Design and Use of Gene-Targeted Antibody Fusion Proteins to Perform Therapeutic Gene Editing in the Body

Abstract

Embodiments of the present disclosure relate to methods and compositions for therapy using CRISPR / Cas9 / targeting antibody / gene-targeting small guide RNA (sgRNA) fusion protein / ribonucleotide complexes. Targeting a polynucleotide of interest, such as a gene, can be effective in eradicating tumors in mammals. In some cases, sgRNA ribonucleotides with sequences designed to target any gene or genomic region are envisioned. In some embodiments, targeting SRC-3, particularly in T regulatory cells, is effective in eradicating tumors in mammals. In certain instances, T regulatory cells undergo CRISPR-based genetic modification in vivo, eliminating the need for adoptive cell therapy. In some cases, a different antibody recognizing another cell surface antigen is used instead of a CD25-targeting antibody. In some cases, sgRNA ribonucleotides with sequences designed to target any gene or genomic region are described.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 377,618, filed September 29, 2022, and U.S. Provisional Patent Application No. 63 / 377,616, filed on the same date, the entire contents of which are incorporated herein by reference.

[0002] Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, immunology and medicine, including cancer care, inborn errors of metabolism, hematopoietic disorders, and / or genetic disorders affecting liver function. [Background technology]

[0003] Immunotherapy for cancer and other diseases represents a promising advance in medicine. There is a need for methods and compositions that specifically act on desired targets, particularly those that act outside the cells of interest and within their nuclei. The present disclosure fulfills this long-standing need in the art. Summary of the Invention

[0004] An embodiment of the present disclosure includes a fusion protein-ribonucleotide complex comprising a fusion protein and at least one RNA, wherein the fusion protein comprises at least one antibody or antibody fragment, and at least one RNA-guided DNA endonuclease.

[0005] Embodiments of the present disclosure include a fusion protein-ribonucleotide complex comprising a fusion protein and at least one RNA targeting SRC-3, wherein the fusion protein comprises at least one antibody or antibody fragment (e.g., scFv), and at least one RNA-guided DNA endonuclease.

[0006] The RNA-guided DNA endonuclease may be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, or a functional derivative thereof. In certain embodiments, the RNA comprises a sequence complementary to one or more polynucleotide targets of interest. In some embodiments, the RNA comprises a sequence complementary to SRC-3. In some embodiments, the fusion protein further comprises a linker region between the antibody or antibody fragment and the RNA-guided DNA endonuclease. The fusion protein may further comprise one or more purification tags, such as a His tag, a FLAG tag, HA, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, an albumin-binding protein, an AU1 epitope, an AU5 epitope, a biotin-carboxy carrier protein, a galactose-binding protein, glutathione S-transferase, Halotag, a streptavidin-binding peptide, or a tandem affinity purification. In some embodiments, the fusion protein may further comprise one or more proteases, such as tobacco etch virus (TEV) protease, enterokinase, PreScission protease, enteropeptidase, thrombin, 3C protease, or Factor Xa. In some embodiments, the antibody fragment is an scFv. In certain embodiments, the antibody or antibody fragment targets a cell surface protein or a cancer antigen. In certain embodiments, the antibody or antibody fragment targets CD25, CD19, CD4, HER2, PD-1, CTLA-4, TGFBR, or EGFR.In some embodiments, the one or more polynucleotide targets of interest are oncogenes, tumor suppressors, proliferation or growth-related genes, invasion or metastasis-related genes, or genes involved in epithelial-mesenchymal transition. Examples of the one or more polynucleotide targets of interest include PTEN, PIK3CA, TP53, VIM, TWIST1, ESR1, or MYC.

[0007] Embodiments of the present disclosure include polynucleotides encoding the fusion proteins, expression constructs comprising at least one nucleotide sequence encoding the fusion protein and the RNA, cells expressing any component of the complex, and / or therapeutic compositions comprising the complex, which may be packaged in a pharmaceutically acceptable carrier.

[0008]

[0010] Embodiments of the present disclosure include methods of treating an individual, comprising administering to the individual a therapeutically effective amount of the complex, polynucleotide, expression construct, therapeutic composition, and / or cells producing all or a portion of the complex. In some embodiments, the individual has or is suspected of having breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, pancreatic cancer, myeloid tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, lung cancer, peritoneal cancer, gastric cancer or tumors, cervical cancer, liver cancer, bladder cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, renal cancer or kidney cancer, vulvar cancer, head and neck cancer, or malignant melanoma. In certain embodiments, the individual has or is suspected of having cancer, including cancers involving cells that express SRC-3. In some embodiments, the individual has or is suspected of having an inflammatory disease, such as allergy, asthma, an autoimmune disease, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, transplant rejection, ankylosing spondylitis (AS), gout, myositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, pelvic inflammatory disease, or vasculitis. In some embodiments, the individual has or is suspected of having a genetic disorder, optionally an inborn error of metabolism, comprising glycogen storage disease, G6PD deficiency, phenylketonuria, maple syrup urine disease, glutaric acidemia type I, carbamoyl phosphate synthase I deficiency, alkaptonuria, mixed malonic and methylmalonic aciduria, 2-hydroxyglutaric aciduria, medium-chain acyl-coenzyme A dehydrogenase deficiency, acute intermittent porphyria, Resch-Nyhan syndrome, lipid-mediated congenital adrenal hyperplasia, congenital adrenal hyperplasia, Cahn-Sayre syndrome, Zellweger syndrome, Gaucher disease, or Niemann-Pick disease.

[0009] In certain embodiments, the cell that produces all or part of the complex is an immune cell such as a T cell, a natural killer (NK) cell, an NKT cell, a B cell, a macrophage, a dendritic cell, or a mixture thereof.

[0010] In certain embodiments, administering to an individual a therapeutically effective amount of the complex, polynucleotide, expression construct, therapeutic composition, and / or cells producing all or part of the complex induces destruction of SRC-3 in immune cells, such as T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, dendritic cells, or mixtures thereof, which may include CD4+ cells, CD25+ cells, and / or FOXP3+ cells.

[0011] In some embodiments, administration is by intravenous, intraperitoneal, intraarterial, topical, inhalation, intramuscular, intrasternal, intraarticular injection, or infusion. Some methods include further administering a second therapeutic intervention, such as surgery, radiation therapy, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof. The second therapeutic intervention may be administered substantially simultaneously with or substantially sequentially to the complex, polynucleotide, expression construct, therapeutic composition, and / or cells producing all or a portion of the complex.

[0012] The complex, polynucleotide, expression construct, therapeutic composition, and / or cells producing all or a portion of the complex may be administered intravenously, intraperitoneally, intra-arterially, topically, by inhalation, intramuscularly, intrasternal, intra-articular injection, or infusion. In some embodiments, the method further comprises administering a second therapeutic intervention, such as surgery, radiation therapy, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof. The second therapeutic intervention may be administered substantially simultaneously with or substantially sequentially to the complex, polynucleotide, expression construct, therapeutic composition, and / or cells producing all or a portion of the complex.

[0013] Embodiments of the present disclosure include methods for producing the complex, including the steps of: a. expressing a fusion protein; b. expressing an RNA; and c. contacting the fusion protein with the RNA. The method may further include purifying the fusion protein, for example, by affinity chromatography. The method may also further include purifying the RNA.

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be more clearly understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows a schematic and sequence details illustrating the construction of an exemplary CRISPR / Cas9 / targeted antibody fusion protein, which in some embodiments is designed for intravenous administration for in vivo gene deletion. The modular components that make up this fusion protein or the cDNA that expresses it include, in some embodiments, a histidine tag (6His tag), a cell-penetrating peptide (CPP), a single-chain variable fragment (scFv) that binds to the CD25 (gene symbol: IL2RA) protein expressed on the surface of regulatory T cells (Tregs), a CRISPR / Cas nuclease protein, a tobacco etch virus (TEV) protease, and a FLAG tag, which are linked via various linker domains to be expressed as a single polypeptide. [Figure 2]Figure 2 shows an example of the production of a CRISPR / Cas9 / CD25-targeting antibody / SRC-3 gene-targeting sgRNA polypeptide / ribonucleotide complex, which is expressed in E. coli or other bacteria, yeast-based expression systems (including Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe), the T7 RNA polymerase-coupled translation (TNT) system, or HEK293T or other mammalian cell expression systems. The SRC-3 gene is merely one example. [Figure 3] Figure 3 shows an example of a route of administration for a CRISPR / Cas9 / CD25-targeting antibody / SRC-3 gene-targeting sgRNA polypeptide / ribonucleotide complex for any type of therapeutic use, with the SRC-3 gene being just one example. DETAILED DESCRIPTION OF THE INVENTION

[0016] [I. Definition] Throughout this specification, the term "about" is meant to encompass the inherent variation of error in any measuring or quantification technique.

[0017] The words "a" or "an," when used in conjunction with the term "comprising," can mean "one," but are also consistent with "one or more," "at least one," or "one or more than one."

[0018] The phrase "and / or" means "and" or "or." For example, A, B, and / or C includes A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together. In other words, "and / or" functions as an inclusive disjunction.

[0019] The words "comprising" (and its variations "comprise" and "comprises"), "having" (and its variations "have" and "has"), "including" (and its variations "includes" and "include") or "containing" (and its variations "contains" and "contain") are inclusive or open-ended and do not exclude the presence of other additional elements or steps.

[0020] Any compositions and / or steps disclosed throughout this specification may be comprised of either "comprises," "consist essentially of," or "consist of" in compositions and methods for their use. Compositions and methods that "consist essentially of" are limited to the specified materials or steps that do not materially affect the basic and novel characteristics of the subject matter of the present disclosure.

[0021] As used herein, a "protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. As used herein, the term "wild-type" refers to a version of an endogenous molecule that occurs naturally in an organism. In some embodiments, a modified / mutant protein or polypeptide has at least one modified activity or function (it is recognized that a protein or polypeptide can have multiple activities or functions). It is expressly contemplated that a modified / mutant protein or polypeptide, while altered with respect to one activity or function, retains wild-type activity or function in other respects, such as immunogenicity.

[0022] As used throughout this specification, the terms "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0023] As used herein, the terms "disruption" or "alteration" refer to the elimination, reduction, or increase in the expression of one or more gene products in a cell compared to the expression of the gene product in the absence of the alteration. Examples of gene products include the mRNA and protein products encoded by the gene. The alteration may be transient or reversible in some cases and permanent in other cases. The alteration may be to a functional or full-length protein or mRNA, even if a truncated or loss-of-function product is produced.

[0024] As used herein, the term "pharmaceutically acceptable carrier" includes any aqueous solvent (e.g., injectable solvents such as water, alcohol / water mixtures, saline, sodium chloride, Ringer's dextrose, etc.), non-aqueous solvent (e.g., injectable organic esters such as propylene glycol, polyethylene glycol, vegetable oils, and ethyl olein), dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases), isotonicity agent, absorption delaying agent, salt, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, coloring agent, fluid and nutrient replenisher, and similar substances and combinations thereof, known to those skilled in the art. The pH and precise concentration of the various components in the therapeutic composition are adjusted according to known parameters.

[0025] As used herein, the terms "subject" or "individual" generally refer to an individual in need of treatment. A subject may be any animal subject to the method or substance, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), household pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals. A subject may be a patient with or suspected of having one or more diseases (which may be referred to as medical conditions), such as cancer. A subject may be undergoing or have previously undergone cancer treatment. In at least some embodiments, the term "individual" may be used interchangeably with "subject." As used herein, a "subject" or "individual" may or may not be housed in a medical facility and may be treated as an outpatient. The individual may have received one or more medical compositions via the internet. The individual may be a human or non-human animal of any age, and thus includes adults and juveniles (e.g., children) and infants, as well as individuals in utero. The individual may be of any sex, race, or ethnicity.

[0026] "Treating" or "treatment" of a disease or condition refers to the implementation of a protocol that involves administering one or more compositions to an individual with the intent of alleviating signs or symptoms of cancer, inborn errors of metabolism, hematopoietic disorders, genetic disorders affecting liver function, and the like. Desirable effects of treatment include slowing the rate of disease progression, amelioration or palliation of the disease state, remission, or improved prognosis. Alleviation may occur before or after the appearance of signs or symptoms of the disease or condition. Thus, "treating" or "treatment" can include "preventing" or "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms or a cure, and expressly includes protocols that provide only a marginal benefit to the patient.

[0027] As used throughout this specification, the term "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or improves the well-being of a subject with respect to the medical treatment of that condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can include reducing tumor size, reducing tumor invasiveness, slowing the rate of cancer growth, preventing metastasis, or delaying the onset of metastasis. Treating cancer can also refer to extending the survival of a subject with cancer.

[0028] As used herein, the term "functionally equivalent codon" refers to a codon that encodes the same amino acid, such as the six different codons for arginine. Also, a "neutral substitution" or "neutral mutation" refers to a codon or change in a codon that encodes a biologically equivalent amino acid.

[0029] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids 100 residues or less in length) and recombinant vectors (including, for example, plasmids, cosmids, phages, viruses, etc.). A polynucleotide, in certain aspects, includes regulatory sequences that are substantially separated from naturally occurring genes or protein-coding sequences. A polynucleotide may be single-stranded (coding or antisense strand) or double-stranded, and may be RNA, DNA (genomic DNA, cDNA, or synthetic DNA), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present within the polynucleotide.

[0030] In this regard, the terms "gene," "polynucleotide," or "nucleic acid" refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including sequences necessary for proper transcription, post-translational modification, or localization). As will be understood by those of skill in the art, this term includes genomic sequences, expression cassettes, cDNA sequences, and smaller artificial nucleic acid fragments that express, or can be tailored to express, proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide may comprise a contiguous nucleic acid sequence that encodes all or a portion of the polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that has a slightly different nucleic acid sequence yet encodes the same or a substantially similar protein.

[0031] As used herein, the terms "sgRNA," "short guide RNA," "small guide RNA," "single guide RNA," "guide RNA," and "gRNA" are used interchangeably.

[0032] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the present disclosure, and vice versa. Additionally, compositions of the present disclosure can be used to practice methods of the present disclosure.

[0033] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given for purposes of illustration and that various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from said detailed description.

[0034] II. General Embodiments The present disclosure relates to systems, methods, and compositions for research purposes or for treating, delaying progression, delaying onset, or reducing the risk of disease. In various embodiments, the disease may be any type of cancer, a genetic disease (e.g., sickle cell disease, beta-thalassemia, retinal dystrophy), an inborn error of metabolism, a hematopoietic disease, a neurodegenerative disease (e.g., acute myeloid leukemia (AML)), an inflammatory disease, and / or a genetic disease (e.g., one affecting liver function). In certain embodiments, the method relates to the specific modification of one or more genes in any type of cell in the human body. The genetic modification may result in the loss and / or modification of expression of at least one target gene. In certain embodiments, the genetic modification is directed to a specific cell based on targeting at least one cell surface antigen of the cell. The genetic modification may result in the loss and / or modification of expression of endogenous SRC-3. In certain embodiments, the genetic modification is directed to a specific cell based on at least one cell surface antigen of the cell. In some embodiments, the cell surface antigen is CD25, CD4, and / or FOXP3.

[0035] In certain embodiments, the present disclosure encompasses methods and compositions that utilize cell surface proteins to deliver synthetically designed CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complexes to cells to specifically disrupt one or more target genes in the cells. In certain embodiments, the protein / ribonucleotide complexes can be administered to an individual in need in any form. The complexes can target any cell or cell population bearing a specifically targetable cell surface antigen, such as T cells, NK cells, NKT cells, B cells, macrophages, dendritic cells, or mixtures thereof. Such complexes may enable gene targeting of Tregs without ex vivo manipulation, such as via leukapheresis.

