Autoregulatory chimeric antigen receptors for natural killer cells.

Abstract

An autoregulatory chimeric antigen receptor and a method for producing the same are disclosed herein. Also disclosed herein are methods for treating cancer through the administration of NK cells and / or NK T cells containing the chimeric antigen receptor.

Description

[Technical Field]

[0001] Statement of government support This invention was made with government support under Grant No. CA232561 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 414,451, filed October 7, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Human peripheral blood natural killer (NK) cells have potent antitumor activity and have been successfully used in several clinical trials. Modifying NK cells with chimeric antigen receptors (CARs) can improve targeting and increase specificity. However, NK cells can be sensitive to some checkpoint inhibitors and to regulatory suppression by suppressor of cytokine signaling 3 (SOCS3), which negatively regulates cytokine signaling via the JAK / STAT pathway. What is needed are new methods to modify NK cells and / or NK T cells for better immunotherapy. Summary of the Invention

[0004] Methods and compositions related to autoregulatory CAR expression in natural killer (NK) cells and natural killer (NK) T cells are disclosed.

[0005] In one aspect, a chimeric antigen receptor (CAR) disclosed herein comprises an endodomain comprising a STAT3 phosphorylation site (e.g., an IL-6, IFN-λR1, IL-2R, IL-10, or IL-21 endodomain, etc.), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain, etc.), and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell (e.g., CD33, etc.). In one aspect, the CAR can further comprise a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0006] Disclosed herein is a plasmid encoding a CAR of any of the aforementioned embodiments.

[0007] In one aspect, also disclosed herein is a plasmid of the aforementioned aspect, wherein the plasmid is for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated integration system, such as a CRISPR / Cas9 integration system, and further comprises, in order, a left homologous arm, a transgene polynucleotide sequence encoding a CAR, and a right homologous arm, wherein each of the left and right homologous arms is 1000 bp or less in length. In one aspect, the plasmid further comprises a polyadenylation signal between the CAR-encoding transgene and the right homologous arm. In some aspects, the left homologous arm and the right homologous arm are different lengths. In other aspects, the left homologous arm and the right homologous arm are the same length.

[0008] Also disclosed herein are plasmids of any of the preceding embodiments, wherein the homology arms are 30 bp, 300 bp, 600 bp, or 1000 bp in length.

[0009] In one aspect, disclosed herein is a plasmid of any of the preceding aspects, wherein the homology arms specifically hybridize to the suppressor of cytokine signaling 3 (SOCS3) locus.

[0010] Also disclosed herein are adeno-associated virus (AAV) vectors (e.g., AAV6 vectors, etc.) comprising the plasmids of any of the foregoing embodiments. In some embodiments, the vectors further comprise plasmids encoding crRNA, tracer RNA (tracrRNA), and CAS endonuclease.

[0011] Also disclosed herein are AAV vectors of any of the foregoing embodiments, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

[0012] In one aspect, disclosed herein is an engineered cell (such as, for example, an engineered NK cell or NK T cell) comprising a CAR of any preceding aspect, a plasmid of any preceding aspect, or an AAV vector of any preceding aspect. For example, in one aspect, disclosed herein is an engineered immune cell (e.g., an engineered NK cell or NK T cell) comprising a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain (e.g., an IL-6, IFN-λR1, IL-2R, IL-10, or IL-21 endodomain) comprising a STAT3 phosphorylation site, a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on a target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus. In one aspect, the CAR of the engineered cell further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0013] Similarly, disclosed herein are methods for treating, suppressing, reducing, decreasing, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., leukemia (including but not limited to acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS))), which comprise administering to the subject an engineered cell of the foregoing embodiment. For example, disclosed herein are methods for treating, suppressing, reducing, decreasing, ameliorating, or preventing cancer and / or metastasis in a subject (e.g., leukemia (including but not limited to acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS))), which comprise administering to the subject engineered cells of the foregoing embodiment. The disclosed methods involve administering to target cells (e.g., T cells), wherein the engineered immune cells comprise a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain comprising a STAT3 phosphorylation site (e.g., an IL-6, IL-10, IFN-λR1, IL-2R, or IL-21 endodomain), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on the target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus. In one aspect, the CAR of the engineered cells used in the disclosed methods further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0014] In one aspect, disclosed herein is a method for promoting proliferation of NK cells and / or NK T cells at the site of an immune response, comprising: a) administering a CRISPR / Cas endonuclease (e.g., Cas obtaining a ribonucleoprotein (RNP) complex complexed with a Class 1 or Class 2 CRISPR / Cas endonuclease, e.g., Cas9) and a corresponding CRISPR / Cas guide RNA, or a nucleic acid encoding the RNP complex, and an AAV vector (e.g., AAV6) comprising a plasmid containing a transgene polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by homologous arms, the homologous arms being 800 bp or less in length, and the CAR comprises an endodomain containing a STAT3 phosphorylation site (e.g., an IL-6, IL-10, IFN-λR1, IL-2R, or IL-21 endodomain), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell (e.g., CD33); and b) injecting the polynucleotide sequence and the RNP complex, or the nucleic acid encoding the RNP complex, into NK cells or NK cells. Injecting the polynucleotide sequence into NK cells or NK T cells (e.g., by electroporation or gene transfer), and injecting the polynucleotide sequence into NK cells or NK T cells with AAV (at a multiplicity of infection (MOI) of about 5x10 3 ~about 5x10 5a) introducing the CAR into a NK cell or NK T cell via a CAR-binding domain (including but not limited to a CAR-binding domain), wherein the RNP complex hybridizes to the suppressor of cytokine signaling 3 (SOCS3) locus in the genomic DNA of the NK cell or NK T cell, and a DNA repair enzyme in the NK cell or NK T cell inserts a transgene encoding a CAR into the host genome at the SOCS3 locus in the genomic DNA of the NK cell or NK T cell, thereby creating an engineered NK cell or an engineered NK T cell; and b) administering the engineered NK cell or the engineered NK T cell to a subject, wherein binding of the CAR to a receptor on the target cell causes phosphorylation via the endodomain of the engineered NK cell or the engineered NK T cell, resulting in the production of more CAR on the surface of the cell and promoting the proliferation of the engineered NK cell or the engineered NK T cell. In some embodiments, the CAR further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0015] Also disclosed herein are methods of increasing proliferation of NK cells or NK T cells according to any of the foregoing embodiments, wherein the RNP complexes are encoded on the same or different AAVs.

[0016] In some embodiments, disclosed herein are methods of increasing NK cell or NK T cell proliferation of any of the preceding embodiments, wherein the left and right homologous arms are of different lengths. In other embodiments, the left and right homologous arms are of the same length.

[0017] Also disclosed herein are methods of increasing proliferation of NK cells or NK T cells according to any of the foregoing embodiments, wherein the homology arms are 30 bp, 300 bp, 600 bp, or 1000 bp in length.

[0018] In some aspects, a method of increasing proliferation of NK cells or NK T cells of any of the preceding aspects is disclosed, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). [Brief explanation of the drawings]

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods.

[0020] [Figure 1] Figure 1 shows a cartoon representation of resting NK cells with a self-driving (autoregulatory) CAR inserted into the SOCS3 locus, which has low baseline expression. The Cas / ribonucleoprotein complex (Cas9) targets the SOCS3 locus, which contains a STAT3 binding site, allowing integration of the CAR transgene delivered by the AAV6 vector into the SOCS3 locus. In the resting state, STAT3 is not phosphorylated; therefore, STAT3-activated gene transcription at the SOCS3 locus does not occur, and CAR expression is minimized. [Figure 2]Figure 2 shows a cartoon representation of activated NK cells in which target binding to a self-driving (autoregulatory) CAR initiates STAT3 phosphorylation, promoting expression of the SOCS3 locus and increasing CAR expression. The Cas9 / ribonucleoprotein complex targets the SOCS3 locus, which contains a STAT3 binding site, allowing the CAR transgene delivered by the AAV6 vector to integrate into the SOCS3 locus. When the membrane-bound CAR binds to its target antigen (e.g., a cancer antigen), cytokine-mediated signaling is initiated at the IL-21 endodomain of the CAR, activating the JAK / STAT pathway and mediating tyrosine phosphorylation. This results in phosphorylation of STAT3 protein, forming STAT dimers, which translocate from the cytoplasm to the nucleus, bind to the SOCS3 locus, and activate transcription of the CAR transgene. In the activated state, expression of the CAR transgene promotes CAR production and expression on the cell surface. Thus, CAR binding to its target antigen promotes additional CAR expression, effectively creating an autoregulatory or autodriving CAR. [Figure 3] Figure 3 shows the overall structure of the AAV packaging plasmid for site-directed insertion of CAR into the AAVS1 locus via 300 bp left and right homology arms. [Figure 4] Figure 4 shows restriction mapping of the CAR transgene with the IL-6R-YMPQ site for STAT3 signaling. [Figure 5] Figure 5 shows restriction mapping of the CAR transgene with a truncated IL-2R-YMPQ site for STAT3 signaling. [Figure 6] Figure 6 shows restriction mapping of the CAR transgene with the IFNLR1-JAK site for STAT3 signaling. [Figure 7]Figure 7 is a graph showing RNA-seq data demonstrating elevated SOCS3 expression in naive NK cells in response to STAT3 signaling initiated by exposure to IL-21, which is maintained in expanded NK cells. Naive-stimulated NK cells showed an average 23-fold increase in STAT3 signaling compared to naive NK cells (p=0.0007). Expansion-stimulated NK cells showed an average 25-fold increase in STAT3 signaling compared to resting, expanded NK cells (p<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0021] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods or to particular recombinant biotechnology methods, unless otherwise specified, and are not limited to particular reagents (which may, of course, vary), unless otherwise specified. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0023] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint. Additionally, certain values ​​are disclosed herein, and it is understood that each value is also disclosed "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of ordinary skill in the art, when a value is disclosed "less than or equal to," it is understood that "greater than or equal to" and possible ranges between those values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, less than 15, less than or equal to 15, and equal to 15, are considered to be disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0024] As used herein and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0025] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0026] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, so long as the increase is statistically significant.

