Targeting of SRC-3 in immune cells as an immunomodulatory therapeutic agent for cancer treatment

Abstract

Methods and compositions related to cancer treatment are provided that involve targeting SRC-3 in immune cells, including T cells, such as T regulatory cells. [Solution] The present disclosure relates to methods and compositions related to cancer treatment, including targeting SRC-3 in immune cells, including T cells, such as T regulatory cells. Targeting SRC-3 in T regulatory cells, in particular, is effective in eradicating tumors in mammals. In certain embodiments, T regulatory cells are subjected to CRISPR ex vivo to produce cells suitable for adoptive cell transfer. In some cases, one or more agents targeting SRC-3 are also administered to an individual and / or exposed to the cells prior to administration.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 106,770 filed on October 28, 2020, the entire contents of which are incorporated herein by reference. Statement regarding federally sponsored research or development

[0002] This invention was made with government support under W81XWH-13-1-0285 awarded by the Department of Defense and under HD008188 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Embodiments of the present disclosure relate to the fields of medicine, including at least cell biology, molecular biology, immunology, and oncology.

Background Art

[0004] Steroid receptor coactivator-3 (SRC-3) functions as a major driver of breast cancer (BC) proliferation, metastasis, and resistance to standard cancer therapies and endocrine-based cancer therapies. SRC-3, an oncogene, acts as a pleiotropic coactivator for nuclear receptors and other transcription factors that drive programs required for cancer cell proliferation and metastasis. Importantly, SRC-3 also plays a critical role in the regulation of the host immune system.

[0005] The present disclosure addresses the long-felt need in the art to provide effective cancer therapies by targeting SRC-3 in a unique manner.

Summary of the Invention

[0006] This disclosure is directed toward systems, methods, and compositions for treating, slowing the progression of, delaying the onset of, or reducing the risk of developing cancer of any kind. In certain embodiments, this disclosure relates to adoptive cell transfer in which the cells administered to an individual in need are immune cells having (1) disruption of endogenous SRC-3 expression; and / or (2) exposure to one or more agents targeting SRC-3. In certain embodiments, the immune cells are CD4+ cells, including T regulatory cells (Treg) which are CD4.

[0007] Current Treg-targeted immune checkpoint inhibitors primarily focus on surface proteins (receptors) that disrupt cell surface interactions with other immune cells. This disclosure is distinct from this because SRC-3, as a Treg gene target, is a nuclear protein that functions to regulate the Treg nuclear gene expression program. As a result of its role as a transcriptional master regulator, the loss of SRC-3 in a mouse gene model modulates Treg function to promote tumor eradication while avoiding the usual serious side effects frequently observed with established immune checkpoint inhibitors, as is shown herein. In certain embodiments, this disclosure relates to a CRISPR-based approach targeting the SRC-3 gene in immune cells, including the use of genetically modified Treg cells for adoptive transfer into individuals with cancer. Thus, this disclosure provides a unique method to support immune system-based tumor eradication.

[0008] In one embodiment, there are engineered immune cells that include the disruption of steroid receptor coactivator 3 (SRC-3). In certain embodiments, the immune cells are T cells, such as regulatory T cells. The T cells may be CD4+, CD25+, and / or FOXP3+. In certain embodiments, the disruption is further defined as immune cells that are genetically modified to have a reduced level of SRC-3 expression or that essentially lack SRC-3 expression. In certain cases, the immune cells are engineered using one or more guide RNAs and Cas9 enzymes. The immune cells may be autogenic, allogeneic, or syngeneic to an individual.

[0009] In certain embodiments, there exists a composition comprising (a) any immune cells included herein, and (b) one or more agents targeting SRC-3. In some cases, (a) and (b) may be the same formulation but are different formulations. In certain embodiments, the agents targeting SRC-3 are small molecule inhibitors, antibodies, proteins, nucleic acids, or combinations thereof. Small molecule inhibitors of SRC-3 may be bufarin, gossypol, vercarin A, SI-2, SI-10, SI-12, their functional derivatives, or combinations thereof. Antibodies may be monoclonal or polyclonal antibodies, with specific examples including 5E11, LS-C801929, PA1-845, AX15.3, PA5-29854, EPR4374(3), or combinations thereof.

[0010] In one embodiment, there exists a method for treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective dose of any cells included herein. The cancer may be SRC-3+ cancer. The cancer may be breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, or pancreatic cancer.

[0011] In some cases, prior to the administration step, cells are ex vivo exposed to one or more agents targeting SRC-3, such as small molecule inhibitors (bufarin, gossypol, vercarin A, SI-2, SI-10, SI-12, their functional derivatives, or combinations thereof), antibodies (monoclonal or polyclonal antibodies, or fragments such as scFv), proteins, nucleic acids, or combinations thereof. Specific antibodies include 5E11, LS-C801929, PA1-845, AX15.3, PA5-29854, EPR4374(3), or combinations thereof. In some cases, the individual is administered an effective amount of additional cancer treatment, such as surgery, radiation, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof. Additional cancer treatment may include one or more agents targeting SRC-3. Cells and additional cancer treatment may be administered to the individual substantially simultaneously or at different time points. Cells and additional cancer therapies may or may not be in the same formulation. Cells may be administered intravenously, intraperitoneally, intra-arterially, topically, by inhalation, intramuscularly, intrasternally, intra-articularly, or by infusion. Cells may be administered once or multiple times, and if administered multiple times, the interval between administrations may be within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months.

[0012] In one embodiment, there exists a method for treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of any composition included herein, such as a composition comprising (a) any immune cells included herein and (b) one or more agents targeting SRC-3. In such a case, (a) and (b) may or may not be administered to the individual in different formulations and / or simultaneously or at different time points. If administered at different time points, the interval between administrations of (a) and (b) may be within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months. The composition may be administered to the individual once or multiple times, and if the composition is administered to the individual multiple times, the interval between administrations may be within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months. The cancer may be SRC-3+. The cancer may be breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, or pancreatic cancer. In certain cases, prior to the administration step, the cells of (a) are ex vivo exposed to one or more effective doses of agents targeting SRC-3.

[0013] In one embodiment, there exists a method for generating any immune cells as defined herein, the method comprising the step of disrupting the expression of SRC-3 in the immune cells.

[0014] The immune cells may be T cells, including Tregs. In certain cases, the method is further defined as (a) obtaining CD4+ T cells or CD4+ Tregs, respectively, and (b) exposing the CD4+ T cells or CD4+ Tregs, respectively, to one or more agents that disrupt the expression of endogenous SRC-3 in the T cells or Tregs. In certain embodiments, the T cells or Tregs are obtained from the spleen, bone marrow, blood, plasma, or a combination thereof. The method may further include the step of obtaining regulatory T cells from CD4+ T cells. In certain cases, the regulatory T cells are CD25+ and / or Fox3p+. In certain cases, the one or more agents that disrupt the expression of endogenous SRC-3 in the T cells include nucleic acids. In certain embodiments, the one or more agents that disrupt the expression of endogenous SRC-3 in the T cells include CRISPR reagents. The method may further include the step of ex vivo exposing immune cells to one or more agents that target SRC-3 in an effective amount.

[0015] The above provides a fairly broad overview of the features and technical advantages of this disclosure so that the following detailed description may be better understood. Additional features and advantages that form the subject matter of the claims are described hereafter in this specification. It should be understood by those skilled in the art that the disclosed concepts and particular embodiments may readily be used as a basis for modifying or designing other structures to accomplish the same objectives of this design. It should also be understood that such equivalent configurations will not deviate from the spirit and scope set forth in the appended claims. Novel features that are considered to be features of the designs disclosed herein, both in terms of configuration and operation, along with further objectives and advantages, will be better understood from the following description when considered in relation to the appended drawings. However, it should be clearly understood that each drawing is provided for illustrative and explanatory purposes only and is not intended to define any limitations of this disclosure. [Brief explanation of the drawing]

[0016] Next, in order to better understand this disclosure, refer to the following description in conjunction with the attached drawings.

[0017] [Figure 1] This figure reveals widespread lymphocytosis in comprehensive immunophenotyping of Src-3 gene knockout (KO) mice. Immunophenotyping of peripheral blood from aged (16-20 month) Src-3 KO mice (- / -, n=6) and wild-type littermates (+ / +, n=7). SRC-3 knockout mice showed significant elevations in T cells and B cells, as well as specific subtypes. There were no statistically significant differences in granulocytes, monocytes, eosinophils, macrophages, dendritic cells, and NK cells (data not shown).

