Modeling the terminal dysfunction of car t cells

Abstract

The present disclosure provides a method of inducing terminal exhaustion in a population of CAR-T cells, as well as populations of terminally exhausted CAR-T cells prepared by these methods. Also provided are methods of using these methods and these populations of terminally exhausted CAR-T cells for screening for agents that can reverse or prevent terminal dysfunction in CAR T cells.

Description

[0001] MODELING THE TERMINAL DYSFUNCTION OF CAR T CELLS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to, and the benefit of, U.S. Provisional Application No. 63 / 635,870, filed April 18, 2024, and U.S. Provisional Application No. 63 / 642,339, filed on May 3, 2024, each of which is hereby expressly incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] CAR T cells offer tremendous therapeutic potential against cancer. However, CAR T cell therapies face many challenges in practice, including challenges associated with the tumor microenvironment (e.g., limitations to CAR-T infiltration) and tumor responses such as antigen-positive relapse and antigen-negative relapse. However, CAR-T cell dysfunction — in which initially activated T cells acquire a hyporesponsive state that progressively diminishes their effector function upon exposure to chronic antigen stimulation or immunosuppressive tumor microenvironment (TME) — significantly limits the efficacy of many CAR-T cell therapies. CAR-T cell therapies could potentially benefit from active agents that prevent and / or reverse the terminal dysfunction of CAR-T cells.

[0006] The methods described herein address these and other needs.

[0007] SUMMARY

[0008] In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to methods of inducing terminal exhaustion in a population of CAR-T cells, as well as populations of terminally exhausted CAR-T cells prepared by these methods.

[0009] For example, provided herein are methods of inducing terminal exhaustion in a population of CAR-T cells that comprise culturing a population of CAR-T cells in a media; and chronically simulating the population of CAR-T cells by exposing the population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule.

[0010] Also provided herein are populations of terminally exhausted CAR T cells prepared by the methods described herein, as well as methods of using these populations of terminally exhausted CAR T cells in an assay to screen an agent for its ability to reverse to prevent terminal dysfunction in a population of CAR-T cells.

[0011] Also provided are methods of screening an agent for its ability to reverse and / or prevent terminal dysfunction in a population of CAR-T cells that comprise preparing a control population of CAR-T cells by a method that comprises culturing a first population of CAR-T cells in a media; and chronically simulating the first population of CAR-T cells by exposing the first population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule; preparing a screening population of CAR-T cells by a method that comprises culturing a second population of CAR-T cells in a media; and chronically simulating the second population of CAR-T cells by exposing the second population of CAR-T cells to a population of cancer cells expressing the CAR-specific antigen in the presence of a tumor microenvironment signaling molecule and an active agent; and evaluating the degree of terminal exhaustion in the control population of CAR-T cells and the screening population of CAR-T cells; wherein a lower degree of terminal exhaustion in the screening population of CAR-T cells as compared to the control population of CAR-T cells indicates that the active agent can reverse and / or prevent terminal dysfunction in a population of CAR-T cells.

[0012] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0015] Figure l is a schematic illustration of some of the challenges associated with CAR-T cell therapies, including CAR-T cell intrinsic challenges such as dysfunction.

[0016] Figure 2 is a schematic illustration of CAR-T cell dysfunction.

[0017] Figure 3 illustrates an approach involving the co-culturing of triple-target CAR T cells with B cell lymphoma lines. Figure 4 illustrates the characterization of the triple CAR T cell product, including the CD4 vs. CD8 distribution. Panel A shows the frequency distribution of CD4+, CD8+ or CD8-intermediate Triple CAR T cells within the total CD3+ cells on Day 0. Products 1, 2 and 3 represent different source donors. Panel B includes flow cytometry plots showing CD4 versus CD8 expression level and gating of the three subsets in Panel A. Panel C includes a flow cytometry plot showing interferon gamma (IFNG) cytokine expression levels. Panel D shoes granzyme B (GZMB) expression level within the three subsets of Triple CAR T cells after PMA / Ionomycin stimulation on Day 0. Orange / yellow color indicates higher expression and green indicates lower expression.

[0018] Figure 5 illustrates the characterization of the triple CAR T cell product, which reveals that the CD8intermediatecell subset is “dysfunctional" on day 0. Panel A shows the expression level (mean fluorescence intensity “MFI”) for IFNG cytokine after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel B shows the expression level for CD 107a degranulation marker after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel C shows the expression level for tumor necrosis factor alpha (TNFa) cytokine after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel D shows the expression level for interleukin-2 (IL-2) cytokine after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel E shows the expression level for granzyme B after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel F shows the expression level for LAG3 inhibitory receptor after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel G shows the expression level for PD-1 inhibitory receptor after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0. Panel H shows the expression level for CD39 exhaustion marker after PMA / Ionomycin stimulation of CD4+, CD8+ or CD8-int Triple CAR T cells on Day 0.

[0019] Figure 6 shows that strong chronic CAR signaling only drives terminal differentiation and activation-induced cell death. Panel A shows a schematic for repeated co-culture of Triple CAR T cells with human B cell lymphoma cell lines that have varying degrees of CD 19, CD20 and CD22 expression level (JeKo-1 - low, Mino - intermediate, Raji - high). Panels B and C show the tracking of CD8+ (Panel B) or CD4+ (Panel C) CAR T cell numbers per 200 ul wells over 12 days of repeated co-culture with either Jeko-1 (green), Mino (yellow) or Raji (red) cells in the presence or absence of low-dose TGFpi cytokine (0.5 ng / ml). Figure 7 shows an optimized modeling of terminal dysfunction in human CAR T cells, in this case an optimized model for repeated co-culture of triple CAR T cells or CD19-CAR T cells with human B cell lymphoma cell lines. In brief, CAR T cells are cocultured with tumor cells for either 7 days (“Acute Stim”) or 21 days (“Chronic Stim”) plus TGFpi cytokine as a tumor microenvironmental signal (TME) at 0.5 ng / ml concentration. Cells are tracked by flow cytometry once weekly for expansion, function, and tumor killing capacity during repeated co-culture.

[0020] Figure 8 illustrates the development of dysfunction features after 1 week CAR stimulation + TGFpi. Panel A shows a schematic for the repeated co-culture of triple CAR- T cells (Product 2 shown) with JeKo-1 lymphoma cells for 8 days in the presence of TGFpi, followed by PMA / Ionomycin stimulation to measure functionality. Panel B shows the tracking of cell numbers of CD8+ (blue), CD8-int (purple) and CD4+ (green) Triple CAR-T cells over 8 days of repeated co-culture with JeKo-1 cells and TGFpi. Plots tracking of the frequency of “Functional” IFNG and CD 107a co-producing CAR T cells (Panel C), the expression level (MFI) of CD39 exhaustion marker (Panel D), and the expression level of LAG3 exhaustion marker (Panel E) over 8 days of repeated co-culture with JeKo-1 cells and TGFpi are also included.

[0021] Figure 9 illustrates that CD8intermediatecells show the most “dysfunctional” state on day 8. Panel A shows the expression level (MFI) of IFNG cytokine on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture. Panel B shows the expression level (MFI) of TNFa cytokine on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture. Panel C shows the expression level (MFI) of IL-2 cytokine on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture. Panel D shows the expression level (MFI) of granzyme B on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture. Panel E shows the expression level (MFI) of CD 107a degranulation marker on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture. Panel F shows the expression level (MFI) of CD39 exhaustion marker on CD4+, CD8+ and CD8-int triple CAR-T cells after PMA / Ionomycin stimulation on Day 8 of co-culture.