[0036] In certain embodiments, it is desired to disrupt one or more specific genes in one or more specific cell types. The complex comprises an antibody or antibody fragment for delivering CRISPR / sgRNA to the cell via a cell surface protein, where it is desired to disrupt the gene of interest with the CRISPR / sgRNA. In certain embodiments, the targeting antibody or antibody fragment targets one or more cell surface proteins, thereby delivering the CRISPR complex to the cell and enabling targeted gene disruption in cells contacted with the complex. Other embodiments of the present disclosure include replacing a cell-targeting antibody with any other antibody targeting a different cell surface antigen. Further embodiments include replacing an sgRNA targeting a specific gene with any other sgRNA designed to target another gene or other region within the genome. Those skilled in the art will understand that the modular components of the complex can be replaced with other functional components. For example, the scFv of the complex described herein, such as an scFv targeting a cancer antigen, can be replaced with an scFv targeting a different surface antigen, including any surface antigen encompassed herein. As another example, the sgRNA of the complexes described herein, e.g., the sgRNA targeting a gene of interest, can be replaced with an sgRNA targeting any other gene described herein. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sgRNAs are used to target 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes.

[0037] In certain embodiments, the present disclosure encompasses methods and compositions that utilize cell surface proteins to deliver synthetically designed CRISPR / Cas9 / targeting antibody / SRC-3-targeting sgRNA fusion protein / ribonucleotide complexes to specifically disrupt SRC-3 (gene symbol: NCOA3). In certain embodiments, the complexes can be administered to individuals in need in any form. The complexes can target any cell or cell population bearing a specifically targetable cell surface antigen, such as T cells, NK cells, NKT cells, B cells, macrophages, dendritic cells, or mixtures thereof. Such complexes may avoid ex vivo gene targeting of Tregs, such as via leukapheresis. In certain embodiments, the targeting antibody targets CD25, thereby enabling targeted gene destruction in cells contacted with the complex. Other embodiments of the present disclosure include replacing a cell-targeting antibody with any other antibody targeting a different cell surface antigen. Those skilled in the art will appreciate that the modular components of the complexes can be replaced with other functional components. For example, the scFv of the conjugates described herein, such as an scFv targeting CD25, can be substituted for an scFv targeting any other surface antigen described herein.

[0038] In some embodiments, a fusion protein-ribonucleotide complex is provided that includes a fusion protein and at least one short guide RNA (sgRNA) (e.g., at least one SRC-3 short guide RNA (sgRNA)). The fusion protein includes at least one antibody or antibody fragment and at least one RNA-guided DNA endonuclease. In certain embodiments, two or more antibodies or antibody fragments are used to enhance the ability to target specific cells that have two different cell surface proteins. In additional or alternative embodiments, the complex includes two or more different sgRNAs that target the same gene or different genes.

[0039] In some embodiments, contacting a cell with a complex described herein disrupts the expression of a gene (including at least SRC-3) in the cell. In some embodiments, the activity or function of the gene is disrupted, rather than the expression of the gene. Gene disruption or modification can be induced by artificial means, i.e., by adding or introducing an RNA-guided DNA endonuclease enzyme (e.g., CRISPR-associated protein 9 (Cas9)) or any CRISPR protein, complex, or composition at the DNA level. Methods of genetic modification herein include any protein that interacts with DNA in a nucleotide-specific manner, including CRISPR proteins (e.g., nuclease-deficient CRISPR mutants), zinc finger nucleases, TALENs, and other gene editing techniques (including cleavage and / or induction of homologous recombination, or chemical modification of DNA or associated chromatin proteins) that result in the inactivation or modification of a target gene. CRISPR proteins can include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein from S. pyogenes is contained in the SwissProt database under accession number Q99ZW2, which is incorporated herein by reference. Modifications can include insertions, mutations, and / or deletions in DNA, DNA methylation, and post-translational modifications to histones associated with the target DNA. These disruptions or alterations typically result in the silencing and / or complete loss of the normal or "wild-type" product encoded by the gene, e.g., SRC-3.In some embodiments, gene disruption or modification is insertion, frameshift mutation, missense mutation, deletion, knock-in and knock-out (including deletion of a gene or a part thereof). Such modification can occur in the coding region, for example, in one or more exons. This can result in the inability to produce a full-length product, a functional product, or any product, for example, through the insertion of a stop codon. Such modification can also occur through modification of promoters, enhancers, or other regions involved in transcription activation, thereby preventing gene transcription. Gene disruption or modification includes gene targeting, including the inactivation of target genes by homologous recombination.

[0040] Current cell-targeted immune checkpoint inhibitors primarily focus on cell surface proteins (receptors) that inhibit cell surface interactions with other immune cells. Embodiments of the present disclosure are distinct from such cell surface interactions in that they ultimately target genetic targets. Certain embodiments relate to immune cells, specifically to approaches that use CRISPR / Cas9 / cell surface protein-targeting antibody / gene-of-interest sgRNA fusion protein-ribonucleotide complexes to target specific genes in immune cells in vivo and generate genetically modified immune cells for the treatment of cancer, genetic diseases, inflammatory disorders, neurodegenerative diseases, inborn errors of metabolism, hematopoietic disorders, and / or genetic diseases affecting liver function. Thus, in certain embodiments, the present disclosure provides a unique method for achieving immune-based tumor elimination without the need for ex vivo manipulation of immune cells. In certain embodiments, the fusion protein-ribonucleotide complex targets other genes to elicit a response within the cell. In certain embodiments, the complex targets other cell surface antigens to elicit a response in cell types other than Tregs.

[0041] Embodiments of the present disclosure distinguish from cell-surface interactions by targeting SRC-3 as a Treg gene target. SRC-3 is a nuclear protein that functions to regulate the nuclear gene expression program of Tregs. Due to its role as a transcriptional master regulator, ablation of SRC-3 in genetic mouse models can modulate Treg function to promote tumor elimination while avoiding the severe side effects commonly seen with established immune checkpoint inhibitors. Certain embodiments relate to an approach using CRISPR / Cas9 / targeting antibody / SRC-3 gene-sgRNA fusion protein-ribonucleotide complexes to target the SRC-3 gene in immune cells in vivo to generate genetically modified Treg cells for the treatment of cancer, genetic diseases, inflammatory disorders, neurodegenerative diseases, inborn errors of metabolism, hematopoietic disorders, and / or genetic diseases affecting liver function. Thus, in certain embodiments, the present disclosure provides a unique method for achieving immune-based tumor elimination without the need for ex vivo manipulation of immune cells. In certain embodiments, the fusion protein-ribonucleotide complex targets other genes to trigger a response within the cells. In certain embodiments, the fusion protein ribonucleotide complexes target other cell surface antigens to elicit responses in cell types other than Tregs. Certain embodiments relate to the use of the fusion protein ribonucleotide complexes for the treatment or prevention of other diseases, such as inborn errors of metabolism, hematopoietic disorders, genetic disorders, and liver diseases.

[0042] Certain embodiments relate to methods and compositions for providing effective treatment, e.g., cancer treatment, while avoiding serious effects on the treated individual. In certain embodiments, the treatment involves modifying immune cells present in the body. In certain embodiments, immune cells may be modified ex vivo and administered to an individual, including an individual with cancer. In certain embodiments, the immune cells are of a specific type, and the modification of the cells may be such that it does not adversely affect the normal function of the modified cells. Certain embodiments also relate to the ability of CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting small guide sgRNA fusion protein / ribonucleotide complexes to specifically target proteins expressing the CD25 protein on the cell surface. In various embodiments, the CD25scFv portion of the CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting small guide RNA (sgRNA) fusion protein / ribonucleotide complex is designed to bind to cell surface CD25 expressed on Tregs.

[0043] Certain embodiments of the present disclosure encompass SRC-3 as a key target in immune cells, including at least Tregs. With regard to Tregs, compartment-specific disruption can result in tumor elimination, including in breast cancer tumor models. In certain embodiments, the disclosed strategy for disrupting SRC-3 in Tregs differs from other immune checkpoint inhibitors primarily because SRC-3 is a nuclear protein, and certain modifications in the present disclosure can modulate its function without abolishing its overall function, which could otherwise cause severe side effects. Furthermore, the present disclosure establishes an approach for specifically deleting SRC-3 in Treg cells in vivo using CRISPR-based gene targeting. In certain embodiments, when applied to human CD4+ lymphocytes, for example, the present disclosure provides an in vivo Treg-based therapy for the treatment of cancer.

[0044] Certain embodiments relate to an engineered cell or cells thereof comprising a disruption of one or more endogenous genes, such as SRC-3. The immune cell may be any immune effector cell. In some embodiments, the disruption is further defined by the immune cell being genetically engineered to reduce or substantially eliminate expression of the endogenous gene. In certain embodiments, the immune cell is engineered with one or more guide RNAs and an RNA-guided DNA endonuclease, or a protein comprising a sequence encoding all or part of the enzyme. In some embodiments, the immune cell is autologous to the individual. In some embodiments, the immune cell is allogeneic to the individual.

[0045] In various embodiments, the immune cells may be any immune effector cell, including a T cell. In some cases, the immune cells may be regulatory T cells (Tregs). The immune cells may be CD4+, CD25+, and / or FOXP3+. In some embodiments, the disruption is further defined by the immune cells being genetically modified to have increased or decreased expression of SRC-3, or to have substantially no expression of SRC-3. In certain embodiments, the immune cells are modified with one or more guide RNAs and a Cas9 enzyme, or a protein comprising a sequence encoding all or a portion of the Cas9 enzyme. In some embodiments, the immune cells are autologous to the individual. In some embodiments, the immune cells are allogeneic to the individual.

[0046] Embodiments herein relate to compositions comprising any of the cells described herein and a CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex, in some embodiments, the cell and complex interact in vitro or ex vivo.

[0047] Embodiments herein relate to cells expressing a CD25 cell surface protein that can be bound by a CD25-targeting scFV antibody.Embodiments herein relate to cells expressing a membrane protein that can be bound by a monoclonal antibody or scFV antibody.

[0048] Embodiments herein relate to compositions comprising any of the cells described herein and a CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting sgRNA fusion protein / ribonucleotide complex, in some embodiments, the cell and complex interact in vitro or ex vivo.

[0049] Certain embodiments relate to methods of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the compositions described herein, in some embodiments, the cancer expresses one or more deleterious genes that are therapeutically targeted by the compositions encompassed herein.

[0050] In some embodiments, the individual is administered a therapeutically effective amount of an additional treatment, e.g., in the case of cancer, one or more additional cancer treatments. The additional cancer treatment may include surgery, radiation therapy, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof. In some embodiments, the cells and / or complexes and the additional cancer treatment are administered to the individual substantially simultaneously or at different times. The cells and / or complexes and the additional cancer treatment may be contained in the same formulation or in different formulations.

[0051] In some embodiments, the cells and / or complexes are administered intravenously, intraperitoneally, intraarterially, transcutaneously, by inhalation, intramuscularly, intrasternally, intraarticularly, or by infusion. In some embodiments, the T cells, Tregs, or other cells are obtained from the spleen, bone marrow, blood, plasma, tissue, or a combination thereof.

[0052] [III. Immune cells] In the present disclosure, certain cells, including immune cells, are modified to disrupt expression of one or more endogenous genes, thereby enabling the cells to be effective in treating disease, including cancer, in the individual receiving the modified cells. In various embodiments, certain cells, including immune cells, are modified to disrupt expression of endogenous SRC-3, thereby enabling the cells to be effective in treating disease, including cancer, in the individual receiving the modified cells. While the cells may be of any type, in certain embodiments, the cells (of any type) express one or more specific cell surface proteins on their cell surface.

[0053] The disclosed methods and compositions can utilize and / or modify any cell that expresses a particular cell surface protein of interest. The term "T cell" refers to a T lymphocyte, including, but not limited to, CD4+ T cells, CD8+ T cells, γ:δ+ T cells, or NKT cells. CD4+ T cells include TH0, TH1, and TH2 cells, as well as regulatory T cells (Tregs). There are at least three types of regulatory T cells: CD4+CD25+ Tregs, CD25 TH3 Tregs, and CD25 TR1 Tregs. "Cytotoxic T cell" refers to a T cell that can kill other cells. While the majority of cytotoxic T cells are CD8+ MHC class I-restricted T cells, some cytotoxic T cells are CD4+. In certain embodiments, the T cell is CD4+.

[0054] Any cell of interest herein can be targeted, optionally via any cell surface marker, using any type of antibody, ligand, or other protein with affinity for the cell surface marker. In certain embodiments, the cell is CD4+, CD25+, and / or FOXP3+, CTL-associated protein 4 (CTLA4)+, CC chemokine receptor type 7 (CCR7)+, and / or CD62 antigen ligand (CD62L)+. Also included herein are cells that may be Tregs, but which, due to disruption of the SRC-3 gene, result in the cessation of FOXP3, CD25, and / or CD4 expression.

[0055] In some embodiments, ex vivo therapy using CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion proteins is utilized, and CD4+ immune cells (including CD4+ T cells) are targeted by the methods and compositions of the present disclosure to disrupt gene expression, and in at least some cases, the cells may be further selected for use as therapeutic cells. In some embodiments, ex vivo therapy using CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting sgRNA fusion proteins is utilized, and CD4+ immune cells (including CD4+ T cells) are targeted by the methods and compositions of the present disclosure to disrupt gene expression, such as SRC-3, and in at least some cases, the cells may be further selected for use as therapeutic cells. In other cases, various CD4+ cells are modified and used collectively as therapeutic cells without further steps to separate the cells.

[0056] When cells, such as immune cells, are used for ex vivo modification, the cells may be from any suitable source, including spleen, bone marrow, blood, plasma, or a combination thereof. In some cases, the cells may be commercially available. In certain embodiments, the cells may be autologous or allogeneic to the recipient. The cells may be engineered prior to modifying the cells to disrupt a gene of interest, such as SRC-3. In some cases, the cells are processed from their source, such as to remove unwanted components. Prior to use, the cells may be exposed to one or more compositions that enhance their activity in the recipient, such as one or more agents or other compositions that target the gene of interest.

[0057] In certain embodiments, immune cells engineered to disrupt a gene (e.g., SRC-3) include B cells. While B cells engineered via scFVs in a CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex are used for any medical condition for which the B cells may be effective, in certain embodiments, the engineered B cells are used to treat one or more inflammatory diseases or any type of cancer, including B cell lymphoma (including those cancers described elsewhere herein). The B cells may be engineered ex vivo to disrupt expression of an endogenous gene (e.g., SRC-3). In some cases, the engineered B cells are exposed to one or more agents, including TGF-β or a tumor-specific antigen, that help increase the effectiveness of the B cells after administration to an individual.

[0058] In certain embodiments, modified B cells, i.e., B cells lacking expression of a particular endogenous gene (e.g., SRC-3) or having reduced expression of the particular endogenous gene compared to unmodified B cells, are administered in an effective amount to an individual having or at risk for one or more inflammatory diseases. The inflammatory disease can be of any type, but in certain embodiments, the disease is allergy, asthma, autoimmune disease, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, transplant rejection, ankylosing spondylitis (AS), gout, myositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE, lupus), pelvic inflammatory disease, or vasculitis, by way of example only. Treatment with the B cells can have the effect of reducing or delaying the severity of the disease, delaying the onset of the disease, ameliorating one or more symptoms of the disease, etc.