[0018] [Figure 2] Figure 2A shows highly enriched SRC-3 expression, correlated with Foxp3 in regulatory T cells (Tregs). Figure 2A is a representative plot of Ncoa3 (Src-3) probe intensity (using two probes) from selected tissues in the BioGPS genomics platform (10), showing that Src-3 is highly expressed in T cell lineages, with the highest expression in Foxp3+CD4+ T cells (Tregs). Figure 2B shows the correlation of 296 selected co-regulators of NURSA with Foxp3 expression in Tregs, performed using the batch-corrected co-expression database Immuno-Navigator (12). Ncoa3 is strongly correlated with Foxp3 expression in Tregs.

[0019] [Figure 3]This figure illustrates how SRC-3 regulates FOXP3 expression in human Tregs. Figure 3A shows that silencing SRC-3 in human Tregs (from healthy donors) reduces mRNA expression of Treg marker genes and immune checkpoint mediators. Donor Tregs were infected with lentiviral vectors expressing luciferase (shLuc) or shRNA directed towards SRC-3, and mRNA levels were measured by RT-qPCR. Figure 3B shows that SRC-3 can stimulate FOXP3 promoter function. SRC-3 co-activates FoxP3 promoter-driven reporter activity in a transient transduction assay. 293T cells were transductioned with a FoxP3 promoter luciferase reporter (donated from GR Lee lab, Hwang et al 2016, Nat. Commun.) and a concentration-escalating SRC-3 expression plasmid.

[0020] [Figure 4] Figure 4A shows the eradication of E0771 BC tumors in a genetically engineered mouse model in which SRC-3 is specifically deleted in Foxp3-expressing Treg cells. Floxed SRC-3 mice were backcrossed to a C57BL / 6J background for 10 generations and also crossed with Foxp3-EGFP / CRE / ERT2 knock-in mice in a C57BL / 6J background. Treatment with tamoxifen resulted in Cre activation and excision of exons 11 and 12 of the SRC-3 gene. Figure 4B shows C57BL / 6J control mice and Treg:SRC-KO mice treated with tamoxifen for 5 days, followed by injection of 1 × 10⁶ luciferase-expressing E0771 BC tumor cells into cleared mammary fat bodies 3 weeks later. Figure 4C shows tumor volume measured over 32 days (left). Images of the tumor and spleen at the end of the study are shown on the right.

[0021] [Figure 5]This is a diagram of the quantification of SRC-3(NcoA3) mRNA in bulk T lymphocytes after CRISPR-based targeting of the SRC-3 gene. The following primers were used in the previously performed RT-qPCR assay. NCoA3 primer (Probe 103): Left: AAG ACT CTT TAG GAC CGC TTT TAC T (SEQ ID NO: 1). Right: ACA CTG CGC CAT GGT TAA T (SEQ ID NO: 2). GAPDH primer (Probe 52): Left: GGG TTC CTA TAA ATA CGG ACT GC (SEQ ID NO: 3). Right: CCA TTT TGT CTA CGG GAC GA (SEQ ID NO: 4). (PE-)-Non-targeted cells; (PE+) SRC-3 gene-targeted cells.

[0022] [Figure 6] This is a diagram of the experimental design of the adoptive SRC-3 KO Treg mouse tumor model.

[0023] [Figure 7] This is a diagram showing that adoptive transfer of SRC-3 KO Treg cells eliminates E0771 tumors. In Figure 7A, luciferase-labeled breast cancer E0771 tumor cells were transplanted into the cleared fat pad of wild-type C57BL / 6 mice. Thereafter, animals (2 per experimental group) were injected with SRC-3 KO Treg, control (Cont) Treg, or no Treg (No ACT, corresponding to wild-type cells), and tumor growth was monitored by luciferase tumor imaging. Tumor elimination in animals that received SRC-3 KO Treg is indicated by arrows. Figure 7B shows the quantification of tumor luciferase imaging. [Modes for Carrying Out the Invention]

[0024] I. Examples of Definitions In accordance with long-standing patent law practice, the words “a” and “an,” when used herein in conjunction with the word “comprising,” including in the claims, mean “one or more.” Where used herein and in the claims, the singular “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “nucleic acid” includes multiple nucleic acids, including mixtures thereof. Some embodiments of the present disclosure may consist of, or essentially consist of, one or more elements, method steps, and / or methods of the present disclosure. Any method or composition described herein may be practiced in relation to any other method or composition described herein, and different embodiments may be combined.

[0025] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to mean that they include the stated step or element or group of steps or elements, but not that they exclude other steps or elements or groups of steps or elements. “Consisting of” means that it includes and limits what follows the phrase “consisting of.” Thus, the expression “consisting of” indicates that the stated element is essential or required, and other elements may not be present. “Essentially consisting of” means that it includes all elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed element. Thus, the expression “essentially consisting of” indicates that the listed element is essential or required, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed element.

[0026] As used herein, the terms “or” and “and / or” are used to describe multiple components, either in combination with each other or exclusively. For example, “x, y, and / or z” may mean “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is particularly intended that x, y, or z may be specifically excluded from embodiments.

[0027] Throughout this application, the term “about” is used in accordance with its obvious and common sense in the field of cell and molecular biology, indicating that the value includes the standard deviation of errors in the method or approach employed to determine the value.

[0028] Throughout this specification, any reference to “one embodiment,” “a certain embodiment,” “a specific embodiment,” “a related embodiment,” “a given embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the appearance of the aforementioned phrases in various places within this specification does not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0029] As used herein, “disruption” or “modification” of a gene means the elimination or reduction of the expression of one or more gene products encoded by a target gene within a cell, compared to the expression level of the gene product without modification. Exemplary gene products include mRNA and protein products encoded by the gene. Modifications in some cases are transient or reversible, while those in other cases are permanent. Modifications in some cases are functional or full-length proteins or mRNAs, despite the fact that cleaved or non-functional products may be produced. In some embodiments herein, the activity or function of a gene is disrupted, as opposed to expression. Gene disruption or modification is generally induced by artificial methods, i.e., by the addition or introduction of compounds, molecules, complexes, or compositions, and / or by modification of the nucleic acid of a gene or a nucleic acid related to a gene, such as at the DNA level. Exemplary methods for gene modification include gene modification techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense technologies such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in a transient reduction in expression, as well as gene editing technologies, which result in the inactivation or modification of a target gene, for example, by cleavage and / or induction of homologous recombination. Examples include insertions, mutations, and / or deletions. Disruption or modification typically results in the suppression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene disruption or modification include insertions of a gene or part of a gene, frameshifts and missense mutations, deletions, knock-ins, and knockouts, including deletions of the entire gene. Such modifications can occur in the coding region, for example, one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, such as through the insertion of a stop codon. Such modifications can also occur through modifications of the promoter or enhancer or other regions that affect transcriptional activation, thereby interfering with gene transcription.Gene disruption or modification includes gene targeting, which involves inactivation of targeted genes by homologous recombination.

[0030] As used herein, “pharmaceutically acceptable carriers” include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral vehicles, e.g., sodium chloride, Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and organic esters for injection, e.g., ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, pigments, liquids, and nutritional supplements, and combinations thereof, which will be known to those skilled in the art. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0031] As used herein, the term “subject” generally refers to an individual in need of treatment. A subject may be any animal subject to which a method or substance is applied, such as mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, chickens), domestic pets (e.g., dogs, cats, mice), horses, and transgenic non-human animals. A subject may be a patient who has or is suspected of having a disease (which may be called a medical condition), such as one or more cancers. A subject may be receiving or have received cancer treatment. The term “individual” may be used interchangeably in at least some embodiments. “Subject” or “individual” as used herein may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. An individual may receive one or more medical compositions via the internet. An individual may include any age of human or non-human animal and therefore include both adults and young children (e.g., children) and infants, and includes individuals in utero. Individuals may be of any sex, race, or ethnicity.

[0032] "Treatment" or "treatment" of a disease or condition refers to the implementation of a protocol that may involve administering one or more drugs to a patient in an effort to alleviate the signs or symptoms of a disease, including cancer. Desired effects of treatment include a slowing of disease progression, improvement or alleviation of the disease state, remission, or improved prognosis. Alleviation can occur not only before the signs or symptoms of the disease or condition appear, but also after they appear. Therefore, "treating" or "treatment" may include "preventing" or "preventing" a disease or undesirable condition. Furthermore, "treating" or "treatment" may include protocols that do not require complete elimination of signs or symptoms, do not require a cure, and in particular have only a slight effect on the patient.

[0033] As used throughout this application, the terms “therapeutic benefit” or “therapeutic effect” refer to any medical treatment of the condition that promotes or enhances the health of the subject. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may include a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, a prevention of metastasis, or a delay in the onset of metastasis. Cancer treatment may also refer to an extension of the survival time of a subject with cancer. II. Embodiments of the Disclosure

[0034] This disclosure relates to methods and compositions for effective cancer treatment, including cancer treatment that avoids serious effects on the treated individual. In certain embodiments, the treatment includes modification of immune cells, including ex vivo methods, and administration of the modified immune cells to an individual with cancer. The immune cells in certain embodiments are certain types of T cells, such as Tregs, and the modification is such that the standard function of the Tregs is not adversely affected.