[0022] Figure 10 is a schematic showing an optimized in vitro model of triple CAR-T cell chronic stimulation. Cells underwent repeated co-culture with JeKo-1 cells for 7 days (“Acute Stim”), or 21 days (“Chronic Stim”) in the presence or absence of TGFpi cytokine. On days 15 and 21 of co-culture, CAR T cells were challenged with Raji lymphoma cells for 6 hours to provide strong, short-term stimulation for measuring phenotype, functionality and tumor-killing capacity after challenge with target tumor cells.

[0023] Figure 11 shows that the CD8intermediatedysfunctional population emerges under chronic CAR stimulation (Product 3). Panel A is a plot tracking cell numbers of either CD8+ or CD8-int Triple CAR T cells after 7 days (Acute, Blue) or 21 days (Chronic, Grey or Chronic+TGFpi, Orange) of repeated co-culture with JeKo-1 tumor cells. Panel B is a flow cytometry plot and bar graph showing frequency of IFNG and CD 107a co-producing CD8+ (grey) or CD8-int (red) chronically stimulated Triple CAR T cells after 6hr Raji stimulation on Day 21 of repeated co-culture. Panel C is a flow cytometry plot and bar graph showing frequency of IFNG and CD 107a co-producing CD8+ (grey) or CD8-int (red) chronic + TGFpi stimulated CAR T cells after 6hr Raji stimulation on Day 21 of repeated co-culture. Panel D is plot showing Granzyme B expression level for chronically stimulated Triple CAR T cells on Day 21 after 6hr Raji stimulation on Day 21 of repeated co-culture. Black line indicates CD8+ and red line indicates CD8-int CAR T cells. Panel E is plot showing Granzyme B expression level for chronic + TGFpi stimulated CAR T cells on Day 21 after 6hr Raji stimulation on Day 21 of repeated co-culture. Black line indicates CD8+ and red line indicates CD8-int CAR T cells.

[0024] Figure 12 demonstrates that chronic CAR + TGFpi stimulation of Triple CAR T cells drives development of dysfunction. Panel A includes flow cytometry plots showing IFNG and CD 107a expression on CD8+ Triple CAR T cells that underwent Acute (7 days) or Chronic (21 days) co-culture with JeKo-1 cells followed by 6hr-challenge with Raji cells on Day 21. Panel B is a histogram showing expression level of Granzyme B on CD8+ Triple CAR T cells after 6hr-challenge with Raji cells on Day 21. Blue line shows Acute, grey line shows Chronic, and orange line shows Chronic+TGFpi. Panels C and D are plots showing the frequency of IFNG and CD 107a co-producing (Panel C) and IFNG and TNFa co-producing (Panel D) CD8+ Triple CAR T cells on Day 21 after 6hr-challenge with Raji cells. Panels E and F are plots showing the expression level (MFI) of CD39 (Panel E) or Granzyme B (Panel F) on CD8+ Triple CAR T cells on Day 21 after 6hr-challenge with Raji cells.

[0025] Figure 13 demonstrates that chronic CAR+TGFpi -stimulated CAR-T cells lose their cytotoxicity against tumor targets. Panels A and B show the number of dead Raji tumor cells per 100 CAR T cells after 6hr-challenge with Raji cells on Day 15 (Panel A) and Day 21 (Panel B) of repeated co-culture with JeKo-1 cells for Acute (Blue), Chronic (Grey), or Chronic+TGFpi (Orange). Panel C is a plot tracking of dead Raji tumor cell numbers per 100 CAR T cells between Day 15 and Day 21 of repeated co-culture with JeKo-1 cells.

[0026] Figure 14 demonstrates that chronically stimulated CAR T cells develop a stable dysfunctional state. Panel A is a schematic for resting of chronically stimulated Triple CAR T cells beginning on day 21 of repeated co-culture until day 28 in the absence of tumor cells and TGFpi. Rested CAR T cells were then rechallenged with Raji tumor cells for 6 hours or 24 hours followed by functional analysis. Panels B and C show the number of dead Raji tumor cells per 100 CD8+ CAR T cells after 6hr-challenge (Panel B) and 24hr-challenge (Panel C) with Raji cells for Acute (Blue), Chronic (Grey), or Chronic+TGFpi (Orange) on Day 28. Panel D is a plot tracking of dead Raji tumor cell numbers per 100 CAR T cells between Day 15 and Day 21 of repeated co-culture with JeKo-1 cells and the resting phase from Day 21 through Day 28.

[0027] Figure 15 demonstrates that chronically stimulated CAR T cells develop a stable dysfunctional state. Panel A includes flow cytometry plots showing expression of IFNG and CD107a on CD8+ Triple CAR T cells after 6hr-stimulation with Raji cells on Day 28 following the resting phase from chronic co-culture. Panels B and C plot the frequency of IFNG and CD 107a co-producing CD8+ (Panel B) and CD8-int (Panel C) Triple CAR T cells after 6hr-stimulation with Raji cells on Day 28 following the resting phase.

[0028] Figure 16 demonstrates that CAR T cell dysfunction is stable upon rest from chronic stimulation. Panels A and B are histograms and bar graphs showing Granzyme B expression level on CD8+ (Panel A) and CD8-int (Panel B) Triple CAR T cells after 6hr- stimulation with Raji cells on Day 28 following the resting phase. Panels C and D are histograms and bar graphs showing Perforin expression level on CD8+ (Panel C) and CD8- int (Panel D) Triple CAR T cells after 6hr-stimulation with Raji cells on Day 28 following the resting phase.

[0029] Figure 17 demonstrates that dysfunctional CAR T cells have limited survival and / or homeostatic proliferation. Panel A is a plot tracking the frequency of dead CD3+ Triple CAR T cells over the course of Acute (blue) or Chronic co-culture with JeKo-1 cells (Grey - without TGFpi, Orange + TGFpi) from Day 7-21 followed by the resting phase from JeKo-1 cells and TGFpi until Day 28. Panel B is a plot tracking the frequency of dead CD3+ CAR T cells on Day 28 after the resting phase.

[0030] Figure 18 is a schematic for repeated co-culture of CD19-specific CAR T cells with JeKo-1 tumor cells for 7 days (Acute) or 21 days (chronic) in the presence or absence of TGFpi cytokine (0.5 ng / ml) as a tumor microenvironmental (TME) signal. On days 15 and 21 of co-culture, CAR T cells were challenged with Raji lymphoma cells for 6 hours or overnight to provide strong, short-term stimulation for measuring phenotype, functionality and tumor-killing capacity after challenge with target tumor cells.

[0031] Figure 19 demonstrates that the surface expression level of CD 19 CAR impacts the degree of CAR-T cell activation. Panel A is a histogram showing expression level for the CD19-CAR receptor on either Untransduced (Grey) or CD19-CAR transduced CD8+ T cells. Cells were grouped based on relative CAR expression level: CD19-CAR-high (Red), CD19-CAR-int (Purple), and CD19-CAR-low (Blue). Panels B-K are histograms and bar graphs showing the expression level of CD107a (Panels B and C), CD39 (Panels D and E), Granzyme B (Panels F and G), IL-7-receptor (Panels H and I), and IL-2 cytokine (Panels J and K) on CD19-CAR-expressing subsets or Untransduced CD8+ T cells after PMA / Ionomycin stimulation on Day 7 of repeated co-culture with JeKo-1 cells.

