WO2025151706A1 - Epigenetic reagents and utilization of car nk cells - Google Patents

Abstract

Embodiments of the disclosure include methods and compositions for treatment of cancer, autoimmune disorders, and/or infectious disease. In particular embodiments, antigen expression on target cells is increased with epigenetic modulators in conjunction with immune effector cells engineered to express a heterologous molecule that targets the target cells. In specific embodiments, the epigenetic modulators are used in conjunction with NK cells expressing a chimeric antigen receptor that targets a cancer antigen, an antigen associated with an autoimmune disorder, or an antigen associated with a pathogen.

Description

EPIGENETIC REAGENTS AND UTILIZATION OF CAR NK CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application

Serial No. 63/620,531, filed January 12, 2024, hereby incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 10, 2025, is named “MDACP1380 Sequence Listing” and is 10,335 bytes in size.

BACKGROUND

I. Technical Field

[0003] This disclosure relates at least to the fields of immunology, cell biology, molecular biology, and medicine, including cancer medicine.

II. Background

[0004] Several studies have explicitly demonstrated that the effectiveness of chimeric antigen receptor (CAR)-designed effector cells is highly dependent on the density of the target antigen expression. In addition, the inadequate immune response towards tumor cells with low antigen density results in the recurrence or development of resistance to CAR therapy. The inclusion of a preconditioning regimen, which entails lymphodepleting chemotherapy prior to the infusion of CAR natural killer (NK)-cells, is a widely adopted approach in conventional treatment protocols. However, the reduced responsiveness caused by low levels of antigen density needs to be addressed to provide efficacious therapies to the clinic. In addition, tumor cells express NK ligands on their surface that could be modulated to enhance their susceptibility to NK mediated killing. The present disclosure provides solutions to such a need.

SUMMARY

[0005] Embodiments of the disclosure encompass methods and compositions for effective treatment of a medical condition for an individual. In particular embodiments, the medical condition is cancer, an autoimmune disorder, or the individual has both. In specific embodiments, therapy for an individual in need thereof includes combination therapy in which at least one of the components of the combination therapy directly or indirectly increases the quantity of certain antigens on cells, and in specific embodiments the antigen-expressing cells are targeted by at least another component of the therapy. In specific embodiments, the antigen(s) of interest are antigens on cancer cells targeted by immune effector cells of any kind that are specifically engineered to target the antigens.

[0006] Although in some embodiments the methods and compositions of the disclosure improve adoptive cell therapy by increasing the density of the target antigen expression on cells that are targeted by the adoptive cell therapy, in other embodiments the methods and compositions of the disclosure improve adoptive cell therapy by an alternative mechanism or in addition to increasing the density of the target antigen expression on cells targeted by the adoptive cell therapy.

[0007] In particular embodiments, at least one of the components of the combination therapy comprises engineered cells, including engineered immune effector cells of any kind. In specific embodiments, the engineered cells are engineered such that they express a heterologous protein that targets a specific antigen, and the antigen target may be soluble or may be present on certain cells, including cancer cells or immune cells of any kind. In particular embodiments, the antigen is an NK cell ligand such as NKG2-ligands, ligands for NK natural cytotoxicity receptors (NCRs), DNAM-ligands, NKG2C ligands, etc., although in alternative embodiments the antigen is not an NK cell ligand. In particular embodiments, at least one of the components of the combination therapy comprises one or more epigenetic reagents, including one or more epigenetic modulators. An epigenetic reagent may be a small molecule, a protein, a peptide, a nucleic acid, or a combination thereof, in some embodiments. In at least some cases, the epigenetic modulators comprise one or (a) one or more hypomethylating agents; (b) one or more histone deacetylase (HD AC) inhibitors; (c) one or more enhancer of zeste 2 (EZH2) inhibitors; (d) one or more meningitis inhibitors; (e) one or more lysine- specific histone demethylase 1A (LSD1) inhibitors; and (f) one or more bromodomain and extra- terminal domain (BET) inhibitors.. In particular embodiments, the disclosure concerns utilization of one or more epigenetic reagents as a preconditioning regimen prior to the infusion of the adoptive cell therapy (such as CAR NK cells), although in other embodiments the order of administration of any components of therapy may be of any order. In specific embodiments, the disclosure concerns exposure of one or more epigenetic reagents to cancer cells prior to the delivery of the adoptive cell therapy (such as CAR NK cells). [0008] In certain embodiments, to address the issue of reduced responsiveness of cancer cells to the adoptive cell therapy, such as caused by low levels of antigen density or NK-ligand density, the disclosure provides utilization of one or more epigenetic modulators to enhance the adoptive cell therapy, such as enhance production of the target antigen or NK-ligand density (including at least increasing its quantity on cells already expressing the antigen). In some embodiments, the exposure of cells to one or more epigenetic modulators results in the upregulation of one or multiple target antigen expressions for the cells. In some embodiments, the exposure of the cells to one or more epigenetic modulators such as decitabine, 5-AZA, and/or any HD AC inhibitor (as examples only) results in the up-regulation of one or multiple target antigen or NK-ligand density expressions. Without being bound by theory, in certain embodiments this effect may be achieved by a process that involves the reduction of antigen promoter methylation and/or the enhancement of transcription factor activity associated with the relevant genes.

[0009] In particular embodiments, the disclosure concerns methods that utilize use of one or more epigenetic modulators optionally in addition to lymphodepletion, e.g., prior to, subsequent to, or at the same time as adoptive cell therapy. In specific embodiments, the adoptive cell therapy utilizes cells that encompass adoptive cell therapy, such as CAR NK cell therapies and/or T cell therapies) to augment the presentation of certain, or any, antigens (including diverse antigens) on the tumor cell membrane. The implementation of this strategy is useful to decrease the probability of relapse or resistance to cells of adoptive cell therapy (such as CAR NK cells), in specific embodiments. The implementation of this strategy is useful to increase efficacy of cells of adoptive cell therapy (such as CAR NK cells), in specific embodiments.

[0010] In specific embodiments, epigenetic modulators enhance the susceptibility of tumor cells to killing by non-engineered NK cells, and in some embodiments this is the direct or indirect result of modulation of NK ligands on tumor cells.

[0011] Embodiments of the disclosure comprise a composition comprising (1) engineered or non-engineered immune effector cells; and (2) one or more epigenetic modulators. In specific embodiments, the one or more epigenetic modulators comprises: (1) engineered immune effector cells, non-engineered immune effector cells, or a mixture thereof; and (2) one or more epigenetic modulators, wherein the epigenetic modulators comprise one or more of the following: (a) one or more hypomethylating agents; (b) one or more histone deacetylase (HD AC) inhibitors; (c) one or more enhancer of zeste 2 (EZH2) inhibitors; (d) one or more meningitis inhibitors; (e) one or more lysine-specific histone demethylase 1A (LSD1) inhibitors; (f) one or more bromodomain and extra- terminal domain (BET) inhibitors; and (g) one or more IDH1 and/or IDH2 inhibitors. The hypomethylating agent may be a DNA methyltransferase (DNMT) inhibitor. Examples of DNMT inhibitors include at least 5- azacytidine, cytidine, decitabine, 5-aza-2-deoxycytidine, deoxyguanosine, guadecitabine, zebularine, clofarabine, arsenic trioxide curcumin, resveratrol, epigallocatechin-3-gallate, or a combination thereof. The HD AC inhibitor may inhibit a Class I, Class IIA, Class III, or Class IV HD AC. In specific embodiments, the HD AC inhibitor is a hydroxamatecyclic tetrapeptide, depsipeptide, benzamide, electrophilic ketone, aliphatic acid compound, or a mixture thereof. The hydroxamatecyclic tetrapeptide may be tricho statin A. The cyclic tetrapeptide may be trapoxin B. In some embodiments, the aliphatic acid compound is phenylbutyrate and/or valproic acid. Examples of HD AC inhibitors include at least phenylbutyrate, valproic acid, trichostatin A, vorinostat, romidepsin, belinostat, givinostat, panobinostat, tucidinostat, entinostat, mocentinostat, TYO-018, sanacruzamate A, scriptaid, and/or abexinostat. Examples of EZH2 inhibitors may include at least tazemotostat, GSK126, CPI-1205, PF- 06821497, SHR2554, UNC1999, valemotostat tosylate, and/or MAK683. In some embodiments, the meningitis inhibitor is selected from the group consisting of vancomycin, but teicoplanin, linezolid, fucidic acid, daptomycin, and a combination thereof. In some embodiments, the LSD1 inhibitor is selected from the group consisting of ladademstat, pulrodemstat benzenesulfonate, tranylcypromine, pargyline, GSK-LSD1, and a combination thereof. In certain embodiments, the BET inhibitor is selected from the group consisting of I- BET 151 (GSK1210151A), I-BET 762 (GSK525762), OTX-015, TEN-010, CPI-203, CPI- 0610, olinone, RVX-208, ABBV-744, LY294002, AZD5153, MT-1,MS645, pelabresib, and a combination thereof. In some embodiments, the IDH1 and/or IDH2 inhibitors comprise enasidenib, ivosidenib, vorisidenib, NI-1, BAY 1436032, Compound 6, or a combination thereof.

