WO2024107410A1

WO2024107410A1 - Cd39 selection for cytotoxicity of genetically engineered t regulatory cells

Info

Publication number: WO2024107410A1
Application number: PCT/US2023/037215
Authority: WO
Inventors: Everett Hurteau Meyer; Xiangni WU; Pin-l CHEN
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2022-11-14
Filing date: 2023-11-13
Publication date: 2024-05-23

Abstract

Compositions and methods are provided herein relating to Treg cells modified by introduction of a chimeric antigen receptor (CAR), referred to herein as CAR-Treg cells. Populations of CAR-Treg cells are bimodal with respect to expression of the surface marker CD39; where the cytotoxic potential of the cell population segregates with respect to such CD39 expression. Selection for a CD39⁺ CAR-Treg population provides for a population with significantly reduced cytotoxic risk toward the cell population targeted by the CAR. In contrast, selection for a CD39⁺ CAR-Treg population provides for a population with enhanced cytotoxicity toward the cell population targeted by the CAR deleting antigen.

Description

CD39 SELECTION FOR CYTOTOXICITY OF GENETICALLY ENGINEERED T REGULATORY CELLS

CROSS REFERENCE TO OTHER APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/425,109, filed November 14, 2022, the contents of which are hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS

[0002] This invention was made with Government support under contracts DK132549 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0003] Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Different subsets with various functions of Treg cells exist. Tregs can be usually identified by flow cytometry. The most specific marker for these cells is FoxP3, which is localized intra-cellularly. Dysregulation in Treg cell frequency or functions may lead to the development of autoimmune disease. Therapeutic Treg modulation is being developed to treat certain immune disorders, for example to prevent graft v host disease, or allograft rejection.

[0004] Promising results of initial studies using Treg as a clinical intervention have increased interest in this type of the cellular therapy. As such, methods of isolation and expansion of Tregs have been studied and optimized. Further, preclinical studies have shown antigenspecific Tregs may be more efficient due to their homing ability towards the cognate antigen. Antigen specificity can be conferred to regulatory T cells by transducing them with recombinant TCR. TCR-engineered T cells recognize peptides from both intracellular and surface derived proteins. Moreover, TCR-T cells have high affinity to cognate major histocompatibility complex (MHC)-peptide and induce a potent immune synapse formation. However, TCR-engineered Tregs are MHC dependent. Chimeric antigen receptor (CAR) technology offers a non-MHC- dependent approach. Such CAR-Treg cells utilize an extracellular antigen recognition domain from a single-chain variable fragment (scFv) of an antibody combined with an intracellular signaling domain. Many other creative receptor-targeted engineered fusion proteins are also being explored to direct and activate T regulatory cells to specific tissue or secreted targets. This combination allows for the construct to activate a T-cell response without interacting with the antigen in the context of MHC. [0005] T regulatory cells can also be present in T cell cultures for CAR T cells expanded and transduced for cancer targeting such as targeting B cells through CD19. T regulatory cells in bulk are less effective in general in cytotoxic killing of target cancer and may affect the success of CAR T therapy by interfering with conventional T cells mediating anti-cancer responses. Subsets that do not mediate cytotoxic killing but are immunoregulatory may contribute exceptionally to reducing the efficacy of CAR T therapy. Likewise, subsets of T regulatory cells that are cytotoxic may be preferred as CAR T therapeutics because they are likely to function when CD8 or other T cell subsets become exhausted, anergic or otherwise hyper-activated and lose efficacy.

SUMMARY

[0006] Compositions and methods are provided herein relating to Treg cells modified by introduction of a chimeric antigen receptor (CAR), referred to herein as CAR-Treg cells. The CAR selectively binds to an antigen present on a target cell population. It is shown herein that populations of CAR-Treg cells are bimodal with respect to expression of the surface marker CD39; and that the cytotoxic potential of the cell population segregates with respect to such CD39 expression. Selection for a CD39⁺ CAR-Treg population provides for a population with significantly reduced cytotoxic risk toward the cell population targeted by the CAR, and may improve immunoregulatory function. These CD39⁺ CAR-Treg cell populations are useful in the treatment of conditions where it is desirable for the Treg cells to enhance immune tolerance, e.g. in the treatment of autoimmune disease, to reduce graft v host disease, to provide tolerance in transplantation, to promote wound healing or tissue regeneration and the like. In contrast, selection for a CD39' CAR-Treg population provides for a population with enhanced cytotoxicity toward the cell population targeted by the CAR deleting antigen, which can find use in depletion of targeted populations, e.g. in cancer, depletion of undesirable antigen- presenting cells such as those present in transplantation rejection, autoimmune or inflammatory diseases, and the like.

[0007] In an embodiment, a composition is provided of CAR-Treg cells with reduced cytotoxic activity against the CAR-targeted cell population, relative to the unselected CAR-Treg population from which it is derived. The CAR-Treg cells are characterized by CD39 expression, i.e. the cells are CD39⁺. The reduction in cytotoxicity may be measured, e.g. by an assay for apoptosis of target cells, where the reduction is at least 25%, at least 50%, at least 75% decrease, and may be a two-fold decrease or greater decrease in cytotoxicity relative to an unselected population. The cell population may be provided as a composition suitable for therapeutic administration. A therapeutic composition may comprise a therapeutically effective dose of CD39⁺ CAR-Treg cells, in a physiologically acceptable excipient. [0008] In an embodiment, a composition is provided of CAR-Treg cells with enhanced cytotoxic activity against the CAR-targeted cell population, relative to the unselected CAR- Treg population from which it is derived. The CAR-Treg cells are characterized low levels or the absence of CD39 expression. The enhancement in cytotoxicity may be measured, e.g. by an assay for apoptosis of target cells, where the enhancement is at least 25%, at least 50%, at least 75% increase, and may be a two-fold increase or greater in cytotoxicity, relative to unselected cells. The cell population may be provided as a composition suitable for therapeutic administration. A therapeutic composition may comprise a therapeutically effective dose of CD39" CAR-Treg cells, in a physiologically acceptable excipient. CD39- CAR Treg cells can regain some CD39+ expression with ex vivo cell culture expansion but retain the ability to have increased cytotoxicity.

[0009] In some embodiments, a method of treating an individual with a therapeutically effective dose of CAR-Treg cells or other genetically modified Treg cells, e.g. T cell receptor transduced cells, is provided, comprising administering to an individual in need thereof an effective dose of a population of genetically modified Treg cells, e.g. CAR-Treg cells that have been selected based on CD39 expression. Conditions where the cells are used may include, for example, GVHD, type 1 diabetes, autoimmunity, wound healing, tissue regeneration and transplantation tolerance.

[0010] In an embodiment, methods of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof are provided, comprising administering an effective dose of a CD39 selected CAR-Treg cell population to an individual in need there.

[001 1] In an embodiment, methods of reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ are provided, comprising administering an effective dose of a CD39 selected CAR-Treg cell population to an individual in need there.

[0012] In some embodiments, a method is provided for the in vitro culture and genetic modification of T cells to generate a population of CAR-Treg cells with reduced cytotoxic activity against the CAR-targeted cell population. An initial T cell population is isolated from a donor, which may be allogeneic or autologous relative to the recipient. The T cells may be a population comprising CD4⁺ T cells, optionally a population selected for Treg cells. In populations that are not selected Tregs, the cells can be engineered by transducing with CAR constructs and FoxP3 coding sequences. Treg populations are transduced with CAR constructs but do not require FoxP3. The population is sorted based on expression of CD39, e.g. by affinity selection such as flow cytometry, MACs, and the like. Selection may be performed or after transductions, usually after. The population of CD39 selected, CAR-Treg cells may be expanded in culture, activated, cryopreserved, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0014] FIG. 1 Co-culture of human CAR Treg targeting human islets in cell culture. Cytotoxic killing of islets can occur as identified by flow cytometric assessment of annexin V and 7AAD in human islet beta cells specifically. Unselected Tregs from HLA-A2 negative donors identified as CD4+CD25+CD17lo were culture expanded and transduced with CAR construct with A2-specific scFv and CD3zeta and CD28 internal stimulatory domain. The HLA-A2 specific CAR Treg specifically caused a decrease in the number of normal healthy islet beta cells that did not express Annexin V or incorporate 7 AAD (normalized to islets in cell culture alone as), as compared to untransduced Treg (mock), or Treg with irrelevant scFv binding domains (1 X9Q that binds FITC) and FRB which binds a myeloid-specific cell surface marker.

