Enhanced Anti-cancer t-cell therapy

Abstract

Provided herein are methods, compositions and kits to enhance anti-cancer T-cell therapy. In particular, provided herein are reagents and methodologies to promote anti-cancer T-cell therapy comprising inhibition of retinoic acid receptor alpha (RARα) in chimeric antigen receptor (CAR) T cells.

Description

[0001] ENHANCED ANTI-CANCER T-CELL THERAPY

[0002] STATEMENT REGARDING FEDERAL FUNDING

[0003] This invention was made with government support under AI148898 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD

[0005] Provided herein are methods, compositions and kits to enhance anti-cancer T-cell therapy. In particular, provided herein are reagents and methodologies to promote anti-cancer T- cell therapy comprising inhibition of retinoic acid receptor alpha (RARoc) in chimeric antigen receptor (CAR) T cells.

[0006] BACKGROUND

[0007] Chimeric antigen receptors (CARs) are genetically engineered receptors that provide specific properties to an immune effector cell (e.g., a lymphocyte). CAR receptors gain the specificity of a monoclonal antibody targeted against specific tumor cells. The term “chimeric” indicates different sources of composing parts of the receptor. Lymphocytes with engineered CARs acquire potent immunological properties by redirecting the immune system to eliminate malignant cells. They act as a living drug, expanding in the patient and ensuring long-term antitumor memory. CAR technology includes use of CAR-T cells in hematological malignancies and solid tumors that may undergo rapid progression, poor prognosis and high mortality.

[0008] SUMMARY

[0009] Provided herein are methods, compositions and kits to enhance anti-cancer T-cell therapy. In particular, provided herein are reagents and methodologies to promote anti-cancer T- cell therapy comprising inhibition of retinoic acid receptor alpha (RARa) in chimeric antigen receptor (CAR) T cells.

[0010] In some embodiments, the present invention provides a method of treating a subject having a cancer, comprising: providing a subject having a cancer; collecting, leukophoresising and purifying T cell lymphocytes from the subject; genetically modifying the purified T cell lymphocytes to express a nucleic acid molecule encoding a chimeric antigen receptor (CAR) specific for the cancer to generate CAR T cell lymphocytes; genetically modifying the CAR T cell lymphocytes to generate CAR T cells with reduced retinoic acid receptor alpha (RARoc) expression; and administering a therapeutically effective amount of the CAR T cells with reduced RARoc expression to the subject having the cancer.

[0011] In some embodiments, the present invention provides a method of stimulating a cell- mediated immune response to a cancer in a subject, comprising administering to a subject having a cancer a therapeutically effective dose of CAR T cells specific for the cancer with reduced RARoc expression.

[0012] In some embodiments, the present invention provides a composition, comprising a T cell comprising one or more chimeric antigen receptors (CARs) and reduced RARoc expression and / or activity.

[0013] In some embodiments, the present invention provides a pharmaceutical composition, comprising an effective amount of CAR T cells specific for a cancer with reduced RARoc expression and / or activity and a pharmaceutically acceptable carrier. In some embodiments, the composition is in dosage form.

[0014] In some embodiments, the present invention provides a kit for treating and / or preventing a cancer, comprising CAR T cells specific for a cancer with reduced RARoc expression.

[0015] In some embodiments, the present invention provides use of a composition or kit of the present invention. In some embodiments, the present invention provides use of a composition or kit of the present invention for treatment of a hematologic cancer or a solid cancer.

[0016] DESCRIPTION OF THE FIGURES

[0017] Figures 1 A and IB show that RARoc-deficiency enhances CAR T cell performance with killing of MC38-hCD19 cells (FIG. 1A) and B16-hCD19 cells (FIG. IB) by wild-type (WT), retinoic acid receptor alpha knock-out (RARa-KO), and retinoic acid receptor alpha high transgene-expressing (RARa-TG) chimeric antigen receptor (CAR) T cells. CAR-T cells and tumor cells were co-cultured at 1: 1 ratio. Caspase-positive tumor cells (size > 150 pm2) are shown in magenta or red.

[0018] Figure 1C shows growth of B16-hCD19 tumor cells in host mice injected with PBS or CAR T cells generated from WT, RARa-KO, or RARa-TG CD8 T cells. Figure ID shows a Kaplan-Meier survival curve of Bl 6-hCD 19 tumor-bearing mice injected with PBS or CAR T cells generated from WT, RARa-KO, or RARa-TG CD8 T cells.

[0019] Figure IE shows tumor size on day 13 post-tumor injection.

[0020] Figure IF shows numbers of CD8 Thyl.l+CAR-T cells in tumors of B16-hCD19 tumor-bearing mice.

[0021] Figure 1G shows that RARa-KO CAR-T cells have elevated levels of IFNg, and GzmB.

[0022] Figures 2A-2C show that RARa-deficiency improves in vitro CAR T cell cytotoxicity. Figure 2A shows images of co-culture of CAR CD8 T cells with MC38-hCD19 tumor cells at an effector to target ratio of 0.5. Caspase-positive MC38 cells (size > 150 pm2) are shown in red. The cytotoxic activity of WT, RARa-KO, or RARa-TG CAR T cells against MC38-hCD19 (Figure 2B) and B16-hCD19 (Figure 2C) tumor cells are shown at effector to target ratios of 1, 0.5, and 0.1.