[0012] The immune effector cells and one or more epigenetic modulators may or may not be in the same formulation.

[0013] In specific embodiments, engineered immune effector cells express one or more heterologous proteins, such as a therapeutic gene product that may or may not be an engineered receptor, an antibody, or the cells express both. The engineered receptor is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor, in specific embodiments. In some aspects, the immune effector cells express one or more heterologous cytokines, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-10, IL-4, IGNy, IFNoc, IL- 33, IL-27, and/or GMCSF.

[0014] Embodiments of the disclosure include any method of treating a medical condition, comprising the step of administering to an individual with the medical condition a therapeutically effective amount of any composition encompassed herein. Embodiments of the disclosure include any method of treating a medical condition, comprising the step of administering to an individual with the medical condition a therapeutically effective amount of any combination therapy encompassed herein. The individual may have cancer and/or an autoimmune disorder and/or an infectious disease. The one or more epigenetic modulators may be administered to the individual prior to, subsequent to, or at substantially the same time as administering the engineered immune effector cells (such as NK cells) to the individual. The one or more epigenetic modulators and the engineered cells may or may not be in the same formulation. The one or more epigenetic modulators and the engineered cells may or may not be provided at the same time to an individual in need of both.

[0015] Embodiments of the disclosure include methods of increasing efficacy of nonengineered NK cells or antigen-targeting NK cells as adoptive cell therapy for an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a composition comprising engineered NK cells (or non-engineered cells) and one or more epigenetic modulators.

[0016] Embodiments of the disclosure include kits comprising any composition encompassed herein.

[0017] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The subject matter of the disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0019] FIGS. 1A-1F. The induction of CD70 with the use of hypomethylating drugs (Decitabine, 5-AZA, as examples) in dose dependent manner on AML cell lines and primary AML blasts.

[0020] FIG. 2. The induction of CD70 with the use of hypomethylating drugs (Decitabine, 5-AZA) and HDAC inhibitor (Vorinostat) (each as examples) in dose dependent manner on MOLM-14 AML cell line.

[0021] FIG. 3. The induction of HLA-G with the use of hypomethylating drugs (Decitabine, 5-AZA) and HDAC inhibitor (Vorinostat) (each as examples) in dose dependent manner on MOLM-14 AML cell line.

[0022] FIG. 4. The induction of HLA-G with the use of hypomethylating drugs (Decitabine, 5-AZA) and HDAC inhibitor( Vorinostat) (each as examples) in dose dependent manner on MOLM-14 AML cell line.

[0023] FIG. 5. The induction of HLA-G with the use of hypomethylating drugs (Decitabine, 5-AZA) and HDAC inhibitor (Vorinostat) (each as examples) in dose dependent manner on THP-1 AML cell line.

[0024] FIG. 6. The induction of HLA-G with the use of hypomethylating drug (Decitabine) and HDAC inhibitor (Vorinostat) (each as examples) in dose dependent manner on THP-1 AML cell line.

[0025] FIG. 7. The induction of Neo peptides(NY-ESOl) with the use of hypomethylating drug (Decitabine) and HDAC inhibitor (Vorinostat), each as examples.

[0026] FIG. 8. Enhanced killing of THP1 AML cell line treated with hypomethylating drugs (Decitabine, as an example) by HLA-G CAR NK cells.

[0027] FIG. 9. Pretreatment with Enasidenib or Decitabine, as examples, enhances NK cell cytotoxicity against UMRC3 (RCC) and BCX010 (triple negative breast cancer (TNBC)) cell lines in a long-term spheroid assay.

[0028] FIGS. 10A-10B shows that pretreatment with Enasidenib or Decitabine (as examples) enhances NK cell cytotoxicity against BCX010 (TNBC) (FIG. 10A) and UMRC3 (renal cell carcinoma (RCC)) (FIG. 10B) cell lines in a long-term xCelligence assay. The y- axis represents quantitative measure of cell growth, adhesion, and morphology changes over time.

[0029] FIG. 11 shows that pretreatment with Enasidenib or Decitabine enhances NK cell cytotoxicity against MDAMB231 (TNBC) cell Lines in long term xCelligence assay. DETAILED DESCRIPTION

I. Examples of Definitions

[0030] It is to be understood that the present disclosure is not limited solely to the particular aspects described herein, as such may, of course, vary, while still being encompassed by the teachings provided herein. 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, since the scope of the present disclosure will be limited only by the appended claims.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, certain embodiments of methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0032] The practice of the present disclosure may employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Ereshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition; all of which are incorporated herein by reference.

[0033] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0034] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0035] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0036] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0037] The term “antigen presenting cells (APCs)” refers to a class of cells capable of presenting one or more antigens in the form of a peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. The term “APC” encompasses intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells, or molecules, naturally occurring or synthetic capable of presenting antigen, such as purified MHC Class I molecules complexed to |32-microglobulin.

[0038] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific embodiments, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins. Cells may be engineered to have reduced or eliminated expression of one or more endogenous genes.

[0039] An “immune disorder,” “immune-related disorder,” or “immune-mediated disorder” refers to a disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft versus host disease and inflammatory and allergic conditions.

[0040] An “immune response” is a response of a cell of the immune system, such as a B cell, or a T cell, or innate immune cell to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen- specific response”).

[0041] An “autoimmune disease” refers to a disease in which the immune system produces an immune response (for example, a B cell or a T cell response) against an antigen that is part of the normal host (that is, an autoantigen), with consequent injury to tissues. An autoantigen may be derived from a host cell, or may be derived from a commensal organism such as the microorganisms (known as commensal organisms) that normally colonize mucosal surfaces. [0042] As used herein, a “disruption” of a gene refers to the elimination or reduction of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the disruption. Exemplary gene products include mRNA and protein products encoded by the gene. Disruption in some cases is transient or reversible and in other cases is permanent. Disruption in some cases is of a functional or full length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, gene activity or function, as opposed to expression, is disrupted. Gene disruption is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene silencing, knockdown, knockout, and/or gene disruption techniques, such as gene editing. Examples include antisense technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and/or complete absence of expression of a normal or “wild type” product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and mis sense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

[0043] The term “heterologous” when used in reference to a gene refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise gene sequences that comprise cDNA forms of a gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or antisense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally found and/or expressed).

[0044] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer. [0045] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0046] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0047] The term “subject,” as used herein, generally refers to an individual having a that has or is suspected of having cancer. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g.. cows, sheep, goats, pigs, turkeys, and chickens), household pets e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., have or be suspected of having a disease (that may be referred to as a medical condition), such as benign or malignant neoplasias, or cancer. The subject may being undergoing or having undergone treatment. The subject may be asymptomatic. The subject may be healthy individuals but that are desirous of prevention of cancer. The term “individual” may be used interchangeably, in at least some cases. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0048] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. Treating may mean alleviation of at least one symptom of the disease or condition.

