Modulator for promoting t cell memory

Abstract

The present disclosure relates to the field of adoptive immune cells such as T cells, and particularly relates to compounds and compositions for generating cell population comprising T memory stem cells, and methods thereof of improving T cell memory. The present disclosure also relates to cell populations generated by methods of the present disclosure and use thereof in immunotherapy such as CAR-T cell therapy.

Description

Mintz Ref. No.: 064362-501001WO MODULATOR FOR PROMOTING T CELL MEMORY CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional PatentApplication No. 63 / 687,640, filed on August 27, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. FIELD

[0002] The present disclosure relates to the field of immunology and cell therapy, andparticularly relates to methods and compounds for generating T memory cells (e.g. T memory stem cell (TSCM)) and for promoting T cell memory. The present disclosure also relates to methods of generating T memory cells using compounds of the present disclosure and use thereof in immunotherapy such as adoptive immune cell therapy. BACKGROUND

[0003] Adoptive immune cell therapies have shown considerable efficacy in the treatment ofcancers and autoimmune related diseases. Immune cells such as T cells can be harvested and modified to express chimeric antigen receptors (CARs) and other synthetic receptors that can recognize antigens on target cells (i.e., malignant or cancer cells) and activate the T cells to induce T cell cytotoxicity toward the malignant cells expressing the target antigen. Current adoptive T cell therapies such as CAR-T cell therapy has significantly advanced the treatment of hematopoietic cancers, yet its application to solid tumors is hindered by challenges such as T cell exhaustion and limited persistence. CAR-T cells lose their anti-tumor activity over time, which may limit their effectiveness for long term. Thus, there is a need for more effective method of generating T memory cells with enhanced persistence and anti-tumor activity. T memory stem cell (TSCM) cells exhibit stem cell-like qualities that enable enhanced engraftment, persistence, and have the ability to sustain long-term immune responses against tumors.

[0004] The present disclosure provides solutions to the problems existing with cell therapy,such as CAR-T cell therapy and provided added benefits as well. SUMMARY

[0005] The present disclosure relates generally to compositions and methods for generatingpopulations of T memory stem cells (TSCM), including engineered TSCM, methods of improving immunotherapy and / or adoptive cell therapy (e.g. CAR-T cell therapy and or tumorMintz Ref. No.: 064362-501001WO infiltrating lymphocyte (TIL) therapy) using the compounds of the present disclosure, methods of generating CAR-T cell populations comprising TSCM, and cell populations generated by methods of the present disclosure.

[0006] In an aspect, the present disclosure includes a compound of Formula I or a salt or solvatethereof whereinR1is selected from H, 3 R2is each independently selected from H and halo, and n is 0 to 3.

[0007] The present disclosure includes methods of generating cell populations comprising Tmemory stem cells (TSCM). The methods of generating cell populations comprising T memory stem cells (TSCM) include contacting a starting population of T cells with a compound of Formula I as described herein, or a salt or solvate thereof to obtain the cell population.

[0008] The present disclosure also includes methods of generating engineered T cellpopulations. The engineered T cell populations comprise engineered TSCM. The methods of generating engineered T cell populations include introducing a starting population of T cells with a CAR construct comprising one or more CARs and contacting the starting population with a compound of Formula I as described herein, or a salt or solvate thereof.

[0009] The engineered TSCM comprises a CAR construct comprising one or more CARs. Assuch, the engineered TSCM can be used in CAR-T cell therapy. Accordingly. the present disclosure also includes methods for improving CAR-T cell therapy.

[0010] The compound of Formula I as described herein or a salt or solvate thereof is shownherein in more detail to affect expression of T cell markers compared to a T cell that is untreated with the compound of Formula I or a salt or solvate thereof. Accordingly, the present disclosure also includes methods of increasing expression of a marker in a T cell. The methods of increasing expression of a marker in a T cell include contacting the T cell with a compound ofMintz Ref. No.: 064362-501001WO Formula I as described herein or a salt or solvate thereof. The marker(s) whose expression is increased are markers for more stem cell like features that are related to improved T cell memory and / or to cell proliferation. The marker(s) whose expression is increased can be selected from CD45RA, CD62L, CD95, CCR7, CD69, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof. The present disclosure also includes methods of reducing a marker in a T cell. The methods of reducing a marker in a T cell include contacting the T cell with a compound of Formula I as described herein or a salt or solvate thereof. The marker(s) with reduced expression are markers related to T cell exhaustion and / or terminal stage differentiation. The marker(s) with reduced expression can be selected from LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform and combinations thereof.

[0011] The methods of the present disclosure generate cell populations comprising TSCM.Accordingly, also included herein in the present disclosure is a cell population generated by a method of the present disclosure.

[0012] The foregoing summary is illustrative only and is not intended to be in any way limiting.In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects, and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The features of the present disclosure are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0014] FIG. 1A is histograms showing mitochondrial content in CD4 and CD8 T cells treatedwith compound I-1, using DMSO as a negative control. FIG.1B is bar graphs of mitochondrial mass quantification shown as mean fluorescence intensity (MFI) in compound I-1 treated CD4 and CD8 T cells. P value was determined by unpaired t test. FIG.1C is contour graphs of flow cytometry analysis of CD45RA+ cell frequency of CD4 and CD8 T cells treated with compound I-1 or DMSO as a negative control. FIG. 1D is bar graphs of the flow cytometry results shown in FIG.1C. P value was determined by unpaired t test.Mintz Ref. No.: 064362-501001WO

[0015] FIG. 2A are contour graphs of flow cytometry results of expression of surfacebiomarkers of TSCM treated with compound I-1 or DMSO (negative control). FIG.2B shows the percentage increase of CD45RA+CD26L+CD95+ TSCM cells in T cell populations treated with compound I-1 vs DMSO.

[0016] FIG.3A shows contour graphs of flow cytometry analysis of CD45RA+ population ofCD8+ T cells treated with compound I-1, TWS119, (E / Z)-GSK-3β inhibitor 1 or DMSO (negative control). FIG. 3B shows the percentage increase of CD45RA+ CD8+ T cells in T cell population treated with compound I-1, TWS119, (E / Z)-GSK-3β inhibitor 1 or DMSO (negative control). FIG. 3C shows contour graphs of flow cytometry analysis of CD45RA+ population of CD8+ T cells treated with compound I-1, KY-02061 or DMSO (negative control). FIG. 3D shows the percentage increase of CD45RA+ CD8+ T cells in T cell population treated with compound I-1, KY-02061 or DMSO (negative control). FIG.3E shows contour graphs of flow cytometry analysis of CD62L+ population of CD44- mouse T cells treated with compound I-1, TWS119, (E / Z)-GSK-3β inhibitor 1, TDZD-8, SB216763, PF- 04802367, AR-A014418, CP21R7 or DMSO (negative control). FIG.3F shows the percentage increase of CD3+CD62L+CD44- T cells in mouse T cell population treated with the compound of Formula I, TWS119, (E / Z)-GSK-3β inhibitor 1, TDZD-8, SB216763, PF-04802367, AR- A014418, CP21R7 or DMSO (negative control).

[0017] FIG. 4A is contour graphs of flow cytometry of mouse T cell population treated withcompound I-1, TWS119, (E / Z)-GSK-3β inhibitor 1 or DMSO (negative control) selected for CD8, CD44, CD4 and CD62L. The third column shows data corrected for Sca1+ T cells. FIG. 4B is histograms of percentage increase of CD8+Sca1+ mouse T cells in cell population treated with compound I-1, TWS119, (E / Z)-GSK-3β inhibitor 1 or DMSO (negative control).

[0018] FIG.5A is histograms of CTV cell proliferation assay of CD4+ and CD8+ cells treatedwith compound I-1, TWS119 or DMSO (negative control). FIG. 5B shows bar graphs of proliferation, expansion and division indices in CD4+ T cells treated with TWS119, compound I-1 or DMSO (negative control).

[0019] FIG.6A shows contour graphs and histograms of transduction efficiency of CD4+ andCD8+ cells with CAR construct. FIG. 6B shows contour graphs of flow cytometry results of cryopreserved and reactivated CAR-T cells treated with compound I-1or DMSO (negative control) gated on CD4, CD8, and CD45RA.

[0020] FIG 7A is histograms of target (NALM6 cells) and non-target cells (K562 cells)population after culture with no T cells, control T cells, CAR-T cells, and CAR-T cells treated with compound I-1. FIG. 7B shows bar graphs of percentage of specific killing of target cellMintz Ref. No.: 064362-501001WO (NALM6 cells) by control T cells, CAR-T cells, and CAR-T cells treated with compound I-1. FIG.7C is contour graphs showing CAR expression measured by flow cytometry in control T cells, CAR-T cells and CAR-T cells treated with compound I-1 pre and post exposure to target cell (NALM6 cells). FIG. 7D is graphs showing percentage increase of CAR expression in control T cells, CAR-T cells and CAR-T cells treated with compound I-1 pre and post exposure to target cell (NALM6 cells). FIG. 7E is contour graphs of flow cytometry results showing CD69+ CD8+ control T cells, CAR-T cells, and CAR-T cells treated with compound I-1, at different effector to target ratios. FIG.7F is bar graphs showing percentage increase of CD69 expression in CD8+ control T cells, CAR-T cells, and CAR-T cells treated with compound I- 1 at different effector to target ratios.