[0015] FIG. 2. Co culture of expanded allogeneic human Tregulatory cells as a group or as CD39+ versus CD39- with human islets in vivo does not result in any changes in Treg cytotoxic killing of islets, so the discovery that CD39+ and CD39= CAR Treg behave differently is not anticipated.

[0016] FIG. 3. When looking at ex vivo culture expanded total Treg as a whole, those CAR Treg with the lowest CD39 expression had the highest cytotoxic activity as measured by islet cell death.

[0017] FIG. 4. CD39+ Treg show distinct features cmTreg: the cells of this weakly proliferating population circulate between secondary lymphoid organs and express anti-apoptotic factors Bcl-2 and Mcl-1 at a high level. Not prone to apoptosis, great potential to have a longer in vivo survival time after injection.

[0018] FIG. 5. CD39+ Treg show more stable and higher FOXP3 expression and more phenotypic stability in cell culture expansion compared to CD39-

[0019] FIG. 6. CD39+ expanded Tregs have statistically lower expression of granzyme when compared to bulk/whole Treg versus CD39- Treg. The difference in the ratio of central memory to effector memory in these cells becomes more pronounced after expansion.

[0020] FIG. 7. CD39 Treg have higher LAG3 expression, suggesting a more suppressive phenotype.

[0021] FIG. 8. |3lox5 cells were derived from purified adult pancreatic beta cells and microscopic evaluation confirms HLA-A2 CAR Treg mediate cytotoxic killing of tissue targets. [0022] FIG. 9. Primary pancreatic islet cells were derived from purified adult pancreatic beta cells and microscopic evaluation confirms HLA-A2 CAR Treg mediate cytotoxic killing of tissue targets.

[0023] FIG. 10. A2 CAR CD39 negative T reg cells directly induce islet cytolysis.

[0024] FIG. 1 1. A2 CAR Treg that are CD39+ preserve glucose-stimulated insulin secretion in target organs, versus CD39- cells that had reduced islet functionality.

DETAILED DESCRIPTION

[0025] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. 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 invention.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. 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. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0028] It must be noted that as used herein and in 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 cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth. [0029] 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 may be different from the actual publication dates which may need to be independently confirmed.

[0030] 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 a range 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 % about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0031] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0032] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0033] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0034] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of disease prior to onset or recurrence.

[0035] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” is the dose of a cell population that achieves a beneficial or desired prophylactic or therapeutic result, including clinical results. [0036] A “therapeutically effective amount” of a cell population may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0037] An “increased” or “enhanced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include an increase that is 1.1 , 1 .2, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1.5, 1.6, 1 .7. 1 .8, etc.) the level of activity in an untreated cell.

[0038] A “decrease” or “reduced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include an decrease that is 1.1 , 1 .2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1 .5, 1 .6, 1 .7. 1 .8, etc.) the level of activity in an untreated cell.

[0039] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule/cell composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0040] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0041] Regulatory T cells (“Tregs”) are a specialized subpopulation of T cells that can suppress activation of the immune system and thereby maintain immune tolerance. Tregs are share the phenotype of being CD4⁺ CD25⁺ FOXP3+. There are various types of Tregs, for example, TCRa|3+CD4+ regulatory T cells, which include natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs). nTregs are T cells produced in the thymus and delivered to the periphery as a long-lived lineage of self-antigen-specific lymphocytes. iTregs are recruited from circulating lymphocytes and acquire regulatory properties under particular conditions of stimulation in the periphery. nTregs and iTregs are CD4+CD25+; both can inhibit proliferation of CD4+CD25- T cells in a dose-dependent manner, and both are anergic and do not proliferate upon TOR stimulation. Tregs can be identified or selected based on various marker expression profiles. Non-limiting examples of marker expression profiles that can be used to select Tregs include (1 ) CD4+CD25+CD127dim, (2) CD4+FOXP3+, (3) CD3+CD4+CD25+, (5) CD3+ CD4+ CD25+ CD127dim, (6) CD3+ CD4+ CD25+ CD127dim F0XP3+, (7) CD3+FOXP3+, (8) CD3+CD4+FOXP3+, (9) CD3+ CD4+CD25+FOXP3+, (10) CD3+CD25+FOXP3+, (11 ) CD3+CD25+CD127dim, (12) CD4+CD25+, (13)

CD4+CD25+CD127dimFOXP3+, (14) FOXP3+, CD4+FOXP3+, (15) CD4+CD25+FOXP3+, (16) CD25+FOXP3+, or (17) CD25+ CD127dim. In some embodiments, the Treg cells are CD4⁺CD25⁺CD127^loFoxP3⁺ Treg. Selection based on certain expression profiles can be achieved based on extracellular markers and without requiring cell permeabilization, for example, selection based on CD4+CD25+CD127¹⁰ phenotype.

[0042] In some embodiments a T cell, e.g. a CD4+ T cell, is engineered to express FoxP3 to achieve a Treg phenotype. Foxp3 plays a crucial role in development and function of Treg cells. Constructs and methods from expressing Foxp3 in T cells are described in, for example, WO 2007/065957, which is incorporated herein in its entirety. In some embodiments the Foxp3 is wild-type (WT) Foxp3. Exemplary wild type human Foxp3 sequences are described in NP_054728.2, the contents of which are incorporated herein by reference.

[0043] In some embodiments, the Foxp3 is a minimal Foxp3. A “minimal Foxp3” is engineered to mimic N-terminally, C-terminally, or N-and C-terminally cleaved Foxp3 forms, see for example WO2019241549A1 , herein specifically incorporated by reference. A minimal FOXP3 may be truncated to start at residue 52 relative to the wild-type protein, and to end at residue 413 (relative to Genbank NP_054728.2). Minimal Foxp3 is, in some contexts, more active than wild-type (WT) Foxp3. In some embodiments, the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated. In some embodiments, the Foxp3 is constitutively active.

[0044] In some embodiments, a polynucleotide encoding the CAR and the polynucleotide encoding the Foxp3 are configured for translation as a fusion protein comprising the CAR and the Foxp3. The CAR polynucleotide may be 5’ to the Foxp3 polynucleotide, in which case the CAR is expressed as a N-terminal fusion to the Foxp3, or vice-versa. In an embodiment, the CAR is N-terminal to the Foxp3 in the fusion protein. In an embodiment, the fusion protein comprises a cleavage site between the CAR and the Foxp3. In an embodiment, the cleavage site is a 2 A peptide.

[0045] The Treg T-cell is modified to surface express a chimeric antigen receptor (a CAR- Treg cell). As used herein, the terms “chimeric antigen receptor T-cell” and “CAR-Treg cell” are used interchangeably to refer to a T-cell that has been recombinantly modified to express a CAR. As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably to refer to a polyprotein comprising multiple functional domains arranged from amino to carboxy terminus in the sequence: (a) an antigen binding domain (ABD), (b) a transmembrane domain (TD); (c) one or more cytoplasmic signaling domains (CSDs) wherein the foregoing domains (a) - (c) may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence which is conventionally removed during post-translational processing and presentation of the CAR on the cell surface. CARs useful in the practice of the present invention are prepared in accordance with principles well known in the art.

[0046] Engineering cells to express CAR constructs is commonly accomplished through viral vector systems such as lentivirus, Gamma-retroviral, and adeno-associated viral (AAV) vectors. Additionally, viral-free systems such as the Sleeping Beauty (SB), or piggyBac transposon have been used to integrate CAR encoding DNA with favorable integration into the target genome. Furthermore, Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein 9 (Cas9) gene-editing technology, which allows for the insertion of DNA at specific locations directed by RNA, has also been used to engineer CAR T cells. CRISPR-engineered CAR T cells, which express CAR from an endogenous TCR locus remain active for longer periods than their virus-transduced counterparts.

[0047] As used herein, the term antigen binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. The ABD may be any polypeptide that specifically binds to one or more antigens expressed on the surface of a target cell.