[0049] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

II. Cellular Therapies

[0050] Cellular therapies encompassed herein include those that may be part of a combination therapy for an individual in need of the combination therapy. In particular embodiments, the cellular therapy comprises engineered immune effector cells that target an antigen of interest. In embodiments, the efficacy of the cells are enhanced at least for cy toxicity because of the action of one or more epigenetic modulators. In some embodiments, the order of delivery of the cells and the epigenetic modulator(s) may be of any order or substantially at the same time.

A. Cell Culture

[0051] In embodiments of the disclosure, engineered immune effector cells are employed to target an antigen(s) expressed by a deleterious cell. The engineered immune effector cells are part of a bipartite therapy in which the therapy also includes epigenetic modulator(s) that increase expression of the antigen expressed by the deleterious cell.

[0052] In some embodiments, cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some embodiments, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and/or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. In some embodiments, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, 100 pg/ml streptomycin and 2 mmol/L L-glutamine. Other embodiments may employ further basal media formulations, such as chosen from the ones above.

[0053] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0054] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0055] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular embodiments, cells are cultured in a cell culture system comprising a cell culture medium, such as in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and/or inducing in an unspecific or specific reprogramming.

B. Cell Generation

[0056] Certain methods of the disclosure concern culturing the engineered immune effector cells obtained from human tissue samples, and the obtained cells may be autologous or allogeneic to a recipient individual of the cells. In particular embodiments of the present disclosure, cells may be plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, in certain embodiments cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such embodiments encompass plating onto a substrate, which allows adherence of cells thereto.

[0057] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation of these cells can be achieved, for example, through flow cytometry, ELISA, and/or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and/or to monitor changes in gene expression in response to differentiation. In certain embodiments, the marker proteins used to identify and characterize the cells are selected from the group consisting of c-Kit, Nanog, Sox2, Heyl, SMA, Vimentin, Cyclin D2, Snail, E-cadherin, Nkx2.5, GATA4, CD 105, CD90, CD29, CD73, Wtl, CD34, CD45, and a combination thereof.

C. Pharmaceutical Compositions

[0058] In certain aspects, the compositions or agents for use in the methods, such as the engineered immune effector cells and/or the epigenetic modulator(s), are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (z.e., to a specific location of the body, such as a tumor or other tissue) or systemic delivery, such as in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0059] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0060] The carrier may also comprise a delivery vehicle to sustain (z.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0061] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0062] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0063] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0064] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0065] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0066] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0067] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0068] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

III. Epigenetic Modulators

[0069] In some embodiments, one or more epigenetic modulators are provided to an individual in need thereof, such as an individual receiving or will receive adoptive cell therapy, including engineered immune cell therapy. The individual may be in need of upregulation of one or more antigens on cells in the individual. In particular embodiments, the epigenetic modulator(s) facilitates upregulation of expression of one or more antigens on cells directly or indirectly deleterious to the individual.

[0070] In certain embodiments, the epigenetic modulator may comprise one or more of the following: (a) one or more hypomethylating agents; (b) one or more histone deacetylase (HD AC) inhibitors; (c) one or more enhancer of zeste 2 (EZH2) inhibitors; (d) one or more meningitis inhibitors; (e) one or more lysine-specific histone demethylase 1A (LSD1) inhibitors; and (f) one or more bromodomain and extra-terminal domain (BET) inhibitors.

IV. Heterologous Proteins

[0071] In particular embodiments, the engineered immune effector cells are engineered to express 1, 2, 3, 4, 5, or more heterologous proteins. A heterologous protein as referred to herein concerns a protein that is not endogenous to the cell in which is resides. In specific embodiments, the heterologous protein is a therapeutic gene product, whether or not the protein provides therapy itself to a recipient or the protein directly or indirectly provides therapy to a recipient. The heterologous protein may be chimeric, having parts from different proteins and/or sources. The heterologous protein may be one or more recombinant proteins, one or more synthetic protein, one or more engineered receptors, one or more cytokines, one or more growth factors, and so forth. In specific embodiments, the heterologous protein is at least one antigen receptor that is specifically designed to target a desired antigen, such as an antigen on a deleterious cell (including a cancer cell or a cell associated with autoimmune disease). In specific embodiments, an engineered receptor is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor, or there may be more than one kind on the cell. In embodiments wherein the heterologous protein is a cytokine, the cytokine may be of any kind, but in specific embodiments the cytokine is IL-2, IL-7, IL- 12, IL- 15, IL- 18, IL- 21, IL-23, and/or GMCSE. [0072] In some embodiments, the immune effector cells are modified to express one or more bispecific or multi- specific antibodies, although in other cases the immune effector cells do not express the antibodies but the antibodies are utilized in conjunction with the immune effector cells. In cases wherein the immune effector cells are modified to express the antibodies, the antibodies may be engagers that bridge a particular immune effector cell with a particular target cell for destruction of the target cell. In some embodiments, the heterologous proteins are BiTEs or BiKEs. When the proteins are BiKEs, the BiKE may target the NK cells through an antibody an NK surface protein such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, an NCR, or KIR, for example. In such cases, the BiKE used in the disclosure may also target a cancer or viral antigen that may be tailored to the medical condition of an intended recipient individual. For example, the BiKE may be tailored to bind a cancer antigen that is characteristic of the cancer cells of a cancer of the individual.

[0073] In particular embodiments, the heterologous protein is the gene product of a suicide gene that is utilized in conjunction with the therapy of the disclosure to control its use and allow for termination of the cell therapy at a desired event and/or time. The suicide gene is employed in transduced cells for the purpose of eliciting death for the transduced cells when needed. The cells of the present disclosure that have been modified to harbor one or more vectors encompassed by the disclosure that may comprise one or more suicide genes. In some embodiments, the term “suicide gene” as used herein is defined as a gene which, upon administration of a prodrug or other agent, effects transition of a gene product to a compound which kills its host cell. In other embodiments, a suicide gene encodes a gene product that is, when desired, targeted by an agent (such as an antibody) that targets the suicide gene product. [0074] Examples of suicide genes include engineered nonsecretable (including membrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT/US 19/62009, which is incorporated by reference herein in its entirety), and they may be affected by delivery of an antibody that binds the TNF-alpha mutant. Examples of suicide gene/prodrug combinations that may be used are Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5- fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The E.coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6 -methylpurine deoxyriboside to toxic purine 6- methylpurine, may be utilized. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), Cytochrome p450 enzymes (CYP), Carboxypeptidases (CP), Carboxylesterase (CE), Nitroreductase (NTR), Guanine Ribosyltransferase (XGRTP), Glycosidase enzymes, Methionine- a, y-lyase (MET), EGFRv3, and Thymidine phosphorylase (TP), as examples.

[0075] In some embodiments, the heterologous protein may be delivered to the cell on a nucleic acid vector. In some embodiments, one or more heterologous proteins may be delivered to a cell on the same vector. In some embodiments, multiple heterologous proteins are delivered on different vectors.

A. Vectors

[0076] To express any polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides (such as being one or more antigen receptors and/or one or more cytokines) are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete engineered antigen receptor and/or cytokine is configured with appropriate restriction sites so that the sequence of any region of the protein can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete cytokine is configured with appropriate restriction sites so that the cytokine may be interchanged with a different cytokine. In some aspects, a vector that encodes a functionally complete suicide gene is configured with appropriate restriction sites so that the suicide gene may be interchanged with a different suicide gene.

[0077] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., for antibody, the portion thereof may be a functional fragment comprising one or more CDRs or one or more variable region domains). Expression vectors comprising the antibody-encoding nucleic acid molecules may encode the heavy chain, light chain, alpha chain, beta chain, or the antigen-binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0078] Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.

B. Expression Systems

[0079] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.