[0035] Certain embodiments of this disclosure encompass SRC-3 as a key target in immune cells, with at least Tregs being mentioned. With respect to Tregs, specific compartment-specific disruption results in tumor eradication, at least in a breast cancer syngeneic tumor model, as described herein. A key difference that distinguishes this strategy of disrupting SRC-3 in Tregs from other immune checkpoint inhibitors is that SRC3 is a nucleoprotein, and the modification in this disclosure modifies Treg function without losing all activity related to Tregs, which could otherwise result in serious side effects. Furthermore, this disclosure establishes an approach to specifically eliminate SRC3 ex vivo in T cells using CRISPR-based gene targeting. In certain embodiments applied to human CD4+ lymphocytes, for example, this disclosure provides methods for adoptive T cell / Treg-based therapeutics for the treatment of cancer. III. Immune cells

[0036] In this disclosure, immune cells are modified to disrupt the expression of endogenous SRC-3, thereby enabling the cells to be effective in treating cancer in the recipient organism. The immune cells can be of any kind, but in certain embodiments, they are of any kind of T cell, with at least Treg and B cells being examples.

[0037] Embodiments of this disclosure include modifications of T cells, such as Tregs, which, through modification, allow T cells to still perform their desired functions (in the case of Tregs, to control immune responses to self and foreign particles (antigens)). Accordingly, the methods and compositions incorporated herein provide cancer treatments with a lower risk of toxicity compared to other therapies with modified T cells, such as modified regulatory T cells.

[0038] Any type of T cell may be used in the methods and compositions of this disclosure. The term “T cell” refers to a T lymphocyte, and includes, but is 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 cells” refers to T cells that can kill other cells. The majority of cytotoxic T cells are CD8+ MHC class I restricted T cells. However, some cytotoxic T cells are CD4+. In certain embodiments, the T cells of this disclosure are CD4+.

[0039] Any T cell used herein may be selected for and / or for one or more specific markers. Any selection step may be performed by positive selection, negative selection, or both. In certain embodiments, the Treg is CD4+, CD25+, and / or FOXP3+. In certain cases, the Treg is CD4+, CD25+, and FOXP3+. In some embodiments, the Treg is CTL-associated protein 4 (CTLA4)+, CC chemokine receptor 7 (CCR7)+, and / or CD62 antigen ligand (CD62L)+. Also included herein are Tregs whose expression is subsequently interrupted by disruption of the SRC-3 gene, thereby disrupting the expression of FOXP3, CD25, and / or CD4+.

[0040] In some embodiments, CD4+ immune cells, such as CD4+ T cells, are targeted by the methods and compositions of this disclosure to disrupt SRC-3 expression, and at least in some cases, Tregs can be further selected therefrom and used as therapeutic cells. In other cases, various CD4+ cells are modified and used together as therapeutic cells without the further step of isolating Tregs.

[0041] When T cells are utilized, the source of the T cells may be any suitable source, including the spleen, bone marrow, blood, plasma, or a combination thereof. In some cases, T cells are commercially obtained. In any case, the T cells may be autologous to the recipient organism, or allogeneic or syngeneic to the recipient organism. The T cells may be manipulated before the process of manipulating the cells to disrupt SRC-3. In some cases, the cells are processed from the source, for example, to remove unwanted components. Before use, the cells may be exposed to one or more compositions that enhance their activity in the organism, such as one or more agents targeting SRC-3, or other compositions, such as one or more cytokines and / or one or more growth factors. The cells may be exposed to one or more agents targeting SRC-3 before, during, and / or after the modification process to disrupt SRC-3 expression. IV. Modification of gene expression

[0042] In certain embodiments, the immune cells of this disclosure are modified to alter the expression of SRC-3, and such manipulation of cells can be carried out by any appropriate method. The manipulated cells are genetically modified to lack or reduce the expression of endogenous SRC-3 in the cell. The reduction in expression may be at a level undetectable by standard methods in the art.

[0043] In some embodiments, altered gene expression is achieved by disrupting a gene, e.g., knockout, insertion, missense, or frameshift mutation, e.g., bi-allele frameshift mutation, deletion of all or part of a gene, e.g., one or more exons or parts thereof, and / or knock-in. For example, altered gene expression can be achieved by sequence-specific or target nucleases, such as DNA-binding target nucleases like zinc finger nucleases (ZFNs) and transcription activator-like effecta nucleases (TALENs), and RNA-guided nucleases such as CRISPR-related nucleases (Cas) specifically designed to target the sequence of the SRC-3 gene or a part thereof.

[0044] In some embodiments, alteration of gene expression, activity, and / or function is achieved by disrupting the gene. In some embodiments, the gene is modified such that its expression is reduced by at least, or about 20, 30, or 40%, generally at least, or about 50, 60, 70, 80, 90, 95%, 96%, 97%, 98%, 99%, or more, compared to its expression in the absence of the gene modification or the component introduced to achieve the modification.

[0045] In some embodiments, the modification is transient or reversible, such that gene expression is restored at a later point in time. In other embodiments, the modification is neither reversible nor transient, but permanent, for example.

[0046] In some embodiments, the gene modification is typically targeted and carried out by inducing one or more double-strand breaks and / or one or more single-strand breaks in the SRC-3 gene. In some embodiments, the double-strand or single-strand breaks are made by a nuclease, such as an endonuclease, such as a gene-targeting nuclease. In some embodiments, the breaks are induced within the coding region of the gene, such as the SRC-3 exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the SRC-3 coding region, for example, within the first exon, the second exon, or a subsequent exon.

[0047] In some embodiments, double-strand or single-strand breaks are repaired via cellular repair processes such as non-homologous end joining (NHEJ) or homologous recombination repair (HDR). In some embodiments, the repair process is prone to errors, resulting in gene disruption such as frameshift mutations, e.g., bi-allele frameshift mutations, which can lead to complete knockout of the SRC-3 gene. For example, in some embodiments, disruption includes inducing deletions, mutations, and / or insertions. In some embodiments, disruption results in the presence of premature stop codons. In some embodiments, the presence of insertions, deletions, translocations, frameshift mutations, and / or immature stop codons results in disruption of gene expression, activity, and / or function.

[0048] Any agent that disrupts SRC-3 expression can be delivered in any appropriate manner to recipient cells, such as immune cells, including T cells of any kind, including Tregs. In certain embodiments, the agent can be delivered by any kind of nucleofection, lipofectin, any kind of electroporation, cationic polymers, non-viral vectors (such as plasmids), viral vectors (such as adenovirus vectors, retrovirus vectors, lentiviral vectors, or adeno-associated virus vectors), cycloamylose-based carriers, any kind of nanoparticles (such as metal nanoparticles, inorganic nanoparticles, or polymer nanoparticles produced using PLGA / PLA or DODAP bases), lipopolyplexes, etc.

[0049] In some embodiments, gene modification is achieved using antisense techniques, for example, by RNA interference (RNAi), small interfering RNA (siRNA), short hairpin (shRNA), and / or ribozymes used to selectively suppress (repress) gene expression. siRNA technology is RNAi that uses double-stranded RNA molecules having sequences homologous to and complementary to the nucleotide sequence of mRNA transcribed from a gene. siRNA can generally be an siRNA comprising multiple RNA molecules that are homologous / complementary to one region of mRNA transcribed from a gene, or homologous / complementary to different regions. In some embodiments, siRNA is contained within a polycistronic construct.

[0050] Examples of modifying cells to disrupt SRC-3 expression are as follows: A.ZFP and ZFN

[0051] In some embodiments, DNA-targeting molecules that target SRC-3 include one or more DNA-binding proteins such as zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs) fused to effector proteins such as endonucleases. Examples include ZFNs, TALEs, and TALENs.

[0052] In some embodiments, the DNA-targeting molecule comprises one or more zinc finger proteins (ZFPs) or their domains that sequence-specifically bind to DNA. A ZFP or its domain is a protein or domain within a larger protein that sequence-specifically binds to DNA via one or more zinc fingers, which are regions of amino acid sequences within a binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. Among ZFPs are artificial ZFP domains, typically 9–18 nucleotides long, that target specific DNA sequences, generated by the assembly of individual fingers.

[0053] Examples of ZFPs include those containing a single-finger domain approximately 30 amino acids long, containing two immutable histidine residues coordinated via zinc to two cysteine ​​molecules in a single beta turn, and containing an alpha-helix with 2, 3, 4, 5, or 6 fingers. Generally, the sequence specificity of a ZFP can be modified by amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, in some embodiments, ZFPs or ZFP-containing molecules are engineered to bind to, for example, selected target sites that do not exist in nature.