[0021] FIG. 8A shows contour graphs of CD62L and CD44 expression in CD4+ and CD8+ Tcells (Tumor infiltrating T cells isolated from mouse DIPG tumor model) at Day 0 of culture prior to activation. FIG. 8B shows CD44 and CD62L expression on Day 3 after activation in the presence of compound I-1 or DMSO (negative control). FIG. 8C shows the percent increase in CD62L+ and CD44+ expression after activation in the presence of compound I-1 or DMSO (negative control).

[0022] FIG. 9A shows a graph of Principal Component Analysis (PCA) results of T cellstreated with DMSO (negative control), TWS119 or compound I-1. FIG.9B is a Venn diagram showing any overlap of gene expression in DMSO (negative control) or compound I-1. FIG. 9C and FIG. 9D show gene set enrichment analysis results for Lymphoid Progenitor Cell Differentiation and Fatty Acid Beta Oxidation respectively. FIG. 9E is bar graphs showing relative expression of LAG3, CTLA-4 and PD1 genes respectively in DMSO (negative control) or compound I-1 treated T cells. FIG.9F is bar graphs showing relative expression of TERT, TET2, IRF4, CD38, and NR4A genes respectively in DMSO (negative control) or compound I-1 treated T cells.

[0023] FIG. 10A shows contour graphs of flow cytometric analysis of CD45RA+ cellfrequency in CD4 and CD8 T cells treated with DMSO (negative control), compound I-1 or compound I-2. FIG. 10B shows bar graphs quantifying the percentage of CD45RA+ cells in CD4 and CD8 T cells treated with DMSO (negative control), compound I-1 or compound I-2. FIG. 10C shows proportion of TSCM cells in CD8 T cells treated with DMSO (negative control), compound I-1 or compound I-2, where TSCM cells identified as CD45RA+CD62L+CD95+. FIG.10D shows bar graphs quantifying the percentage of TSCM cells identified as CD45RA+CD62L+CD95+, in CD8 T cells treated with DMSO (negative control), compound I-1 or compound I-2.Mintz Ref. No.: 064362-501001WO DETAILED DESCRIPTION OF THE DISCLOSURE

[0024] The present disclosure provides, inter alia, compositions and methods for generatingpopulations of T memory stem cells (TSCM), including engineered TSCM, methods of improving immunotherapy and / or adoptive cell therapy (e.g. CAR-T cell therapy and or tumor infiltrating lymphocyte (TIL) therapy) using the compounds of the present disclosure, methods of generating CAR-T cell population comprising TSCM, and cell populations generated by methods of the present disclosure.

[0025] As described in greater herein, compound of Formula I or a salt or solvate thereof canbe used to generate populations of cells whereby the population has an increased proportion of more stem cell like T cell population (e.g. TSCM). It can be appreciated that stem cell like T cells such as TSCM have the ability to self-renew and to produce effector cells. Relative to T effector cells, TSCM are longer lived and less prone to exhaustion. Further, TSCM have greater ability to migrate and engraft. Accordingly, the cell populations of the present disclosure prepared by methods of present disclosure are useful in immunotherapy and / or adoptive cell therapy and can be engineered to provide therapeutic compositions such as CAR-T cell therapy.

[0026] The section headings used herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

[0027] Although various features of the disclosure can be described in the context of a singleembodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0028] It is intended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.Mintz Ref. No.: 064362-501001WO

[0029] All patent filings, websites, other publications, accession numbers and the like citedabove or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. DEFINITIONS

[0030] Unless otherwise defined, all terms of art, notations, and other scientific terms orterminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0031] The singular form “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0032] It is understood that aspects and embodiments of the disclosure described herein include“comprising,” “consisting,” and “consisting essentially of” aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.Mintz Ref. No.: 064362-501001WO

[0033] As used herein, the term “about” or “approximately” refers to a quantity, level, value,number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term “about” or “approximately” refers to a rage of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0034] The term “chimeric antigen receptors (CARs)” as used herein, may refer to artificialreceptors derived from T-cell receptors and encompasses engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. Exemplary CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In some embodiments, CARs further comprise a hinge domain. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides). In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors. In some embodiments, the extracellular ligand binding domain of the first receptor is fused to the extracellular domain of a CAR. In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo.

[0035] As used herein, a "T-cell" refers to a type of lymphocyte that originates from a bonemarrow precursor that develops in the thymus gland. There are several distinct types of T-cells which develop upon migration to the thymus, which include, helper CD4+ T- cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells. Different types of T-cells can be distinguished by the ordinarily skilled artisan based on their expressionMintz Ref. No.: 064362-501001WO of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.

[0036] It is appreciated that certain features of the disclosure, which are, for clarity, describedin the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. COMPOUND AND COMPOSITIONS OF THE DISCLOSURE

[0037] As described in greater detail below, one aspect of the present disclosure relates to acompound of Formula I or a salt or solvate thereofwherein R1is selected from H, and C1-3alkyl, R2is each independently selected from H and halo, and n is 0 to 3.

[0038] For example, the compound of Formula I is a compound of Formula I-AMintz Ref. No.: 064362-501001WOor a salt or solvate thereof, wherein R1is selected from H, and C1-3alkyl, R2is each independently selected from H and halo.

[0039] In some instances, the compound of Formula I or a salt or solvate thereof is selectedfrom the compound I-1, the compound I-2, a salt or solvate thereof, and combinations thereof .compound I-1 or a salt or solvatethereof. For example, the compound of Formula I is the compound I-2 or a salt or solvate thereof.

[0041] For example, R1 can be H. For example, R1 can be C1-3 alkyl, such as CH3.

[0042] It can be appreciated that halo includes any suitable halogen substituents. For instance,halo can be selected from F, Cl, Br, and I. In some embodiments, R2is each independently halo. For example, R2is Cl.

[0043] It is contemplated that n can be any integer of 0 to 3. For example, n can be 0, 1, 2, or3. In some embodiments, n is 2. In some embodiments, n is 0.

[0044] The compound of Formula I or the salt or solvate thereof can be used in a composition.For example, the compound of Formula I or the salt or solvate thereof can be formulated in aMintz Ref. No.: 064362-501001WO suitable solvent. Accordingly, the present also includes a composition comprising a compound of Formula I or the salt or solvate thereof and a solvent. METHODS OFGENERATINGCELLPOPULATIONS

[0045] The present disclosure includes methods of generating cell populations that are enrichedfor TSCM. Thecell populations comprising T memory stem cell (TSCM) include contacting a starting population of T cells with a compound of Formula I as described herein, or a salt or solvate thereof to obtain the cell populations.

[0046] As exemplified in the Examples, the compound of the present disclosure has beenshown to increase the proportion of TSCM in cell populations treated with the compound of the present disclosure using methods of the present disclosure compared to a starting population or population untreated with the compound of the present disclosure. TSCM can be identified by marker expression in the T cells. Markers indicative of TSCM can be selected from CD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof. For example, the markers indicative of TSCM can be selected from CD45RA, CD62L, CD95, CCR7, and combinations thereof. Generated Cell Populations

[0047] Accordingly, the cell populations (e.g. CAR-T cell population) generated by methodsof the present disclosure compared to a cell population such as the starting population or a population untreated with the compound of the present disclosure comprises a higher proportion of TSCM. For example, about 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, or about 80% to about 99%, or about 80% to about 90% of total cells in the cell population generated by a method of the present disclosure are TSCM. For example, at least about 30%, about 40%, about 50%, about 60%, about 70% or about 80% of total cells in the cell population generated by a method of the present disclosure are TSCM. For example, at most 100%, at most 99%, at most 95%, at most 90%, at most 85%, at most 80% of total cells in the cell populations generated by methods of the present disclosure are TSCM.

[0048] The compound of the present disclosure has been shown herein to increase expressionof T cell markers relevant to stem-cell like features. Accordingly, relative to cell population untreated with the compound of the present disclosure, the TSCM of the cell populations generated by methods of the present disclosure can have increased expression of a marker selected from CD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT),Mintz Ref. No.: 064362-501001WO lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof. For example, the markers can be selected from CD45RA, CD62L, CD95, CCR7, and combinations thereof. The lymphoid progenitor cell differentiation genes can include BMP4, SOX4, BATF,SOS1, NOTCH1, or combinations thereof. For example, in some instances, CD45RA expression can be increased by about 10% to about 99%, about 20% to about 80%, or about 30% to about 70%. In some instances, CD62L expression can be increased by about 5% to about 50%, about 10% to about 45%, or about 15% to about 40%. For example, CD95 expression can be increased by about 10% to about 90%, about 15% to about 80%, or about 20% to about 70%. The fatty acid beta oxidation genes can include MCAT, PEX5, MTOR, MECR, SLC25A17 or combinations thereof.