[0032] Figure 20 demonstrates that chronic tumor plus TGFpi exposure limits CD 19 CAR- T cell expansion but improves survival. Panels A and C-D are plots tracking fold expansion during acute or chronic CAR stimulation of CD8+ (Panel A), CD4+ (panel C), and CD8- intermediate (Panel D) human CD19-CAR T cells or untransduced (UTD) control from day 0 through day 20 of repeated co-culture with JeKo-1 cells. Panel B shows the frequency of dead T cells on day 20 of chronic CAR + / - TGFpi stimulation for Acute (blue), Chronic (grey) or Chronic+TGFpi (Orange) stimulated CD19-CAR T cells.

[0033] Figure 21 demonstrates that chronically stimulated human CD 19 CAR-T cells develop features of dysfunction. Panel A shows flow cytometry plots showing expression level of IFNG and CD 107a for acute or chronically stimulated CD19-CAR T cells after overnight stimulation with Raji cells on Day 20 of repeated co-culture. Panels B and C plot the frequency of IFNG and CD107a co-producing (Panel B) and "dysfunctional" CD8+ CD19-CAR T cells (IFNG- TNF- CD107a-) (Panel C) after overnight stimulation with Raji cells on Day 20 of repeated co-culture. Panels D and E plot the expression level (MFI) of TNF cytokine (Panel D) and CD39 exhaustion marker (Panel E) on CD8+ CD19-CAR T cells after overnight stimulation with Raji cells on Day 20 of repeated co-culture.

[0034] Figure 22 shows that tumor killing capacity is reduced in chronically CAR+ TGFpi stimulated CD19 CAR T cells. Panel A shows a schematic for overnight (19hr) co-culture of Acute or Chronically stimulated CD19-CAR T cells with CD19+ lymphoma cells (Raji) at 1 :3 effector: target ratio to measure phenotype, function and tumor-killing activity of CD19-CAR T cells. Panel B is a plot showing the number of dead tumor cells per 100 total T cells after overnight challenge with Raji tumor cells on Day 14 of repeated co-culture. Panel C is a plot showing the expression level of Granzyme B after overnight challenge with Raji tumor cells on Day 14 of repeated co-culture. Panel D is a plot showing the expression level of CD107a on CD19-CAR T cells after overnight challenge with Raji tumor cells on Day 14 of repeated co-culture.

[0035] Figure 23 shows that dysfunction in chronically stimulated CD19 CAR T Cells is stable upon resting. Panel A shows a schematic for resting phase of CD19-CAR T cells from repeated co-culture with JeKo-1 cells and TGFpi starting from Day 21 through Day 28. Resting phase was followed by overnight re-challenge with CD 19+ Raji lymphoma cells to track T cell function and killing activity after rest. Panel B shows the absolute frequency of viable CD19-CAR T cells from Day 20 to Day 28 during the resting phase after acute or chronic CAR + / - TGFpi co-culture. Panel C shows the fold change of % viable CD19-CAR T cells from Day 20 to Day 28 during the resting phase after acute or chronic CAR + / - TGFpi co-culture. Panel D plots the number of dead Raji tumor cells per 100 T cells for Acute or Chronically stimulated CD19-CAR T cells after overnight rechallenge with Raji cells on Day 28 following the resting phase. Panel E plots the frequency of "dysfunctional" CD8+ CAR T cells (IFNG- TNF- CD107a-) after overnight rechallenge with Raji tumor cells on Day 28. Plot F plots the expression level of CD39 exhaustion marker on CD8+ CD19-CAR T cells after overnight rechallenge with Raji tumor cells on Day 28.

[0036] DETAILED DESCRIPTION

[0037] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0038] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0039] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible nonexpress basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0040] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0041] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0042] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions

[0043] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0044] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like.

[0045] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0046] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0047] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and subrange is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0048] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight or less, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition.

[0049] The term “subject” preferably refers to a human in need of treatment with an anticancer agent or treatment for any purpose, and more preferably a human in need of such a treatment to treat cancer, or a precancerous condition or lesion. However, the term “patient” can also refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others, that are in need of treatment with an anti-cancer agent or treatment.

[0050] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. Furthermore, prevent refers to the lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “prevent T-cell terminal exhaustion” means reducing the rate of terminal exhaustion relative to a standard or a control.

[0051] The term “treating” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0052] "CAR-T cells" refer to a T cell or population thereof, which has been modified through molecular biological methods to express a chimeric antigen receptor (CAR) on the T cell surface.

[0053] Methods

[0054] Provided herein are methods of inducing terminal exhaustion in a population of CAR-T cells. These methods can comprise culturing a population of CAR-T cells in a media; and chronically simulating the population of CAR-T cells by exposing the population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule.

[0055] In some embodiments, chronically simulating the population of CAR-T cells comprises co-culturing the population of CAR-T cells with the population of cancer cells expressing the CAR-specific antigen. The population of CAR-T cells and the population of cancer cells expressing the CAR-specific antigen are co-cultured for a period of from 7 days to 60 days, such as from 7 days to 42 days, from 7 days to 35 days, from 7 days to 28 days, from 7 days to 21 days, from 14 days to 60 days, from 14 days to 42 days, from 14 days to 35 days, from 14 days to 28 days, from 14 days to 21 days, from 21 days to 60 days, from 21 days to 42 days, from 21 days to 35 days, or from 21 days to 28 days.

[0056] The tumor microenvironment signaling molecule can comprise any suitable signaling molecule present (e.g., overexpressed or otherwise at elevated concentrations relative to normal tissue) in the tumor microenvironment. Examples of such molecules include cytokines, chemokines, growth factors, extracellular matrix proteins, and / or other biomolecules. These signaling molecules can facilitate communication between tumor cells and various components within the TME, including stromal cells, immune cells, and endothelial cells. As such, these signaling molecules can play an important role in tumor development and progression. In some embodiments, the tumor microenvironment signaling molecule comprises a cytokine (TGF-P cytokine). In certain embodiments, the TGF-P cytokine comprises a TGF-pi cytokine. In some embodiments, the tumor microenvironment signaling molecule can be present in the media at a concentration of less than 5 ng / mL, such as a concentration of from 0.001 ng / mL to 4 ng / mL, from 0.005 ng / mL to 4 ng / mL, from 0.01 ng / mL to 4 ng / mL, from 0.05 ng / mL to 4 ng / mL, from 0.1 ng / mL to 4 ng / mL, 0.001 ng / mL to 3 ng / mL, from 0.005 ng / mL to 3 ng / mL, from 0.01 ng / mL to 3 ng / mL, from 0.05 ng / mL to 3 ng / mL, from 0.1 ng / mL to 3 ng / mL, 0.001 ng / mL to 2 ng / mL, from 0.005 ng / mL to 2 ng / mL, from 0.01 ng / mL to 2 ng / mL, from 0.05 ng / mL to 2 ng / mL, from 0.1 ng / mL to 2 ng / mL, 0.001 ng / mL to 1 ng / mL, from 0.005 ng / mL to 1 ng / mL, from 0.01 ng / mL to 1 ng / mL, from 0.05 ng / mL to 1 ng / mL, from 0.1 ng / mL to 1 ng / mL, or from 0.25 ng / mL to 0.75 ng / mL. In certain embodiments, the tumor microenvironment signaling molecule is present in the media at a concentration of about 0.5 ng / mL.