C. Methods of Gene Transfer

[0080] Suitable methods for nucleic acid delivery to effect expression of compositions encompassed herein are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

D. Host Cells

[0081] In another aspect, contemplated are the use of host cells (such as engineered immune effector cells) into which a recombinant expression vector encoding, e.g., a heterologous protein, has been introduced. Antigen receptors, cytokines and/or antibodies can be expressed in a variety of cell types. An expression construct encoding an antigen receptor, cytokine, and/or antibody can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT-1 or NF-KB, both of which are transcription factors that can be activated upon T-cell activation. Control of antibody expression allows T cells, such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0082] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.

E. Isolation

[0083] The nucleic acid molecule encoding one or more heterologous proteins may be obtained from any source. Methods of isolating nucleic acid are well known in the art. See e.g., Sambrook et al., supra. Nucleic acid molecules encoding the heterologous proteins may be expressed in a cell into which they have been introduced and the protein isolated.

V. Formulations and Culture of the Engineered Immune Effector Cells

[0084] In particular embodiments, the engineered immune effector cells of the disclosure may be specifically formulated and/or they may be cultured in a particular medium. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects. In specific embodiments, the immune effector cells are NK cells or T cells. In particular embodiments, the immune effector cells are engineered to produce a heterologous gene product, such as an antigen receptor(s), cytokine(s), and/or antibody(ies).

[0085] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0086] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0087] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0088] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and/or T3 (triodo-I-thyronine). . In specific embodiments, one or more of these may be explicitly excluded.

[0089] In some embodiments, the medium further comprises one or more vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alphatocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D- Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno- free B-27® supplement, GS21TM supplement, or combinations thereof. In some embodiments, the medium comprises or futher comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and/or one or more proteins discussed herein, and/or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L- camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I- thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and/or phosphorus) or salts thereof, and/or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be explicitly excluded.

[0090] The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and/or inorganic salts. . In specific embodiments, one or more of these may be explicitly excluded.

[0091] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/ml, pg/ml, mg/ml, or any range derivable therein. [0092] In specific embodiments, the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular embodiments the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing. [0093] In particular embodiments, the cells of the disclosure comprise an exogenous TCR, which may be of a defined antigen specificity. In some embodiments, the TCR can be selected based on absent or reduced alloreactivity to the intended recipient. In the example where the exogenous TCR is non-alloreactive, during T cell differentiation the exogenous TCR suppresses rearrangement and/or expression of endogenous TCR loci through a developmental process called allelic exclusion, resulting in T cells that express only the non-alloreactive exogenous TCR and are thus non-alloreactive. In some embodiments, the choice of exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some embodiments, the endogenous TCR genes have been modified by genome editing so that they do not express a protein. Methods of gene editing such as methods using the CRISPR/Cas9 system are known in the art and described herein.

[0094] In some embodiments, the cells of the disclosure comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which a CAR or TCR may be directed include at least CD40, CD40L, KRAS, 5T4, 8H9, avP6 integrin, BCMA, B7-H3, B7- H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, Folate receptor, GD2, G250/CAIX, GD3, Glypican-3 (GPC3), Her2, HEA-G), IE-13Roc2, Eambda, Eewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEMs, TROP2, carcinoembryonic antigen, HMW- MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO- 1/LAGE-l, PAME, SSX-2, Melan-A/MART-1, GP100/pmell7, TRP-1/-2, P. polypeptide, MC1R, Prostate-specific antigen, P-catenin, BRCA1/2, CML66, Fibronectin, MART-2, TGF- PRII, or VEGF receptors (e.g., VEGFR2), for example. The CAR may be a first, second, third, or more generation CAR. The CAR may be bispecific for any two nonidentical antigens, or it may be specific for more than two nonidentical antigens.

A. Chimeric Antigen Receptors

[0095] In certain embodiments, the present NK cells are genetically modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.

[0096] A CAR recognizes cell-surface tumor-associated antigen independent of human leukocyte antigen (HLA) and employs one or more signaling molecules to activate genetically modified NK cells for killing, proliferation, and cytokine production (Jena et al., 2010). In certain embodiments, the platform technologies disclosed herein to genetically modify NK cells comprise (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28/CD3-C,, CD137/CD3-C,, or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the antigen-recognition domain to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPC) derived from K562 to be able to robustly and numerically expand CAR⁺ NK cells (Singh et al., 2008; Singh et al., 2011).

[0097] Embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding an antigen- specific chimeric antigen receptor (CAR) polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide e.g., cytokine) that binds to a receptor.

[0098] It is contemplated that the human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure provides a full-length CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen- specific antibody. In a more specific embodiment, the fragment is an antigen- specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.

[0099] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and/or dimerizes can serve this purpose. One of the Fc domains, e.g. , either the CH2 or CH3 domain from human immunoglobulin may be used. The hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization may be used. In other aspects, just the hinge portion of an immunoglobulin or portions of CD8a may be used.

[0100] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to a primary signal initiated by CD3^, an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for full activation of NK cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy.

[0101] The intracellular signaling domain of a chimeric antigen receptor is responsible for activation of at least one of the normal effector functions of the immune cell in which the chimeric antigen receptor has been placed. The term “effector function” refers to a specialized function of a differentiated cell, such as a NK cell. In specific embodiments, intracellular receptor signaling domains in the CAR include those of the T-cell antigen receptor complex, such as the zeta chain of CD3, also Fc y RIII costimulatory signaling domains, CD28, CD27, DAP10, CD137, 0X40, CD2, alone or in a series with CD3zeta, for example. In specific embodiments, the intracellular domain (which may be referred to as the cytoplasmic domain) comprises part or all of one or more of TCR zeta chain, CD28, CD27, OX40/CD134, 4- 1BB/CD137, Fc e RI y , ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and CD40. In some embodiments, one employs any part of the endogenous T-cell receptor complex in the intracellular domain. One or multiple cytoplasmic domains may be employed, as so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effect, for example.

[0102] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a tumor associated antigen or a pathogen-specific antigen binding domain. Antigens include carbohydrate antigens recognized by pattern-recognition receptors, such as Dectin- 1. A tumor associated antigen may be of any kind so long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor associated antigens include CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c- Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, and mutated ras. Additional exemplary antigens include CD99, CLL-1, CD47, CD33, CS1, and BCMA.

[0103] In certain embodiments, the CAR may be co-expressed with a cytokine to improve persistence when there is a low amount of tumor-associated antigen. For example, CAR may be co-expressed with IL- 15 and/or IL-21.

[0104] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0105] It is contemplated that the chimeric construct can be introduced into NK cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0106] Alternatively, a viral vector e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into NK cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the NK cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0107] The CAR encoding construct may also comprise a sequence encoding a suicide gene, such as CD20, CD52, EGFRv3, or inducible caspase 9. An engineered NK cell provided herein may comprise a heterologous sequence encoding a suicide gene, such as CD20, CD52, EGFRv3, or inducible caspase 9.

[0108] The CAR may comprise a tumor antigen-binding domain. Any suitable antigen may be targeted in the present method. The antigen may be associated with certain cancer cells but not associated with non-cancerous cells, in some cases. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto-/self-antigens, tumor-/cancer- associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular aspects, the antigens include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL/DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL- HRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, -catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPVmbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor- associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor- Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP- 4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRN1. 1. Signal peptide

[0109] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface. In some embodiments, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0110] In some embodiments, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In some embodiments, a restriction site is at the carboxy end of the signal peptide to facilitate cleavage.

2. Antigen binding domain

[0111] Polypeptides of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). In some embodiments, an antigen binding domain comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. In some embodiments, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0112] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0113] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0114] It is also contemplated that the antigen binding domain may be multi- specific or multivalent by multimerizing the antigen binding domain with VH and VL region pairs that bind either the same antigen (multi- valent) or a different antigen (multi- specific).