[0027] "Reduction" can refer to any change that results in a lower amount of a symptom, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of a gene product containing the substance is less than the output of the gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average decrease in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as the reduction is statistically significant.

[0028] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" means that compounds are administered at the same point in time or essentially immediately after each other. In the latter case, the two compounds are administered sufficiently close in time that the results observed are indistinguishable from the results that would be achieved if the compounds were administered at the same point in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entrance into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​or immediately adjacent to the point of administration and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal portions of the subject's body. Administration includes self-administration and administration by another.

[0029] "Biocompatible" generally refers to a material and any metabolic or breakdown products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects in the subject.

[0030] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements, but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean including the recited elements, but excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharmaceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc., from isolation and purification methods. "Consisting of" shall mean excluding more than trace elements of other components, as well as substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0031] A "control" is a substitute subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0032] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific drug, and so forth. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any subject may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the subject's age, sex, and weight. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0033] A "pharmaceutically acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., an ingredient that can be incorporated into a pharmaceutical formulation provided by the present disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is included. When used in reference to human administration, the term generally means that the ingredient has met the necessary standards of toxicology and manufacturing testing, or that it is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0034] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0035] "Pharmacologically active" (or simply "active") can refer to derivatives or analogs (e.g., salts, esters, amides, complexes, metabolites, isomers, fragments, etc.) that have the same type of pharmacological activity as the parent compound, and to approximately the same extent, in a "pharmacologically active" derivative or analog.

[0036] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., the prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0037] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a drug) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of type 1 diabetes. In some embodiments, the desired therapeutic result is control of obesity. A therapeutically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), and various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition over a period of days, weeks, or years.

[0038] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. The DNA polynucleotide may encode an RNA that is translated into a protein (mRNA) (thus, DNA and mRNA together encode a protein), or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.).

[0039] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0040] "Fragments" can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not associated with other sequences, so long as the activity of the fragment is not significantly altered or impaired compared to the unmodified peptide or protein. These modifications can provide additional properties, such as removing or adding amino acids capable of disulfide bonding, increasing biolongevity, or altering secretion characteristics. In any case, the fragment must possess a biologically active property, such as modulating transcription of a target gene.

[0041] The term "gene" or "gene sequence" refers to a coding sequence or a regulatory sequence, or fragments thereof. A gene can include any combination of coding sequences, regulatory sequences, or fragments thereof. Thus, a "gene" referred to herein can be all or a portion of a naturally occurring gene. The polynucleotide sequences referred to herein are used synonymously with the term "gene" and can include any coding, non-coding, or regulatory sequence, fragments thereof, and combinations thereof. The terms "gene" or "gene sequence" include, for example, regulatory sequences that are upstream of the coding sequence (e.g., a ribosome binding site).

[0042] As used herein, the term "operably linked" can refer to the placement of regulatory sequences useful for expression of a nucleic acid coding sequence in a nucleic acid molecule in an appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition can also apply to the placement of coding sequences and / or transcriptional control elements (e.g., promoters, enhancers, termination elements) and / or selectable markers in an expression vector. The term "operably linked" can also refer to the placement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, proteins, fragments thereof, linking peptides, and / or signal peptides. The term "operably linked" can refer to the direct fusion of different individual polypeptides within a single polypeptide or fragment thereof, whether there are no intervening amino acids between the different segments or when the individual polypeptides are connected to each other via one or more intervening amino acids.

[0043] "Primers" are a subset of probes that can support some type of enzymatic manipulation and can hybridize to a target nucleic acid so that enzymatic manipulation can occur. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with enzymatic manipulation.

[0044] A "probe" is typically a molecule that can interact with a target nucleic acid in a sequence-specific manner, for example, through hybridization. Nucleic acid hybridization is well understood in the art and is discussed herein. Typically, probes can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

[0045] A "protein coding sequence," or a sequence encoding a specific protein or polypeptide, is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA (in the case of DNA) and translated into a polypeptide in vitro or in vivo (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (N-terminus) and a translation stop nonsense codon at the 3'-terminus (C-terminus). Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is typically located 3' to the coding sequence.

[0046] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers.

[0047] The term "polypeptide" refers to a compound consisting of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0048] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0049] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operatively linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, direct expression of the gene product in a tissue-specific manner.

[0050] As used herein, a "transgene" refers to exogenous genetic material (e.g., one or more polynucleotides) that has been or can be artificially provided to a cell. The term can be used to refer to a "recombinant" polynucleotide encoding any of the polypeptides disclosed herein that are the subject of this disclosure. The term "recombinant" refers to a sequence (e.g., a polynucleotide or polypeptide sequence) that is not present in the cell in which the sequence is artificially provided, or a sequence that is joined to other polynucleotides in an arrangement that is not present in the cell in which the sequence is artificially provided. The term "artificial" refers to non-natural occurrence in a host cell and is understood to include manipulation by humans, machines, exogenous agents (e.g., enzymes, viruses, etc.), other non-natural manipulation, or a combination thereof. A transgene can include, but is not limited to, a gene operatively linked to a promoter (e.g., an open reading frame). When a transgene is artificially provided to a cell, the transgene can be integrated into a chromosome of the host cell, present extrachromosomally, or any combination thereof.

[0051] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the sentence in which they are relied upon and are individually and specifically incorporated by reference herein for the material contained therein.

[0052] B. Chimeric Antigen Receptors (CARs), Plasmids, Vectors, and Engineered Cells Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. Where these and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each of the various individual and collective combinations and permutations of these compounds may not be specifically mentioned and explicitly disclosed, but each is specifically contemplated and described herein. For example, where a particular chimeric antigen receptor, plasmid, vector, or engineered NK cell / NK T cell is disclosed and discussed, and many modifications that can be made to many molecules comprising the chimeric antigen receptor, plasmid, vector, or engineered NK cell / NK T cell are discussed, what is specifically contemplated is each combination and permutation and possible modification of the chimeric antigen receptor, plasmid, vector, or engineered NK cell / NK T cell, unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C, and classes D, E, and F are disclosed, and one example of a combination molecule, A-D, is disclosed, each is considered individually and collectively disclosed, even if each is not individually listed, as are combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F. Likewise, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0053] Enhanced cancer immunotherapy through genetic modification of NK cells has potential applications in the treatment of a wide range of cancers. Disclosed herein is a strategy for generating large numbers of genetically modified NK cells by introducing CRISPR / Cas elements, such as CRISPR / Cas9, CRISPR / Cas12, or CRISPR / CasX elements, into NK cells. In certain embodiments, the CRISPR / Cas elements comprise a ribonucleoprotein (RNP) complex in which a CRISPR / Cas endonuclease and a CRISPR / Cas guide RNA form a complex. In certain embodiments, the CRISPR / Cas elements comprise nucleic acids encoding the RNP and the CRISPR / Cas guide RNA. The RNP and the CRISPR / Cas guide RNA can be encoded by a single nucleic acid or separate nucleic acids. This method is used to disrupt suppressor of cytokine signaling 3 (SOCS3) by using the CRISPR / Cas system to integrate a nucleic acid encoding a chimeric antigen receptor and target the integration of DNA into SOCS3. SOCS3 expression is typically induced by various cytokines, including IL-2, IL-6, IL-10, IFN-γ, and IL-21. CARs are designed to bind to target antigens and signal their binding status via STAT3 phosphorylation sites from several cytokine receptor endodomains (such as the IL-2, IL-6, IL-10, IFN-γ, and IL-21 endodomains, which typically induce SOCS3 transcription), inducing transcription and expression of the integrated CAR (Figures 1 and 2). Thus, binding of the CAR to its target resulted in additional CAR production (Figure 2). When no target is available for the CAR to bind, only background levels of CAR production occur (Figure 1). Thus, the chimeric antigen receptor (CAR) disclosed herein comprises an endodomain containing a STAT3 phosphorylation site (e.g., an IL-6, IL-10, IFN-λR1, IL-2R, or IL-21 endodomain), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on a target cell (see Figures 3, 4, 5, and 6).