[0049] Similarly, the compound of the present disclosure has been shown to decreaseexpression of markers for T cell exhaustion and terminal differentiation relative to a cell population untreated by a compound of the present disclosure. Accordingly, in some instances, the cell population can have reduced expression of at least one T cell marker or at least one T cell exhaustion marker compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof. For example, the at least one T cell exhaustion marker is selected from LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform and combination thereof s. For example, NR4A isoform can be NR4A1, NR4A2, NR4A3, or combinations thereof. In some instances, the expression of at least one T cell markers or at least one T cell exhaustion marker can be reduced by about 1.1 fold to about 2 fold, about 1.1 fold to about 1.7 fold, about 1.2 fold to about 1.5 fold. Starting Population

[0050] The cells or population of cells used in the methods of the present disclosure can beobtained from any source, such as a blood bank or a donor. For example, the starting population comprising naïve T cells, pan T cells and / or tumor infiltration T cells can be obtained from cell cultures such as peripheral blood mononuclear cells (PBMC), Leukopak, buffy coat, apheresis product, or combination thereof. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions is within the skill of the art. An effective amount means an amount which provides a therapeutic benefit. The dosage administered will be dependent upon the age, health and weight of the recipient, the kind of concurrent treatment, if any, the frequency of treatment and the nature of the effect desired.

[0051] It can be appreciated that the compound of the present disclosure has shown effects onT cell marker expression and composition. As such, the starting population used in the methodsMintz Ref. No.: 064362-501001WO of the present disclosure comprises T cells. For example, the T cells in the starting population can be activated T cells. For example, the T cells in the starting population can comprise CD8+ and / or CD4+ T cells. In some instances, the starting population can comprise naïve T cells, pan T cells, tumor infiltrating T cells, or combinations thereof. In the situations where the starting population comprises unactivated T cells (e.g. naïve T cells, pan T cells, tumor infiltrating T cells, or combinations thereof), it may be desirable to activate the unactivated T cells. Accordingly, in some instances, the methods of the present disclosure further comprise activating the starting population or activating the naïve T cells, pan T cells, tumor infiltrating T cells, or combinations thereof in the starting population.

[0052] It can be appreciated that the activating can be carried out using techniques known inthe field of T cell activation. For example, the activating can comprise treating the starting population with a T cell activator (e.g. CD3 / CD28 T cell activator beads) and IL-2. The activating can comprise culturing the starting population in a culture medium, the culture medium comprising a T cell activator and IL-2, IL-15, IL-7, or combinations thereof.

[0053] The T cell activator can be any suitable activator known in the field. For instance, theT cell activator can be an anti-CD3 antibody or a CD3 binding fragment thereof, an anti-CD28 antibody or a CD28 binding fragment thereof, or combinations thereof. Optionally, the T cell activator can be bound to a solid support, such as beads (e.g. magnetic beads). As such, the anti-CD3 antibody or a CD3 binding fragment thereof, the anti-CD28 antibody or a CD28 binding fragment thereof, or combinations thereof can be bound to a solid support, such as beads (e.g. magnetic beads). Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth.227(1-2):53-63, 1999).

[0054] It can be understood that the treating with the T cell activator can be carried out usingstandard T cell activation parameters and protocols. For example, the treating can be for about 24 hours to about 96 hours, about 50 hours to about 85 hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours.

[0055] It can be appreciated that the starting population can be in a culture medium. Therefore,in some instances, the contacting of the compound of the present disclosure with the starting population can comprise combining the culture medium with the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can comprise culturing the starting population in a culture medium that comprises the compound of the present disclosure.Mintz Ref. No.: 064362-501001WO Contacting with the Compound of the Present Disclosure

[0056] The contacting of the compound of the present disclosure with the starting populationcan be carried out concurrently with and / or subsequently to the activating of the starting population. In some instances, the contacting of the compound of the present disclosure with the starting population can be carried out subsequently to the activating of the starting population. For example, the contacting can be carried out about 4 days to about 9 days, about 5 days to about 7 days, or about 6 days to about 8 days subsequently to the activating.

[0057] In some instances, the contacting of the compound of the present disclosure with thestarting population can be carried out concurrently with the activating of the starting population. For example, the activating of the starting population can be carried out in the presence of the compound of the present disclosure.

[0058] The contacting can be for about 12 hours to about 96 hours, about 24 hours to about 85hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours.

[0059] The contacting of the compound of the present disclosure with the starting populationcan be carried out at about 0.1 µM to about 10 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at about 1 µM to about 9 µM, about 2 µM to about 8 µM, about 3 µM to about 7 µM, about 4 µM to about 6 µM, or about 5 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at at least about 0.1 µM, at least about 0.5 µM, at least about 1 µM, at least about 2 µM, at least about 3 µM, at least about 4 µM, at least about 4.5 µM, at least about 5 µM, at least about 6 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at less than about 10 µM, less than about 9 µM, less than about 8 µM, less than about 7.5 µM, less than about 7 µM, less than about 6.5 µM, less than about 6 µM, less than about 5.5 µM, less than about 5 µM of the compound of the present disclosure.

[0060] The methods of the present disclosure can further include contacting the startingpopulation with IL-2, IL-15, IL-7 or combinations thereof concurrently with the contacting with the compound of the present disclosure. METHODS OF AFFECTING T CELL MAKER EXPRESSION

[0061] The compound of Formula I as described herein or a salt or solvate thereof is shownherein in more detail to affect expression of T cell markers compared to a T cell that is untreatedMintz Ref. No.: 064362-501001WO with the compound of Formula I or a salt or solvate thereof. Accordingly, the present disclosure also includes methods of increasing expression of a marker in a T cell. The methods of increasing expression of a marker in a T cell include contacting the T cell with a compound of Formula I as described herein or a salt or solvate thereof. The marker(s) whose expression is increased are markers for more stem cell like features that are related to improved T cell memory and / or to cell proliferation. The marker(s) whose expression is increased can be selected from CD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof. The lymphoid progenitor cell differentiation genes can include BMP4, SOX4, BATF,SOS1, NOTCH1, or combinations thereof. The fatty acid beta oxidation genes can include MCAT, PEX5, MTOR, MECR, SLC25A17 or combinations thereof. The present disclosure also includes methods of reducing a marker in a T cell. The methods of reducing a marker in a T cell include contacting the T cell with a compound of Formula I as described herein or a salt or solvate thereof. The marker(s) with reduced expression are markers related to T cell exhaustion and / or terminal stage differentiation. The marker(s) with reduced expression can be selected from LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform and combinations thereof.

[0062] It has been shown herein that the compound of the present disclosure is effective on forexample activated T cells. Therefore, in some instances, the T cell is an activated T cell. For example, the T cell is a CD4+ and / or a CD8+ T cell. The compound of the present disclosure is effective on T cells that are CAR-T cells. Accordingly, in some instances, the T cell is capable of expressing one or more CARs (e.g. is a CAR-T cell).METHODS OF GENERATING ENGINEERED T CELL POPULATION

[0063] The present disclosure also methods of generating engineered T cellpopulations. The methods of generating T cell populations include introducing a starting population with a CAR construct comprising one or more CARs and contacting the starting population with a compound of Formula I as described herein, or a salt or solvate thereof. The engineered T cell populations comprise TSCM. The engineered T cell populations are capable of expressing one or more CARs.

[0064] It has been shown herein that the compound of the present disclosure was able toincrease the killing ability of CAR-T cells where the specific killing at various effector to target ratio increased. Accordingly, the methods of generating cell population comprising TSCM of the present disclosure can be used to generate CAR-T cells. Therefore, in some instances, theMintz Ref. No.: 064362-501001WO methods of the present disclosure can further comprise introducing a CAR construct into the starting population. It can be appreciated that the CAR construct can be introduced into the starting population by any suitable means known in the art. For example, introduction of CAR construct can be achieved by transduction, electroporation, other non-viral delivery system like transposon based approach and CRISPR. For example, the method of the present disclosure can further include transducing the starting population with a CAR construct. In some instances, the cell population comprising T cells is a CAR-T cell population. Introduction of CAR Construct

[0065] It can be appreciated that the transducing or transfecting with the CAR construct canbe performed with any suitable transduction or transfection method known in the field of T cell engineering. Methods transforming populations of immune cells, such as T cells, with the vector constructs comprising one or more CARs will be readily apparent to the person of ordinary skill in the art. For example, the transducing can be carried out with a transduction method selected from lentivirus, retrovirus, sleeping beauty, TALENS, CRISPR-CAS-9, and electroporation-based methods.