[0054] In some embodiments, the DNA targeting molecule is a zinc finger DNA-binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN), or includes such domain. In some embodiments, the fusion protein includes a cleavage domain (or cleavage half-domain) derived from at least one liS restriction enzyme and one or more zinc finger-binding domains, which may or may not be manipulated. In some embodiments, the cleavage domain is derived from the liS restriction endonuclease Fok I. Fok I generally catalyzes double-strand breaks of DNA with 9 nucleotides from a recognition site on one strand and 13 nucleotides from a recognition site on the other strand.

[0055] Many engineered gene-specific zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA), in collaboration with Sigma-Aldrich (St. Louis, MO, USA), has developed a zinc finger construction platform (CompoZr) (allowing researchers to avoid the entire process of constructing and validating zinc fingers), and offers thousands of specific target zinc fingers for 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, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTil-lKT, and PZD0020). B.TAL, TALE, and TALEN

[0056] In some embodiments, the DNA targeting molecule for SRC-3 includes, for example, a transcription activator-like protein effector (TALE) protein containing a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA-binding domain. See, for example, U.S. Patent Application Publication 2011 / 0301073 (which is incorporated herein by reference in its entirety).

[0057] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are involved in the binding of the TALE to its congenerally targeted DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33–35 amino acid long and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. Each TALE repeat unit typically contains one or two DNA-binding residues at repeat positions 12 and / or 13, constituting a repeat variable two residue (RVD). The natural (canonical) code for DNA recognition of these TALEs is determined such that the HD sequences at positions 12 and 13 lead to binding to cytosine (C), with NG binding to T, NI to A, NN to G or A, and NO to T; and non-canonical (atypical) RVDs are also known. In some embodiments, TALEs can be targeted to any gene by designing a TALE array with specificity to the target DNA sequence. The target sequence generally begins with thymidine.

[0058] In some embodiments, the molecule is a DNA-binding endonuclease such as TALE nuclease (TALEN). In some embodiments, TALEN is a fusion protein comprising a DNA-binding domain derived from TALE and a nuclease catalytic domain for cleaving nucleic acid target sequences.

[0059] In some embodiments, TALENs recognize and cleave target sequences within genes. In some embodiments, DNA cleavage results in double-strand breaks. In some embodiments, cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an incomplete repair process that often results in alterations to the DNA sequence at the cleavage site. In some embodiments, the repair mechanism involves the rejoining of the remainder of the two DNA ends via direct religation or so-called microhomology-mediated end joining. In some embodiments, NHEJ-mediated repair results in small insertions or deletions and can be used to suppress gene disruption. In some embodiments, the modification may be a substitution, deletion, or addition of at least one nucleotide. In some embodiments, cells in which a cleavage-induced mutagenic event, i.e., a mutagenic event following an NHEJ event, occurs can be identified and / or selected by methods well known in the art.

[0060] In some embodiments, TALE repeats are assembled to specifically target genes (Gaj et al., 2013). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Specifically, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, for example, in U.S. Patent Application Publication No. 2014 / 0120622 and U.S. Patent Application Publication No. 2013 / 0315884.

[0061] In some embodiments, the TALEN is introduced as a transgene encoded by one or more plasmid vectors. In some embodiments, the plasmid vector may contain a selection marker that enables the identification and / or selection of cells that have received the vector.

[0062] In some embodiments, SRC-3 gene editing is performed using one or more DNA-binding nucleic acids, such as modifications mediated by RNA-guided endonucleases (RGENs). For example, such modifications may be performed using clustered and regularly arranged short palindromic sequence repeats (CRISPR) and CRISPR-related (Cas) proteins. Generally, the “CRISPR system” refers collectively to the CRISPR-related (“Cas”) gene (including the sequence encoding the Cas gene), tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), tracrmate sequence (including “series repeats” in the context of the endogenous CRISPR system, and partial series repeats processed by tracrRNA), guide sequence (also referred to as “spacers” in the context of the endogenous CRISPR system), and / or other sequences and transcripts and other elements that are involved in or direct the activity of transcripts derived from the CRISPR locus.

[0063] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may originate from a type I, type II, or type III CRISPR system, for example, from a specific organism (e.g., Streptococcus pyogenes) that contains an endogenous CRISPR system.

[0064] In some embodiments, a Cas nuclease and gRNA (including a fusion of a target sequence-specific crRNA and a default tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to its target site, e.g., a gene, through complementary base pairing. The target site may be selected based on the position immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system features elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, the “target sequence” refers to a sequence designed so that the guide sequence is complementary, and the hybridization of the target sequence and the guide sequence facilitates the formation of the CRISPR complex. If there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex, complete complementarity is not necessarily required.

[0065] The CRISPR system can induce a double-strand break (DSB) at the target site of SRC-3, followed by disruption or modification as discussed herein. In other embodiments, a Cas9 variant, considered a “nickase,” is used to introduce a nick into the single strand at the target site. For example, to improve specificity, a pair of nickases may be used, each led by a pair of different gRNAs targeting the sequence, and when the nicks are introduced simultaneously, a 5' overhang is introduced. In other embodiments, a catalytically inactive Cas9 is fused to a heterogeneous effector domain, such as a transcription repressor or transcription activator, to affect gene expression.

[0066] The target sequence of SRC-3 may include any polynucleotide, such as DNA polynucleotides or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within a cell organelle. Generally, a sequence or template that can be used for recombination to a targeted locus containing the target sequence is referred to as an “edit template,” “edited polynucleotide,” or “edited sequence.” In some embodiments, an exogenous template polynucleotide may be referred to as the edit template. In some embodiments, the recombination is homologous recombination.

[0067] Typically, in the context of endogenous CRISPR systems, the formation of a CRISPR complex (which includes a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins) results in a cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more from the target sequence). Tracr sequences that may contain or consist of all or part of a wild-type tracr sequence (for example, approximately 20 nucleotides, 26 nucleotides, 32 nucleotides, 45 nucleotides, 48 ​​nucleotides, 54 nucleotides, 63 nucleotides, 67 nucleotides, 85 nucleotides or more, or more than approximately 20 nucleotides, more than approximately 26 nucleotides, more than approximately 32 nucleotides, more than approximately 45 nucleotides, more than approximately 48 nucleotides, more than approximately 54 nucleotides, more than approximately 63 nucleotides, more than approximately 67 nucleotides, more than approximately 85 nucleotides or more) may also form part of a CRISPR complex, such as by hybridization along at least part of the tracr sequence to all or part 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 the CRISPR complex (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned).

[0068] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell such that the expression of one or more elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Alternatively, the components can be delivered to the cell as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more further vectors providing any components of the CRISPR system not included in the first vector. These vectors may contain one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements in one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0069] The vector may contain a regulatory element operably linked to an enzyme-coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins 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, their homologs or modified versions thereof. These enzymes are well known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database as accession number Q99ZW2.

[0070] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct the cleavage of one or both strands at a target sequence location, such as within the target sequence and / or within the complementary strand of the target sequence. A vector may encode a mutated CRISPR enzyme compared to the corresponding wild-type enzyme, which lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of S. pyogenes-derived Cas9 (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). In some embodiments, the Cas9 nickase can be used in combination with two guide sequences, e.g., two guide sequences that target the sense and antisense strands of its DNA target, respectively. This combination can be used to introduce nicks into both strands and to induce NHEJ or HDR.

[0071] In some embodiments, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells of a particular organism (e.g., mammals including, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates) or cells derived from such organisms. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to increase expression in a target host cell by maintaining the native amino acid sequence while replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the host cell's gene. Different species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon usage frequency between organisms) often correlates with messenger RNA (mRNA) translation efficiency, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell usually reflects that it is the codon most frequently used in peptide synthesis. Therefore, based on codon optimization, genes can be adapted to optimal gene expression in a given organism.

[0072] Generally, the guide sequence is any polynucleotide sequence that has sufficient complementarity to the target sequence to hybridize with the target sequence and to direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and the corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or more, or greater than about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, or more, when optimally aligned using a preferred alignment algorithm.