[0057] In some embodiments, the conditions of the co-culture (e.g., oxygen concentration, pH, etc.) can be adjusted to mimic conditions characteristic of the tumor microenvironment (e.g., nutrient deficiency, hypoxia, acidic pH, or a combination thereof). In some embodiments, the concentration of the tumor microenvironment signaling molecule can be reduced when the conditions of the co-culture mimic the characteristic of the tumor microenvironment. In some variants, the tumor microenvironment signaling molecule can be omitted from the co-culture when the conditions of the co-culture mimic the characteristic of the tumor microenvironment.

[0058] In some embodiments, the population of CAR-T cells can comprise a population of human CAR-T cells. In some embodiments, the population of CAR-T cells comprises CD4 T cells, CD8 T cells, or a combination thereof.

[0059] The CAR-specific antigen can comprise any suitable CAR-specific antigen (tumor antigen) known in the art. Examples include, for example, CD 19 (found on the surface of B-cells, including B-cell lymphomas and leukemias); BCMA (B-cell maturation antigen) (primarily found on multiple myeloma cells); CD22 (also found on B-cells, making it another target for B-cell cancers); CD30 (found on Hodgkin's lymphoma cells); EGFRvIII (Variant III of the epidermal growth factor receptor; overexpressed in glioblastomas); IL13Ra2 (Interleukin- 13 receptor alpha 2; found on glioma cells); mesothelin (overexpressed in mesothelioma, ovarian cancer, and pancreatic cancer); MUC1 (Mucin 1; overexpressed in ovarian, breast, and prostate cancers); GD2 (Disialoganglioside 2; found on neuroblastoma and melanoma cells); HER2 (Human epidermal growth factor receptor 2; overexpressed in breast, ovarian, and other cancers); and PSMA (Prostate-specific membrane antigen; primarily found on prostate cancer cells). In some embodiments, the population of cancer cells comprises human cancer cells. For example, in some embodiments, the population of cancer cells comprise carcinoma cells, sarcoma cells, leukemia cells, lymphoma cells, myeloma cells, or a combination thereof. In certain embodiments, the population of cancer cells comprise lung cancer cells (e.g., non-small cell, small cell), breast cancer cells, colorectal cancer cells, prostate cancer cells, pancreatic cancer cells, gastric (stomach) cancer cells, esophageal cancer cells, liver cancer cells (hepatocellular carcinoma), bladder cancer cells, kidney (renal cell) cancer cells, cervical cancer cells, endometrial (uterine) cancer cells, head and neck squamous cell carcinoma cells, ovarian cancer cells, skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma) cells, osteosarcoma cells, liposarcoma cells, leiomyosarcoma cells, rhabdomyosarcoma cells, Ewing sarcoma cells, fibrosarcoma cells, synovial sarcoma cells, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia) cells, lymphoma (e.g., Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma) cells, multiple myeloma cells, myelodysplastic syndrome cells, myeloproliferative neoplasm cells, glioblastoma multiforme cells, astrocytoma cells, oligodendroglioma cells, medulloblastoma cells, ependymoma cells, cutaneous melanoma cells, ocular melanoma cells, mucosal melanoma cells, testicular cancer cells (e.g., seminoma, non-seminoma), ovarian germ cell tumor cells, extragonadal germ cell tumor cells, neuroblastoma cells, pheochromocytoma cells, carcinoid tumor cells, pancreatic neuroendocrine tumor cells, Wilms tumor cells, retinoblastoma cells, medulloblastoma cells, neuroblastoma cells, or pediatric leukemia and lymphoma cells.

[0060] Also provided herein are populations of terminally exhausted CAR T cells prepared by the methods described herein, as well as methods of using these populations of terminally exhausted CAR T cells in an assay to screen an agent for its ability to reverse to prevent terminal dysfunction in a population of CAR-T cells. Such methods can comprise, for example, contacting these populations of terminally exhausted CAR T cells with an active agent, and evaluating the degree of terminal exhaustion in the population of terminally exhausted CAR T cells following contact with the active agent. In some embodiments, these methods can further comprise evaluating the degree of terminal exhaustion in the population of terminally exhausted CAR T cells prior to contact with the active agent. A lower degree of terminal exhaustion in the population of terminally exhausted CAR-T cells following contact with the active agent (e.g., as compared to the population of terminally exhausted CAR-T cells prior to contact with the active agent) can indicate that the active agent can reverse and / or prevent terminal dysfunction in a population of CAR-T cells. Such methods can be performed in vitro using conventional screening strategies, including high throughput screening strategies.

[0061] Also provided are methods of screening an agent for its ability to reverse and / or prevent terminal dysfunction in a population of CAR-T cells that comprise preparing a control population of CAR-T cells by a method that comprises culturing a first population of CAR-T cells in a media; and chronically simulating the first population of CAR-T cells by exposing the first population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule; preparing a screening population of CAR-T cells by a method that comprises culturing a second population of CAR-T cells in a media; and chronically simulating the second population of CAR-T cells by exposing the second population of CAR-T cells to a population of cancer cells expressing the CAR-specific antigen in the presence of a tumor microenvironment signaling molecule and an active agent; and evaluating the degree of terminal exhaustion in the control population of CAR-T cells and the screening population of CAR-T cells; wherein a lower degree of terminal exhaustion in the screening population of CAR-T cells as compared to the control population of CAR-T cells indicates that the active agent can reverse and / or prevent terminal dysfunction in a population of CAR-T cells. In these methods, the control population of CAR-T cells and the screening population of CAR-T cells can be prepared by methods (such as those described above) that are otherwise identical except for the presence of the active agent.

[0062] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0063] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0064] EXAMPLES

[0065] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0066] Example 1: Method to Induce Terminal Disfunction of Human CAR T Cells

[0067] An example protocol for a method of inducing terminal disfunction in human CAR T Cells is detailed below.

[0068] Goal: To establish terminal exhaustion in human CAR T cells by co-culturing human CD19-specific CAR T cells or “triple” CAR T cells (specific to CD 19, CD20 and CD22 antigens) with human B cell lymphoma cell lines (JeKo-1, Raji) that express variable levels of CD 19, CD20, and CD22 antigens (JeKo-1 cells express low-intermediate levels, while Raji cells express high levels), in the presence of low repeated doses of TGFpi (0.5 ng / ml).

[0069] Conditions include:

[0070] 1- Chronic Stimulation group (repeated cocultured with JeKo-1 leukemia cells only) for 3 weeks

[0071] 2- “Terminally Dysfunctional” group — Chronic Stim+ TGFpi (repeated cocultured with JeKo-1 leukemia cells in the presence of 0.5 ng / ml of TGFpi) for 3 weeks

[0072] 3- Acute Stimulation group (repeated cocultured with JeKo-1 leukemia cells for 1 week only then rested afterwards for 2 weeks)

[0073] The method also describes cellular and molecular assays for tracking effector function, expansion and tumor cell killing activity by CAR T cells at different timepoints during chronic stimulation.