[0048] In one embodiment, the ABD is a single chain Fv (ScFv). An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061 . The generation of ScFvs based on monoclonal antibody sequences is well known in the art. See, e.g. The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 “Bacterial Expression, Purification and Characterization of Single-Chain Antibodies” Kipriyanov, S. Antibodies used in the preparation of scFvs may be optimized to select for those molecules which possess particular desirable characteristics (e.g. enhanced affinity) through techniques well known in the art such as phage display and directed evolution. In some embodiments, the ABD comprises an anti-CD19 scFv, an anti-PSA scFv, an anti- HER2 scFv, an anti-CEA scFv, an anti-EGFR scFv, an anti-EGFRvlll scFv, an anti-NY-ESO- 1 scFv, an anti-MAGE scFv, an anti-5T4 scFv, or an anti-Wnt1 scFv. In another embodiment, the ABD is a single domain antibody obtained through immunization of a camel or llama with a target cell derived antigen, in particular a tumor antigen. See, e.g. Muyldermans, S. (2001 ) Reviews in Molecular Biotechnology 74: 277-302. Alternatively, the ABD may be generated wholly synthetically through the generation of peptide libraries and isolating compounds having the desired target cell antigen binding properties in substantial accordance with the teachings or Wigler, et al. United States Patent No. 6303313 B1 issued November 12, 1999; Knappik, et al., United States Patent No 6,696,248 B1 issued February 24, 2004, Binz, et al. (2005) Nature Biotechnology 23:1257-1268, and Bradbury, et al. (2011 ) Nature Biotechnology 29:245-254.

[0049] In some embodiments, the CARs of the present disclosure comprise a target-specific binding element otherwise referred to as an antigen binding moiety. The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR of the invention include those associated with autoimmune disease, transplant rejection, and the like.

[0050] In some embodiments, the CAR targets specific alleles of components of the Major Histocompatibility Complex (MHC) through the extracellular domain. In some embodiments, the resulting CAR-Treg cells downregulate or suppress the immune response to alloantigens.

[0051] As used herein, “an alloantigen” refers to a genetically determined antigen present in some but not all subjects of a species and which is capable of inducing the production of an alloantibody by subjects which lack it. Exemplary alloantigens that can lead to transplant rejection include, but are not limited to, ABO blood group and Rh blood group, MHC class I or MHC class II complexes, where allele mismatches in MHC I or MHC II are significant risk factors for transplant rejection or graft-versus-host disease.

[0052] In some embodiments, the CAR targets specific alleles of components of the Major Histocompatibility Complex (MHC) e.g., HLA class 1 alleles HLA-A1 , HLA-A2, HLA-A1 1 , HLA- B44, HLA-B27, HLA-C07 and HLA-C04. In some embodiments, the MHC is MHC class I or MHC class II. In some embodiments, the CAR targets a human leukocyte antigen, e.g. an allele of human leukocyte antigen A (HLA-A, major histocompatibility complex, class I, A), human leukocyte antigen B (HLA-B, major histocompatibility complex, class I, B) or human leukocyte antigen C (HLA-C, major histocompatibility complex, class I, C).

[0053] In some embodiments, the CAR targets an allele of HLA-A. There are about 2,900 known variants of HLA-A, all of which are envisaged as within the scope of the disclosure. In some embodiments, the HLA-A allele is an HLA-A 1 , HLA-A2, HLA -Al I, HLA-A24, HLA-A26, HLA-A30, HLA-A31 , HLA-A34, HLA-A36, HLA-A66, HLA-A68 or HLA-A69 allele. In some embodiments, the HLA-A allele is an HLA- A2 allele. Exemplary HLA-A2 alleles include, but are not limited to, HLA-A*2:0l, an HLA- A*2:02, HLA-A* 2: 03, HLA-A*2:05, HLA-A*2:06, HLA- A*2:07 or HLA-A*2:0l I . In some embodiments, the extracellular domain of the CAR targets HLA-A*2:0l, an HLA-A* 2: 02, HLA-A* 2: 03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA- A*2:0l I . In some embodiments, the extracellular domain of the CAR targets HLA-A*02:01. [0054] In some embodiments, the CAR targets an allele of HLA-B. There are about 3,600 known variants of HLA-B, all of which are envisaged as within the scope of the disclosure. In some embodiments, the CAR targets an allele of HLA-C. There are about 2,400 known variants of HLA-B, all of which are envisaged as within the scope of the disclosure. In some embodiments, CAR targets an allele of human leukocyte antigen A (HLA-A, major histocompatibility complex, class I, A), human leukocyte antigen B (HLA-B, major histocompatibility complex, class I, B).

[0055] The ABD may have affinity for more than one target antigen. For example, an ABD of the present invention may comprise chimeric bispecific binding members, i.e. have capable of providing for specific binding to a first target cell expressed antigen and a second target cell expressed antigen. Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)₂, bispecific antibody fragments (e.g., F(ab)₂, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann (2012) MAbs. 4(2): 182-197; Stamova et al. (2012) Antibodies, 1 (2), 172-198; Farhadfar et al. (2016) Leuk Res. 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. (2015) J Hematol Oncol. 8:130; May et al. (2016) Am J Health Syst Pharm. 73(1 ):e6-e13. In some embodiments, the chimeric bispecific binding member is a bivalent single chain polypeptides. See, e.g. Thirion, et al. (1996) European J. of Cancer Prevention 5(6) :507-51 1 ; DeKruif and Logenberg (1996) J. Biol. Chem 271 (13)7630-7634; and Kay, et al. United States Patent Application Publication Number 2015/0315566 published November 5, 2015. In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3. In some instances, a chimeric bispecific binding member may be a CAR T cell adapter. As used herein, by “CAR T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR T cell adapter will have to binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR T cell adapters include but are not limited to e.g., those described in Kim et al. (2015) J Am Chem Soc. 137(8):2832-5; Ma et al. (2016) Proc Natl Acad Sci U S A. 1 13(4):E450-8 and Cao et al. (2016) Angew Chem Int Ed Engl. 55(26) :7520-4. [0056] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to facilitate antigen binding. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. In one embodiment, the linker is the hinge region from an immunoglobulin, e.g. the hinge from any one of IgG 1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In those instances where the ABD is an scFv, an IgG hinge is effective. In some embodiments the linker comprises the amino acid sequence (G4S)_n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0057] CARs useful in the practice of the present invention further comprise a transmembrane domain joining the ABD (or linker, if employed) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membrane spanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e.g Pace, etal. (1998) Biophysical Journal 75: 422-427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3^, CD4, CD8, CD28, etc.

[0058] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor ^-chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1 BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), p110a, p, or 5 catalytic subunit of PI3 kinase, the human CD3 - chain, cytoplasmic domain of CD134 (also referred to as 0X40 and TNFRSF4), FceR1 y and chains, MB1 (Iga) chain, B29 (IgP) chain, etc.), CD3 polypeptides (5, A and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.

[0059] In some embodiments, the CAR may also provide a co-stimulatory domain. The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of costimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of members of the TNFR superfamily, CD28, CD137 (4-1 BB), CD134 (0X40), Dap10, CD27, CD2, CD5, ICAM- 1 , LFA-1 (CD1 1 a/CD18), Lek, TNFR-I, TNFR-II , Fas, CD30, CD40 or combinations thereof.

[0060] In some embodiments, the intracellular domain of the CARs of the instant disclosure comprises an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2Rbeta domain is truncated. In some embodiments, the IL- 2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs. In some embodiments, the CAR comprises one or more STAT5-recruitment motifs outside the IL- 2Rbeta cytoplasmic domain. In an embodiment, the IL-2R-beta cytoplasmic domain comprises one or more STAT5- recruitment motifs. Exemplary STAT5 -recruitment motifs are provided by Passerini et al. (2008) STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells. International Immunology, Vol. 20, No. 3, pp. 421 -431 , and by Kagoya et al. (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nature Medicine doi: I0. IO38/nm.4478.

[0061] CARs are often referred to as first, second, third or fourth generation. The term first- generation CAR refers to a CAR wherein the cytoplasmic domain transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FcaRly, or the CD3<J chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigendependent T-cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding. Second- generation CARs include a co-stimulatory signal in addition to the CD3T signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain is usually be membrane proximal relative to the CD3^ domain. Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3^, 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR T-cells are further gene modified to express or block molecules and/or receptors to enhance immune activity.

[0062] Examples of intracellular signaling domains comprising may be incorporated into the CAR of the present invention include (amino to carboxy): CD3^; CD28 - 41 BB - CD3^; CD28 - 0X40 - CD3 ; CD28 - 41 BB - CD3^; 41 BB -CD-28 - CD3 and 41 BB - CD3^.

[0063] The term CAR includes CAR variants including but not limited split CARs, ON-switch CARS, bispecific or tandem CARs, inhibitory CARs (iCARs) and induced pluripotent stem (iPS) CAR-T cells.

[0064] The term “Split CARs” refers to CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014/016527, US1996/017060, US2013/063083; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0065] The term “bispecific or tandem CARs” refers to CARs which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR.

[0066] The term “inhibitory chimeric antigen receptors” or “iCARs” are used interchangeably herein to refer to a CAR where binding iCARs use the dual antigen targeting to shut down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains of a secondary CAR binding domain results in inhibition of primary CAR activation. Inhibitory CARs (iCARs) are designed to regulate CAR- T cells activity through inhibitory receptors signaling modules activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA- 4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility. [0067] The term “tandem CAR” or “TanCAR” refers to CARs which mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens.