[0023] Figures 3A-3E show that nuclear histone acetyltransferase (HAT) activity that is critical for transcriptional factor and trafficking receptor switches is regulated by RARa in cytotoxic T lymphocytes (CTLs). Figure 3A shows total HAT activity in CTLs from WT, RARa-KO, or RARa-TG mice. Figure 3B shows the impact of p300 HATi (C646, 5 mM) on T cell factor 1(TCF1) and basic leucine zipper ATF-like transcription factor (BATF) expression. Figure 3C shows the frequencies of TCF1+BATF", TCF1+BATF+, and TCF1’ BATF+CTLs from cultured OT-1 CTLs. Figure 3D shows the impact of HATi (C646 and anacardic acid / AA) on the C-C chemokine receptor type 7 (CCR7) to chemokine receptor CXCR3 switch. Figure 3E shows the frequencies of CCR7+CXCR3’, CCR7+CXCR3+, and CCR7' CXCR3+CTLs examined in cultured OT-1 T cells. For panels Figure 3B - Figure 3E OT-1 T cells were cultured for 5 days with repeated stimulation with OVA SIINFEKL antigen peptide in the presence of IL-7 and IL- 15. Statistical significance was determined using two-way ANOVA with Tukey's multiple comparisons test (n=4 for Figure 3 A) and Student’s paired t-test in Figure 3C and Figure 3E; *P < 0.05; **P < 0.01; ***P < 0.001.

[0024] Figures 4A-4C show the negative impact of RARa expression on the effector T cell (Teff) trafficking receptor switch in tumor-draining lymph nodes (dLNs). Figure 4A and Figure 4B show expression of CCR7 and CXCR3 by CD44+CTLs in the dLNs of MC38 tumor-bearing control, RARa-KO, and RARa-TG mice. Figure 4C shows the frequencies of CCR7 CXCR3" and CCR7 CXCR3+CTLs in the dLNs of MC38 tumor-bearing control, RARa-KO, and RARa- TG mice 15-25 days post tumor implantation with flow cytometry. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. *P < 0.05; **P < 0.01; *** < 0.001; ****P < 0.0001.

[0025] DEFINITIONS

[0026] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0027] As used herein the terms “disease” and “pathologic condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g., humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such a deviation can manifest as a state, signs, and / or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions. A disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infective agent (e.g., a virus or bacteria)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and / or climate), may be responsive to an inherent or latent defect in the organism (e.g., genetic anomalies) or to combinations of these and other factors.

[0028] The terms “host,” “subject,” or “patient” are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the invention, the term “subject” generally refers to an individual who will be administered or who has been administered one or more compositions of the present invention (e.g., genetically modified immune cells described herein).

[0029] The term “solution” refers to an aqueous or non-aqueous mixture.

[0030] A “disorder” is any condition or disease that would benefit from treatment with a composition or method of the invention. This includes chronic and acute disorders including those pathological conditions which predispose the mammal to the disorder in question. Nonlimiting examples of disorders to be treated herein include conditions such as cancer.

[0031] The terms “cell proliferative disorder,” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. For example, a “hyperproliferative disorder or disease” is a disease or disorder caused by excessive growth of cells. In one embodiment, the cell proliferative disorder is cancer.

[0032] As used herein, the terms “cancer” and “tumor” refer to a cell that exhibits a loss of growth control or tissue of uncontrolled growth or proliferation of cells. Cancer and tumor cells generally are characterized by a loss of contact inhibition, may be invasive, and may display the ability to metastasize. The present invention is not limited by the type of cancer or the type of treatment (e.g., prophylactically and / or therapeutically treated). Indeed, a variety of cancers may be treated with compositions and methods described herein including, but not limited to, brain cancer or other cancers of the central nervous system (e.g., diffuse midline glioma or diffuse intrinsic pontine glioma (DIPG, a highly aggressive glial tumor found at the base of the brain, see, e.g., Louis et al., Acta Neuropathol (2016) 131 :803-820), melanomas, lymphomas, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, sarcomas, carcinomas, and / or a combination thereof.

[0033] “Metastasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[0034] The term “anticancer agent” as used herein, refer to any therapeutic agents (e.g., chemotherapeutic compounds and / or molecular therapeutic compounds), antisense therapies, radiation therapies, or surgical interventions, used in the treatment of hyperproliferative diseases such as cancer (e.g., in mammals, e.g., in humans).

[0035] An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0036] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the rate of tumor growth (e.g., reduces and / or clears tumor burden in the patient (e.g., reduces the number positive cancer cells in a patient)), decreases tumor mass, decreases the number of metastases, decreases tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0037] The terms “sensitize” and “sensitizing,” as used herein, refer to making, through the administration of a first agent, an animal or a cell within an animal more susceptible, or more responsive, to the biological effects (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.

[0038] As used herein, the terms “administration” and “administering” refer to the act of giving a composition of the present invention to a subject. Exemplary routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intraperitoneally, intratumorally, etc.), topically, and the like.

