. The "fourth generation" CAR (also known as a TRUCK or armored CAR) additionally contains additional factors that can further improve function. For example, in some aspects, the fourth generation CAR additionally contains a cytokine that can be released upon CAR signaling in the targeted tumor tissue. In some aspects, the fourth generation CARs include one or more additional elements, such as homing and suicide genes, which can help further modulate the activity of the CAR. In some aspects, the CAR is designed as a split CAR. In a "split CAR" system, one or more components of the CAR (e.g., the extracellular targeting domain, the transmembrane domain, and the intracellular signaling/activation domain) are split into two or more parts, such that it relies on multiple inputs that facilitate the assembly of the complete functional receptor. In some aspects, the CAR is designed as a switchable CAR. For a "switchable CAR," the CAR may be turned on (turning the CAR on) or off (turning the CAR off) in the presence of a stimulus (e.g., instantaneously). Additional examples of CARs that may be used with the present disclosure are described, for example, in US2020/0172879 A1 and US 2019/0183932A1, each of which is incorporated herein by reference in its entirety.

Engineered T cell receptors

In some aspects, ROR1 binding proteins that may be used with the present disclosure comprise engineered T Cell Receptors (TCRs) (also referred to in the art as "transgenic" TCRs). As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to a T Cell Receptor (TCR) that is isolated or engineered to specifically bind to a Major Histocompatibility Complex (MHC)/peptide target antigen with a desired affinity and is introduced into a population of immune cells (e.g., T cells and/or NK cells).

Thus, in some aspects, immune cells that can be cultured using the methods provided herein have been modified to express a transgenic TCR and increased levels of c-Jun protein. For example, in some aspects, the immune cells comprise cd8+ T cells and express a transgenic TCR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd4+ T cells and express a transgenic TCR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd8+ T cells and cd4+ T cells, wherein each of the cd8+ T cells and cd4+ T cells comprise a transgenic TCR and express increased levels of c-Jun protein.

TCRs are molecules visible on the surface of T cells that are responsible for recognizing antigen fragments as peptides bound to Major Histocompatibility Complex (MHC) molecules. TCRs are heterodimers composed of two distinct protein chains. In some aspects, the TCR consists of an alpha (alpha) chain and a beta (beta) chain (encoded by TRA and TRB, respectively). In some aspects, TCRs consist of gamma and delta (gamma/delta) chains (encoded by TRG and TRD, respectively). When the TCR is engaged with an antigenic peptide (peptide/MHC) presented by an MHC molecule, T lymphocytes are activated via signal transduction. In certain aspects, the engineered TCRs described herein comprise an antigen binding domain generally composed of at least a portion of a β chain variable region and at least a portion of an a chain variable region, wherein the antigen binding domain is specific for or binds to a ROR1 peptide/MHC complex.

In certain embodiments, the engineered TCR is MHC class I-restricted. In another embodiment, the engineered TCR is MHC class II-restricted. In certain embodiments, the engineered TCR recognizes ROR1 peptide, MHC complex. In certain embodiments, the engineered TCR comprises a transmembrane domain and a TCR domain that promotes recruitment of at least one TCR-associated signaling molecule. In some embodiments, the engineered TCRs further comprise one or more TCR-derived constant domains, e.g., CH1 and CL.

T cell receptor mimics (TCRm)

In some aspects, ROR1 binding proteins useful for modifying immune cells comprise T cell receptor mimics (TCR mimics). As used herein, the term "T cell receptor mimetic" or "TCR mimetic" refers to an antibody (or fragment thereof) that has been engineered to recognize a tumor antigen, wherein the tumor antigen is presented in the context of an HLA molecule. As will be apparent to those skilled in the art, these antibodies may mimic the specificity of a TCR. Non-limiting examples of TCR mimics are provided, for example, in US2009/0226474 A1; US2019/0092876 A1; and Traneska et al, front. Immunol.8 (1001): 1-12 (2017), each of which is incorporated by reference herein in its entirety. In some aspects, the TCR mimics comprise (i) an antibody moiety that specifically binds to a related peptide, MHC complex, and (ii) a T cell receptor module capable of recruiting at least one TCR-related signaling molecule. In some aspects, the TCR mimetic comprises (i) an antibody moiety that specifically binds to a related peptide, MHC complex, and (ii) a transmembrane domain, one or more intracellular signaling domains (e.g., a CD3 zeta chain domain), and optionally one or more costimulatory domains (e.g., CD28 or 4-1 BB).

Thus, in some aspects, immune cells that can be cultured using the methods provided herein have been modified to express TCR mimics and increased levels of c-Jun protein (e.g., transduced with one or more exogenous nucleotide sequences encoding the c-Jun protein and TCR mimics). In some aspects, the immune cells comprise cd8+ T cells and express TCR mimics and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd4+ T cells and express TCR mimics and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd8+ T cells and cd4+ T cells, wherein the cd8+ T cells and cd4+ T cells each express a TCR mimetic and increased levels of c-Jun protein.

In some aspects, the TCR mimetic comprises a chimeric antibody-T cell receptor (caTCR). As used herein, a "chimeric antibody-T cell receptor" or "caTCR" comprises (i) an antibody portion that specifically binds to a related antigen, and (ii) a T cell receptor module capable of recruiting at least one TCR-related signaling molecule. In some aspects, the antibody moiety and the T cell receptor moiety are fused together. Additional disclosure regarding caTCR useful in the present disclosure is provided, for example, in US10,822,413B2; and Xu et al, cell Discovery 4:62 (2018), each of which is incorporated by reference herein in its entirety.

Thus, in some aspects, immune cells that can be cultured using the methods provided herein have been modified to express caTCR and increased levels of the c-Jun protein (e.g., transduced with one or more exogenous nucleotide sequences encoding c-Jun polypeptides and caTCR). In some aspects, immune cells modified to express caTCR and increased levels of the c-Jun protein are further modified to express chimeric co-stimulatory receptors. In some aspects, immune cells (such as T cells) provided herein express increased levels of c-Jun protein and comprise: caTCR and a chimeric co-stimulatory receptor comprising: i) A ligand binding module capable of binding or interacting with a target ligand; ii) a transmembrane module; and iii) a costimulatory immune cell signaling module capable of providing a costimulatory signal to an immune cell, wherein the ligand binding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the chimeric costimulatory receptor lacks a functional primary immune cell signaling domain. In some aspects, the chimeric co-stimulatory receptor comprises a ligand binding module that binds to a tumor antigen. Exemplary chimeric co-stimulatory receptors are described, for example, in US10,822,413, incorporated herein by reference in its entirety. In some aspects, the immune cells described herein comprise cd8+ T cells and express caTCR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd4+ T cells and express caTCR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd8+ T cells and cd4+ T cells, wherein the cd8+ T cells and cd4+ T cells each express caTCR and increased levels of c-Jun protein.

Chimeric Signaling Receptor (CSR)

In some aspects, the ROR1 binding protein comprises a Chimeric Signaling Receptor (CSR). A "chimeric signaling receptor" or "CSR" comprises a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain capable of providing a stimulation signal to an immune cell expressing the CSR. Chimeric signaling receptors can comprise (1) an extracellular binding domain (e.g., a natural/modified receptor extracellular domain, a natural/modified ligand extracellular domain, scFv, nanobody, fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates a transcription factor, or recruits and/or activates JAK/STAT, kinase, phosphatase, and ubiquitin; SH3; SH2; and PDZ). See, e.g., EP340793B1, US2021/0253665 A1, US10,822,413B2, and Xu et al, cell Discovery 4:62 (2018), each of which is incorporated herein by reference in its entirety.

In some aspects, immune cells (e.g., modified to express increased levels of c-Jun protein) cultured using the methods provided herein express chimeric signaling receptors. In some aspects, the immune cells comprise cd8+ T cells and express CSR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd4+ T cells and express CSR and increased levels of c-Jun protein. In some aspects, the immune cells comprise cd8+ T cells and cd4+ T cells, wherein the cd8+ T cells and cd4+ T cells each express CSR and increased levels of c-Jun protein.

Antigen binding domains

As described herein, ROR1 binding proteins (e.g., CAR, TCR, caTCR, CSR or TCR mimics) useful in the present disclosure comprise an antigen binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or a combination thereof. Additional disclosure regarding transmembrane domains, costimulatory domains, and intracellular signaling domains is provided elsewhere in this disclosure.

As further described elsewhere in this disclosure, the antigen binding domain of the ROR-1 binding protein (e.g., CAR, TCR, caTCR, CSR or TCR mimics) can be any polypeptide capable of binding ROR1 (including fragments or variants thereof). In some aspects, the antigen binding domains comprise or are derived from Ig NAR, fab fragments, fab ' fragments, F (ab) '2 fragments, F (ab) '3 fragments, fv, single chain variable fragments (scFv), bi-scFv, (scFv) 2, minibodies, diabodies, trifunctional antibodies, tetrafunctional antibodies, diabodies, disulfide stabilized Fv proteins (dsFv), diabodies, nanobodies, and antigen binding regions derived from antibodies that can specifically bind to a related protein (e.g., ROR 1), ligand (including natural ligand), receptor fragment, peptide aptamer, or a combination thereof. In some aspects, the antigen binding domain is a single chain Fv (scFv).

Signaling (intracellular), transmembrane and costimulatory domains

In some aspects, ROR1 binding proteins (e.g., CAR, TCR, caTCR, CSR or TCR mimics) described herein comprise an intracellular signaling domain that transduces effector function signals upon binding of an antigen to an extracellular domain and directs a cell (e.g., T cell) expressing the ROR1 binding protein to perform a particular function. Non-limiting examples of intracellular signaling domains include intracellular signaling domain regions derived from: cd3ζ, fcrγ, common FcRγ(FCER1G)、FcγRIIa、FcRβ(FcεRib)、CD3γ、CD3δ、CD3ε、CD22、CD79a、CD79b、CD278("ICOS")、FcεRI、CD66d、CD32、DAP10、DAP12, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain (e.g., the domain described in SEQ ID NO: 90).

In some aspects, the ROR1 binding protein comprises the entire intracellular domain of the proteins disclosed herein. In some aspects, the intracellular domain is truncated. A truncated portion of the intracellular domain may be used in place of the complete chain, so long as it still transduces effector function signals. Thus, the term intracellular domain is intended to include any truncated portion of the intracellular domain sufficient to transduce an effector function signal.

In some aspects, ROR1 binding proteins (e.g., CAR, TCR, caTCR, CSR or TCR mimics) useful in the present disclosure also comprise a transmembrane domain. In some aspects, the antigen binding domain of the ROR1 binding protein is linked to the intracellular domain by a transmembrane domain. In some aspects, the antigen binding domain of the ROR1 binding protein is linked to the transmembrane domain by a linker. In some aspects, including a linker between the antigen binding domain and the transmembrane domain can affect the flexibility of the antigen binding domain and thereby improve one or more properties of the ROR1 binding protein.

Any transmembrane domain known in the art may be used for the ROR1 binding proteins described herein (e.g., CAR, TCR, caTCR, CSR or TCR mimics). In some aspects, the transmembrane domain is artificial (e.g., an engineered transmembrane domain). In some aspects, the transmembrane domain is derived from a naturally occurring polypeptide. In some aspects, the transmembrane domain comprises a transmembrane domain from a naturally occurring polypeptide. Non-limiting examples of transmembrane domains include transmembrane domain region ：KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKG2D、NKG2C、CD19、CD8 of each or any combination thereof. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain (e.g., the domain set forth in SEQ ID NO: 75).

As described herein, in some aspects, ROR1 binding proteins (e.g., CAR, TCR, caTCR, CSR or TCR mimics) useful in the present disclosure comprise one or more costimulatory domains (e.g., second and third generation CARs). Without wishing to be bound by any theory, these co-stimulatory domains may further improve the expansion, activation, memory, persistence, and/or effector function of immune cells engineered to express the ROR1 binding protein (e.g., in combination with the c-Jun polypeptide). In some aspects, the transmembrane domain is fused to a costimulatory domain, optionally the costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, but is not limited to cd3ζ. Non-limiting examples of co-stimulatory domains include interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX, DAP10, or any combination thereof. In some aspects, the costimulatory domain comprises a 4-1BB/CD137 costimulatory domain (e.g., the domain depicted in SEQ ID NO: 76).

c-Jun

As described herein, in some aspects, the immune cells described herein (e.g., modified and cultured using the methods provided herein) comprise or are capable of expressing a c-Jun protein. In the case where immune cells are capable of naturally expressing c-Jun proteins, in some cases, expression of the endogenous c-Jun protein is induced, thereby resulting in an increase or overexpression of the protein. In inducing expression (or overexpression) of the c-Jun protein in a cell, in some aspects, the c-Jun protein is exogenously added. In some aspects, the c-Jun protein is recombinantly expressed in cells. For example, in some aspects, the cells described herein have been modified or engineered (e.g., genetically) to include an exogenous polynucleotide that includes a nucleotide sequence encoding a c-Jun protein (also referred to herein as a "c-Jun nucleotide sequence") such that c-Jun protein expression is increased in the modified cells compared to reference cells (e.g., corresponding cells that are not modified to include the exogenous polynucleotide). In some aspects, the cell has been modified by a transcriptional activator (e.g., a CRISPR/Cas system-based transcriptional activator, e.g., CRISPRa) such that expression of the endogenous c-Jun protein is increased compared to a reference cell (e.g., a corresponding cell not modified by the transcriptional activator).

In some aspects, due to modification (e.g., introduction of exogenously introduced c-Jun nucleotide sequences and/or transcriptional activators), the engineered cells overexpress, i.e., express, c-Jun proteins at a level (e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% higher, or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher) than the corresponding cells without such modification ("reference cells"). The terms "increased level of expression [ or amount ]," over-expression "or increased expression with" … … "(and similar forms of the phrase as used herein) are used interchangeably.

In some aspects, the engineered (or modified) cells described herein express at least about 2-100-fold higher, about 5-50-fold higher, about 5-40-fold higher, about 5-30-fold higher, about 5-20-fold higher, about 8-20-fold higher, or about 10-20-fold higher c-Jun protein than the reference cells. In some aspects, the c-Jun protein expression in a modified cell described herein is increased by at least about 0.5 fold, at least about 1 fold, 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, at least about 10 fold, at least about 12 fold, at least about 14 fold, at least about 16 fold, at least about 18 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, or at least about 50 fold as compared to the c-Jun protein expression in a reference cell.

Furthermore, as described herein, in some aspects, the media of the present disclosure (e.g., comprising potassium ions at a concentration greater than 5 mM) can also help to further increase c-Jun protein (or any other related protein) expression in the modified cells. Thus, in some aspects, the c-Jun protein expression in the modified cell (e.g., caused by the introduction of an exogenous nucleotide sequence encoding a c-Jun protein and/or a transcriptional activator capable of increasing expression of an endogenous c-Jun polypeptide) is further increased by at least 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold when cultured using the methods provided herein compared to the c-Jun protein expression in the reference cell. Thus, in some aspects, the methods provided herein comprise modifying an immune cell (e.g., a T cell) with an exogenous polynucleotide encoding a ROR1 binding protein and a c-Jun polypeptide in a medium comprising potassium ions at a concentration of greater than 5mM, wherein after modification, expression of the c-Jun polypeptide in the immune cell is increased compared to a reference cell. In some aspects, the immune cells can be modified with an exogenous polynucleotide in a separate medium, followed by subsequent transfer and culture in a medium comprising potassium ions at a concentration greater than 5 mM.

As described herein, in some aspects, the reference cell may comprise any of the following: (i) Corresponding cells that are unmodified and not cultured in medium (i.e., do not contain potassium ions at a concentration above 5mM, e.g., TCM); (ii) Corresponding cells that have been modified but have not been cultured in a medium; (iii) the corresponding cells unmodified but cultured in culture medium; or (iv) any combination of (i), (ii), and (iii).

As is apparent from the present disclosure, in some aspects, the immune cells described herein (e.g., cultured using the methods provided herein) have been modified to express one or more additional transgenes as well as increased amounts of c-Jun protein. For example, in some aspects, immune cells useful in the present disclosure have been modified to comprise: (i) A first exogenous nucleotide sequence encoding a c-Jun polypeptide and (ii) a second exogenous nucleotide sequence encoding a ROR1 binding protein. 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 such polycistronic polynucleotides are described further below. As described herein, in some aspects, such modification of immune cells (e.g., to express ROR1 binding protein and have increased levels of c-Jun polypeptide) occurs in a medium comprising potassium ions at a concentration of greater than 5 mM. In some aspects, the immune cells are modified in a reference medium (e.g., a medium that does not contain potassium ions at a concentration above 5 mM), and then cultured in a medium that contains potassium ions at a concentration above 5 mM. In some aspects, T cells may be cultured in a medium comprising potassium ions at a concentration greater than 5mM prior to modification. In some aspects, the modified T cells may be further cultured after modification in a medium comprising potassium ions at a concentration of greater than 5 mM. In some aspects, the immune cells are cultured in a medium comprising potassium ions at a concentration greater than 5mM before, during, and after modification.

C-Jun is an oncogenic transcription factor belonging to the family of activator protein-1 (AP-1). It interacts with a variety of proteins (e.g., c-Fos) to form dimeric complexes that regulate a variety of cell signaling pathways, including cell proliferation and tumor progression. Thus, increased c-Jun expression has been observed in certain cancers, and there is great interest in developing c-Jun antagonists to treat such cancers. See, e.g., brennan, A. Et al, J Exp CLIN CANCER RES (1): 184 (9 months of 2020).

In humans, the c-Jun protein is encoded by the Jun gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047 of GenBank accession nc_000001.11, negative strand orientation). The JUN gene and its synonyms for the encoded protein are known and include the "JUN oncogene, the 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 AP1". The wild type human c-Jun protein sequence is 331 amino acids in length. The amino acid and nucleic acid sequences of wild-type human c-Jun are provided in tables 1 and 2, respectively.

The wild type human c-Jun (UniProt identifier: P05412-1) protein sequence is 331 amino acids in length (SEQ ID NO: 13). The amino acid and nucleic acid sequences are shown in tables 1 and 2, respectively.

TABLE 1 c-Jun protein sequence

TABLE 2 c-Jun nucleic acid sequence

In some aspects, the immune cells disclosed herein have been modified to include an exogenous nucleotide sequence encoding a wild-type c-Jun protein, such as the wild-type nucleotide sequence set forth in SEQ ID NO. 12. Alternatively, in some aspects, the immune cells described herein are modified to include an exogenous nucleotide sequence encoding a mutant c-Jun protein that retains the ability to prevent and/or reduce immune cell depletion. In some aspects, a mutant C-Jun protein that can be expressed on an immune cell 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 to a C-terminal amino acid residue (e.g., 50, 75, 100, 150, 200, or 250 or more residues) of a wild-type C-Jun (i.e., SEQ ID NO: 13), a C-terminal portion (e.g., a quarter, third, or half), or a C-terminal domain (e.g., epsilon, bZIP, and the amino acid C-terminus thereof). In some aspects, the N-terminal amino acid residue (e.g., 50, 75, 100, or 150 or more of the N-terminus), the N-terminal portion (e.g., one-fourth, one-third, or half), or the N-terminal domain (e.g., delta, transactivation (transactivation) domain, and amino acid N-terminus thereof) of wild-type c-Jun (i.e., SEQ ID NO: 13) is deleted, mutated, or otherwise inactivated. In some aspects, the c-Jun is a mutant human c-Jun, optionally comprising an inactivating mutation in his transactivation domain or delta domain. In some aspects, the c-Jun mutant comprises S63A and S73A mutations. In some aspects, the c-Jun mutant comprises a deletion between residues 2 and 102, as compared to wild-type c-Jun (SEQ ID NO: 13). In some aspects, the c-Jun mutant comprises a deletion between residues 30 and 50 as compared to wild-type c-Jun (SEQ ID NO: 13). In some aspects, mutant c-Jun comprises (i) a S63A and S73A mutation, or (ii) a deletion between residues 2 and 102 or between residues 30 and 50, as compared to wild-type c-Jun (SEQ ID NO: 13). Non-limiting examples of mutant c-Jun proteins useful in the present disclosure are provided in US2019/0183932 A1 and US2017/0037376A1, each of which is incorporated herein by reference in its entirety.