Source Cells

[0068] Typically, the chimeric antigen receptor T-cells (CAR-T cells) are T-cells which have been recombinantly modified by transduction with an expression vector encoding a CAR in substantial accordance with the teaching above. As disclosed above, a Treg cell, or a CD4+ T cells modified by introduction of a FoxP3 coding sequence may be used. T cells for engineering as described above are collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation.

[0069] A population of cells comprising T regs can be isolated from whole blood. A population of cells comprising Tregs can be isolated from a peripheral blood apheresis product. A population of cells comprising Tregs can be isolated from a population of cells previously enriched and/or depleted for one or more other cell types, e.g., isolated from a population of cells depleted of CD34+ cells. In some embodiments, Tregs are isolated from the flow-through fraction of a CD34+ MACS selection.

[0070] The number of Tregs in a population of cells can be determined, for example, by flow cytometry, where Tregs can be identified as, for example, CD4+CD25+CD127¹⁰ or CD4+FOXP3+. Dose calculations can be adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0071] An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0072] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS). The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0073] In some embodiments, the engineered cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy- Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0074] In some embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor and a cellular product suitable for all patients independent of HLA type provides an alternative. A universal ‘off the shelf’ CAR T cell product provides advantages in uniformity of harvest and manufacture.

[0075] The Treg cells may be cultured in vitro under various culture conditions, as disclosed herein. The cell population may be conveniently suspended in an appropriate nutrient medium, such as TexMACS, Iscove’s modified DMEM, RPMI-1640, etc., and as disclosed herein are supplemented with human serum. The culture may contain activation agent factors to which the cells are responsive.

Methods

[0076] Treg cells are usually obtained from a single donor, for example, obtained from mobilized peripheral blood apheresis of a single donor, although can be from combined donors. A donor and a recipient of the disclosure can be allogenic. A donor and a recipient of the cryopreserved cells can be HLA matched. A donor and a recipient of the of the cryopreserved cells can be HLA mismatched, e.g. mismatched at 1 , 2, 3, 4, 5, or 6 of the major HLA alleles. A donor and a recipient of the cryopreserved cells can be haploidentical.

[0077] In some embodiments, a cell population of the disclosure can be obtained from whole blood. A cell population of the disclosure can be obtained from a peripheral blood apheresis product, for example, a mobilized peripheral blood apheresis product. A cell population of the disclosure can be obtained from at least one apheresis product, two apheresis products, three apheresis products, four apheresis products, five apheresis products, six apheresis products, or more.

Sorting

[0078] The population of cells, before or after introduction of a CAR, usually after introduction of a CAR, is selected for expression of CD39, where CD39- and CD39⁺ populations find use depending on the requirement for cytotoxicity in the application of the cells. CD39 (ectonucleoside triphosphate diphosphohydrolase-1 , gene: ENTPD1 ; protein: NTPDasel) is a cell surface enzyme with a catalytic site on the extracellular face, that catalyzes the hydrolysis of y- and |3-phosphate residues of triphospho- and diphosphonucleosides to the monophosphonucleoside derivative. The refseq for the human protein may be accessed at Genbank, NP_001091645, NP_001 157650, NP_001 157651 , NP_001 157653,

NP_001 157654.

[0079] Selection methods for cell populations can comprise methods involving positive or negative selection of a cell population of interest. Selection methods for cell populations can comprise affinity reagents, including but not limited to an antibody, a full-length antibody, a fragment of an antibody, a naturally occurring antibody, a synthetic antibody, an engineered antibody, a full-length affibody, a fragment of an affibody, a full-length affilin, a fragment of an affilin, a full-length anticalin, a fragment of an anticalin, a full-length avimer, a fragment of an avimer, a fulllength DARPin, a fragment of a DARPin, a full-length fynomer, a fragment of a fynomer, a full-length kunitz domain peptide, a fragment of a kunitz domain peptide, a full- length monobody, a fragment of a monobody, a peptide, or a polyaminoacid. In some embodiments, the affinity reagent is directly conjugated to a detection reagent and/or purification reagent. In some cases, the detection reagent and purification reagent are the same. In other cases, the detection reagent and purification reagent are different. For example, the detection reagent and/or purification reagent is fluorescent, magnetic, or the like. In some cases, the detection reagent and/or purification reagent is a magnetic particle for column purification. For example, magnetic column purification may be performed using the Miltenyi system (CliniMACs) of columns, antibodies, buffers, preparation materials and reagents [0080] Affinity reagents can comprise immunoaffinity reagents, utilizing the binding specificity of antibodies or fragments or derivatives thereof to positively or negatively select for a cell population of interest. Selection methods for cell populations can comprise an affinity agent and a column, such as magnetic activated cell sorting (MACS) with specific antibodies and microbeads. Selection methods for cell populations can comprise fluorescent activated cell sorting (FACS), with cell populations sorted based on staining profiles with one or more fluorescently-conjugated antibodies. Selection methods for cell populations can comprise physical adsorption, for example, physical adsorption of T cells to protein ligands such as lectins.

[0081] A large number of anti-human CD-39 antibodies are commercially available and can find use for this purpose, including without limitation, antibodies sold by Amcam: Rabbit monoclonal [EPR26473-58] to CD39; Rabbit monoclonal [EPR20627] to CD39; Mouse monoclonal [AC2] to CD39; Rabbit monoclonal [EPR20461 ] to CD39; Rabbit monoclonal [EPR3678(2)] to CD39; Rabbit monoclonal [EPR26473-58] to CD39; Mouse monoclonal [IMG17B5F11] to CD39; Mouse monoclonal [A1] to CD39; APC Mouse monoclonal [TU66] to CD39; FITC Mouse monoclonal [A1] to CD39; and PE Mouse monoclonal [TU66] to CD39; antibodies sold by Invitrogen: CD39 Monoclonal Antibody (eBioAl (A1 )); CD39 Recombinant Rabbit Monoclonal Antibody (JA90-36); sold by LSBio: CD39 Mouse anti-Human Monoclonal (aa38-307) (2B10) Antibody, D39 Mouse anti-Human Monoclonal (BU61 ) Antibody, CD39 Antibody (clone A1 ), CD39 Mouse anti-Human Monoclonal (6H12F9) Antibody, CD39 Antibody (clone BU61 ); and the like.

[0082] A population of cells comprising CAR-Tregs can be selected using magnetic activated cell sorting (MACS). A population of cells comprising Tregs can be selected using fluorescent activated cell sorting (FACS). A population of cells comprising Tregs can be selected using multiple procedures, for example, multiple MACS selections, multiple FACS selections, or a combination of MACS and FACS selections.

[0083] In some embodiments the cells are first enriched by CD25 expression by CliniMACS immunomagnetic selection, followed by flow cytometric sorting for CD4⁺, CD127^l0/ne9 expression. The resulting product is enriched for Tregs defined phenotypically as CD4⁺, CD25⁺, CD127^l0/ne9 and expressing the transcription activator FoxP3. FoxP3 detection is used only to assess the percentage of CAR-Tregs in samples drawn from the final cellular product for infusion. The final population is selected into CD39 positive and negative fractions.

[0084] The cells are characterized by their expression of cell surface markers. For several of these markers, the expression is low or intermediate in level. While it is commonplace to refer to cells as “positive” or “negative” for a particular marker, actual expression levels are a quantitative trait. The number of molecules on the cell surface can vary by several logs, yet still be characterized as “positive”. Characterization of the level of staining permits subtle distinctions between cell populations.

[0085] The staining intensity of cells can be monitored by flow cytometry, where lasers detect the quantitative levels of fluorochrome (which is proportional to the amount of cell surface antigen bound by the antibodies). Flow cytometry, or FACS, can also be used to separate cell populations based on the intensity of antibody staining, as well as other parameters such as cell size and light scatter. Although the absolute level of staining may differ with a particular fluorochrome and antibody preparation, the data can be normalized to a control.

[0086] In order to normalize the distribution to a control, each cell is recorded as a data point having a particular intensity of staining. These data points may be displayed according to a log scale, where the unit of measure is arbitrary staining intensity. In one example, the brightest cells in a bone marrow sample are designated as 4 logs more intense than the cells having the lowest level of staining. When displayed in this manner, it is clear that the cells falling in the highest log of staining intensity are positive, while those in the lowest intensity are negative. The “low” staining cells, may fall in the 2-3rd log of staining intensity. An alternative control may utilize a substrate having a defined density of antigen on its surface, for example a fabricated bead or cell line, which provides the positive control for intensity.