[0115] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and/or light chain variable region), or of the CDRs may be at least 10'⁵M, 10'⁶M, 10'⁷M, 10'⁸M, 10'⁹M, 10'¹⁰M, 10 ¹M, 10-¹²M, or 10'¹³M. In some embodiments, the KD of the antigen binding region, such as the variable regions (heavy chain and/or light chain variable region), or of the CDRs may be at least 10'⁵M, 10'⁶M, 10'⁷M, 10'⁸M, 10'⁹M, 10'¹⁰M, 10'ⁿM, 10'¹²M, or 1O'¹³M (or any derivable range therein).

[0116] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0117] In some embodiments, the polypeptide comprising the humanized binding region has equal, better, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% binding affinity and/or expression level in host cells, compared to a polypeptide comprising a non-humanized binding region, such as a binding region from a mouse.

[0118] In some embodiments, the framework regions, such as FR1, FR2, FR3, and/or FR4 of a human framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,

125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,

144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,

163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a mouse framework.

[0119] In some embodiments, the framework regions, such as FR1, FR2, FR3, and/or FR4 of a mouse framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,

125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,

144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,

163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,

182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a human framework.

[0120] The substitution may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 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 100 of FR1, FR2, FR3, or FR4 of a heavy or light chain variable region.

3. Peptide spacer

[0121] A peptide spacer, such as an extracellular spacer may link an antigen-binding domain to a transmembrane domain. In some embodiments, a peptide spacer is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In one embodiment, the spacer comprises the hinge region from IgG. In some embodiments, the spacer comprises or further comprises the CH2CH3 region of immunoglobulin and portions of CD3. In some embodiments, the CH2CH3 region may have L235E/N297Q or L235D/N297Q modifications, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. In some embodiments, the spacer is from IgG4. An extracellular spacer may comprise a hinge region.

[0122] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0123] The extracellular spacer can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140,

150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,

237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300,

325, 350, or 400 amino acids (or any derivable range therein). In some embodiments, the extracellular spacer consists of or comprises a hinge region from an immunoglobulin e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0124] The length of an extracellular spacer may have effects on the signaling activity of the CAR and/or the expansion properties of the CAR T cells in response to antigen-stimulated CAR signaling. In some embodiments, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some embodiments, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro. [0125] When the extracellular spacer comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some embodiments, the extracellular spacer consists essentially of a hinge, CH2, and/or CH3 region, meaning that the hinge, CH2, and/or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.

4. Transmembrane domain

[0126] Polypeptides of the present disclosure may comprise a transmembrane domain. In some embodiments, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0127] In some embodiments, the transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is interposed between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0128] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta, CD 134, or CD7.

5. Cytoplasmic region

[0129] After antigen recognition, receptors of the present disclosure may cluster and a signal transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domains described herein are part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains.

[0130] Cytoplasmic regions and/or costimulatory regions suitable for use in the polypeptides of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen binding domain. In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes DAP10/CD28 type signaling chains.

[0131] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L/I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L/I))(X3)n(YXlX2(L/I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0132] A suitable cytoplasmic region may be an TTAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP 12, DAP 10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0133] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that may be incorporated in a CAR of the disclosure:

[0134] In some embodiments, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length DAP12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length FCERI G amino acid sequence. [0135] In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3gamma; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3 > ; T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3-epsilon, T3e, etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3y, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3^, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 zeta amino acid sequence.

[0136] In some embodiments, the cytoplasmic region is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membranebound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD79A amino acid sequence.

6. Costimulatory region

[0137] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD 137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, HVEM, DAP10, and/or DAP12.

[0138] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3- 395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

7. Detection peptides

[0139] In some embodiments, the polypeptides described herein may further comprise a detection peptide. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:1); FLAG (e.g., DYKDDDDK (SEQ ID NO:2); c-myc (e.g., EQKLISEEDL; SEQ ID NOG), and the like. Other suitable detection peptides are known in the art.

8. Peptide linkers

[0140] In some embodiments, the polypeptides of the disclosure include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain/regions described herein. As an example, a linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, flanking the costimulatory region or on the N- or C- region of the costimulatory region, and/or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0141] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0142] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0143] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0144] Example flexible linkers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:4), (G4S)n and (GGGS)n (SEQ ID NO:5), where n is an integer of at least one. In some embodiments, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine- serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:6), GGSGG (SEQ ID NO:7), GSGSG (SEQ ID NO:8), GSGGG (SEQ ID NO:9), GGGSG (SEQ ID NO: 10), or GSSSG (SEQ ID NO: 11).

VI. Antigen Presenting Cells [0145] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular major histocompatibility complex (MHC) molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The MHC is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans, the MHC is referred to as the HLA complex and in mice the H-2 complex.

[0146] In some cases, aAPCs are useful in preparing therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. W02007/103009.

[0147] In certain embodiments, methods of the disclosure comprise expansion of NK cells in the presence of aAPCs for at least a first culture condition. In certain embodiments, methods of the disclosure comprise expansion of NK cells in the presence of aAPCs for a first culture condition and a second culture condition. In some embodiments, expansion of NK cells with a culture condition that does not comprise aAPCs is for less than about 4 or 5 days. In certain embodiments, if a culture condition will last for longer than 4 or 5 days, aAPCs are utilized to promote NK cell viability.

[0148] aAPC systems may comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co- stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcyRI), 41BB ligand (CD137 ligand), and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell- to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001. [0149] In particular embodiments, the aAPCs have been engineered to express CD 137 ligand, such as by retroviral vectors. The aAPCs may further express membrane-bound cytokines, such as membrane -bound IL-21 (mIL-21) or membrane-bound IL- 15 (mIL-15). In particular aspects, the aAPCs express CD137 ligand and mIL-21. The aAPCs may be K562 leukemia cells engineered to express CD137 and mIL-21. The aAPCs may be developed to express a desired antigen, such as CD 19. As needed, additional stimulation cycles can be undertaken to generate larger numbers of NK cells.

VII. Gene Editing

[0150] In specific embodiments, the NK cells are modified by editing of one or more of their endogenous genes. In specific embodiments, the editing encompasses disruption of one or more endogenous genes in the cells, such as 1, 2, 3, 4, or more endogenous genes. In specific embodiments, the gene editing may occur by any mechanism. The disruption may encompass partial or complete reduction in expression of the endogenous gene(s).

[0151] In particular embodiments, one or more of the following genes are modified in the NK cells, such as by knockdown or knockout: NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1 (GR), PD1, PDL- 1, PDL-2, CD47, SIRPA, SHIP1, ADAM 17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and/or CD7.

[0152] In specific embodiments, an engineered mutation in an endogenous gene can be a mutation in GR, TGFBR2, CISH, and/or CD38. In specific embodiments, an NK cell is engineered to comprise a mutation in the endogenous genes GR and TGFBR2.

[0153] In certain embodiments, an engineered mutation is in an endogenous gene mutation as described in international PCT patent application publication WO 2020/113029 A2, published June 4, 2020; international PCT patent application publication WO 2021/146719 Al, published July 22, 2021; international PCT patent application publication WO 2021/108671 Al, published June 3, 2021; and/or international PCT patent application publication WO 2023/245041 A2, published December 21, 2023; each of which are hereby incorporated in their entirety for any purpose described herein.

VIII. Administration of Therapeutic Compositions

[0154] Any therapy provided herein may comprise administration of engineered immune effector cells and one or more epigenetic modulators. The bipartite therapy may be administered in any suitable manner known in the art. For example, the cells and the modulators may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the cells and the modulators are administered in a separate composition. In some embodiments, the cells and the modulators are in the same composition.

[0155] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed. In some cases, the cells and the modulators are not given the same number of times to the individual. For example, the individual may receive more doses of cells than doses of modulators, although the individual may receive more disease of modulators than doses of cells. In specific embodiments, the individual receives the same number of doses of the cells and of the modulators. In some cases [0156] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0157] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0158] In some embodiments, the therapy comprises a population of cells and one or more small inhibitors and/or proteins. In some embodiments, a single dose of the cells and the epigenetic modulator(s) is administered. In some embodiments, multiple doses of the cells and the epigenetic modulator(s) are administered. In some embodiments, the modulator is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some embodiments, the modulator is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108,

109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,

185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,

204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,

223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,

242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,

261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,

280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,

299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,

318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,

337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,

356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,

375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,

394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,

413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,

432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,

451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,

470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,

489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,

508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526,

527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,

546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564,

565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg/kg. [0159] In some embodiments, a single dose of the therapy is administered. In some embodiments, multiple doses of the therapy are administered. In some embodiments, the therapy is administered at a dose of between 1 mg/kg and 100 mg/kg. In some embodiments, the therapy is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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 100 mg/kg.