[0074] Day 0- starting co-culture experiment:

[0075] Human T Cell Media preparation:

[0076] 45 mL of complete RPMI (with 10% FBS and IX P / S)

[0077] 45 pL of human IL-15

[0078] 20 pL of human IL-2 A. Prepare CAR T cells:

[0079] 1. Thaw 1 tube of frozen cells each: CD19-CAR, Untransduced, and Triple-CAR T Cells - in Liquid Nitrogen Freezer. a. Place in the 37 degree C water bath for a few minutes until partially thawed with a frozen core. b. Transfer thawed cells to a 50-ml tube containing ~20 ml of cRPMI media. c. Spin down the cells to wash at 400xg for 10 minutes at 20 degrees.

[0080] 2. Aspirate the supernatant, then resuspend the pellet in 0.5-1 ml of Human T Cell Media.

[0081] 3. Count the cells using the hemacytometer.

[0082] 4. For Day 0 stim, take 40K cells from each cell type into a separate tube - increase volume to 200 pL of T Cell Media, then seed 50 pL / well into 4 wells total per cell type in a 96-well U-bottom plate. Return plate to incubator to rest cells for ~2-3hr before stimulation.

[0083] 5. For the remaining CAR T cells, increase volume so that there are 50K viable cells per 100 pL of T Cell Media.

[0084] 6. Seed the cells at 100 pL / well in a 96-well flat-bottom plate.

[0085] 7. Return the plate to the 37 degree C incubator & rest the cells for ~2-3hr before starting stimulation or tumor cell co-culture.

[0086] B. Start Raji-6hr stimulation after resting CAR T cells (Day 0 Analysis):

[0087] 1. During the rest, harvest 1 flask of Raji tumor cells (collect in a 50-ml conical tube, then spin down at 200xg for 6 minutes at RT).

[0088] 2. Resuspend the pellet in 1 ml of cRPMI-10. Take viable count by hemacytometer.

[0089] 3. For ~16 wells for Day 0 stimulation, need -500K Raji cells. Take 500K cells into an Eppendorf tube, then spin down in sterile PBS for 5 minutes.

[0090] 4. Use the cell pellet for CFSE labeling: resuspend in 384 pL of sterile PBS, add 16 pL of 1 : 1000 diluted CFSE (prepared from stock in -20). Cover w / foil & incubate for 4 minutes at room temp, vortex, then incubate another 3 minutes. Add 100 pL of FBS, vortex & incubate another 3 minutes before spinning down again.

[0091] 5. Resuspend the CFSE-labeled Raji pellet in 833 pL of cRPMI-10 (to final cone, of 30K cells / 50 pL). Then add Golgi-Plug + Golgi-Stop both at 1 :500. Vortex to mix.

[0092] 6. Add 50 pL of Raji cells to the 50 pL of 10K CAR T cell wells, then resuspend with multichannel pipette.

[0093] 7. After 3hr, add aCD107a-BV605 (1 :200) antibody: a. Total volume per stim well is 100 pL, so need to add 0.5 pL aCD107a to each stim well.

[0094] 8. Incubate for another 3hr (total 6hr) followed by staining w / Triple CAR T Experiment Stim panel. In the meantime, set up the co-cultures for the remaining wells.

[0095] C. Prepare tumor cell line for co-culture:

[0096] 1. Use JeKo-1 cells that have been growing in culture at least 1 week before starting co-culture: a. Media for JeKo-1 cell line: RPMI with 20% FBS and IX P / S.

[0097] 2. On day of co-culture, harvest the JeKo-1 cell line flask by transferring the suspended cells to a 50-ml tube. Then spin down the cells at 200xg for 6 minutes at 20 degrees.

[0098] 3. Aspirate the old media, then resuspend the cell pellet in 1 ml of complete RPMI.

[0099] 4. Take viable count for each cell line using the hemacytometer.

[0100] 5. To prepare wells with 25K tumor cells each, take the corresponding number of JeKo-1 cells into a new tube: a. 25K cells X ~82 wells = 2.05eA6 tumor cells needed total. b. Increase the volume of the 2.05eA6 tumor cells to 8.2 ml of T Cell Media.

[0101] Vortex to mix.

[0102] *Conditions: Chronic Stim (Ch.) (JeKo-1 cells only) and Chronic Stim (Ch.) +TGFB1 (JeKo-1 + TGFpi) i. Ch.+TGFBl : Take 4.2 ml of the above JeKo-1 cells into a separate tube:

[0103] 1. Add 4.2 pL of TGFpi (2X = Ing / ml, so dilute the 1 ug / ml stock 1 : 1000 in the media. Final IX = 0.5 ng / ml).

[0104] 2. Vortex to mix, then add 100 pL / well to the corresponding CH.+ TGFpi wells. ii. Ch.: Use the remaining 4.0 ml of JeKo-1 cells without TGFpi. c. Seed 100 pL of tumor cells per well (contains 25K tumor cells for every 100 pL) on top of the corresponding wells of 100 pL of CAR T cells in the 96- well flat-bottom plate.

[0105] 6. Resuspend wells with multichannel pipette. Return the plate to the 37 degree C incubator.

[0106] 7. For the extra tumor cells, continue growing in T75 culture flasks as below: a. JeKo-1 cell line: Add 2-4e6 tumor cells + 15 ml of cRPMI-20 (w / 20% FBS & 1X P / S)

[0107] Day 2 and Day 5- replacing media:

[0108] • On Day 2, harvest the JeKo-1 tumor cells & take viable count by hemacytometer.

[0109] • To prepare conditions with only 15K JeKo-1 cells / well, take the corresponding number of JeKo-1 cells into a new tube: o 15K cells X ~82 wells = 1.23e6 tumor cells needed total. o Increase the volume of the 1.23e6 tumor cells to 8.2 ml of T Cell Media. Vortex to mix.

[0110] • Prepare the following treatment conditions: o Treatments: Ch. (JeKo-1 cells only) and Ch.+ TGFpi (JeKo-1 + TGFpi) o Ch.+ TGFpi (Bl-12, C7-12, Dl-12, Gl-10): Take 4.2 ml of the above JeKo- 1 cells into a separate tube:

[0111] ■ Add 4.2 pL of TGFpi (2X = Ing / ml, so dilute the 1 ug / ml stock 1 : 1000 in the media. Final IX = 0.5 ng / ml).

[0112] ■ Vortex to mix, then add 100 pL / well to the corresponding CH.+ TGFpi wells. o Ch.: Use the remaining 4.0 ml of JeKo-1 cells without TGFpi.

[0113] • Take the co-culture plate & carefully aspirate 100 pL of old media from each well. Then add 100 pL of the corresponding media + tumor cells per well and resuspend. Return plate to incubator.

[0114] • For the extra tumor cells, continue growing in T75 culture flasks as below: o JeKo-1 cell line: Add 2-4 e6 JeKo-1 cells + 15 ml of cRPMI-20 (w / 20% FBS & 1X P / S)

[0115] Day 7- 6hr Raji stimulation, changing media:

[0116] Raji-6hr stimulation (starting Day 7):

[0117] • In the morning on Day 7, take portion from all conditions to use for Raji stim: o Pool 25 pL from 4 wells each condition (Ch. and Ch.+TGFBl, 2 total groups):

[0118] ■ Take viable count on ViCell, then prepare to seed 10K cells per stimulation well at 50 pL / well (4 per group - 24 total wells). • Harvest Raji cells & perform CFSE labeling as on Day 0. Resuspend labeled cells in cRPMI to concentration of 30K Raji cells per 50 pL media (1 :3 E:T ratio). Then add G.P. / G.S. at 1 :500 for 2X.