[0059] [IV. Gene Disruption] In various embodiments, one or more endogenous genes (e.g., SRC-3) are disrupted within the cell, such as by disrupting expression, and components used for such disruption are delivered to the cell based on their binding sites to the cell's surface, such as via cell surface proteins. The cells may be used for research and / or therapeutic purposes, and the gene of interest may be selected based on the desired use of the cells. In some embodiments, altered gene expression (e.g., SRC-3) is achieved by disrupting the gene, e.g., by knockout, insertion, missense or frameshift mutation (e.g., biallelic frameshift mutation), deletion of all or part of the gene (e.g., deletion of one or more exons or portions thereof), and / or knock-in. For example, altered gene expression can be achieved by RNA-guided nucleases, such as CRISPR / Cas9, or variants thereof, or sequence-specific or targeted nucleases, including DNA-binding targeted nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). In some embodiments, the sequence-specific or targeted nuclease, or a fusion protein or complex comprising same, is designed to specifically bind to a sequence of a target gene or a portion thereof.Examples of genes that may be targeted for disruption include at least the following: SRC-3, CD19, CD319 / CS1, ROR1, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, and CD3. 0, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL / DR4, VEGFR2, fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3, FGFR4), E-cadherin, collagen-binding integrins (α1β1, α2β1, α10β1, α11β1), and / or leukocyte-binding integrins (e.g., αLβ2, αMβ2, α4β1).

[0060] In some embodiments, the alteration of gene expression, activity, and / or function is achieved by disrupting the gene. In some aspects, the gene is altered to reduce its expression by at least about 20%, 30%, or 40%, typically at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, compared to expression in the absence of the genetic alteration or the components introduced to effect the alteration.

[0061] In some embodiments, the modification is transient or reversible, allowing expression of the gene to be restored at a later time. In other embodiments, the modification is neither reversible nor transient. In some embodiments, the modification is permanent.

[0062] In some embodiments, gene modification (e.g., SRC-3) is carried out by inducing one or more double-strand breaks and / or one or more single-strand breaks in the gene. These double-strand or single-strand breaks can be made in a targeted manner. In some embodiments, the double-strand or single-strand breaks are caused by a nuclease, such as an endonuclease, more particularly a gene-targeting nuclease. In some aspects, the breaks are induced in the coding region of the gene, for example, within an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the gene coding region, i.e., the first exon, the second exon, or the subsequent exon.

[0063] In some aspects, the double-stranded or single-stranded break is repaired via an intracellular repair process, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some aspects, the repair process is error-prone, resulting in a gene disruption, e.g., a frameshift mutation (e.g., a biallelic frameshift mutation), which can result in a complete knockout of the gene (e.g., SRC-3). For example, in some aspects, the disruption involves the introduction of a deletion, mutation, and / or insertion. In some embodiments, the disruption results in the appearance of a premature stop codon. In some aspects, the presence of an insertion, deletion, translocation, frameshift mutation, and / or premature stop codon causes disruption of the expression, activity, and / or function of the gene. In some embodiments, a nuclease-deficient CRISPR / Cas9 protein is used fused to a protein that alters DNA chemical structure, e.g., cytosine methylation, or post-translational modification of histones (e.g., histone methylation or demethylation).

[0064] As an alternative to CRISPR / Cas9, any DNA targeting method known to those skilled in the art can be used to disrupt the expression of a gene of interest (e.g., SRC-3). Such methods include the use of DNA targeting molecules, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease, to target the gene. Examples include ZFNs (zinc finger nucleases), TALEs (TAL effectors), and TALENs (TAL effector nucleases).

[0065] In some embodiments, the DNA targeting molecule comprises one or more ZFPs or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers (amino acid sequence regions within the binding domain whose structure is stabilized by the coordination of zinc ions). ZFPs include artificial ZFP domains created by combining individual fingers that target specific DNA sequences (typically 9 to 18 nucleotides).

[0066] ZFPs include those with a finger domain consisting of approximately 30 amino acids, an α-helix containing two invariant histidine residues, and zinc-coordinated β-turns with two cysteine ​​residues, and have two, three, four, five, or six fingers. Generally, the sequence specificity of ZFPs can be altered by amino acid substitutions at four positions (-1, 2, 3, and 6) along the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, i.e., designed to bind to any target site.

[0067] In some embodiments, the DNA targeting molecule comprises a zinc finger DNA binding domain fused to a DNA cleavage domain, thereby forming a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) derived from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may be engineered or unengineered. In some embodiments, the cleavage domain is derived from the type IIS restriction endonuclease FokI. FokI catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0068] Many gene-specific pre-designed zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, California, USA) has partnered with Sigma-Aldrich (St. Louis, Missouri, USA) to develop a platform for zinc finger construction (CompoZr), allowing researchers to bypass the entire zinc finger construction and validation process and providing specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom-designed. (See, e.g., Sigma-Aldrich catalog numbers: CSTZFND, CSTZFN, CTil-1KT, PZD0020.)

[0069] In some embodiments, the DNA targeting molecule comprises the DNA binding domain of a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL), such as found in a transcription activator-like effector (TALE) protein (see, e.g., U.S. Patent Publication No. 2011 / 0301073, which is incorporated herein by reference).

[0070] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. These repeat domains are responsible for the binding of TALEs to their corresponding target DNA sequences. A "repeat unit" (also called a "repeat") typically consists of 33-35 amino acids and shares at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit contains one or two DNA-binding residues that constitute a Repeat Variable Diresidue (RVD), usually located at positions 12 and / or 13 in the repeat. The natural (canonical) code for DNA recognition by these TALEs has been elucidated as follows: HD sequences at positions 12 and 13 bind to cytosine (C), NG to thymine (T), NI to adenine (A), NN to guanine (G) or adenine (A), and NO to thymine (T). Non-canonical (atypical) RVDs are also known. In some embodiments, TALEs can be targeted to any gene by designing a TAL sequence specific to the target DNA sequence, which generally begins with a thymidine.

[0071] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, the TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0072] In some embodiments, the TALEN recognizes and cleaves a target sequence in the gene. In some aspects, DNA cleavage causes double-strand breaks. In some aspects, these breaks promote the rate of homologous recombination or non-homologous end joining (NHEJ). NHEJ is generally an imperfect repair process, often resulting in alterations of the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves direct ligation of the remaining sequences at the two DNA ends or rejoining via so-called microhomology-mediated end joining. In some embodiments, NHEJ repair results in small insertions or deletions, which can be used to disrupt genes and silence their expression. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event subsequent to an NHEJ event, has occurred can be identified and / or selected using methods well known to those of skill in the art.

[0073] In some embodiments, TALE repeats are assembled to target specific genes (Gaj et al., 2013). A TALEN library targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE sequences are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Kentucky, USA), and Life Technologies (Grand Island, New York, USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3), and representative molecules are described, for example, in U.S. Patent Publication Nos. US2014 / 0120622 and 2013 / 0315884.

[0074] In some embodiments, the TALENs are introduced as transgenes encoded by one or more plasmid vectors, which in some aspects may contain a selection marker that allows for identification and / or selection of cells that have received the vector.

[0075] In some embodiments, the gene of interest is modified using one or more DNA-binding nucleic acids, including, for example, modification by RNA-guided endonucleases (RGENs). For example, the modification can be performed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated (Cas) proteins. Generally, the term "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated (Cas) genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0076] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide RNA) that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system, for example, from a particular organism (e.g., Streptococcus pyogenes) that has an endogenous CRISPR system.

[0077] In some aspects, Cas nuclease and gRNA (comprising a fusion of target sequence-specific crRNA and fixed tracrRNA) are introduced into cells. Generally, the target site at the 5' end of gRNA guides Cas nuclease to the target site (e.g., gene) through complementary base pairing. The target site can be selected to be located just before (5') a protospacer adjacent motif (PAM) sequence, such as NGG or NAG. In this regard, gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the target sequence site. As used herein, the term "target sequence" generally refers to a sequence to which a guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not required; sufficient complementarity is sufficient to cause hybridization and the formation of a CRISPR complex.

[0078] In certain embodiments, the CRISPR system induces a double-strand break (DSB) at the target site, i.e., the target sequence in a desired gene (e.g., SRC-3), followed by disruption or modification as described herein. In other embodiments, a Cas9 mutant called a "nickase" is used to nick one strand at the target site. To improve specificity, paired nicases can be used, guided by two gRNAs, each targeting a different sequence, simultaneously introducing a nick to create a 5' overhang. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0079] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the cell nucleus or cytoplasm, e.g., within an organelle. Generally, a sequence or template that can be used to direct recombination at a target site that contains a target sequence is referred to as an "editing template," "editing polynucleotide," or "editing sequence." In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0080] In some embodiments, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and one or more Cas proteins) causes cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 base pairs of the target sequence). The tracr sequence may consist of or include all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more bases), and may form part of a CRISPR complex by hybridizing to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, the complementarity being at least 50%, 60%, 70%, 80%, 90%, 95% or 99% over the length of the tracr mate sequence when optimally aligned.

[0081] One or more vectors driving the expression of one or more CRISPR system components may be introduced into a cell, such that expression of the CRISPR system components induces the formation of CRISPR complexes at one or more target sites. Components can be introduced into a cell as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence may each be placed on separate vectors operably linked to separate regulatory sequences. Alternatively, two or more components expressed from the same or different regulatory sequences may be combined into a single vector, with additional vectors providing components of the CRISPR system not included in the initial vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also known as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to direct CRISPR activity at multiple different corresponding target sequences within a cell.

[0082] The vector may comprise a regulatory sequence operably linked to an enzyme-coding sequence that encodes a CRISPR enzyme (e.g., a Cas protein). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also called Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes can be found in the SwissProt database under accession number Q99ZW2.

[0083] In some embodiments, the CRISPR enzyme is Cas9 (e.g., derived from S. pyogenes or S. pneumoniae). CRISPR enzymes can direct single-strand or double-strand cleavage at the location of a target sequence, e.g., within the target sequence and / or its complementary strand. The vector can encode a CRISPR enzyme having a mutation compared to the corresponding wild-type enzyme, where the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a mutation (D10A) that substitutes aspartic acid for alanine in the RuvC I catalytic domain of S. pyogenes-derived Cas9 converts Cas9 from a double-strand cleavage nuclease to a single-strand cleavage nuclease (nickase). In some embodiments, Cas9 nickase is used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination can nick both strands, inducing NHEJ or HDR.

[0084] In some embodiments, the enzyme coding sequences encoding CRISPR enzymes and scFv antibodies are codon-optimized for expression in specific cells, such as eukaryotic cells. The eukaryotic cells may be cells derived from or belonging to a specific organism, such as a mammal (e.g., human, mouse, rat, rabbit, dog, non-human primate, etc.). Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon in the native sequence with a codon more or most frequently used in the host cell's genes while retaining the amino acid sequence, in order to enhance expression in the host cell of interest. Some species exhibit codon bias, which favors certain codons for certain amino acids. Codon bias (differences in codon usage between species) generally correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend on multiple factors, such as the nature of the codon being translated and the availability of certain transfer RNAs (tRNAs). The prevalence of certain tRNAs in a cell typically reflects the codons most frequently used in peptide synthesis in that cell. Thus, genes can be tailored for optimal gene expression in a particular organism based on codon optimization.

[0085] Generally, a guide sequence refers to any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence, and can hybridize with the target sequence to allow a CRISPR complex to bind to the target sequence in a sequence-specific manner. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0086] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, ClustalX, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0087] The CRISPR enzyme itself and the entire CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA (small guide RNA) fusion protein may be part of a fusion protein containing one or more heterologous protein domains. The CRISPR enzyme fusion protein may contain any additional protein sequence and may have a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional dissociation factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include glutathione-S-transferase (GST), peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). CRISPR enzymes may be fused to gene sequences encoding proteins or fragments thereof that bind to DNA molecules or other intracellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, herpes simplex virus (HSV) BP16 protein fusions, etc. Additional domains that can be part of fusion proteins containing CRISPR enzymes are described in U.S. Patent Publication No. US2011 / 0059502, which is incorporated herein by reference in its entirety.

[0088] [V. Complexes] Certain embodiments herein relate to fusion proteins complexed with ribonucleotides, i.e., fusion protein-ribonucleotide complexes. The ribonucleotide may be an sgRNA that interacts with the fusion protein or a domain thereof to affect a target gene (e.g., SRC-3). The target gene may be any gene, and in some embodiments, the target gene is an oncogene, a tumor suppressor gene, a mutated gene, an immunostimulatory gene, an immunomodulatory gene, or a gene associated with a disease.

[0089] The fusion protein may comprise at least one scFv (single-chain fragment variable region). The scFv may target any surface antigen, for example, any tumor-associated antigen. In some embodiments, the surface antigen may comprise an immune cell marker, an estrogen receptor, a tyrosine kinase receptor, a growth factor receptor, or an immune checkpoint receptor. The scFv may comprise all or a portion of a therapeutic antibody, including but not limited to: 3F8, Abagovomab, Abciximab, Abituzumab, Abrezekimab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Alacizumab pegol, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Amivantamab, Anatumomab mafenatox, Andecaliximab, Anetumab ravtansine, Anifrolumab, Ansuvimab, Anrukinzumab, Apolizumab, Aprutumab ixadotin, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atidortoxumab, Atinumab,Atoltivimab, Atoltivimab / Maftivimab / Odesivimab, Atorolimumab, Avelumab, Azintuxizumab vedotin, Bamlanivimab, Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bebtelovimab, Bectumomab, Begelomab, Belantamab mafodotin, Belimumab, Bemarituzumab, Benralizumab, Berlimatoxumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Virtamimab, Bivatuzumab, Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, (anaplastic large-cell lymphoma), Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab,Burosumab, Cabiralizumab, Camidanlumab tesirine, Camrelizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Casirivimab, Capromab, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate Immunoconjugate, Cedelizumab, Cemiplimab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetrelimab, Cetuximab, Cibisatamab, Cilgavimab, Cirmtuzumab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan tetraxetan, Codrituzumab, Cofetuzumab pelidotin, Coltuximab ravtansine, Conatumumab, Concizumab, Cosfroviximab, Crenezumab, Crizanlizumab, Clotedumab, CR6261, Cusatuzumab,Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Derlotuximab biotin, Detumomab, Dezamizumab, Dinutuximab, Dinutuximab Dinutuximab beta, Diridavumab, Domagrozumab, Dorlimomab aritox, Dostarlimab, Drozitumab, DS-8201, Durigotuzumab, Dupilumab, Durvalumab, Dusigitumab, Duvortuximab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elezanumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emapalumab, Emibetuzumab, Emicizumab, Enapotamab vedotin, Enavatuzumab, EnfotumabEnfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epcoritamab, Epitumomab cituxetan, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etaracizumab, Etesevimab, Etigilimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fivatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Flotetuzumab, Fontolizumab, Foralumab, Foravirumab, Fremanezumab, Fresolimumab, Frovocimab, Flunevetmab, Fulranumab, Futuximab, Galcanezumab, Galiximab,Gancotamab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavilimomab, Gedivumab, Gemtuzumab ozogamicin, Gevokizumab, Gilvetmab, Gimsilumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Gosuranemab, Guselkumab, Ianalumab, Ibalizumab, Sintilimab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Ifabotuzumab, Igovomab, Iladatuzumab vedotin, Imalumab, Imaprelimab, Imciromab, Imdevimab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iomab-B, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Ladiratuzumab vedotin, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab emtansine,Larcaviximab, Lebrikizumab, Lemalesomab, Lendalizumab, Lenvervimab, Lenzilumab, Lerdelimumab, Leronlimab, Lesofavumab, Letolizumab, Lexatumumab, Ribivirumab, Rifastuzumab vedotin, Ligelizumab, Loncastuximab tesirine, Rosatuximab Losatuxizumab vedotin, Lilotomab satetraxistan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Rupartumab, Lupartumab amadotin, Lutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, MirvetuximabMirvetuximab soravtansine, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximab Naratuximab emtansine, Narnatumab, Natalizumab, Navixixizumab, Navivimab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001, Nerelimomab, Nesvacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odesivimab, Odulimomab, Ofatumumab, (multiple sclerosis), Olaratumab, Oleclumab, Olendalizumab, Olokizumab, Omalizumab,Omburtamab, OMS721, Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otilimab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pancomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Prezalumab, Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Porgaviximab, Prasinezumab, Prezalizumab, Priliximab, Pritoxaximab,Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetmab, Ranibizumab, Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, Regavirumab, Regdanvimab, Relatlimab, Remtolumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab, Alumab, Roledumab, Romilkimab, Romosozumab, Rontalizumab, Rosmantuzumab, Rovalpituzumab tesirine, Rovelizumab, Rozanolixizumab, Ruplizumab, SA237, Sacituzumab govitecan, Samalizumab, Samrotamab vedotin, Sarilumab, Satralizumab, Satumomab pendetide, Secukinumab, Selicrelumab, Seribantumab,Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirtratumab vedotin, Sirukumab, Sofituzumab vedotin, Solanez Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Sotrovimab, Spartalizumab, Spesolimab, Stamulumab, Sulesomab, Saptavumab, Sutimlimab, Suvizumab, Suvratoxumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Tafasitamab, Talacotuzumab, Talizumab, Talquetamab, Tamtuvetmab, Tanezumab, Taplitumomab paptox, Tarextumab, Tavolimab, Teclistamab, Tefibazumab, Telimomab aritox, Telisotuzumab, Telisotuzumab vedotin, Tenatumomab, Teneliximab, Teplizumab, Tepoditamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tibulizumab, Tildrakizumab, Tigatuzumab, Timigutuzumab, Timolumab, Tiragolumab, Tiragotumab,Tislelizumab, Tisotumab vedotin, Tixagevimab, TNX-650, Tocilizumab, Tomuzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab duocarmazine, Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximab talirine, Vanalimab, Vandortuzumab vedotin, Vantictumab Vanucizumab, Vapaliximab, Varisacumab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Voloximab,Vonlerolizumab, Vopratelimab, Vorsetuzumab mafodotin, Votumumab, Vunakizumab, Xentuzumab, XMAB-5574, Zalutumumab, Zanolimumab, Zatuximab, Zenocutuzumab, Ziralimumab, Zolbetuximab, or Zolimomab aritox.