[0066] The CAR construct can be any suitable CAR construct desired by the user. The CARconstruct can comprise one or more CARs. For instance, the CAR construct can target growth factor receptors (GFR) / Receptor tyrosine kinases (RTK), cell surface receptor / enzymes, tumor-associated antigens (TAAs), cell adhesion molecules (CAMs), immune checkpoint molecules / receptors, cytokines and cytokine receptors, stem-cell surface proteins, tumor necrosis factor associated ligand and receptor, B cell receptors, SLAM family receptors, or combinations thereof. GFR / RTK targets can be but are not limited to HER2, ERBB2, EGFR, TRKB, C-MET, BRAF, or combinations thereof. Cell surface receptor / enzymes targets can be but are not limited to PSMA, ROR1, FOLR1, CD73, GD2, CA-IX, CD147, DLL3, FAP (Fibroblast Activation Protein, GUCY2C (Guanylyl Cyclase C), PSMA, NKG2DL (Natural Killer Group 2D Ligands), CD70, CD133 (Prominin-1), GPRC5D, or combinations thereof. TAAs targets can be but are not limited to MSLN, GPC3, CEA, PSCA, Muc1, Trop2, TAG72 (Tumor-associated glycoprotein 72) or combinations thereof. CAMs targets can be but are not limited to Nectin-4, Claudin 18.2, ROBO1 (Roundabout Guidance Receptor 1), CD171 (L1- CAM), CD138, CD38, or combinations thereof. Immune checkpoint molecules / receptors targets can be but are not limited to B7-H3 / CD276, SLAMF7, or combinations thereof. Cytokines and cytokine receptors targets can be but are not limited to IL13Rα2 (Interleukin 13 Receptor Subunit Alpha 2), CD123, or combinations thereof. Stem-cell surface proteins targets can be but are not limited to PRTG. Tumor Necrosis factor associated ligand and receptorMintz Ref. No.: 064362-501001WO targets can be but are not limited to BCMA.CD30, APRIL, or combinations thereof. B cell receptors targets can be but are not limited to CD19, CD20, CD22, or combinations thereof. SLAM family receptor targets can be but is not limited to SLAMF7, CD229, CS1, or combinations thereof. Engineered T Cell Populations

[0067] The compound of the present disclosure has been shown herein to not affectproliferation of CD4+ and CD8+ T cells. The compound of the present disclosure has been shown to increase markers for proliferation for T cells. Accordingly, the methods of the present disclosure do not affect proliferation of T cells and can improve proliferation of T cells.

[0068] As exemplified in the Examples, the compound of the present disclosure can be usedwith a T cell comprising a CAR construct to increase the killing ability of CAR-T cells where the specific killing at various effector to target ratio increased. The compound of the present disclosure has also been shown herein to increase expression of markers such as early activation markers (e.g. CD69) upon exposure to tumor cells in CAR-T cells compared to untreated CAR-T cells. Accordingly, the present disclosure also includes methods of generating engineered T cell populations comprising TSCM, the methods including introducing a starting population of T cells with a CAR construct comprising one or more CARs and contacting the starting population with a compound of Formula I as described herein, or a salt or solvate thereof. Similarly, since the engineered T cell populations comprising TSCM are capable of expressing one or more CARs, the T cell population can be used in CAR- T cell therapy. Accordingly, the present disclosure also includes methods for improving CAR- T cell therapy.

[0069] The engineered T cells can have an increased expression of a marker selected fromCD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof, compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof. The lymphoid progenitor cell differentiation genes can include BMP4, SOX4, BATF,SOS1, NOTCH1, or combinations thereof. The fatty acid beta oxidation genes can include MCAT, PEX5, MTOR, MECR, SLC25A17 or combinations thereof. The marker with increased expression can be related to activation of T cells, and / or increasing stem cell like features of the T cells to improve T cell memory.

[0070] The engineered T cell populations can have a reduced expression of a marker selectedfrom LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform (e.g. NR4A1, NR4A2 andMintz Ref. No.: 064362-501001WO NR4A3) and combinations thereof compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof.

[0071] The engineered T cell populations generated by a method of the present disclosurecompared to a cell population such as the starting population or a population untreated with the compound of the present disclosure comprises a higher proportion of TSCM. For example, about 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, or about 80% to about 99%, or about 80% to about 90% of total cells in the engineered T cell populations generated by a method of the present disclosure are TSCM. For example, at least about 30%, about 40%, about 50%, about 60%, about 70% or about 80% of total cells in the engineered T cell population generated by a method of the present disclosure are TSCM. For example, at most 100%, at most 99%, at most 95%, at most 90%, at most 85%, at most 80% of total cells in the engineered T cell population generated by a method of the present disclosure are TSCM. Contacting with the Compound of the Present Disclosure

[0072] When using the methods of the present disclosure for generating engineered T cellpopulations, the contacting of the starting population with the compound of the present disclosure can be carried out concurrently and / or subsequently to the introducing of the CAR construct into the T cells of the starting population. For example, the starting population can be transfected or transduced with the CAR construct and then be contacted with the compound of the present disclosure. Alternatively or additionally, the introduction of the CAR construct into the T cells of the starting population can be carried out in the presence of the compound of the present disclosure. The contacting with the compound of the present disclosure can be carried out in the presence of IL-2, IL-15, and / or IL-7.

[0073] The contacting of the compound of the present disclosure with the starting populationcan be carried out for about 12 hours to about 96 hours, about 24 hours to about 85 hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours. The contacting with the compound of Formula I of a salt or solvate thereof cam be carried out at a concentration of the compound of Formula I of about 0.1 µM to about 10 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at about 1 µM to about 9 µM, about 2 µM to about 8 µM, about 3 µM to about 7 µM, about 4 µM to about 6 µM, or about 5 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at at least about 0.1 µM, at least about 0.5 µM, at least about 1 µM, at least about 2 µM, at least about 3 µM, atMintz Ref. No.: 064362-501001WO least about 4 µM, at least about 4.5 µM, at least about 5 µM, at least about 6 µM of the compound of the present disclosure. For example, the contacting of the compound of the present disclosure with the starting population can be carried out at less than about 10 µM, less than about 9 µM, less than about 8 µM, less than about 7.5 µM, less than about 7 µM, less than about 6.5 µM, less than about 6 µM, less than about 5.5 µM, less than about 5 µM of the compound of the present disclosure. CELL POPULATIONS OF THE DISCLOSURE

[0074] The methods of the present disclosure generate cell populations comprising TSCM.also included herein in the present disclosure is a cell population generated by a method of the present disclosure.

[0075] In some instances, the cell population generated can be an engineered T cell populationas described herein. The engineered T cell population comprises TSCM. METHODS OF USING THE CELL POPULATIONS

[0076] The engineered T cell populations comprising one or more CARs of the presentdisclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired.

[0077] In general, administration may be parenteral.

[0078] Methods for administration of cells for adoptive cell therapy are known and may beused in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al and U.S. Pat. No.4,690,915 to Rosenberg.

[0079] The compositions of the disclosure are suitable for parenteral administration. As usedherein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracerebroventtrivular, intracerebral, intracranial, intratumoral, intrasynovial injection or infusions; and kidney dialytic infusionMintz Ref. No.: 064362-501001WO techniques. In some embodiments, parenteral administration of the compositions of the present disclosure comprises intravenous or intraarterial administration.

[0080] The disclosure provides pharmaceutical compositions comprising a plurality ofimmune cells of the disclosure, and a pharmaceutically acceptable carrier, diluent or excipient.

[0081] Formulations of a pharmaceutical composition suitable for parenteral administrationtypically generally comprise of immune cells combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0082] In some embodiments, the formulated composition comprising the immune cells issuitable for administration via injection. In some embodiments, the formulated composition comprising the immune cells is suitable for administration via infusion.

[0083] The pharmaceutical compositions of the present disclosure, which may conveniently bepresented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the immune cells with the pharmaceutical carrier(s) or excipient(s), such as liquid carriers.

[0084] Aqueous suspensions may further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0085] The compositions of the present disclosure may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as,Mintz Ref. No.: 064362-501001WO for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the immune cells of the compositions of the present disclosure.

[0086] The formulation or composition may also contain more than one active ingredientuseful for the particular indication, disease, or condition being treated with the immune cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents.

[0087] The pharmaceutical composition in some aspects can employ time-released, delayedrelease, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0088] Administration can be effected in one dose, continuously or intermittently throughoutthe course of treatment. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0089] The pharmaceutical composition in some embodiments contains the immune cells inamounts effective to treat or prevent a cancer, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over days, weeks or months, depending on the condition, the treatment can be repeated until a desired suppression of cancer signs or symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration or infusion of the composition or by multiple bolus administrations or infusions of the composition.

[0090] The cells or population of cells can be administrated in one or more doses. In someembodiments, an effective amount of cells can be administrated as a single dose. In some embodiments, an effective amount of cells can be administrated as more than one doses over aMintz Ref. No.: 064362-501001WO period time. Timing of administration is within the judgment of a managing physician and depends on the clinical condition of the patient.

[0091] The cells or population of cells may be obtained from any source, such as a blood bankor a donor, or the patient themselves.