[0119] • Add 50 pL of Raji cells to the 50 pL of T cell wells, mix w / multichannel pipette & incubate for 6 hours at 37 degrees C. o After 3hr, add aCD107a antibody.

[0120] Media replacement (starting Day 7):

[0121] • Harvest the JeKo-1 tumor cells & take viable count by hemacytometer.

[0122] • To prepare conditions with only 15K JeKo-1 cells / well, take the corresponding number of JeKo-1 cells into a new tube: o 15K cells X ~68 wells = 1.02e6 tumor cells needed total. o Increase the volume of the 1.02e6 tumor cells to 6.8 ml of T Cell Media. Vortex to mix.

[0123] • Prepare the following treatment conditions: o Conditions: Ch. (JeKo-1 cells only), Ch.+ TGFpi (JeKo-1 + TGFpi), Acute Stim (starting from day 7 add T cell media only, no tumor cells). o Ch.+ TGFpi : Take 4.2 ml of the above JeKo-1 cells into a separate tube:

[0124] ■ Add 4.2 pL of TGFpi (2X = Ing / ml, so dilute the 1 ug / ml stock 1 : 1000 in the media. Final IX = 0.5 ng / ml).

[0125] ■ Vortex to mix, then add 100 pL / well to the corresponding CH.+ TGFpi wells. o Ch.: Use the remaining 2.6 ml of JeKo-1 cells without TGFpi. o Acute Stim: Use T cell media only.

[0126] • Take the co-culture plate & carefully aspirate 100 pL of old media from each well. Then add 100 pL of the corresponding media + tumor cells per well and resuspend. Return plate to incubator.

[0127] • For the extra tumor cells, continue growing in T75 culture flasks as below: o JeKo-1 cell line: Add 2-4 e6 JeKo-1 cells + 15 ml of cRPMI-20 (w / 20% FBS & 1X P / S)

[0128] Day 14- Overnight Raji stimulation, changing media:

[0129] Raji-overnight stimulation (~19hr):

[0130] • In the early afternoon on Day 14, take portion from all conditions to use for Raji stim: o Pool 25 pL from 4 wells each condition (Acute Stim, Ch. and Ch.+TGFBl, 9 total groups): o **For Ch.+ TGFpi on Day 14 for CD19-CAR and TCAR, pool ALL wells together (rest will be split for new ttt).

[0131] ■ Take viable count on ViCell, then prepare to seed 10K cells per stimulation well at 100 pL / well (4 per group - 36 total wells).

[0132] • Harvest Raji cells & perform CFSE labeling as on Day 7. Resuspend labeled cells in cRPMI to concentration of 30K Raji cells per 50 pL media (1 :3 E:T ratio). Then add G.P. / G.S. at 1 :500 for 2X, and aCD107a-BV605 (1 :200).

[0133] • Add 100 pL of Raji cells to the 100 pL of T cell wells, mix w / multichannel pipette & incubate for ~18 hours at 37 degrees C (overnight stimulation).

[0134] Media replacement (starting Day 14):

[0135] • Harvest the JeKo-1 tumor cells & take viable count by hemacytometer.

[0136] • To prepare conditions with only 15K JeKo-1 cells / well, take the corresponding number of JeKo-1 cells into a new tube: o 15K cells X ~80 wells = 1.2e6 tumor cells needed total. o Increase the volume of the 1.02e6 tumor cells to 8.0 ml of T Cell Media. Vortex to mix.

[0137] • Prepare the following treatment conditions: o Treatments: Ch. (JeKo-1 cells only), Ch.+TGFBl (JeKo-1 + TGFpi), Acute Stim (Acute, T cell media only, no tumor cells). o Ch.+TGFBl : Take 5.4 ml of the above JeKo-1 cells into a separate tube:

[0138] ■ Add 5.4 pL of TGFpi (2X = Ing / ml, so dilute the 1 ug / ml stock 1 : 1000 in the media. Final IX = 0.5 ng / ml).

[0139] ■ Vortex to mix. o Ch.: Use the remaining 2.6 ml of JeKo-1 cells without TGFpi. o Acute Stim: Use T cell media only.

[0140] • Take the co-culture plate & carefully aspirate 100 pL of old media from each well. Then add 100 pL of the corresponding media + tumor cells per well and resuspend. Return plate to incubator.

[0141] • For the extra tumor cells, continue growing in T75 culture flasks as below: o JeKo-1 cell line: Add 2-4 e6 JeKo-1 cells + 15 ml of cRPMI-20 (w / 20% FBS & 1X P / S) Day 20- PMA and Overnight Raji Stimulation:

[0142] • On Day 20, resuspend 3-4 wells per condition and pool 25 pL from each well into an Eppendorf tube (1 tube per condition, = 100 pL total for all except those with 3 wells only - 75 pL total).

[0143] • Spin down & resuspend in 300 pL of Human T Cell Media, then take 10 pL for ViCell count.

[0144] • Used 100 pL per condition from the above cells for PMA stimulation (split into 25 u / well for 4 wells per condition).

[0145] • Remaining -190 pL: Used to set up Raji co-culture stimulation. o **DAY 20 - changed input to 30K T cells + 90K Raji cells (1 :3 E:T ratio) o Take 120K cells per condition, then increase volume to 400 pL of Human Recovery Media (cRPMI + IL- 15 only). o **For UTD conditions, used all of the remaining cells (to increase input accounting for JeKo-1 cells in the viable count). o Seed 100 pL / well, keep in incubator until afternoon when Raji cells are added.

[0146] • In the afternoon, harvest Raji cells & label with 0.5 pM CFSE. Then resuspend labeled cells in Human Recovery Media and add G.P. / G.S. (1 :500 each for 2X) and aCD107a-BV605 (1 :200).

[0147] • Seed 90K labeled Raji cells per 100 pL in a 96-well flat-bottom plate. Then transfer the 30K T cells on top of the Raji wells.

[0148] • Place plate in incubator overnight (-19hr), followed by staining.

[0149] Day 21- Sorting & starting recovery plate:

[0150] Recovery plate + media prep (starting Day 21):

[0151] • On Day 21, pool all remaining wells into a 15-ml tube per each condition. Then spin down the cells at 400xg for 6 minutes, room temp.

[0152] • Resuspend pellet in 1 ml Human Recovery Media & take 10 pL for ViCell count. Recovery Media was prepared by combining 45 mL of complete RPMI (with 10%

[0153] FBS and IX P / S) with 45 pL of human IL-15.

[0154] • For the Recovery plate, each condition will have 4 wells of 50K cells each (so 200K total needed). o Take 400K - make 8 wells of 50K cells each. • Take the 200K or 400K cells into an Eppendorf tube, then increase volume to 400 pL of Recovery Media. Mix & seed 100 pL per well.

[0155] • Add another 100 pL of Recovery Media to the seeded wells.

[0156] Day 21 sorting:

[0157] • For the remaining cells, spin down & resuspend in FACS buffer. Then use for sorting.

[0158] Day 28- Final Raji overnight stimulation:

[0159] Raji-overnight stimulation (~19hr):

[0160] • In the early afternoon on Day 28, take portion from all conditions to use for Raji stim: o Pool all wells per condition, then take viable count on ViCell. o Prepare to seed 30K cells per stimulation well at 100 pL / well.

[0161] • Harvest Raji cells & perform CFSE labeling with 0.5 pM CFSE. Resuspend labeled cells in cRPMI to concentration of 90K Raji cells per 100 pL media (1 :3 E:T ratio). Then add G.P. / G.S. at 1 :500 for 2X, and aCD107a-BV605 (1 :200).