[0090] In certain embodiments, the surface antigen is selected from the group consisting of CD319 / CS1, ROR1, CD20, carcinoembryonic antigen, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD 56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL / DR4, VEGFR2, fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3, FGFR4), E-cadherin, collagen-binding integrins (α1β1, α2β1, α10β1, α11β1), leukocyte-binding integrins (e.g. αLβ2, αMβ2, α4β1), TGFBR, 4-1BB (CD137), 5'-nucleotidase, 5T4, activated F9, F10, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, alpha-fetoprotein, α-synuclein, amyloid, angiopoietin 2, angiopoietin 3, anthrax toxin (protective antigen), AOC3, AOC3 (VAP-1), AXL, Bacillus anthracis (Bacillus anthracis), BAFF-R, B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), CD3, B lymphoma cells, C242 antigen, C5, CA-125, CA-125 (mimetic), calcitonin, calcitonin gene-related peptide (α and β), calcitonin gene-related peptide receptor (CGRP), CanAg (glycoform of MUC1), dog (Canis lupusfamiliaris) IL-31, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA)-related antigen, cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD123, CD125, CD134, CD147 (basigin), CD4, CD15, CD152, CD154 (CD40L), CD19, CD3E, CD2, CD20, CD200, CD22, CD23 (IgE receptor), CD25 (IL-2 receptor α chain), CD27, CD276, CD278 (also called ICOS), CD28, CD3, CD3, CD20, CD30 (TNFRSF8), CD319, CD33, CD37, CD38, CD3E, CD3E, MS4A1, CD20, CD40, CD41 (integrin α-IIb), CD44 v6, CD45, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEACAM5, claudin 18 isoform 2, Clostridium difficile, clumping factor A, c-Met, coagulation factor III, complement C5a, complement component 1s (C1s), complement factor D (CFD), connective tissue growth factor (CTGF), CSF1 (macrophage colony-stimulating factor MCSF), CSF1R, CSF2, CTLA-4, CXCL10 (IP-10), CXCR4 (CD184), cytomegalovirus, Itomegalovirus glycoprotein B, dabigatran, dendritic cell-associated lectin 2, DLL3, DLL4, DLL4 and VEGFA, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, Ebola virus glycoprotein, EGFL7, EGFR, EGFR extracellular domain III, endoglin, endotoxin, EpCAM, EpCAM, CD3, EPHA3 (ephrin receptor A3), epidermal growth factor receptor (EGFR), cMet, HER1 (ERBB1), episialin, ERBB3 (HER3), Escherichia coli (Escherichiacoli), respiratory syncytial virus F protein, FAP (FAP gene), FCGRT, FGF23, FGFR2, fibrin II (β chain), fibronectin extra domain B, folate hydrolase, folate receptor 1, folate receptor α, frizzled receptor, GD2 ganglioside, GDF-8, gelatinase B, glucagon receptor (GCGR), glutamate carboxypeptidase II, glypican 3, GMCSF, GMCSF receptor α chain, GPNMB, GPR C5D, CD3, growth differentiation factor 8, GUCY2C, hemagglutinin (influenza), hepatitis B surface antigen, hepatitis B virus, hepatocyte growth factor (HGF), HER1, HER2, HER2 / neu, HER2 / neu, CD3, HGFR, histone complex, HIV-1, HLA-DR, Hsp90, human scatter factor receptor kinase, ICAM-1 (CD54), IFN receptor, IFN-γ, IFN-α / β receptor, IFN-α, IgE, IgEFc region, IGF-1 receptor (CD221), IGF-1, IGF-2, IGHE, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-17F, IL-17AF, IL -1α, IL-1β, IL-2, IL-20, IL-22, IL-23A, IL-3 receptor, IL-31 receptor A, IL-4, IL-4Rα, IL-5, IL-6, IL-6 receptor, IL-9, inducible T cells Cellular costimulatory ligand (ICOSL), integrin α4, integrin α4β7, integrin α5β1, integrin αIIbβ3, integrin αvβ3, integrin β7, interleukin 36 receptor (IL1RL2 / IL1RAP), ITGA2 (CD49b), ITGB2 (CD18), kallikrein, KIR2D, LAG3, Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoprotein Ichoic acid, LIV-1, LOXL2, LRRC15, L-selectin (CD62L), lymphotoxin α (LTA), LYPD3, macrophage migration inhibitory factor (MIF), MASP-2, MCP-1, melanoma cell adhesion molecule (MCAM), mesothelin (MSLN), MS4A1, MST1R (also known as RON), MUC1, MUC5AC, mucosal adductor cell adhesion molecule, myelin-associated glycoprotein, myoglobin Statins, NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), NGNA ganglioside, NKG2A, NOGO-A, Notch1, Notch receptor, NRP1, OX-40, oxidized LDL, PCDC1 (CD279), PCDP1, PCSK9, PD-1, PDGFRA, PDGFRB, PD-L1, sodium phosphate cotransporter, phosphatidylserine, Pseudomonas aeruginosaaeruginosa, Pseudomonas aeruginosa type III secretion system, PTK7, rabies virus G glycoprotein, RANKL, repulsion-inducing molecule A (RGMA), respiratory syncytial virus, RHD (gene) (Rh factor), root plate-specific spondin 3, ROR1, RSV fusion glycoprotein (RSVFR), RTN4, sclerostin (SOST), SDC1, selectin P, serum amyloid A protein, serum amyloid P component, SLAMF7, SLITRK6, sphingosine-1-phosphate, SARS-CoV-2 spike protein receptor-binding domain (RBD), Staphylococcus aureus aureus and its alpha toxin / bicomponent leukocidin, STEAP1, TAG-72, tau protein, T cell receptor, TEM1, tenascin-C, TGF-β (including TGF-β1 and TGF-β2), thymic stromal lymphopoietin (TSLP), TIGIT, tissue factor pathway inhibitor (TFPI), TNF (including TNF-α), TRAIL-R1, TRAIL-R2, TROP-2, tumor antigen CTAA16.88, tumor necrosis factor receptor (TNFR) These include superfamily 4, tumor necrosis factor-related activating protein (TRAP), tumor-specific glycosylation of MUC1, tumor-associated glycoprotein 72 (TAG-72), TWEAK receptor, TYRP1 (glycoprotein 75), vascular endothelial growth factor A (VEGFA), VEGF-A and Ang-2, VEGFR-1, VEGFR2, vimentin, VISTA (VSIR), VWF, Zaire Ebola virus glycoprotein, or β-amyloid (including 1-40 and 1-42).

[0091] The antibody is targeted to TGFBR, 4-1BB (CD137), 5'-nucleotidase, 5T4, activated F9, F10, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, alpha-fetoprotein, alpha-synuclein, amyloid, angiopoietin 2, angiopoietin 3, anthrax toxin (protective antigen), AOC3, AOC3 (VAP-1), AXL, Bacillus anthracis, BAFF-R, B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), CD3, B lymphoma cells, C242 antigen, C5, CA-125, CA-125 (mimetic), calcitonin, calcitonin gene-related peptide (α and β), calcitonin gene-related peptide receptor (CGRP), CanAg (glycoform of MUC1), and dog (Canis lupus) familiaris), IL-31, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA)-related antigen, cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD123, CD125, CD134, CD147 (basigin), CD4, CD15, CD152, CD154 (CD40L), CD19, CD3E, C D2, CD20, CD200, CD22, CD23 (IgE receptor), CD25 (IL-2 receptor α chain), CD27, CD276, CD278 (also called ICOS), CD28, CD3, CD3, CD20, CD30 (TNFRSF8), CD319, CD33, CD37, CD38, CD3E, CD3E, MS4A1, CD20, CD40, CD41 (integrin α-IIb), CD44v6, CD45, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEACAM5, claudin 18 isoform 2, Clostridium difficile, clumping factor A, c-Met, coagulation factor III, complement C5a, complement component 1s (C1s), complement factor D (CFD), connective tissue growth factor (CTGF), CSF1 (macrophage colony-stimulating factor MCSF), CSF1R, CSF2, CTLA-4, CXCL10 (IP-10), CXCR4 (CD184), cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, dendritic cell-associated lectin 2, DLL3, DLL4, DLL4 and VEGFA, DPP4, DR5, E. coli Shiga toxin type-1, E. coli Shiga toxin type-2, Ebola virus glycoprotein, EGFL7, EGFR, EGFR extracellular domain III, endoglin, endotoxin, EpCAM, EpCAM, CD3, EPHA3, ephrin receptor A3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor (EGFR), cMet, epidermal growth factor receptor (EGFR), HER1, epidermal growth factor receptor (EGRF), ERBB1 HER1, episialin, ERBB3 (HER3), Escherichia coli, respiratory syncytial virus F protein, FAP (FAP gene), FCGRT, FGF 23, FGFR2, fibrin II (β chain), fibronectin Extra domain-B, folate hydrolase, folate receptor 1, folate receptor α, frizzled receptor, GD2 ganglioside, GDF-8, gelatinase B, glucagon receptor (GCGR), glutamate carboxypeptidase II, glypican 3, GMCSF, GMCSF receptor α chain, GPNMB, GPRC5D, CD3, growth differentiation factor 8, GUCY2C, hemagglutinin (influenza), hepatitis B surface antigen, hepatitis B virus, hepatocyte growth factor (HGF), HER1, HER2, HER2 / neu, HER2 / neu, CD3, HGFR, histone complex, HIV-1, HLA-DR, Hsp90, human scatter factor receptor kinase, ICAM-1, ICAM-1 (CD54), IFN receptor, IFN-γ, IFN-α / β receptor, IFN-α, IgE, IgEFc region, IGF-1 receptor (CD221), IGF-1, IGF-2, IGF-2, IGHE, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-17A, IL-17F, IL-17A, IL-17F, IL-17AF, IL-17A, TNF, IL- 1α, IL-1β, IL-2, IL-20, IL-22, IL-23, IL-23A, IL-3 receptor, IL-31 receptor A, IL-4, IL-4Rα, IL-5, IL-6, IL-6 receptor, IL-9, inducible T cell costimulatory ligand (ICOSL), integrin α4, integrin α4 β7, integrin α5 β1, integrin αIIbβ3, integrin αvβ3, integrin β7, interleukin 36 receptor (IL1RL2 / IL1RAP), ITGA2 (CD49b), ITGB2 (CD18), kallikrein, KIR2D, LAG3, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LIV-1, LOXL2, LRRC15, L-selectin (CD62L), lymphotoxin α (LTA), LYPD3, macrophage migration Mitotic inhibitory factor (MIF), MASP-2, MCP-1, melanoma cell adhesion molecule (MCAM), mesothelin, mesothelin (MSLN), MS4A1, MST1R (also known as RON), MUC1, MUC5AC, mucosal adductor cell adhesion molecule, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), NGNA ganglioside, NKG2A, NOGO-A, Notch 1, Notch receptor, NRP1, OX-40, oxidized LDL, PCDC1, PCDC1, CD279, PCDP1, PCSK9, PD-1, PDGFRA, PDGFRB, PD-L1, sodium phosphate cotransporter, phosphatidylserine, Pseudomonas aeruginosa, Pseudomonas aeruginosa type III secretion system, PTK7, rabies virus G glycoprotein, rabies virus glycoprotein, RANKL, repulsion-inducing molecule A (RGMA), respiratory syncytial virus, RHD (gene) (Rh factor), Rhesus factor, root plate-specific spondin 3, ROR1, RSV fusion glycoprotein, RSVFR, RTN4, sclerostin, sclerostin (SOST), SDC1, selectinP, serum amyloid A protein, serum amyloid P component, SLAMF7, SLITRK6, SOST, sphingosine-1-phosphate, SARS-CoV-2 spike protein receptor-binding domain (RBD), Staphylococcus aureus, Staphylococcus aureus alpha-toxin, Staphylococcus aureus bicomponent leukocidin, STEAP1, TAG-72, tau protein, T cell receptor, TEM1, tenascin The target antigen may be any antigen, including C, TGF-β, TGF-β1, TGF-β2, thymic stromal lymphopoietin (TSLP), TIGIT, tissue factor pathway inhibitor (TFPI), TNF, TNF-α, TRAIL-R1, TRAIL-R2, TROP-2, tumor antigen CTAA16.88, tumor necrosis factor receptor (TNFR) superfamily 4, tumor necrosis factor-related activating protein (TRAP), tumor-specific glycosylation of MUC1, tumor-associated glycoprotein 72 (TAG-72), TWEAK receptor, TYRP1 (glycoprotein 75), vascular endothelial growth factor A (VEGFA), VEGF-A, VEGF-A and Ang-2, VEGFR-1, VEGFR2, vimentin, VISTA (VSIR), VWF, Zaire Ebola virus glycoprotein, β-amyloid, or β-amyloid (1-40 and 1-42).

[0092] (A. Protein) Proteins specifically referred to herein generally refer to native (wild-type) or recombinant (modified) proteins, or proteins from which signal sequences may optionally be removed. Proteins may be isolated directly from their native organisms, produced by recombinant DNA or exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. Specific embodiments include isolated nucleic acid fragments and recombinant vectors incorporating nucleic acid sequences encoding a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant," sometimes used in conjunction with a polypeptide or the name of a specific polypeptide, generally refers to a polypeptide produced from nucleic acid molecules manipulated in vitro, or the product of replication of such molecules.

[0093] In certain embodiments, the size of the protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,275,300,325,350,375,400,425,450,475,500,525,550,575,600,625,650,675,700,725,750,775,80 Polypeptides may be mutated by truncation to be shorter than their wild-type counterparts. They may also be modified by fusion or conjugation with heterologous protein or polypeptide sequences that have a specific function, such as targeting, localization, enhanced immunogenicity, or purification. As used herein, the term "domain" refers to a structurally or functionally distinct unit of a protein or polypeptide, generally referring to an amino acid sequence whose structure or function can be identified by one of ordinary skill in the art.