[0092] An effective amount means an amount which provides a therapeutic or prophylacticbenefit. The dosage administered will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of cells or composition comprising those cells are administrated parenterally. In some embodiments, administration can be an intravenous administration. In some embodiments, administration can be directly done by injection within a tumor.

[0093] For purposes of the disclosure, an assay, which comprises, for example, comparing theextent to which target cells are lysed or one or more cytokines are secreted by immune cells expressing the receptors, upon administration of a given dose of such immune cells to a mammal, among a set of mammals of which is each given a different dose of the immune cells, can be used to determine a starting dose to be administered to a mammal.

[0094] In some embodiments, the cells are administered as part of a combination treatment,such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The immune cells of the disclosure are in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the immune cells are co-administered with another therapy sufficiently close in time such that the immune cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the immune cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the immune cells are administered after to the one or more additional therapeutic agents.

[0095] In embodiments, a lymphodepleting chemotherapy is administered to the subject priorto, concurrently with, or after administration (e.g., infusion) of adoptive immune cells. In an example, the lymphodepleting chemotherapy is administered to the subject prior to administration of the immune cells. For example, the lymphodepleting chemotherapy ends 1- 4 days (e.g., 1, 2, 3, or 4 days) prior to adoptive cell infusion. In embodiments, multiple doses of adoptive cells are administered, e.g., as described herein. In embodiments, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, orMintz Ref. No.: 064362-501001WO after administration (e.g., infusion) of the immune cells described herein. Examples of lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc. Examples of lymphodepleting agents include, but are not limited to, antithymocyte globulin, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD52 antibodies, anti- CD2 antibodies, TCRαβ blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD 154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, OKT3, OKT4, OKT8, OKT11, fingolimod, anti-CD40 antibodies, anti-BR3 antibodies, Campath-1H, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which may be used alone or in combination. As a further example, a lymphodepletion regimen can include, administration of alemtuzumab, cyclophosphamide, benduamustin, rituximab, pentostatin, and / or fludarabine. Lymphodepletion regimen can be administered in one or more cycles until the desired outcome of reduced circulating immune cells. In some embodiments, the lymphodepletion comprises administering an agent that specifically targets, and reduces or eliminates CD52+ cells in the subject, and the immune cells are modified to reduce or eliminate CD52 expression.

[0096] In some embodiments, an immune stimulating therapy is administered to the subjectprior to, concurrently with, or after administration (e.g. infusion) of adoptive immune cells. In some embodiments, the immune stimulating therapy comprises homeostatic cytokines. In some embodiments, the immune stimulating therapy comprises immune-stimulatory molecules. In some embodiments, the immune stimulating therapy comprises IL-2, IL-7, IL-12, IL-15, IL- 21, IL-9, or a functional fragment thereof. In some embodiments, the immune stimulating therapy comprises IL-2, IL-7, IL-12, IL-15, IL-21, IL-9, or combinations thereof. In some embodiments, the immune stimulating therapy comprises IL-2, or a functional fragment thereof.

[0097] Methods for adoptive cell therapy using autologous cells includes isolating immunecells from patient blood, performing a series of modifications on the isolated cells including transducing the cells with one or more vectors encoding the dual receptor system described herein, and administering the cells to a patient. Providing immune cells from a subject suffering from or at risk for cancer or a hematological malignancy requires isolation of immune cell from the patient’s blood, and can be accomplished through methods known in the art, for example, by leukapheresis. During leukapheresis, blood from a subject is extracted and the peripheral blood mononuclear cells (PBMCs) are separated, and the remainder of the blood is returned toMintz Ref. No.: 064362-501001WO the subject’s circulation. The PBMCs are stored either frozen or cryopreserved as a sample of immune cells and provided for further processing steps, such as, e.g. the modifications described herein.

[0098] In some embodiments, the method of treating a subject described herein comprisesmodifications to immune cells from the subject comprising a series of modifications comprising enrichment and / or depletion, activation, genetic modification, expansion, formulation, and cryopreservation.

[0099] The disclosure provides enrichment and / or depletion steps that can be, for example,washing and fractionating methods known in the art for preparation of subject PBMCs for downstream procedures, e.g. the modifications described herein. For example, without limitation, methods can include devices to remove gross red blood cells and platelet contaminants, systems for size-based cell fractionation for the depletion of monocytes and the isolation of lymphocytes, and / or systems that allow the enrichment of specific subsets of T cells, such as, e.g. CD4+, CD8+, CD25+, or CD62L+ T cells. Following the enrichment steps, a target sub-population of immune cells will be isolated from the subject PMBCs for further processing. Those skilled in the art will appreciate that enrichment steps, as provided herein, may also encompass any newly discovered method, device, reagent or combination thereof.

[0100] The disclosure provides activation steps that can be any method known in the art toinduce activation of immune cells, e.g. T cells, required for their ex vivo expansion. Immune cell activation can be achieved, for example, by culturing the subject immune cells in the presence of dendritic cells, culturing the subject immune cells in the presence of artificial antigen-presenting cells (AAPCs), or culturing the immune cells in the presence of irradiated K562-derived AAPCs. Other methods for activating subject immune cells can be, for example, culturing the immune cells in the presence of isolated activating factors and compositions, e.g. beads, surfaces, or particles functionalized with activating factors. Activating factors can include, for example, antibodies, e.g. anti-CD3 and / or anti-CD28 antibodies. Activating factors can also be, for example, cytokines, e.g. interleukin (IL)-2 or IL-21. Activating factors can also be costimulatory molecules, such as, for example, CD40, CD40L, CD70, CD80, CD83, CD86, CD137L, ICOSL, GITRL, and CD134L. Those skilled in the art will appreciate that activating factors, as provided herein, may also encompass any newly discovered activating factor, reagent, composition, or combination thereof that can activate immune cells.

[0101] The disclosure provides genetic modification steps for modifying the subject immunecells. In some embodiments, the genetic modification comprises transducing the immune cell with a vector comprising a shRNA described herein complementary to B2M or HLA-A. InMintz Ref. No.: 064362-501001WO some embodiments, the genetic modification comprises modifying the genome of the immune cells to induce mutations in B2M or HLA-A using CRISPR / Cas mediated genome engineering. In some embodiments, the method comprises transducing the immune cell with one or more vectors encoding the activator and inhibitory receptors, thereby producing immune cells expressing the activator and inhibitory receptors.

[0102] The disclosure provides expansion steps for the genetically modified subject immunecells. Genetically modified subject immune cells can be expanded in any immune cell expansion system known in the art to generate therapeutic doses of immune cells for administration. For example, bioreactor bags for use in a system comprising controller pumps, and probes that allow for automatic feeding and waste removal can be used for immune cell expansion. Cell culture flasks with gas-permeable membranes at the base may be used for immune cell expansion. Any such system known in the art that enables expansion of immune cells for clinical use is encompassed by the expansion step provided herein. Immune cells are expanded in culture systems in media formulated specifically for expansion. Expansion can also be facilitated by culturing the immune cell of the disclosure in the presence of activation factors as described herein. Those skilled in the art will appreciate that expansion steps, as provided herein, may also encompass any newly discovered culture systems, media, or activating factors that can be used to expand immune cells.

[0103] The disclosure provides formulation and cryopreservation steps for the expandedgenetically modified subject immune cells. Formulation steps provided include, for example, washing away excess components used in the preparation and expansion of immune cells of the methods of treatment described herein. Any pharmaceutically acceptable formulation medium or wash buffer compatible with immune cell known in the art may be used to wash, dilute / concentration immune cells, and prepare doses for administration. Formulation medium can be acceptable for administration of the immune cells, such as, for example crystalloid solutions for intravenous infusion. Cryopreservation can optionally be used to store immune cells long-term. Cryopreservation can be achieved using known methods in the art, including for example, storing cells in a cryopreservation medium containing cryopreservation components. Cryopreservation components can include, for example, dimethyl sulfoxide or glycerol. Immune cells stored in cryopreservation medium can be cryopreserved by reducing the storage temperature to -80oC to -196oC.

[0104] In some embodiments, a therapeutically effective dose of the immune cells describedherein are administered. In some embodiments, the immune cells of the disclosure are administered by intravenous injection. In some embodiments, the immune cells of theMintz Ref. No.: 064362-501001WO disclosure are administered by intraperitoneal injection. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells, about 1×106cells, about 2×106cells, about 3×106cells, 4×106cells, about 5×106cells, about 6×106cells, about 7×106cells, about 8×106cells, about 9×106cells, about 1×107, about 2×107, about 3×107, about 4×107, about 5×107, about 6×107, about 7×107, about 8×107, about 9×107, about 1×108cells, about 2×108cells, about 3×108cells, about 4×108cells, about 5×108cells, about 6×108cells, about 7×108cells, about 8×108cells, about 9×108cells, about 1×109cells, about 2×109cells, about 3×109cells, about 3×109cells, about 4×109cells, about 5×109cells, about 5×109cells, about 6×109cells, about 7×109cells, about 8×109cells, about 9×109cells, about 1×1010cells, about 2×1010cells, about 3×1010cells, about 4×1010cells, about 5×1010cells, about 6×1010cells, about 7×1010cells, about 8×1010cells, or about 9×1010cells.