[0039] As used herein, the terms “co-admini strati on” and “co-administering” refer to the administration of at least two agent(s) (e.g., genetically modified immune cells and one or more other agents - e.g., anti-cancer agents) or therapies to a subject. In some embodiments, the coadministration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. In some embodiments, coadministration can be via the same or different route of administration. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are coadministered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co- administration of the other agent.

[0040] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or other immunologic reactions) when administered to a subject.

[0041] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, and the like. The compositions also can include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference). As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of immunotherapeutic agents, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and / or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant immunotherapeutic agents (e.g., genetically modified immune cells and / or supporting materials). As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising an immunotherapeutic composition for a particular use, while a second container contains a second agent (e.g., a chemotherapeutic agent). Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of an immunogenic agent needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0042] As used herein, the term “gene transfer system” refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue. For example, gene transfer systems include, but are not limited to, vectors (e.g., retroviral, adenoviral, lentiviral, adeno- associated viral, and other nucleic acid-based delivery systems), microinjection of naked nucleic acid, polymer-based delivery systems (e.g., liposome-based and metallic particle-based systems), biolistic injection, and the like. As used herein, the term “viral gene transfer system” refers to gene transfer systems comprising viral elements (e.g., intact viruses, modified viruses and viral components such as nucleic acids or proteins) to facilitate delivery of the sample to a desired cell or tissue. Non-limiting examples of viral gene transfer systems useful in the compositions and methods of the invention are lentiviral- and retroviral-gene transfer systems.

[0043] As used herein, the term “site-specific recombination target sequences” refers to nucleic acid sequences that provide recognition sequences for recombination factors and the location where recombination takes place.

[0044] As used herein, the term “nucleic acid molecule” refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2- thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6- methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2- thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil -5 -oxy acetic acid, oxybutoxosine, pseudouracil, queosine, 2 thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4- thiouracil, 5 -methyluracil, N-uracil-5-oxyacetic acid methylester, and 2,6-diaminopurine.

[0045] The term “gene” refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e g., mRNA, rRNA, tRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length gene product or fragment thereof are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA specifies the sequence or order of amino acids in a nascent polypeptide during translation (e.g., protein synthesis). As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment. 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 non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the 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 expressed).

[0046] As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

[0047] As used herein, the terms “an oligonucleotide having a nucleotide sequence encoding a gene” and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence that encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be singlestranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[0048] “Amino acid sequence” and terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0049] “Percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0050] “Sequence identity” refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

[0051] The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded) but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0052] As used herein, the term “purified” or “to purify” refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0053] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0054] The term “about” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0055] Compositions, methods, and practice of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. laneway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobdized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0056] DETAILED DESCRIPTION

[0057] Provided herein are methods, compositions and kits to enhance anti-cancer T-cell therapy. In particular, provided herein are reagents and methodologies to promote anti-cancer T- cell therapy comprising inhibition of retinoic acid receptor alpha (RARoc) in chimeric antigen receptor (CAR) T cells.

[0058] In some embodiments, the present invention provides methods, compositions and kits to enhance the anti-tumor activity of T cells comprising one or more chimeric antigen receptors (CARs) that increase target-specific cytotoxicity by inhibition of RARoc expression and activity. In some embodiments, cancer-specific CAR T cells comprising reduced RARoc expression and activity elevate the anti-tumor activity of non-CAR T cells. In some embodiments, the methods, compositions and kits of the present invention comprise effector T cells engineered to have reduced RARoc expression with enhanced effective tumor infiltration and cytotoxic activity, tumor amelioration and eradication. In some embodiments, the present invention provides methods, compositions and kit comprising regulation of CD8 T cell functions in other conditions including, for example, autoimmune diseases, inflammatory diseases and other conditions comprising CD8 T cell activity.

[0059] Engineered Cell Types

[0060] An engineered cell or isolated cell of the present disclosure can be a human cell. An engineered cell or isolated cell can be a human primary cell. An engineered primary cell can be a tumor infiltrating primary cell. An engineered primary cell can be a primary T cell. An engineered primary cell can be a hematopoietic stem cell (HSC). An engineered primary cell can be a natural killer cell. An engineered primary cell can be any somatic cell. An engineered primary cell can be an mesenchymal stromal cell (MSC). In some embodiments, the engineered cell is derived from the subject. In some embodiments, the engineered cell is allogeneic with reference to the subj ect.

[0061] An engineered cell of the present disclosure can be isolated from a subject, such as a subject known or suspected to have cancer. Cell isolation methods include, but are not limited to, sorting techniques based on cell-surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA-matched to the subject being administered a treatment. An engineered cell can be a cultured cell, such as an ex vivo cultured cell. An engineered cell can be an ex vivo cultured cell, such as a primary cell isolated from a subject. Cultured cell scan be cultured with one or more cytokines.

[0062] In some embodiments, an engineered or isolated cell of the present disclosure is selected from: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell. In some embodiments, the engineered cell is a Natural Killer (NK) cell. In some embodiments, an engineered cell is autologous. In some embodiments, an engineered cell is allogeneic.

[0063] In some embodiments, an engineered cell of the present disclosure is a tumor cell selected from: an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colorectal tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a gastric tumor cell, a testicular yolk sac tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell. Also provided herein are methods that include culturing the engineered cells of the present disclosure. One skilled in the art will recognize that culturing conditions will depend on the particular engineered cell of interest. One skilled in the art will further recognize that culturing conditions will depend on the specific downstream use of the engineered cell, for example, specific culturing conditions for subsequent administration of the engineered cell to a subject.