In some aspects, the immune cells described herein have 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 of the nucleic acid sequences set forth in SEQ ID NOS.1-11. In some aspects, the exogenous nucleotide sequence encoding a c-Jun polypeptide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 1 to 11.

In some aspects, the 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. 1. In some aspects, the 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. 1. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 1.

In some aspects, the 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. 2. In some aspects, the 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. 2. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 2.

In some aspects, the 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. 3. In some aspects, the 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. 3. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 3.

In some aspects, the 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. 4. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide has 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. 4. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 4.

In some aspects, the 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. 5. In some aspects, the 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. 5. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 5.

In some aspects, the 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. 6. In some aspects, the 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. 6. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO. 6.

In some aspects, the 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. 7. In some aspects, the 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. 7. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO. 7.

In some aspects, the 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. 8. In some aspects, the 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. 8. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO. 8.

In some aspects, the 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. 9. In some aspects, the 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. 9. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO. 9.

In some aspects, the 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. 10. In some aspects, the 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. 10. In some aspects, the exogenous nucleotide comprises the nucleotide sequence set forth in SEQ ID NO. 10.

Exemplary c-Jun nucleotide sequences are provided in table 3 (below).

Table 3.c-Jun nucleotide sequence

The c-Jun nucleotide sequences disclosed herein may be codon optimized using any method known in the art. For example, in some aspects, the codons of the c-Jun nucleotide sequences disclosed herein have been optimized to modify (e.g., increase or decrease) one or more of the following parameters as compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 11): (i) Codon adaptation index (i.e., codon usage preference); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and labile motifs; (iv) Repeat sequences (e.g., forward repeat, reverse repeat, dyad repeat); (v) a restriction enzyme recognition site; or (vi) combinations thereof.

In some aspects, exogenous polynucleotides encoding the c-Jun polypeptides provided herein are capable of increasing expression of the encoded c-Jun protein when transfected, transduced, or otherwise introduced into immune cells (e.g., human immune cells), as compared to corresponding expression in cells transfected with a wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 11). In some aspects, c-Jun protein expression in immune cells modified to include an exogenous polynucleotide is increased by at least about 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold as compared to corresponding expression in cells transfected, transduced or otherwise genetically modified with a wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 11) for expression.

While certain of the disclosure provided above generally relates to modifying immune cells to include exogenous nucleotide sequences encoding a c-Jun protein (wild-type c-Jun or variant thereof), it should be apparent to those of skill in the art that other suitable methods may be used to induce and/or increase c-Jun protein expression (wild-type or variant thereof) in cells. For example, as described herein, in some aspects, an endogenous c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa). The disclosure provided above using exogenous nucleotide sequences applies equally to other methods of inducing and/or increasing c-Jun protein expression in cells provided herein (e.g., transcriptional activators such as CRISPRa), unless otherwise indicated.

In some aspects, increased c-Jun protein expression may improve and/or enhance one or more characteristics of a modified immune cell (e.g., a T cell, such as a cd4+ and/or cd8+ T cell). Non-limiting examples of such characteristics include: resistance to depletion (e.g., as indicated by reduced expression of depletion markers such as PD-1, CD39, TIM-3, and/or LAG-3, increased persistence/survival, delay in onset of a dysfunctional state, and/or increased cytokine production), increased amplification/proliferation, increased antigen sensitivity, improved effector function (in particular, improved effector function after repeated antigen stimulation (e.g., cytokine production after antigen stimulation, lysis of cells expressing a target antigen, or both)), or a combination thereof.

Assays useful for measuring depletion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, cytokine production/release and gene expression patterns are known in the art and include, for example, flow cytometry, intracellular Cytokine Staining (ICS),Immune cell killing assays, mesoscale discovery (Meso Scale Discovery, MSD) or similar assays, continuous antigen stimulation assays, batch and single cell RNAseq (see, e.g., Fron Genet.2020;11:220;2019Bioinformatics 35:i436-445;2019Annual Review of Biomed.Data Sci.2:139-173)、 cytotoxicity/killing assays, ELISA, western blot, and other standard molecular and cell biology methods, such as described herein or as described, e.g., in Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, inc., 1999-2021) or elsewhere.

In some aspects, increased c-Jun protein expression increases immune cell resistance to depletion. In some aspects, the depletion resistance is increased by at least about 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold as compared to a reference cell (e.g., a corresponding cell that has not been modified to have increased c-Jun protein expression).

In some aspects, increased c-Jun protein expression can reduce depletion in depleted cells. In some aspects, increased c-Jun protein expression can reduce depletion 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% as measured, for example, using one or more assays as described herein, as compared to a reference cell (e.g., a corresponding depleted cell that is not modified to have increased c-Jun protein expression).

In some aspects, increased c-Jun protein expression delays the onset of cell depletion. In some aspects, increased c-Jun protein expression delays the onset of depletion 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% as measured, for example, using one or more assays as described herein, as compared to a reference cell (e.g., a corresponding cell that is not modified to have increased c-Jun protein expression). In some aspects, increased c-Jun protein expression delays onset of depletion for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days or more.

Thus, in some aspects, expression of one or more of the depletion markers (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3) in the cells described herein is reduced 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% as compared to a reference cell (e.g., a corresponding cell that is not modified to have increased c-Jun protein expression).

In some aspects, expression of one or more depletion markers (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3) in a cell described herein is reduced 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 than a reference cell (e.g., a corresponding cell not engineered to overexpress-Jun).

In some aspects, the depletion state of a population of immune 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 immune cells that express a given depletion marker (e.g., TIGIT, PD-1, TIM-3, and/or LAG-3). For example, when an immune cell population is modified to express increased levels of c-Jun protein (e.g., in combination with ROR1 binding protein), the amount (e.g., number and/or percentage) of cells expressing a given depletion marker is reduced compared to the amount in a corresponding immune cell population that is not modified as described herein. Thus, in some aspects, the amount of cells expressing a given depletion marker in a modified immune cell population as described herein is reduced 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 immune cell population not modified as described herein.

In some aspects, increased c-Jun protein expression may increase persistence/survival of immune cells, e.g., when administered to a subject in vivo. In some aspects, the persistence/survival of a cell is increased by at least about 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold as compared to a reference cell (e.g., a corresponding cell that has not been modified to have increased c-Jun protein expression).

In some aspects, the persistence/survival of an 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% as compared to the amount in a corresponding population of immune cells not modified as described herein.

Thus, in some aspects, the immune cells of the present disclosure are modified to: (i) Expression of a ROR1 binding protein (e.g., an anti-ROR 1 CAR) and (ii) having an increased level of a c-Jun polypeptide (e.g., with an exogenous nucleotide sequence encoding the c-Jun polypeptide and/or a transcriptional activator capable of increasing endogenous c-Jun expression) and culturing in a medium comprising potassium ions at a concentration of greater than 5mM such that after modification and culturing, persistence/survival of immune cells is increased compared to reference cells. As described herein, in some aspects, the reference cells comprise the following corresponding immune cells: (i) Culturing in a medium that is not modified to overexpress c-Jun, but that comprises potassium ions at a concentration above 5 mM; (ii) Is modified to overexpress c-Jun, but is not cultured in a medium comprising potassium ions at a concentration above 5 mM; or (iii) both (i) and (ii).

In some aspects, increased c-Jun protein expression may increase expansion/proliferation of immune cells, e.g., following antigen stimulation. In some aspects, the expansion/proliferation of the cells is increased by at least about 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold as compared to a reference cell (e.g., a corresponding cell that is not modified to have increased c-Jun protein expression).

In some aspects, for example, the expansion/proliferation of immune cells after antigen stimulation 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 not modified as described herein.

Thus, in some aspects, the immune cells of the present disclosure are modified to: (i) Expression of a ROR1 binding protein (e.g., an anti-ROR 1 CAR) and (ii) having an increased level of a c-Jun polypeptide (e.g., with an exogenous nucleotide sequence encoding the c-Jun polypeptide and/or a transcriptional activator capable of increasing endogenous c-Jun expression) and culturing in a medium comprising potassium ions at a concentration of greater than 5mM such that after modification and culturing, expansion/proliferation of immune cells is increased compared to reference cells. As described herein, in some aspects, the reference cells comprise the following corresponding immune cells: (i) Culturing in a medium that is not modified to overexpress c-Jun, but that comprises potassium ions at a concentration above 5 mM; (ii) Is modified to overexpress c-Jun, but is not cultured in a medium comprising potassium ions at a concentration above 5 mM; or (iii) both (i) and (ii).

In some aspects, increased c-Jun protein expression may increase effector functions of the cell, such as increased cytokine (e.g., IFN- γ, TNF- α, and/or IL-2) production, granzyme release, and/or cytotoxicity. In some aspects, the increase in effector function is in response to a sustained antigen stimulus. As used herein, the term "sustained antigen stimulation" or "long term antigen stimulation" refers to repeated exposure of an immune cell (e.g., a T cell) to its cognate antigen such that the immune cell is stimulated or activated. In some aspects, sustained antigen stimulation comprises exposing an immune cell (e.g., a T cell) to its cognate antigen for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year. In some aspects, the sustained antigen stimulation may be continuous. In some aspects, the sustained antigen stimulation may include multiple rounds of antigen stimulation, wherein each round of antigen stimulation is followed by a period of non-antigen stimulation. In some aspects, the sustained antigen stimulation comprises at least about 2 rounds, at least about 3 rounds, at least about 4 rounds, at least about 5 rounds, or at least about 6 rounds or more of antigen stimulation. As is apparent from the present disclosure and known in the art, such sustained antigen stimulation of immune cells can lead to immune cell depletion.

In some aspects, the effector function of the cell is increased by at least about 0.5-fold, at least about 1-fold, 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, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-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, or at least about 50-fold as compared to a reference cell (e.g., a corresponding cell that is not modified to have increased c-Jun protein expression).

In some aspects, increased c-Jun protein expression may increase effector function of a 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% as compared to a reference cell.

Thus, in some aspects, the immune cells of the present disclosure are modified to: (i) Expression of a ROR1 binding protein (e.g., an anti-ROR 1 CAR) and (ii) having an increased level of a c-Jun polypeptide (e.g., with an exogenous nucleotide sequence encoding the c-Jun polypeptide and/or a transcriptional activator capable of increasing endogenous c-Jun expression) and culturing in a medium comprising potassium ions at a concentration of greater than 5mM such that after modification and culturing, effector function of immune cells is increased, e.g., in response to sustained antigen stimulation, as compared to reference cells. As described herein, in some aspects, the reference cells comprise the following corresponding immune cells: (i) Culturing in a medium that is not modified to overexpress c-Jun, but that comprises potassium ions at a concentration above 5 mM; (ii) Is modified to overexpress c-Jun, but is not cultured in a medium comprising potassium ions at a concentration above 5 mM; or (iii) both (i) and (ii).

In some aspects, cells modified to express increased levels of c-Jun (e.g., as described herein), such as increased cytokine (e.g., IFN-gamma, TNF-alpha, and/or IL-2) production, granzyme release, and/or cytotoxicity (e.g., the ability to kill associated target cells), are subjected to at least one, at least two, at least three, or more additional rounds of continuous, long-term, or sequential stimulation assays (such as described in example 3 or, e.g., zhao et al, 2015Cancer Cell 28 (4): 415-428; kunkele et al, 2015Cancer 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 that do not overexpress c-Jun).

In some aspects, immune cells cultured in a metabolic reprogramming medium of the present disclosure (e.g., comprising potassium ions at a concentration of greater than 5 mM) are capable of producing higher amounts of cytokines (e.g., IFN- γ and/or IL-2) after at least two rounds of antigen stimulation, after at least three rounds of antigen stimulation, after at least five rounds of antigen stimulation, after at least six rounds of antigen stimulation, as compared to corresponding immune cells cultured in a reference medium. Thus, in some aspects, provided herein is a method of increasing cytokine production by an immune cell in response to antigen stimulation, wherein the method comprises culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5 mM. As described herein, in some aspects, the immune cells have been modified to include ROR1 binding proteins (e.g., anti-ROR 1 CARs) and to have increased levels of c-Jun polypeptides as compared to reference cells (e.g., corresponding immune cells that have not been modified to have increased levels of c-Jun polypeptides).

In some aspects, after culturing, the modified immune cells produce at least about a 1-fold, at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about a 8-fold, at least about a 9-fold, at least about a 10-fold, at least about a 11-fold, at least about a 12-fold, at least about a 13-fold, at least about a 14-fold, at least about a 15-fold, at least about a 16-fold, at least about a 17-fold, at least about a 18-fold, at least about a 19-fold, at least about a 20-fold, at least about a 25-fold, at least about a 30-fold, at least about a 35-fold, at least about a 40-fold, at least about a 45-fold, at least about a 50-fold, at least about a 75-fold, at least about a 100-fold, at least about a 200-fold, at least about a 300-fold, at least about a 400-fold, at least about a 500-fold, at least about a 750-fold, or at least about a1,000-fold or more in comparison to a reference cell (e.g.g.described herein). In some aspects, the cytokine produced by the modified immune cells in response to antigen stimulation 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 at least about 100% after culturing, as compared to a reference cell.

Increased c-Jun expression in T cells can help maintain the active state of the cells by, for example, alleviating or preventing T cell dysfunction (e.g., T cell depletion). Thus, different methods of increasing c-Jun protein expression in a cell provided herein (e.g., modifying a cell with an exogenous polynucleotide encoding a c-Jun polypeptide and/or a transcriptional activator capable of increasing endogenous c-Jun expression) can be used to engineer immune cells (such as T cells) that in turn exhibit sustained, potent cytotoxicity to a desired target cell (e.g., a target of an endogenous TCR or a target of a ROR1 binding protein as described herein). The engineered T cells disclosed herein (which have increased c-Jun protein expression) exhibit little evidence of T cell depletion as described above, as compared to T cells that do not overexpress c-Jun.

Furthermore, as is apparent from the present disclosure, in some aspects, one or more of the above properties are further enhanced when any of the modified immune cells provided herein (e.g., expressing ROR1 binding protein and having increased levels of c-Jun protein) are cultured using the methods provided herein (e.g., in metabolic reprogramming media comprising potassium ions at a concentration greater than 5 mM). For example, in some aspects, immune cells of the disclosure (modified to express ROR1 binding protein and having increased levels of c-Jun protein and cultured in metabolic reprogramming media (e.g., comprising potassium ions at a concentration greater than 5 mM)) can exhibit one or more of the following compared to reference cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, but not cultured in metabolic reprogramming media described herein, and/or cultured in metabolic reprogramming media described herein, but not modified to express ROR1 binding protein and having increased levels of c-Jun protein): (i) Increased resistance to depletion (e.g., as indicated by reduced expression of depletion markers (such as PD-1, CD39, TIM-3, and/or LAG-3), increased persistence/survival, delay in onset of a dysfunctional state, and/or increased cytokine production), (i) increased amplification/proliferation, (iii) increased antigen sensitivity, (iv) increased effector function (especially after repeated antigen stimulation) (e.g., cytokine production after antigen stimulation, lysis of cells expressing a target antigen, or both), or (v) any combination thereof.

II.C.2. additional translatable sequences

In some aspects, the immune cells described herein (e.g., modified and cultured using the methods provided herein) can express one or more additional related proteins. For example, in some aspects, the modified immune cells described herein further comprise one or more exogenous nucleotide sequences encoding additional related proteins. Thus, in some aspects, the immune cells disclosed herein comprise an exogenous nucleotide sequence encoding a ROR1 binding protein, and one or more additional exogenous nucleotide sequences encoding additional related proteins (e.g., c-Jun and/or EGFRt). Non-limiting examples of such additional translatable sequences are described below.

Truncated EGFR

In some aspects, the immune cells disclosed herein (e.g., modified and cultured using the methods provided herein) further comprise an exogenous nucleotide sequence encoding a truncated epidermal growth factor receptor (EGFRt) such that the EGFRt comprises only a partial sequence of a full length EGFR protein (e.g., SEQ ID NO: 19). In some aspects, EGFRt comprises EGFR extracellular domains III and IV and EGFR transmembrane domains, but lacks EGFR extracellular domains I and II and EGFR intracellular sequences. Thus, in some aspects, the immune cells disclosed herein have been modified to comprise: (i) an exogenous nucleotide sequence encoding a c-Jun polypeptide, (ii) an exogenous nucleotide sequence encoding a ROR1 binding protein, and (iii) an exogenous nucleotide sequence encoding an EGFRt. As described herein, in some aspects, transcriptional activators (e.g., CRISPRa) may be used to increase expression of an endogenous c-Jun protein. Thus, in some aspects, the immune cells described herein have been modified to comprise: (i) a transcriptional activator capable of increasing expression of an endogenous c-Jun protein, (ii) an exogenous nucleotide sequence encoding a ROR1 binding protein, and (iii) an exogenous nucleotide sequence encoding an EGFRt. In each of the above aspects, one or more of the plurality of exogenous nucleotide sequences may be part of a single polycistronic polynucleotide.

EGFR is a 180kDa monomeric glycoprotein comprising a large extracellular region, a single transmembrane domain, an intracellular membrane proximal region, a tyrosine kinase domain and a C-terminal regulatory region. The extracellular region comprises four domains: domains I and III are cognate ligand binding domains, and domains II and IV are cysteine-rich domains (Ferguson, annu Rev Biophys. (2008) 37:353-3). EGFR as used herein refers to human EGFR unless indicated otherwise. Due to alternative splicing, there are at least four known human EGFR isoforms. The sequences of the different EGFR isoforms are provided in table 4 (below).

Table 4: human EGFR sequences

In the EGFR canonical sequence described above (i.e., isoform 1), the individual EGFR domains are described below. The signal peptide spans amino acids 1-24. The extracellular sequence spans amino acids 25-645, with domain I, domain II, domain III, and domain IV spanning amino acids 25-188, 189-333, 334-504, and 505-645, respectively. The transmembrane domain spans amino acids 646-668. The intracellular domain spans amino acids 669-1,210, wherein the membrane proximal domain spans amino acids 669-703 and the tyrosine kinase domain spans amino acids 704-1,210.

In some aspects, EGFRt useful in the present disclosure comprises an amino acid sequence having 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 amino acid sequence set forth in SEQ ID NO. 19.

In some aspects, EGFR useful in the present disclosure 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% or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 21. In some aspects, the EGFRt comprises the amino acid sequence set forth in SEQ ID NO. 21 (see Table 6). In some aspects, EGFR useful in the present disclosure 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% or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 24. In some aspects, the EGFRt comprises the amino acid sequence set forth in SEQ ID NO:24 (see Table 5).

Table 5: truncated EGFR sequences

In some aspects, the EGFRt described herein further comprises a membrane proximal domain. As used herein, the term "membrane-proximal domain" refers to the intracellular portion of a cell surface protein (e.g., EGFR) immediately C-terminal to the transmembrane domain. Without wishing to be bound by any theory, in some aspects, the addition of a membrane proximal domain may increase the expression of a protein encoded by a polynucleotide of the present disclosure.

In some aspects, the membrane proximal domain can be about 1 to about 20 (e.g., 2-20, 3-20, 4-20, 5-20, 2-18, 3-18, 4-18, or 5-18) amino acids long. In some aspects, the membrane proximal domain can be longer than 20 amino acids. In some aspects, the first 1 or more (e.g., the first 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids of the membrane proximal domain are net neutral or net positive charge sequences (e.g., the number of arginine and lysine residues is greater than or equal to the number of aspartic acid and glutamic acid residues). In some aspects, those preceding amino acids contain more than about 30% (e.g., more than 40%, 50%, 60%, 70%, 80%, or 90%) hydrophilic amino acids. Non-limiting examples of membrane proximal domains useful in the present disclosure are provided in table 6 (below).