[0105] In some embodiments, a therapeutically effective dose comprises about 0.5×106 cellsto about 9×1010cells, about 1×106cells to about 5×1010cells, about 2×106cells to about 5×109cells, about 3×106cells to about 5×109cells, about 4×106cells to about 3×109cells, about 5×106cells to about 2×109cells, about 6×106cells to about 1×109cells, 0.5×106cells to about 6×109cells, about 1×106cells to about 5×109cells, about 2×106cells to about 5×109cells, about 3×106cells to about 4×109cells, about 4×106cells to about 3×109cells, about 5×106cells to about 2×109cells, about 6×106cells to about 1×109cells, 0.5×106cells to about 6×108cells, about 1×106cells to about 5×108cells, about 2×106cells to about 5×108cells, about 3×106cells to about 4×108cells, about 4×106cells to about 3×108cells, about 5×106cells to about 2×108cells, about 6×106cells to about 1×108cells, about 7×106cells to about 9×108cells, about 8×106cells to about 8×108cells, about 9×106cells to about 7×108cells, about 1×107cells to about 6×108cells, about 2×107cells to about 5×108cells, about 7×106cells to about 9×107cells, about 8×106cells to about 8×107cells, about 9×106cells to about 7×107cells, about 1×107cells to about 6×107cells, or about 2×107cells to about 5×107cells.

[0106] In some embodiments, a therapeutically effective dose comprises about 0.5×105 cellsto about 9×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells to about 1×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells to about 5×109cells. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells to about 1×109cells. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells to about 6×108cells. In some embodiments, a therapeutically effective dose comprises about 0.5×106cells to about 9×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×107cells toMintz Ref. No.: 064362-501001WO about 1×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×107cells to about 5×109cells. In some embodiments, a therapeutically effective dose comprises about 0.5×107cells to about 1×109cells. In some embodiments, a therapeutically effective dose comprises about 0.5×107cells to about 6×108cells. In some embodiments, a therapeutically effective dose comprises about 0.5×108cells to about 9×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×108cells to about 1×1010cells. In some embodiments, a therapeutically effective dose comprises about 0.5×108cells to about 5×109cells. In some embodiments, a therapeutically effective dose comprises about 0.5×108cells to about 1×109cells. The term “about” as referred to in a therapeutically dose, can be, for example, ± 0.5×106cells, ± 0.5×107cells, or ± 0.5×108cells.

[0107] Additional embodiments are disclosed in further detail in the following examples,which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLES

[0108] The practice of the present invention will employ, unless otherwise indicated,conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in MolecularBiology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al.(1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. &Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher;Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides,Mintz Ref. No.: 064362-501001WOS. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: ElsevierSciences B.V., the disclosures of which are incorporated herein by reference.

[0109] Additional embodiments are disclosed in further detail in the following examples,which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1: Identification of the Compound of Formula I as Modulator for Improving T cell

[0110] A small molecule library was screened to assess the molecule’s ability to modulatebiomarkers in T cell in order to promote T cell memory and activation. Total T cells were purified from PBMC using a Pan T Cell Isolation Kit by a negative selection approach to preserve their natural state and functionality. Purified T cells were activated with anti- CD3 / anti-CD28 Dynabeads in T cell media supplemented with 50IU / ml of human recombinant IL-2 in the presence of various molecules from the small molecule library dissolved in DMSO. After 72 hours, T cells were analyzed for mitochondrial content and markers for TSCM (CD45RA+CD62L+CD95+). Compound of Formula I (e.g. compounds I-1 and I-2) was identified to modulate T cells as described in detail below.

[0111] Mitochondrial content in CD4 and CD8 T cells treated with compound I-1 is shown inFIG.1A. The treated T cells were analyzed using flow cytometry analysis. T cells were stained with 100 nM MitoTracker Red CMXRos (which fluoresces upon oxidation in respiring mitochondria). Mitochondrial mass quantification, represented by mean fluorescence intensity (MFI), showed a significant increase in compound I-1-treated cells compared to the vehicle control (DMSO).

[0112] Flowcytometric analysis of CD45RA+ cell frequency was performed in compound I-1or DMSO (negative control) treated CD4 and CD8 T cells. Results are shown in FIG. 1C. Proportion of TSCM cells, identified as CD45RA+CD62L+CD95+, was markedly higher in the compound I-1-treated CD4 and CD8 cells. Bar graph (FIG. 1D) showing quantitative difference in percentage of CD45RA+ cells in both compound I-1 or DMSO treated cells.EXAMPLE 2: EFFECT OF COMPOUND OF FORMULA I IN AUGMENTING TSCM(CD45RA+CD62L+CD95+)

[0113] Expression of key surface markers of TSCM cells was determined using flowcytometryin T cells treated with compound I-1. Human T cells were activated with anti-CD3 / anti-CD28 Dynabeads in T cell media supplemented with 50IU / ml of human recombinant IL-2 either in the presence of DMSO (negative control) or compound I-1 (5µM). After 72 hours, T cells wereMintz Ref. No.: 064362-501001WO stained and analyzed for surface markers for TSCM (CD45RA+CD62L+CD95+). The results are shown in FIG.2A in contour graphs and in FIG.2B as bar graphs.

[0114] FIG. 2B shows a significant increase in percentage of TSCM(CD45RA+CD62L+CD95+) cells in compound I-1 treated group as compared to DMSO as control. P value was determined by unpaired t test.EXAMPLE 3: EFFECT OF COMPOUND OF FORMULA I IN HUMAN AND MOUSE T CELLS

[0115] Compound I-1 is known as inhibitor of both GSK3Beta and DVL-CXXC5 interaction.(e.g. TWS119, E / Z-GSK 3β inhibitor 1, TDZD-8, SB216783, PF-04802367, AR-A014418, CP21R7, KY-02061) were used to assess any effects on CD45RA expression in human T cells. As described here, it was shown that the compound of Formula I such as compound I-1 was unique in its enhancement of CD45RA+ cells. Unlike other inhibitors targeting these pathways, compound I-1significantly enhanced the prevalence of CD45RA+ cells. Results are shown in FIGs.3A to 3F.

[0116] Flowcytometric analysis was performed for the expression of CD45RA+ cells in humanCD8+ T cells following activation and treatment with various inhibitors. As shown in FIG. 3A, maintenance of human CD45RA+ cells was unique to the compound of Formula I such as compound I-1. Other GSK3B inhibitors, such as TWS119 and (E / Z)-GSK-3β inhibitor 1, did not show any significant increase in CD45RA+ in CD8+ T cells.

[0117] FIG. 3B represents the percentage increase in CD8+CD45RA+ cells upon treatmentwith compound I-1 compared to other GSK3B inhibitors (such as TWS119 and (E / Z)-GSK-3β inhibitor 1). (P value determined by one-way ANOVA test)

[0118] Flowcytometric analysis of CD45RA+ cells in human CD8+ T cells was also conductedfollowing activation and treatment with KY-02061 (DVL-CXXC5 interaction inhibitor) and compared to treatment with compound I-1. (FIG.3C)

[0119] FIG. 3D represents the percentage increase in CD8+CD45RA+ cells upon treatmentwith compound I-1compared with other DVL-CXXC5 interaction inhibitor (KY-02061). (P value determined by one-way ANOVA test)

[0120] Comparative experiments were also performed in human and mouse T cells usingcompound I-1 and other GSK3B and CXX5-DVL interaction inhibitors. The effect on augmenting TSCM like properties was assessed using flow cytometry through CD26L expression. Except for the compound of Formula I such as compound I-1, no other GSK3B inhibitors was seen to increase Stem cell memory like T cells (CD62L+CD44-) followed byMintz Ref. No.: 064362-501001WO activation with anti-CD3 / anti-CD28 Dynabeads. Flowcytometric analysis of CD62L+ CD44- T cells upon activation (72 hours) and treatment with various GS3B inhibitors (5µM) is shown in FIGs.3E and 3F. DMSO was used a vehicle control.

[0121] FIG. 3F represents the percentage increase in CD3+CD62L+CD44- cells upontreatment with compound I-1 compared with other GSK3B inhibitors. (P value determined by one-way ANOVA test and for comparison Tukey's multiple comparisons test was used)EXAMPLE 4: EFFECT OF COMPOUND OF FORMULA I ON PROLIFERATION AND MAINTENANCE OFTSCM (CD45RA+ SCA1+) CELLS

[0122] Stem Cell Antigen 1 (Sca1) was used as a marker of hematopoietic stem cells todifferentiate between the naïve T cells (TN) and Stem cell memory T cells (TSCM) to analyze the percentage of TSCM cells. The functional maintenance of T cell memory requires the expression of Sca1 on a specialized population of memory T cells termed “memory stem cells”. The results of Example 3 were therefore corrected for Sca1+ expression. Corrected results are shown in Fis.4A and 4B.