[0162] • Add 100 pL of Raji cells to the 100 pL of T cell wells, mix w / multichannel pipette & incubate for ~19 hours at 37 degrees C (overnight stimulation).

[0163] The development and characteristics of this protocol is further illustrated in Figures 3-23, which are described in detail in the Brief Description of the Drawings. All statistical analyses were performed using ordinary one-way ANOVA test with multiple comparisons (for three or more groups) or unpaired Mann-Whitney t-test (for two groups). P-value is indicated as * = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001. Comparisons are shown relative to the Chronic+TGFpi group or as indicated. All experiments were performed with n>4 technical replicates per group and repeated using biologically independent samples.

[0164] In sum, as illustrated in Figures 3-23, the repeated exposure to B cell lymphoma cells and low TGFpi dose establishes a stable terminal dysfunctional state in CAR T cells (e.g., human CAR T cells). Further, the in dysfunctional CAR T cells have limited survival and / or homeostatic proliferation capacity. These cells can serve as a platform for drug screenings / discovery of new therapeutics to reverse or prevent terminal dysfunction in human CAR T cells. Likewise, methods exemplified above can also be used to screen for agents that can reverse or prevent terminal dysfunction in human CAR T cells. For example, potential agents can be added to the co-culture to evaluate their ability to prevent terminal dysfunction.

[0165] In this system, we provided chronic stimulation of CAR T cells (including both CD4 and CD8 T cells) via repeated exposure to human B cell lymphoma cells expressing CAR- specific antigen(s), such as CD 19, CD20, and CD22, over 3 weeks. While this repeated coculture system is similar in some respects to previously explored models, we found that the chronically stimulated CAR T cells are quickly lost due to activation-induced cell death, while the remaining surviving cells retain the ability to recover their effector functions and cytotoxic activity after resting from chronic stimulation. In this model system, we optimized the culture condition by including a small dose of TGFP cytokine (0.5 ng / mL) during the repeated coculture with tumor cells (day 0-21), in addition to optimizing the ratio of CAR T cells-to-tumor cells. This method enhanced the survival of repeatedly stimulated human CAR T cells, while accelerating the acquisition of a terminal dysfunctional state that remained stable even after resting from chronic stimulation for 7 days. In addition, our in vitro-<i.QnQva Qd dysfunctional CAR T cells exhibit poor survival and homeostatic proliferation capacity — features that recapitulate the major limitation of currently approved CAR T cell therapy showing poor in vivo persistence in patients.

[0166] Likewise, a model that induces dysfunction in human CD8 T cells, rather than CAR T cells, via prolonged TCR stimulation using soluble anti-CD3 antibody (day 0-28) in the presence of high doses of TGFpi cytokine (5 ng / mL) from day 7-28 has also been described. While both models include a chronic antigen plus TGFpi stimulation, the technical approaches in both systems are different with regards to the source of antigen stimulation, TGFP dosing, and signaling duration.

[0167] Other advantages which are obvious, and which are inherent to the invention, will be evident to one skilled in the art. It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of inducing terminal exhaustion in a population of CAR-T cells, the method comprising: culturing a population of CAR-T cells in a media; and chronically simulating the population of CAR-T cells by exposing the population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule.

2. The method of claim 1, wherein chronically simulating the population of CAR-T cells comprises co-culturing the population of CAR-T cells with the population of cancer cells expressing the CAR-specific antigen.

3. The method of claim 2, wherein the population of CAR-T cells and the population of cancer cells expressing the CAR-specific antigen are co-cultured for a period of from 7 days to 60 days, such as from 7 days to 42 days, from 7 days to 35 days, from 7 days to 28 days, from 7 days to 21 days, from 14 days to 60 days, from 14 days to 42 days, from 14 days to 35 days, from 14 days to 28 days, from 14 days to 21 days, from 21 days to 60 days, from 21 days to 42 days, from 21 days to 35 days, or from 21 days to 28 days.

4. The method of any one of claims 1-3, wherein the tumor microenvironment signaling molecule comprises a cytokine, a chemokine, a growth factor, an extracellular matrix protein, or a combination thereof.

5. The method of any one of claims 1-4, wherein the tumor microenvironment signaling molecule comprises a cytokine6. The method of claim 5, wherein the cytokine comprises a TGF-P cytokine.

7. The method of claim 6, wherein the TGF-P cytokine comprises a TGF-P 1 cytokine.

8. The method of any one of claims 1-7, wherein the tumor microenvironment signaling molecule is present in the media at a concentration of less than 5 ng / mL, such as aconcentration of from 0.001 ng / mL to 4 ng / mL, from 0.005 ng / mL to 4 ng / mL, from 0.01 ng / mL to 4 ng / mL, from 0.05 ng / mL to 4 ng / mL, from 0.1 ng / mL to 4 ng / mL, 0.001 ng / mL to 3 ng / mL, from 0.005 ng / mL to 3 ng / mL, from 0.01 ng / mL to 3 ng / mL, from 0.05 ng / mL to 3 ng / mL, from 0.1 ng / mL to 3 ng / mL, 0.001 ng / mL to 2 ng / mL, from 0.005 ng / mL to 2 ng / mL, from 0.01 ng / mL to 2 ng / mL, from 0.05 ng / mL to 2 ng / mL, from 0.1 ng / mL to 2 ng / mL, 0.001 ng / mL to 1 ng / mL, from 0.005 ng / mL to 1 ng / mL, from 0.01 ng / mL to 1 ng / mL, from 0.05 ng / mL to 1 ng / mL, from 0.1 ng / mL to 1 ng / mL, or from 0.25 ng / mL to 0.75 ng / mL.

9. The method of claim 8, wherein the tumor microenvironment signaling molecule is present in the media at a concentration of about 0.5 ng / mL.

10. The method of any one of claims 1-9, wherein the population of CAR-T cells comprise a population of human CAR-T cells.

11. The method of any one of claims 1-10, wherein the CAR-specific antigen comprises CD19, BCMA, CD22, CD20, CD33, CD123, EGFRvIII, IL13Ra2, mesothelin, HER2, MUC1, prostate-specific membrane antigen (PSMA), or a combination thereof.

12. The method of any one of claims 1-11, wherein the population of CAR-T cells comprises CD4 T cells, CD8 T cells, or a combination thereof.

13. The method of any one of claims 1-12, wherein the population of cancer cells comprise human cancer cells.

14. The method of any one of claims 1-13, wherein the population of cancer cells comprise carcinoma cells, sarcoma cells, leukemia cells, lymphoma cells, myeloma cells, or a combination thereof.