Table 6: membrane proximal domain sequence

Net charge	Sequence(s)
		+1	K
+2	KR
		+3	KRK
+2	KSR
		+1	KSGSGS(SEQ ID NO:25)
+2	SKR
		+1	KRSD(SEQ ID NO:26)
+2	KRSDK(SEQ ID NO:27)
		0	SGGGG(SEQ ID NO:28)
0	SGAGG(SEQ ID NO:29)
		+2	KRADK(SEQ ID NO:30)
+3	RRRSGGGGSGGGGS(SEQ ID NO:31)
		0	SGGGGSGGGGS(SEQ ID NO:32)
0	(GGGGS)n，n>1(SEQ ID NO:33)

In some aspects, the membrane-proximal domains useful in the present disclosure may be derived from a membrane-proximal region of a native cell surface protein, such as a membrane-proximal region of a human receptor tyrosine kinase (e.g., all or part of the sequence of the first 20 membrane-proximal amino acids) that interacts with Phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol-4, 5-bisphosphate (PIP 2) (see, e.g., hedger et al, sci rep (2015) 5:9198). Non-limiting examples of receptor tyrosine kinases are ERBB1(EGFR)、ERBB2(HER2)、ERBB3(HER3)、ERBB4(HER4)、INSR、IGF1R、INSRR、PGFRA、PGFRB、KIT、CSF1R、FLT3、VGFR1、VGFR2、VGFR3、FGFR1、FGFR2、FGFR3、FGFR4、PTK7、NTRK1、NTRK2、NTRK3、ROR1、ROR2、MUSK、MET、RON、UFO、TYRO3、MERTK、TIE1、TIE2、EPHA1、EPHA2、EPHA3、EPHA4、EPHA5、EPHA6、EPHA7、EPHA8、EPHAA、EPHB1、EPHB2、EPHB3、EPHB4、EPHB6、RET、RYK、DDR1、DDR2、ROS1、LMTK1、LMTK2、LMTK3、LTK、ALK and STYK1. In some aspects, the membrane proximal domain may comprise one or more mutations (e.g., substitutions or deletions) that remove residues that are known to phosphorylate in order to circumvent any unintended signaling capacity of the proteins encoded by the polynucleotides of the present disclosure.

In some aspects, the membrane-proximal domain is derived from the membrane-proximal region of EGFR. A non-limiting example of an EGFR-derived membrane proximal domain comprises one of the sequences provided in table 7 (below). In some aspects, the membrane proximal domain comprises the amino acid sequence RRR. In some aspects, EGFRt comprising such a membrane proximal domain comprises the sequence set forth in SEQ ID NO. 24.

Table 7: EGFR-derived membrane-proximal domain sequences

Net charge	Sequence(s)
		+6	RRRHIVRKR(SEQ ID NO:34)
+5	RRRHIVRK(SEQ ID NO:35)
		+4	RRRHIVR(SEQ ID NO:36)
+3	RRRHIV(SEQ ID NO:37)
		+3	RRRHI(SEQ ID NO:38)
+3	RRRH(SEQ ID NO:39)
		+3	RRR
+2	RR
		+1	R

As is apparent from the present disclosure, modifying immune cells described herein (e.g., expressing ROR1 binding proteins and having increased levels of c-Jun polypeptides) to also include exogenous nucleotide sequences encoding EGFRt provides certain advantages. For example, in some aspects, EGFRt may act as a kill switch (KILL SWITCH). In some aspects, when the engineered cells described herein are no longer needed in vivo, a pharmaceutical grade anti-EGFR antibody, such as cetuximab (cetuximab), panitumumab (panitumumab), nimotuzumab (nimotuzumab) or toxazumab (necitumumab), may be administered to a subject who has received the engineered cells, thereby removing the engineered cells, e.g., via antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular phagocytosis (ADCP).

Spacer

In some aspects, an immune cell described herein (e.g., modified and cultured using the methods provided herein) further comprises an exogenous nucleotide sequence encoding a spacer. Thus, in some aspects, the immune cells described herein have been modified to express increased levels of a c-Jun protein (e.g., having an exogenous nucleotide sequence encoding a c-Jun protein and/or a transcriptional activator capable of increasing expression of an endogenous c-Jun protein) and comprise: an exogenous nucleotide sequence encoding a c-Jun protein, an exogenous nucleotide sequence encoding a ROR1 binding protein, and an exogenous nucleotide sequence encoding a spacer. In some aspects, the immune cells have been modified to express increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a c-Jun protein, an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding EGFRt, and an exogenous nucleotide sequence encoding a spacer. In some aspects, the one or more exogenous nucleotide sequences are part of a single polycistronic polynucleotide. As used herein, the term "spacer" refers to a polypeptide sequence that is capable of covalently linking two spacer portions (e.g., a P2A linker and ROR1 binding protein) together.

In some aspects, the spacer is derived from an immunoglobulin (e.g., from a hinge region or a loop region). In some aspects, the spacer comprises an IgA1, igA2, igG1, igG2, igG3, igG4, igD, igE, or IgM hinge region, fragments thereof (alone or capped with additional sequences, such as CH1 or CH2 region sequences), or a combination of fragments from an IgA1, igA2, igG1, igG2, igG3, igG4, igD, igE, or IgM hinge region (referred to herein as a "hinge region-derived spacer"). In some aspects, the spacer comprises an IgA1, igA2, igG1, igG2, igG3, igG4, igD, igE, or IgM constant domain loop region, a fragment thereof (alone or capped by additional sequences, e.g., from adjacent β chains), or a combination of fragments from IgA1, igA2, igG1, igG2, igG3, igG4, igD, igE, or IgM loop regions (referred to herein as "loop region-derived spacers"). In some aspects, the spacers include hinge region source spacers, loop region source spacers, or both (e.g., two or more connected hinge region source spacers and loop region source spacers).

Thus, in some aspects, a polynucleotide described herein encodes a polypeptide comprising: (i) a c-Jun protein, (ii) a first linker (e.g., a P2A linker), (iii) a signal peptide (e.g., hIgkappa), (iv) an antigen binding domain (e.g., scFv), (v) a second linker (e.g., GGGSG; SEQ ID NO: 40), (vi) a spacer (e.g., an IgG2 hinge-derived spacer), (vii) a transmembrane domain (e.g., CD 28), (viii) a co-stimulatory domain (e.g., 4-1 BB), (ix) an intracellular signaling domain (e.g., CD3 zeta), (x) a third linker (e.g., a P2A linker), and (xi) EGFRt.

In some aspects, a spacer useful in the present disclosure comprises a subsequence of an immunoglobulin heavy chain selected from the group consisting of: HUMAN IgA1 (Uniprot: P01876, IGHA 1. Sup. HUMAN constant α1; SEQ ID NO: 41), HUMAN IgA2 (Uniprot P01877, IGHA 2. Sup. HUMAN constant α2; SEQ ID NO: 42), murine IgG2A (Uniprot P01665, GCAM. Sup. MOUSE, immunoglobulin gamma.2A chain C region; SEQ ID NO: 43), HUMAN IgG1 (Uniprot P01857, IGHG1_HUMAN, immunoglobulin heavy chain constant γ1; SEQ ID NO: 44), HUMAN IgG2 (Uniprot P01859, IGHG2_HUMAN, immunoglobulin heavy chain constant γ2; SEQ ID NO: 45), HUMAN IgG3 (Uniprot P01860, IGHG3_HUMAN, immunoglobulin heavy chain constant γ3; SEQ ID NO: 46), HUMAN IgG4 (Uniprot P01861, IGHG4, immunoglobulin heavy chain constant γ4; SEQ ID NO: 47), HUMAN IgD (Uniprot P01880, IGHD _HUMAN, immunoglobulin heavy chain constant δ; SEQ ID NO: 48), HUMAN IgE (Uniprot P01854, IGHE _HUMAN, immunoglobulin heavy chain constant ε; SEQ ID NO: 49) or (Uniprot P01871, HM_HU, immunoglobulin heavy chain constant μ; SEQ ID NO: 50), the hinge region or the hinge region thereof comprising a portion thereof in SEQ 1-CH 2. In some aspects, the subsequence further comprises adjacent portions of CH1 and/or CH2 constant domains.

In some aspects, the spacer comprises a subsequence of an immunoglobulin heavy chain selected from the group consisting of: HUMAN IgA1 (Uniprot: P01876, IGHA 1. Sup. HUMAN constant α1; SEQ ID NO: 41), HUMAN IgA2 (Uniprot P01877, IGHA 2. Sup. HUMAN constant α2; SEQ ID NO: 42), murine IgG2A (Uniprot P01665, GCAM. Sup. MOUSE, immunoglobulin gamma.2A chain C region; SEQ ID NO: 43), HUMAN IgG1 (Uniprot P01857, IGHG1_HUMAN, immunoglobulin heavy chain constant γ1; SEQ ID NO: 44), HUMAN IgG2 (Uniprot P01859, IGHG2_HUMAN, immunoglobulin heavy chain constant γ2; SEQ ID NO: 45), HUMAN IgG3 (Uniprot P01860, IGHG3_HUMAN, immunoglobulin heavy chain constant γ3; SEQ ID NO: 46), HUMAN IgG4 (Uniprot P01861, IGHG4, immunoglobulin heavy chain constant γ4; SEQ ID NO: 47), HUMAN IgD (Uniprot P01880, IGHD _HUMAN, immunoglobulin heavy chain constant δ; SEQ ID NO: 48), HUMAN IgE (Uniprot P01854, IGHE _HUMAN, immunoglobulin heavy chain constant ε; SEQ ID NO: 49) or (Uniprot P01871, HM_HU, immunoglobulin heavy chain constant μ; SEQ ID NO: 50), the sequence comprising a constant domain from which part of the sequence or a constant domain thereof. In some aspects, the subsequence further comprises a contiguous portion of a β chain.

In some aspects, the spacers useful in the present disclosure are derived from IgG, e.g., igG1, igG2, igG3, or IgG4. In some aspects, the spacer is derived from an IgG2 hinge. In some aspects, the IgG2 hinge-derived spacer comprises at least five, six, or seven consecutive amino acids of SEQ ID NO. 51 (KPCPPCKCP). In some aspects, the spacer comprises an amino acid sequence that is 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%, at least about 99%, or about 100% identical to the sequence set forth in SEQ ID NO. 51 (KPCPPCKCP). In some aspects, the spacer comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO. 51 (KPCPPCKCP). In some aspects, the spacer comprises the sequence set forth in SEQ ID NO. 51 (KPCPPCKCP), except for 1,2,3,4,5,6,7, 8, 9, or 10 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some aspects, the amino acid substitutions comprise at least one non-conservative amino acid substitution.

In some aspects, the spacers of the present disclosure comprise the sequence set forth in SEQ ID NO. 51, wherein the spacer sequence further comprises an optional flexible linker (e.g., a linker of GGGSG (SEQ ID NO: 40)). Thus, in some aspects, the spacers of the present disclosure comprise a spacer sequence (e.g., SEQ ID NO: 51) and optionally a C-terminal or N-terminal flexible linker. In some aspects, any optional flexible linker disclosed herein (e.g., gly/ser-rich linker) can be attached to the C-terminus and/or N-terminus of the spacer.

Signal peptides

As described herein, in some aspects, the immune cells provided herein have been modified to further express a signal peptide (e.g., comprising an exogenous nucleotide sequence encoding the signal peptide). The signal peptide may promote cell surface expression of the encoded protein and may then subsequently be cleaved from the mature protein. In some aspects, such immune cells have been modified to have increased levels of a c-Jun protein (e.g., to have an exogenous nucleotide sequence encoding a c-Jun protein and/or a transcriptional activator capable of increasing expression of an endogenous c-Jun protein) and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein and an exogenous nucleotide sequence encoding a signal peptide. In some aspects, the immune cells have been modified to express increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding an EGFRt, and an exogenous nucleotide sequence encoding a signal peptide. In some aspects, the immune cells have been modified to express increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding EGFRt, an exogenous nucleotide sequence encoding a spacer, and an exogenous nucleotide sequence encoding a signal peptide. In some aspects, the one or more exogenous nucleotide sequences are part of a single polycistronic polynucleotide.

Any suitable signal peptide known in the art may be used in the present disclosure. Non-limiting examples of signal peptides are provided in table 8 (below). In some aspects, the signal peptide is derived from human igκ. In some aspects, the signal peptide 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 54 (MVLQTQVFISLL LWISGAYG). In some aspects, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 54 (MVLQTQVFISLLLWISGAYG). In some aspects, the signal peptide is derived from GM-CSF. In some aspects, such signal peptides comprise 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 the amino acid sequence set forth in SEQ ID NO. 53 (MLLLVTSLLLCELPHPAFLLIP). In some aspects, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO 53 (M LLLVTSLLLCELPHPAFLLIP).

Table 8: signal peptide sequences

In some aspects, polynucleotides useful for modifying immune cells described herein comprise a single signal peptide (e.g., SEQ ID NO:53 or 54). In some aspects, the polynucleotide comprises a plurality of signal peptides (e.g., at least two, three, four, or more). Where multiple signal peptides are involved, in some aspects, each of the multiple signal peptides is different. In some aspects, two or more of the plurality of signal peptides are identical.

Joint

In some aspects, immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein and cultured using the methods provided herein) have been modified to additionally comprise an exogenous nucleotide sequence encoding a linker. Thus, in some aspects, the immune cells described herein have been modified to have increased levels of a c-Jun protein (e.g., having an exogenous nucleotide sequence encoding a c-Jun protein and/or a transcriptional activator capable of increasing expression of an endogenous c-Jun protein) and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein and an exogenous nucleotide sequence encoding a linker. In some aspects, the immune cells have been modified to express increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding EGFRt, and an exogenous nucleotide sequence encoding a linker. In some aspects, the immune cells have been modified to express increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding EGFRt, an exogenous nucleotide sequence encoding a spacer, and an exogenous nucleotide sequence encoding a linker. In some aspects, the modified immune cells described herein have increased levels of c-Jun protein and comprise: an exogenous nucleotide sequence encoding a ROR1 binding protein, an exogenous nucleotide sequence encoding an EGFRt, an exogenous nucleotide sequence encoding a spacer, an exogenous nucleotide sequence encoding a signal peptide, and an exogenous nucleotide sequence encoding a linker.

Where multiple exogenous nucleotide sequences are involved, in some aspects, the one or more exogenous nucleotide sequences are part of a single polycistronic polynucleotide. For such aspects, the linker may be between any of the different components of the polynucleotides described herein. For example, in some aspects, a polynucleotide (e.g., polycistronic) comprises: (i) a first exogenous nucleotide sequence encoding a c-Jun polypeptide, (ii) a second exogenous nucleotide sequence encoding a linker, and (iii) a third nucleotide sequence encoding a ROR1 binding protein (e.g., CAR, TCR, caTCR, CSR or TCR mimetic), wherein the second nucleotide sequence is between the first and third nucleotide sequences such that the c-Jun protein is linked to the ROR1 binding protein by the linker. In some aspects, a polynucleotide of the disclosure may comprise a plurality of nucleotide sequences (e.g., at least two separate nucleotide sequences) encoding a linker. In some aspects, the plurality of joints are identical. In some aspects, the plurality of joints are different.

In some aspects, the linker is a peptide linker. In some aspects, the linker comprises at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, or at least about 30 amino acids. In some aspects, the linker is glycine-rich (e.g., for flexibility). In some aspects, the linker comprises serine and/or threonine (e.g., for solubility). In some aspects, the linker is a Gly/Ser linker.

In some aspects, the glycine/serine linker is according to the formula [ (Gly) n-Ser ] m (SEQ ID NO: 77), wherein n is any integer from 1 to 100 and m is any integer from 1 to 100. In some aspects, the glycine/serine linker is according to the formula [ (Gly) x- (Ser) y ] z (SEQ ID NO: 78), wherein x is an integer from 1 to 4, y is 0 or1, and z is an integer from 1 to 50. In some aspects, the Gly/Ser linker comprises the sequence Gn (SEQ ID NO: 79), wherein n may be an integer from 1 to 100. In some aspects, the optional linker may comprise the sequence (GlyAla) n (SEQ ID NO: 80), wherein n is an integer between 1 and 100.

In some aspects, the sequence of the optional linker is GGGG (SEQ ID NO: 81). In some aspects, the sequence of the optional linker is GGGSG (SEQ ID NO: 82).

In some aspects, the optional linker comprises the sequence (GGGSG) n (SEQ ID NO: 64). In some aspects, the optional linker comprises the sequence (GGGGS) n (SEQ ID NO: 65). In some aspects, the optional linker may comprise the sequence (GGGS) n (SEQ ID NO: 66). In some aspects, the optional linker may comprise the sequence (GGS) n (SEQ ID NO: 67). In these cases, n may be an integer from 1 to 100. In other cases, n may be an integer from 1 to 20, i.e., 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some aspects, n is an integer from 1 to 100.

Examples of optional linkers include, but are not limited to, for example GSGSGS(SEQ ID NO:68)、GGSGG(SEQ ID NO:69)、SGGSGGS(SEQ ID NO:70)、GGSGGSGGSGGSGGG(SEQ ID NO:71)、GGSGGSGGGGSGGGGS(SEQ ID NO:72)、GGSGGSGGSGGSGGSGGS(SEQ ID NO:73) or GGGGSGGGGSGGGGS (SEQ ID NO: 74).

In some aspects, the optional linker comprises the sequence PGG. In some aspects, the optional linker comprises additional amino acids in addition to glycine and serine. In some aspects, the optional linker comprises 1,2,3, 4, or 5 non-gly/non-ser amino acids. In some aspects, the Gly/Ser linker comprises at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least 95% glycine or serine amino acids.

In some particular aspects, the length of the optional linker is between 1 and 10 amino acids. In some aspects, the length of the optional linker is between about 5 and about 10 amino acids, between about 10 and about 20 amino acids, between about 20 and about 30 amino acids, between about 30 and about 40 amino acids, between about 40 and about 50 amino acids, between about 50 and about 60 amino acids, between about 60 and about 70 amino acids, between about 70 and about 80 amino acids, between about 80 and about 90 amino acids, or between about 90 and about 100 amino acids.

In some aspects, the linker is a non-cleavable linker such that the linker and the different components of the polynucleotides provided herein (e.g., c-Jun protein and ROR1 binding protein) are expressed as a single polypeptide. In some aspects, the linker is a cleavable linker. As used herein, the term "cleavable linker" refers to a linker that comprises a cleavage site such that upon expression, it can be selectively cleaved to yield two or more products. In some aspects, the linker is selected from a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof (see table 9 below). In some aspects, the linker further comprises a GSG linker sequence. In some aspects, linkers useful in the present disclosure comprise an Internal Ribosome Entry Site (IRES) such that separate polypeptides encoded by the first and second genes are produced during translation. Additional descriptions of joints that may be used in the present disclosure are provided, for example, in WO 2020/223625 A1 and US2019/0276801A1, each of which is incorporated herein by reference in its entirety.

Table 9: linker sequences

In some aspects, the linker comprises a P2A linker. In some aspects, the linker 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO. 14.

In some aspects, the linker comprises a T2A linker. In some aspects, the linker 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 15. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO. 15.

In some aspects, the linker comprises an F2A linker. In some aspects, the linker 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO. 16.

In some aspects, the linker comprises an E2A linker. In some aspects, the linker 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO. 17.

In some aspects, the linker comprises an amino acid sequence comprising a furin cleavage site. In some aspects, the linker 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%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO. 18.

As is apparent from the above disclosure, in some aspects, an immune cell described herein (e.g., modified and cultured using the methods provided herein) comprises an exogenous polynucleotide comprising (from 5 'to 3'): (i) a first nucleotide sequence encoding a c-Jun polypeptide, (ii) a second nucleotide sequence encoding a first linker (e.g., a P2A linker), (iii) a third nucleotide sequence encoding a first signal peptide (e.g., higκ), (iv) a fourth nucleotide sequence encoding a ROR1 binding protein (e.g., scFv), (v) a fifth nucleotide sequence encoding a second linker (e.g., GGGSG; SEQ ID NO: 40), (vi) a sixth nucleotide sequence encoding a spacer (e.g., an IgG2 hinge-origin spacer), (vii) a seventh nucleotide sequence encoding a transmembrane domain (e.g., CD 28), (viii) a ninth nucleotide sequence encoding a co-stimulatory domain (e.g., 4-1 BB), (ix) a ninth nucleotide sequence encoding an intracellular signaling domain (e.g., CD 3), (x) a tenth nucleotide sequence encoding a third linker (e.g., a P2A linker), (xi) a second signal peptide (e.g., GMCSFR SP alpha) and (xij) a twelfth nucleotide sequence encoding xij.