[0094] Polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the present disclosure may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more mutated amino acids or nucleotides. The amino acid sequence may contain nucleotide substitutions or have 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similarity, identity, or homology.This is at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 0,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,14 5,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205 , 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, 1500 or more consecutive amino acids or nucleotides, or any range contained in any of SEQ ID NOs: 1-10.

[0095] SEQ ID NO: 1: Amino acid sequence of 6xHis purification tag (6His) HHHHHH

[0096] SEQ ID NO: 2: Amino acid sequence of cell penetrating peptide (CPP) KLALKLALKALKAALK

[0097] SEQ ID NO: 3: Amino acid sequence of linker 1 G

[0098] SEQ ID NO: 4: Amino acid sequence of linker 2 LFPTI

[0099] SEQ ID NO: 5: Amino acid sequence of CRISPR / Cas9 nuclease

[0100] SEQ ID NO: 6: Amino acid sequence of linker 3 GPG

[0101] SEQ ID NO: 7: Amino acid sequence of TEV protease ENLYFQG

[0102] SEQ ID NO: 8: Amino acid sequence of linker 4 AG

[0103] SEQ ID NO: 9: Amino acid sequence of the FLAG tag DYKDDDDKG

[0104] SEQ ID NO: 10: Amino acid sequence of CD25 scFv QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSTLSASVGDRVITITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVKR

[0105] SEQ ID NO: 11: Amino acid sequence of CRISPR / Cas9 nuclease

[0106] Nucleotide sequences and protein, polypeptide, and peptide sequences for various genes have been previously disclosed and are available in publicly known computerized databases. Two commonly used databases are the GenBank® and GenPept® databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0107] It is contemplated that compositions of the present disclosure contain a range of about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per mL. The concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / mL or more (or any range derivable therein).

[0108] <1. Mutant Polypeptide> The following is a discussion of altering the amino acid subunits of proteins to generate equivalent or even improved second-generation mutant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence without significantly impairing its interactive binding ability with structures such as the antigen-binding site of an antibody or a binding site on a substrate molecule. Because the interactive ability and properties of a protein define its functional activity, specific amino acid substitutions in a protein sequence and its corresponding DNA coding sequence can also produce proteins with similar or desirable properties. Therefore, the inventors contemplate that various changes can be made to the DNA sequences of genes encoding proteins without significantly impairing their biological utility or activity.

[0109] The amino acid sequence variants of the present disclosure may be substitutional, insertional, or deletional variants. Mutations in the polypeptides of the present disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues of the protein or polypeptide compared to the wild-type. Variants may include amino acid sequences that share at least 50%, 60%, 70%, 80%, or 90% identity to any of the sequences disclosed or referenced herein (including all values ​​and ranges therebetween). The variant may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0110] It is also understood that amino acid and nucleic acid sequences may contain additional residues, such as N- or C-terminal amino acids, or 5' or 3' terminal sequences, and still be considered substantially identical to any of the sequences disclosed herein, so long as they meet the above conditions, particularly the condition of maintaining biological activity with respect to protein expression. The addition of terminal sequences applies particularly to nucleic acid sequences, and may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0111] Deletion mutants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or multiple consecutive amino acids may be deleted. A stop codon may also be introduced (substitution or insertion) into the encoding nucleic acid sequence to produce a truncated protein.

[0112] Insertional variants typically involve the addition of amino acid residues to a non-terminal position in the polypeptide. This can involve the insertion of one or more amino acid residues. Terminal additions can also be produced, which can include fusion proteins (which are multimers or concatamers of one or more peptides or polypeptides described or referenced herein).

[0113] Substitution variants typically involve the substitution of one amino acid for another at one or more sites in a protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., where one amino acid is replaced with another amino acid having similar chemical properties. "Conservative amino acid substitutions" can include exchanges between members of the same amino acid class. Conservative substitutions are well known to those skilled in the art and include, for example, the following substitutions: alanine for serine, arginine for lysine, asparagine for glutamine or histidine, aspartic acid for glutamic acid, cysteine ​​for serine, glutamine for asparagine, glutamic acid for aspartic acid, glycine for proline, histidine for asparagine or glutamine, isoleucine for leucine or valine, leucine for valine or isoleucine, lysine for arginine, methionine for leucine or isoleucine, phenylalanine for tyrosine, leucine or methionine, serine for threonine, threonine for serine, tryptophan for tyrosine, tyrosine for tryptophan or phenylalanine, valine for isoleucine or leucine, etc. Conservative amino acid substitutions may encompass unnatural amino acid residues, typically introduced by chemical peptide synthesis rather than synthesis in a biological system. This includes peptide mimetics and reversed or inverted amino acid substructures.

[0114] Alternatively, substitutions may be "non-conservative," in which case the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing an amino acid residue with one that is chemically dissimilar, for example, a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0115] <2. Considerations for substitution> Suitable variants of the polypeptides described herein can be determined by those skilled in the art using well-known techniques. By targeting regions not believed to be important for activity, one skilled in the art can identify sites on the molecule that can be altered without impairing activity. One skilled in the art can also identify amino acid residues and portions of molecules that are conserved among similar proteins or polypeptides. In yet other embodiments, regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without significantly altering the biological activity or adversely affecting the structure of the protein or polypeptide.

[0116] Such modifications can be made by taking into account the hydrophobicity index of amino acids, which is calculated by assigning a numerical value (hydrophobicity index) to each amino acid and averaging these values ​​repeatedly along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). The importance of the hydrophobic amino acid index in conferring interactive biological function to a protein is widely understood by those skilled in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). The relative hydrophobicity of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn determines its interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. It is also known that substitution of certain amino acids with other amino acids having similar hydrophobicity indices or scores can retain biological activity. When modifications are made based on the hydrophobicity index, certain embodiments include substitutions between amino acids with hydrophobicity indices within ±2. Some aspects of the present disclosure include substitutions within ±1, and other aspects include substitutions within ±0.5.

[0117] Those skilled in the art also understand that substitution of similar amino acids can be effectively performed based on hydrophilicity. U.S. Patent No. 4,554,101 (incorporated herein by reference) describes that the greatest local average hydrophilicity of a protein is governed by the hydrophilicity of its adjacent amino acids, and that this correlates with the biological properties of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, can correlate with immunogenicity and antigen-binding ability, i.e., the biological properties of the protein. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0 ± 1), glutamic acid (+3.0 ± 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 ± 1), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4). When changes are made based on similar hydrophilicity values, certain embodiments involve substitutions of amino acids with hydrophilicity values ​​within ±2, other embodiments involve substitutions within ±1, and still other embodiments involve substitutions within ±0.5. In some cases, epitopes can be identified by hydrophilicity based on the primary amino acid sequence. These regions are also referred to as "epitope core regions." It is also understood that substitution of one amino acid with another amino acid with a similar hydrophilicity value will result in a biologically equivalent and immunologically equivalent protein.

[0118] Additionally, one skilled in the art can refer to structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. Based on such comparisons, one skilled in the art can predict the importance of amino acid residues in a protein that correspond to amino acid residues identified as important for activity or structure in similar proteins. One skilled in the art can select chemically similar amino acids to substitute for such predicted important amino acid residues.

[0119] Those skilled in the art can also analyze the three-dimensional structure of similar proteins or polypeptides and the amino acid sequences associated with that structure. Based on this information, those skilled in the art can predict the placement of amino acid residues relative to the three-dimensional structure of the antibody. Those skilled in the art may choose not to alter amino acid residues predicted to be present on the surface of the protein because they may be involved in important interactions with other molecules. Furthermore, those skilled in the art can generate test variants with a single amino acid substitution at each desired amino acid residue. These variants can be screened using standard binding and / or activity assays, and the information gained from such routine experiments allows those skilled in the art to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure are available at www.expasy.org / proteomics / protein_structure.

[0120] In some embodiments of the present disclosure, amino acid substitutions are made to (1) reduce susceptibility to protease degradation, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity for a ligand or antigen, and / or (5) confer or modify other physicochemical or functional properties on the polypeptide. For example, one or more amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made to a naturally occurring sequence. Substitutions can be made in regions of the antibody outside the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially alter the structural features of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure of the wild-type antibody) can be used.

[0121] (B. Nucleic acid) In certain embodiments, nucleic acid sequences may exist in various forms, including isolated fragments and sequences incorporated into recombinant vectors; recombinant polynucleotides encoding one or both chains of an antibody, or fragments, derivatives, muteins, or variants thereof; polynucleotides usable as hybridization probes, PCR primers, or sequencing primers used to identify, analyze, mutate, or amplify polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides; and sequences complementary to the above sequences. Nucleic acids encoding epitopes bound by specific antibodies provided herein are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Such nucleic acids may be single-stranded or double-stranded and may contain RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0122] In certain embodiments, polynucleotide variants are present that are substantially identical to the sequences disclosed herein, i.e., have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity (as measured using methods such as BLAST analysis with standard parameters) to the polynucleotide sequences disclosed herein, including these values ​​and all ranges therebetween. In certain aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 90%, preferably 95% or more identity over its entire length to the amino acid sequences described herein, or a nucleotide sequence complementary to the isolated polynucleotide.

[0123] The overall length of the nucleic acid fragments of the present invention can vary widely, regardless of the length of the coding sequence itself, as they may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme recognition sites, multiple cloning sites, and other coding sequence fragments. The nucleic acids may be of any length, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more bases. They may also contain one or more additional sequences, e.g., regulatory sequences, or may be part of a larger nucleic acid, such as a vector. Thus, nucleic acid fragments of almost any length are contemplated, with the total length preferably being limited only by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence that includes additional heterologous coding sequences for purification, transport, secretion, post-translational modification, or therapeutic benefits such as targeting or efficacy. As noted above, tags or other heterologous polypeptides may be added to the sequence encoding the modified polypeptide. Here, "heterologous" refers to a polypeptide that is not identical to the modified polypeptide.

[0124] <1. Mutation> Mutations can be introduced into nucleic acids to produce changes in the amino acid sequence of the polypeptide (e.g., an antibody or antibody derivative) encoded thereby. Mutations can be introduced using any technique known to those of skill in the art. In certain embodiments, one or more specific amino acid residues can be altered, for example, using site-directed mutagenesis protocols. In other embodiments, one or more randomly selected residues can be altered, for example, using random mutagenesis protocols. Regardless of how they are produced, the mutant polypeptides can be expressed and screened for desired properties.

[0125] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the polypeptides encoded thereby. For example, nucleotide substitutions resulting in amino acid substitutions at non-essential amino acid residues can be made. Alternatively, one or more mutations can be introduced into nucleic acids that selectively alter the biological activity of the polypeptides encoded thereby (see, for example, RomainStuder et al., Biochem. J. 449:581-594 (2013)). For example, mutations can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include enhancing, reducing, or eliminating activity. Examples of qualitative changes include changing the antigen specificity of an antibody.

[0126] <2. Short guide RNA> In certain embodiments, the composition comprising any of the fusion protein-ribonucleotide complexes described herein comprises at least one short guide RNA (sgRNA). The sgRNA may form a complex with a nuclease, i.e., any of the CRISPR proteins, at a specific sequence. The sgRNA may target a specific gene, i.e., any of the genes described herein.

[0127] VI. Obtaining Embodiments of the Encoded Polypeptides In some aspects, there are nucleic acid molecules encoding fusion protein-ribonucleotide complex polypeptides. These are generated by methods known to those skilled in the art, such as phage display, expressed in a suitable recombinant expression system, and assembled to form the complex molecule. Nucleic acids encoding such polypeptides are described elsewhere herein.

[0128] SEQ ID NO: 12: Nucleotide sequence of 6xHis purification tag (6His) CACCACCATCATCATCAT

[0129] SEQ ID NO: 13: Nucleotide sequence of cell-penetrating peptide (CPP) AAGCTGGCCCTGAAGCTGGCCCTGAAGGCCCTGAAGGCCGCCCTGAAG

[0130] SEQ ID NO: 14: Nucleotide sequence of linker 1 GGC

[0131] SEQ ID NO: 15: Nucleotide sequence of linker 2 CTGTTCCCCACCATC

[0132] SEQ ID NO: 16: Nucleotide sequence of CRISPR / Cas9 nuclease

[0133] SEQ ID NO: 17: Nucleotide sequence of linker 3 GGCCCCGGC

[0134] SEQ ID NO: 18: Nucleotide sequence of TEV protease GAGAACCTGTACTTCCAGGGC

[0135] SEQ ID NO: 19: Nucleotide sequence of linker 4 GCCGGC

[0136] SEQ ID NO: 20: Nucleotide sequence of the FLAG tag GACTACAAGGACGACGACGACAAGGGCTGA

[0137] SEQ ID NO: 21 Nucleotide sequence of CD25 scFv CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCACCGGCTACACCGAGTACAACCAGAAGTTCAAGGACAAGGCCACCATCACCGCCGACGAGAGCACCAACACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGGCGGCGTGTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGATCACCATCACCTGCAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCACCAGCAACCTGGCCAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCACCAGAGGAGCACCTACCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGGTGAAGAGG

[0138] Sequence number 22: Nucleotide sequence of CRISPR / Cas9 nuclease

[0139] SEQ ID NO: 23: Nucleotide sequence of CD25 scFc-Cas9 fusion protein

[0140] (A. Expression) The nucleic acid molecules disclosed herein can be used to express large amounts of fusion proteins, and when these nucleic acid molecules are derived from non-human, non-transgenic animals, they can be used for antibody humanization.

[0141] <1. Vector> In some embodiments, expression vectors containing nucleic acid molecules encoding a polypeptide of a desired sequence, or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains) are contemplated. In addition to sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions.

[0142] To express fusion proteins, DNA encoding partial or full-length light and heavy chains may be inserted into an expression vector such that the gene regions are operably linked to transcriptional and translational control sequences. Expression vectors used in any host cell typically contain sequences for plasmid or viral maintenance and for cloning and expression of exogenous nucleotide sequences. These sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Sequences such as these and methods for their use are widely known to those skilled in the art.

[0143] <2. Expression system> Numerous expression systems exist that contain at least some or all of the expression vectors described above. In this embodiment, prokaryotic and / or eukaryotic expression systems can be used to produce nucleic acid sequences or their corresponding polypeptides, proteins, and peptides. Commercially available and widely available expression systems include, but are not limited to, bacterial, mammalian, yeast, and insect cell systems. Each host cell has its own unique mechanism for post-translational modification and processing of proteins. An appropriate cell line or host system can be selected to ensure that the foreign protein is correctly modified and processed. Those skilled in the art can express the vector using an appropriate expression system to produce the nucleic acid sequence or its corresponding polypeptide, protein, or peptide.

[0144] <3. Gene transfer method> As described herein or known to those of skill in the art, suitable methods for introducing nucleic acids to achieve expression of the compositions contemplate virtually any method for introducing nucleic acids (e.g., including DNA, viral vectors and non-viral vectors) into cells, tissues or individuals.These methods include direct introduction of DNA, e.g., by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, all of which are incorporated herein by reference), by microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference), by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference), calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al. al., 1990), DEAE-dextran treatment followed by polyethylene glycol (Gopal, 1985), direct sonophoresis (Fechheimer et al., 1987), and liposome-mediated gene transfer (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wonget et al., 1980; Kaneda et al., 1989; Katoet al., 1991), microprojectile bombardment (International Patent Applications WO 94 / 09699 and WO 95 / 06128; U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, all of which are incorporated by reference), agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, all of which are incorporated by reference), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, all of which are incorporated by reference), PEG-mediated transfer of protoplasts (Omirulleh et al., 1996). al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, both of which are incorporated by reference), and the desiccation / inhibition-mediated DNA uptake method (Potrykus et al., 1985).Other methods include viral vector-mediated gene transfer, such as lentivirus or retrovirus-mediated gene transfer.