[0064] Methods of Engineering Cells

[0065] Also provided herein are compositions and methods for engineering cells to produce the chimeric antigen receptors (CAR). In general, cells are engineered to produce CARs through introduction of one or more polynucleotides comprising a promoter and an exogenous polynucleotide sequence encoding a CAR into the cell’s cytosol and / or nucleus. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0066] In some embodiments, the engineered cell is transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof.

[0067] The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes a CAR (and, e.g., one more transgenes encoding one or more immunomodulating effector molecules). The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes a CAR. In some embodiments, the cell is engineered via transduction with an oncolytic virus.

[0068] Viral-Mediated Delivery

[0069] Viral vector-based delivery platforms can be used to engineer cells. In general, a viral vector-based delivery platform engineers a cell through introducing a virus into a host cell. A viral vector-based delivery platform can be a nucleic acid, and as such, an engineered nucleic acid can also encompass an engineered virally-derived nucleic acid. Such engineered virally- derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0070] A viral vector-based delivery platform can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, an engineered virally-derived nucleic acid, e.g., a recombinant virus or an engineered virus, can encode one or more transgenes. The one or more transgenes encoding the CARs can be configured to express the CARs. A viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes (e.g., transgenes encoding the CARs), such as viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

[0071] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and / or viral production on one or more additional separate vectors in addition to the vector encoding the CAR. One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acids that are configured to produce CARs. More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acid configured to produce CARs. The number of viral vectors used can depend on the packaging capacity of the above-mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0072] In general, any of the viral vector-based systems can be used for the in vitro production of chimeric antigen receptors or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding CARs. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.

[0073] Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, an adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second / third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing- mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0074] The sequences may be preceded with one or more sequences targeting a subcellular compartment. Upon introduction into a host cell, infected cells can express the CARs. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351 :456-460 (1991)). A wide variety of other vectors useful for the introduction of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

[0075] The viral vector-based delivery platforms can be a virus that targets a tumor cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella vims, an oncolytic dengue vims, an oncolytic chikungunya vims, an oncolytic respiratory syncytial vims, an oncolytic lymphocytic choriomeningitis vims, an oncolytic morbillivims, an oncolytic lentivims, an oncolytic replicating retrovims, an oncolytic rhabdovims, an oncolytic Seneca Valley vims, an oncolytic sindbis virus, and any variant or derivative thereof. Any of the oncolytic vimses described herein can be a recombinant oncolytic vims comprising one more transgenes (e.g., an engineered nucleic acid) encoding CARs.

[0076] In some embodiments, the vims is selected from: a lentivims, a retrovims, an oncolytic vims, an adenovims, an adeno-associated vims (AAV), and a vims-like particle (VLP).

[0077] The viral vector-based delivery platform can be retrovims-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding CARs into the target cell to provide permanent transgene expression. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, vims (MuLV), gibbon ape leukemia vims (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et ah, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retrovims system.

[0078] The viral vector-based delivery platform can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). Lentiviral-based delivery platforms can be SIV, or FIV-based. Other exemplary lentivims-based delivery platforms are described in more detail in U.S. Pat. Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721 ; 7,078,031 ; 7,070,993; 7,056,699; 6,955,919, each herein incorporated by reference for all purposes.

[0079] The viral vector-based delivery platform can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO96 / 13597, each herein incorporated by reference for all purposes.

[0080] The viral vector-based delivery platform can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; US patent publications US 2003-0138772, US 2007 / 0036760, and US 2009 / 0197338; Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No, 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each herein incorporated by reference for all purposes. In general, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.RhlO, AAV11 and variants thereof.

[0081] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

[0082] The viral vector-based delivery platform can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell. In general, the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. The viral vector-based delivery platform can be pantropic and infect a range of cells. For example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. The viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting a desired cell type.

[0083] Lipid Structure Delivery Systems

[0084] Engineered nucleic acids can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo / payload in vitro, in vivo, or ex vivo. A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. As used herein, a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.

[0085] A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing self-rearrangement. A desired cargo (e.g., a polypeptide, a nucleic acid, a small molecule drug, and an engineered nucleic acid) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0086] A liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art. Preparations of liposomes are described in further detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each herein incorporated by reference for all purposes.

[0087] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications W003 / 015757A1, WO04029213A2, and W002 / 100435A1, each hereby incorporated by reference in their entirety.

[0088] Lipid-mediated gene delivery methods are described, for instance, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; W091 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.

[0089] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell's cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Exosomes useful for the delivery of nucleic acids are known to those skilled in the art, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.

[0090] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The cargo can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0091] As used herein the term “exosome” refers to a cell-derived small (e.g., between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA) a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is hereby incorporated by reference in its entirety.

[0092] As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (e.g., between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide, a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. Lipid nanoparticles (LNPs), in general, are synthetic lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures. (Riley et al. Recent Advances in Nanomaterials for Gene Delivery — A Review. Nanomaterials 2017, 7(5), 94. ), herein incorporated by reference for all purposes. In general, these vesicles deliver cargo / payloads by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability. In an example, the lipid composition comprises dilinoleylmethyl-4- dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0093] Micelles, in general, are spherical synthetic lipid structures that are formed using singlechain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.