Delivery vehicle

In some aspects, provided herein are vectors (e.g., expression vectors) useful for modifying immune cells described herein (e.g., cultured using the methods provided herein). In some aspects, the vectors described herein comprise a plurality (e.g., 2, 3, or 4 or more) of polynucleotides, wherein each of the plurality of polynucleotides encodes a protein described herein (e.g., a c-Jun protein, a ROR1 binding protein (e.g., CAR), or EGFRt). Thus, in some aspects, the vector comprises a polycistronic vector (e.g., a bicistronic vector or a tricistronic vector). In some aspects, the polynucleotides described herein are contained on the same vector (e.g., a polycistronic expression vector). In some aspects, polynucleotides encoding a protein described herein (e.g., a c-Jun protein, ROR1 binding protein (e.g., CAR), or EGFRt) are provided on one or more separate vectors.

Such vectors are useful for recombinant expression in host cells and cells targeted for therapeutic intervention, as described herein. As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is 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 may 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. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA techniques are typically in the form of plasmids. In this disclosure, "plasmid" and "vector" are sometimes used interchangeably, as the plasmid is the most commonly used form of vector, depending on the context. However, other forms of expression vectors are also disclosed herein, such as viral vectors (e.g., lentiviruses, replication defective retroviruses, poxviruses, herpesviruses, baculoviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

In some aspects, the vector comprises a polynucleotide (e.g., encoding a ROR1 binding protein, a c-Jun protein, and/or EGFRt) and a regulatory element described herein. For example, in some aspects, a vector comprises a polynucleotide described herein (e.g., encoding a ROR1 binding protein, a c-Jun protein, and/or EGFRt) operably linked to a promoter. In some aspects, the vector may comprise a plurality of promoters (e.g., at least two, at least three, at least four, at least five, or more). For example, in some aspects, the nucleotide sequence encoding the c-Jun protein may be under the control of a first promoter, and the nucleotide sequence encoding one or more additional components of the polynucleotide (e.g., ROR1 binding protein) may be under the control of a second promoter. In some aspects, each of the plurality of promoters is the same. In some aspects, one or more of the plurality of promoters are different.

Any suitable promoter known in the art may be used in the present disclosure. In some aspects, promoters useful in the present disclosure include mammalian or viral promoters, such as constitutive or inducible promoters. In some aspects, the promoters used in the present disclosure comprise at least one constitutive promoter and at least one inducible promoter, e.g., a tissue-specific promoter.

Constitutive mammalian promoters include, but are not limited to, promoters of the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters for constitutive use in eukaryotic cells include, for example, promoters from Cytomegalovirus (CMV), simian viruses (e.g., SV 40), papillomaviruses, adenoviruses, human Immunodeficiency Virus (HIV), rous sarcoma viruses, cytomegaloviruses, long Terminal Repeat (LTR) and other retroviruses of moloney mouse leukemia virus, and thymidine kinase promoters of herpes simplex virus. As described herein, in some aspects, promoters useful in the present disclosure are inducible promoters. Inducible promoters are expressed in the presence of an inducer. For example, metallothionein promoters are induced in the presence of certain metal ions to promote transcription and translation. When multiple inducible promoters are present, they may be induced by the same inducer molecule or by different inducers.

In some aspects, the promoter comprises a myeloproliferative sarcoma virus enhancer, a deleted negative control region, a dl587rev primer binding site substituted (MND) promoter, an EF1a promoter, or both.

In some aspects, vectors useful in the present disclosure (e.g., comprising one or more nucleotide sequences encoding a c-Jun protein and/or a ligand binding protein) further comprise one or more additional regulatory elements. Non-limiting examples of regulatory elements include Translational Enhancer Elements (TEEs), translation initiation sequences, microrna binding sites or seeds thereof, 3' tail regions of linked nucleosides, AU-rich elements (ars), post-transcriptional control regulators, 5' utrs, 3' utrs, localization sequences (e.g., membrane localization sequences, nuclear exclusion sequences, or proteasome targeting sequences), post-translational modification sequences (e.g., ubiquitination, phosphorylation, or dephosphorylation), or combinations thereof.

In some aspects, the vector may additionally comprise a transposable element. Thus, in some aspects, the vector comprises a polynucleotide described herein (e.g., encoding a c-Jun protein and/or a ligand binding protein) flanked by at least two transposon specific Inverted Terminal Repeats (ITRs). In some aspects, the transposon specific ITR is recognized by a DNA transposon. In some aspects, the transposon-specific ITR is recognized by a retrotransposon. Any transposon system known in the art may be used to introduce a nucleic acid molecule into the genome of a host cell (e.g., an immune cell). In some aspects, the transposon is selected from the group consisting of 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 herein by reference in its entirety.

In some aspects, the vector is a transfer vector. The term "transfer vector" refers to a composition of matter that comprises an isolated nucleic acid (e.g., a polynucleotide as described herein) and that can be used to deliver the isolated nucleic acid into the interior of a cell. A variety of 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 autonomously replicating plasmids or viruses. The term should also be construed to also include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

In some aspects, the vector is an expression vector. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements sufficient for expression; other elements for expression may be provided 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) incorporating the recombinant polynucleotide.

In some aspects, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some aspects, the vector is selected from the group consisting of: adenovirus vectors, lentiviruses, sendai virus vectors, baculovirus vectors, epstein Barr virus vectors, papova virus vectors, vaccinia virus vectors, herpes simplex virus vectors, heterozygous virus vectors and adeno-associated virus (AAV) vectors.

In some aspects, the adenovirus vector is a third generation adenovirus vector. ADEASY ^TM is the most popular method to date for generating adenovirus vector constructs. The system consists of two types of plasmids: shuttle (or transfer) vectors and adenovirus vectors. The relevant transgene was cloned into a shuttle vector, validated, and linearized with the restriction enzyme PmeI. This construct was then transformed into ADEASIER-1 cells, which were BJ5183 E.coli cells containing PADEASY ^TM. PADEASY ^TM is an adenovirus plasmid of about 33Kb, which contains adenovirus genes necessary for viral production. The shuttle vector and adenovirus plasmid have matched left and right homology arms that facilitate homologous recombination of the transgene into the adenovirus plasmid. Standard BJ5183 can also be co-transformed with supercoiled PADEASY ^TM and shuttle vectors, but this approach results in a higher background for non-recombinant adenovirus plasmids. Next, the size and appropriate restriction digestion pattern of the recombinant adenovirus plasmid was verified to confirm that the transgene had been inserted into the adenovirus plasmid and that no other recombination pattern occurred. Once validated, the recombinant plasmid was linearized with PacI to produce linear dsDNA constructs flanking the ITR. 293 or 911 cells were transfected with the linearization constructs and virus could be collected after about 7-10 days. In addition to such methods, other methods known in the art for producing adenoviral vector constructs at the time of application of the application may also be used to practice the methods disclosed herein.

In some aspects, the viral vector is a retroviral vector, such as a lentiviral vector (e.g., a third generation or fourth generation lentiviral vector). The term "lentivirus" refers to a genus of the retrovirus family. Lentiviruses are unique among retroviruses in that they are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, and therefore they are one of the most effective methods of gene delivery vectors. 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 lentiviral genome, and includes in particular a self-inactivating lentiviral vector as provided in Milone et al mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, those such as those from Oxford BioMedicaGene delivery techniques, LENTIMAX ^TM vector systems from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

Lentiviral vectors are typically produced in transient transfection systems, in which a cell line is transfected with three independent plasmid expression systems. These systems include transfer vector plasmids (part of the HIV provirus), packaging plasmids or constructs, and plasmids with heterologous envelope genes (env) of different viruses. The three plasmid components of the vector are placed into packaging cells, which are then inserted into the HIV shell. The viral portion of the vector contains an insertion sequence such that the virus cannot replicate in the cellular system. Current third generation lentiviral vectors encode only three of nine HIV-1 proteins (Gag, pol, rev), expressed by separate plasmids to avoid recombination-mediated replication competent viral production. In fourth generation lentiviral vectors, the retroviral genome has been further reduced (see, e.g.LENTI-X ^TM fourth generation packaging System).

In some aspects, non-viral methods can be used to deliver polynucleotides described herein (e.g., encoding the c-Jun protein and/or ROR1 binding protein) into immune cells. In some aspects, the non-viral method comprises using a transposon. In some aspects, use of a non-viral delivery method allows reprogramming of cells (e.g., T cells or NK cells), and infusion of cells directly into a subject. In some aspects, polynucleotides can be inserted into the genome of a target cell (e.g., T cell) or host cell (e.g., cell for recombinant expression of the encoded protein) by using CRISPR/Cas systems and genome editing alternatives, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Meganucleases (MNs). Non-viral delivery systems also include electroporation, cell extrusion, nanoparticles (including lipid nanoparticles), gold nanoparticles, polymer nanoparticles. An illustrative non-viral delivery system includes and is described, for example, in EbioMedicine, 2021, month 5; 67:103354.

In some aspects, vectors disclosed herein (e.g., lentiviral vectors) comprise a polynucleotide comprising one or more nucleotide sequences encoding (i) a c-Jun protein and (ii) an antigen binding domain (e.g., an anti-ROR 1 scFv). In some aspects, the vector comprises a polynucleotide comprising one or more nucleotide sequences encoding (i) a c-Jun protein, (ii) an antigen binding domain (e.g., an anti-ROR 1 scFv), and (iii) EGFRt. In some aspects, the vector comprises a polynucleotide comprising one or more nucleotide sequences encoding (i) a c-Jun protein, (ii) an antigen binding domain (e.g., an anti-ROR 1 scFv), (iii) a transmembrane domain (e.g., CD 28), (iv) a costimulatory domain (4-1 BB), (v) an intracellular signaling domain (cd3ζ), and (vi) EGFRt. In some aspects, the one or more nucleotide sequences additionally encode a linker, a spacer, a signal peptide, or a combination thereof. For example, in some aspects, the vectors described herein comprise a polynucleotide comprising (from 5 'to 3'): (i) a first nucleotide sequence encoding a c-Jun protein, (ii) a second nucleotide sequence encoding a first linker (e.g., a P2A linker), (iii) a third nucleotide sequence encoding a first signal peptide (e.g., hIgκ), (iv) a fourth nucleotide sequence encoding an antigen binding domain (e.g., an anti-ROR 1 scFv), (v) a fifth nucleotide sequence encoding a second linker (e.g., GGGSG; SEQ ID NO: 40), (vi) a sixth nucleotide sequence encoding a spacer (e.g., an IgG2 hinge-derived spacer), (vii) a seventh nucleotide sequence encoding a transmembrane domain (e.g., CD 28), (viii) an eighth nucleotide sequence encoding a co-stimulatory domain (e.g., 4-1 BB), (ix) a ninth nucleotide sequence encoding an intracellular signaling domain (e.g., CD3 ζ), (x) a tenth nucleotide sequence encoding a third linker (e.g., a P2A linker), (xi) a second signal peptide (e.g., GM-CSF) and (xij) a twelfth nucleotide sequence.

In some aspects, a polynucleotide disclosed herein (e.g., encoding a c-Jun protein and/or ligand binding protein) is DNA (e.g., a DNA molecule or combination thereof), RNA (e.g., an RNA molecule or combination thereof), or any combination thereof. In some aspects, the polynucleotide is single-or double-stranded RNA or DNA (e.g., ssDNA or dsDNA) in genomic or cDNA form, or a DNA-RNA hybrid, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. As described herein, such nucleic acid sequences may comprise additional sequences useful in 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 and secretion signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those skilled in the art, based on the teachings herein, what nucleotide sequences will encode the different polypeptides described herein (e.g., ROR1 binding protein, c-Jun protein, and/or EGFRt).

III compositions of the present disclosure

Certain aspects of the present disclosure are directed to a cell composition comprising a population of immune cells (e.g., T cells and/or NK cells) cultured according to the methods disclosed herein. Certain aspects of the present disclosure are directed to a cell composition comprising a population of immune cells (e.g., T cells and/or NK cells) modified to express increased levels of a c-Jun polypeptide as compared to a reference immune cell (e.g., a corresponding cell that has not been modified to have increased levels of a c-Jun polypeptide) and cultured according to the methods disclosed herein. The cell populations according to the methods disclosed herein and/or cultured in the metabolic reprogramming media disclosed herein have an increased number of poorly differentiated cells as compared to comparable cells cultured according to conventional methods (e.g., in media containing less than 5mM K ⁺). In some aspects, cells cultured according to the methods disclosed herein exhibit increased expression of one or more markers characteristic of a stem cell-like phenotype. In some aspects, a population of cells according to the methods disclosed herein and/or cultured in the metabolic reprogramming media disclosed herein has an increased number of effector-like cells as compared to comparable cells cultured according to conventional methods (e.g., in media containing less than 5mM K ⁺). In some aspects, a population of cells cultured according to the methods disclosed herein and/or in the metabolic reprogramming media disclosed herein has an increased number of stem cell-like and effector-like cells as compared to comparable cells cultured according to conventional methods (e.g., in media containing less than 5mM K ⁺). In some aspects, cells cultured according to the methods disclosed herein exhibit greater proliferation potential than cells cultured according to conventional methods. In some aspects, cells cultured according to the methods disclosed herein exhibit increased transduction efficiency. In some aspects, cells cultured according to the methods disclosed herein exhibit increased in vivo viability after transplantation in a subject. In some aspects, cells cultured according to the methods disclosed herein exhibit increased cellular potency. In some aspects, cells cultured according to the methods disclosed herein exhibit reduced cell depletion. In some aspects, cells cultured according to the methods disclosed herein exhibit increased in vivo persistence after transplantation into a subject. In some aspects, cells cultured according to the methods disclosed herein exhibit increased in vivo activity after transplantation into a subject. In some aspects, cells cultured according to the methods disclosed herein exhibit a more durable in vivo response after transplantation into a subject. In some aspects, the subject is a human.

In some aspects, at least about 5% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 10% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 15% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 20% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 25% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 30% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 35% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 40% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 45% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 50% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 55% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 60% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 65% of the cells in the cell composition have a stem cell-like phenotype. In some aspects, at least about 70% of the cells in the cell composition have a stem cell-like phenotype.

In some aspects, after culturing T cells according to the methods disclosed herein, the stem cell-like T cells comprise at least about 10% to at least about 70% of the total number of T cells in the culture. In some aspects, after culturing T cells according to the methods disclosed herein, the stem cell-like T cells comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD8 ⁺ T cells in the culture. In some aspects, after culturing T cells according to the methods disclosed herein, the stem cell-like T cells comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD4 ⁺ T cells in the culture.

In some aspects, the increase in the proportion of progenitor cells depleted of T cells (i.e., T cells enriched in TPE gene imprints) is between about 1.5-fold and about 20-fold after culturing T cells according to the methods disclosed herein. In some aspects, the progenitor cells deplete T cells at a rate that is between about 2-fold and about 10-fold after culturing T cells according to the methods disclosed herein. In some aspects, the progenitor cells deplete T cells at a rate that is between about 2-fold and about 5-fold after culturing T cells according to the methods disclosed herein.

In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 2-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 2.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 3-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 3.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 4-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 4.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 5.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 6-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 6.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 7-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 7.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 8-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 8.5-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 9-fold after culturing T cells according to the methods disclosed herein. In some aspects, the proportion of progenitor cells depleted of T cells increases by at least about 10-fold after culturing T cells according to the methods disclosed herein.

In some aspects, after culturing T cells according to the methods disclosed herein, the proportion of T cells depleted (e.g., T cells enriched in TTE gene imprinting) is reduced by at least about 1/4 and the proportion of T cells depleted by progenitor cells is increased by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing T cells according to the methods disclosed herein, the proportion of T cells depleted is reduced by at least about 1/3 and the proportion of progenitor cells depleted of T cells is increased by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing T cells according to the methods disclosed herein, the proportion of T cells depleted is reduced by at least about 1/2 and the proportion of progenitor cells depleted of T cells is increased by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing T cells according to the methods disclosed herein, the proportion of T cells depleted is reduced by at least about 3/4 and the proportion of progenitor cells depleted of T cells is increased by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold.

In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 1.5-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 2-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 2.5-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 3-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 3.5 fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, or at least about 10 fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 4-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 5-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold. In some aspects, after culturing the T cells according to the methods disclosed herein, the proportion of stem cell-like T cells increases by at least about 6-fold and the proportion of progenitor cells depleted of T cells increases by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least about 10-fold.

In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers, and an increased percentage of immune cells that express one or more TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least two stem cell-like markers, and an increased percentage of immune cells that express one or more TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least three stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least four stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least two TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least three TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least four TPE markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers, and an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least five TPE markers.

In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein between about 4% and about 10% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein between about 4% and about 9% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein between about 4% and about 8% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein between about 4% and about 7% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein between about 4% and about 6% of the cells are progenitor cell depleted T cells.

In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor depleted T cells and at least about 4% of the cells are stem cell-like T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor depleted T cells and at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are stem cell-like T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor depleted T cells and at least about 4% are stem cell-like T cells. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor cell depleted T cells and at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are stem cell-like T cells.

In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor cell depleted T cells and less than about 20% of the cells are terminal depleted cells (TTEs). In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor cell depleted T cells. In some aspects, the cell compositions herein comprise a population of immune cells wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor cell depleted T cells and less than about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% of the cells are terminal depleted cells (TTEs).

In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor depleted T cells, at least about 4% of the cells are stem cell-like T cells and less than about 20% of the cells are TTE. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4% of the cells are progenitor depleted T cells, at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are stem cell-like T cells and less than about 20% of the cells are TTE. In some aspects, the cell compositions herein comprise a population of immune cells, wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor depleted T cells, at least about 4% are stem cell-like T cells, and less than about 20% of the cells are TTE. In some aspects, the cell compositions herein comprise a population of immune cells wherein at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are progenitor cell depleted T cells, at least about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the cells are stem cell-like T cells and less than about 20% of the cells are TTE.

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

In some aspects, after culturing the T cells according to the methods disclosed herein, at least about 10% to at least about 70% of the total number of T cells in the culture are CD39 ^-/TCF7⁺ T cells. In some aspects, after culturing the T cells according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 ^-/TCF7⁺ T cells. In some aspects, the T cell is a CD4 ⁺ T cell. In some aspects, the T cell is a CD8 ⁺ T cell.

In some aspects, the cell composition comprises immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that do not express CD45RO. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA. in some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CCR7. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD62L. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express TCF7. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD3. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD27. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95 and CD45RA. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA and CCR7. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95, CD45RA, and CCR7. in some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA, CCR7, and CD62L. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95, CD45RA, CCR7, and CD62L. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA, CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95, CD45RA, CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA, CCR7, CD62L, TCF, and CD27. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD45RA, CCR7, CD62L, TCF, and CD27, and that do not express CD45RO or are CD45RO ^{Low and low}. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27, and that do not express CD45RO or are CD45RO ^{Low and low}.

In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that do not express CD39 and CD69. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express CD8 and do not express CD39 and CD69. In some aspects, after culturing the T cells according to the methods disclosed herein, at least about 10% to at least about 40% of the total number of T cells in the culture are CD39 ^-/CD69^- T cells. In some aspects, after culturing the T cells according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 ^-/CD69^- T cells.

In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express (i) one or more stem cell-like markers and (ii) one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least two stem cell-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least three stem cell-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express at least four stem cell-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells (e.g., T cells and/or NK cells) that express one or more stem cell-like markers and at least two effector-like markers.

In some aspects, the stem cell-like marker is selected from the group consisting of cd45ra+, cd62l+, CCR7+, cd27+, cd28+, bach2+, lef1+, tcf7+, and any combination thereof. In some aspects, the stem cell-like markers comprise cd45ra+, cd62l+, ccr7+ and tcf7+, or any combination thereof. In some aspects, the cell expresses CD45RO ^{Low and low}. In some aspects, the stem cell-like markers comprise one or more genes listed herein as part of a genetic stamp (see, e.g., gattinoni, L. Et al, nat Med 17 (10): 1290-97 (2011) or Galletti et al, nat Immunol 21,1552-62 (2020)).