[0123] FIG. 4B represents the percentage increase in CD8+CD45RA+Sca1+ cells upontreatment with the compound of Formula I compared to other GSK3B inhibitors. (P value determined by one-way ANOVA test) All data shown are representative of at least three independently performed experiments. As shown in the data, compound I-1 specifically increased proliferation and maintenance of TSCM cells., whereas other GSK3B inhibitors had no effect.EXAMPLE 5: EFFECT OF COMPOUND OF FORMULA I ON PROLIFERATION AND EXPANSION OFHUMAN ACTIVATED T CELLS

[0124] CD4 and CD8 T cells treated with compound I-1 or DMSO (control) were then treatedwith Cell Trace Violet (CTV) fluorescent dye. Distribution of the dye as cell proliferated was measured by flow cytometry. The histogram in FIG. 5A represents the distribution of cell proliferation in CTV assay, with no difference between compound I-1-treated CD4 and CD8 T cells and control (DMSO). While treatment with other GSK3B inhibitor (TWS119) significantly impaired proliferation of CD4 and CD8 cells.

[0125] As shown in FIG. 5B, the quantitative comparison of proliferation, expansion anddivision index of CD4 and CD8 T cells treated with DMSO, TWS119 or compound I-1. Compound I-1 treatment did not show any reduction in proliferation, expansion and division indexes in both CD4 + and CD8 + T cells. (P value determined by one-way ANOVA test and for comparison Tukey's multiple comparisons test was used)Mintz Ref. No.: 064362-501001WOEXAMPLE 6: EFFECT OF COMPOUND OF FORMULA I ON STEM CELL MEMORY-LIKE FEATURESIN NEWLY GENERATED AND CRYOPRESERVED CAR-T CELLSmarker of TSCM, in human T cells regardless of their differentiation state. Flow cytometric analysis showed that compound I-1 did not impact the transduction efficiency of the CAR construct in both CD4+ and CD8+ cells during CAR-T cell generation. (FIG.6A)

[0127] CAR-T cells, cryopreserved for 60 days without prior exposure to compound I-1, werethawed and reactivated with anti-CD3 / anti-CD28 Dynabeads. Following reactivation, the cells were treated with compound I-1 (5µM) for 72 hours. Flow cytometric analysis of CD45RA surface expression (FIG. 6B) revealed a significant increase in CD45RA+ cells within both CD4+ and CD8+ CAR-expressing T cells. Notably, the extreme right panel in FIG. 6B indicates that 97.8% of CD8 T cells expressing CAR were also CD45RA+, compared to only 15.7% in those treated with DMSO.EXAMPLE 7: EFFECT OF COMPOUND OF FORMULA I ON CAR-T CELLS IN VITROCAR-T cells waswith compound I-1. Untransduced T cells served as the control group. For the in vitro killing assay, equal numbers of NALM6 (target cells: CD19+) and K562 (non-target cells: CD19-) were co- cultured with CD19 CAR-T cells at various effector to target ratios for 16 hours. Following the incubation, cells were washed and stained with a Live / Dead dye to assess specific lysis. The percentage of cell killing was quantified using the formula: Specific Lysis % = [(Target cell control - Target cells with effector) / Target cell control] * 100

[0129] FIG. 7A shows histograms demonstrating significant antigen-specific killing ofNALM6 cells (target cells) by CD19 CAR-T cells generated both with and without compound I-1across various effector-to-target (E:T) ratios. Negligible cell death was observed in K562 (non-target) cells when cultured with the CD19 CAR-T cells. The data, representative of triplicate wells for each effector-to-target cell ratio, underline the specificity and effectiveness of the CD19 CAR-T cell response.

[0130] FIG. 7B shows bar graph representation of % specific killing of NALM6 cells (targetcell) at various effector-to-target (E:T) ratios. (P value determined by one-way ANOVA test and for comparison Tukey's multiple comparisons test was used)

[0131] FIG.7C shows a comparative analysis of percentage of CAR expressing CD4 and CD8cells before and after tumor exposure (target cells) via flowcytometry. CAR-T cells generatedMintz Ref. No.: 064362-501001WO in the presence of compound I-1 exhibited a significant increase in the percentage of CAR+ cells within both the CD4 and CD8 subsets, compared to those generated without compound I-1. This data was quantified and shown in FIG.7D.

[0132] FIG.7D shows quantitative analysis of increase in percentage of CAR expressing CD4and CD8 T cells between CAR-T cells generated in the presence and absence of compound I- 1 before and after target cell exposure. Untransduced T cells were used as control. CAR-T cells generated in the presence of compound I-1 exhibited a significant increase in the percentage of CAR+ cells within both the CD4 and CD8 subsets, compared to those generated without compound I-1. (P value determined by two-way ANOVA test and for comparison Tukey's multiple comparisons test was used)

[0133] As shown in FIGs. 7E and 7F, flow cytometric analysis revealed that CAR-T cellsgenerated with compound I-1 exhibited increased expression of CD69, an early activation marker, upon exposure to tumor cells. This increase indicates that the CAR-T cells were actively recognizing and responding to their target antigens on cancer cells. Specifically, a higher percentage of CAR+ cells within both CD4 and CD8 subsets showed elevated CD69 expression when generated with compound I-1, compared to those produced without compound I-1, across all effector-to-target (E:T) ratios using Raji cells as targets.

[0134] The bar graph (FIG. 7F) illustrates a significant increase in CD69 expression in CAR-T cells generated with compound I-1 compared to those generated without the compound of Formula I at all effector-to-target (E:T) ratios. Untransduced T cells served as the control group. Statistical significance was determined using a one-way ANOVA, with Tukey’s multiple comparisons test for post-hoc analysis.EXAMPLE 8: EFFECT OF COMPOUND OF FORMULA I ON STEM CELL MEMORY IN TUMOR-evaluated.Usually, TILs are fully differentiated T cells and lacks TSCM marker like CD62L. A mouse model of DIPG (Diffuse Intrinsic Pontine Glioma, a type of brain tumor) was used for isolating TILs. TILs were isolated and activated with anti-CD3 / anti-CD28 Dynabeads in T cell media supplemented with 50IU / ml of mouse recombinant IL-2 in the presence of compound I-1 (5µM). DMSO was used as vehicle control.

[0136] FIG. 8A shows results of flowcytometric analysis of CD62L and CD44 surfaceexpression in CD4+ and CD8+ T cells (isolated and purified from TILs) before activation at day 0. FIG. 8B shows flowcytometric analysis of CD62L and CD44 surface expression inMintz Ref. No.: 064362-501001WO CD4+ and CD8+ T cells (TILs) followed by activation and treatment with compound I-1 at day 3. A significant increase was observed in CD62L surface expression in CD8 TILs in presence of compound I-1 (79.5%) as compared to DMSO (0.74%).

[0137] As evidenced in the bar graph of FIG. 8C, a significant increase was observed in thepercentage of CD62L expression both CD4 and CD8+ TILs in presence of compound I-1. Statistical significance was determined using unpaired t test.EXAMPLE 9: RNA SEQUENCING IDENTIFIES IMPORTANT GENES FOR TSCM-LIKE PROPERTIESIN HUMAN T CELLS

[0138] RNA sequencing was performed for human T cells activated in presence of compoundI-1 or DMSO (negative control) to see the differential expression of gene set. Genes such as TET2,TERT,IRF4, NR4A, etc. were seen to be differentially regulated in response to compound I-1. This change in gene expression can highlight potentially relevant gene pathways that can augment TSCM.

[0139] Principal component analysis (PCA) RNA-seq data from treatment with compound I-1, control (DMSO) or TWS119 (n = 3 per group) shown in FIG. 9A demonstrated that there was a primary separation of samples by treatment status. Common and unique genes observed between compound I-1 and DMSO treated groups are shown in the Venn diagrams in FIG.9B.

[0140] Gene Set Enrichment Analysis determined significant enrichment for LymphoidProgenitor Cell Differentiation (FIG.9C) and Fatty Acid Beta Oxidation (FIG.9D) associated genes in compound I-1 treated samples.

[0141] A significant reduction of immune checkpoint associated genes LAG3, CTLA4 andPD1 was observed in compound I-1-treated cells as compared to control. (FIG.9E) Statistical significance was determined using unpaired t test.