[0073] The optimal alignment can be determined using any suitable algorithm for aligning sequences. Non-restrictive examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0074] A CRISPR enzyme can be part of a fusion protein containing one or more heterologous protein domains. A CRISPR enzyme fusion protein may contain any further protein sequences and, optionally, linker sequences 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 having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules (including, but not limited to, maltose-binding protein (MBP), S-tags, Lex A DNA-binding domain (DBD) fusions, GAL4A DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions). Further domains that may form part of a fusion protein containing a CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, incorporated herein by reference. V. How to use

[0075] In some embodiments, the Disclosure provides a method for treating cancer using immunotherapy, comprising administering an effective amount of the SRC-3-destroying immune cells of the Disclosure. In one embodiment, a method for treating cancer, slowing the progression of cancer, delaying the onset of cancer, or reducing the risk of developing cancer in an individual by administering an effective amount of SRC-3-destroying cell therapy to the individual is encompassed herein. The method may be used to treat solid tumors or hematological malignancies. In certain embodiments, the cancer is SRC-3 positive. The cancer may be primary, metastatic, treatment-resistant, etc. The cancer may be of any type and at any stage. Individuals may be at risk of cancer, for example, across the general population, and individuals at risk of cancer may be so due to personal or family medical history, such as being a tobacco user, being obese, consuming excessive alcohol, having some type of viral infection such as human papillomavirus (HPV), being exposed to one or more carcinogens, or being excessively exposed to radiation such as ultraviolet light from the sun. A quantity can be considered excessive if it is greater than the average individual in the population.

[0076] Tumors for which the disclosed treatment methods are useful include all malignant cell types, such as those found in solid tumors or hematological malignancies. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of breast, ovary, pancreas, colon, cecum, stomach, brain, head, neck, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, gastric tissue, and endometrium. Exemplary hematological malignancies include bone marrow tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, and myeloma. Specific examples of cancers that can be treated using 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 (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0077] Cancer can be, in specific terms, of the following histological types, but is not limited to these: Neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatous carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumor; carcinoid tumor, malignant; branch alveolar adenocarcinoma; papillary adenocarcinoma; pigment-averse carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary adenocarcinoma and follicular adenocarcinoma; non Encapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; otogonial gland carcinoma; mucosal epidermal 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; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; meningioma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma , malignant; pheochromocytoma; mammary angiosarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acromial lens melanoma; nodular melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Müller mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated germ cell tumor; embryonic carcinoma; Teratoma, malignant; ovarian stroma, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; periangiocarcinoma, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; wing sarcoma; odontogenic tumor, malignant; ameloblastic gingivoma; ameloblastoma, malignant; ameloblastofibrosarcoma; pineal glandoma, malignant; chordoma tendinea; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; protoneurectoderm;Cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin lymphomas; B-cell lymphoma; low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade Small, unsevered cell NHL; giant lesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; 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.

[0078] In certain embodiments of this disclosure, immune cells are delivered to an individual in need, for example, an individual who has or is suspected of having cancer. The cells then enhance the individual's immune system to attack the respective cancer. In some cases, the individual is provided with one or more doses of immune cells. If the individual is provided with two or more doses of immune cells, the interval between doses should be sufficient to allow time for proliferation in the individual, and in certain embodiments, the interval between doses is one day, two days, three days, four days, five days, six days, seven days, or more. In some cases, the interval between doses is one to 24 hours, one to seven days, one to four weeks, one to twelve months, or more, or any range that can be derived between them.

[0079] In some embodiments, the subject may be administered non-myeloablative lymphocyte depletion chemotherapy prior to immunotherapy. Non-myeloablative lymphocyte depletion chemotherapy can be any appropriate therapy that can be administered by any appropriate route. Non-myeloablative lymphocyte depletion chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, especially if the cancer is melanoma, which may be metastatic. An exemplary route for administering cyclophosphamide and fludarabine is intravenous. Similarly, any appropriate dose of cyclophosphamide and fludarabine can be administered. In a particular embodiment, approximately 60 mg / kg of cyclophosphamide is administered for 2 days, followed by approximately 25 mg / m². 2 Fludarabine is administered for 5 days.

[0080] In certain embodiments, one or more growth factors and / or one or more cytokines that promote the proliferation and activation of immune cells are administered to the target simultaneously with or following the immune cells. The immune cell growth factors can be any suitable growth factors that promote the proliferation and activation of immune cells. 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.

[0081] A therapeutically effective dose of immune cells can be administered parenterally, for example, by several routes including intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection, intranasal, or intra-arterial injection, or by infusion.

[0082] The therapeutically effective dose of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in the target being treated. For example, this could be the amount of immune cells essential to inhibit proliferation, induce cancer regression, or improve at least one symptom of cancer.

[0083] The immune cell population can be administered in a disease-appropriate treatment regimen, such as a single or multiple doses over one to several days to improve the disease state, or a long-term, regular dose to inhibit disease progression and prevent disease recurrence. The exact dose to be used in formulation form will depend on the route of administration and the severity of the disease or disorder, and should be determined according to the practitioner's judgment and the individual patient's condition. The therapeutically effective dose of immune cells will depend on the target being treated, the severity and type of distress, and the mode of administration. In some embodiments, doses that may be used for the treatment of human subjects range from at least 3.8 × 10⁴, at least 3.8 × 10⁵, at least 3.8 × 10⁶, at least 3.8 × 10⁷, at least 3.8 × 10⁸, at least 3.8 × 10⁹, or at least 3.8 × 10¹⁰ immune cells / m². In specific embodiments, doses used for the treatment of human subjects range from approximately 3.8 × 10⁹ to approximately 3.8 × 10¹⁰ immune cells / m². In additional embodiments, the therapeutically effective dose of immune cells may vary from approximately 5 × 10⁶ cells to approximately 7.5 × 10⁸ cells per kg of body weight, for example, from approximately 2 × 10⁷ cells to approximately 5 × 10⁸ cells per kg of body weight, or from approximately 5 × 10⁷ cells to approximately 2 × 10⁸ cells per kg of body weight. The exact amount of immune cells can be readily determined by those skilled in the art based on the age, weight, sex, and physiological state of the subject. The effective dose can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0084] Immune cells may be administered in combination with one or more other therapeutic agents for treating cancer. Combination therapies may include, but are not limited to, one or more antitumor agents or vaccines. In certain cases, chemokines (e.g., methotrexate, treosulfan, busulfan), irradiation, 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 pharmaceutical agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. This administration of cells and one or more additional anticancer agents may be via the same or different routes, and may be at the same or different sites. VI. Pharmaceutical Compositions

[0085] Provided herein are pharmaceutical compositions and formulations comprising immune cells (SRC-3-destroying immune cells, such as T cells, including Treg cells) and pharmaceutically acceptable carriers. In some embodiments, there exist compositions comprising (a) any immune cells incorporated herein, and (b) one or more agents targeting SRC-3. In such cases, (a) and (b) may or may not be in the same formulation form and may or may not be configured to be delivered by the same route of administration.

[0086] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (such as an antibody or polypeptide) of desired purity in the form of a lyophilized formulation or aqueous solution with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include, but are not limited to, buffers such as phosphoric acid, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); and low molecular weight (approximately 10 residues). Polypeptides (less than 100%); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or 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 / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersants, such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and uses, including rHuPH20, are described in U.S. Patent Application Publication No. 2005 / 0260186 and U.S. Patent Application Publication No. 2006 / 0104968.In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase. VII. Combination Therapy

[0087] In certain embodiments, the compositions and methods of this embodiment include a population of immune cells combined with at least one additional therapy. The additional therapy may be radiotherapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, protein therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. The additional therapy may be one or more agents targeting SRC-3, such as antibodies of any kind, small molecule inhibitors, nucleic acids, proteins, or a combination thereof.

[0088] In some embodiments, additional therapy is the administration of small molecule inhibitors or anti-metastatic agents of SRC-3. In some embodiments, additional therapy is the administration of side effect limiting agents (e.g., agents intended to reduce the occurrence and / or severity of side effects of the procedure, e.g., antiemetics, etc.). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation. In some embodiments, additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. Additional therapy may be one or more chemotherapeutic agents known in the art.

[0089] Immunotherapy can be administered before, during, or after additional cancer treatment, or in various combinations. Doses may be administered simultaneously, at intervals of minutes, hours, days, or weeks. In embodiments where immunotherapy is provided to the patient separately from additional therapeutic agents, those skilled in the art will generally ensure that no critical period expires between each delivery time so that the two compounds can still exert a beneficially combined effect on the patient.