[0145] <4.Host cells> Another aspect is the use of a host cell into which a recombinant expression vector has been introduced. Fusion proteins can be expressed in a variety of cell types. Expression constructs encoding fusion proteins can be transfected into cells by a variety of methods well known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. Some vectors utilize control sequences that allow replication and / or expression in both prokaryotic and eukaryotic cells.

[0146] In stable transfection of mammalian cells, it is known that only a small number of cells integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants (integrated cells), a selectable marker (e.g., an antibiotic resistance marker) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by methods well known to those skilled in the art, such as drug selection (e.g., cells that have incorporated the selectable marker gene survive, while other cells die).

[0147] VII. Additional Therapeutic Interventions Certain embodiments relate to administering the fusion protein-ribonucleotide complexes or disrupted immune cells described herein and an additional therapeutic intervention, which may be any intervention useful in treating a disease in an individual, including, but not limited to, a small molecule, a biologic, an immunotherapy, surgery, radiation therapy, or a combination thereof.

[0148] The additional treatment may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, protein therapy, or a combination thereof. The additional treatment may be in the form of adjuvant therapy or neoadjuvant therapy. The additional treatment may be one or more agents targeting the gene of interest (e.g., SRC-3) or its gene product (protein of interest), and the agents may include any type of antibody, small molecule inhibitor, nucleic acid, protein, or a combination thereof.

[0149] In some embodiments, the additional treatment is administration of a small molecule inhibitor of a protein of interest or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a small molecule inhibitor of SRC-3 or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side effect-reducing agent (e.g., an agent aimed at reducing the occurrence and / or severity of side effects of treatment, such as an antiemetic). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a therapy targeting the PBK / AKT / mTOR pathway, treatment with an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional treatment may be one or more chemotherapeutic agents known to those skilled in the art.

[0150] The CRISPR / Cas9 / targeting antibody-gene-targeting sgRNA fusion protein / ribonucleotide complex may be administered before, during, or after an additional cancer treatment, or in various combinations thereof. The CRISPR / Cas9 / targeting antibody-SRC-3 gene-targeting sgRNA fusion protein / ribonucleotide complex may also be administered before, during, or after an additional cancer treatment, or in various combinations thereof. These administrations may occur simultaneously or within intervals ranging from minutes, hours, days, or weeks. In embodiments in which the CRISPR / Cas9 / targeting antibody-gene-targeting sgRNA fusion protein / ribonucleotide complex treatment is provided to the patient separately from the additional therapeutic agent, it is generally desirable not to allow a significant time lapse between administrations to allow the compounds to exert their beneficial synergistic effects on the patient.

[0151] When administering any of the compounds or treatments according to the embodiments of the present invention to an individual, general protocols for the administration of such compounds should be followed, taking into account the toxicity, if any, of the compound, and thus, in some embodiments, include monitoring for toxicity resulting from the combination therapy.

[0152] A. Checkpoint Inhibitors and Combination Therapies Embodiments of the present disclosure can include the administration of immune checkpoint inhibitors, as described in further detail below. In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints function to enhance (e.g., costimulatory molecules) or suppress signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), natural killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), programmed cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. In other embodiments, another immune checkpoint that can be targeted by the methods described herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0153] 1. PD-1, PDL1, and PDL2 inhibitors PD-1 may act in the tumor microenvironment where T cells encounter infections and tumors. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-γ induce PDL1 expression in epithelial cells and tumor cells. PDL2 is expressed in macrophages and dendritic cells. The primary role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during immune responses. The inhibitors disclosed herein can inhibit one or more functions of PD-1 and / or PDL1 activity.

[0154] Aliases for "PD-1" include CD279 and SLEB2. Aliases for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Aliases for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, human PDL1, and human PDL2.

[0155] In some embodiments, the PD-1 inhibitor is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In certain embodiments, the ligand-binding partner of PD-1 is PDL1 and / or PDL2. In another embodiment, the PDL1 inhibitor is a molecule that inhibits PDL1 from binding to its binding partner. In certain embodiments, the binding partner of PDL1 is PD-1 and / or B7-1. In yet another embodiment, the PDL2 inhibitor is a molecule that inhibits PDL2 from binding to its binding partner. In certain embodiments, the binding partner of PDL2 is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions disclosed herein are known to those of skill in the art and are described, for example, in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0156] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin in which the extracellular domain or a PD-1-binding portion of PDL1 or PDL2 is fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PDL1 inhibitor comprises AMP-224 (AMP-224). Nivolumab, also known as MDX-1106-04 (MDX-1106-04), MDX-1106 (MDX-1106), ONO-4538 (ONO-4538), BMS-936558 (BMS-936558), and OPDIVO® (OPDIVO®), is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475 (MK-3475), Merck 3475 (Merck 3475), lambrolizumab, KEYTRUDA® (KEYTRUDA®), and SCH-900475 (SCH-900475), is an anti-PD-1 antibody described in WO 2009 / 114335. Pidilizumab, also known as CT-011 (CT-011), hBAT (hBAT), or hBAT-1 (hBAT-1), is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224 (AMP-224), also known as B7-DCIg (B7-DCIg), is a PDL2-Fc fusion soluble receptor and is described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680 (MEDI0680) (also known as AMP-514 (AMP-514)) and REGN2810 (REGN2810).

[0157] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as durvalumab (also known as MEDI4736 (MEDI4736)), atezolizumab (also known as MPDL3280A (MPDL3280A)), avelumab (also known as MSB00010118C (MSB00010118C)), MDX-1105 (MDX-1105), BMS-936559 (BMS-936559), or a combination thereof. In certain embodiments, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7 (rHIgM12B7).

[0158] In some embodiments, the inhibitor comprises the heavy and light chain CDRs (CDRs) or variable regions (VRs) of nivolumab, pembrolizumab, or pidilizumab. Thus, in certain embodiments, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for or binds to the same epitope as the above-mentioned antibody. In yet another embodiment, the antibody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derived therefrom) variable region amino acid sequence identity with the above-mentioned antibody.

[0159] 2. CTLA-4, b7-1, and B7-2 Another immune checkpoint that can be targeted herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152 (CD1h52). The complete cDNA sequence of human CTLA-4 is recorded in GenBank accession number L15006. CTLA-4 is present on the surface of T cells and acts as an "off" switch upon binding to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA-4 resembles the T cell costimulator CD28; both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found on regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors according to the present disclosure can inhibit the function of any of CTLA-4, B7-1, or B7-2. In one embodiment, the inhibitor inhibits the interaction between CTLA-4 and B7-1. In another embodiment, the inhibitor inhibits the interaction between CTLA-4 and B7-2.

[0160] In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0161] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods known to those skilled in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in the following documents can be used in the methods of the present disclosure: U.S. Pat. No. 8,119,129, International Publication Nos. WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, or tremelimumab, formerly known as ticilimumab), U.S. Pat. No. 6,207,156; Hurwitz et al., 1998, and the like. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized anti-CTLA-4 antibodies are described in International Patent Publication Nos. WO2001 / 014424, WO2000 / 037504, and US Pat. No. 8,017,114, which are also incorporated herein by reference.

[0162] Another anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, Yervoy®), or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0163] (B. Immunotherapy) In one embodiment, the inhibitor comprises the heavy and light chain CDRs (CDRs) or VRs (variable regions) of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody binds to or competes for binding with PD-1, B7-1, or B7-2 to the same epitope as the above-described antibodies. In yet another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any range derivable therein) variable region amino acid sequence identity to the above-described antibodies.

[0164] In some embodiments, the method includes administering cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) utilizes the immune system to treat cancer. Immunotherapies can be classified as active, passive, or a hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface known as tumor-associated antigens (TAAs) that can be detected by the immune system. These molecules are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting tumor-associated antigens. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapies are known to those skilled in the art, and some are described below.

[0165] Those skilled in the art will understand that in addition to the immunotherapies encompassed herein, other immunotherapies can be used in combination or in conjunction with the methods of the present embodiments. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can function alone as an effector of treatment or can recruit other cells to actually perform cell killing. The antibody can also be conjugated to a drug or toxin (e.g., a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0166] Antibody-drug conjugates (ADCs) have emerged as a groundbreaking approach in the development of cancer therapeutics. Antibody-drug conjugates (ADCs) are monoclonal antibodies (MAbs) covalently linked to cell-killing agents. This approach combines the high specificity of MAbs for target antigens with highly potent cytotoxic agents, resulting in "armed" MAbs that deliver payloads to tumor cells with high antigen expression. Targeted drug delivery minimizes drug exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. In some embodiments, ADCs are used as an additional therapeutic intervention. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1), in 2011 and 2013, respectively, is just one example of the effectiveness of this approach.

[0167] In one aspect of immunotherapy, tumor cells must have a targetable marker, i.e., a marker not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy is the combination of anti-cancer and immunostimulatory effects. Immune stimulatory molecules also exist, including cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, γ-IFN), chemokines (e.g., MIP-1, MCP-1, IL-8), and growth factors (e.g., FLT3 ligand).

[0168] 1. Inhibition of costimulatory molecules In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0169] (C.CAR-T cell therapy) Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptors engineered to confer new specificity to immune cells for targeting cancer cells. Typically, these receptors transfer the specificity of monoclonal antibodies onto T cells. These receptors are called "chimeric" because they combine components from different sources. CAR-T cell therapy refers to the use of such engineered cells to treat cancer.

[0170] The basic principle of CAR-T cell design is the use of recombinant receptors that combine antigen-binding and T cell activation functions. The general premise of CAR-T cells is to engineer T cells to target markers present on cancer cells. Scientists can extract T cells from an individual, genetically modify them, and then inject them back into the patient to attack cancer cells. Once engineered into CAR-T cells, they function as "living drugs." CAR-T cells activate T cells by forming a link between their extracellular ligand-recognition domain and an intracellular signaling molecule. The extracellular ligand-recognition domain is typically a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is ensuring that it targets only cancerous tumor cells and not normal cells. The specificity of CAR-T cells is determined by the choice of targeting molecule.

[0171] Examples of CAR-T cell therapies include tisagenlecleucel (Kymriah) and axicabtageneciloreucel (Yescarta). In some embodiments, the CAR-T cell therapy targets CD19.

[0172] (D. Cytokine Therapy) Cytokines are proteins produced by a wide variety of cells present within tumors and can modulate the immune response. Tumors often use them to promote their own growth and suppress the immune response. These immunomodulatory effects allow cytokines to be used as agents to induce immune responses. Two commonly used cytokines are interferons and interleukins.

[0173] Interferons are produced by the immune system. They are usually involved in antiviral responses but can also be used against cancer. Interferons are classified into three groups: Type I (IFNα and IFNβ), Type II (IFNγ), and Type III (IFNλ).

[0174] Interleukins have diverse effects on the immune system. IL-2 is a representative interleukin-based cytokine therapy.

[0175] (E.Adoptive T cell therapy) Adoptive T cell therapy is a form of passive immunization through the infusion of T cells (adoptive cell transfer). T cells are present in the blood and tissues and are typically activated upon detecting a foreign pathogen. Specifically, activation occurs when the T cell's surface receptor encounters a cell that presents a portion of the foreign protein as its surface antigen. These cells may be infected cells or antigen-presenting cells (APCs). They are present in both normal and tumor tissues, where they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs, such as dendritic cells, that present tumor antigens. Although these cells can attack tumors, the environment within the tumor is highly immunosuppressive, inhibiting tumor cell death through the immune response.

[0176] Several methods have been developed to generate and obtain tumor-targeting T cells. Tumor antigen-specific T cells can be obtained by depletion from tumor samples (TILs) or by filtration from the blood. They are then activated and cultured ex vivo, and the resulting cells are reinfused. Activation can be achieved by gene therapy or by exposing T cells to tumor antigens.

[0177] Cancer treatments that exclude any of the cancer treatments described herein are also contemplated. Furthermore, embodiments of the present disclosure include patients who have previously received, are currently receiving, or have not received a treatment described herein. In some embodiments, the patient is determined to be resistant to a treatment described herein. In some embodiments, the patient is determined to be sensitive to a treatment described herein.

[0178] (F.Chemotherapy) In some embodiments, the additional treatment comprises chemotherapy. A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents are classified by their mode of action within cells, for example, whether or not they affect the cell cycle and the stage at which it occurs. Alternatively, they may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or affect nucleic acid synthesis to induce chromosomal or mitotic abnormalities. Suitable classes of chemotherapeutic agents include: alkylating agents (nitrogen mustards: mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, etc.), ethylenimines and methylmelamines (hexamethylmelamine, thiotepa, etc.), alkylsulfonic acids (busulfan, improsulfan, piposulfan, etc.), nitrosoureas (carmustine, lomustine, chlorozoticin, streptozocin, etc.), triazines (dacarbazine, etc.), antimetabolites (folic acid analogs: methotrexate, etc., pyrimidine analogs: 5-fluorouracil, floxuridine, cytarabine, azauridine, etc., purine analogs and related compounds: 6-mercaptopurine, 6-thioguanine, pentostatin, etc.), natural products (vinca alkaloids drugs: vinblastine, vincristine, etc., epipodophyllotoxins: etoposide, teniposide, etc., antibiotics: dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitoxantrone, etc., enzymes (L-asparaginase, etc.), biological response modifiers (interferon-α, etc.), others (platinum complexes: cisplatin, carboplatin, etc., substituted ureas: hydroxyurea, etc., methylhydrazine derivatives: procarbazine, etc., adrenocortical suppressants: taxol, mitotane, etc.), aziridines (benzodopa, carboquone, meturedopa, uredopa, etc.), ethyleneimines and methylmelamines (arretamine, triethylenemelamine, triethylenephosphoramide, triethylenetriphosphoramide, trimethylolmelamine, etc.),Acetogenins (especially bullatacin and bullatacinone), camptothecin (including the synthetic analogue topotecan), bryostatin, kallistatin, CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin), cryptophycins (especially cryptophycins 1 and 8), dolastatin, duocarmycins (including the synthetic analogues KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictin, spongistatin, nitrogen mustards (chlorambucil, chlornaphazine, colofos) Famide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.), nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.), enene antibiotics (calicheamicin, especially calicheamicin γI and calicheamicin ωI1), dynemicins (including dynemicin A), bisphosphonates (clodronate, etc.), esperamicin, ne Ocarzinostatin chromophore and related chromoproteins Enene antibiotic chromophores, aclacinomycin, actinomycin, auxaramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, deoxydoxorubicin), epirubicin, eso Rubicin, idarubicin, marcelomycin, mitomycin (mitomycin C, etc.), mycophenolic acid, nogalamycin, olivomycin, peplomycin, portifromycin, ptomycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubericidin, ubenimex, zinostatin, zorubicin, antimetabolites (methotrexate, 5-fluorouracil (5-FU), etc.), folic acid analogues (denopterin, pteropterin, trimetrexate, etc.), purine analogues (fludarabine, 6-mercaptopurine, thiamiprine,thioguanine, etc.), pyrimidine analogues (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.), androgens (calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.), adrenal suppressants (mitotane, trilostane, etc.), folic acid supplements (florinic acid, etc.), aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, Bestravcil, Bisantre acetaminophen, edatrexate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilones, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (maitansin, ansamitocins, etc.), mitoguazone, mitoxantrone, mopidanol, nitraelin, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofiran, spiroglimani methicone, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veraculin A, roridin A, anguidine, etc.), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, taxanes (paclitaxel, docetaxel), gemcitabine, 6-thioguanine, mercaptopurine, platinum complexes (cisplatin, oxaliplatin, carboplatin, etc.), vinplas tin, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronic acid, irinotecan (CPT-11, etc.), topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids (retinoic acid, etc.), capecitabine, carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum,and pharmaceutically acceptable salts, acids, or derivatives of any of the above agents. In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0179] Cisplatin has been widely used to treat, for example, metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer, or other tumors. Cisplatin is not absorbed orally and must be delivered by other routes, such as intravenous, subcutaneous, intratumoral, or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and has been clinically effective in certain embodiments at doses of about 15 mg / m for 5 days every 3 weeks. 2 to approximately 20 mg / m 2 In some embodiments, the amount of cisplatin delivered to a cell and / or subject in combination with a construct in which the Egr-1 promoter is operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount delivered when cisplatin is used alone.