[0094] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and / or neutralization of the viral delivery system. Therefore, encapsulation of an engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off-target affects. In certain examples, an engineered nucleic acid and / or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of an engineered nucleic acid and / or viral delivery system within an LNP can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, the desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles encapsulating the cargo / payload (e.g., an engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration.

[0095] Genomic Editing Systems

[0096] A genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vectorbased delivery platform.

[0097] A transposon system can be used to integrate an engineered nucleic acid into a host genome. Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the TIR-flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo / payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tcl / mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes.

[0098] A nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid. Without wishing to be bound by theory, in general, the nuclease- mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT (e.g., a gene or a portion of a gene) can be incorporated (i.e., “integrated”) into the given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid.

[0099] In some examples, an HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

[0100] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of the endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

[0101] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.

[0102] A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator-like effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0103] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by reference for all that it teaches. In general, a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and an RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 systems comprised of a Cas9 nuclease and an RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with and thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpfl system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double-stranded break typically produced by Cas9 enzymes.

[0104] In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPR systems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced into a cell’s cytosol and / or nucleus, e.g., a T cell’s cytosol and / or nucleus. The in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro using a variety of protein production techniques known to those skilled in the art. CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro using a variety of RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0105] An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can also be used at different amounts in a CRISPR- mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

[0106] In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

[0107] Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

[0108] The engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.

[0109] In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

[0110] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a doublestranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12 / 965,590; U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No. 13 / 738,381 , all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties for all purposes.

[0111] Other Engineering Delivery Systems

[0112] Various additional means to introduce engineered nucleic acids into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0113] Electroporation can used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo / payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i .e. permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the efficiency of internalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art. A variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

[0114] Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means. Compositions and methods for delivering engineered mRNAs in vivo, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10; 27(4): 710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60.), each herein incorporated by reference for all purposes.

[0115] Methods of Use

[0116] Methods for treatment of diseases are also encompassed by this disclosure. The methods include administering a therapeutically effective amount of an engineered nucleic acid, engineered cell, or isolated cell as described above. In some embodiments, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0117] In some embodiments, provided herein are methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0118] In some embodiments, provided herein are methods of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0119] In some embodiments, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0120] In some embodiments, provided herein are methods of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the engineered cells, isolated cells, or compositions disclosed herein.

[0121] In some embodiments, the isolated cell is derived from the subject. In some embodiments, the isolated cell is allogeneic with reference to the subject.

[0122] In some embodiments, the method further comprises administering a checkpoint inhibitor, the checkpoint inhibitor is selected from: an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti- TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti- CD27 antibody, an anti-TNFa antibody, an anti-TREMl antibody, and an anti-TREM2 antibody. In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0123] In some embodiments, the tumor is selected from: an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colorectal tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, a hematologic cancer and a uterine tumor.

[0124] In some embodiments, the method comprises selecting a subject (or patient population) having a tumor or cancer and treating that subject with engineered cells or delivery vehicles that modulate tumor-mediated immunosuppressive mechanisms. In some embodiments, the methods provided herein also include delivering a preparation of engineered cells or delivery vehicles. A preparation, in some embodiments, is a substantially pure preparation, containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than engineered cells. A preparation may comprise IxlO5cells / kg to IxlO7cells / kg cells. The step of administering may include the placement (e.g., transplantation) of the therapeutic T cells into a subject by a method or route that results in at least partial localization of the therapeutic T cells at a desired site, such as a tumor site, such that a desired effect(s) can be produced. Therapeutic T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty -four hours, to a few days, to as long as several years, or even the lifetime of the subject, z.e., long-term engraftment. For example, in some embodiments, an effective amount of the therapeutic T cells can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0125] In some embodiments, the therapeutic T cells are administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. In some embodiments, the administering comprises intratumoral administration

[0126] A subject may be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some instances, the human patient has a cancer. As described herein, the therapeutic T cells may be autologous (“self’) to the subject, i.e., the cells are from the same subject. Alternatively, the therapeutic T cells can be non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic) to the subject. “Allogeneic” means that the therapeutic T cells are not derived from the subject who receives the treatment but from different individuals (donors) of the same species as the subject. A donor is an individual who is not the subject being treated. A donor is an individual who is not the patient. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors, e.g., two or more donors, are used.