In some aspects, the stem cell-like marker comprises a gene expressed in the WNT signaling pathway. In some aspects, the stem cell-like markers comprise one or more genes ：GNG2、PSMC3、PSMB10、PSMC5、PSMB8、PSMB9、AKT1、MYC、CLTB、PSME1、DVL2、PFN1、H2AFJ、LEF1、CTBP1、MOV10、HIST1H2BD、FZD3、ITPR3、PARD6A、LRP5、HIST2H4A、HIST2H3C、HIST1H2AD、HIST2H2BE、HIST3H2BB、DACT1 selected from the group consisting of and any combination thereof. In some aspects, the stem cell-like marker comprises one or more genes selected from the group consisting of: MYC, AKT1, LEF1, and any combination thereof.

In some aspects, the effector-like marker is selected from pstat5+, tat5+, pstat3+, tat3+, and any combination thereof. In some aspects, the effector-like marker comprises STAT targets ：AKT1、AKT2、AKT3、BCL2L1、CBL、CBLB、CBLC、CCND1、CCND2、CCND3、CISH、CLCF1、CNTF、CNTFR、CREBBP、CRLF2、CSF2、CSF2RA、CSF2RB、CSF3、CSF3R、CSH1、CTF1、EP300、EPO、EPOR、GH1、GH2、GHR、GRB2、IFNA1、IFNA10、IFNA13、IFNA14、IFNA16、IFNA17、IFNA2、IFNA21、IFNA4、IFNA5、IFNA6、IFNA7、IFNA8、IFNAR1、IFNAR2、IFNB1、IFNE、IFNG、IFNGR1、IFNGR2、IFNK、IFNL1、IFNL2、IFNL3、IFNLR1、IFNW1、IL10、IL10RA、IL10RB、IL11、IL11RA、IL12A、IL12B、IL12RB1、IL12RB2、IL13、IL13RA1、IL13RA2、IL15、IL15RA、IL19、IL2、IL20、IL20RA、IL20RB、IL21、IL21R、IL22、IL22RA1、IL22RA2、IL23A、IL23R、IL24、IL26、IL2RA、IL2RB、IL2RG、IL3、IL3RA、IL4、IL4R、IL5、IL5RA、IL6、IL6R、IL6ST、IL7、IL7R、IL9、IL9R、IRF9、JAK1、JAK2、JAK3、LEP、LEPR、LIF、LIFR、MPL、MYC、OSM、OSMR、PIAS1、PIAS2、PIAS3、PIAS4、PIK3CA、PIK3CB、PIK3CD、PIK3CG、PIK3R1、PIK3R2、PIK3R3、PIK3R5、PIM1、PRL、PRLR、PTPN11、PTPN6、SOCS1、SOCS2、SOCS3、SOCS4、SOCS5、SOCS7、SOS1、SOS2、SPRED1、SPRED2、SPRY1、SPRY2、SPRY3、SPRY4、STAM、STAM2、STAT1、STAT2、STAT3、STAT4、STAT5A、STAT5B、STAT6、TPO、TSLP、TYK2 selected from the group consisting of and any combination thereof.

In some aspects, the effector-like marker is an effector memory-related gene comprising one or more genes ：TBCD、ARL4C、KLF6、LPGAT1、LPIN2、WDFY1、PCBP4、PIK343、FAS、LLGL2、PPP2R2B、TTC39C、GGA2、LRP8、PMAIP1、MVD、IL15RA、FHOD1、EML4、PEA15、PLEKHA5、WSB2、PAM、CD68、MSC、TLR3、S1PR5、KLRB1、CYTH3、RAB27B、SCD5 selected from the group consisting of and any combination thereof. In some aspects, the effector-like marker comprises one or more genes selected from the group consisting of: KLF6, FAS, KLRB1, TLR3 and any combination thereof.

In some aspects, the cell composition comprises an increased percentage of cd45ra+, stat5+, and stat3+ immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of cd62l+, stat5+, and stat3+ immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of tcf7+, stat5+, and stat3+ immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of cd45ra+, cd62l+, ccr7+, cd27+, cd28+, bach2+, lef1+, tcf7+, stat5+, and stat3+ immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of cd45ra+, cd62l+, ccr7+, cd27+, cd28+, bach2+, lef1+, tcf7+, pstat5+, stat5+, pstat3+ and stat3+ immune cells (e.g., T cells and/or NK cells). In some aspects, the cell composition comprises an increased percentage of cd45ra+, cd45ro-, cd62l+, ccr7+, cd27+, cd28+, bach2+, lef1+, tcf7+, pstat5+, stat5+, pstat3+ and stat3+ immune cells (e.g., T cells and/or NK cells).

In some aspects, immune cells (e.g., T cells and/or NK cells) comprise one or more markers selected from the group consisting of cd45ra+, cd62l+, ccr7+, cd27+, cd28+, bach2+, lef1+, tcf7+, and any combination thereof, and one or more markers selected from the group consisting of pstat5+, tat5+, pstat3+, stat3+, and any combination thereof. In some aspects, the immune cells (e.g., T cells and/or NK cells) express CD45RO ^{Low and low}. In some aspects, immune cells (e.g., T cells and/or NK cells) comprise one or more markers selected from the group consisting of cd45ra+, cd62l+, ccr7+, cd27+, cd28+, bach2+, lef1+, tcf7+, and any combination thereof, and one or more effector-like markers. In some aspects, the immune cells (e.g., T cells and/or NK cells) comprise one or more stem cell-like markers and one or more markers selected from pstat5+, stat5+, pstat3+, stat3+, and any combination thereof. In some aspects, the immune cells (e.g., T cells and/or NK cells) express CD45RO ^{Low and low}.

Some aspects of the present disclosure are directed to a cell composition comprising an immune cell population, wherein the immune cell population comprises (i) a first subpopulation of immune cells (e.g., stem cell-like immune cells) that express one or more stem cell-like markers and (ii) a second subpopulation of immune cells (e.g., effector-like immune cells) that express one or more effector-like markers, wherein the immune cell population comprises a higher percentage (i.e., number of stem cell-like immune cells/total number of immune cells) of the first subpopulation of immune cells that express the one or more stem cell-like markers as compared to an immune cell population cultured using conventional methods (e.g., in a medium having less than 5mM potassium ions). In some aspects, the immune cell is a T cell. In some aspects, the immune cells are NK cells. In some aspects, immune cells (e.g., T cells and/or NK cells) cultured according to the methods disclosed herein produce these cell compositions.

In some aspects, immune cells (e.g., T cells and/or NK cells) cultured according to the methods disclosed herein have increased expression (e.g., a higher percentage of immune cells (e.g., T cells and/or NK cells) that express GZMB, MHC-II, LAG3, TIGIT, and/or NKG 7), and decreased expression (e.g., a lower percentage of immune cells (e.g., T cells and/or NK cells) that express IL-32). The highest cells of NKG7 have been shown to be the preferred killers (Malarkannan et al, 2020Nat. Immuno.) whereas the higher IL-32 cells have been shown to have activation-induced cell death (Goda et al, 2006Int. Immunol). In some aspects, immune cells (e.g., T cells and/or NK cells) with higher GZMB, MHC-II, LAG3, TIGIT, and/or NKG7 expression are cd8+ T cells that express effector-like markers. In some aspects, immune cells (e.g., T cells and/or NK cells) with lower IL-32 expression are cd8+ T cells that express effector-like markers.

In some aspects, the cell composition obtained by any of the methods described herein (e.g., yield of the final cell product for use as a therapy) comprises at least about 1×10⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹ or 5x 10 ⁹ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 1×10³、5×10³、1×10⁴、5×10⁴、1×10⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹ or 5x 10 ⁹ stem cell-like cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、10×10¹⁰、11×10¹⁰、12×10¹⁰、13×10¹⁰、14×10¹⁰ or 15 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 1 x 10 ⁶ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 1 x 10 ⁶ stem cell-like cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 1 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 2 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 3x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 4 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 5 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 6 x10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 7 x10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 8 x10 ¹⁰ cells. in some aspects, the cell composition obtained by any of the methods described herein comprises at least about 9 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 10 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 11 x 10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 12 x10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 13 x10 ¹⁰ cells. In some aspects, the cell composition obtained by any of the methods described herein comprises at least about 14 x10 ¹⁰ cells. in some aspects, the cell composition obtained by any of the methods described herein comprises at least about 15 x 10 ¹⁰ cells. In some aspects, the cell yield represents the total number of cd3+ cells.

In some aspects, the methods disclosed herein result in a composition comprising at least about 1×10 ¹⁰, at least about 1.1×10 ¹⁰, at least about 1.2×10 ¹⁰, at least about 1.3×10 ¹⁰, at least about 1.4×10 ¹⁰, at least about 1.5×10 ¹⁰, at least about 1.6×10 ¹⁰, at least about 1.7×10 ¹⁰, at least about 1.8×10 ¹⁰, at least about 1.9×10 ¹⁰, or at least about 2.0×10 ¹⁰ cells by culturing in the presently disclosed media for at least about 10 days. In some aspects, the methods disclosed herein produce a composition comprising at least about 1.8x10 ¹⁰ cells by culturing in the presently disclosed medium for at least about day 10.

In some aspects, the cell composition comprises at least about 1×10 ¹⁰, at least about 1.1×10 ¹⁰, at least about 1.2×10 ¹⁰, at least about 1.3×10 ¹⁰, at least about 1.4X10 ¹⁰, at least about 1.5X10 ¹⁰, at least about 1.6X10 ¹⁰, at least about 1.7X10 ¹⁰, At least about 1.8X10 ¹⁰, at least about 1.9X10 ¹⁰, or at least about 2.0X10 ¹⁰ stem cell-like cells. In some aspects, the methods disclosed herein result in a composition comprising at least about 1×10 ¹⁰, at least about 1.1×10 ¹⁰, at least about 1.2×10 ¹⁰, at least about 1.3×10 ¹⁰, by at least about 10 days of culture, at least about 1.4X10 ¹⁰, at least about 1.5X10 ¹⁰, at least about 1.6X10 ¹⁰, at least about 1.7X10 ¹⁰, A composition of at least about 1.8x10 ¹⁰, at least about 1.9x10 ¹⁰, or at least about 2.0x10 ¹⁰ stem cell-like cells. in some aspects, the methods disclosed herein produce a composition comprising at least about 1.8x10 ¹⁰ stem cell-like cells by culturing in the presently disclosed medium for at least about day 10.

In some aspects, the methods disclosed herein result in a composition comprising at least about 80%, at least about 85%, at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% viable immune cells. In some aspects, the methods disclosed herein produce a composition comprising at least about 1.8x10 ¹⁰ stem cell-like cells having at least about 94% cell viability.

IV. method of treatment

Some aspects of the disclosure are directed to methods of administering an immune cell described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, 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, the methods comprising administering to the subject an immune cell described herein. For example, in some aspects, disclosed herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an immune cell that has been engineered to express a ROR1 binding protein (e.g., CAR) and over-express a c-Jun protein. In some aspects, the disease or condition comprises a tumor (i.e., cancer). In some aspects, the methods comprise stimulating a T cell-mediated immune response in a subject to a target cell population or tissue, comprising administering an immune cell as described herein. In some aspects, the target cell population comprises a tumor. In some aspects, the tumor is a solid tumor.

In some aspects, administration of an immune cell described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) reduces the tumor volume of a subject as compared to a reference tumor volume. In some aspects, the reference tumor volume is the tumor volume of the subject prior to administration. In some aspects, the reference tumor volume is the tumor volume of the corresponding subject not receiving administration. In some aspects, the tumor volume of 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 administration as compared to a reference tumor volume.

In some aspects, treating the tumor comprises reducing the tumor weight of the subject. In some aspects, when administered to a subject, administration of an immune cell described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) can reduce tumor weight in 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 administration as compared to a reference tumor weight. In some aspects, the reference tumor weight is the tumor weight of the subject prior to administration. In some aspects, the reference tumor weight is the tumor weight of the corresponding subject not receiving the administration.

In some aspects, administering immune cells described herein (e.g., modified to express a ROR1 binding protein (e.g., an anti-ROR 1 CAR) and having increased levels of c-Jun protein, and cultured using the methods provided herein) to, for example, a subject suffering from a tumor, can increase the number and/or percentage of T cells (e.g., CD4 ⁺ or CD8 ⁺) in the blood of the subject. In some aspects, the T cell is a modified immune cell. In some aspects, the number and/or percentage of T cells in blood (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided 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%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more, as compared to a reference (e.g., a subject not receiving the administration or a corresponding value in the same subject prior to administration). In some aspects, the number and/or percentage of T cells in blood 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 as compared to a reference (e.g., a corresponding subject not receiving administration).

In some aspects, administering immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) to, for example, a subject afflicted with a tumor, can increase the number and/or percentage of T cells (e.g., CD4 ⁺ or CD8 ⁺) in the tumor and/or Tumor Microenvironment (TME) of the subject. In some aspects, the T cell is a modified immune cell. In some aspects, the number and/or percentage of T cells (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein) in a tumor and/or TME 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%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290% or at least about 300% or more, as compared to a reference (e.g., a subject not receiving the administration or a corresponding value in the same subject prior to administration). In some aspects, the number and/or percentage of T cells in a tumor and/or TME 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 as compared to a reference (e.g., a corresponding subject not receiving administration).

In some aspects, administering an immune cell described herein (e.g., modified to express a ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) to a subject, e.g., suffering from a tumor, can increase the duration of the immune response in the subject relative to the duration of the immune response in a corresponding subject not receiving administration (e.g., treated with a corresponding cell lacking c-Jun protein expression). In some aspects, the immune response duration 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 as compared to a reference (e.g., a corresponding subject not receiving administration). In some aspects, the immune response duration 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 as compared to a reference (e.g., a corresponding subject not receiving administration). In some aspects, the duration of the immune response is increased by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years as compared to a reference (e.g., a corresponding subject not receiving administration).

As described herein, the immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) can be used to treat a variety of cancers. Non-limiting examples of cancers that can be treated include adrenocortical cancer, advanced cancer, anal cancer, aplastic anemia, cholangiocarcinoma, bladder cancer, bone metastases, brain tumors, brain cancer, breast cancer, childhood cancer, primary unknown cancer, castleman's disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, ewing's tumor family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hodgkin's disease, kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal carcinoma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, and combinations thereof small cell lung cancer, lung carcinoid tumors, cutaneous lymphomas, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and sinus cancers, nasopharyngeal cancers, neuroblastoma, non-hodgkin's lymphoma, oral and oropharyngeal cancers, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue sarcoma, basal cell and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulval cancer, fahrenheit macroglobulinemia, wilms' tumors, secondary cancers caused by cancer treatment, and combinations thereof. In some aspects, the cancer is associated with a solid tumor.

In some aspects, immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) are used in combination with other therapeutic agents (e.g., anti-cancer agents and/or immunomodulators). Thus, in some aspects, a method of treating a disease or disorder disclosed herein (e.g., a tumor) comprises administering an immune cell described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) in combination with one or more additional therapeutic agents. Such agents may include, for example, chemotherapeutic drugs, targeted anti-cancer therapies, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgery, radiation therapies, co-stimulatory molecule activators, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof.

In some aspects, the immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) are administered to a subject prior to or after administration of an additional therapeutic agent. In some aspects, an immune cell described herein (modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) is administered to a subject concurrently with an additional therapeutic agent. In some aspects, the immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) and additional therapeutic agents can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In some aspects, the immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) and additional therapeutic agent are administered concurrently as separate compositions.

In some aspects, an immune cell described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) is administered to a subject prior to or after administration of a BCR-ABL/Src kinase inhibitor, such as dasatinib (dasatinib) or pluratinib (ponatinib). In some aspects, dasatinib or pluratinib can be administered to reduce cytotoxicity (e.g., cytokine storm) that can sometimes occur in CAR-T cell therapies. Src kinases are known to play an important role in physiological T cell activation. In agreement with this, dasatinib has been shown to significantly inhibit antigen-specific physiological T cell activation, proliferation, cytokine production and degranulation in a dose-dependent manner (Schade et al Blood 111:1366-77,2008; weichsel et al CLIN CANCER RES14:2484-91, 2008), and has been shown to reduce cytotoxicity in CAR-T cell therapies (see, e.g., US 2021032363).

In some aspects, the immune cells described herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) are used in combination with standard of care therapies (e.g., surgery, radiation, and chemotherapy). The methods described herein may also be used as maintenance therapies, for example therapies intended to prevent tumorigenesis or recurrence.

In some aspects, the immune cells provided herein (e.g., modified to express ROR1 binding protein and having increased levels of c-Jun protein, and cultured using the methods provided herein) are 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: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secreting cell vaccines, cpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, such as by inhibiting CTLA-4 and/or PD 1/PD-L2 pathways and/or clearing or blocking tregs or other immunosuppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immunomodulation, e.g., using agonists that stimulate CD-137, OX-40, and/or CD40 or GITR pathways and/or that stimulate T cell effector function; a therapy to increase the frequency of anti-tumor T cells systemically; therapies that eliminate or inhibit tregs, such as tregs in tumors, for example, using CD25 antagonists (e.g., daclizumab) or by ex vivo anti-CD 25 bead elimination; therapies that affect the inhibition of bone marrow cell function in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically engineered cells, e.g., cells engineered to express a chimeric antigen receptor (CAR-T therapy); therapies that inhibit metabolic enzymes such as Indoleamine Dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse/prevent T cell anergy or depletion; a therapy that triggers innate immune activation and/or inflammation at a tumor site; administering an immunostimulatory cytokine; blocking immunosuppressive cytokines; or any combination thereof.

In some aspects, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors include the following: anti-PD 1 antibodies (e.g., nivolumab,) Pembrolizumab (pembrolizumab,MK-3475), pilizumab (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-L1 antibodies (e.g., atizulizumab,RG7446; MPDL3280A; RO 5541267), devaluzumab (durvalumab, MEDI4736,) BMS-936559, avermectin (avelumab,) LY3300054, CX-072 (procaim-CX-072), FAZ053, KN035, MDX-1105 and combinations thereof); and anti-CTLA-4 antibodies (e.g., ipilimumab (ipilimumab,) Tremelimumab (tremelimumab, tiximab (ticilimiumab); CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof).

In some aspects, the anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through a particular immune checkpoint pathway). In some aspects, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX 40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD 137) agonist (e.g., an anti-CD 137 antibody), a GITR agonist (e.g., an anti-GITR antibody), a TIM3 agonist (e.g., an anti-TIM 3 antibody), or a combination thereof.

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 well described in the literature. See, e.g., sambrook et al (1989) Molecular Cloning A Laboratory Manual (2 nd edition; cold Spring Harbor Laboratory Press); sambrook et al (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); glover code, (1985) DNA Cloning, roll I and roll II; gait, 1984, oligonucleotide Synthesis; mullis et al, U.S. Pat. nos. 4,683,195; hames and Higgins, inc. (1984) Nucleic Acid Hybridization; hames and Higgins, chem. (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; paper Methods In Enzymology (ACADEMIC PRESS, inc., n.y.); miller and Calos et al (1987) GENE TRANSFER Vectors For MAMMALIAN CELLS, (Cold Spring Harbor Laboratory); wu et al, methods In Enzymology, volume 154 and volume 155; mayer and Walker, inc. (1987) Immunochemical Methods IN CELL AND Molecular Biology (ACADEMIC PRESS, london); weir and Blackwell, inc. (1986) Handbook Of Experimental Immunology, volume I-IV ;Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,(1986);Crooks,Antisense drug Technology:Principles,strategies and applications,, version 2 CRC Press (2007), ausubel et al (1989) Current Protocols in Molecular Biology (John Wiley and Sons, baltimore, md.).

All references cited above and all references and amino acid or nucleotide sequences (e.g., genBank numbers and/or Uniprot numbers) cited herein are incorporated by reference in their entirety.

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

Examples

Example 1: CAR transduction efficiency analysis

To assess the effect of metabolic reprogramming media on CAR transduction efficiency, human cd4+ and cd8+ T cells were transduced with an anti-ROR 1 CAR construct in Metabolic Reprogramming Media (MRM) or T cell conditioned media (i.e., TCM). An exemplary method for carrying out the present embodiment is provided below.