Activation

[0087] The sorted CAR-Treg cells may be activated in vitro for a period of time of at least about 24 hours, and not more than about 48 hours at 37°C. The in vitro activation can be from about 24 hours, from about 26 hours, from about 28 hours, from about 30 hours, from about 32 hours, from about 34 hours from about 36 hours, up to about 48 hours, up to about 46 hours, up to about 44 hours, up to about 42 hours, up to about 40 hours, up to about 38 hours, and may be for about 24, about 30, about 36, about 42, about 48 hours. For activation the cells are suspended in medium at a concentration of from about 0.9 x 10⁵ viable cells/ml from about 1 x 10⁶, up to about 2 x 10⁶; up to about 3 x 10⁶ and may be at a concentration of from about 1.1 x 10⁶ to about 2 x 10⁶ viable cells/ml, e.g. at about 1.1 x 10⁶; 1 .2 x 10⁶; 1 .3 x 10⁶; 1 .4 x 10⁶; 1.5 x 10⁶; 1 .6 x 10⁶; 1.7 x 10⁶; 1.8 x 10⁶; 1 .9 x 10⁶; 2 x 10⁶.

[0088] The medium comprises an suitable cell medium, e.g. TexMACs™, etc., and effective dose of human serum, a T cell activating agent, e.g. TransAct™ (Miltenyi); and IL-2. Each of the reagents is generally GMP grade. Human serum may be present at a concentration of from about 5% to about 15% vol/vol, e.g. from about 6%, about 7%, about 8% about 9%, about 10% about 1 1 % about 12%, up to about 15%, up to about 14%, up to about 13%, up to about 13%, up to about 12%, and may be present at about 10% concentration. IL-2 is present at a concentration of from about 100 lU/ml up to about 5000 lU/ml, e.g. from about 100 IU, from about 200 IU, from about 300 IU, from about 400 IU, from about 500 IU, up to about 5000 IU, up to about 4000 IU, up to about 3000 IU, up to about 2000 IU, up to about 1000 IU, and may be present at about 500 IU. TransAct™ may be added in accordance with the manufacturers’ guideline, e.g. diluted from about 1 :20 to about 1 :10, and may be 1 :17.5, 1 :15, 1 :12.5, etc.

Freezing

[0089] The activated Treg calls are optionally cryopreserved. Cryopreservation can comprise addition of a preservative agent (e.g., DMSO), and gradual cooling of cells in a controlled-rate freezer to prevent osmotic cellular injury during ice crystal formation. Cryopreservation can comprise commercial cryopreservation reagents and materials, for example, Cryobags and CryoStor® CS10. In some embodiments the cells are resuspended in Normosol-R, pH 7.4; with hydroxyethyl starch 30.5%; human serum albumin (HSA) 12%; and DMSO 7.5%. Cryopreserved cells can be stored for periods of time ranging from hours to years at low temperatures. Cryopreserved cells can be stored at ultralow temperatures, for example, -50 aC, -60 ⁹C, -70 ⁹C, -80 ^aC, -90 ⁹C, -100 ⁹C, -110 ^eC, -120 ⁹C, -130 ⁹C, -140 ^eC, -150 ⁹C, -160 9C, 170⁹C, -180 ⁹C, -190 ^aC, -196 ^eC, or less. Cryopreserved cells can be stored in storage devices comprising liquid nitrogen.

[0090] Post-thaw the cells are placed in a suitable medium comprising human serum and IL- 2, and may be activated a second time, in the absence of the T cell activating agent. For activation the cells are suspended in medium at a concentration of from about 0.9 x 10⁵ viable cells/ml from about 1 x 10⁶, up to about 2 x 10⁶; up to about 3 x 10⁶ and may be at a concentration of from about 1 .1 x 10⁶ to about 2 x 10⁶ viable cells/ml, e.g. at about 1 .1 x 10⁶;

1 .2 x 10⁶; 1 .3 x 10⁶; 1 .4 x 10⁶; 1 .5 x 10⁶; 1 .6 x 10⁶; 1 .7 x 10⁶; 1 .8 x 10⁶; 1 .9 x 10^s; 2 x 10⁶. After the initial culture, a T cell activating agent, e.g. TransAct™ is added to the cells and the cell suspension returned to 37°C, 5% CO₂ for a period of from about 12 to about 24 hours, e.g. from about 12 hours, from about 14 hours, from about 16 hours, from about 18 hours, from about 20 hours, up to about 24 hours, up to about 22 hours, up to about 20 hours, up to about 18 hours, up to about 16 hours.

Administration

[0091] A population of cells comprising CD39⁺ CAR-Tregs or CD39 CAR-Tregs can be administered to a subject at a dose greater than at least about 1 x 10⁴, 1 x 10⁵, 2 x 10⁵, 3 x

10⁵, 4 x 10⁵, 5 x 10⁵, 6 x 10⁵, 7 x 10⁵, 8 x 10⁵, 9 x 10⁵, 1 x 10⁶, 1.1 x 10⁶, 1.2 x 10⁶, 1 .3 x 10⁶, 1 .4 x 10⁶, 1.5 x 10⁶, 1.6 x 10⁶, 1 .7 x 10⁶, 1.8 x 10⁶, 1 .9 x 10⁶, 2 x 10⁶, 2.1 x 10⁶, 2.2 x 10⁶, 2.3 x 10⁶, 2.4 x 10⁶, 2.5 x 10⁶, 2.6 x 10⁶, 2.7 x 10⁶, 2.8 x 10⁶, 2.9 x 10⁶, 3 x 10⁶, 3.1 x 10⁶, 3.2 x

10⁶, 3.3 x 10⁶, 3.4 x 10⁶, 3.5 x 10⁶, 3.6 x 10⁶, 3.7 x 10⁶, 3.8 x 10⁶, 3.9 x 10⁶, 4 x 10⁶, 4.1 x 10⁶,

4.2 x 10⁶, 4.3 x 10⁶, 4.4 x 10⁶, 4.5 x 10⁶, 4.6 x 10⁶, 4.7 x 10⁶, 4.8 x 10⁶, 4.9 x 10⁶, 5 x 10⁶, 5.1 x 10⁶, 5.2 x 10⁶, 5.3 x 10⁶, 5.4 x 10⁶, 5.5 x 10⁶, 5.6 x 10⁶, 5.7 x 10⁶, 5.8 x 10⁶, 5.9 x 10⁶, 6 x 10⁶, 6.5 x 10⁶, 7 x 10⁶, 7.5 x 10®, 8 x 10⁶, 8.5 x 10⁶, 9 x 10⁶, 9.5 x 10⁶, 1 x 10⁷, 1.5 x 10⁷, 2 x

10⁷, 2.5 x 10⁷, 3 x 10⁷, 3.5 x 10⁷, 4 x 10⁷, 4.5 x 10⁷, 5 x 10⁷, 5.5 x 10⁷, 6 x 10⁷, 6.5 x 10⁷, 7 x

10⁷, 7.5 x 10⁷, 8 x 10⁷, 8.5 x 10⁷, 9 x 10⁷, 9.5 x 10⁷, 1 x 10^s, 1 x 10⁸, 1.5 x 10⁸, 2 x 10⁸, 2.5 x

10⁸, 3 x 10⁸, 3.5 x 10⁸, 4 x 10⁷, 4.5 x 10⁸, 5 x 10⁸, 5.5 x 10⁸, 6 x 10⁸, 6.5 x 10⁸, 7 x 10⁸, 7.5 x

10⁸, 8 x 10⁸, 8.5 x 10⁸, 9 x 10⁸, 9.5 x 10⁸, 1 x 10⁹, or more cells per kg of recipient body weight.