[0127] An effective amount refers to the amount of a population of engineered T cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject’s signs or symptoms of cancer. An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0128] The efficacy of a treatment using the therapeutic T cells disclosed herein can be determined by a skilled clinician. A treatment is considered “effective”, if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease ( .g., cancer) are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0129] In some embodiments, the methods provided herein also include administering a drug or pharmaceutical composition in combination with a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein. For example, tamoxifen or a metabolite thereof (e.g., 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen) can be administered. The drug or pharmaceutical can be administered prior to, concurrently with, simultaneously with, and / or subsequent to administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical can be administered serially. The drug or pharmaceutical can be administered concurrently or simultaneously with administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical can be administered at separate intervals than (e.g., prior to or subsequent to) administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical can be administered both concurrently / simultaneously as well as at separate intervals than any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical composition and the engineered cells, isolated cells, or compositions can be administered via different routes, e.g., the drug or pharmaceutical composition can be administered orally and the engineered cells, isolated cells, or compositions can be administered intraperitoneally, intravenously, subcutaneously, or any other route appropriate for administration, as will be appreciated by one skilled in the art. Thus, some embodiments of the present disclosure provide methods for administering an effective amount of CAR T cells and / or antibodies and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and / or therapeutic technique (e.g., surgical intervention, and / or radiotherapies). In a particular embodiment, the additional therapeutic agent(s) is an anticancer agent. The disclosure is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to a subject. For example, a subject may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to a subject using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

[0130] In some embodiments of the present disclosure, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein are administered prior to the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 hours or more, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6 or more weeks prior to the administration of the therapeutic or anticancer agent. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein are administered after the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 or more hours, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6, or more weeks after the administration of the anticancer agent. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., modified immune cells are administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, once every four weeks, or more. In other embodiments, modified immune cells are administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, once every four weeks, or more.

[0131] In some embodiments, the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0132] In vivo Expression

[0133] The methods provided herein also include delivering a composition in vivo capable of producing the engineered cells described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo. Such compositions include any of the viral- mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems, or any of the other engineering delivery systems described herein capable of engineering a cell in vivo.

[0134] Kits

[0135] The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses. In some embodiments, a kit provided herein may comprise a population of genetically engineered T cells as disclosed herein, and one or more components for producing the therapeutic T cells as also disclosed herein. Such components may comprise a nucleic acid coding for a CAR construct of interest. In some instances, the donor template may be carried by a viral vector such as a retroviral vector, a lentiviral vector, or other vector described herein or known in the art. In yet other embodiments, the kit disclosed herein may comprise a population of therapeutic T cells as disclosed for the intended therapeutic purposes. In some embodiments, a kit provided herein comprises a component for knocking down or knocking out RARoc. In some embodiments, a kit of the present invention comprises one or more cells in which RARoc is knocked down or knocked out. Any of the kits disclosed herein may comprise instructions for making the therapeutic T cells, or therapeutic applications of the therapeutic T cells. In some embodiments, the included instructions may comprise a description of how to introduce a nucleic acid encoding a CAR construct into the T cells for making therapeutic T cells.

[0136] In some embodiments, a kit as disclosed herein may comprise a population of genetically engineered T cells ( .g., CAR-T cells) for use to eliminate undesired cells targeted by the CAR construct (e.g., for treatment of cancer such as a solid tumor). Such a kit may comprise one or more containers in which the genetically engineered T cells can be placed. The kit may further comprise instructions for administration of the therapeutic T cells as disclosed herein to achieve the intended activity, e.g., eliminating disease cells targeted by the CAR expressed on the therapeutic T cells. Alternatively, or in addition, the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions relating to the use of the therapeutic T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g, multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the therapeutic T cells are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0137] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device for administration of the therapeutic T cells. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0138] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0139] One of ordinary skill in the art, based on the present disclosure, can utilize the compositions and methods described to their fullest extent. The specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions.

[0140] EXPERIMENTAL EXAMPLES

[0141] EXAMPLE 1 - Enhanced Anti-Tumor cytotoxicity in vitro

[0142] To test if CAR T cell function is improved by RARa deficiency WT, RARa-KO, and RARa-TG CD8 T cells were transduced with a retroviral vector expressing a CAR against hCD19, and the cells were co-cultured with hCD19-expressing MC38 or B16 cells at varying effector-to-target (E:T) ratios (Figures IB and 2A-C). At the ratios tested in vitro cytotoxicity of RARa-KO CAR T cells was greater than that of WT CAR T cells. In contrast, RARa-TG CAR T cells were less efficient than WT T cells in killing tumor cells in vitro.

[0143] EXAMPLE 2 - Enhanced Anti-Tumor cytotoxicity in vivo

[0144] To test the anti -turn or efficacy of WT, RARa-KO, and RARa-TG CAR T cells in vivo, (Figure 1C), 1.5 million CAR T cells were administered to B16- hCD19 tumor-bearing mice. (Figures ID (percent survival) and IE (tumor size)). RARa-KO CAR-T cells were effective in delaying tumor growth. RARa-TG CAR-T cells were ineffective in eliminating tumors. These data indicate that RARa deficiency increases anti-tumor CAR T activity. Flow analysis of tumor infiltrating lymphocytes (TILs) detected numerous RARa-KO CAR-T cells at the termination of the experiments with few WT and RARa-TG CAR-T cells found in tumors (Fig. 7F). As well, RARa-KO CAR-T cells changed the effector phenotype of host non-CAR T cells (Figure 1G). Host TIL non-CAR CD8 T cells in mice treated with RARa-KO CAR-T cells had elevated expression of ZFNy, GzmB and Perforin. These data indicate that RARa deficiency enhances CAR-T cell tumor infiltration, cytotoxicity, and tumor microenvironment (TME)- modifying properties.