[0054] As used herein, "chimeric antigen receptor" or "CAR" refers to a chimeric receptor that targets a cancer antigen and attracts receptor-expressing cells to cancer cells expressing the target antigen. Typically, a CAR comprises a molecule that recognizes a peptide derived from a tumor antigen presented by an MHC molecule, or an antibody or fragment thereof (e.g., Fab', scFv, or Fv) expressed on the surface of the CAR cell that targets the cancer antigen. The receptor is fused via a linker to a signaling domain (e.g., the CD3ζ domain of a T cell or the NKG2C, NKp44, or CD3ζ domain of an NK cell or NK T cell). Tumor antigen targets are proteins produced by tumor cells that elicit an immune response, particularly an NK cell- or NK T cell-mediated immune response. The choice of antigen-binding domain will depend on the specific type of cancer being treated. Tumor antigens are known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CALX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUL, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl-GM1, GloboH, MN-CAIX, EPCAM, EVT6-AML, TGS5, human telomere Reverse transcriptase, polysialic acid, PLAC1, RUL, RU2 (AS), intestinal carboxylesterase, lewisY, sLE, LY6K, muthsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPVE6, E7, sperm protein 17, SSEA-4, tyrosinaseTARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma metastasis breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, These include CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TNAg, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include: differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal rearrangements such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, IL13Ra2, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, and p1 6, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3 / CA27.29 / BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag,These include MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilum C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0055] The CAR polypeptide can also include a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD28 transmembrane domain, a CD3ξ transmembrane domain, etc.) and a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of a 2B4 domain, a CD28 costimulatory domain, and / or a 4-1BB costimulatory domain).

[0056] The endodomain of the CAR promotes signal transduction to the SOCS3 promoter. In normal NK cells and NK T cells, when a cytokine binds to its specific cytokine receptor, the endodomain of the cytokine receptor, such as IL-10, IL-6, IFN-λR1, IL-2R, or IL-21, is activated, activating STAT3 and promoting gene transcription in the nucleus, including upregulation of SOCS3 (see Figure 7). However, by inserting the CAR transgene into SOCS3, it is the CAR that is upregulated.

[0057] Disclosed herein is a plasmid encoding any of the CARs disclosed herein. For example, disclosed herein is a plasmid encoding a CAR comprising an endodomain (such as an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain) containing a STAT3 phosphorylation site, a transmembrane domain (such as a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (such as CD33) on a target cell, wherein the plasmid is for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated integration system, such as a CRISPR / Cas9 integration system, and comprises, in order, a left homologous arm, a polynucleotide sequence encoding the CAR, and a right homologous arm, each of which is 1000 bp or less in length. In one aspect, the plasmid further comprises a polyadenylation signal between the polynucleotide sequence encoding the CAR and the right homologous arm.

[0058] In general, a "CRISPR system" or "CRISPR integration system" collectively refers to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated "Cas" genes. In some embodiments, a CRISPR / Cas integration system comprises a Class 1, Class 2, or Class 3 CRISPR / Cas system. In some embodiments, one or more elements of the CRISPR system are derived from a Type I, Type II, or Type III CRISPR system. CRISPR systems are known in the art. See, e.g., U.S. Patent No. 8,697,359, incorporated herein by reference in its entirety.

[0059] An endonuclease / RNP (e.g., Cas / RNP) is composed of a recombinant endonuclease protein (e.g., Cas9, Cas12a, or CasX endonuclease) complexed with a CRISPR locus. The endonuclease complexed with a CRISPR locus may be referred to as a CRISPR / Cas guide RNA. The CRISPR locus contains a synthetic single guide RNA (gRNA) consisting of an RNA that can hybridize to a complementary repeat RNA (crRNA) and a trans-complementary repeat RNA (tracrRNA) complexed with a target sequence. Thus, the CRISPR / Cas guide RNA hybridizes to a target sequence within the genomic DNA of a cell. In some cases, the Class 2 CRISPR / Cas endonuclease is a Type II CRISPR / Cas endonuclease. In some cases, the Class 2 CRISPR / Cas endonuclease is a Cas9 polypeptide, and the corresponding CRISPR / Cas guide RNA is a Cas9 guide RNA. In some cases, the CRISPR / Cas endonuclease is a CasX polypeptide, and the corresponding CRISPR / Cas guide RNA is a CasX guide RNA. These Cas / RNPs can cleave genomic targets with higher efficiency than approaches that rely on foreign DNA because they are delivered as a functional complex. In addition, the rapid clearance of Cas / RNPs from cells can reduce off-target effects, such as the induction of apoptosis.

[0060] To generate the RNP complex, the crRNA and tracrRNA can be mixed in a 1:1, 2:1, or 1:2 ratio at a concentration of about 50 μM to about 500 μM (e.g., 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, 425 μM, 450 μM, 475 μM, or 500 μM), preferably between 100 μM and about 300 μM, most preferably about 200 μM, at 95°C for about 5 minutes to form the crRNA:tracrRNA complex (i.e., guide RNA). The crRNA:tracrRNA complex can be mixed with a Cas endonuclease (e.g., Cas9) at a final dilution of about 20 μM to about 50 μM (e.g., 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or 50 μM).

[0061] Upon binding to a target sequence in a target cell, the CRISPR locus can modify the genome by introducing one or more base pair insertions or deletions, insertion of a heterologous DNA fragment (e.g., a donor polynucleotide), deletion of an endogenous DNA fragment, inversion or translocation of an endogenous DNA fragment, or a combination thereof, into the target DNA. Thus, the disclosed method can be used to generate knockouts or knock-ins when combined with DNA for homologous recombination. Herein, it is shown that adeno-associated virus (AAV)-mediated transfer of Cas / RNPs is an efficient method that overcomes previous limitations of genetic modification in NK cells and NK T cells.

[0062] The maximum AAV packaging capacity of approximately 4.5 kilobases limits the size of the donor, including homologous arms. Thus, in certain embodiments, any transcript and any gene larger than 100 bp can have homologous arms, each of which is at least 800 bp, with many systems using 800 bp and 1000 bp asymmetric arms for a total of 1800 bp. Therefore, AAV vectors cannot carry transgenes larger than approximately 2.5 kb. In one aspect, disclosed herein is an AAV CRISPR / CAS9 nucleotide delivery system comprising a donor assembly plasmid with homology arms of 30 bp to 1000 bp, and the donor assembly plasmid may be 30 bp, 50 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp, 260 bp, 270 bp, 280 bp, 290 bp, 300 bp, 310 bp, 320 bp, 330 bp, 340 bp, 350 bp, 360 bp, 370 bp, 380 bp, 390 bp, 400 bp, 410 bp, 420 bp, 430 bp, 440 bp, 450 bp, 460 bp, 470 bp, 480 bp, 490 bp , 500bp, 510bp, 520bp, 530bp, 540bp, 550bp, 560bp, 570bp, 580bp, 590bp, 600bp, 610bp, 620bp, 63 0bp, 640bp, 650bp, 660bp, 670bp, 680bp, 690bp, 700bp, 710bp, 720bp, 730bp, 740bp, 750bp, 760bp , 770bp, 780bp, 790bp, 800bp, 810bp, 820bp, 830bp, 840bp, 850bp, 860bp, 870bp, 880bp, 890bp, 900bp, 910bp, 920bp, 930bp, 940bp, 950bp, 960bp, 970bp, 980bp, 990bp, or 1000bp.For example, the homology arms can be symmetric 30 bp homology arms, symmetric 300 bp homology arms, symmetric 500 bp homology arms, symmetric 600 bp homology arms, symmetric 800 bp homology arms, symmetric 1000 bp homology arms, or asymmetric 800 bp homology arms comprising an 800 bp left homology arm (LHA) and a 1000 bp right homology arm (RHA) for homologous recombination (HR), or no homology arms at all for non-homologous end joining using a homology-independent targeted integration (HITI) plasmid.

[0063] It is understood and contemplated herein that the homology arms can be symmetric (same length on both sides) or asymmetric (different lengths on both sides) to accommodate different transgene lengths. That is, the lengths of the homology arms can have any combination of left homology arm (LHA) length and right homology arm (RHA) length, such as LHA 30 bp and RHA 30 bp, LHA 30 bp and RHA 100 bp, LHA 30 bp and RHA 300 bp, LHA 30 bp and RHA 500 bp, LHA 30 bp and RHA 800 bp, LHA 30 bp and RHA 1000 bp, LHA 100 bp and RHA 30 bp, LHA 100 bp and RHA 30 bp, LHA 100 bp and RHA 500 bp, LHA 100 bp and RHA 800 bp, LHA 100 bp and RHA 100 bp, LHA 100 bp and RHA 30 bp, LHA 100 bp and RHA 10 ... 0bp and RHA100bp, LHA100bp and RHA300bp, LHA100bp and RHA500bp, LHA100bp and RHA800bp, LHA100bp and RHA1000bp, LHA300bp and RHA30bp, LHA300bp and RHA100bp, LHA300bp and RHA300bp, LHA300bp and RHA500bp, LHA300bp and RHA800bp, LHA30 0bp and RHA1000bp, LHA500bp and RHA30bp, LHA500bp and RHA100bp, LHA500bp and RHA300bp, LHA500bp and RHA500bp, LHA500bp and RHA800bp, LHA500bp and RHA1000bp, LHA800bp and RHA30bp, LHA800bp and RHA100bp, LHA800bp and RHA300bp, LHA80 LHA1000bp and RHA500bp, LHA800bp and RHA800bp, LHA800bp and RHA1000bp, LHA1000bp and RHA30bp, LHA1000bp and RHA100bp, LHA1000bp and RHA300bp, LHA1000bp and RHA500bp, LHA1000bp and RHA800bp, and LHA1000bp and RHA1000bp.

[0064] There are several methods for delivering DNA templates, including viral and non-viral methods. In non-viral approaches, single- or double-stranded DNA templates are typically electroporated with Cas / RNPs, but this approach is less efficient than viral transduction. For viral gene delivery, adeno-associated viruses (AAVs), including AAV6, have been safely used in clinical trials and are useful vectors for sensitive primary immune cells, including T cells. Alternatively, Cas / RNPs can be replaced with nucleic acids encoding the Cas / RNPs.