[0090] To administer any compound or therapy of this embodiment to a patient, a general protocol for the administration of such compounds will be followed, taking into account the toxicity of the agent, if any. Therefore, in some embodiments, there is a step to monitor for toxicity resulting from the combination therapy. A. Chemotherapy

[0091] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. Such agents or drugs are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at what stage they affect it. In addition, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0092] Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and picosulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine); acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and Cryptophycin 1 and Cryptophycin 1). Liptophycin 8); Dorastatin; Duocalmycin (including synthetic analogues, KW-2189, and CB1-TM1); Eleuterobin; Pancratistatin; Sarcodicin; Spongestatin; Nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard; Nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; Antibiotics, e.g., engineeric antibiotics (e.g., calicheamicin, in particular calicheamicin gamma II) (gamma-II), and calicheamicin omega-II); dynemycin (dynemycin A is an example); bisphosphonates, e.g., clodronate; esperamycin;Furthermore, neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore, acrasinomycin, actinomycin, autolarmycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin) Examples include cereals, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalarnycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolbicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, pterop Therin and trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents, e.g., mitotane and trilostane; Folic acid supplements, e.g., floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diaziquan; elformitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamine; mytansinoids, e.g., mytansin and anthamitosin; mitogwazone; mitoxantrone; mopidanmol; nitraerine;Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veraculin A, loridine A, and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ar a-C); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinated complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeroda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS Examples include difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans platinum, and any of the pharmaceutically acceptable salts, acids, or derivatives thereof. B. Radiation therapy

[0093] Other widely used factors that cause DNA damage include gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also considered, such as microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are most likely to affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from a daily dose of 50–200 roentgens over long periods (3–4 weeks) to a single dose of 2000–6000 roentgens. Radioisotope dose ranges vary widely and depend on the isotope half-life, the intensity and type of radiation emitted, and uptake by neoplastic cells. C. Immunotherapy

[0094] Those skilled in the art will understand that additional immunotherapies to those encompassed herein may be used in combination with or in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Immune effectors may be, for example, antibodies specific to certain markers on the surface of tumor cells. Antibodies can function alone as effectors of therapy or can mobilize other cells to actually influence cell death. Antibodies may also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) to function as targeting agents. In addition, effectors may be lymphocytes carrying surface molecules that directly or indirectly interact with tumor cell targets. Examples of various effector cells include cytotoxic T cells and NK cells.

[0095] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer treatments. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently bound to a cell-killing drug. This approach combines the high specificity of the MAb to an antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells enriched with antigen levels. Targeted drug delivery also minimizes exposure to normal tissue, leading to reduced toxicity and improved therapeutic index. The FDA's approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, merely serve as examples to validate this approach.

[0096] In one embodiment of immunotherapy, tumor cells must possess some marker suitable for targeting, i.e., markers not present in most other cells. Many tumor markers exist, all of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative embodiment of immunotherapy combines anticancer effects with immunostimulatory effects. Immunostimulatory molecules include cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and growth factors such as FLT3 ligand.

[0097] In some embodiments, immunotherapy may be immune checkpoint inhibitors. Immune checkpoints either enhance or degrade signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and the V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. Another immune checkpoint that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. D. Surgery

[0098] Approximately 60% of people with cancer will undergo some form of surgery, including prophylactic surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in combination with other therapies such as the treatment of this embodiment, chemotherapy, radiotherapy, 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 microsurgery (Mohs surgery).

[0099] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be performed with varying dosages. VIII. Manipulated B cells and their use

[0100] In certain embodiments, B cells are examples of immune cells that are engineered to have SRC-3 destruction. In certain embodiments, SRC-3-destroyed B cells are utilized for any medical condition in which B cells are effective, while in certain embodiments, modified B cells are utilized for the treatment of one or more inflammatory diseases or cancers of any kind, including, for example, B-cell lymphoma and cancers listed elsewhere herein.

[0101] In certain embodiments, B cells are obtained commercially or from individuals requiring treatment. B cells may be obtained from healthy individuals for allogeneic purposes. B cells can be manipulated ex vivo by standard methods that disrupt endogenous SRC-3 expression in the B cells. In some cases, the manipulated B cells are exposed to one or more agents, such as TGF-□ or tumor-specific antigens, which enhance the efficacy of the B cells when administered to individuals in need.

[0102] In certain embodiments, engineered B cells lacking SRC-3 expression or exhibiting reduced SRC-3 expression compared to unengineered B cells are administered in an effective dose to individuals having or at risk of having one or more inflammatory diseases. While inflammatory diseases can be of any kind, in certain embodiments, the diseases are, to name just a few, allergies, asthma, autoimmune diseases, 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. B cell treatment may reduce or delay the severity of the disease, delay the onset of the disease, improve one or more signs of the disease, etc. IX. Manufactured goods or kits

[0103] Furthermore, manufactured products or kits containing immune cells are also provided herein. The immune cells may be T cells, such as Treg cells. The kit may include cells without SRC-3 disruption, one or more reagents that induce SRC-3 disruption (e.g., nucleic acids and proteins that specifically promote SRC-3 knockout or knockdown), cells with SRC-3 disruption, one or more agents targeting SRC-3, buffers, salts, instructions for use, or a combination thereof. In certain embodiments, the kit includes a CRISPR reagent that targets SRC-3, regulatory T cells, or both.

[0104] The manufactured product or kit may further include a package insert containing instructions for using immune cells to treat cancer in an individual or to slow the progression of cancer. Any immune cells described herein may be included in the manufactured product or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or alloy (e.g., stainless steel or nickel-molybdenum alloy). In some embodiments, the container may hold the formulation and label on the container or be associated with the container to display instructions for use. The manufactured product or kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and a package insert containing instructions for use. In some embodiments, the manufactured product further includes one or more other agents (e.g., chemotherapeutic agents and anti-cancer drugs). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes. [Examples]

[0105] The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples constitute a particular form for implementation, as they demonstrate that the techniques discovered by the inventors function well in carrying out the methods and compositions of the present disclosure. However, those skilled in the art will understand that many modifications can be made in light of the present disclosure to the particular embodiments disclosed, and similar or analogous results can still be obtained without departing from the spirit and scope of the present disclosure. Example 1 SRC-3 controls immune cell populations.

[0106] The cell-autonomous oncogenic function of SRC-3 (gene name: NCOA3) in breast cancer cells is well established (1-9), but the role of SRC-3 expressed in immune cells is largely unknown. Gene deletion of the Src-3 gene in mice leads to an expansion of the lymphoid lineage and CD4 + and CD8 + This results in an increase in the number of T cells and B lymphocytes (first reported in (11), and recent data summarizing this observation are shown in Figure 1). In the peripheral blood of Src-3 KO mice, levels of several cytokines, including CXCL2, IL2, IL1α and β, CCL2, and GCSF, were elevated (data not shown). These findings reveal the immunosuppressive function of SRC-3, highlighting it as a novel transcriptional immune checkpoint regulator, strongly suggesting that pharmacological inhibition of SRC-3 in immune cells can enhance their ability to attack cancer cells. However, the precise immune cell population in which SRC-3 exerts this role remained undefined. Gene expression analysis of publicly available datasets revealed that SRC-3 expression was very high in Treg cells and correlated with Foxp3 expression (Figure 2). Among the 296 carefully selected co-regulators from the Nuclear Receptor Signaling Atlas (NURSA), NCOA3 (Src-3) was the second most strongly correlated co-activator of Foxp3 expression in Treg cells (Figure 3). Example 2 Genetic disruption of SRC-3 in Tregs promotes robust breast cancer tumor clearance.

[0107] Given previously reported data showing that SRC-3- / - mice exhibit a lymphoproliferative phenotype and that disruption of SRC-3 expression in Treg cells leads to decreased expression of immunosuppressive proteins in this cell type, we characterized SRC-3 as an immune checkpoint regulatory protein specifically in the "Treg compartment" using a mouse gene model system. We backcrossed SRC-3flox / flox mice to a pure C57BL / 6J background for 10 generations, and then crossed these mice with Foxp3:EGFP-Cre-ERT2 mice already in a C57BL / 6J background to produce Treg:SRC-3KO mice.

[0108] Tamoxifen treatment activates Cre, leading to specific disruption of the SRC-3 gene in Tregs. Using this genetically engineered mouse model system, the effects of SRC-3 gene disruption were tested in a broad range of immunocompetent syngeneic tumor cell lines compatible with the C57BL / 6J background (including the E0771 mouse mammary tumor cell line shown below). E0771 tumors are particularly aggressive, and it is well known that controlling tumor growth in this model system is difficult. However, in host mice in which SRC-3 was specifically disrupted in Tregs, the tumors were essentially eradicated, whereas in wild-type host animals, the tumors rapidly grew to a large size (Figure 4).

[0109] The results of this study strongly demonstrate that SRC-3 is a newly discovered and important immune checkpoint modulator. Another unexpected and important result from this animal model experiment was that the animals appeared healthy, with normal-sized spleens and no significant changes in body weight or activity. This is in contrast to Foxp3 mutant scurfy mice, which have a severe autoimmune phenotype and die at a young age. In certain embodiments, Treg:SRC-3 KO mice are different from scurfy mice because genetic disruption of SRC-3 in Foxp3 cells disrupts only a subset of the immunosuppressive activity of Tregs. Existing immune checkpoint modulators in clinical practice have potent, dose-limited, and life-threatening side effect profiles. Therefore, in certain embodiments, inhibition of only specific aspects of Treg biology by SRC-3 SMI allows targeting only its tumor-associated immunosuppression and does not generally overstimulate undesirable immune events in other tissues. Example 3 CRISPR-based targeting of SRC-3 in Tregs

[0110] Genetically modified mouse models have demonstrated that aggressive mammary cancer can be eradicated by specifically targeting SRC-3 within Treg cells. To translate this finding into a clinically interpretable therapeutic intervention, the inventors developed a method to target the SRC-3 gene in mouse immune cells using the CRISPR-based approach described below. In certain embodiments, genetically modified cells are used as a source of T cells / Tregs for adoptive therapy to eliminate mammary tumors in a syngeneic mouse model system. This process is adaptable to (e.g.) human CD4+ cells useful for treating human patients with breast cancer or other cancers.