[0160] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[0161] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0162] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0163] It will be understood by those skilled in the art and made aware that dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0164] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 day intervals, including all ranges there between. In some embodiments, any part of the therapy of the disclosure includes (such as is preceded by) lymphodepleting chemotherapy of any kind. In some embodiments, the lymphodepleting therapy includes Fludarabine and/or Cyclophosphamide. In some embodiments, Fludarabine at a dose of 30 mg/m² is provided intravenously. In some embodiments, Cyclophosphamide is given intravenously at a dose of 300 mg/m².

[0165] In a specific embodiment, engineered immune effector cells are provided at Day 0. The next 1-4 days of the regimen, the individual may receive no therapy, such as Day 1 and Day 2. Following this, the individual may receive on consecutive days 1-5 administrations of lymphodepleting chemotherapy, such as on Day 3, Day 4, and Day 5. In some embodiments, there is overlap of administration of lymphodepleting chemotherapy and the epigenetic modulator(s), although in some embodiments there is no overlap. When there is overlap of administration of lymphodepleting chemotherapy and the epigenetic modulator(s), the overlap may be for 1, 2, 3, 4, or more days. In some embodiments, on Day 4 and Day 5 there is overlap of lymphodepleting chemotherapy and the epigenetic modulator(s). Following overlap, the epigenetic modulator(s) may continue to be administered, such as for 1, 2, 3, 4, 5, or more days. [0166] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0167] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0168] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0169] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0170] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum mono stearate and gelatin.

[0171] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0172] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0173] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0174] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cancer treatments are administered in a separate composition. In some embodiments, the first and second cancer treatments are in the same composition. In some embodiments, an individual receiving and/or who has received and/or who will receive the combinatorial engineered immune effector cells/epigenetic modulators are also given an additional therapy. The additional therapy may be given at any time in comparison to the combinatorial engineered immune effector cells/epigenetic modulators, including before, during, and/or after. The additional therapy may comprise surgery, radiation, immunotherapy, chemotherapy, hormone therapy, or a combination thereof.

IX. Kits

[0175] Certain aspects of the present disclosure also concern kits comprising compositions of the disclosure and/or compositions to implement methods of the invention. In particular embodiments, the kit comprises immune effector cells, fresh or frozen, and that may or may not have been engineered. The immune effector cells may or may not have been pre-activated or expanded. The immune effector cells may or may not already express one or more heterologous proteins. In cases wherein the immune effector cells do not already express one or more heterologous proteins, the kit may comprise reagents for corresponding transfection or transduction of the immune effector cells, including reagents such as vectors that express the component(s), primers for amplification of the component(s), and so forth. In some cases, the kit may comprise vectors that express heterologous protein(s), primers for amplification of the heterologous protein(s), and so forth.

[0176] In certain embodiments, the kit comprises one or more epigenetic modulators, including one or more of (a) one or more hypomethylating agents; (b) one or more histone deacetylase (HD AC) inhibitors; (c) one or more enhancer of zeste 2 (EZH2) inhibitors; (d) one or more meningitis inhibitors; (e) one or more lysine-specific histone demethylase 1A (LSD1) inhibitors; and (f) one or more bromodomain and extra-terminal domain (BET) inhibitors.. The one or more epigenetic modulators may or may not be provided in the kit in the same formulation as the cells. In some embodiments, the cells are in a separate container as the epigenetic modulator(s). In some embodiments, the cells are in the same container as the epigenetic modulator(s). Pharmaceutically acceptable carriers may or may not be provided in the kit.

[0177] Kits may comprise components which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

X. Examples [0178] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the subject matter of the disclosure.

EXAMPLE 1

UTILIZATION OF EPIGENETIC REAGENTS IN COMBINATION WITH CAR NK CELLS

[0179] Induction of expression of a target antigen in the presence of hypomethylating reagents was examined.

[0180] As shown in FIG. 1, hypomethylating reagents dose-dependently induce CD70 expression. CD70 mean fluorescence intensity (MFI) expression in 3 AML cell lines (M0LM13, M0LM14 and MV4-11) was measured before and after treatment with the hypomethylating agents (HMA) 5-Azacytidine and Decitabine for 24hrs. Left panel of FIG. IE shows representative CD70 expression histograms, and the right panel shows combined CD70 expression analysis data from all the AML cell lines. Moreover, to further investigate the association between HMA therapy and CD70 expression, 3 naive primary AML blasts and 3 AML cell lines (M0LM13, M0LM14 and MV4-11) were treated with 5-azacytidine (AZA) and Decitabine (DEC) in-vitro for 24 hrs and there was a significant increase in CD70 expression, FIGS. IE, IF).

[0181] In FIG. 2, it is shown that the hypomethylating reagent (Decitabine) and HDAC inhibitor (Vorinostat) induce CD70 expression. Western blot analysis of the CD70 protein level in AML line(M0LM14) 2 days after Decitabine and Vorinostat treatment is shown. DMSO was used as a negative control. Vinculin was used as protein loading control.

[0182] A hypomethylating reagent (Decitabine) and an HDAC inhibitor (Vorinostat) dose- dependently induce HLA-G expression (FIG. 3). Western blot analysis of the HLA-G protein level in an acute myeloid leukemia (AML) line (M0LM14) 2 days after Decitabine/5-AZA (left) and Vorinostat (right) treatment. DMSO was used as a negative control. Actin was used as protein loading control. DNMT-1 is DNA-methyltransferase 1, an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, a process called DNA methylation. 5AZA and Decitabine target and inhibit DNMT-1 and cause DNA hypomethylation. Acetyl histone H3 is a DNA packaging protein that has been acetylated at several lysine positions in the histone tail. Acetylation of histones is often associated with transcriptional activation. Inhibition of histone deacetylases (HDACs) via an HD AC inhibitor increases Acetyl histone H3 as well as the transcription process.

[0183] FIG. 4 also shows induction of HLA-G expression by a hypomethylating reagent and an HD AC inhibitor dose-dependently. Decitabine and Vorinostat dose-dependently induce HLA-G expression. Flow cytometry analysis of the HLA-G protein level in the AML line MOLM-14 2 days after Decitabine/5-AZA (upper) and Vorinostat (bottom) treatment. DMSO was used as a negative control. Western blot analysis of the HLA-G protein level in AML line (THP-1) 2 days after Decitabine/5-AZA(left) and Vorinostat (right) treatment is shown in FIG. 5. DMSO was used as a negative control. Actin was used as protein loading control. FIG. 6 shows induction of HLA-G protein level in the AML line (THP-1) 2 days after Decitabine/5- AZA (upper) and Vorinostat (bottom) treatment. DMSO was used as a negative control.

[0184] In FIG. 7, a hypomethylating reagent (Decitabine) and an HD AC inhibitor (Vorinostat) induce Neo peptide expression. Western blot analysis of the NY-ESO1 protein level in the AML line (M0LM14) 2 days after Decitabine and Vorinostat treatment is provided. DMSO was used as a negative control, and Vinculin was used as protein loading control.

[0185] Decitabine enhances the cytotoxicity of HLA-G CAR NK cells (as an example) against the M0LM14 cell line. Incucyte-based live imaging analysis shows the application of decitabine therapy prior to NK cell challenge results in an increased susceptibility of THP- 1 cell lines to killing by HLA-G CAR-NK cells (FIG. 8).