[0065] In some embodiments, the homology arms specifically hybridize to the suppressor of cytokine signaling 3 (SOCS3) locus.

[0066] The plasmids disclosed herein can also be integrated into the genome of transfected cells via HITI, CRISPaint, or other non-homologous end joining (NHEJ) techniques, thus offering the advantage of higher integration efficiency. In some examples, the NHEJ plasmid is disclosed in International Publication No. WO 2020 / 198675, the entire contents of which are incorporated herein by reference. To aid in identifying the cleavage site for transgene removal upon integration, the plasmid may contain a protospacer adjacent motif (PAM) and a crRNA (i.e., gRNA) to target the integration of the donor transgene. In some examples, in an NHEJ DNA template (e.g., a CRISPaint DNA template), a single (PAMg) or double (PAMgPAMg) Cas target sequence (e.g., Cas9, Cas12, or CasX target sequence) is integrated around the transgene (e.g., a polynucleotide encoding a CAR, such as the CD33CAR disclosed herein), but within the inverted terminal repeats (ITRs). Thus, Cas can simultaneously cleave the gDNA and the CRISPaint DNA template, allowing integration at the double-strand break in the genome.

[0067] As mentioned above, the use of AAV as a vector for delivering the disclosed CRISPR / Cas9 plasmid and any donor gene is limited to a maximum of approximately 4.5 kb. One way to increase the allowable size of the transgene is understood and contemplated herein to generate additional space by replacing the typically used Cas9 from Streptococcus pyogenes (SpCas9) with a synthetic Cas9 or Cas9 from a different bacterial source. Cas9 substitutions can also be used to increase target specificity, thereby reducing the need for gRNAs. Thus, for example, Cas9 can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus spp. (AsCpf1), Lachnospiracasebacterium (LbCpf1), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacterjejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HF1, Fokl-fused dCas9, multiplying Cas9 (xCas9), and / or catalytically inactive Cas9 (dCas9).

[0068] It is understood and contemplated herein that the disclosed plasmids and vectors can be incorporated into cells (e.g., NK cells or NK T cells) for use in employed immunotherapy, and the NK cells or NK T cells incorporating the plasmids and / or vectors express the CAR. Accordingly, disclosed herein are engineered cells (e.g., engineered NK or NK T cells) comprising any of the CARs disclosed herein, any of the plasmids disclosed herein, and / or any of the AAV vectors disclosed herein. For example, in one aspect, disclosed herein is an engineered immune cell (e.g., an engineered NK cell or NK T cell) comprising a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain comprising a STAT3 phosphorylation site (e.g., an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on a target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus. In one aspect, the CAR of the engineered cell further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0069] Prior to transduction of cells (e.g., NK cells and / or NK T cells), the cells can be incubated in a medium suitable for cell growth. It is understood and contemplated herein that the culture conditions can include the addition of cytokines, antibodies, and / or feeder cells. Thus, in one aspect, disclosed herein is a method of genetically modifying cells (e.g., NK cells and / or NK T cells), further comprising incubating the cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to transduction of the cells in a medium that supports cell growth, wherein the medium further comprises cytokines, antibodies, and / or feeder cells. For example, the medium can include IL-2, IL-12, IL-15, IL-18, and / or IL-21. In one aspect, the medium can also include an anti-CD3 antibody. In one aspect, the feeder cells can be purified from the feeder cells that stimulate the cells. For example, NK cell-stimulating feeder cells for use in embodiments disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or PBMCs, RPMI8866, HFWT, K562, K562 cells transformed with membrane-bound IL-15 and 4-1BBL, or membrane-bound IL-21 and 4-1BBL, cell membrane vesicles containing membrane-bound IL-15 and 4-1BBL, or membrane-bound IL-21 and 4-1BBL, e.g., cell membrane vesicles obtained from K562 cells, or any combination thereof, or EBV-LCL. In some aspects, the feeder cells can be provided in combination with a solution of IL-21, IL-15, and / or 4-1BBL. The feeder cells can be seeded into the cell culture at a ratio of 1:2, 1:1, or 2:1. It is understood and contemplated herein that the culture period can be 1 to 14 days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after AAV infection, preferably 3 to 7 days, and most preferably 4 to 6 days.

[0070] It is understood and contemplated herein that culture conditions for primary and expanded cells (including, but not limited to, primary and expanded NK cells and / or NK T cells) may vary. In one aspect, culture of primary NK cells or NK T cells prior to AAV infection includes medium, cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21), and / or anti-CD3 antibodies for less than 5 days (e.g., 1, 2, 3, or 4 days). For expanded NK cells, culture may be performed in the presence of NK feeder cells (e.g., at a 1:1 ratio) in addition to, or in place of, cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21) and / or anti-CD3 antibodies. Culture of expanded NK cells may be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to transduction. Thus, disclosed herein are methods for genetically modifying cells (e.g., T cells, B cells, macrophages, NK cells, NK T cells, fibroblasts, neurons, osteoblasts, hepatocytes, epithelial cells, and / or muscle cells), comprising culturing primary cells in the presence of IL-2 for 4 days prior to infection with an AAV vector and / or electroporation (if the RNP complex is introduced via electroporation), or culturing expanded cells in the presence of irradiated feeder cells for 4, 5, 6, or 7 days prior to infection with an AAV vector and / or electroporation (if the RNP complex is introduced by electroporation).

[0071] Following transduction of cells (e.g., NK cells and / or NK T cells) (e.g., by AAV infection or electroporation), the now engineered cells can be grown in medium containing feeder cells that stimulate the modified cells (e.g., NK cells and / or NK T cells). Thus, the engineered cells retain viability and proliferation potential and can be grown after AAV infection and / or electroporation using irradiated feeder cells (e.g., via electroporation when an RNP complex or nucleic acid encoding an RNP is introduced). For example, NK cell-stimulating feeder cells for use in embodiments disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or PBMCs, RPMI8866, HFWT, K562, K562 cells transformed with membrane-bound IL-15 and 4-1BBL, or membrane-bound IL-21 and 4-1BBL, cell membrane vesicles containing membrane-bound IL-15 and 41BBL, or membrane-bound IL-21 and 4-1BBL, e.g., cell membrane vesicles obtained from K562 cells, or any combination thereof, or EBV-LCL. In some aspects, NK cell feeder cells can be provided in combination with a solution of IL-21, IL-15, and / or 41BBL. Feeder cells can be seeded into NK cell cultures at a ratio of 1:2, 1:1, or 2:1. It is understood and contemplated herein that the culture period can be 1 to 14 days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) post-infection and / or electroporation, preferably 3 to 7 days, and most preferably 4 to 6 days. In some embodiments, the medium for culturing the modified NK cells can further comprise cytokines such as, for example, IL-2, IL-12, IL-15, IL-18, and / or IL-21.

[0072] In one aspect, it is understood and contemplated herein that expression of the disclosed CARs not only creates a feedback loop of CAR expression, but also promotes proliferation of NK cells and / or NK T cells, and further suppresses SOCS3-mediated suppression of NK cell and NK T cell regulators. In one aspect, disclosed herein is a method of promoting proliferation of NK cells and / or NK T cells at the site of an immune response, comprising: a) obtaining a ribonucleoprotein (RNP) complex with a CRISPR / Cas endonuclease (e.g., CasX or a class 2 CRISPR / Cas endonuclease, e.g., Cas9) and a corresponding CRISPR / Cas guide RNA, or a nucleic acid encoding the RNP complex, and an AAV vector (e.g., AAV6) comprising a plasmid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is expressed by a homologous CAR. a) introducing (e.g., by electroporation or gene transfer) a polynucleotide sequence and an RNP complex, or a nucleic acid encoding the RNP complex, into NK cells or NK T cells, wherein the polynucleotide sequence is transduced into the NK cells or NK T cells by AAV infection (multiplicity of infection (MOI) of about 5x10) 3 ~about 5x10 5a) introducing the CAR into a NK cell or NK T cell via a CAR-binding domain (e.g., a CAR-binding domain, a CD28 domain, a CD32 domain, a 4-1BB domain, or any combination thereof), wherein the RNP complex hybridizes to the suppressor of cytokine signaling 3 (SOCS3) locus in the genomic DNA of the NK cell or NK T cell, and a DNA repair enzyme in the NK cell or NK T cell inserts the transgene into the host genome at the SOCS3 locus in the genomic DNA of the NK cell or NK T cell, thereby creating an engineered NK cell or an engineered NK T cell; and b) administering the engineered NK cell or the engineered NK T cell to a subject, wherein binding of the CAR to a receptor on the target cell causes phosphorylation via the endodomain of the engineered NK cell or the engineered NK T cell, resulting in the production of more CAR on the surface of the cell and promoting the proliferation of the engineered NK cell or the engineered NK T cell. In some embodiments, the CAR further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0073] 1. Hybridization / Selective Hybridization The term hybridization typically refers to a sequence-driven interaction between at least two nucleic acid molecules, such as a primer or probe and a gene. A sequence-driven interaction refers to an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide-specific manner. For example, G interacting with C, or A interacting with T, is a sequence-driven interaction. Typically, a sequence-driven interaction occurs on the Watson-Crick or Hoogsteen face of a nucleotide. The hybridization of two nucleic acids is affected by several conditions and parameters known to those skilled in the art. For example, the salt concentration, pH, and temperature of the reaction all affect whether two nucleic acid molecules hybridize.