Preparation of the culture Medium

T cell conditioned medium (TCM) used as a control was supplemented with immune cell serum replacement (Thermo Fisher), 2mM L-glutamine (Gibco), 2mM Glutamax (Gibco), MEM nonessential amino acid solution (Gibco), sodium pyruvate (Gibco); IL-2, 200IU/mL; IL-7, 1200IU/ml; IL-15, 200IU/ml.

TCM supplemented with varying concentrations of sodium, potassium, glucose and calcium was used to generate Metabolic Reprogramming Media (MRM). The final concentration is in the following range: naCl (40-80 mM), KCl (40-80 mM), calcium (0.5-2.8 mM), glucose (10-24 mM) and osmolality (about 250-260 mOsmol). See table 10.

TABLE 10 Medium with varying concentrations of Potassium, sodium, glucose and calcium

* The tension is calculated based on the following formula: 2× (K concentration+NaCl concentration)

Lentiviral vector (LVV) construction and lentiviral production

Generating an anti-ROR 1CAR construct comprising: (i) an anti-ROR 1CAR (derived from an R12 antibody) (referred to herein as "R12 CAR") (SEQ ID NO: 83), (ii) a truncated EGFR ("EGFRt") (SEQ ID NO: 24), and (iii) a wild-type c-Jun protein (SEQ ID NO: 13) (referred to herein as "c-Jun-R12 CAR"; SEQ ID NO: 86). See table 14 (below). The c-Jun-R12 CAR construct is designed such that when transduced in a cell (e.g., T cell), the transduced cell will exhibit increased c-Jun protein expression as well as surface expression of the anti-ROR 1CAR and EGFRt. As a control, a corresponding anti-ROR 1CAR construct comprising truncated CD19 ("CD 19 t") instead of c-Jun (referred to herein as a "control CD19t-R12 CAR") was also generated. See Terakura, S. et al, blood 119 (1): 72-82 (2012), which is incorporated herein by reference in its entirety.

Lentiviral vectors were pseudotyped with VSV-G envelope and generated by transient transfection of HEK293T cells. The semi-finished product was kept at 2-8 ℃ for no more than 24 hours, then 1mL LVV aliquots were filled and stored at-80 ℃. LVV aliquots were thawed on ice followed by T cell transduction.

T cell isolation

Cd4+ and cd8+ T cells were isolated from three healthy donors and frozen using suppliers BloodWorks (Seattle, WA, USA) and AllCells (Alameda, CA, USA). The supplier obtains and maintains all appropriate consent from the donor. To isolate cd4+ and cd8+ T cells, samples were collected via hemocytometer in which the cd4+ and cd8+ cells were isolated separately, in the order of first positively selecting cd8+ T cells followed by positive selection of cd4+ T cells from the effluent of the CD8 selection (flow-through). Isolated cd4+ or cd8+ T cells were frozen in 20e+06 cells (AllCells) or 50e+06 cells (BloodWorks) per vial.

Cell culture and transduction

Human cd4+ and cd8+ T cells (i.e., from the supplier) were thawed in an appropriate medium (i.e., TCM or MRM) and combined at a 1:1 ratio. The combined donor CD4+ and CD8+ T cells were centrifuged at 300x g for 5 minutes and resuspended in an appropriate medium (i.e., T cell conditioned medium or MRM) supplemented with IL-2, IL-7 and IL-15. The T cells were then activated using CD3/CD28 TRANSACT ^TM (Miltenyi Biotec Inc.). After 24 hours of activation in TCM or MRM (i.e., day 1), T cells were transduced with LVV as described above comprising an anti-ROR 1 CAR construct (i.e., "c-Jun-R12 CAR" and "control CD19T-R12 CAR"). Untransduced T cells were used as controls. On the next day after transduction (i.e., day 2), fresh medium (i.e., TCM or MRM) was added to dilute TRANSACT ^TM and terminate T cell activation. The transduced T cells were further expanded for another five days (i.e., day 7) and then subsequently analyzed or cryopreserved in liquid nitrogen for long term storage.

Transduction efficiency analysis

To compare CAR transduction efficiencies of the different groups (see table 11), flow cytometry was used to determine the percentage of cd4+ and cd8+ T cells expressing: (i) c-Jun, anti-ROR 1R12 scFv and EGFRt comprising the sequence set forth in SEQ ID NO:24, or (ii) c-Jun, anti-ROR 1R12 scFv and truncated CD19.

TABLE 11 Experimental group

Group number	Description of the invention
		1	Untransduced T cells cultured in TCM
2	T cells transduced with control CD19T-R12 CAR and cultured in TCM
		3	T cells transduced with c-Jun-R12 CAR and cultured in TCM
4	Untransduced T cells cultured in MRM
		5	T cells transduced with control CD19T-R12 CAR, cultured in MRM
6	T cells transduced with c-Jun-R12 CAR, cultured in MRM

As shown in fig. 1A-1C, transduction efficiencies between control CD19t-R12 CAR and C-Jun-R12 CAR were comparable in TCM and MRM groups and in each donor. Also, as between TCM and MRM groups, the percentage of transduced T cells (i.e., expressing both EGFRt and R12 CAR) is comparable. No significant differences in the percentage of transduced cd4+ and cd8+ T cells from the different groups were observed within each donor (see fig. 2A-2C). Interestingly, C-Jun-R12 CAR transduced T cells from the MRM group expressed significantly higher levels of C-Jun protein expression than corresponding transduced T cells from the TCM group (see fig. 3A-3C). The increased expression was specific for the c-Jun protein and not global for other transgenes (i.e., R12 CAR and EGFRt).

These results indicate that the anti-ROR 1 CAR constructs described herein are able to transduce into T cells to a similar extent under both culture conditions. The results further demonstrate that metabolic reprogramming media conditions may be suitable for selectively increasing c-Jun protein expression of the anti-ROR 1 CAR constructs provided herein.

Example 2: analysis of Stem cell-like phenotypic expression

To assess the effect of metabolic reprogramming media on stem cell-like properties of c-Jun-overexpressing transduced T cells, human cd4+ and cd8+ T cells were transduced with an anti-ROR 1 CAR construct (i.e., c-Jun-R12 CAR or control CD19T-R12 CAR) as described in example 1. Next, after allowing the cells to re-expand for four to five days (i.e., day 6 or day 7), stem properties of the transduced T cells were assessed using flow cytometry.

Briefly, T cells were first washed with cell staining buffer and stained with anti-CCR 7 at 37 ℃ for 15 minutes. Next, T cells were washed again and then a master mix of antibodies to several other antigens (as detailed below) was added to the cells and incubated in the dark for 25 minutes at room temperature. Cells were then washed with cell staining buffer and permeabilized with FOXP3 staining kit (ebioscience) according to the manufacturer's protocol. After fixation, cells were blocked with pre-diluted normal mouse serum (Jackson Immuno Research- # 015-000-120) and normal rabbit serum (Jackson ImmunoResearch- # 011-000-120) in the dark at room temperature for 15 minutes. Cells were then stained with a 2x antibody mixture of TCF7 and c-Jun in the dark at room temperature for 30 minutes. After thoroughly washing the cells, they were analyzed by flow cytometry on a Cytek Aurora Spectral flow cytometer and analyzed using FlowJo software (TreeStar, ashland, OR).

The following is a list ：CD8(Thermo-#58-0088-42)、CD4(BD-#612936)、CD27(BD-#612829)、CD3(Thermo-#612896)、CD28(Biolegend-#302936)、CD62L(BD-#740301)、R12Anti-Id(Genscript-#48F6H5E1)、EGFR(ΒioLegend-#98812)、CD 45RO(BioLegend-#566143)、CD39(BioLegend-#328236)、TCF7(C ell Signaling-#9066S)、c-Jun(Cell Signaling-#15683S)、CCR7(BD-#562381)、CD45RA(BD-#560673)、LAG-3(Thermo-#67-2239-42)、TIM-3(Thermo-#78-3109-42)、TIGIT(Thermo-#46-9500-42)、PD-1(Thermo-#25-2799-42). for evaluating dry labeled antibodies specifically, "stem cell-like" cells are defined as described herein: CD45RO ^-CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺TCF7⁺.

As shown in fig. 4A-4C, cd4+ T cells transduced with the anti-ROR 1 CAR construct were more stem cell-like in MRM with respect to their phenotypic expression compared to cells from the TCM group. This is generally true whether the cd4+ T cells were transduced with either a C-Jun-R12 CAR or a control CD19T-R12CAR (see the last two bars in fig. 4A-4C). Likewise, cd8+ T cells transduced in MRM are generally more stem-like (at least for cd8+ T cells derived from donors #1 and # 2; see fig. 4D and 4E) compared to corresponding cells transduced in TCM. However, unlike cd4+ T cells, a consistent increase in stem cell-like cells was observed when cd8+ T cells were transduced with c-Jun-R12 CARs, as compared to control CD19T-R12 CARs. Thus, among cd8+ T cells, the largest percentage of stem cell-like cells was observed when cd8+ T cells were transduced with c-Jun-R12 CAR in MRM. As shown in fig. 4G-4I, cd4+ T cells transduced with the anti-ROR 1 CAR construct contained a higher proportion of naive and stem cell memory T cells in MRM (as demonstrated by CCR7 ⁺ and CD45RA ⁺ expression) than cells from the TCM group (comparing the first and last two bars, respectively). In general, an increase in the proportion of naive and stem cell memory T cells was also observed when cd4+ T cells were transduced with c-Jun-anti-ROR 1 CAR as compared to control anti-ROR 1 CAR (compare the second and fourth bars in fig. 4G-4I with the first and third bars). Similar results were observed in cd8+ T cells (see fig. 4J-4L). Thus, among cd4+ T cells and cd8+ T cells, the greatest percentage of naive and stem cell memory T cells are generally observed when transduced with a c-Jun-anti-ROR 1 CAR in MRM.

These results highlight the usefulness of the metabolic reprogramming media described herein in generating poorly differentiated (i.e., more stem cell-like) transduced cd4+ and cd8+ T cells. Also, at least for cd8+ T cells, the results further indicate that overexpression of transcription factors such as c-Jun can further improve the stem cell-like properties of transduced T cells.

Example 3: phenotypic and functional analysis

To further assess the effect of MRM on ROR1 CAR T cells, human cd4+ and cd8+ T cells were transduced with the anti-ROR 1 CAR construct and expanded as described in example 1. On day 6 or 7, transduced cd4+ and cd8+ T cells were analyzed phenotypically (e.g., expressing different surface markers such as CD39, LAG3, PD1, TIGIT, and TIM 3) and functionally (e.g., IL-2 and/or IFN- γ production and in vitro killing following primary and/or long-term antigen stimulation).

Phenotypic expression

Expression of surface markers on transduced cd4+ and cd8+ T cells was assessed using Cytek Aurora Spectral flow cytometry and analyzed using FlowJo software (TreeStar, ashland, OR).

Cytotoxicity and cytokine secretion

Cytolytic activity of transduced T cells was measured using an in vitro killing assay. Briefly, transduced T cells ("effector") were co-cultured with target tumor cells ("target") at effector to target ratios of 1:4, 1:16, 1:64, and 1:128, and scanned every 6 hours using IncuCyte at 4x magnification (cytolytic activity was measured by tracking the number of red nuclei representing target tumor cells). After 24 hours of co-culture, supernatants were collected from different conditions and frozen at-80 ℃ for later cytokine analysis. The cell-containing plates were then returned to IncuCyte for continued periodic scanning.

For cytokine secretion analysis, previously frozen supernatants were thawed and levels of certain cytokines (e.g., IL-2 and IFN-g) were assessed using MesoScaleDiscovery (MSD) multiplexing platform and measured on a MSD Meso Sector S600 machine according to the manufacturer's protocol.

Continuous re-stimulus analysis

In this analysis, transduced cd4+ and cd8+ T cells were serially re-stimulated with a549 NLR target cells every three or four days. The T cells were seeded at an E:T ratio of 1:1 for a total of 2 to 4 rounds of stimulation. The density of 3×10 ⁵ transduced T cells/mL was maintained throughout the study. To set up each round of stimulation, T cells were stained with the following markers and analyzed using flow cytometry to calculate the proportion of transduced T cell populations present in the co-culture: CD45, CD3, CD4, CD8, CAR and EGFRt (SEQ ID NO: 24). An aliquot of each sample was retained for titration Incucyte killing analysis as described above.

Results

As shown in fig. 5A-5E, 6A-6E, and 7A-7E, significant differences in the expression levels of various surface markers were observed between cd4+ T cells transduced with the anti-ROR 1 CAR construct in MRM as compared to the corresponding cells transduced in TCM. Similar results were observed for cd8+ T cells (see fig. 5F-5J, 6F-6J, and 7F-7J), further confirming that potassium concentrations present during transduction can have an effect on transduced T cells.

Furthermore, in addition to the phenotypic differences, significant functional differences were also observed in transduced T cells. As shown in fig. 8A-8C, T cells transduced and cultured in MRM produced higher amounts of IL-2 after primary antigen stimulation than the corresponding cells transduced and cultured in TCM. Also, as previously observed in example 1, increased c-Jun protein expression in transduced T cells also resulted in more IL-2 secretion. For example, in the TCM and MRM groups, T cells transduced with c-Jun-R12 CAR produced higher levels of IL-2 after primary stimulation than corresponding cells transduced with control CD19T-R12 CAR. Thus, maximum IL-2 production is generally observed in T cells modified to overexpress c-Jun and cultured in MRM.

Similar results were observed after continuous/long term antigen stimulation. As shown in fig. 9A-9C, T cells transduced and cultured in MRM maintained their ability to produce IFN- γ after the last round of antigen stimulation compared to the corresponding cells transduced and cultured in TCM. Also, c-Jun-R12 CAR transduced T cells from the MRM group maintained the ability to produce IFN- γ much longer than transduced cells from other groups. Regarding cytotoxicity of transduced cells after multiple antigen stimulations, T cells transduced and cultured in MRM maintained their ability to kill tumor cells much longer than corresponding cells from TCM group (fig. 10A-10E).

In summary, the above results demonstrate that CAR T cells modified to overexpress c-Jun (e.g., with ROR1 CAR and c-Jun overexpression) cultured in MRM allow for the generation of stem cell-like transduced T cells that remain functional even after long term antigen stimulation.

Example 4: analysis of in vivo anti-tumor efficacy

To assess whether the above-described anti-ROR 1 CAR T cells (i.e., transduced and cultured in MRM) are functional in vivo, anti-tumor activity of the anti-ROR 1 CAR T cells was tested in a mouse animal model. Briefly, tumor cells (i.e., ROR1 positive H1975NSCLC cell line) were subcutaneously implanted with NOD SCID GAMMA having MHC class I and II knockouts; NOD.Cg-PRKDCSCID IL2rgtm1Wjl/SzJ (NSG) mice in the flank. When the tumor reached about 125mm ³, mice were infused intravenously with one of the following: (i) A mock (non-transduced) T cell cultured in MRM, (ii) a T cell transduced with a control CD19T-R12 CAR (i.e., R12CAR without c-Jun) and cultured in MRM; and (3) T cells transduced with a c-Jun-R12CAR (i.e., an R12CAR with c-Jun) and cultured in MRM. The T cells were administered to mice at one of two doses: (a) 5 x 10 ⁶ car+ T cells or (b) 2.5 x 10 ⁶ car+ T cells. Antitumor activity was assessed by measuring tumor volume (using calipers) and survival, and treatment-related toxicity was assessed by measuring animal body weight.

As shown in fig. 11A and 11B, animals treated with either the mock T cells or the control anti-ROR 1 CAR T cells (i.e., not overexpressing c-Jun) failed to control tumor growth and eventually died from the tumor (see survival data of fig. 11E and 11F). In contrast, animals treated with anti-ROR 1 CAR T cells overexpressing c-Jun (at both doses) showed significantly greater tumor control and survived the entire experimental duration (see fig. 11A, 11B, 11E and 11F). In addition, there was no treatment-related toxicity, as animals treated with anti-ROR 1 CAR T cells overexpressing C-Jun maintained their body weight throughout the duration of the experiment (see fig. 11C and 11D). Consistent with the improved anti-tumor data, anti-ROR 1 CAR T cells transduced and cultured in MRM that overexpressed c-Jun also exhibited long-term persistence in vivo (fig. 12).

These results confirm earlier in vitro data and demonstrate that the methods described herein (e.g., transduction and culture in medium comprising potassium ions at concentrations above 5 mM) can be used to generate potent tumor-specific T cells.

Example 5: clinical development

FIH, phase 1, single arm, open label, dose escalation and expansion, multicenter studies designed to assess safety, PK and anti-tumor activity of anti-ROR 1CAR T cells overexpressing c-Jun in patients with ROR1 positive recurrent and/or refractory TNBC and NSCLC will be performed. The main objective of the phase 1 study was to evaluate the safety and tolerability of c-Jun overexpressing anti-ROR 1CAR T cells in patients with recurrent/refractory TNBC and NSCLC and to determine RP2D of c-Jun overexpressing anti-ROR 1CAR T cells. A secondary objective of the phase 1 study was to evaluate the anti-tumor activity of the anti-ROR 1CAR T cells overexpressing c-Jun, and to evaluate PK (e.g., expansion and persistence) of the anti-ROR 1CAR T cells overexpressing c-Jun in peripheral blood samples.

Proposed phase 1 design

This would be a single arm, open label, dose escalation and expansion, multicenter study designed to assess the safety, PK and anti-tumor activity of anti-ROR 1 CAR T cells overexpressing c-Jun in patients with recurrent and/or refractory TNBC and NSCLC. During the dose escalation phase, only TNBC participants will be enrolled; during the amplification phase, TNBC and NSCLC study groups will be enrolled.

Participants who were found to be ROR1 positive by immunohistochemistry will be eligible for enrollment, with TNBC participants not passing 2-line therapy including checkpoint inhibitors and albumin-bound paclitaxel (abraxane), or NSCLC participants not passing 2-line therapy including targeted therapy of those patients with EGFR ⁺ and ALK ⁺ disease, as well as other qualifying criteria. Participants meeting all qualifying criteria will be enrolled and will undergo leukapheresis to enable product production. After successful product manufacture, the participants will enter the treatment phase and will receive 1 treatment cycle. The treatment cycle will involve lymphocyte scavenging chemotherapy with fludarabine and cyclophosphamide for 3 days, followed by IV administration of a single dose of the cell product at one of the dose levels prescribed by the regimen. The cell product will be administered several days after completion of lymphocyte removal chemotherapy, except after discussion with medical monitoring personnel, where clinical or logistical conditions require modification of this schedule to a later date.

The safety, disease state, additional anti-cancer therapies and survival of the participants will be followed up to 2 years after administration of the cell product. All participants receiving anti-ROR 1CAR T cells over-expressing c-Jun will be required to register in sponsored long-term follow-up (LTFU) studies at the completion or cessation of this study.

Example 6: transcriptome analysis

To assess the effect of MRM on T cells at the genetic level, single cell RNA-seq analysis was performed on T cells following continuous antigen stimulation analysis (see e.g. example 3). Prior to antigen stimulation, the T cells were either (i) transduced with a CD19T-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in control medium ("control ROR1 CAR") or (ii) transduced with a c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in MRM ("c-Jun ROR1 CAR"). At various time points in the continuous stimulation assay, RNA was extracted and T cell clusters enriched for stem cell-like genes and T cell terminal depletion (TTE) genes were assessed. For stem cell-like genes, the gene set described in Caushi et al, nature 596:126-132 (2021) was used. For the TTE gene, the gene set described in Oliveira et al, nature596:119-125 (2021) was used. Caushi et al and Oliveira et al are incorporated by reference herein in their entirety. The identification of stem cell-like clusters indicates the presence of relatively poorly differentiated cd8+ T cells, and the identification of TTE clusters indicates the presence of depleted/dysfunctional cd8+ T cells, particularly after continuous antigen stimulation.