[0186] FIG. 9 shows that pretreatment with Enasidenib or Decitabine enhances NK cell cytotoxicity against UMRC3 (RCC) and BCX010 (TNBC) Cell Lines. UMRC3 and BCX010 cells were treated with Decitabine (2.5 pM) or Enasidenib (1 pM) for 24 hours, followed by NK cell treatment of the spheroids. Shown are representative images of 3D tumor spheroids treated with NK cells, either with or without prior treatment with Decitabine or Enasidenib. IncuCyte-based live imaging analysis demonstrates that pretreating the spheroids with Decitabine or Enasidenib, as examples of epigenetic modulators, before NK cell challenge increases the susceptibility of both NT and CAR NK cells to these tumor cells, as examples of cancer cells.

[0187] FIG. 10 demonstrates that pretreatment with Enasidenib or Decitabine enhances NK cell cytotoxicity against UMRC3 (RCC) and BCX010 (TNBC) cell lines. UMRC3 and BCX010 cells were treated with Decitabine (2.5 |aM), Enasidenib (1 |aM), or the EZH2 inhibitor Tazemostat (luM) for 24 hours, followed by NK cell treatment. Representative realtime impedance data from the xCELLigence system treated with NK cells, either with or without prior treatment with Decitabine, Tazemostat or Enasidenib, are shown. Data analysis demonstrates that pretreating the cells with Decitabine, Tazemostat, or Enasidenib before NK cell challenge increases the susceptibility of both NT and CAR NK cells to these tumor cells. [0188] Pretreatment with Enasidenib or Decitabine enhances NK cell cytotoxicity against MDAMB231(TNBC) Cell Lines. MDSMB231 cells were treated with Decitabine (2.5 pM for 24 hours), followed by NK cell treatment. Representative real-time impedance data from the xCELLigence system treated with NK cells, either with or without prior treatment with Decitabine, are shown. Data analysis demonstrates that pretreating the cells with Decitabine before NK cell challenge increases the susceptibility of both NT and CAR NK cells to these tumor cells.

I. Aspects

[0189] Aspect 1. A regimen comprising (1) engineered immune effector cells, nonengineered immune effector cells, or a mixture thereof; and (2) one or more epigenetic modulators, wherein the epigenetic modulators comprise one or more of the following: [0190] (a) one or more hypomethylating agents;

[0191] (b) one or more histone deacetylase (HD AC) inhibitors;

[0192] (c) one or more enhancer of zeste 2 (EZH2) inhibitors;

[0193] (d) one or more meningitis inhibitors;

[0194] (e) one or more lysine-specific histone demethylase 1A (LSD1) inhibitors;

[0195] (f) one or more bromodomain and extra-terminal domain (BET) inhibitors; and

[0196] (g) one or more IDH1 and/or IDH2 inhibitors.

[0197] Aspect 2. The regimen of aspect 1, wherein the hypomethylating agent is one or more DNA methyltransferase (DNMT) inhibitors.

[0198] Aspect 3. The regimen of claim 2, wherein the DNMT is DNMT1, DNMT2, DNMT3A, and/or DNMT3B.

[0199] Aspect 4. The regimen of aspect 2 or 3, wherein the DNMT inhibitor is selected from the group consisting of 5-azacytidine, cytidine, decitabine, 5-aza-2-deoxycytidine, deoxyguanosine, guadecitabine, zebularine, clofarabine, arsenic trioxide curcumin, resveratrol, epigallocatechin-3-gallate, and a combination thereof.

[0200] Aspect 5. The regimen of any one of aspects 1-4, wherein the HD AC inhibitor inhibits a Class I, Class IIA, Class III, or Class IV HD AC.

[0201] Aspect 6. The regimen of any one of aspects 1-5, wherein the HDAC inhibitor is a hydroxamatecyclic tetrapeptide, depsipeptide, benzamide, electrophilic ketone, aliphatic acid compound, or a mixture or combination thereof.

[0202] Aspect 7. The regimen of any one of aspects 1-6, wherein the HDAC inhibitor is selected from the group consisting of trichostatin A, trapoxin B, phenylbutyrate, valproic acid, vorinostat, romidepsin, belinostat, givinostat, panobinostat, tucidinostat, entinostat, mocentinostat, TYO-018, sanacruzamate A, scriptaid, abexinostat, and a combination thereof. [0203] Aspect 8. The regimen of any one of aspects 1-7, wherein the EZH2 inhibitor is selected from the group consisting of tazemotostat, GSK126, CPI- 1205, PF-06821497, SHR2554, UNC1999, valemotostat tosylate, MAK683, and a combination thereof.

[0204] Aspect 9. The regimen of any one of aspects 1-8, wherein the meningitis inhibitor is selected from the group consisting of vancomycin, but teicoplanin, linezolid, fucidic acid, daptomycin, and a combination thereof.

[0205] Aspect 10. The regimen of any one of aspects 1-9, wherein the LSD1 inhibitor is selected from the group consisting of ladademstat, pulrodemstat benzenesulfonate, tranylcypromine, pargyline, GSK-LSD1, and a combination thereof.

[0206] Aspect 11. The regimen of any one of aspects 1-10, wherein the BET inhibitor is selected from the group consisting of I-BET 151 (GSK1210151A), I-BET 762 (GSK525762), OTX-015, TEN-010, CPI-203, CPI-0610, olinone, RVX-208, ABBV-744, LY294002, AZD5153, MT-1,MS645, pelabresib, and a combination thereof.

[0207] Aspect 12. The regimen of any one of aspects 1-11, wherein the IDH1 and/or IDH2 inhibitor is selected from the group consisting of enasidenib, ivosidenib, vorisidenib, NI-1, BAY1436032, Compound 6, and a combination thereof.

[0208] Aspect 13. The regimen of any one of aspects 1-12, wherein the immune effector cells and one or more epigenetic modulators are in the same formulation.

[0209] Aspect 14. The regimen of any one of aspects 1-12, wherein the immune effector cells and one or more epigenetic modulators are not in the same formulation.

[0210] Aspect 15. The regimen of any one of aspects 1-14, wherein the immune effector cells express one or more heterologous proteins. [0211] Aspect 16. The regimen of aspect 15, wherein the heterologous protein is a therapeutic gene product and/or directly or indirectly enhances the therapeutic efficacy of the immune effector cells.

[0212] Aspect 17. The regimen of aspect 15, wherein the therapeutic gene product comprises an engineered receptor, an antibody, a cytokine, or the cells express 1, 2, or all 3 of them.

[0213] Aspect 18. The regimen of aspect 17, wherein the engineered receptor is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor.

[0214] Aspect 19. The regimen of aspect 17 or 18, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-10, IL-4, IGNg, IFNa, IL-33, IL- 27, GMCSF, and a combination thereof.

[0215] Aspect 20. A method of treating a medical condition, comprising the step of administering to an individual with the medical condition a therapeutically effective amount of the regimen of any one of aspects 1-19.

[0216] Aspect 21. The method of aspect 20, wherein the individual has cancer, an autoimmune disorder, an infectious disease, or a combination thereof.

[0217] Aspect 22. The method of aspect 20 or 21, wherein the one or more epigenetic modulators are administered to the individual prior to administering the engineered NK cells to the individual.

[0218] Aspect 23. The method of aspect 20 or 21, wherein the one or more epigenetic modulators are administered to the individual subsequent to administering the engineered NK cells to the individual.

[0219] Aspect 24. The method of aspect 20 or 21, wherein the one or more epigenetic modulators are administered to the individual at substantially the same time as administering the engineered NK cells to the individual.

[0220] Aspect 25. The method of aspect 24, wherein the one or more epigenetic modulators and the engineered NK cells are in the same formulation.

[0221] Aspect 26. The method of aspect 24 or 25, wherein the one or more epigenetic modulators and the engineered NK cells are not in the same formulation.

[0222] Aspect 27. The method of any one of aspects 20-26, wherein the one or more epigenetic modulators comprise one or more HD AC inhibitors, one or more DNMT inhibitors, or a mixture thereof.