[0074] Parameters for selective hybridization between two nucleic acid molecules are known to those of skill in the art. For example, in some embodiments, selective hybridization conditions can be defined as stringent hybridization conditions. For example, the stringency of hybridization is controlled by both the temperature and salt concentration of either or both of the hybridization and washing steps. For example, hybridization conditions to achieve selective hybridization can involve hybridization in a high ionic strength solution (6X SSC or 6X SSPE) at a temperature approximately 12-25°C below Tm (the melting temperature at which half of the molecules dissociate from their hybridization partner), followed by washing at a temperature and salt concentration combination selected so that the wash temperature is approximately 5-20°C below Tm. Temperature and salt conditions are easily determined empirically in preliminary experiments in which a sample of reference DNA immobilized on a filter is hybridized to a target nucleic acid, followed by washing under conditions of different stringency. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. Stringency can be achieved using these conditions as described above or as known in the art. Preferred stringent hybridization conditions for DNA:DNA hybridizations are approximately 68°C (in aqueous solution) in 6X SSC or 6X SSPE, followed by a wash at 68°C. If desired, the stringency of the hybridization and wash can be reduced as the desired degree of complementarity decreases, and depending on the GC or AT richness of any region where variability is sought. If desired, the stringency of the hybridization and wash can be increased as the desired degree of complementarity increases, and depending on the GC or AT richness of any region where high complementarity is desired, all as known in the art.

[0075] Another way to define selective hybridization is by looking at the amount (percentage) of one nucleic acid bound to the other nucleic acid. For example, in some embodiments, selective hybridization conditions are when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in, for example, 10-, 100-, or 1000-fold excess. This type of assay is useful when both the limiting and non-limiting primers are in, for example, their k d or only one of the nucleic acid molecules is 10-fold, 100-fold, or 1000-fold less than their k d The reaction can be carried out under conditions exceeding 100°C.

[0076] Another way to define selective hybridization is by examining the percentage of primers that are enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments, selective hybridization conditions are when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the primers are enzymatically manipulated under conditions that promote enzymatic manipulation. For example, if the enzymatic manipulation is DNA extension, selective hybridization conditions are those in which at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or art-specific conditions appropriate for the enzyme performing the manipulation.

[0077] As with homology, it is understood that various methods for determining the level of hybridization between two nucleic acid molecules are disclosed herein. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise specified, meeting the parameters of any method will be sufficient. For example, if 80% hybridization is required, as long as hybridization occurs within the required parameters in any one of these methods, it will be considered disclosed herein.

[0078] One of skill in the art will understand that if a composition or method, either collectively or individually, meets any one of these criteria for determining hybridization, it is a composition or method disclosed herein.

[0079] 2. Nucleic acid The various molecules disclosed herein are based on nucleic acids. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, when a vector is expressed in a cell, it is understood that the expressed mRNA is typically composed of A, C, G, and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment, for example, through exogenous delivery, it is understood that it is advantageous for the antisense molecule to be composed of nucleotide analogs, which reduces degradation of the antisense molecule in the cellular environment.

[0080] Nucleotides and related molecules Nucleotides are molecules containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside linkage. The base moieties of nucleotides are adenine-9-yl (A), cytosin-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are a wide variety of these types of molecules available in the art and available herein.

[0081] A nucleotide analog is a nucleotide that contains some type of modification in either the base, sugar, or phosphate moiety. Modifications to nucleotides are well known in the art and may include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties. There are a wide variety of these types of molecules available in the art and available herein.

[0082] Nucleotide substitutes are molecules that have similar functional properties as nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNAs). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes can adopt a double helix type structure when interacting with an appropriate target nucleic acid. There are a wide variety of these types of molecules available in the art and available herein.

[0083] Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analog. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are a wide variety of these types of molecules available in the art and available herein.

[0084] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine-based nucleotide, nucleotide analog, or nucleotide substitute, and the C2, N3, and C4 positions of a pyrimidine-based nucleotide, nucleotide analog, or nucleotide substitute.

[0085] A Hoogsteen interaction is an interaction that occurs on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the reactive groups (NH or O) at the N7 and C6 positions of purine nucleotides.

[0086] B. Array There are various sequences related to protein molecules involved in the signaling pathways disclosed herein, such as CD33, 4-1BB, NKG2D, or 2B4, all of which are encoded by or are nucleic acids. Sequences of human and other analogs of these genes, as well as alleles, splice variants, and other types of variants of these genes, are available in various protein and gene databases, including Genbank. Those skilled in the art will understand how to analyze sequence discrepancies and differences and how to adjust compositions and methods related to a particular sequence to other related sequences. Primers and / or probes can be designed for any given sequence given the information disclosed herein and known in the art.

[0087] c. Primers and probes Compositions comprising primers and probes are disclosed and can interact with the disclosed nucleic acids, such as CD33, disclosed herein. In certain embodiments, primers are used to support DNA amplification reactions. Typically, primers may be extended in a sequence-specific manner. Extension of a primer in a sequence-specific manner includes any method in which the sequence and / or composition of the nucleic acid molecule to which the primer hybridizes or otherwise associates induces or influences the composition or sequence of the product produced by primer extension. Thus, extension of a primer in a sequence-specific manner includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence-specific manner are preferred. In certain embodiments, primers are used in DNA amplification reactions, such as PCR or direct sequencing. It should be understood that in certain embodiments, primers can also be extended using non-enzymatic techniques, for example, nucleotides or oligonucleotides used to extend the primer are modified so that they chemically react to extend the primer in a sequence-specific manner. Typically, the disclosed primers hybridize to the disclosed nucleic acids or regions of the nucleic acids, or they hybridize to the complement of the nucleic acid or the complement of a region of the nucleic acid.

[0088] In certain embodiments, the size of the primer or probe for interacting with a nucleic acid can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification, or simple hybridization of the probe or primer. Exemplary primers or probes comprise at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 8, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0089] In other embodiments, the primers or probes are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, It can be up to 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0090] Primers for the CD33 gene can typically be used to generate an amplified DNA product containing a region of the CD33 gene or the full-length gene, generally such that the size of the product can be accurately determined to within 3 nucleotides, or within 2 nucleotides, or within 1 nucleotide.

[0091] In certain embodiments, the product comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0092] In other embodiments, the product is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0093] 3. Delivery of Compositions to Cells Several compositions and methods exist that can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can be broadly divided into two categories: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered through several direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, and cosmids, or via the transfer of genetic material into cells or carriers, such as cationic liposomes. Suitable means for gene transfer, including viral vectors, chemical transfectants, or physical and mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, by Wolff, J.A., et al., Science, 247, 1465-1468, (1990), and Wolff, J.A. Nature, 352, 815-818, (1991). Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. In certain cases, the methods will be modified to work specifically with large DNA molecules. Furthermore, these methods can be used to target specific diseases and cell populations by using the targeting properties of the carrier.

[0094] a) Nucleic acid-based delivery systems A transfer vector can be any nucleotide construct used to deliver genes to cells (e.g., a plasmid) or used as part of a general strategy for delivering genes, for example, as part of a recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0095] As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acid without being degraded by cells and contains a promoter that drives gene expression in the delivered cells. Viral vectors include, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Sindbis virus, and other RNA viruses, including those with an HIV backbone. Also preferred are viral families that share the properties of these viruses and are suitable for use as vectors. Retroviruses include murine moloney leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as vectors. Retroviral vectors are commonly used vectors because they can carry larger genetic payloads, i.e., transgenes or marker genes, than other viral vectors. However, they are less useful in non-proliferating cells. Adenoviral vectors are relatively stable, easy to handle, highly concentrated, can be administered in aerosol formulations, and are capable of gene transfer into non-dividing cells. Poxvirus vectors are large, have several sites for gene insertion, are thermostable, and can be stored at room temperature. A preferred embodiment is a viral vector designed to suppress the immune response of the host organism caused by viral antigens. Preferred vectors of this type carry the coding region for interleukin 8 or 10.

[0096] Viral vectors can have a higher transfection capacity than chemical or physical methods for introducing genes into cells. Viral vectors typically contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and a promoter controlling the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more early genes removed, and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. This type of construct can carry up to approximately 8 kb of foreign genetic material. The necessary functions of the removed early genes are usually supplied by cell lines engineered to express the gene products of the early genes in trans.

[0097] (1) Adeno-associated virus vector Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably integrate into chromosome 19 (e.g., AAV integration site 1 (AAVS1)). Vectors with this site-specific integration property are preferred. The AAV used can be derived from any AAV serotype, including, but not limited to, AAC1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and recombinant (rAAV) AAVs such as AAV-Rh74, and / or synthetic AAVs (e.g., AAV-DJ, Anc80). AAV serotypes can be selected based on cell or tissue tropism. AAV vectors used in the disclosed compositions and methods can be single-stranded (SS) or self-complementary (SC).

[0098] Transcripts delivered via AAV vectors can be packaged as linear single-stranded (ss) DNA (ssAAV) or linear self-complementary (sc) DNA (scAAV) approximately 4.7 kb in length. The advantage of scAAV vectors is that they contain mutated inverted terminal repeats (ITRs) required for replication, helping to avoid the rate-limiting step of double-strand generation compared to ssDNA vectors. Due to the limited packaging capacity of scAAV, homology arms (HAs) of 30 bp, 300 bp, 500 bp, and 800-1000 bp have been designed for the right and left sides of the Cas9 target site, providing researchers with the ability to find optimal HA lengths and select HA lengths based on the size of the transgene. Furthermore, due to the limited packaging capacity compared to ssAAV, scAAV may not be suitable for large transgenes, such as chimeric antigen receptors (CARs) targeting CD33. Therefore, based on the size of the transgene, both ssAAV and scAAV have been designed and tested, offering a wide range of options for gene insertion in primary NK cells and / or NK T cells.