As shown in fig. 13A and 13B, in control ROR1 CAR T cells cultured in control medium, the proportion of clusters enriched for stem cell-like gene sets was still low on days 7 and 10 of continuous antigen stimulation, while the proportion of clusters enriched for TTE gene sets increased with further stimulation (e.g., compare days 7 and 10 in fig. 13B). These results indicate that control ROR1 CAR T cells cultured in control medium differentiated to a greater extent and were depleted/dysfunctional following prolonged antigen stimulation. In contrast, c-Jun ROR1 CAR T cells cultured in MRM had a significantly higher proportion of stem cell-like enriched clusters (see fig. 13A) and a reduced proportion of TTE enriched clusters (see fig. 13B) on days 7 and 10 of continuous antigen stimulation, indicating the continued presence of a higher stem cell-like population.

In summary, the results provided above further confirm the therapeutic potential of the modified immune cells described herein (e.g., modified to overexpress c-Jun (e.g., with ROR1 CAR and c-Jun overexpression), cultured in MRM).

Example 7: additional transcriptome profiling of MRM and c-Jun overexpression against the effects of ROR1 CAR T cells

With respect to example 6 provided above, single cell RNA-seq analysis was then performed on transduced T cells described herein (e.g., anti-ROR 1 CAR T cells) following adoptive transfer into NSG MHC dKO mice bearing H1975 xenograft tumors. Specifically, when the tumor reached about 400mm ³, the animals received either c-Jun ROR1 CAR T cells (i.e., overexpressing c-Jun) cultured (i.e., transduced and expanded) in MRM (one of the following doses: 1e6, 2.5e6, and 5e6 cells/animal) or control ROR1 CAR T cells (i.e., not overexpressing c-Jun) (2.5e6 cells/animal) cultured in MRM. On day 13 after T cell injection, mice were sacrificed and excised tumors were dissociated using GENTLEMACS ^TM Octo Dissociator with heater and a human tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol, with the amount of enzyme R in the enzyme mixture reduced to 20%. The detached tumor cells were then understood for FACS sorting and single cell RNA sequencing was performed on Live mCD45 ^-hCD45⁺ T cells sorted from c-Jun ROR1 CAR T cells dosed with 2.5e6 cells/animal (n=5) and 5e6 cells/animal (n=2) and control ROR1 CAR T cells dosed with 2.5e6 cells/animal (n=5).

Samples were processed for single cell RNA sequencing using cell index (CITE-Seq) analysis of transcriptomes and epitopes by sequencing. The CITE-Seq analysis allows for simultaneous measurement of single cell RNA and cell surface proteins. Cells were stained with a mixture of fluorescent dye-binding antibodies for FACS sorting, total-SeqC antibodies for DNA binding to cell surface proteins of CITE-seq, and unique tag antibodies for cell barcodes (cell barcoding). Live mCD45 ^-hCD45⁺ T cells sorted from all unique bar coded samples were pooled together and loaded into Chromium Next GEM chip K using Chromium Next GEM single cell 5' v2 kit (10 x Genomics). After single cell capture and lysis, cDNA was synthesized and amplified and DNA was bound to antibodies that bind to cell surface proteins, ultimately generating a 5 'Gene Expression (GEX) and antibody-derived tag (ADT) library according to the manufacturer's protocol (10 x Genomics). Using NovaSeq 6000 system (Illumina), GEX and ADT libraries prepared from multiple channels of Chromium Next GEM chip K were quantified, pooled together, and sequenced.

Single Cell CITE-Seq data was processed using 10X Cell range software version 6.1.2 (10X Genomics), with GRCh38 (and the control R12 vector sequence and c-Jun sequence from the c-Jun ROR1 CAR vector added) as reference genome and default parameters. The cell-gene matrix was further processed using Seurat software package (Hao et al, 2021cell,184 (13): 3573-3587). Briefly, cells were first filtered (using a threshold of percent mitochondria, ncount_rna, nfeature_rna, and hashtag doublet), and then CD8 ⁺ and CD4 ⁺ T cells were isolated by gating on CD8 and CD4 protein expression measured with a CITE-Seq. CD8 ⁺ T cells from control ROR1 CAR T cells or c-Jun ROR1 CAR T cells from tumor treatment were combined for single cell transcriptome analysis. The filtered cell-gene matrix was normalized and scaled with variable feature selection. The effect of cell cycle heterogeneity was corrected by calculating cell cycle stage scores (g2m.score, s.score) using the CellCycleScoring function in Seurat, and then regressing the cell cycle stage scores. Genes associated with either of the two cell cycle stage scores (where Pearson correlation coefficient is greater than 0.3) were excluded from the selected features to further minimize the effects of cell cycle heterogeneity. Mitochondrial, ribosomal, TCR and IG complex related genes are also excluded from the selected characteristics. Next, the first 50 PCs were calculated by RunPCA functions using the filtered features. The cells were then mapped to two-dimensional space for visualization using a unified manifold approximation and projection (Uniform Manifold Approximation and Projection, UMAP) obtained by the RunUMAP function in Seurat, where each point represents one cell. Cells were subjected to cluster analysis using the FindClusters function in Seurat, and cluster identification was refined using the FindSubCluster function. The CITE-Seq assay is also referred to as a single cell RNA-Seq assay because the assay (UMAP, clustering) is done using RNA expression in CD8 ⁺ T cells, rather than protein expression.

Details of single cell cluster analysis are described in the previous paragraph and earlier examples (see e.g., example 6). Clusters enriched for stem cell-like genes, T cell activation (Tact) genes, T cell progenitor depletion (TPE) genes, and T cell terminal depletion (TTE) genes were evaluated. For stem cell-like genes, the gene set described in Caushi et al, nature 596:126-132 (2021) was used. For the Tact gene, TPE gene and TTE gene, the gene set described in Oliveira et al, nature 596:119-125 (2021) was used. Caushi et al and Oliveira et al are incorporated by reference herein in their entirety. Non-limiting examples of Tact, TPE, TTE and stem cell-like genes are provided elsewhere in this disclosure. The identification of stem cell-like clusters indicates the presence of relatively poorly differentiated CD8 ⁺ T cells, and the identification of Tact clusters indicates the presence of activated CD8 ⁺ T cells in the tumor. Identification of the TPE cluster indicates the presence of progenitor cells to deplete CD8 ⁺ T cells, and identification of the TTE cluster indicates the presence of depleted/dysfunctional CD8 ⁺ T cells in the tumor.

As shown in fig. 14A-14E, T cells sorted from tumors treated with C-Jun ROR1 CAR T cells had a significantly reduced proportion of clusters enriched for TTE gene sets (fig. 14B) and a significantly higher proportion of clusters enriched for TPE gene sets (fig. 14C) compared to T cells sorted from tumors treated with control ROR1 CAR T cells. TPE cells are known to be less depleted than TTE cells, with expression of memory-related transcripts (TCF 7, CCR7 and IL 7R) that are likely to expand upon activation and acquire a re-oscillating CD39 memory phenotype associated with long-term persistence. [ Oliveira et al, nature 596:119-125 (2021) ]. The decrease in TTE cluster and the increase in TPE cluster demonstrate the role of c-Jun overexpression in combating depletion. Interestingly, T cells sorted from tumors treated with c-Jun ROR1 CAR T cells had a significantly higher proportion of stem cell-like enriched clusters (fig. 14D), indicating the presence and increased persistence of stem cell-like populations. The proportion of T cell activating enriched clusters was also higher in T cells sorted from tumors treated with c-Jun ROR1 CAR T cells (fig. 14E).

These results demonstrate that over-expression of c-Jun provides additional benefits compared to the effect of MRM in increasing stem cell-like populations, thus demonstrating the benefits of the combination of MRM and c-Jun over-expression.

Example 8: further phenotypic and functional analysis of immune cells transduced and cultured in various metabolic reprogramming media carrying anti-ROR 1CAR

To further evaluate the effect of MRM on modified cells described herein (e.g., anti-ROR 1CAR T cells), several MRMs with different concentrations of potassium ions were prepared as described in example 1. Specifically, the final concentrations of the different components of the MRM are within the following ranges: naCl (55-90 mM), KCl (40-80 mM) and osmolality (about 250-260 mOsmol). Next, human cd4+ and cd8+ T cells (isolated from three donors) were transduced with anti-ROR 1CAR constructs with and without c-Jun and expanded using different MRMs or TCMs essentially as described in example 1. Next, as described in the earlier examples (e.g., examples 2 and 3), transduced T cells were analyzed for various characteristics such as transduction efficiency, c-Jun expression, stem cell-like phenotype expression and function (e.g., IL-2 and IFN- γ production following primary and/or long term antigen stimulation, and in vitro killing).

Results:

Similar to the earlier examples (see e.g. example 1), transduced cells were cultured and expanded for a total of 7 days after transduction and prior to analysis in TCM or in different MRM formulations with different concentrations of potassium ions (i.e. between 40-80 mM).

As shown in fig. 15A-15C, and consistent with the data previously described (see, e.g., fig. 3A-3C), T cells transduced with the C-Jun-R12 CAR construct and subsequently cultured in the MRM have higher C-Jun expression for all MRMs tested, as compared to the corresponding T cells transduced and cultured in TCM. The highest increase in c-Jun expression was observed in MRM with the highest potassium concentration.

Regarding the stem nature of the modified cells, and again in agreement with the data previously described (see, e.g., fig. 4A-4C), cd4+ T cells transduced with the anti-ROR 1 CAR construct were more stem-like in all MRM formulations with respect to their phenotypic expression (see, e.g., fig. 16A-16C and table 12 below) compared to cells from the TCM group. This is generally true whether the cd4+ T cells are transduced with a c-Jun-R12 CAR or a control CD19T-R12 CAR. The percentage of cd4+ T cells in MRM-cultured cells was highest compared to TCM-cultured T cells, with MRM potassium concentration having a dose-dependent effect on dryness (highest dryness at highest potassium concentration).

Likewise, cd8+ T cells transduced in MRM were generally more stem cell-like than the corresponding cells transduced in TCM (see fig. 17A-17C and table 13). Unlike cd4+ T cells, a consistent increase in stem cell-like cells was observed when cd8+ T cells were transduced with c-Jun-R12 CAR, as compared to control CD19T-R12 CAR. Thus, among cd8+ T cells, the largest percentage of stem cell-like cells was observed when cd8+ T cells were transduced with c-Jun-R12 CARs in MRM at various potassium concentrations. Like cd4+ T cells, a dose-dependent effect of MRM concentration on dryness was observed, with the highest dryness observed in MRM with the highest potassium concentration.

To assess the ability of T cells transduced and cultured in different MRMs to produce cytokines following antigen stimulation, ifnγ, IL-2 and tnfα cytokine secretion (after day 7 of expansion) of non-transduced (simulated, data not shown), ROR1 CAR with c-Jun (black squares) and ROR1 CAR without c-Jun T cell products were assessed after co-culturing with a549 NLR cancer cell line for 20 to 22 hours at E: T at 1:1 and 1:4. As shown in fig. 18A-18I (1:1 effector: target ratio) and fig. 19A-19I (1:4 effector: target ratio), anti-ROR 1 CAR T cells from the MRM group (with and without c-Jun overexpression) produced higher levels of IL2, tnfα (from all donors) and ifnγ (from 2 out of 3 donors) as compared to T cells modified and cultured in TCM. Also, the highest cytokine production was observed in MRM with the highest potassium concentration.

To assess the cytolytic capacity of the modified cells, an in vitro killing assay substantially as described in example 3 was used. The IncuCyte killing assay was established in 96-well flat bottom well assay plates using T cells from day 0, 7 or 14 of the serial re-stimulation assay and NucLight Red (NLR) target cell line a549 at E: T ratios of 1:1 and 1:4. The percent tumor viability was calculated using the area under the curve (AUC) (treaty low, cytotoxicity about high) of the incycyte killing curve obtained from control R12CAR and c-Jun-R12 CAR T cell products cultured in TCM or MRM (high to low) on days 0, 7, and 14 (continuous stimulation (SS) 1, 3, and 5) of the continuous re-stimulation assay after co-culturing with the a549 NLR target cell line at E: T ratios of 1:1 and 1:4. Data for the last round of stimulation (168 h to 300 h) are shown.

Consistent with the cytokine data described immediately above, anti-ROR 1 CAR T cells from different MRM groups generally exhibited increased killing as compared to those from TCM groups. As shown in fig. 20A-20D, a great improvement in cytolytic activity was observed in T cells transduced in MRM and subsequently cultured after 2 rounds of antigen stimulation, as compared to corresponding T cells from the control group. Improved cytolytic activity was observed in both anti-ROR 1 CAR T cells overexpressing c-Jun and anti-ROR 1 CAR T cells not modified to overexpress c-Jun.

In summary, the above results further confirm the therapeutic benefit of the culture methods provided herein. More specifically, the above results further demonstrate that T cells modified (e.g., to express ligand binding proteins and having increased c-Jun protein expression levels) in the presence of a medium comprising potassium ions at a concentration above 5mM (e.g., between 40-80 mM) can greatly increase the stem properties of the cells and allow the cells to exhibit potent functional activity even in the presence of prolonged antigen stimulation.

TABLE 14 c-Jun-anti-ROR 1 CAR sequence

Claims

1. A method of increasing the stem properties of an immune cell during in vitro or in vitro culture, the method comprising culturing an immune cell in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have increased levels of the c-Jun polypeptide.

2. A method of increasing yield of immune cells during in vitro or in vitro culture, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of the c-Jun polypeptide.

3. A method of preparing a population of immune cells for immunotherapy, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of the c-Jun polypeptide.

4. A method of increasing stem performance of immune cells for immunotherapy, while increasing yield of immune cells, during in vitro or in vitro culture, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of the c-Jun polypeptide.

5. A method of expanding a population of stem cell-like immune cells ex vivo or in vitro, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cells have been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to corresponding immune cells not modified to have increased levels of the c-Jun polypeptide.

6. A method of increasing cytokine production by an immune cell in response to antigen stimulation, the method comprising culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide.

7. The method of claim 6, wherein the cytokine comprises IL-2.

8. The method of claim 6 or 7, wherein the cytokine produced in response to the antigen stimulation is increased at least about 1-fold, 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, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-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 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more after the culturing, as compared to a reference immune cell.

9. A method of increasing effector function of an immune cell in response to a persistent antigen stimulus, the method comprising culturing an immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have increased levels of the c-Jun polypeptide.

10. The method of claim 9, wherein the immune cells retain effector function in at least one, at least two, or at least three additional rounds of antigen stimulation analysis as compared to a reference immune cell.

11. The method of claim 9 or 10, wherein the effector function comprises the following capabilities: (i) killing target cells (e.g., tumor cells), (ii) producing cytokines upon further antigen stimulation, or (iii) both (i) and (ii).

12. The method of claim 11, wherein the cytokine comprises IFN- γ.

13. The method of any one of claims 9 to 12, wherein after the culturing, the effector function of the immune cells in response to persistent antigen stimulation is increased at least about 1-fold, 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, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-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 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a reference immune cell.

14. A method of altering the phenotypic expression of one or more markers on an immune cell, the method comprising culturing the immune cell in a medium comprising potassium ions at a concentration greater than 5mM, wherein the immune cell has been modified to (i) express a chimeric polypeptide comprising a ROR1 binding protein and (ii) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell not modified to have increased levels of the c-Jun polypeptide.

15. The method of claim 14, wherein the one or more markers comprise CD39, LAG3, PD1, TIGIT, TIM3, or a combination thereof.

16. The method of claim 14 or 15, wherein expression of the one or more markers is reduced 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 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to a reference immune cell.

17. The method of claim 14 or 15, wherein expression of the one or more markers is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least about 5-fold as compared to a reference immune cell.

18. The method of any one of claims 8, 10-13, 16 and 17, wherein the reference immune cells comprise the following corresponding immune cells: (i) The c-Jun polypeptide that has been modified to have increased levels and is cultivated in a medium that does not comprise potassium ions at a concentration above 5 mM; (ii) Unmodified to have increased levels of the c-Jun polypeptide and cultured in a medium comprising potassium ions at a concentration above 5 mM; (iii) A culture medium that is unmodified to have increased levels of the c-Jun polypeptide and that does not comprise potassium ions at a concentration above 5 mM; or (iv) any combination of (i) to (iii).

19. The method of any one of claims 1 to 18, wherein the immune cell has been modified by an exogenous polynucleotide encoding the chimeric polypeptide, the c-Jun polypeptide, or both, such that after the modification, the immune cell has an increased level of the c-Jun polypeptide as compared to a corresponding immune cell.

20. The method of any one of claims 1 to 18, wherein the c-Jun polypeptide is endogenous to the immune cell, and wherein the immune cell has been modified by a transcriptional activator capable of increasing expression of the endogenous c-Jun polypeptide.

21. The method of claim 20, wherein the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity.

22. A method of preparing an immune cell for immunotherapy ex vivo or in vitro, the method comprising modifying an immune cell with an exogenous polynucleotide encoding (i) a c-Jun polypeptide and (ii) a chimeric polypeptide comprising a ROR1 binding protein in a medium comprising potassium ions at a concentration above 5 mM.

23. A method of preparing immune cells ex vivo or in vitro for immunotherapy, the method comprising using, in a medium comprising potassium ions at a concentration of greater than 5 mM: (i) A transcriptional activator capable of increasing expression of said endogenous c-Jun polypeptide; and (ii) an exogenous polynucleotide encoding a chimeric polypeptide comprising a ROR1 binding protein.

24. The method of claim 23, wherein the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity.

25. The method of any one of claims 1 to 24, wherein the chimeric polypeptide comprises a Chimeric Antigen Receptor (CAR).

26. The method of any one of claims 1 to 25, wherein the c-Jun polypeptide is capable of preventing or reducing depletion of the immune cells.

27. A method of increasing the stem properties of an immune cell during in vitro and in vitro culture, the method comprising culturing the immune cell in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cell has been modified to express a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

28. A method of increasing the yield of immune cells during in vitro or in vitro culture, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

29. A method of preparing a population of immune cells for immunotherapy, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

30. A method of increasing the stem properties of immune cells for immunotherapy while increasing the yield of immune cells during ex vivo or in vitro culture expansion, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

31. A method of expanding a population of stem cell-like immune cells ex vivo or in vitro, the method comprising culturing immune cells in a medium comprising potassium ions at a concentration of greater than 5mM, wherein the immune cells have been modified to express a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain.

32. The method of any one of claims 27 to 31, further comprising modifying the immune cell with an exogenous polynucleotide encoding the chimeric polypeptide.

33. A method of preparing an immune cell for immunotherapy ex vivo or in vitro, the method comprising modifying an immune cell with an exogenous polynucleotide encoding a chimeric polypeptide comprising (i) a ROR1 binding protein, (ii) a spacer comprising an amino acid sequence as set forth in SEQ ID NO:51, and (iii) a transmembrane domain in a medium comprising potassium ions at a concentration of greater than 5 mM.

34. The method of any one of claims 27 to 33, wherein the chimeric polypeptide comprises a Chimeric Antigen Receptor (CAR).

35. The method of any one of claims 27-34, wherein the immune cell further overexpresses a c-Jun polypeptide.

36. The method of claim 35, wherein the immune cell has been modified by an exogenous polynucleotide encoding the chimeric polypeptide, the c-Jun polypeptide, or both, such that upon the modification, the immune cell has an increased level of the c-Jun polypeptide as compared to a corresponding immune cell not modified to have an increased level of the c-Jun polypeptide.

37. The method of claim 35, wherein the c-Jun polypeptide is endogenous to the immune cell, and wherein the immune cell has been modified by a transcriptional activator capable of increasing expression of the endogenous c-Jun polypeptide.

38. The method of claim 37, wherein the transcriptional activator is linked to a Cas protein that has been modified to lack endonuclease activity.

39. The method of any one of claims 35 to 38, wherein the c-Jun polypeptide is capable of preventing or reducing depletion of the immune cells.

40. The method of any one of claims 1-26 and 35-39, wherein the c-Jun polypeptide comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID No. 13.

41. The method of any one of claims 1-26 and 35-40, wherein the c-Jun polypeptide is encoded by an exogenous polynucleotide comprising a nucleotide sequence having at least about 60%, at least about 70%, at least about 80%, 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 the nucleotide sequence of any one of SEQ ID NO:12、SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9 or SEQ ID NO: 10.

42. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 12.

43. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 1.

44. The method of claim 43, wherein said exogenous polynucleotide encoding said c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 1.

45. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having 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 a nucleic acid sequence as set forth in SEQ ID NO. 2.

46. The method of claim 45, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 2.

47. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having 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%, 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 as set forth in SEQ ID NO. 4.

48. The method of claim 45, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 4.

49. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having 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 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 as set forth in SEQ ID NO. 5.

50. The method of claim 49, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 5.

51. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having at least 88%, at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 6.

52. The method of claim 51, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 6.

53. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having at least 82%, at least 83%, at least 84%, 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 a nucleic acid sequence as set forth in SEQ ID NO. 7.

54. The method of claim 53, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 7.

55. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having 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 a nucleic acid sequence as set forth in SEQ ID NO. 8.

56. The method of claim 55, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 8.

57. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having at least 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 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 as set forth in SEQ ID NO 9.

58. The method of claim 57, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 9.

59. The method of claim 41, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 a nucleic acid sequence as set forth in SEQ ID NO 10.

60. The method of claim 59, wherein the exogenous polynucleotide encoding the c-Jun polypeptide comprises the nucleotide sequence set forth in SEQ ID NO. 10.

61. The method of any one of claims 1-26 and 35-60, wherein the c-Jun polypeptide and the chimeric polypeptide are on the same vector.

62. The method of any one of claims 1-26 and 35-60, wherein the c-Jun polypeptide and the chimeric polypeptide are on different vectors.

63. The method of any one of claims 1 to 62, wherein the ROR1 binding protein comprises an antibody or antigen binding portion thereof that specifically binds to ROR 1.

64. The method of claim 63, wherein the ROR1 binding protein specifically binds to the same epitope as an R12 antibody.

65. The method of claim 63 or 64, wherein said ROR1 binding protein comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 of said R12 antibody and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 of said R12 antibody.

66. The method of claim 65, wherein the VH CDR1 comprises the amino acid sequence set forth in SEQ ID No. 57, VH CDR2 comprises the amino acid sequence set forth in SEQ ID No. 58, and VH CDR3 comprises the amino acid sequence set forth in SEQ ID No. 59.

67. The method of claim 65 or 66, wherein said VL CDR1 comprises an amino acid sequence as set forth in SEQ ID NO:61, VL CDR2 comprises an amino acid sequence as set forth in SEQ ID NO:62, and VL CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 63.

68. The method of any one of claims 65 to 67, wherein said VH of said ROR1 binding protein comprises the amino acid sequence set forth in SEQ ID No. 56 and said VL of said ROR1 binding protein comprises the amino acid sequence set forth in SEQ ID No. 60.

69. The method of claim 68, wherein the ROR1 binding protein comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence as set forth in SEQ ID No. 83.

70. The method of any one of claims 1 to 69, wherein the chimeric polypeptide further comprises a Transmembrane (TM) domain.

71. The method of claim 70, wherein said TM domain is derived from CD8a, CD2, CD4, CD28, CD45, PD1, CD152, or any combination thereof.

72. The method of claim 71, wherein the TM domain is derived from CD28.

73. The method of claim 72, wherein said TM domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID No. 75.

74. The method of any one of claims 70-73, wherein the chimeric polypeptide further comprises a spacer between the ROR1 binding protein and the TM domain.

75. The method of claim 74, wherein the spacer is derived from an immunoglobulin hinge region or CD8.

76. The method of claim 74, wherein the spacer comprises the amino acid sequence set forth in SEQ ID NO. 51.

77. The method of any one of claims 74-76, wherein the spacer further comprises a linker.

78. The method of claim 77, wherein said linker comprises GGGSG (SEQ ID NO: 40).

79. The method of any one of claims 1-78, wherein the chimeric polypeptide further comprises an intracellular signaling domain.

80. The method of claim 79, wherein the intracellular signaling domain comprises a cd3ζ activation domain, a cd3δ activation domain, a cd3ε activation domain, a cd3η activation domain, a CD79A activation domain, a DAP 12 activation domain, a FCER1G activation domain, a DAP10/CD28 activation domain, a ZAP70 activation domain, or any combination thereof.

81. The method of claim 80, wherein the intracellular signaling domain comprises a cd3ζ activating domain.

82. The method of claim 81, wherein the cd3ζ activation domain comprises amino acid sequence having 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%, at least about 99%, or at least about 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 84.

83. The method of any one of claims 1-82, wherein the chimeric polypeptide further comprises an intracellular co-stimulatory domain.

84. The method of claim 83, wherein the intracellular co-stimulatory domain comprises a co-stimulatory domain of: interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, ligand that binds specifically to CD83, CD28 (ICA) that lacks Lck binding, OX40, BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, fc receptor gamma chain, fc receptor epsilon chain, ligand that binds specifically to CD83, or any combination thereof.

85. The method of claim 83, wherein the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

86. The method of claim 84, wherein the 4-1BB co-stimulatory domain comprises an amino acid sequence having 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%, at least about 99% or at least about 100% sequence identity to the amino acid sequence as set forth in SEQ ID No. 76.

87. The method of any one of claims 1-26 and 35-86, wherein the c-Jun polypeptide and the ROR1 binding protein are linked by a linker.

88. The method of claim 87, wherein the linker is a cleavable linker.

89. The method of claim 88, wherein the cleavable linker comprises a P2A linker, T2A linker, F2A linker, E2A linker, furin cleavage site, or any combination thereof.

90. The method of any one of claims 1-89, wherein the chimeric polypeptide further comprises a truncated EGF receptor (EGFRt).

91. The method of claim 90, wherein the EGFRt comprises an amino acid sequence with at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID No. 24.

92. The method of claim 91 or 91, wherein the EGFRt is linked to the c-Jun polypeptide and/or the ROR1 binding protein by a linker.

93. The method of claim 92, wherein the linker is a cleavable linker.

94. The method of claim 93, wherein the cleavable linker comprises a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof.

95. The method of any one of claims 1-94, wherein the chimeric polypeptide further comprises a signal peptide.

96. The method of claim 95, wherein the signal peptide is derived from hIgK.

97. The method of claim 96, wherein the hIgK signal peptide comprises the amino acid sequence set forth in SEQ ID No. 54.

98. The method of any one of claims 1 to 97, wherein the chimeric polypeptide comprises 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 the amino acid sequence set forth in SEQ ID No. 54.

99. The method of any one of claims 1 to 98, wherein the chimeric polypeptide is encoded by an exogenous polynucleotide comprising a nucleotide sequence having 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%, at least about 99% or about 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO 86.

100. The method of claim 99, wherein the exogenous polynucleotide comprises a vector.

101. The method of claim 99 or 100, wherein the exogenous polynucleotide further comprises a myeloproliferative sarcoma virus enhancer, a deleted negative control region, a dl587rev primer binding site substitution (MND) promoter, an EF1a promoter, a ubiquitin promoter, or any combination thereof.

102. The method of claim 101, wherein the MND promoter comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, 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 the amino acid sequence set forth in SEQ ID No. 92.

103. The method of any one of claims 1-102, wherein the concentration of potassium ions is greater than about 10mM, greater than about 15mM, greater than about 20mM, greater than about 25mM, greater than about 30mM, greater than about 35mM, greater than about 40mM, greater than about 45mM, greater than about 50mM, greater than about 55mM, greater than about 60mM, greater than about 65mM, greater than about 70mM, greater than about 75mM, greater than about 80mM, greater than about 85mM, or greater than about 90mM.

104. The method of any one of claims 1-102, wherein the concentration of potassium ions is selected from the group consisting of about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, and about 80 mM.

105. The method of any one of claims 1-102, wherein the concentration of potassium ions is between about 30mM and about 80mM, between about 40mM and about 80mM, between about 50mM and 80mM, between about 60mM and about 80mM, between about 70mM and about 80mM, between about 40mM and about 70mM, between about 50mM and about 70mM, between about 60mM and about 70mM, between about 40mM and about 60mM, between about 50mM and about 60mM, or between about 40mM and about 50 mM.

106. The method of any one of claims 1-102, wherein the concentration of potassium ions is about 50mM, about 60mM, or about 70mM.

107. The method of any one of claims 1-106, wherein the medium further comprises sodium ions.

108. The method of any one of claims 1-107, wherein the medium further comprises NaCl.

109. The method of any one of claims 1-108, wherein the culture medium comprises less than about 140mM, less than about 130mM, less than about 120mM, less than about 110mM, less than about 100mM, less than about 90mM, less than about 80mM, less than about 70mM, less than about 60mM, less than about 50mM, or less than about 40mM NaCl.

110. The method of any one of claims 1-109, wherein the medium is hypotonic or isotonic.

111. The method of any one of claims 107-110, wherein the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration multiplied by 2 is less than 280mM.

112. The method of any one of claims 107-110, wherein the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration multiplied by 2 is greater than 240mM and less than 280mM.

113. The method of any one of claims 107-110, wherein the medium is isotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration multiplied by 2 is greater than or equal to 280mM and less than 300mM.

114. The method of any one of claims 108-113, wherein the concentration of potassium ions is about 60mM and the concentration of NaCl is less than about 80mM, less than about 75mM, less than about 70mM, less than about 65mM, or less than about 60mM.

115. The method of any one of claims 108-113, wherein the concentration of potassium ions is about 55mM and the concentration of NaCl is less than about 85mM, less than about 80mM, less than about 75mM, less than about 70mM, or less than about 65mM.

116. The method of any one of claims 108-113, wherein the concentration of potassium ions is about 50mM and the concentration of NaCl is less than about 90mM, less than about 85mM, less than about 80mM, less than about 75mM, or less than about 70mM.

117. The method of any one of claims 1 to 116, wherein the immune cells comprise T cells, B cells, regulatory T cells (tregs), tumor Infiltrating Lymphocytes (TILs), natural Killer (NK) cells, natural Killer T (NKT) cells, or any combination thereof.

118. The method of any one of claims 1-117, wherein the immune cells have been engineered in vitro or ex vivo.

119. The method of any one of claims 1-118, wherein the medium further comprises one or more cytokines.

120. The method of claim 119, wherein the one or more cytokines comprise interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-21 (IL-21), interleukin-15 (IL-15), or any combination thereof.

121. The method of claim 120, wherein the one or more cytokines comprise IL-2, IL-7, and IL-15.

122. The method of claim 119 or 121, wherein the medium comprises IL-2 at a concentration of about 50IU/mL to about 500 IU/mL.

123. The method of claim 122, wherein the concentration of IL-2 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL.

124. The method of claim 122 or 123, wherein the concentration of IL-2 is between about 100IU/mL to about 300 IU/mL.

125. The method of any one of claims 122-124, wherein the concentration of IL-2 is about 200IU/mL.

126. The method of any one of claims 120 and 122-125, wherein the medium comprises IL-21 at a concentration of about 50IU/mL to about 500 IU/mL.

127. The method of claim 126, wherein the concentration of IL-21 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL.

128. The method of claim 126 or 127, wherein the concentration of IL-21 is between about 100IU/mL to about 300 IU/mL.

129. The method of any one of claims 126-128, wherein the concentration of IL-21 is about 200IU/mL.

130. The method of any one of claims 120-129, wherein the medium comprises IL-7 at a concentration of about 500IU/mL to about 1,500IU/mL.

131. The method of claim 130, wherein the concentration of IL-7 is about 500IU/mL, about 550IU/mL, about 600IU/mL, about 650IU/mL, about 700IU/mL, about 750IU/mL, about 800IU/mL, about 850IU/mL, about 900IU/mL, about 950IU/mL, about 1,000IU/mL, about 1,050IU/mL, about 1,100IU/mL, about 1,150IU/mL, about 1,200IU/mL, about 1,250IU/mL, about 1,300IU/mL, about 1,350IU/mL, about 1,400IU/mL, about 1,450IU/mL, or about 1,500IU/mL.

132. The method of claim 130 or 131, wherein the concentration of IL-7 is about 1,000iu/mL to about 1,400IU/mL.

133. The method of any one of claims 130-132, wherein the concentration of IL-7 is about 1,200IU/mL.

134. The method of any one of claims 130-133, wherein the medium comprises IL-15 at a concentration of about 50IU/mL to about 500 IU/mL.

135. The method of claim 134, wherein the concentration of IL-15 is about 50IU/mL, about 60IU/mL, about 70IU/mL, about 80IU/mL, about 90IU/mL, about 100IU/mL, about 125IU/mL, about 150IU/mL, about 175IU/mL, about 200IU/mL, about 225IU/mL, about 250IU/mL, about 275IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, or about 500IU/mL.

136. The method of claim 134 or 135, wherein the concentration of IL-15 is between about 100IU/mL to about 300 IU/mL.

137. The method of any one of claims 134-136, wherein the concentration of IL-15 is about 200IU/mL.

138. The method of any one of claims 1-137, wherein the medium further comprises a cell expansion agent.

139. The method of claim 138, wherein the cell expansion agent comprises a GSK3B inhibitor, ACLY inhibitor, PI3K inhibitor, AKT inhibitor, or any combination thereof.

140. The method of claim 139, wherein the PI3K inhibitor is selected from hydroxycitrate, LY294002, pitiriprist (pictilisib), CAL101, IC87114, or any combination thereof.

141. The method of claim 139, wherein the AKT inhibitor is selected from MK2206, a443654, AKTi-VIII, or any combination thereof.

142. The method of any one of claims 1-141, wherein the medium is capable of being in a final cell product as compared to immune cells cultured in a medium that does not contain the high concentration of potassium ions and/or immune cells that do not contain the c-Jun polypeptide, as compared to starting immune cells:

a. increasing the number and/or percentage of poorly differentiated and/or undifferentiated cells;

b. increasing transduction efficiency;

c. increasing stem cell-like immune cells;

d. increasing the activity in vivo;

e. Increasing cellular potency;

f. Preventing cell depletion; or (b)

G. Any combination thereof.

143. The method of any one of claims 1-142, wherein the medium further comprises calcium ions, glucose, or both.

144. The method of claim 143, wherein the medium further comprises glucose, and wherein the concentration of glucose is greater than about 10mM.

145. The method of claim 144, wherein the concentration of glucose is about 10mM to about 25mM, about 10mM to about 20mM, about 15mM to about 25mM, about 15mM to about 20mM, about 15mM to about 19mM, about 15mM to about 18mM, about 15mM to about 17mM, about 15mM to about 16mM, about 16mM to about 20mM, about 16mM to about 19mM, about 16mM to about 18mM, about 16mM to about 17mM, about 17mM to about 20mM, about 17mM to about 19mM, or about 17mM to about 18mM.

146. The method of claim 144, wherein the concentration of glucose is about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, or about 25mM.

147. The method of claim 144, wherein the concentration of glucose is about 15.4mM, about 15.9mM, about 16.3mM, about 16.8mM, about 17.2mM, or about 17.7mM.

148. The method of any one of claims 143-147, wherein the medium further comprises calcium ions, and wherein the concentration of calcium ions is greater than about 0.4mM.

149. The method of claim 148, wherein the concentration of the calcium ion is about 0.4mM to about 2.8mM, about 0.4mM to about 2.5mM, about 0.5mM to about 2.0mM, about 1.0mM to about 2.0mM, about 1.1mM to about 2.0mM, about 1.2mM to about 2.0mM, about 1.3mM to about 2.0mM, about 1.4mM to about 2.0mM, about 1.5mM to about 2.0mM, about 1.6mM to about 2.8mM, about 1.7mM to about 2.0mM, about 1.8mM to about 2.0mM, about 1.2 to about 1.3mM, about 1.2 to about 1.4mM, about 1.2 to about 1.5mM, about 1.2 to about 1.6mM, about 1.2 to about 1.7mM, about 1.8mM to about 1.3mM, about 1.3mM to about 1.5mM, about 1.3mM to about 1.7mM, about 1.3mM to about 1.5mM, about 1.7mM to about 1.7mM, about 1.6mM to about 1.8mM, about 1.7mM to about 1.8mM.

150. The method of claim 148, wherein the concentration of calcium ions is about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, about 2.0mM, about 2.1mM, about 2.2.mm, about 2.3mM, about 2.4mM, about 2.5mM, about 2.6mM, about 2.7mM, about 2.8mM, about 2.9mM, or about 3.0mM.

151. The method of any one of claims 1-150, wherein the immune cells are cd3+, CD45RO-, ccr7+, cd45ra+, cd62l+, cd27+, cd28+, or tcf7+, or any combination thereof after the culturing.

152. The method of any one of claims 1-151, wherein the immune cell is formulated in a pharmaceutical composition and a pharmaceutically acceptable carrier.

153. A population of immune cells prepared by the method of any one of claims 1 to 152.

154. A population of immune cells capable of achieving long-term persistence in vivo, wherein the immune cells have: (i) Culturing in a medium comprising potassium ions at a concentration of greater than 5mM, and (ii) modifying to express a chimeric polypeptide comprising a ROR1 binding protein and having an increased level of the c-Jun polypeptide as compared to a corresponding immune cell not modified to have an increased level of the c-Jun polypeptide.

155. The population of immune cells of claim 154, which are capable of lasting at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months in a subject in need thereof when administered to the subject.

156. A pharmaceutical composition comprising the population of immune cells of any one of claims 153-155 and a pharmaceutically acceptable carrier.

157. A method of treating a tumor in a subject in need thereof, the method comprising administering to the subject the population of immune cells of any one of claims 153-155 or the pharmaceutical composition of claim 156.

158. The method of claim 157, wherein the tumor is derived from a cancer comprising breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (stomach/cancer) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

159. The method of claim 157 or 158, wherein the tumor is a solid tumor.

160. The method of any one of claims 156-159 wherein the tumor size (tumor size) is reduced after the administration as compared to a reference tumor size.

161. The method of claim 160, wherein the reference tumor size comprises: (i) tumor size prior to said administration, (ii) tumor size of the corresponding subject not receiving said administration (e.g., receiving administration of the corresponding immune cells transduced and cultured in a medium not comprising potassium ions at a concentration above 5 mM), or (iii) both (i) and (ii).

162. The method of claim 160 or 161, wherein the tumor size is reduced 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 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference tumor size.

163. The method of any one of claims 157-162, wherein after the administration, the subject's survival duration is increased compared to a reference survival duration.

164. The method of claim 163, wherein the reference survival duration comprises the survival duration of a corresponding subject who did not receive the administration (e.g., received administration of a corresponding immune cell transduced and cultured in a medium that does not comprise potassium ions at a concentration greater than 5 mM).

165. The method of claim 163 or 164, wherein the survival duration is increased by at least about one week, at least about two weeks, at least about three weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about 10 months, at least about 11 months, or at least about one year compared to the reference survival duration.

166. The method of any one of claims 157-165, further comprising administering to the subject at least one additional therapeutic agent.

167. The method of claim 166, wherein the at least one additional therapeutic agent comprises a chemotherapeutic drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, a co-stimulatory molecule activator, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

168. The method of claim 167, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, or any combination thereof.

169. A composition comprising a population of cd4+ T cells and cd8+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein: (i) At least about 20% of the modified cd4+ T cells are surface positive for CCR7 and CD45 RA; (ii) At least about 20% of the modified cd8+ T cells are surface positive for CCR7 and CD45 RA; or (iii) both (i) and (ii).

170. A composition comprising a population of cd4+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein at least about 20% of the modified cd4+ T cells are surface positive for CCR7 and CD45 RA.

171. A composition comprising a population of cd8+ T cells that have been modified to (a) express a chimeric polypeptide comprising a ROR1 binding protein and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein at least about 20% of the modified cd8+ T cells are surface positive for CCR7 and CD45 RA.

172. A composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein at least about 4% of the cells are progenitor depleted T cells.

173. A composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein between about 4% and about 6% of the cells are progenitor depleted T cells.

174. A composition comprising a population of immune cells that have been modified to (a) express an engineered Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) and (b) have increased levels of a c-Jun polypeptide as compared to a corresponding immune cell that has not been modified to have increased levels of the c-Jun polypeptide, wherein at least about 4% of the cells are progenitor depleted T cells and at least about 4% of the cells are stem cell-like T cells.