[0111] Spleens were collected from 12-week-old male C57BL / 6 mice. A single-cell suspension of splenocytes was prepared by crushing the spleen with a 40 μm cell strainer using the plunger end of a syringe. The splenocyte suspension was placed in a conical tube and centrifuged (1500 rpm, 5 min). The erythrocyte pellet was debridemented using RBC removal buffer (Sigma R 7757) to obtain a beige lymphocyte pellet. The lymphocytes were plated onto CD3-coated plates using complete RPMI medium (L-glu, FBS, antibiotics) supplemented with CD28 / IL2 / MeSH. After 3 days of culture, cell viability was evaluated (80%), and bulk lymphocytes were CD4-enriched using the Miltenyi CD4 enrichment kit (130-104-545). The enriched CD4 cells (40M) were plated for another two days under the above conditions to proliferate the cell population to over 100 million cells.

[0112] Nucleofection: CD4 cell enrichment and proliferation populations were nucleofected with Cas9-NCoA3 guide RNA (gRNA) ribonucleoprotein (RNP) using the Lonza 4D nucleofector and primary cell nucleofection kit (Lonza V4XP-3024). RNP was prepared by mixing fluorescently labeled trcrRNA (IDT#1075928) with crRNA (IDT) in a 1:1 ratio (11.25 μl of TrcrRNA-ATTO 550 with three different crRNAs (3.75 μl each)). The gRNA (composed of trcrRNA + crRNA) was incubated at 37°C for 15 minutes. Next, gRNA (concentration 50 μM) was mixed with Cas9 protein (IDT#1081059, concentration 60 μM) in a 3:1 ratio to obtain 15 μM RNP with an excess of gRNA. The RNP was incubated at 37°C for 15 minutes and then mixed with nucleofection buffer in a 1:5 ratio. Thirteen nucleofection reaction solutions were prepared. Each solution contained 8 to 10 million CD4 cells and 130 μl of RNP-nucleofection buffer mix (the final concentration of RNP was 3.5 μM, with a 3-fold excess of gRNA). Nucleofection was performed in a 4D nucleofector by subjecting each nucleo cuvette to the manufacturer's EN138 pulse program.

[0113] After nucleofection, the cells were supplied with culture medium and transferred to a CD3-covered culture dish. After overnight recovery, the cells were sorted by flow cytometry to allow for the isolation of the nucleofected population (by tracking fluorescently labeled gRNA).

[0114] Sorted populations (positive and negative) were cultured for 5 days to induce gene editing, protein turnover, and cell proliferation. Subsequently, total RNA was isolated from these cells, and two-step RT-qPCR was performed to test for disruption of SRC-3 (gene symbol Ncoa3) expression.

[0115] These results demonstrate that CRISPR-based disruption of SRC-3 in T lymphocytes is robust and generates high-yield viable, proliferative cells for further downstream applications such as adoptive T cell transfer.

[0116] The data strongly suggest that SRC-3 is a critical target in Treg cells, and its specific disruption in this cellular compartment leads to tumor eradication in a breast cancer syngeneic tumor model. This strategy of disrupting SRC-3 in Treg cells distinguishes it from other immune checkpoint inhibitors because SRC-3 is a nuclear protein that modulates Treg function to promote tumor eradication without completely eliminating its function, which is associated with potentially serious side effects. This disclosure provides an approach to specifically eliminate SRC-3 ex vivo in mouse T cells using CRISPR-based gene targeting. When administered using human CD4+ lymphocytes, this method has clear translational potential as an adoptive T cell / Treg-based therapeutic for the treatment of human cancers. Example 4 Transfer of adoptive SRC-3 KO Treg cells to mice with breast cancer

[0117] Given previously reported data showing that SRC-3- / - mice exhibit a lymphoproliferative phenotype and that disruption of SRC-3 expression in Treg cells leads to decreased expression of immunosuppressive proteins in this cell type, we characterized SRC-3 as an immune checkpoint regulatory protein specifically in the "Treg compartment" using a mouse gene model system. We backcrossed SRC-3flox / flox mice to a pure C57BL / 6J background for 10 generations, and then crossed these mice with Foxp3:EGFP-Cre-ERT2 mice already in a C57BL / 6J background to produce Treg:SRC-3KO C57BL / 6J mice.

[0118] Tamoxifen treatment activates Cre, specifically disrupting the SRC-3 gene in Treg cells. Using this genetically engineered mouse model system, SRC-3 KO Treg cells were generated from the spleen of SRC-3 knockout (KO) Treg animals. Approximately 1.6 million SRC-3 KO Treg cells were isolated from the spleen. 92% of the isolated SRC-3 KO Treg cells were viable. As a control, SRC-3 flox / flox(SRC-3) F / F Treg cells (approximately 1.9 million) were isolated from the spleen removed from a mouse. 93% of the isolated control cells were viable.

[0119] The effects of isolated SRC-3 KO Treg cells were tested in C57BL / 6J mice carrying tumors derived from E0771 cells, a mouse mammary cancer cell line first isolated from spontaneously occurring tumors in C57BL / 6 mice (Figure 6). E0771 tumors are particularly aggressive, and suppressing E0771 tumor growth in immune-intact C57BL / 6J mice is challenging. However, E0771 tumors were essentially eradicated in mice treated with isolated SRC-3 KO Treg cells (900,000 cells) compared to E0771 tumor growth in C57BL / 6J mice without adoptive Treg cell transfer (No AT). In contrast, SRC-3 F / F Transplantation of 900,000 Treg cells derived from control mice (Cont Treg, control SRC-3+Treg) did not suppress E0771 cell proliferation compared to E0771 tumor growth in C57BL / 6J mice without adoptive Treg cell transplantation (Figure 7). Furthermore, adoptive SRC-3 KO Treg cell transplantation did not cause apparent toxicity, and animals that received SRC-3 KO Treg cells remained fertile (data not shown).

[0120] The results of this study demonstrate that adoptive transfer of SRC-3 KO Treg cells is useful for cancer treatment, and that in certain embodiments, SRC-3 KO Treg cells can be used to control and / or eliminate tumor growth. References