15. The method of any one of claims 1-14, wherein the population of cancer cells comprise lung cancer cells (e.g., non-small cell, small cell), breast cancer cells, colorectal cancer cells, prostate cancer cells, pancreatic cancer cells, gastric (stomach) cancer cells, esophageal cancer cells, liver cancer cells (hepatocellular carcinoma), bladder cancer cells,kidney (renal cell) cancer cells, cervical cancer cells, endometrial (uterine) cancer cells, head and neck squamous cell carcinoma cells, ovarian cancer cells, skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma) cells, osteosarcoma cells, liposarcoma cells, leiomyosarcoma cells, rhabdomyosarcoma cells, Ewing sarcoma cells, fibrosarcoma cells, synovial sarcoma cells, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia) cells, lymphoma (e.g., Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma) cells, multiple myeloma cells, myelodysplastic syndrome cells, myeloproliferative neoplasm cells, glioblastoma multiforme cells, astrocytoma cells, oligodendroglioma cells, medulloblastoma cells, ependymoma cells, cutaneous melanoma cells, ocular melanoma cells, mucosal melanoma cells, testicular cancer cells (e.g., seminoma, non-seminoma), ovarian germ cell tumor cells, extragonadal germ cell tumor cells, neuroblastoma cells, pheochromocytoma cells, carcinoid tumor cells, pancreatic neuroendocrine tumor cells, Wilms tumor cells, retinoblastoma cells, medulloblastoma cells, neuroblastoma cells, or pediatric leukemia and lymphoma cells.

16. A population of terminally exhausted CAR T cells prepared by the method of any one of claims 1-15.

17. The use of the population of terminally exhausted CAR T cells of claim 16 in an assay to screen an agent for its ability to reverse to prevent terminal dysfunction in a population of CAR-T cells.

18. A method of screening an agent for its ability to reverse and / or prevent terminal dysfunction in a population of CAR-T cells, the method comprising: preparing a control population of CAR-T cells by a method that comprises culturing a first population of CAR-T cells in a media; and chronically simulating the first population of CAR-T cells by exposing the first population of CAR-T cells to a population of cancer cells expressing a CAR-specific antigen in the presence of a tumor microenvironment signaling molecule; preparing a screening population of CAR-T cells by a method that comprises culturing a second population of CAR-T cells in a media; and chronically simulating the second population of CAR-T cells by exposing the second population of CAR-T cells to apopulation of cancer cells expressing the CAR-specific antigen in the presence of a tumor microenvironment signaling molecule and an active agent; and evaluating the degree of terminal exhaustion in the control population of CAR-T cells and the screening population of CAR-T cells; wherein a lower degree of terminal exhaustion in the screening population of CAR- T cells as compared to the control population of CAR-T cells indicates that the active agent can reverse and / or prevent terminal dysfunction in a population of CAR-T cells.

19. The method of claim 18, wherein the control population of CAR-T cells and the screening population of CAR-T cells are prepared by methods that are otherwise identical except for the presence of the active agent.

20. The method of any one of claims 18-19, wherein chronically simulating the first population of CAR-T cells comprises co-culturing the first population of CAR-T cells with the population of cancer cells expressing the CAR-specific antigen; and wherein chronically simulating the second population of CAR-T cells comprises co-culturing the second population of CAR-T cells with the population of cancer cells expressing the CAR-specific antigen.

21. The method of claim 20, wherein the first population of CAR-T cells and the population of cancer cells expressing the CAR-specific antigen, and the second population of CAR-T cells and the population of cancer cells expressing the CAR-specific antigen are co-cultured for a period of from 7 days to 60 days, such as from 7 days to 42 days, from 7 days to 35 days, from 7 days to 28 days, from 7 days to 21 days, from 14 days to 60 days, from 14 days to 42 days, from 14 days to 35 days, from 14 days to 28 days, from 14 days to21 days, from 21 days to 60 days, from 21 days to 42 days, from 21 days to 35 days, or from 21 days to 28 days.22 The method of any one of claims 18-21, wherein the tumor microenvironment signaling molecule comprises a cytokine, a chemokine, a growth factor, an extracellular matrix protein, or a combination thereof.

23. The method of any one of claims 18-22, wherein the tumor microenvironment signaling molecule comprises a cytokine24. The method of claim 23, wherein the cytokine comprises a TGF-P cytokine.

25. The method of claim 24, wherein the TGF-P cytokine comprises a TGF-pi cytokine.

26. The method of any one of claims 18-25, wherein the tumor microenvironment signaling molecule is present in the media at a concentration of less than 5 ng / mL, such as a concentration of from 0.001 ng / mL to 4 ng / mL, from 0.005 ng / mL to 4 ng / mL, from 0.01 ng / mL to 4 ng / mL, from 0.05 ng / mL to 4 ng / mL, from 0.1 ng / mL to 4 ng / mL, 0.001 ng / mL to 3 ng / mL, from 0.005 ng / mL to 3 ng / mL, from 0.01 ng / mL to 3 ng / mL, from 0.05 ng / mL to 3 ng / mL, from 0.1 ng / mL to 3 ng / mL, 0.001 ng / mL to 2 ng / mL, from 0.005 ng / mL to 2 ng / mL, from 0.01 ng / mL to 2 ng / mL, from 0.05 ng / mL to 2 ng / mL, from 0.1 ng / mL to 2 ng / mL, 0.001 ng / mL to 1 ng / mL, from 0.005 ng / mL to 1 ng / mL, from 0.01 ng / mL to 1 ng / mL, from 0.05 ng / mL to 1 ng / mL, from 0.1 ng / mL to 1 ng / mL, or from 0.25 ng / mL to 0.75 ng / mL.

27. The method of claim 26, wherein the tumor microenvironment signaling molecule is present in the media at a concentration of about 0.5 ng / mL.

28. The method of any one of claims 18-27, wherein the first population of CAR-T cells and the second population of CAR-T cells comprise human CAR-T cells.

29. The method of any one of claims 18-28, wherein the CAR-specific antigen comprises CD19, BCMA, CD22, CD20, CD33, CD123, EGFRvIII, IL13Ra2, mesothelin, HER2, MUC1, prostate-specific membrane antigen (PSMA), or a combination thereof.

30. The method of any one of claims 18-29, wherein the first population of CAR-T cells and the second population of CAR-T cells comprises CD4 T cells, CD8 T cells, or a combination thereof.

31. The method of any one of claims 18-30, wherein the cancer cells comprise human cancer cells.

32. The method of any one of claims 18-31, wherein the cancer cells comprise carcinoma cells, sarcoma cells, leukemia cells, lymphoma cells, myeloma cells, or a combination thereof.

33. The method of any one of claims 18-32, wherein the cancer cells comprise lung cancer cells (e.g., non-small cell, small cell), breast cancer cells, colorectal cancer cells, prostate cancer cells, pancreatic cancer cells, gastric (stomach) cancer cells, esophageal cancer cells, liver cancer cells (hepatocellular carcinoma), bladder cancer cells, kidney (renal cell) cancer cells, cervical cancer cells, endometrial (uterine) cancer cells, head and neck squamous cell carcinoma cells, ovarian cancer cells, skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma) cells, osteosarcoma cells, liposarcoma cells, leiomyosarcoma cells, rhabdomyosarcoma cells, Ewing sarcoma cells, fibrosarcoma cells, synovial sarcoma cells, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia) cells, lymphoma (e.g., Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma) cells, multiple myeloma cells, myelodysplastic syndrome cells, myeloproliferative neoplasm cells, glioblastoma multiforme cells, astrocytoma cells, oligodendroglioma cells, medulloblastoma cells, ependymoma cells, cutaneous melanoma cells, ocular melanoma cells, mucosal melanoma cells, testicular cancer cells (e.g., seminoma, non-seminoma), ovarian germ cell tumor cells, extragonadal germ cell tumor cells, neuroblastoma cells, pheochromocytoma cells, carcinoid tumor cells, pancreatic neuroendocrine tumor cells, Wilms tumor cells, retinoblastoma cells, medulloblastoma cells, neuroblastoma cells, or pediatric leukemia and lymphoma cells.

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