[0099] In another type of AAV virus, the AAV has a pair of inverted terminal repeats (ITRs) flanking at least one cassette having a promoter that directs cell-specific expression operably linked to a heterologous gene, heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

[0100] Typically, the AAV and B19 coding regions are deleted, resulting in a safe, non-cytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration but not cytotoxicity, and the promoter drives cell-specific expression.

[0101] Thus, the disclosed vectors provide DNA molecules that can be integrated into mammalian chromosomes without substantial toxicity.

[0102] The inserted gene in viral and retroviral systems usually contains a promoter and / or enhancer that helps control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when in a relatively fixed location with respect to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0103] It is understood that the packaging capacity of AAV is limited, and this is taken into account herein. One way to overcome the loading capacity of AAV vectors is to use two vectors, where the transgene is split between two plasmids and a 3' splice donor and 5' splice acceptor are used to join the two portions of the transgene into a single full-length transgene. Alternatively, the two transgenes can be engineered to have significant overlap, and the two segments are joined into a full-length transcript by homologous recombination.

[0104] In some embodiments, the methods disclosed herein involve infecting NK cells with an MOI of AAV ranging from about 1 to about 1000K (e.g., about 5-500K MOI). For example, the methods disclosed herein involve infecting NK cells with AAV at an MOI of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500.

[0105] 4. Expression System The nucleic acid delivered to a cell typically contains an expression control system. For example, inserted genes in viral and retroviral systems usually contain promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when it is in a relatively fixed position with respect to the transcription start site. A promoter contains core elements required for the basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0106] a) Viral promoters and enhancers Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, including the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus, or heterologous mammalian promoters such as the beta-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Of course, promoters from host cells or related species are also useful herein.

[0107] Enhancers generally refer to sequences of DNA that function at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, M. L. et al., Mol. Cell Bio. 3:1108 (1983)). Furthermore, enhancers can be located within introns (Banerji, J. Lett. et al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, T. F. et al., Mol. Cell Bio. 4:1293 (1984)). They are typically 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers often also contain response elements that mediate transcriptional regulation. Promoters may also contain response elements that mediate transcriptional regulation. Enhancers often determine the regulation of gene expression. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but typically, enhancers from eukaryotic cell viruses will be used for general gene expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0108] The promoter and / or enhancer can be specifically activated either by light or by specific chemical events that trigger their function. The system can be regulated by agents such as tetracycline and dexamethasone. There are also ways to enhance gene expression of viral vectors by exposure to radiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0109] In certain embodiments, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in certain types of cells at certain times. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, the cytomegalovirus (full-length promoter), and the LTR of retroviral vectors.

[0110] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0111] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) may also contain sequences necessary for transcription termination, which can affect mRNA expression. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also includes a transcription termination site. The transcription unit preferably also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. A homologous polyadenylation signal is preferably used in transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of approximately 400 bases. The transcription unit also preferably contains other standard sequences that, alone or in combination with the above sequences, improve expression or stability from the construct.

[0112] b) Marker Viral vectors can contain nucleic acid sequences encoding marker products. This marker product is used to determine whether the gene has been delivered to a cell and is being expressed once delivered. Preferred marker genes are the E. coli lacZ gene (encoding β-galactosidase) and green fluorescent protein.

[0113] In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. There are two widely used categories of selection regimes. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independently of a supplemented medium. Two examples include CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without added nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium is to alter the growth requirements of cells lacking the respective genes by introducing an intact DHFR or TK gene: Individual cells not transformed with the DHFR or TK gene will not be able to survive in unsupplemented medium.

[0114] The second category is dominant selection, which refers to selection schemes that can be used with any cell type and do not require the use of mutant cell lines. These schemes typically use drugs to stop host cell growth. Those cells carrying the novel gene will express a protein that conveys drug resistance and survive selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P. Science 209:1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). Three examples use bacterial genes under eukaryotic control to convey resistance to the appropriate drugs, G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0115] 5. Peptides a) Protein variants Protein variants and derivatives are well understood by those skilled in the art and can include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional variants. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions will usually be smaller than those of amino- or carboxyl-terminal fusions, e.g., on the order of one to four residues. Derivatives of immunogenic fusion proteins, such as those described in the Examples, are generated by fusing a polypeptide of sufficient size to confer immunogenicity to a target sequence by in vitro crosslinking or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about two to six residues are deleted at any one site within the protein molecule. These variants are typically prepared by site-directed mutagenesis of nucleotides within the DNA encoding the protein, thereby producing DNA encoding the variant, followed by expression of the DNA in recombinant cell culture. Well-known techniques for making substitution mutations at predetermined sites in DNA with a known sequence include M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues but can occur at several different locations at once; insertions are usually on the order of about 1 to 10 amino acid residues, and deletions range from about 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final construct. Mutations should not shift the sequence out of reading frame and preferably do not create complementary regions that could produce secondary mRNA structure. Substitution variants are those in which at least one residue has been removed and a different residue has been inserted in its place. Such substitutions are generally made in accordance with Tables 1 and 2 below, and are referred to as conservative substitutions. TIFF2025535720000001.tif111170TIFF2025535720000002.tif110170

[0116] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., by selecting residues that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains. The substitutions generally expected to result in the greatest changes in protein properties would be those resulting in (a) a hydrophilic residue, e.g., seryl or threonyl, being substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline being substituted for (or by) any other residue; (c) a residue with an electropositive side chain, e.g., lysyl, arginyl, or histidyl, being substituted for (or by) an electronegative residue, e.g., glutamyl or aspar; or (d) a residue with a bulky side chain, e.g., phenylalanine, being substituted for a residue without a side chain, e.g., glycine in this case; or (e) increasing the number of sites for sulfation and / or glycosylation.

[0117] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue with another, or replacing one polar residue with another. Substitutions include, for example, combinations of glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine, and the like. Such conservatively substituted variations of each explicitly disclosed sequence are included in the mosaic polypeptides provided herein.

[0118] Substitutional or deletional mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteines or other labile residues may also be desirable. Deletion or substitution of potential proteolysis sites, e.g., Arg, is accomplished, for example, by deleting one of the basic residues or substituting one with a glutaminyl or histidyl residue.

[0119] Certain post-translational derivatizations are the result of the action of recombinant host cells on expressed polypeptides. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the O-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] ), acetylation of N-terminal amines, and, in some cases, amidation of C-terminal carboxyls.

[0120] It should be understood that one way of defining variants and derivatives of the proteins disclosed herein is by defining them in terms of homology / identity to a particular known sequence. Specifically disclosed are variants of these and other proteins disclosed herein that have at least 70%, 75%, 80%, 85%, 90%, or 95% homology to the set forth sequence. Those skilled in the art can readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0121] Other methods for calculating homology can be performed using published algorithms. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0122] For nucleic acids, the same type of homology can be obtained, for example, by the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, and Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0123] It is understood that the descriptions of conservative variation and homology can be combined together in any combination, such as in an embodiment where a variant has at least 70% homology to a particular sequence, where the variant is a conservative variation.

[0124] Because this specification discusses various proteins and protein sequences, it is understood that the nucleic acids capable of encoding those protein sequences are also disclosed. This would include all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that encodes a particular protein sequence, as well as all nucleic acids containing degenerate nucleic acids that encode disclosed variants and derivatives of a protein sequence. Thus, while each specific nucleic acid sequence may not be described herein, it is understood that each and every sequence is actually disclosed and described herein through the disclosed protein sequences. It is also understood that, although there is no amino acid sequence that represents the specific DNA sequence encoding the protein in an organism in which a particular variant of a disclosed protein is disclosed herein, known nucleic acid sequences encoding that protein are also known, disclosed, and described herein.

[0125] It is understood that there are numerous amino acid and peptide analogs that can be incorporated into the disclosed compositions. For example, there are numerous D-amino acids or amino acids with different functional substituents than those shown in Tables 1 and 2. Opposite stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogs, are disclosed. These amino acids can be readily incorporated into a polypeptide chain by charging a tRNA molecule with the amino acid of choice and engineering a genetic construct that inserts the analog amino acid into the peptide chain in a site-specific manner, e.g., using an amber codon.

[0126] Molecules can be produced that resemble peptides but are not linked via natural peptide bonds. For example, amino acid or amino acid analog linkages can include CH2NH--, --CH2S--, --CH2--CH2--, --CH=CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CHH2SO-- (these and others are described in Spatola, A. F., In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends PharmSci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185(1979)(--CH2NH--,CH2CH2--);Spatola et al.Life Sci38:1243-1249(1986)(--CHH2--S);Hann J.Chem.Soc Perkin Trans.I307-314(1982)(--CH--CH--,cis and trans);Almquist et al.J.Med.Chem.23:1392-1398(1980)(--COCH2--);Jennings-White et al.Tetrahedron Lett 23:2533(1982)(--COCH2--);Szelke et al.European Appln,EP 45665 CA (1982):97:39405 (1982) (--CH(OH)CH--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH--); and Hruby Life Sci 31:189-199 (1982) (--CH--S--), each of which is incorporated herein by reference.A particularly preferred non-peptide bond is --CH2NH--. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0127] Amino acid analogs and analogs, as well as peptide analogs, often have more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broader spectrum of biological activity), reduced antigenicity, etc.