[0092] In some embodiments, a population of cells comprising CD39⁺ CAR-Tregs or CD39' CAR-Tregs administered to a subject can be administered at a dose of at most about 1 x 10⁴, 1 x 10⁵, 2 x 10⁵, 3 x 10⁵, 4 x 10⁵, 5 x 10⁵, 6 x 10⁵, 7 x 10⁵, 8 x 10⁵, 9 x 10⁵, 1 x 10⁶, 1 .1 x 10⁶, 1 .2 x 10⁶, 1.3 x 10⁶, 1.4 x 10⁶, 1 .5 x 10⁶, 1.6 x 10⁶, 1 .7 x 10⁶, 1 .8 x 10⁶, 1.9 x 10⁶, 2 x 10⁶, 2.1 x 10⁶, 2.2 x 10⁶, 2.3 x 10⁶, 2.4 x 10⁶, 2.5 x 10®, 2.6 x 10®, 2.7 x 10®, 2.8 x 10®, 2.9 x 10®, 3 x 10®, 3.1 x 10®, 3.2 x 10®, 3.3 x 10®, 3.4 x 10®, 3.5 x 10®, 3.6 x 10®, 3.7 x 10®, 3.8 x 10®, 3.9 x 10®, 4 x 10®, 4.1 x 10®, 4.2 x 10®, 4.3 x 10®, 4.4 x 10®, 4.5 x 10®, 4.6 x 10®, 4.7 x 10®, 4.8 x 10®, 4.9 x 10®, 5 x 10®, 5.1 x 10®, 5.2 x 10®, 5.3 x 10®, 5.4 x 10®, 5.5 x 10®, 5.6 x 10®, 5.7 x 10®, 5.8 x 10®, 5.9 x 10®, 6 x 10®, 6.5 x 10®, 7 x 10®, 7.5 x 10®, 8 x 10®, 8.5 x 10®, 9 x 10®, 9.5 x 10®, 1 x

10⁷, 1.5 x 10⁷, 2 x 10⁷, 2.5 x 10⁷, 3 x 10⁷, 3.5 x 10⁷, 4 x 10⁷, 4.5 x 1 O⁷, 5 x 10⁷, 5.5 x 1 O⁷, 6 x

10⁷, 6.5 x 10⁷, 7 x 1 O⁷, 7.5 x 10⁷, 8 x 1 O⁷, 8.5 x 10⁷, 9 x 10⁷, 9.5 x 1 O⁷, 1 x 10⁸, 1 x 10⁸, 1.5 x

10⁸, 2 x 10⁸, 2.5 x 10⁸, 3 x 10⁸, 3.5 x 10⁸, 4 x 10⁷, 4.5 x 10⁸, 5 x 10⁸, 5.5 x 10⁸, 6 x 10⁸, 6.5 x

10⁸, 7 x 10⁸, 7.5 x 10⁸, 8 x 10⁸, 8.5 x 10⁸, 9 x 10⁸, 9.5 x 10⁸, 1 x 10⁹, or less cells per kg of recipient body weight, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim FOXP3+,

CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+ CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, FOXP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+ CD127dim.

[0093] A population of cells comprising CD39⁺ CAR-Tregs or CD39’ CAR-Tregs of the disclosure can comprise, for example, greater than at least about 50%, 51%, 52%, 53%, 54%,

55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,

87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more FOXP3+ cells as a percentage of total cells, nucleated cells, or CD45+ cells, or CD4+CD25+CD127dim cells as a percentage of total cells, nucleated cells, or CD45+ cells, or as a percentage of total cells, nucleated cells, or CD45+ cells.

[0094] A population of cells comprising CD39⁺ CAR-Tregs or CD39’ CAR-Tregs of the disclosure can have a defined level of contaminating non-Treg cells. For example, greater than at least about 1 x 10², 2 x 10², 3 x 10², 4 x 10², 5 x 10², 6 x 10², 7 x 10², 8 x 10², 9 x 10², 1 x 10³, 2 x 10³, 3 x 10³, 4 x 10³, 5 x 10³, 6 x 10³, 7 x 10³, 8 x 10³, 9 x 10³, 1 x 10⁴, 2 x 10⁴, 3 x 10⁴, 4 x 10⁴, 5 x 10⁴, 6 x 10⁴, 7 x 10⁴, 8 x 10⁴, 9 x 10⁴, 1 x 10⁵, or more non-T reg cells per kg of recipient body weight can be present in population of cells comprising Tregs of the disclosure, where non-Treg cells are FOXP3- or CD127+/bright.

[0095] In some embodiments, at most about 1 x 10², 2 x 10², 3 x 10², 4 x 10², 5 x 10², 6 x 10², 7 x 10², 8 x 10², 9 x 10², 1 x 10³, 2 x 10³, 3 x 10³, 4 x 10³, 5 x 10³, 6 x 10³, 7 x 10³, 8 x 10³, 9 x 10³, 1 x 10⁴, 2 x 10⁴, 3 x 10⁴, 4 x 10⁴, 5 x 10⁴, 6 x 10⁴, 7 x 10⁴, 8 x 10⁴, 9 x 10⁴, 1 x 10⁵, or less non- comprising CD39⁺ CAR-Tregs or CD39’ CAR-Tregs cells per kg of recipient body weight are present in a population of cells comprising Tregs of the disclosure, where non-Treg cells are FOXP3- or CD127+/bright.

[0096] A population of cells comprising CD39⁺ CAR-Tregs or CD39" CAR-Tregs of the disclosure can comprise, for example, greater than at least about 0.001 %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008% 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1 .1 %, 1 .2%, 1 .3%, 1 .4%, 1 .5%, 1 .6%, 1 .7%, 1 .8%, 1 .9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1 %, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more non-Treg cells as a percentage of total cells, nucleated cells, or CD45+ cells, where non-Treg cells are FOXP3- or CD127+/bright.

[0097] In some embodiments, a population of cells comprising CD39⁺ CAR-Tregs or CD39" CAR-Tregs of the disclosure comprises at most about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008% 0.009%, 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.1 %, 1 .2%, 1 .3%, 1 .4%, 1 .5%, 1 .6%, 1 .7%, 1 .8%, 1 .9%, 2%, 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or less non-Treg cells as a percentage of total cells, nucleated cells, or CD45+ cells, where non-Treg cells are FOXP3- or CD127+/bright.

Conditions for treatment

[0098] CAR Treg cells are useful in a variety of conditions, including, for example, treatment of cancer, in association with hematopoietic stem cell transplantation (HCT), for example to reduce graft v host disease, to reduce allograft rejection, and the like. In some embodiments a condition to be treated is an autoimmune disease, allergic disease, or inflammatory disease. For example, a population of cells comprising CD39 selected CAR-Tregs can be administered to an HCT recipient subject, and the population of CD39 selected CAR-Tregs can serve to reduce or prevent GVHD in the HCT recipient subject.

[0099] Graft-versus-host disease (GVHD) is an inflammatory disease that can occur in the allogenic transplant setting. GVHD involves donor cells (graft) attacking recipient cells (host). GVHD can be classified into acute GVHD (aGVHD) and chronic GVHD (cGVHD). aGVHD typically occurs in the first 3 months after transplantation. aGVHD can be life-threatening and can involve, for example, the skin, the intestines, and/or the liver. cGVHD typically occurs after the first 3 months following transplant. cGVHD is a major source of late treatment-related complications, and can be life-threatening. In addition to inflammation, cGVHD can lead to the development of fibrosis, which can result in functional disability.

[00100] The early morbidity and mortality associated with acute graft versus host disease (aGVHD) is a major factor limiting the success of HCT, as is the long-term morbidity associated with chronic GVHD (cGVHD). The incidence of aGVHD following allogeneic HCT from an HLA-matched sibling donor (MSD) is 20 to 60%, despite the use of various immunosuppressive agents such as tacrolimus, cyclosporine, methotrexate, mycophenolate, anti-thymocyte globulin and corticosteroids. Approximately one-third of patients who undergo allogeneic HCT using a MSD and a T cell replete graft will develop chronic GVHD.

[00101] GVHD severity can be graded, for example, using the Glucksberg grade (l-IV) or the International Bone Marrow Transplant Registry (IBMTR) grading system (A-D). The severity of acute GVHD is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined with (Glucksberg) or without (IBMTR) the patient’s performance status to produce an overall grade, which can have prognostic significance. Grading is important in terms of assessing the response to prophylaxis or treatment, impact upon survival, and association with graft-versus- leukemia effect.

[00102] Grade l(A) GVHD is characterized as mild disease, grade ll(B) GVHD as moderate, grade lll(C) as severe, and grade IV(D) life-threatening. The IBMTR grading system defines the severity of acute GVHD as follows: Grade A: stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement; Grade B: stage 2 skin involvement, stage 1 to 2 gut or liver involvement; Grade C: stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg/dL; diarrhea 1500 to 2000 mL/day); Grade D: stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg/dL; diarrhea >2000 mL/day OR pain OR ileus). Patients with moderate to severe GVHD have a significantly higher mortality rate compared with those with milder disease, for example, estimated five year survival for patients with grade III (C) aGVHD is 25%, while for patients with grade IV (D) estimated five year survival is 5%.