[0145] EXAMPLE 3 - RARa and retinoic acid regulate histone acetyl transferase (HAT) activity and the expression of TCF1 and BATF

[0146] RARa recruits HATs, for example p300 / CBP, for gene expression. (Bastien J, Rochette- Egly C. Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene. 2004 Mar 17;328 : 1 -16.) Total nuclear HAT activity measured in 20h-stimulated CTLs from WT, RARa- KO, and RARa-TG mice, showed high and low nuclear HAT activity, respectively, in RARa- KO and RARa-TG CTLs indicating that RARa decreases nuclear HAT activity (Figure 3A). A p300 HAT-specific inhibitor (C646) was used in the OVA-specific OT-1 CTL culture system with repeated stimulation with the OVA257-264 peptide to mimic TME to determine if p300 HAT is required for BATF up-regulation. C646 increased TCF1 expression and the generation of less differentiated early stage TCF1+BATF' CTLs mirroring the RARa-TG CTLs in tumors, but suppressed BATF expression and the generation of differentiated TCF1 BATF+cells mirroring the phenotype of RARa-KO CTLs in tumors (Figure 3B and Figure 3C). p300 HAT inhibition with C646 increased CCR7-expressing but suppressed CXCR3 -expressing OT-1 T cells after repeated stimulation with the cognate OVA257-264 peptide for 5 days (Figure 3D and Figure 3E). Anacardic acid (AA), an inhibitor of the co-activators p300 and p300 / CREB-binding protein- associated factor (PCAF), showed similar effects as C646 on the trafficking receptor switch. A similar effect of the HAT inhibitors was observed on 2 day-cultured OT-1 T cells with acute stimulation. These data show that p300 HAT activity regulated by RARa expression participates in transcription factors and Teff trafficking receptor switches in primed CTLs.

[0147] EXAMPLE 4 - Upregulated T cell-expressed RARa inhibits Teff trafficking receptor expression

[0148] CTL subsets in RARa-KO mice do not accumulate the CD62L CD44+effector-like CTLs in draining lymph nodes (dLNs) in keeping with movement of effector T cells to effector sites following their generation. Migration of CTLs requires a trafficking receptor switch from lymphoid tissue (i.e., CD62L and CCR7) to effector site-homing receptors (e.g., CXCR3, CCR8, CCR5, CCR2, and CCR1) upon antigen priming. (Woodland DL, Kohlmeier JE. Migration, maintenance and recall of memory T cells in peripheral tissues. Nat Rev Immunol. 2009 Mar;9(3): 153-61.) Expression of CCR7 and CXCR3, a major Teff-associated chemokine receptor, by CTLs in the dLNs of tumor-bearing control, RARa-KO, and RARa-TG mice was examined (Figure 4A, Figure 4B, Figure 4C). RARa-TG mice showed decreased frequencies of CXCR3+CTLs but increased frequencies of unswitched CCR7+CXCR3‘ CTLs in dLNs, denoting a defect in the trafficking receptor switch. RARa-TG mice showed significant differences from RARa-KO mice in the frequencies of CCR7 CXCR3' and CCR7 CXCR3+CTLs in dLNs. These data show that the trafficking receptor switch during Teff differentiation is suppressed by upregulated RARa in keeping with low infiltration of CTLs in tumors of RARa-TG mice.

[0149] Experimental Methods

[0150] Animals

[0151] C57BL / 6 mice (Taconic Biosciences), Ragl ^ (The Jackson Laboratory, stock 002216), Lck-CrexRara f / f (RARa-KO) and CD2-Rara (RARa-TG) mice were kept under a specific pathogen-free condition on a regular rodent chow ad libitum on the 12-hour dark and 12-hour light cycle. (Friesen LR, Gu B, Kim CH. A ligand-independent fast function of RARa promotes exit from metabolic quiescence upon T cel I activation and controls T cell differentiation.

[0152] Mucosal Immunol. 2021 Jan; 14(1): 100-112.) Experiments were performed on age- matched 6- to 10-week-old male and female mice.

[0153] Cells and cell culture

[0154] 11CD19-MC38 and hCD19-B16 were provided by Dr. Anjana Rao ((Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530-534 (2019).) Tumor cells were thawed and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 1% L-glutamine and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged once after thawing before implantation in mice. Platinum-E packaging cells were cultured Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 1% L-glutamine and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator.

[0155] Naive CD8 T cells were isolated from spleens using the Naive CD8 T cell isolation kit using the AutoMACS system (Miltenyi Biotech). CD8 T cells were cultured on 96 or 48-well plates coated with anti-CD3 (1 ug / l) in RPMI supplemented with anti-CD28 (2 pg / ml) and IL- 2 (100 U / ml). Either 10% FBS or 10% charcoal-stripped FBS were used for the medium. OT-1 T cells were similarly isolated from the spleen and cultured at 150,000 cells per well (48-well plates) in the presence of the OVA peptide (SIINFEKL, 10 ng / ml) and cytokines (IL- 7 and IL-15 at 20 ng / ml) for 2 days, and the 2 day-activated OT-1 cells were repeatedly activated thereafter every day, and the cultures were terminated on day 5 or 6 for flow cytometry analysis. When indicated, the histone acetyl transferase inhibitors, anacardic acid and C646 (Cayman Chemical), were added to the culture at an optimal concentration (5 pM).