[0128] Because D-amino acids are not recognized by peptidases and the like, D-amino acids can be used to generate more stable peptides. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together. This can be beneficial for constraining peptides into a particular conformation.

[0129] 6. Pharmaceutical Carriers / Pharmaceutical Product Delivery As mentioned above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier will necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as will be well known to those skilled in the art.

[0130] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically (including topical intranasal administration or administration by inhalant), etc. As used herein, "topical intranasal administration" means delivery of a composition to the nose and nasal passages via one or both nostrils and can include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalant can be through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, etc. Thus, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0131] Parenteral administration of compositions, when used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution in liquid prior to injection, or as emulsions. A recent revision of parenteral administration approaches involves the use of slow or sustained release to maintain a constant dosage. See, for example, U.S. Patent No. 3,610,795, incorporated herein by reference.

[0132] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992), and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in pathways of either constitutive or ligand-induced endocytosis. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0133] a) Pharmaceutically acceptable carrier The present compositions comprising antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0134] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the administered composition.

[0135] Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0136] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetic agents, etc.

[0137] The pharmaceutical compositions can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, or intranasal), oral, inhalation, or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0138] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0139] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0140] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0141] Some of the present compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and arylamines, and substituted ethanolamines.

[0142] b) Therapeutic use Effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. The dosage range for administering the compositions is large enough to produce the desired effect of affecting the symptoms of the disorder. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. Generally, dosages will vary depending on the patient's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosages can be adjusted by individual physicians in the event of any contraindications. Dosages can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceutical products. For example, guidance in selecting an appropriate dose for an antibody can be found in literature on the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and 303-357, and Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) 365-389. A typical daily dosage of an antibody used alone can range from about 1 μg / kg body weight up to 100 mg / kg body weight, or more, depending on the factors mentioned above.

[0143] C. Methods of Treating Cancer Cancer immunotherapy has advanced in recent years. Genetically modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells that have been successfully deployed in cancer immunotherapy. While these cells were recently approved by the FDA for the treatment of CD19+ B-cell malignancies, success to date has been limited to diseases with a small number of targetable antigens, and targeting such a limited antigen repertoire is prone to failure due to immune escape. Furthermore, CAR T cells have focused on the use of autologous T cells due to the risk of graft-versus-host disease caused by allogeneic T cells. In contrast, natural killer (NK) cells can kill tumor targets in an antigen-independent manner and do not cause graft-versus-host disease (GvHD), making them excellent candidates for cancer immunotherapy. It is understood and contemplated herein that the disclosed plasmids and methods can be used, for example, to generate cancer-targeting chimeric antigen receptor natural killer (CAR NK) T cells and chimeric antigen receptor natural killer (CAR NK) cells. The disclosed plasmids, chimeric antigen receptor natural killer (CAR NK) T cells, and chimeric antigen receptor natural killer (CAR NK) cells can be used to manufacture or prepare a medicament for the treatment of cancer and / or metastasis. For example, one aspect of the present disclosure includes the use of engineered immune cells, such as engineered NK cells or NK T cells, in the preparation or manufacture of a medicament for the treatment of cancer, wherein the engineered immune cells comprise a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain (e.g., an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain) containing a STAT3 phosphorylation site, a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on a target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus.In one aspect, the CAR of the engineered cells used in the disclosed methods further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0144] Similarly, disclosed herein are methods for treating, reducing, decreasing, suppressing, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS) and the like), the methods comprising administering to a subject having cancer any of the modified cells (e.g., engineered NK cells and NK T cells) disclosed herein. Accordingly, in one aspect, disclosed herein are methods for treating, suppressing, reducing, decreasing, suppressing, ameliorating, or preventing cancer and / or metastasis in a subject (e.g., leukemia, including but not limited to acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS) and the like), the methods comprising administering to the subject a therapeutically effective amount of engineered immune cells (e.g., engineered NK cells or NK T cells). The disclosed methods involve administering to target cells (e.g., T cells), wherein the engineered immune cells comprise a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain comprising a STAT3 phosphorylation site (e.g., an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain), a transmembrane domain (e.g., a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain), and a single-chain variable fragment (scFV) that specifically binds to a receptor (e.g., CD33) on the target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus. In one aspect, the CAR of the engineered cells used in the disclosed methods further comprises a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof).

[0145] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, a reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to native or control levels.

[0146] "Reduce" or other forms of this term, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value; in other words, it is relative, but it is understood that reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0147] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction and therefore does not require a comparison to a control. As used herein, something may be reduced but not prevented, although something that is reduced may be prevented. Similarly, something may be prevented but not reduced, although something that is prevented may be reduced. It is understood that where reduction or prevention is used, the use of other words is expressly disclosed unless specifically specified otherwise.

[0148] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. In one aspect, a subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0149] The term "therapeutically effective" refers to the amount of the composition used being sufficient to alleviate one or more causes or symptoms of a disease or disorder. Such alleviation need only be a reduction or alteration, not elimination.

[0150] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder; preventative treatment, i.e., treatment directed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed at ameliorating the associated disease, condition, or disorder.

[0151] As noted above, the plasmids, vectors, engineered NK cells and NK T cells disclosed herein can be used to treat, suppress, reduce, decrease, ameliorate, or prevent cancer. A representative, but non-limiting list of cancers that can be treated using the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), myelodysplastic syndromes (MDS), myeloid leukemia (including, but not limited to, acute myeloid leukemia (AML) and chronic myeloid leukemia (CML)), bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, cancer of the mouth, pharynx, larynx, and squamous cell carcinoma of the lung, cervical cancer, carcinoma), breast cancer, as well as epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0152] In one aspect, the engineered cells (e.g., NK cells and / or NK T cells) used in the immunotherapy methods of the present disclosure can be primary cells from a donor source (e.g., an allogeneic or autologous donor source for adoptive transfer therapy (i.e., the ultimate recipient of the modified cells), a cell line (including, but not limited to, the NK cell lines NKRPMI8866, HFWT, K562, and EBV-LCL), or a source of expanded cells derived from a primary cell source or cell line. While primary cells can be used, it is understood and contemplated herein that the disclosed modifications of cells can occur ex vivo or in vitro.

[0153] While the disclosed treatments provide a complete and effective method for treating cancer, it is understood and contemplated herein that a comprehensive treatment plan may include the administration of additional anticancer agents. Accordingly, the disclosed treatments include abemaciclib, abiraterone acetate, avitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ad-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), afatinib dimalate, Afinitor (everolimus), and Aquinzeo (netpitant and palonosetron hydrochloride). , Aldara (imiquimod), Aldesulquin, Alecenza (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copalisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Ambochlorin chlorambucil, amifostine, aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidro) disodium nitrate), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab) Ozogamicin), bevacizumab, bexarotene, Bexar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Brincito (blinatumomab), bortezomib, Bosurif (bosutinib), bosutinib, brentaximab vedotin, brigatinib, Bumel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF,Campus (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Caracca (topical fluorouracil), carboplatin, carboplatin-Taxol, carfilzomib, Carumbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine , Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clor (clofarabine), CMF, cobimetinib, Cometrik (cabozantinib-S-maleate), copanilisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmigen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyphos (ifosfamide), Ciramaza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytaxan (cyclo Fosfamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denyluekin diftitoxin, denosumab, Depocyte (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutaximab, docetaxel, Doxil (doxorubicin hydrochloride liposome) doxorubicin hydrochloride, doxorubicin hydrochloride liposome, DoxSL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), darvalamab, Efudex (fluorouracil-topical), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab),Eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase Erwinia chrysanthemi), Essiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Ebacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fareston (toremifene), Faridak (panobinostat) t), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (pralatexate), FU -LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nanovalent vaccine), Gadiva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimalate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), Glucarpi Dase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nanovalent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper CVAD, Ibrance (palbociclib), ibrutumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride,Idelalisib, Idefifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidom (ifosfamide), IL-2 (aldesluikine), imatinib mesylate, Imbrivica (ibrutinib), Imfinzi (darvalamab), imiquimod, Imlizic (talimogene laherparepec), Inrita (axitinib), inotuzumab ozogamicin, interferon alfa-2b, recombinant, interleukin-2 (aldesluikine), Intron A (recombinant interferon alfa-2b) ), iodine I131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib acid), JEB, Jeftana (cabazitaxel), Kadsila (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Muria (tisagenlecleucel), Cyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Raltruvo (oratumumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), luprolide acetate, Lustatin (cladribine), Revlan (aminolevulinic acid), Rhinfolidine (chlorambucil), Lipodox (doxorubicin hydrochloride liposomal), lomustine, Lonsuf (trifluridine and tyrosine) Piracil hydrochloride), Lupron (leuprorelin acetate), Lupron Depot (leuprorelin acetate), Lupron Depo-Ped (leuprorelin acetate), Lynparza (olaparib), Marquibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), metazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate),Methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Myrosal (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine) tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib malate, Nerlynx (neratinib malate), netupitant and palonosetron hydrochloride, Nulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab , Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaparib, omacetaxine mepescicinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onibide (irinotecan hydrochloride liposomal), Ontak (denilquindiftitoxin), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netopitant, pamidro disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Interon (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide),Ponatinib hydrochloride, Portorazza (necitumumab), pralatexate, prednisone, procarbazine hydrochloride, Prolequin (aldesulquin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipulcel-T), prinetol (mercapto, toprine), Pulixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Lilyst (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R- ICE, Rituxan (rituximab), Rituxan Hycera (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase human, rolapitant hydrochloride, romidepsin, romiplast, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Ridapt (midostaurin), Skerosol intrapleural aerosol (talc), siltoximab, Sipulcel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Scalp Pricel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc), Stivalga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Silatron (peginterferon alfa-2b), Silvant (siltoximab), Synribo (omacetaxine mepescicinate), Tabloid (thioguanine), TAC, Tafinra (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tesenriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Talimoide (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Trac (fluorouracil-topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I131 tositumomab, Totect (dexrazoxane hydrochloride),TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbi (rolapitan hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Belsar (vinblastine sulfate), Vem Rafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine liposomal sulfate, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxase (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin) Hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (dibasic aflibercept), Zarxio (filgrastim), Zejula (nirapaributyric acid monohydrate), Zelboraf (bevacizumab), Therapies may also include administration of any anti-cancer therapy known in the art, including, but not limited to, rafenib, Zevalin (ibrutumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Therapies may include those targeting PD-1 (pembrolizumab, nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559),MPDL3280A, or MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016), or further including, but not limited to, checkpoint inhibitors.