[00103] Management of GVHD may require immunosuppressive therapy (for example, high dose corticosteroids, prolonged administration of immunosuppressants) or cytotoxic mediations, all of which are associated with toxicity. In many cases, immunosuppressive therapies can fail to effectively treat GVHD, or can result in increased susceptibility to infection, or blunted anti-tumor immunity. [00104] In some embodiments, administration of the CD39 selected CAR-Treg cells, particularly CD39⁺ CAR-Treg cells of the disclosure disclosed herein prevent or reduce GVHD in an HCT recipient subject. For example, the methods disclosed herein can prevent any manifestation of GVHD in a subject receiving HCT. In some embodiments the CAR of such cells is targeted to a transplantation alloantigen, e.g. an HLA Class I or HLA Class II antigen.

[00105] The methods disclosed herein can prevent, for example, any GVHD of stage 1 or above, any GVHD of stage 2 or above, any GVHD of stage 3 or above, or any GVHD of stage 4 in subjects receiving HCT.

[00106] Type 1 diabetes (T1 D) is an autoimmune disorder characterized by insulin deficiency due to the destruction of pancreatic p cells. Studies have shown reduced immune suppressive functionality of Tregs in patients with T1 D. This observation and the success of Treg transplantation in maintaining immunologic tolerance has led to the application of Treg infusion in T1 D patients to rescue remaining cells. Infusion of expanded antigen-specific Tregs showed promising results in animal models in blocking and reversing diabetes.

[00107] In some embodiments, administration of the CD39 selected CAR-Treg cells, particularly CD39⁺ CAR-Treg cells of the disclosure disclosed herein prevent or reduce T1 D in a recipient. For example, the methods disclosed herein can prevent any manifestation of T1 D in a subject. In some embodiments the CAR of such cells is targeted to a pancreatic antigen, e.g. glutamic acid decarboxylase (GAD), insulin, HPi2, immunodominant GAD65 beta-cell epitopes, etc.

[00108] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is transplant rejection. In some embodiments, the transplant rejection occurs in response to transplanted blood, bone marrow, bone, skin, heart, kidney, lung, muscle, heart or liver. In some embodiments, the transplant rejection is hyperacute rejection. In some embodiments, the transplant rejection is acute rejection. In some embodiments, the transplant rejection is chronic rejection. In some embodiments the transplant is allogeneic islet transplantation.

[00109] In some embodiments, the CD39 selected CAR-Tregs of the disclosure comprise a CAR that targets a human leukocyte antigen. In some embodiments, the HLA targeted by the CAR is the same HLA allele as a donor. For example, the donor is HLA-A*2:0l and the CAR- Treg targets HLA- A* 2:0.1 In some embodiments, the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient. All alleles of HLA-A, HLA-B and HLA-C are envisaged as within the scope of the disclosure.

[001 10] In some embodiments, the method increases CD45+ cell engraftment, increases overall survival, decreases serum inflammatory cytokines or a combination thereof. In some embodiments, the method decreases spleen inflammation, liver inflammation, lung inflammation, inflammation of the central nervous system (CNS), inflammation of the skin, inflammation of the pancreas, inflammation of the kidney, inflammation of the pleural cavity, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or inflammation of the pelvis. In some embodiments, the disclosure provides a method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising providing administering the CD39 selected CAR- Tregs to the patient.

[001 11] In some cases, the method prevents allograft rejection, improving upon concepts described in Noyan et al. (2017) Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor. Am. J. Transplantation 2017; 17: 917-930.

EXPERIMENTAL

Example 1

Materials and methods

[001 12] Cell purification, in vitro expansion, and characterization. In this study, Fresh peripheral blood mononuclear cell (PBMC) samples were isolated from LRS chambers/buffy coats of healthy donors purchased from Stanford Blood Centers (Palo Alto, CA) or and 50 to 70 mL freshly drawn peripheral blood from patients with Type 1 diabetes(T1 D). Human peripheral blood mononuclear cells (PBMC) were isolated from blood by gradient centrifugation on Ficoll-Hypaque gradients (GE Healthcare Bioscience). Natural regulatory T cells (nTregs; CD3+ CD4+ CD8-CD25highCD127low) and autologous CD4+ T cells required for suppression assays were isolated by enrichment from PBMCs using EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions.

[001 13] The isolated Treg then were purified with fluorescence-activated cell sorting using the following gates: lymphocytes, single cells, live (aqua-), whole Treg CD3+CD4+CD8- CD25hiCD127-, CD39+ Treg CD3+CD4+CD8-CD25hiCD127-CD39+ and CD39- Treg CD3+CD4+CD8-CD25hiCD127-CD39- The monoclonal antibody combinations were as listed on Table 1. The FACS-based cell sorting was performed at Stanford Shared FACS Facility using a FACSAria™ II Cell Sorter (Beckton Dickinson) and had a purity of >99.0%.

[001 14] Sorted populations were expanded in X-VIVO™ 15 Chemically Defined Medium, with gentamicin and phenol red (Lonza) supplemented with 10% heat inactivated Human male AB serum (Sigma-Aldrich) and 10% L-glutamine(HyClone™), 10% Sodium Pyruvate(Gibco) and 10% non-essential amino acids(NEAA)(Fisher) in the presence of recombinant human IL-2 (rlL-2, PeproTech) (100 lU/ml for Teff; 500 lU/ml for Tregs), expanded by using Dynabeads® Human T-Activator CD3/CD28(Gibco) according to the manufacturer’s instructions. Tregs have been cultured for 12 consecutive days, checked every 6 days for purity by FACS analysis (CD4+FoxP3+ cells constantly > 90%), washed, and then transfected. To evaluate the purity of Tregs isolated by this cell-sorting scheme, they were stained with anti-FoxP3 (Clone 206D, BioLegend) in FoxP3 staining buffer (eBioscience). Flow cytometry data were acquired on an LSRII or FACS Caliber (Becton Dickinson); FACS Diva software (BD) and FlowJo version v10 software (Treestar) were used for analysis.

[001 15] Transfection of Tregs. For T cell activation, expanded Treg cells were activated using Dynabeads® Human T-Activator CD3/CD28(Gibco) in a bead: cell=1 : 3 ratio for 1 -3 days at 37°C with 5% CO2 in the presence of 500 lU/ml recombinant human IL-2 (rhlL-2) (R&D Systems). Treg were harvested and washed with PBS. A total of 2 x 10⁶ activated Treg was electroporated with a Neon Transfection System (Invitrogen) under the following conditions: voltage (2400 V), width (10 ms), pulses (three), 100-pil tip, and Buffer T. Cells were transfected with 2 pg of HLA-A2 CAR mRNA or 2 pg of 1 x9Q mRNA. After electroporation, cells were plated in a 6-well plate in 2 ml of CM RPMI 1640 with 500 IU rhlL-2/ml without antibiotics. The CAR construct expression efficacy was checked using Flag or Tetra HLA-A2. Cells were then used for functional assay described below.

[001 16] Glucose-induced insulin-secretion test. Islets after coculture with CAR-Treg for 48 hours were preincubated in a modified Krebs-Ringer Bicarbonate buffer (KRBB) containing 1 15 mmol/l NaCI, 5 mmol/l KCI, 24 mmol/l NaHCOs, 2.2 mmol/l CaCL, 1 mmol/l MgCL, 20 mmol/l HEPES, 2 g/l human serum albumin (Cealb, Sanquin, The Netherlands), pH 7.4 for 2 hours. Subsequently, islets were cultured in Krebs-Ringer bicarbonate buffer (KRBB) with 1 % bovine serum albumin (BSA) and with 2.8 mM glucose for 1 hour. Then islets were cultured in KRBB containing 2.8 mM or 16.7 mM glucose for additional 2 hours at 37°C. Afterwards, the supernatants were collected. Insulin concentrations were determined in the supernatants using Human Insulin ELISA Kit(Sigma-Aldrich)

[001 17] Purified Human Pancreatic Islet Viability Assessment Using Fluorescent Dyes and microscope. The viability was assessed fluorescein diacetate (FDA) and propidium iodide(PI). The concentrations are FDA(5mg/ml), Pl(2mg/ml). FDA passes through plasma membranes and is hydrolyzed by intracellular esterase to produce bright green fluorescent and identify viable cells. PI enters dead and dying cells and produces orange/red fluoresces. Briefly, the islet were collected in a tube, centrifuged, and suspended in 1 mL PBS. Afterwards, working solutions (1 .5 pL each) of FDA and PI were added to each tube, incubated for 15 min at room temperature, and the cells were washed with PBS to remove the unbound dye. Then, live dead imaging was performed using a fluorescence microscope (Leika in Dr. Justin Annes’ lab). CAR-Treg were labeled with CellTrace™ Violet (ThermoFisher Scientific) to be identified as blue. In addition, quantitative assessment of the viability was performed by using cell counting kit-8 (Dojindo Molecular Technologies, Inc, Rockville, Maryland, USA). [001 18] Purified Human Pancreatic Islet Viability Assessment Using flow. In a separate series of studies, islets were dispersed with Accumax - Cell Aggregate Dissociation Medium (Invitrogen™) and cocultured with CAR-Treg in 96-well plates at a density of 1 *10⁵ dispersed islet cells for 48 hours. Then cells were collected and stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD(BioLegend) according to the manufacturer’s instructions. Data were collected using a FACS Aria II analyzer and analyzed using Flowjo v10 software.