[0180] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("Taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector with doxorubicin has been shown to be effective in overcoming resistance to chemotherapy and / or TNFα, suggesting that combined treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNFα.

[0181] Doxorubicin is poorly absorbed, so intravenous administration is preferred. In certain embodiments, a suitable intravenous dose for adults is about 60 mg / m 2 to approximately 75 mg / m 2 at intervals of about 21 days, or about 25 mg / m per day for 2 or 3 consecutive days. 2 to approximately 30 mg / m2 and repeat at intervals of about 3 to 4 weeks, or about 20 mg / m 2 The lowest doses should be used in elderly patients, those with myelosuppression from previous chemotherapy, those with neoplastic bone marrow infiltration, or those given in combination with other myelosuppressive agents.

[0182] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards may include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®, available from Mead Johnson; NEOSTAR®, available from Adria) is another suitable chemotherapeutic agent. Suitable oral dosages for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day. Intravenous dosages include, for example, an initial dose of about 40 mg / kg to about 50 mg / kg administered in divided doses over two to five days, or about 10 mg / kg to about 15 mg / kg administered about every seven to about ten days, or about 3 mg / kg to about 5 mg / kg administered twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse effects on the gastrointestinal system, intravenous administration is preferred. The drug may also be administered intramuscularly, invasively, or into a body cavity.

[0183] Other suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU is administered to a subject at a dose of approximately 7.5 mg / m 2 to approximately 1000 mg / m 2 Furthermore, the administration schedule for 5-FU can be for a variety of periods, for example up to six weeks, or as determined by one of ordinary skill in the art of this disclosure.

[0184] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine") is another suitable chemotherapeutic agent that is recommended for the treatment of advanced or metastatic pancreatic cancer and is therefore useful for these cancers in the present disclosure.

[0185] The amount of chemotherapeutic agent delivered to a patient can vary. In a suitable embodiment, the chemotherapeutic agent can be administered in an amount effective to cause the arrest or regression of cancer in a host when administered with the construct. In other embodiments, the chemotherapeutic agent can be administered at an amount about 2 to about 10,000 times lower than its chemotherapeutic effective dose. For example, the chemotherapeutic agent can be administered at an amount about 20 times lower, about 500 times lower, or about 5,000 times lower than its chemotherapeutic effective dose. The chemotherapeutic agents of the present disclosure can be tested in vivo to determine the desired therapeutic activity and effective dose in combination with the construct. For example, these compounds can be tested in appropriate animal model systems, including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc., prior to human testing. In vitro testing can also be used to determine appropriate combinations and doses, as described in the Examples.

[0186] (G.Surgery) Approximately 60% of cancer patients will undergo some form of surgery, including preventative surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be combined with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery).

[0187] Removal of part or all of the cancer cells, tissue, or tumor may result in the formation of a cavity within the body. Treatment may be achieved by administering additional anticancer therapy to the area via perfusion, direct injection, or local application. Such therapy may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days; every 1, 2, 3, 4, or 5 weeks; or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These therapy may also be administered at various doses.

[0188] (H. Radiation Therapy) In some embodiments, the additional or preceding treatment comprises radiation, such as ionizing radiation. As used herein, "ionizing radiation" refers to radiation that includes particles or photons and has sufficient energy to cause ionization (gain or loss of electrons) or can generate sufficient energy through nuclear reactions. An exemplary and preferred ionizing radiation is X-ray radiation. Means for delivering X-ray radiation to target tissues or cells are well known to those skilled in the art.

[0189] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in a single dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three fractions. In some embodiments, the amount of ionizing radiation can be at least, at most, or exactly 2, 4, 6, 8, 10, 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, or 50 Gy (or any derivable range therein). In some embodiments, the ionizing radiation can be administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractionated doses (or any derivable range therein). When administered in multiple doses, the interval between each dose may be about 1, 4, 8, 12, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks, or any interval derivable therein.

[0190] In some embodiments, the amount of ionizing radiation (IR) is presented as a total dose, which is administered in fractions. For example, in some embodiments, the total dose is 50 Gy, administered in 10 fractions of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractions of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 1 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some embodiments, the total administered dose can consist of fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein).In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractional doses may be administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses may be administered per day (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 fractionated doses may be administered per week (or any derivable range therein).

[0191] Other widely used agents that cause DNA damage include the commonly known gamma rays, X-rays, and / or radioisotopes, which are delivered to tumor cells in a targeted manner. Other forms of DNA damaging agents include microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and ultraviolet (UV) radiation. All of these agents likely cause widespread damage to DNA itself, its precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dosages range from 50–200 roentgens per day for 3–4 weeks to single doses of 2000–6000 roentgens. The dosage range for radioisotopes is very broad and depends on the half-life of the isotope, the strength and type of radiation emitted, and its uptake by tumor cells.

[0192] VIII. Treatment of Disease In some embodiments, the present disclosure provides methods for treating cancer using immunotherapy, or for treating metabolic inborn errors, hematopoietic disorders, inflammatory disorders, and / or genetic disorders affecting liver function. In certain embodiments, the methods and compositions described herein are used to treat, delay progression, delay onset, or reduce the risk of developing a disease, such as cancer. In some embodiments, the methods and compositions include administering to an individual a therapeutically effective amount of a fusion protein-ribonucleotide complex, such as a CRISPR / Cas9 / targeting antibody / gene-targeting guide RNA (sgRNA) fusion protein / ribonucleotide complex.

[0193] In certain embodiments, metabolic inborn errors include, but are not limited to, glycogen storage disease, G6PD deficiency, phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, carbamoyl phosphate synthase I deficiency, alkaptonuria, combined malonic-methylmalonic aciduria, 2-hydroxyglutaric aciduria, medium-chain acyl-coenzyme A dehydrogenase deficiency, acute intermittent porphyria, Resch-Nyhan syndrome, lipid-type congenital adrenal hyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher disease, or Niemann-Pick disease.

[0194] In certain embodiments, hematopoietic disorders include, but are not limited to, bone marrow failure disorders such as Fanconi anemia, thrombocytopenia with radial defect, Diamond-Blackfan anemia, Shwatchman-Diamond syndrome, and cartilage-hair hypoplasia; hemoglobinopathies such as sickle cell disease and thalassemia; neutrophil and lymphocyte dyscrasias such as chronic granulomatous disease and X-linked agammaglobulinemia; and monocyte-macrophage system disorders such as Gaucher disease.

[0195] In certain embodiments, inflammatory diseases include, but are not limited to, allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, transplant rejection, ankylosing spondylitis (AS), gout, myositis, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, pelvic inflammatory disease, or vasculitis.

[0196] In certain embodiments, genetic diseases affecting liver function include, but are not limited to, hemochromatosis and alpha-1 antitrypsin deficiency.

[0197] This method can be applied to the treatment of solid cancers or blood cancers. The cancer may be primary, metastatic, or resistant to treatment. The type and stage of cancer are not important. The individuals may be at higher risk for cancer than the general population, including those with personal or family medical history, smoking history, obesity, excessive alcohol consumption, certain viral infections such as human papillomavirus (HPV), exposure to one or more carcinogens, or excessive radiation exposure, including ultraviolet radiation from the sun. An amount of exposure is considered "excessive" if it is greater than the average individual in that population.

[0198] Tumors for which the therapeutic methods disclosed herein are useful include any malignant cell type, such as those found in solid tumors or hematological tumors. In some embodiments, solid tumors include, but are not limited to, tumors of organs selected from the group consisting of breast, ovary, pancreas, colon, appendix, stomach, brain, head, neck, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, gastric tissue, and endometrium. In some embodiments, hematological tumors include bone marrow tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Examples of specific cancers that can be treated by the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer or tumors (including gastrointestinal cancer and gastrointestinal stromal tumors), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various head and neck cancers, melanoma, and the like.

[0199] Cancers encompassed herein may specifically refer to the following histological types, including, but not limited to, malignant tumor, carcinoma, undifferentiated carcinoma, giant cell carcinoma and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, mixed hepatocellular and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenomatous intrapolypoid adenocarcinoma, adenocarcinoma in familial adenomatous polyposis, solid carcinoma, malignant carcinoid tumor, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, eosinophilic carcinoma, eosinophilic adenocar ... Eosinophilic adenocarcinoma, Basophilic carcinoma, Clear cell adenocarcinoma, Granular cell carcinoma, Follicular adenocarcinoma, Papillary and follicular adenocarcinoma, Non-capsular sclerosing carcinoma, Adrenal cortical carcinoma, Endometrial carcinoma, Adnexal carcinoma of the skin, Apocrine adenocarcinoma, Sebaceous gland carcinoma, Auricular gland adenocarcinoma, Mucoepidermoid carcinoma, Cystadenocarcinoma, Papillary cystadenocarcinoma, Papillary serous cystadenocarcinoma, Mucinous cystadenocarcinoma, Mucinous adenocarcinoma, Signet ring cell carcinoma, Invasive ductal carcinoma, Medullary carcinoma, Lobular carcinoma, Inflammatory carcinoma, Paget's disease (breast), Acinic cell carcinoma, Adenosquamous carcinoma, Adenocarcinoma with squamous metaplasia, Malignant thymoma, Malignant ovarian stromal tumor, Malignant secoma, Malignant granulosa cell tumor, Malignant androblastoma, Sertoli cell carcinoma, Malignant Leydi's carcinoma Histoma, malignant lipocytoma, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomus angiosarcoma, malignant melanoma, non-melanotic melanoma, superficial spreading melanoma, lentigo melanoma, acral lentigo melanoma, nodular melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell melanoma, blue nevus melanoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed malignant tumor, Müllerian mixed malignant tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor Tumors, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant struma ovary, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, subcortical osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, blastic odontogenic sarcoma, malignant ameloblastoma, blastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astrocytoblastoma, glioblastoma, oligodendroglioma, oligodendroglioma,Primitive neuroectodermal tumor, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory nerve tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's granuloma, malignant lymphoma (small lymphocytic), malignant lymphoma (large cell / diffuse), malignant lymphoma (follicular), mycosis fungoides, other specified non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-Hodgkin's lymphoma cleaved cell NHL, mass-forming disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, malignant histiocytosis, multiple myeloma, mast cell sarcoma, small intestinal immunoproliferative disorder, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroblastic leukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0200] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need, such as an individual with or suspected of having cancer. In some embodiments, the administered cells enhance the individual's immune system, thereby attacking the cancer. In some cases, the individual receives one or more administrations of immune cells and / or the CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complexes described herein. When an individual receives more than one administration, the intervals are sufficient to allow time for targeted Treg proliferation within the individual; in certain embodiments, the interval between administrations is 1, 2, 3, 4, 5, 6, 7, or more days. In some cases, the interval between administrations may be 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or longer, or any range derivable therein.

[0201] In some embodiments, the subject may be administered non-myeloablative lymphodepleting chemotherapy prior to immune cell therapy and / or conjugate therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable therapy and may be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, and may also be applied when the cancer is potentially metastatic melanoma. In certain embodiments, the administration route of cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and / or fludarabine may be administered. In certain aspects, about 60 mg / kg of cyclophosphamide is administered over two days, followed by about 25 mg / m 2 of fludarabine is administered for 5 days.

[0202] In certain embodiments, a growth factor or cytokine that promotes immune cell proliferation and activation is administered to a subject simultaneously with the CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex. The immune cell growth factor can be any suitable growth factor that promotes immune cell proliferation and activation. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7, and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[0203] A therapeutically effective amount of the CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex can be administered by several routes, including, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection, intranasal, intraarterial, or by infusion.

[0204] In some embodiments, a therapeutically effective amount of a CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex is an amount that achieves a desired effect in a subject being treated. For example, this may be the amount of a CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex required to inhibit cancer growth, cause cancer regression, or improve at least one symptom of cancer.

[0205] The immune cell population can be administered in a treatment regimen appropriate for the disease. For example, one or more administrations can be administered over one to several days to ameliorate the disease state, or they can be administered periodically over a long period of time to suppress disease progression and prevent recurrence. The exact dosage used in the prescription will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the individual's circumstances. The amount of immune cells that will be therapeutically effective will depend on the subject being treated, the severity and type of the disease, and the method of administration. In some embodiments, a dosage that can be used to target Tregs present in a human subject is at least 3.8 x 10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 pieces / m 2 In certain embodiments, the dose used to treat a human subject may be about 3.8 x 10 immune cells. 9 pieces / m 2 to approximately 3.8 x 10 10 pieces / m 2 In additional embodiments, the range is 5 x 10 immune cells per kg body weight. 6 pieces to approximately 7.5 x 10 8A therapeutically effective amount of the CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex that can target Treg cells is, for example, about 2 x 10 per kg body weight. 7 From about 5 x 10 8 pieces, or approximately 5 x 10 7 pieces to approximately 2 x 10 8 The effective dose is in the range of 100-150 cells. The exact amount of CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex can be easily determined by those skilled in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated based on dose-response curves derived from in vitro or animal model test systems.

[0206] The CRISPR / Cas9 / targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex may be administered in combination with one or more other therapeutic agents for the treatment of cancer. The CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting sgRNA fusion protein / ribonucleotide complex may also be administered in combination with one or more other therapeutic agents for the treatment of cancer. Combination therapy may include, but is not limited to, one or more anti-tumor agents or vaccines. In certain cases, chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan), radiation, or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) may be administered in addition to the immune cells. Such additional agents may be administered before, during, or after the administration of the immune cells, depending on the desired effect. The immune cells and one or more additional anti-cancer agents may be administered by the same route or by different routes, at the same site or at different sites.

[0207] IX. ADMINISTRATION OF THERAPEUTIC COMPOSITIONS The treatments provided herein may include the combined administration of a first fusion protein-ribonucleotide complex therapy and a second therapy (including other therapeutic interventions described herein). These treatments may be administered in any suitable manner known to those skilled in the art. For example, the first therapy and the second therapy may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first therapy and the second therapy are administered as separate compositions. In some embodiments, the first therapy and the second therapy are contained in the same composition.

[0208] In some embodiments, the first therapy and the second therapy are administered substantially simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy, the second therapy, and the third therapy are administered sequentially. In some embodiments, the first therapy and the second therapy are administered.

[0209] The present disclosure relates to compositions and methods, including therapeutic compositions. Different treatments can be administered in one composition or in multiple compositions, such as two, three, or four compositions. Combinations of various agents can be used.

[0210] The therapeutic agents of the present disclosure can be administered by the same or different routes of administration. In some embodiments, the cancer treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage is determined based on the type, severity, and course of the disease being treated, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0211] The treatment can include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount to be administered, as well as the particular route of administration and formulation, are within the discretion of one skilled in the art of clinical medicine. The unit dose does not necessarily have to be administered as a single injection, but may consist of a continuous infusion over a period of time. In some embodiments, the unit dose comprises a single administrable dose.