[0142] Significant increase in TERT (Telomerase Reverse Transcriptase) expression incompound I-1 treated cells and significant reduced expression of TET2, IRF4, CD38 and NR4A isoform (NR4A1, NR4A2 and NR4A3) in compound I-1 treated cells were observed as compared to control. (FIG.9F)EXAMPLE 10: EFFECT OF COMPOUNDS I-1 AND I-2 ON TSCM MARKERS IN T CELLS

[0143] Compound I-2 was assessed for its effects on augmenting TSCM biomarkers such asCD45RA, CD62L and CD95 using flow cytometry based on protocol similar to Examples 2 and 3. DMSO was used as negative control. Compound I-1 data is shown alongside data for compound I-2 in FIGs.10A to 10D.Mintz Ref. No.: 064362-501001WO

[0144] As shown in FIG. 10A, compound I-2 increased CD45RA+ expressing cell frequencyin CD4 and CD8 T cells compared to DMSO. The percentage CD45A+ cells is shown in FIG. 10B. A significant increase was observed in compound I-1 and compound I-2 treated CD4 and CD8 T cells.

[0145] Further, increase in other TSCM markers was observed in CD8 T cells treated withcompounds I-1 and I-2 compared to DMSO. FIG. 10C shows the T cell population that is CD45RA+, CD62L+, and CD95+, all of which are known markers for TSCM. The percentage CD45RA+, CD62L+, and CD95+ CD8 TSCM is shown in FIG.10D. Accordingly, compound I-2 was also shown to be effective in promoting differentiation of T cells into TSCM.

[0146] While particular alternatives of the present disclosure have been disclosed, it is to beunderstood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

Mintz Ref. No.: 064362-501001WO WHAT IS CLAIMED IS:

1. A method of generating a cell population comprising T memory stem cell (TSCM),the method comprising contacting a starting population of T cells with a compound of Formula I or a salt or solvate thereof to obtain the cell population whereinR1is selected from H, and C1-3 alkyl, R2is each independently selected from H and halo, and n is 0 to 3.

2. The method of claim 1, wherein n is 0.

3. The method of claim 1, wherein n is 2.

4. The method of claim 1, wherein the compound of Formula I is a compound ofFormula I-A or a salt or solvate thereof5. The method of any oneis C1-3 alkyl.

6. The method of any one of claims 1 to 4, wherein R1 is H.Mintz Ref. No.: 064362-501001WO7. The method of any one of claims 1 to 6, wherein R2 is halo.

8. The method of any one of claims 1 to 6, wherein R2 is selected from F, Br, Cl, and I.

9. The method of claim 1, wherein the compound of Formula I is compound I-1 orcompound I-2 110. The method of any one of claims 1 to 9, wherein at least about 30% to about 99%, orabout 80% to about 90% of total cells in the cell population are TSCM.

11. The method of claim 10, wherein the TSCM are CD45RA+CD62L+CD95+.

12. The method of any one of claims 1 to 11, wherein the TSCM of the cell populationhas increased expression of a marker selected from CD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof, compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof.

13. The method of claim 12, wherein the expression of the marker CD45RA is increasedby about 10% to about 99%, about 20% to about 80%, or about 30% to about 70%, wherein the expression of the marker CD62L is increased by about 5% to about 50%,Mintz Ref. No.: 064362-501001WO about 10% to about 45%, or about 15% to about 40%, and / or wherein the expression of the marker CD95 is increased by about 10% to about 90%, about 15% to about 80%, or about 20% to about 70%.

14. The method of any one of claims 1 to 13, wherein the cell population has reducedexpression of at least one T cell exhaustion marker compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof, wherein the at least one T cell exhaustion marker is selected from LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform and combinations thereof.

15. The method of claim 14, wherein the expression of the at least one T cell exhaustionmarker is reduced by about 10% to about 60% or to about 20 % to about 50%.

16. The method of any one of claims 1 to 15, wherein the starting population comprisesactivated T cells.

17. The method of any one of claims 1 to 15, wherein the starting population comprisesnaïve T cells, pan T cells, tumor infiltrating T cells, or combinations thereof.

18. The method of claim 17, wherein the starting population is obtained from PBMCcells, Leukopak, buffy coat, apheresis product, or combination thereof.

19. The method of claim 17, wherein the method further comprises activating the naïve Tcells, pan T cells, tumor infiltrating T cells, or combinations thereof in the starting population to obtain activated T cells.

20. The method of claim 19, wherein the naïve T cells, pan T cells, tumor infiltrating Tcells, or combinations thereof comprise CD8+ and / or CD4+ T cells.

21. The method of claim 19 or 20, wherein the activating comprises treating the startingpopulation with a T cell activator and IL-2.

22. The method of claim 21, wherein the T cell activator is an anti-CD3 antibody or aCD3 binding fragment thereof, an anti-CD28 antibody or a CD28 binding fragment thereof, or combinations thereof.

23. The method of claim 22, wherein the anti-CD3 antibody or the CD3 binding fragmentthereof, the anti-CD28 antibody or the CD28 binding fragment thereof, or the combinations thereof is on a solid support.Mintz Ref. No.: 064362-501001WO24. The method of any one of claims 21 to 23, wherein the treating is for about 24 hoursto about 96 hours, about 50 hours to about 85 hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours.

25. The method of any one of claims 21 to 24, wherein the contacting is carried outsubsequently to the activating.

26. The method of claim 25, wherein the contacting is carried out about 4 days to about 9days, about 5 days to about 7 days, or about 6 days to about 8 days subsequently to the activating.

27. The method of any one of claims 19 to 26, wherein the activating comprises culturingthe starting population in a culture medium and wherein the culture medium comprises a T cell activator, and at least one of IL-2, IL-15 and IL-7.

28. The method of any one of claims 1 to 27, wherein the starting population is in aculture medium.

29. The method of claim 28, wherein the contacting comprises combining the culturemedium with the compound of Formula I or a salt or solvate thereof.

30. The method of claim 28, wherein the contacting comprises culturing the startingpopulation in the culture medium and wherein the culture medium comprises the compound of Formula I or a salt or solvate thereof.

31. The method of any one of claims 1 to 30, wherein the contacting is for about 12 hoursto about 96 hours, about 24 hours to about 85 hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours.

32. The method of any one of claims 1 to 31, wherein the method further comprisescontacting the starting population with at least one of IL-2, IL-15 and IL-7 concurrently with the contacting with the compound of Formula I or a salt or solvate thereof.

33. The method of any one of claims 1 to 32, wherein the contacting with the compoundof Formula I of a salt or solvate thereof is carried out at a concentration of the compound of Formula I of about 0.1 µM to about 10 µM.

34. The method of any one of claims 1 to 33, wherein the TSCM comprises a CARconstruct comprising one or more chimeric antigen receptors (CARs).Mintz Ref. No.: 064362-501001WO35. The method of claim 34, wherein the method further comprises introducing a CARconstruct into the T cells of the starting population.

36. The method of claim 35, wherein the introducing is carried out in the presence of thecompound of Formula I.

37. The method of any one of claims 1 to 36, wherein the method does not affectproliferation of T cells.

38. A cell population generated by a method as defined in any one of claims 1 to 37.

39. The cell population of claim 38, wherein about 30% to about 99%, or about 80% toabout 90% of the total T cells in the cell population are TSCM.

40. A method of generating an engineered T cell population, the method comprisingintroducing a starting population of T cells with a CAR construct comprising one or more CARs; contacting the staring population with a compound of Formula I or a salt or solvate thereof, wherein the compound of Formula I is as defined in any one of claims 1 to 9 to obtain the engineered T cell population; wherein the engineered T cell population comprises T stem cell memory (TSCM).

41. The method of claim 40, wherein the introducing and the contacting are carried outsubstantially concurrently.

42. The method of claim 40, wherein the contacting is carried out subsequently to theintroducing.

43. The method of any one of claims 40 to 42, wherein the method further comprisescontacting of the starting population of T cells with IL-2, IL-15, and / or IL-7 concurrently to the contacting of the starting population with the compound of Formula I of a salt or solvate thereof.

44. The method of any one of claims 40 to 43, wherein the engineered T cell populationhas increased expression of a marker selected from CD45RA, CD62L, CD95, CCR7, Telomerase reverse transcriptase (TERT), lymphoid progenitor cell differentiation genes, fatty acid beta oxidation genes, and combinations thereof, compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof.Mintz Ref. No.: 064362-501001WO45. The method of any one of claims 40 to 44, wherein the engineered T cell populationhas reduced expression of a marker selected from LAG3, CTLA-4, PD1, TET2, IRF4, CD38, NR4A isoform and combinations thereof compared to a starting population that is untreated with the compound of Formula I or a salt or solvate thereof.

46. The method of any one of claims 40 to 45, wherein the contacting of the startingpopulation with the compound of Formula I or a salt or solvate thereof is carried out at for about 12 hours to about 96 hours, about 24 hours to about 85 hours, about 60 hours to about 80 hours, about 65 to about 75 hours, about 70 to about 75 hours, or about 72 hours.

47. The method of any one of claims 40 to 46, wherein the contacting with the compoundof Formula I of a salt or solvate thereof is carried out at a concentration of the compound of Formula I of about 0.1 µM to about 10 µM.

48. The method of any one of claims 40 to 46, wherein about 30% to about 99%, or about80% to about 90% of the total T cells in the engineered T cell population are TSCM.

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