[001 19] Monocyte Viability Assessment Using flow. Monocytes were isolated by enrichment from PBMCs using EasySep Human Monocyte Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. The isolated monocyte were then purified with fluorescence-activated cell sorting using the following gates: lymphocytes, single cells, live (aqua-), CD14+HLA-DR+CD11 b+CD3-CD56-CD19- The monoclonal antibody combinations were as listed on Table 1. Monocyte were cocultured with CAR-Treg in 48-well plates at a density of 1 x10⁶/ml for 48 hours. Then cells were collected and stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD(BioLegend) according to the manufacturer's instructions, monocyte were gated on CD14+HLA-DR+CD11 b+lineage(CD3,CD56,CD19)-. Data were collected using a FACS Aria II analyzer and analyzed using Flowjo v10 software.

[00120] Tunel assay: Coverslips were coated (High performance coverslip 12mm dia 1.5h treated sterile super resolution, Neuvitro) with Poly-D-Lysine(Gibco). The coated sterile coverslips were placed into 12-well plate using sterile forceps, p Lox5 cells were plated onto sterilized glass coverslips after labeled with CellTrace™ Far Red Cell (Thermo Fisher Scientific) and cultured for 24 hours. Then CAR-Treg were seeded and cocultured with pLox5 cells for 24 hours. For detection of apoptosis, the coverslips were stained using the In situ Cell Death detection kit (Roche). Briefly, the coverslips were washed once gently with PBS, pH 7.2, avoiding removal of dying cells. The cells were fixed in room-temperature 4% paraformaldehyde (PFA) for one hours. And samples then were permeabilized with 0.1 % Triton X-100, 0.1 % sodium citrate, for 2 minutes at room temperature. 50 pl of TUNEL reaction solution (In Situ Cell Death Detection Kit, Fluorescein, Roche; Enzyme solution+Label solution) was added onto each coverslip and incubate at 37°C for 1 hour. Nuclei were stained with DAPI (100 ng/mL; MilliporeSigma). Negative controls for TUNEL staining included only 50ul label solution. Finally, the sections were analyzed using Leica SP8 White Light Confocal microscope with a 40x HC PL APO, CS2 oil objective lens (Stanford University Cell Sciences Imaging Facility). Images were evaluated for TUNEL staining using Leica LAS X software. The percentage of TUNEL positive cells was determined as the ratio of TUNEL positive cells to total nuclei x 100.

Table 1 : Antibodies used for flow cytometry. Target Clone Fluorochrome Vendor RRID

CD3 OKT3 Alexa Fluor 488 BioLegend AB_571877

CD3 OKT3 APC BioLegend AB_1937212

CD3 OKT3 BV785 BioLegend AB_2563507

CD4 SK3 Alexa Fluor 700 BioLegend AB_2563150

CD4 OKT4 Alexa Fluor 488 BioLegend AB_571939

CD8 SK1 BUV805 BD Biosciences AB_2833078

CD8 SK1 APC/Cyanine7 BioLegend AB_2044006

CD25 BC96 PE/Dazzle™ 594 BioLegend AB 2734260

CD25 2A3 BUV395 BD Biosciences AB_2738556

CD127 A019D5 PerCP/Cyanine5.5 BioLegend AB_10897104

CD39 eBioAl BV605 BioLegend AB_2750430

CD45RO UCHL1 BUV395 BD Biosciences AB_2744410

CD45RO UCHL1 Alexa Fluor 488 BioLegend AB 528823

CD62L DREG-56 PE-Cy7 BioLegend AB_830801

PD-1 NAT105 BV785 BioLegend AB_2721562

Tim -3 F38-2E2 PE/Dazzle™ 594 BioLegend AB_2565886

LAG-3 11 C3C65 BV650 BioLegend AB_2632951

F0XP3 206D BV421 BioLegend AB_2565972

IL- 10 JES3-19F1 APC-R700 BD Biosciences AB_2870004

IFN-Y 4S.B3 BV605 BioLegend AB_2563881

TNF-a Mab11 APC BioLegend AB_315264

IL-4 MP4-25D2 PE BioLegend AB_315129

Granzyme B GB11 Alexa Fluor 647 BioLegend AB_2566333

Granzyme B QA16A02 APC/Fire™ 750 BioLegend AB 2728377

HLA-A2 BB7.2 PE BioLegend AB_1877227

HLA-A2 BB7.2 APC BioLegend AB_2561567

PD-L1 29E.2A3 PE-Cy7 BioLegend AB_2561687

CD209 9E9A8 Alexa Fluor 647 BioLegend AB_1 186092

CD14 M5E2 APC/Fire™ 750 BioLegend AB_2632660

CD163 GHI/61 PerCP/Cyanine5.5 BioLegend AB_2228986

CD16 G8 BUV395 BD Biosciences AB_2744293

CD206 19.2 BUV805 BD Biosciences AB_2871334

CD11c Bu15 BV421 BioLegend AB 2564485 Target Clone Fluorochrome Vendor RRID

CD11b ICRF44 BV605 BioLegend AB_2562021

CD86 IT2.2 BV785 BioLegend AB_2616794

HLA-DR L243 PE/Dazzle™ 594 BioLegend AB_2563646

HPx1 HIC0-3B3 Dy Light 405 Novus Biologicals

HPi2 HIC1 -2B4.2B Alexa Fluor 647 Novus Biologicals

[00121] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of selecting chimeric antigen receptor (CAR) regulatory T cells (Tregs) for reduced cytotoxicity, the method comprising: introducing into a population of Tregs a CAR targeted to an antigen of interest to generate a CAR-Treg population; selecting the CAR-Treg population for expression of CD39, wherein CD39⁺ cells have decreased cytotoxicity to the targeted cell population.

2. The method of claim 1 , wherein the reduction in cytotoxicity is at least 25%, at least 50%, at least 75% decrease, and may be a two-fold decrease in cytotoxicity or more.

3. The method of claim 1 or claim 2, wherein the T regs are administered to an individual in need thereof.

4. The method of claim 3, wherein the composition comprising Tregs are administered at a dose of from 10⁴ cells/kg body weight to about 10⁹ cells/kg body weight.

5. The method of claim 3 or claim 4, wherein the composition comprising Tregs comprises at least about 50% CD4⁺ CD25⁺ CD127^l0 cells.

6. The method of any of claims 3-5, wherein the cells are administered for the treatment of an autoimmune disease or graft versus host disease.

7. A method of selecting chimeric antigen receptor (CAR) regulatory T cells (Tregs) for increased cytotoxicity, the method comprising: introducing into a population of Tregs a CAR targeted to an antigen of interest to generate a CAR-Treg population; selecting the CAR-Treg population for expression of CD39, wherein CD39- cells have increased cytotoxicity to the targeted cell population.

8. The method of claim 7, wherein the increase in cytotoxicity is at least 25%, at least 50%, at least 75% increase, and may be a two-fold increase in cytotoxicity or more.

9. The method of claim 7 or claim 8, wherein the T regs are administered to an individual in need thereof.

10. The method of claim 9, wherein the composition comprising T regs are administered at a dose of from 10⁴ cells/kg body weight to about 10⁹ cells/kg body weight.

1 1 . The method of claim 9 or claim 10, wherein the composition comprising Tregs comprises at least about 50% CD4⁺ CD25⁺ CD127^l0 cells.

12. The method of any of claims 3-5, wherein the cells are administered for the treatment of transplant rejection or cancer.

13. A composition comprising a population of Tregs obtained by the method according to any of claims 1 -2 or 7-8.

14. A method of selecting genetically modified regulatory T cells (Tregs) for reduced cytotoxicity, the method comprising: introducing into a population of Tregs a construct targeted to an antigen of interest to generate a genetically modified Treg population; selecting the Treg population for expression of CD39, wherein CD39+ cells have decreased cytotoxicity to the targeted cell population.