[0212] In some embodiments, treatment with the fusion protein-ribonucleotide complex is administered in a single dose. In some embodiments, the fusion protein-ribonucleotide complex is administered in multiple doses. In some embodiments, the fusion protein-ribonucleotide complex is administered at a dose ranging from 1 mg / kg to 5000 mg / kg. In some embodiments, the fusion protein-ribonucleotide complex is administered in a dose ranging from at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 21,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173, 174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515 ,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554 ,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2 It is administered at doses of 300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0213] In some embodiments, the additional therapeutic intervention is administered in a single dose. In some embodiments, the additional therapeutic intervention is administered in multiple doses. In some embodiments, the additional therapeutic intervention is administered at a dose ranging from 1 mg / kg to 100 mg / kg. In some embodiments, the additional therapeutic intervention is administered in a dose ranging from at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, Administered at a dose of 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg / kg.

[0214] The amount to be administered, both in terms of the number of treatments and the unit dose, depends on the desired therapeutic effect. An effective dose is understood to mean the amount required to achieve a specific effect. In certain embodiments, a dose ranging from 10 mg / kg to 200 mg / kg is expected to affect the protective capacity of these agents. Thus, doses including about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day (or any derivable range therein) are contemplated. Furthermore, such doses may be administered multiple times a day, or over multiple days, weeks, or months.

[0215] In certain embodiments, an effective dose of a therapeutic composition is one that can provide a blood concentration of about 1 μM to 150 μM, hi other embodiments, an effective dose can provide a blood concentration of about 4 μM to 100 μM, or about 1 μM to 100 μM, about 1 μM to 50 μM, about 1 μM to 40 μM, about 1 μM to 30 μM, about 1 μM to 20 μM, about 1 μM to 10 μM, about 10 μM to 150 μM, about 10 μM to 100 μM, about 10 μM to 50 μM, about 25 μM to 150 μM, about 25 μM to 100 μM, about 25 μM to 50 μM, about 50 μM to 150 μM, or about 50 μM to 100 μM (or any derivable range therein). In another embodiment, the dose is about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM (or any derivable range therein). In certain embodiments, if a therapeutic agent administered to a subject is metabolized in the body to produce a therapeutic agent as a metabolite, blood levels may refer to the amount of the metabolized therapeutic agent. Alternatively, if a therapeutic agent is not metabolized by the subject, blood levels referred to herein may refer to the unmetabolized therapeutic agent.

[0216] The precise amount of the therapeutic composition depends on the judgment of the practitioner and is peculiar to each individual patient. Factors affecting the dosage include the patient's physical and clinical condition, the route of administration, the therapeutic objectives (palliative or curative), the efficacy, stability, and toxicity of the particular therapeutic agent, and other treatments the subject may be undergoing.

[0217] Those skilled in the art will understand that dosage units of μg / kg or mg / kg body weight can be expressed in terms of blood concentration units of μg / mL or mM, e.g., 4 μM to 100 μM. It will also be understood that uptake is species- and organ / tissue-dependent. Applicable conversion factors and physiological assumptions that should be made regarding uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy, and results described herein.

[0218] In certain cases, it may be desirable to administer the composition multiple times, for example, 2, 3, 4, 5, 6, or more times. These administrations may be given at 1, 2, 3, 4, 5, 6, 7, 8, 5, 6, 7, 8, 9, 10, 11, or 12 week intervals (and any derivable ranges therein).

[0219] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmacologically active substances is well known to those skilled in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, for example, other anti-infective agents or vaccines, can also be incorporated into the compositions.

[0220] The active compound can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous, or intraperitoneal route. Typically, such compositions can be prepared as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions by adding liquid before injection can also be prepared; furthermore, these preparations can be emulsified.

[0221] The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, such as formulations containing aqueous propylene glycol, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and fluid enough to be easily syringable.It must also be stable under the conditions of manufacture and storage, and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0222] Proteinaceous compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed by reaction with free amino groups of the protein), for example, with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed by reaction with free carboxyl groups can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0223] Therapeutic compositions may contain solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using in the composition absorption delaying agents, for example, aluminum monostearate or gelatin.

[0224] Sterile injectable solution is prepared by dissolving the required amount of active compound in a suitable solvent together with various other ingredients as listed above as necessary, and then sterilizing by filtration or equivalent means.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and the other required ingredients as listed above.For the preparation of sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which is a method to obtain a powder of active ingredient and any additional ingredients in the solution that has been previously sterile-filtered.

[0225] Administration of the compositions is typically via any common route of administration, including, but not limited to, oral or intravenous administration. Alternatively, administration may be via intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions are typically administered as pharmaceutically acceptable compositions containing physiologically acceptable carriers, buffers, or other excipients.

[0226] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically or prophylactically effective. These formulations can be readily administered in a variety of dosage forms, such as the types of injectable solutions described above.

[0227] (A. Therapeutic composition) In some embodiments, provided herein are therapeutic compositions and formulations comprising a fusion protein-ribonucleotide complex, such as a CRISPR / Cas9 / CD25-targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex and a pharmaceutically acceptable carrier. In some embodiments, provided herein are therapeutic compositions and formulations comprising a CRISPR / Cas9 / targeting antibody / SRC-3 gene-targeting sgRNA fusion protein / ribonucleotide complex and a pharmaceutically acceptable carrier. The therapeutic compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an active ingredient (e.g., a CRISPR / Cas9 / CD25-targeting antibody / gene-targeting sgRNA fusion protein / ribonucleotide complex) having a desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd Edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol). low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as polyethylene glycol (PEG).Additionally, exemplary pharmaceutically acceptable carriers herein include interstitial drug diffusion facilitators, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoprotein (rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of their use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0228] In some embodiments, the carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the agent in the carrier. In some aspects of the present disclosure, the agent is formulated for local delivery (i.e., delivery to a specific site in the body) or systemic delivery in solid, semi-solid, gel, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants, and injections, allowing for oral, parenteral, or surgical administration. In certain aspects of the disclosure, local administration by coating the composition on a medical device or the like is also contemplated.

[0229] Suitable carriers for parenteral administration by injection, infusion, or irrigation, and for topical administration, include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. Additionally, sterile, fixed oils can be used as solvents or suspension media. For this purpose, any biocompatible oil, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The carrier and the agent can be prepared as a liquid, suspension, polymeric or non-polymeric gel, paste, or ointment.

[0230] Carriers may also include delivery vehicles to sustain (i.e., extend, delay, or modulate) drug delivery or to enhance the delivery, uptake, stability, or pharmacokinetics of therapeutic agents. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, polymeric micelles.

[0231] In certain embodiments, the actual dosage of a composition administered to a patient or subject may be determined by physical and physiological factors such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, patient-specific disease factors, and route of administration. The clinician responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and the appropriate dose for the individual subject.

[0232] Solutions of the therapeutic composition can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0233] In certain embodiments, therapeutic compositions are advantageously administered in the form of injectable compositions, as liquid solutions or suspensions.They can also be prepared in solid form, suitable for dissolving or suspending in liquid before injection.These preparations can also be emulsified.Typical compositions for this purpose include a pharmaceutically acceptable carrier.For example, the composition can contain 10mg or less, 25mg, 50mg, or up to about 100mg of human serum albumin per mL of phosphate-buffered saline.Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients (including salts, preservatives, buffers, etc.).

[0234] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include parenteral vehicles such as water, alcohol / water mixtures, saline, sodium salts, and Ringer's dextrose. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antibacterial agents, antifungals, antioxidants, chelating agents, inert gases, and the like. The pH and precise concentration of each component in the therapeutic composition are adjusted according to well-known parameters.

[0235] There are also additional formulations suitable for oral administration. Oral formulations include typical excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0236] In yet another embodiment, the therapeutic composition may comprise a classical pharmaceutical formulation. Administration of the therapeutic composition according to certain embodiments may be via any common route of administration, so long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal, or transdermal administration. Alternatively, administration may be via orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are typically administered as pharmaceutically acceptable compositions containing physiologically acceptable carriers, buffers, or other excipients. Aerosol administration may be used to treat pulmonary diseases. The volume of the aerosol may be, for example, about 0.01 mL to 0.5 mL.

[0237] The effective amount of a therapeutic composition is determined based on the intended purpose. The term "unit dose" or "dose" refers to a physically discrete unit suitable for administration to a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response associated with said administration, i.e., an appropriate administration route and treatment regimen. The amount administered, both in terms of the number of administrations and the unit dose, depends on the desired preventive or therapeutic effect.

[0238] The precise dose of a therapeutic composition will also depend on the judgment of the practitioner and is peculiar to each individual patient. Factors affecting the dose include the physical and clinical condition of the patient, the route of administration, the therapeutic objectives (e.g., symptomatic relief or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

[0239] [X. Manufactured Product or Kit] Articles of manufacture or kits are provided that include any of the fusion protein / ribonucleotide complexes disclosed herein, and may include one or more reagents for producing targeted gene disruption (e.g., including nucleic acids and proteins that specifically promote gene knockout or knockdown), one or more agents that target the gene product of interest, buffers, salts, instructions for use, or combinations thereof.

[0240] The article of manufacture or kit may further comprise a package insert containing instructions for using the fusion protein / ribonucleotide complex to treat or delay the progression of cancer in an individual. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or nickel-molybdenum alloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. In some embodiments, the article of manufacture may further include one or more other agents (e.g., chemotherapeutic or anti-tumor agents). Suitable containers for the agents include, for example, bottles, vials, bags, and syringes.

[0241] All methods disclosed and claimed herein can be carried out and made without undue experimental effort in light of the disclosure herein. Although the compositions and methods of the present disclosure have been described in preferred embodiments, it will be apparent to those skilled in the art that various modifications can be made to the methods and the steps or order of steps therein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein with the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the claims appended hereto.

Claims

1. 1. A fusion protein-ribonucleotide complex comprising a fusion protein and at least one RNA, wherein said fusion protein comprises: at least one antibody or antibody fragment; and at least one RNA-guided DNA endonuclease, Optionally, the RNA targets SRC-3.

2. The RNA-guided DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Cs 2. The complex of claim 1, wherein the complex is selected from the group consisting of Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, and functional derivatives thereof.

3. 3. The complex of claim 1 or 2, wherein the RNA-guided DNA endonuclease is Cas9.

4. The complex of any one of claims 1 to 3, wherein the RNA comprises a sequence complementary to one or more target polynucleotides of interest. Optionally, the polynucleotide is SRC-3.

5. The conjugate of claim 1 , wherein the fusion protein further comprises a linker domain between the antibody or antibody fragment and the RNA-guided DNA endonuclease.

6. The complex of any one of claims 1 to 5, wherein the fusion protein further comprises one or more purification tags.

7. 7. The complex of claim 6, wherein the purification tag is a His tag, a FLAG tag, HA, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, an albumin-binding protein, an AU1 epitope, an AU5 epitope, a biotin-carboxy carrier protein, a galactose-binding protein, glutathione S-transferase, a Halotag, a streptavidin-binding peptide, or a tandem affinity purification tag.

8. The conjugate of any one of claims 1 to 7, wherein the fusion protein further comprises one or more proteases.

9. 9. The conjugate of claim 8, wherein the protease is tobacco etch virus (TEV) protease, enterokinase, PreScission protease, enteropeptidase, thrombin, 3C protease, or Factor Xa.

10. The complex of any one of claims 1 to 9, wherein the RNA targets SRC-3.

11. The conjugate of any one of claims 1 to 10, wherein the antibody fragment is an scFv.

12. The conjugate of claim 11 , wherein the antibody or antibody fragment targets a cell surface protein.

13. The conjugate of claim 11 or 12, wherein the antibody or antibody fragment targets a cancer antigen.

14. 2. The conjugate of claim 1, wherein the antibody or antibody fragment targets CD19, CD4, HER2, PD-1, CTLA-4, TGFBR, or EGFR.

15. 15. The conjugate of any one of claims 1 to 14, wherein the one or more target polynucleotides of interest are oncogenes, tumor suppressor genes, genes associated with growth or proliferation, genes associated with invasion or metastasis, or genes involved in epithelial-mesenchymal transition.

16. 15. The complex of any one of claims 1 to 14, wherein the one or more target polynucleotides of interest are PTEN, PIK3CA, TP53, VIM, TWIST1, ESR1, or MYC.

17. A nucleotide encoding the fusion protein according to any one of claims 1 to 16.

18. An expression construct comprising at least one nucleotide sequence encoding the fusion protein and / or said RNA according to any one of claims 1 to 16.

19. A therapeutic composition comprising the conjugate of any one of claims 1 to 16.

20. 20. The therapeutic composition of claim 19, wherein the therapeutic composition is contained in a pharmaceutically acceptable carrier.

21. A method for treating an individual, comprising administering a therapeutically effective amount of the complex of any one of claims 1 to 16, the nucleotide of claim 17, the expression construct of claim 18, the therapeutic composition of claim 19 or 20, or a cell that produces part or all of the complex.

22. 22. The method of claim 21, wherein the individual has or is suspected of having cancer.

23. 23. The method of claim 21 or 22, wherein the cancer comprises breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, pancreatic cancer, myeloid tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, lung cancer, peritoneal cancer, gastric cancer or tumors, cervical cancer, liver cancer, bladder cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, head and neck cancer, or melanoma.

24. 22. The method of claim 21, wherein the individual has or is suspected of having an inflammatory disease.

25. 25. The method of claim 24, wherein the inflammatory disease is allergy, asthma, an autoimmune disease, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, transplant rejection, ankylosing spondylitis (AS), gout, myositis, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, pelvic inflammatory disease, or vasculitis.

26. 22. The method of claim 21, wherein the individual has or is suspected of having a genetic disease. Optionally, said genetic disease is an inborn error of metabolism.

27. 27. The method of claim 26, wherein the congenital metabolic disorder is glycogen storage disease, G6PD deficiency, phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, carbamoyl phosphate synthase I deficiency, alkaptonuria, combined malonic acid-methylmalonic acid aciduria, 2-hydroxyglutaric aciduria, medium-chain acyl-CoA dehydrogenase deficiency, acute intermittent porphyria, Restsch-Nyhan syndrome, lipid-type congenital adrenal hyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher disease, or Niemann-Pick disease.

28. 19. The method of claim 18, wherein the cell that produces some or all of the complex is an immune cell.

29. 29. The method of claim 28, wherein the immune cells comprise T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, dendritic cells, or a mixture thereof.

30. 30. The method of any one of claims 21 to 29, wherein the conjugate is administered intravenously, intraperitoneally, intra-arterially, topically, by inhalation, intramuscularly, intrasternally, intra-articular injection, or by infusion.

31. 31. The method of any one of claims 21 to 30, further comprising administering a second therapeutic intervention.

32. 32. The method of claim 31, wherein the second therapeutic intervention comprises surgery, radiation, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof.

33. 33. The method of claim 31 or 32, wherein the second therapeutic intervention is administered substantially simultaneously with the complex of any one of claims 1 to 16, the nucleotide of claim 17, the expression construct of claim 18, the therapeutic composition of claim 19 or 20, or a cell producing part or all of the complex.

34. 34. The method of any one of claims 31 to 33, wherein the second therapeutic intervention is administered sequentially with the complex of any one of claims 1 to 16, the nucleotide of claim 17, the expression construct of claim 18, the therapeutic composition of claim 19 or 20, or cells producing part or all of the complex.

35. A method for producing the composite of any one of claims 1 to 17, comprising the steps of: a. expressing the fusion protein; b. expressing the RNA; c) contacting the fusion protein with the RNA.

36. 36. The method of claim 35, further comprising purifying the fusion protein.

37. 37. The method of claim 36, wherein the fusion protein is purified by affinity chromatography.

38. 39. The method of any one of claims 35 to 38, further comprising the step of purifying the RNA.

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