Claims

1. A chimeric antigen receptor (CAR) comprising an endodomain containing a STAT3 phosphorylation site, a transmembrane domain, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell.

2. The CAR of claim 1, wherein the endodomain comprises an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain.

3. The CAR of claim 1 or 2, wherein the transmembrane domain of the CAR comprises a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

4. The CAR according to any one of claims 1 to 3, further comprising a costimulatory domain.

5. The CAR of claim 4, wherein the costimulatory domain of the CAR comprises a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof.

6. The CAR according to any one of claims 1 to 5, wherein the scFv of the CAR specifically binds to CD33.

7. A plasmid encoding the CAR according to any one of claims 1 to 6.

8. 8. The plasmid of claim 7, wherein the plasmid is used in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) integration system, and further comprises, in order, a left homologous arm, a polynucleotide sequence encoding the CAR, and a right homologous arm, each of which is 1000 bp or less in length, and which specifically hybridizes to adeno-associated virus integration site 1 (AAVS1) of suppressor of cytokine signaling 3 (SOCS3) locus.

9. 9. The plasmid of claim 8, wherein the CRISPR / Cas integration system comprises a Class 2 CRISPR / Cas integration system.

10. 10. The plasmid of claim 9, wherein the Class 2 CRISPR / Cas system comprises an integrated CRISPR / Cas9 system.

11. The plasmid of any one of claims 7 to 10, wherein the plasmid further comprises a polyadenylation signal between the polynucleotide sequence encoding the CAR and the right homologous arm.

12. The plasmid according to any one of claims 7 to 10, wherein the left homologous arm and the right homologous arm are of different lengths.

13. The plasmid according to any one of claims 7 to 10, wherein the left homologous arm and the right homologous arm are the same length.

14. 14. The plasmid of any one of claims 7 to 13, wherein the homology arms are 30 bp, 300 bp, 600 bp, or 1000 bp in length.

15. An adeno-associated virus (AAV) vector comprising the plasmid of any one of claims 7 to 14.

16. The AAV vector of claim 15, wherein the AAV serotype comprises AAV6.

17. 17. The AAV vector of claim 15 or 16, wherein the vector further comprises a plasmid encoding crRNA, a tracer RNA (tracrRNA), and a CAS endonuclease.

18. The AAV vector according to any one of claims 15 to 17, wherein the vector is a single-stranded AAV (ssAAV).

19. The AAV vector according to any one of claims 15 to 17, wherein the vector is a self-complementary AAV (scAAV).

20. An engineered cell comprising the CAR of any one of claims 1 to 6, the plasmid of any one of claims 6 to 13, or the AAV vector of any one of claims 14 to 18.

21. 21. The engineered cell of claim 20, wherein the cell comprises a natural killer (NK) cell or an NK T cell.

22. 1. An engineered immune cell comprising a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain comprising a STAT3 phosphorylation site, a transmembrane domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus of the immune cell.

23. 23. The engineered cell of claim 22, wherein the endodomain comprises an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain.

24. 24. The engineered cell of claim 22 or 23, wherein the transmembrane domain of the CAR comprises a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

25. 25. The engineered cell of any one of claims 22-24, wherein the CAR further comprises a costimulatory domain.

26. 26. The engineered cell of claim 25, wherein the costimulatory domain of the CAR comprises a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof.

27. 27. The engineered cell of any one of claims 22 to 26, wherein the scFv of the CAR specifically binds to CD33.

28. 28. The engineered cell of any one of claims 22 to 27, wherein the engineered cell comprises a NK cell or a NK T cell.

29. 29. A method of treating cancer in a subject, comprising administering to said subject an engineered cell of any one of claims 21 to 28.

30. 1. A method of treating cancer in a subject, comprising administering to the subject engineered immune cells comprising a transgene encoding a chimeric antigen receptor (CAR) comprising an endodomain comprising a STAT3 phosphorylation site, a transmembrane domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell, wherein the transgene is inserted into the suppressor of cytokine signaling 3 (SOCS3) locus.

31. 31. The method of treating cancer of claim 30, wherein the endodomain comprises an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain.

32. 32. The method of treating cancer of claim 30 or 31, wherein the transmembrane domain of the CAR comprises a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

33. The method for treating cancer according to any one of claims 30 to 32, wherein the CAR further comprises a costimulatory domain.

34. 34. The method of treating cancer of claim 33, wherein the costimulatory domain of the CAR comprises a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof.

35. The method of treating cancer according to any one of claims 30 to 34, wherein the scFv of the CAR specifically binds to CD33.

36. 36. The method of treating cancer according to any one of claims 29 to 35, wherein the engineered cells are natural killer (NK) cells or NK T cells.

37. 37. The method of treating cancer according to any one of claims 29 to 36, wherein the cancer comprises leukemia.

38. 1. A method for increasing proliferation of NK cells and / or NK T cells at the site of an immune response in a subject, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a CRISPR / Cas endonuclease complexed with a corresponding CRISPR / Cas guide RNA, or a nucleic acid encoding the RNP complex, and an AAV vector comprising a plasmid comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by homologous arms, and the length of the homologous arms is 800 bp or less, and the CAR comprises an endodomain comprising a STAT3 phosphorylation site, a transmembrane domain, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell; b) introducing the polynucleotide sequence and the RNP complex, or a nucleic acid encoding the RNP complex, into the NK cell or NK T cell, wherein the RNP complex hybridizes to the suppressor of cytokine signaling 3 (SOCS3) locus in the genomic DNA of the NK cell or NK T cell, and wherein DNA repair enzymes in the NK cell or NK T cell insert the polynucleotide sequence into the host genome at the SOCS3 locus in the genomic DNA of the NK cell or NK T cell, thereby producing an engineered NK cell or an engineered NK T cell; and c) administering the engineered NK cells or the engineered NK T cells to a subject, wherein when the CAR binds to a receptor on the target cells, phosphorylation of the engineered NK cells or the engineered NK T cells occurs via the endodomain, resulting in the production of more CAR on the surface of the cells and proliferation of the engineered NK cells or the engineered NK T cells.

39. 39. The method of claim 38, wherein the endodomain comprises an IL-10, IL-6, IFN-λR1, IL-2R, or IL-21 endodomain.

40. 40. The method of claim 38 or 39, wherein the transmembrane domain of the CAR comprises a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

41. The method of any one of claims 38 to 40, wherein the CAR further comprises a costimulatory domain.

42. 42. The method of claim 41, wherein the costimulatory domain of the CAR comprises a 2B4 domain, a CD28 costimulatory domain, a CD32 domain, a 4-1BB costimulatory domain, or any combination thereof.

43. The method of any one of claims 38 to 42, wherein the scFv of the CAR specifically binds to CD33.

44. 44. The method of any one of claims 38 to 43, wherein the cells are infected with the AAV at a multiplicity of infection (MOI) of about 5 to about 500,000.

45. The method of any one of claims 38 to 44, wherein the RNP complex is introduced into the cell via infection with AAV.

46. 45. The method of any one of claims 38 to 44, wherein the RNP complex is introduced into the cell via electroporation.

47. 47. The method of any one of claims 38 to 46, wherein the RNP complex is introduced into the cell via gene transfer, and the RNP complex is encoded on the same or a different AAV.

48. 48. The method of any one of claims 38 to 47, wherein the CRISPR / Cas endonuclease is a class 2 CRISPR / Cas endonuclease.

49. 49. The method of Claim 48, wherein the Class 2 CRISPR / Cas endonuclease is a Cas9 endonuclease.

50. 50. The method of any one of claims 38 to 49, wherein the left homologous arm and the right homologous arm are the same length.

51. 51. The method of claim 50, wherein the homology arms are 600 bp or less in length.

52. The method of any one of claims 38 to 49 and 51, wherein the left homologous arm and the right homologous arm are different in length.

53. The method of any one of claims 38 to 52, wherein the AAV serotype comprises AAV6.

54. 54. The method of any one of claims 38 to 53, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

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