[0121] All patents and publications referenced herein represent the level of skill of those skilled in the art in which the present invention relates. All patents and publications are incorporated herein by reference to the same extent that each individual publication is specifically and individually indicated to be incorporated by reference. 1.Geng C, He B, Xu L, Barbieri CE, Eedunuri VK, Chew SA, Zimmermann M, Bond R, Shou J, Li C, Blattner M, Lonard DM, Demichelis F, Coarfa C, Rubin MA, Zhou P, O'Malley BW, Mitsiades N. Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulates steroid receptor coactivator 3 protein turnover. Proc Natl Acad Sci US A. 2013;110(17):6997-7002. doi: 10.1073 / pnas.1304502110. PubMed PMID: 23559371; PMCID: PMC3637757. 2.Eedunuri VK, Rajapakshe K, Fiskus W, Geng C, Chew SA, Foley C, Shah SS, Shou J, Mohamed JS, Coarfa C, O'Malley BW, Mitsiades N. miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation. Mol Endocrinol. 2015;29(8):1170-83. doi: 10.1210 / me.2015-1080. PubMed PMID: 26066330; PMCID: PMC4518002. 3.Coarfa C, Fiskus W, Eedunuri VK, Rajapakshe K, Foley C, Chew SA, Shah SS, Geng C, Shou J, Mohamed JS, O'Malley BW, Mitsiades N. Comprehensive proteomic profiling identifies the androgen receptor axis and other signaling pathways as targets of microRNAs suppressed in metastatic prostate cancer. Oncogene. 2016;35(18):2345-56. doi: 10.1038 / onc.2015.295. PubMed PMID: 26364608; PMCID: PMC5915337. 4.Wang Y, Lonard DM, Yu Y, Chow DC, Palzkill TG, O'Malley BW. Small molecule inhibition of the steroid receptor coactivators, SRC-3 and SRC-1. Mol Endocrinol. 2011;25(12):2041-53. Epub 2011 / 11 / 05. doi: me.2011-1222 [pii] 10.1210 / me.2011-1222. PubMed PMID: 22053001; PMCID: 3231837. 5.Tien JC, Liu Z, Liao L, Wang F, Xu Y, Wu YL, Zhou N, Ittmann M, Xu J. The steroid receptor coactivator-3 is required for the development of castration-resistant prostate cancer. Cancer Res. 2013;73(13):3997-4008. Epub 2013 / 05 / 08. doi: 0008-5472.CAN-12-3929 [pii] 10.1158 / 0008-5472.CAN-12-3929. PubMed PMID: 23650284; PMCID: 3732785. 6.Zhou HJ, Yan J, Luo W, Ayala G, Lin SH, Erdem H, Ittmann M, Tsai SY, Tsai MJ. SRC-3 is required for prostate cancer cell proliferation and survival. Cancer Res. 2005;65(17):7976-83. Epub 2005 / 09 / 06. doi: 65 / 17 / 7976 [pii] 10.1158 / 0008-5472.CAN-04-4076. PubMed PMID: 16140970. 7.Yan J, Yu CT, Ozen M, Ittmann M, Tsai SY, Tsai MJ. Steroid receptor coactivator-3 and activator protein-1 coordinately regulate the transcription of components of the insulin-like growth factor / AKT signaling pathway. Cancer Res. 2006;66(22):11039-46. Epub 2006 / 11 / 17. doi: 66 / 22 / 11039 [pii] 10.1158 / 0008-5472.CAN-06-2442. PubMed PMID: 17108143. 8.Ayala G, Yan J, Li R, Ding Y, Thompson TC, Mims MP, Hayes TG, MacDonnell V, Lynch RG, Frolov A, Miles BJ, Wheeler TM, Harper JW, Tsai MJ, Ittmann MM, Kadmon D. Bortezomib-mediated inhibition of steroid receptor coactivator-3 degradation leads to activated Akt. Clin Cancer Res. 2008;14(22):7511-8. Epub 2008 / 11 / 18. doi: 14 / 22 / 7511 [pii] 10.1158 / 1078-0432.CCR-08-0839. PubMed PMID: 19010869; PMCID: 2820291. 9.Yan J, Erdem H, Li R, Cai Y, Ayala G, Ittmann M, Yu-Lee LY, Tsai SY, Tsai MJ. Steroid receptor coactivator-3 / AIB1 promotes cell migration and invasiveness through focal adhesion turnover and matrix metalloproteinase expression. Cancer Res. 2008;68(13):5460-8. Epub 2008 / 07 / 03. doi: 68 / 13 / 5460 [pii] 10.1158 / 0008-5472.CAN-08-0955. PubMed PMID: 18593949. 10.Wu C, Orozco C, Boyer J, Leglise M, Goodale J, Batalov S, Hodge CL, Haase J, Janes J, Huss JW, 3rd, Su AI. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol. 2009;10(11):R130. doi: 10.1186 / gb-2009-10-11-r130. PubMed PMID: 19919682; PMCID: PMC3091323. 11.Coste A, Antal MC, Chan S, Kastner P, Mark M, O'Malley BW, Auwerx J. Absence of the steroid receptor coactivator-3 induces B-cell lymphoma. EMBO J. 2006;25(11):2453-64. Epub 2006 / 05 / 06. doi: 7601106 [pii] 10.1038 / sj.emboj.7601106. PubMed PMID: 16675958; PMCID: 1478181. 12.Vandenbon A, Dinh VH, Mikami N, Kitagawa Y, Teraguchi S, Ohkura N, Sakaguchi S. Immuno-Navigator, a batch-corrected coexpression database, reveals cell type-specific gene networks in the immune system. Proc Natl Acad Sci U S A. 2016;113(17):E2393-402. doi: 10.1073 / pnas.1604351113. PubMed PMID: 27078110; PMCID: PMC4855614.

[0122] While the present disclosure and its merits have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the design defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, material compositions, means, methods, and steps described herein. As will be readily apparent to those skilled in the art from this disclosure, existing or subsequently developed processes, machines, manufactures, material compositions, means, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with this disclosure. Accordingly, the scope of the appended claims is intended to include such processes, machines, manufactures, material compositions, means, methods, or steps within its scope.

Claims

1. (a) Manipulated T-regulatory (Treg) cells, including the disruption of steroid receptor coactivator 3 (SRC-3), and (b) One or more agents that target SRC-3 and A pharmaceutical composition containing the following:

2. (a) Manipulated T-regulatory (Treg) cells, including the disruption of steroid receptor coactivator 3 (SRC-3), and (b) One or more agents that target SRC-3 A combination drug.

3. The pharmaceutical composition according to claim 1, wherein the agent targeting SRC-3 is a small molecule inhibitor, an antibody, a protein, a nucleic acid, or a combination thereof.

4. The pharmaceutical composition according to claim 3, wherein the agent targeting SRC-3 is a small molecule inhibitor, and the small molecule inhibitor is bufarin, gossypol, belcarin A, SI-2, SI-10, SI-12, their functional derivatives, or a combination thereof.

5. The pharmaceutical composition according to claim 3, wherein the agent targeting SRC-3 is an antibody, and the antibody is a monoclonal antibody or a polyclonal antibody.

6. A composition administered to an individual to treat cancer, comprising engineered T-regulatory (Treg) cells, which includes the disruption of steroid receptor coactivator 3 (SRC-3).

7. The composition according to claim 6, wherein the cancer is SRC-3+ cancer.

8. The composition according to claim 6 or 7, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, or pancreatic cancer.

9. The composition according to any one of claims 6 to 8, wherein the cells are ex vivo exposed to one or more agents that target SRC-3 in an effective amount.

10. The composition according to claim 9, wherein the agent targeting SRC-3 is a small molecule inhibitor, an antibody, a protein, a nucleic acid, or a combination thereof.

11. The composition according to claim 10, wherein the small molecule inhibitor of SRC-3 is bufarin, gossypol, belcarin A, SI-2, SI-10, SI-12, their functional derivatives, or a combination thereof.

12. The composition according to claim 10 or 11, wherein the agent targeting SRC-3 is an antibody, and the antibody is a monoclonal antibody or a polyclonal antibody.

13. The composition according to any one of claims 6 to 12, to be used in combination with additional cancer treatment.

14. The composition according to claim 13, wherein the additional cancer treatment includes surgery, radiation, chemotherapy, hormone therapy, drug therapy, protein therapy, immunotherapy, or a combination thereof.

15. The composition according to claim 13 or 14, wherein the additional cancer treatment comprises one or more agents targeting SRC-3.

16. The composition according to claim 15, wherein the agent targeting SRC-3 is a small molecule inhibitor, an antibody, a protein, a nucleic acid, or a combination thereof.

17. The composition according to claim 16, wherein the agent targeting SRC-3 is a small molecule inhibitor, and the small molecule inhibitor is bufarin, gossypol, belcarin A, SI-2, SI-10, SI-12, functional derivatives thereof, or a combination thereof.

18. The composition according to claim 16 or 17, wherein the agent targeting SRC-3 is an antibody, and the antibody is a monoclonal antibody or a polyclonal antibody.

19. The composition according to any one of claims 13 to 18, wherein the composition and the additional cancer treatment are administered to the individual substantially simultaneously.

20. The composition according to any one of claims 13 to 18, wherein the composition and the additional cancer treatment are administered to the individual at different time points in time.

21. The composition according to any one of claims 6 to 20, wherein the composition is administered by intravenous, intraperitoneal, intraarterial, local, inhalation, intramuscular, intrasternal, intra-articular injection, or infusion.

22. The composition according to any one of claims 6 to 21, wherein the composition is administered once or multiple times.

23. The composition according to claim 22, wherein, when the composition is administered to the individual multiple times, the interval between administrations is within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months.

24. A combination pharmaceutical for treating cancer in an individual, comprising (a) engineered T-regulatory (Treg) cells, including the disruption of steroid receptor coactivator 3 (SRC-3), and (b) one or more agents targeting SRC-3.

25. The combination pharmaceutical according to claim 24, wherein when (a) and (b) are administered to the individual at different time points, the interval between the administrations of (a) and (b) is within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months.

26. The combination drug according to claim 24, wherein the combination drug is administered to the individual once or multiple times.

27. The combination pharmaceutical according to claim 26, wherein when (a) and (b) are administered to the individual at different time points, the interval between administrations is within 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months.

28. The combination pharmaceutical according to any one of claims 24 to 27, wherein the cancer is SRC-3+ cancer.

29. The combination pharmaceutical according to any one of claims 24 to 28, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, gastric cancer, multiple myeloma, thyroid cancer, or pancreatic cancer.

30. The combination pharmaceutical according to any one of claims 24 to 29, wherein, prior to the administration, the cells of (a) are ex vivo exposed to one or more agents that target SRC-3.

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