[0156] Tumor growth and animal survival

[0157] Mice were injected with 2*105of hCD19-MC38 cells subcutaneously into the right shaved flank. Tumors were measured every 2-3 days for at least 20 days if not otherwise indicated. Animals were sacrificed and considered dead once the tumor reached a volume of >1000 mm3, and these data were plotted as Kaplan -Meier survival curves. Production and application of CAR T cells

[0158] For production of retroviral particles, Plantinum-E cells were co-transfected with pCL- Eco (2 pg, (Addgene #12371) and MSCV- RARa-FLAG-Thyl.l or MSCV-myc-CAR-2A- Thyl.l (5 pg, Addgene #127890) with Lipofectamine 3000 (Thermo Fisher) according to manufacturer’s instruction. The culture medium was changed after 7 h post transfection and viral particles were in culture supernatant were harvested 36 h later. For generation of transduced CD8 T cells, 24 h- activated CD8 T cells were spin-infected with retroviral particles in a medium supplemented with polybrene (8 pg / ml) by centrifugation at 3200rpmi at 32°C for 90 min. The infected cells were rested for 1 h at 37°C and cultured in RPMI medium supplemented with IL-2 (100 U / ml) and FBS (10%). The retroviral transduction was repeated the next day and rested for 24 h and examined for viability and transduction efficiency.

[0159] In vitro CAR T cell cytotoxicity assay hCD19-expressing MC38 or hCD19-expressing B16 cells (1.5* 104per well) were seeded on 96- well plates. After 24 h, cells were moved to an IncuCyte S3 Live Cell Analysis System (Sartorius) and imaged at 10x magnification (tO). Next, CAR T cells were added at the indicated ratios, assuming that there were 1.5 x 104MC38 cells per well at the time of adding T cells. The caspase- 3 / 7 reagent (2.5 pM, Essen Bioscience) was added visualize dying cells. Images were acquired every hour for 24-40 h. Data was analyzed using the IncuCyte analysis software to detect and quantify the number of green (apoptotic) cells per image. A size threshold of 150 pm2was applied to exclude dead T cells.

[0160] In Vivo CAR T cell tumor size and survival assay

[0161] For in vivo CAR T cell experiments, mice were injected with 2xl05hCD19-expressing B16 cells subcutaneously into the right shaved flank. After 7 days, the B16-hCD19 tumorbearing mice were injected intravenously with 1.5xl06CAR T cells. Tumors were measured every 2-3 days for ~25 days. CD8 T cells were transduced with ecotropic retrovirus particles harboring the MSCV-RARa- FLAG-Thyl. l construct as previously described (Friesen LR, Gu B, Kim CH. A ligand-independent fast function of RARa promotes exit from metabolic quiescence upon T cell activation and controls T cell differentiation. Mucosal Immunol. 2021 Jan;14(l): 100-112.) Nuclear HAT assay for CTLs.

[0162] Isolated naive spleen CD8 T cells from control, RARa-KO, or RARa-TG mice were cultured plate-bound in anti-CD3 (1 mg / ml), soluble anti-CD28 (2 mg / ml) and IL-2 (20 ng / ml) for 20h in RPMI1640 supplemented with 10% charcoal -treated FBS. All-trans-retinoic acid (RA) was added at 20 nM. Nuclear lysates were prepared after cell membrane lysis, and total HAT activity was measured using EpiQuik HAT Activity / Inhibition Assay Kit (Epigentek, Cat# P- 4003-96).

[0163] Statistical analysis

[0164] P values were calculated using paired and unpaired two-tailed Student’s t tests. Significances for Kaplan-Meier survival curves were calculated with a log-rank (Mantel-Cox) test with a degree of freedom of 1. At least 3 independent experiments were performed unless otherwise indicated. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

[0165] INCORPORATION BY REFERENCE

[0166] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

Claims

CLAIMSWe claim:

1. A method of treating a subject having a cancer, comprising: a) collecting, leukophoresising and purifying T cell lymphocytes from a subject; b) genetically modifying said purified T cell lymphocytes to express a nucleic acid molecule encoding a chimeric antigen receptor (CAR) specific for said cancer to generate CAR T cell lymphocytes; c) genetically modifying said CAR T cell lymphocytes to generate CAR T cells with reduced retinoic acid receptor alpha (RARa) expression; and d) administering a therapeutically effective amount of said CAR T cells with reduced RARa expression to said subject having said cancer.

2. A method of stimulating a cell-mediated immune response to a cancer in a subject, comprising administering to said subject having a cancer a therapeutically effective dose of CAR T cells specific for said cancer with reduced RARa expression.

3. A composition, comprising a T cell comprising one or more chimeric antigen receptors (CARs) and reduced RARa expression and / or activity.

4. A pharmaceutical composition comprising an effective amount of CAR T cells specific for a cancer with reduced RARa expression and / or activity and a pharmaceutically acceptable carrier.

5. The composition of any of claims 3 or 4 in dosage form.

6. A kit for treating and / or preventing a tumor, comprising CAR T cells specific for a cancer with reduced RARa expression specific for a cancer.

7. Use of a kit or composition of any of claims 3-6.

9. Use of a kit or composition of any of claims 3-6 for treatment of a hematologic cancer or a solid cancer.

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