[0223] Aspect 28. The method of any one of aspects 20-27, wherein the engineered NK cells express one or more heterologous proteins. [0224] Aspect 29. The method of aspect 28, wherein the heterologous protein is an engineered receptor that is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor.

[0225] Aspect 30. A method of increasing efficacy of non-engineered NK cells or antigentargeting NK cells as adoptive cell therapy for an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a regimen comprising engineered NK cells and one or more epigenetic modulators.

[0226] Aspect 31. The method of aspect 30, wherein the individual has cancer and/or an autoimmune disorder and/or an infectious disease.

[0227] Aspect 32. The method of aspect 30 or 31, wherein the one or more epigenetic modulators are administered to the individual prior to administering the engineered NK cells to the individual.

[0228] Aspect 33. The method of aspect 30 or 31, wherein the one or more epigenetic modulators are administered to the individual subsequent to administering the engineered NK cells to the individual.

[0229] Aspect 34. The method of aspect 30 or 31, wherein the one or more epigenetic modulators are administered to the individual at substantially the same time as administering the engineered NK cells to the individual.

[0230] Aspect 35. The method of aspect 34, wherein the one or more epigenetic modulators and the engineered NK cells are in the same formulation.

[0231] Aspect 36. The method of aspect 34 or 35, wherein the one or more epigenetic modulators and the engineered NK cells are not in the same formulation.

[0232] Aspect 37. A kit comprising the regimen of any one of aspects 1-19.

* * *

[0233] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A regimen comprising (1) engineered immune effector cells, non-engineered immune effector cells, or a mixture thereof; and (2) one or more epigenetic modulators, wherein the epigenetic modulators comprise one or more of the following:

(a) one or more hypomethylating agents;

(b) one or more histone deacetylase (HD AC) inhibitors;

(c) one or more enhancer of zeste 2 (EZH2) inhibitors;

(d) one or more meningitis inhibitors;

(e) one or more lysine-specific histone demethylase 1A (LSD1) inhibitors;

(f) one or more bromodomain and extra-terminal domain (BET) inhibitors; and

(g) one or more IDH1 and/or IDH2 inhibitors.

2. The regimen of claim 1, wherein the hypomethylating agent is one or more DNA methyltransferase (DNMT) inhibitors.

3. The regimen of claim 2, wherein the DNMT is DNMT1, DNMT2, DNMT3A, and/or DNMT3B.

4. The regimen of claim 2 or 3, wherein the DNMT inhibitor is selected from the group consisting of 5-azacytidine, cytidine, decitabine, 5-aza-2-deoxycytidine, deoxyguanosine, guadecitabine, zebularine, clofarabine, arsenic trioxide curcumin, resveratrol, epigallocatechin-3-gallate, and a combination thereof.

5. The regimen of any one of claims 1-4, wherein the HDAC inhibitor inhibits a Class I, Class IIA, Class III, or Class IV HDAC.

6. The regimen of any one of claims 1-5, wherein the HDAC inhibitor is a hydroxamatecyclic tetrapeptide, depsipeptide, benzamide, electrophilic ketone, aliphatic acid compound, or a mixture or combination thereof.

7. The regimen of any one of claims 1-6, wherein the HDAC inhibitor is selected from the group consisting of trichostatin A, trapoxin B, phenylbutyrate, valproic acid, vorinostat, romidepsin, belinostat, givinostat, panobinostat, tucidinostat, entinostat, mocentinostat, TYO- 018, sanacruzamate A, scriptaid, abexinostat, and a combination thereof.

8. The regimen of any one of claims 1-7, wherein the EZH2 inhibitor is selected from the group consisting of tazemotostat, GSK126, CPI-1205, PF-06821497, SHR2554, UNC1999, valemotostat tosylate, MAK683, and a combination thereof.

9. The regimen of any one of claims 1-8, wherein the meningitis inhibitor is selected from the group consisting of vancomycin, but teicoplanin, linezolid, fucidic acid, daptomycin, and a combination thereof.

10. The regimen of any one of claims 1-9, wherein the LSD1 inhibitor is selected from the group consisting of ladademstat, pulrodemstat benzenesulfonate, tranylcypromine, pargyline, GSK-LSD1, and a combination thereof.

11. The regimen of any one of claims 1-10, wherein the BET inhibitor is selected from the group consisting of I-BET 151 (GSK1210151A), I-BET 762 (GSK525762), OTX-015, TEN- 010, CPI-203, CPI-0610, olinone, RVX-208, ABBV-744, LY294002, AZD5153, MT- 1,MS645, pelabresib, and a combination thereof.

12. The regimen of any one of claims 1-11, wherein the IDH1 and/or IDH2 inhibitor is selected from the group consisting of enasidenib, ivosidenib, vorisidenib, NI-1, BAY1436032, Compound 6, and a combination thereof.

13. The regimen of any one of claims 1-12, wherein the immune effector cells and one or more epigenetic modulators are in the same formulation.

14. The regimen of any one of claims 1-12, wherein the immune effector cells and one or more epigenetic modulators are not in the same formulation.

15. The regimen of any one of claims 1-14, wherein the immune effector cells express one or more heterologous proteins.

16. The regimen of claim 15, wherein the heterologous protein is a therapeutic gene product and/or directly or indirectly enhances the therapeutic efficacy of the immune effector cells.

17. The regimen of claim 15, wherein the therapeutic gene product comprises an engineered receptor, an antibody, a cytokine, or the cells express 1, 2, or all 3 of them.

18. The regimen of claim 17, wherein the engineered receptor is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor.

19. The regimen of claim 17 or 18, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-10, IL-4, IGNy, IFNoc, IL-33, IL-27, GMCSF, and a combination thereof.

20. A method of treating a medical condition, comprising the step of administering to an individual with the medical condition a therapeutically effective amount of the regimen of any one of claims 1-19.

21. The method of claim 20, wherein the individual has cancer, an autoimmune disorder, an infectious disease, or a combination thereof.

22. The method of claim 20 or 21, wherein the one or more epigenetic modulators are administered to the individual prior to administering the engineered NK cells to the individual.

23. The method of claim 20 or 21, wherein the one or more epigenetic modulators are administered to the individual subsequent to administering the engineered NK cells to the individual.

24. The method of claim 20 or 21, wherein the one or more epigenetic modulators are administered to the individual at substantially the same time as administering the engineered NK cells to the individual.

25. The method of claim 24, wherein the one or more epigenetic modulators and the engineered NK cells are in the same formulation.

26. The method of claim 24 or 25, wherein the one or more epigenetic modulators and the engineered NK cells are not in the same formulation.

27. The method of any one of claims 20-26, wherein the one or more epigenetic modulators comprise one or more HD AC inhibitors, one or more DNMT inhibitors, or a mixture thereof.

28. The method of any one of claims 20-27, wherein the engineered NK cells express one or more heterologous proteins.

29. The method of claim 28, wherein the heterologous protein is an engineered receptor that is a chimeric antigen receptor, a T cell receptor, a chimeric cytokine receptor, or a chemokine receptor.

30. A method of increasing efficacy of non-engineered NK cells or antigen-targeting NK cells as adoptive cell therapy for an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a regimen comprising engineered NK cells and one or more epigenetic modulators.

31. The method of claim 30, wherein the individual has cancer and/or an autoimmune disorder and/or an infectious disease.

32. The method of claim 30 or 31, wherein the one or more epigenetic modulators are administered to the individual prior to administering the engineered NK cells to the individual.

33. The method of claim 30 or 31, wherein the one or more epigenetic modulators are administered to the individual subsequent to administering the engineered NK cells to the individual.

34. The method of claim 30 or 31, wherein the one or more epigenetic modulators are administered to the individual at substantially the same time as administering the engineered NK cells to the individual.

35. The method of claim 34, wherein the one or more epigenetic modulators and the engineered NK cells are in the same formulation.

36. The method of claim 34 or 35, wherein the one or more epigenetic modulators and the engineered NK cells are not in the same formulation.

37. A kit comprising the regimen of any one of claims 1-19.