Large-scale combination of CAR transduction and CRISPR gene editing in B cells

Abstract

Methods and compositions are provided that can be used for any purpose, including the treatment of medical conditions such as cancer, infectious diseases and / or immune-related disorders. [Solution] Embodiments of the present disclosure include methods and compositions for producing engineered B cells. The disclosure relates to a large-scale process for producing B cells that have been engineered to disrupt expression of one or more genes, such as using CRISPR, and that are engineered to express at least one heterologous antigen receptor. Certain embodiments include specific parameters for the process.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 941,651, filed on November 27, 2019. This provisional application is hereby incorporated by reference in its entirety into this specification.

[0002] The present disclosure generally relates to the fields of immunology, cell biology, molecular biology, and medicine.

Background Art

[0003] B cells can be broadly classified into effector cells and regulatory cells. Effector B cells are important factors driving humoral immunity because they have the ability to produce antibodies specific to pathogens. Regulatory B cells have recently been shown to control the inflammatory response in multiple diseases through the production of anti - inflammatory cytokines such as IL - 10, IL - 35, and TGF - beta. Due to the important effects these B - cell subsets exert on immune regulation and humoral immunity, these subsets are candidates for valuable cell therapies for various immune disorders. Thus, the need to improve methods of B - cell immunotherapy remains unaddressed. The fact that B cells are difficult to expand ex vivo and are prone to apoptosis when cultured is a major factor limiting the potential for treatment. Creating a successful expansion protocol with excellent survival rates opens the way to directly using B cells in treatment or using them ex vivo as a source of antibody production. Furthermore, strategies for manipulating B cells can further enhance their utility for treatment. Thus, the need for improvement in methods of cell immunotherapy, particularly using B cells, remains unaddressed.

Summary of the Invention

[0004] Embodiments of the present disclosure include methods and compositions for enhancing the survival rate and persistence of B cells, wherein the B cells are specifically engineered to have such activity compared to B cells that have not been so engineered. Specific embodiments include methods and compositions for reducing apoptosis of B cells, wherein the B cells are specifically engineered to reduce the risk of apoptosis compared to B cells that have not been so engineered. The methods of the present disclosure use specific parameters that improve the expansion of B cells and enhance their effectiveness for use as a treatment for an individual in need thereof.

[0005] Embodiments of the present disclosure include novel large-scale approaches (including GMP grade) for the expansion of B cells, with or without CAR transduction and / or gene editing (including CRISPR gene editing). The present disclosure provides engineered B cells that express one or more heterologous antigen receptors and / or are gene edited such that one or more endogenous genes in the B cells are disrupted. In some embodiments, B cells transduced with a CAR are engineered such that the expression of one or more endogenous genes in the B cells is disrupted, and in other embodiments, B cells in which the expression of one or more endogenous genes in the B cells is disrupted are transduced or transfected to express one or more heterologous antigen receptors. Specific embodiments provide a large-scale CRISPR / Cas9-mediated engineering strategy for primary B cells transduced with a CAR.

[0006] In certain embodiments, the process is such that the process is large-scale (up to 1×10 9 、1×10 10 、1×10 11Use specific parameters (including conditions and / or reagents) that enable the manipulation of one or more cells. In certain cases, the process includes one or more expansion steps and a series of steps that include modifying the cells to enhance the proliferation of B cells as follows: (1) modifying the cells to have one or more heterologous antigen receptors; (2) modifying the cells such that the expression of one or more endogenous genes in the B cells is eliminated or reduced; and / or (3) modifying the cells to express one or more cytokine genes. The modification of B cells with respect to (1), (2), and (3) can be in any order. In some cases, one or more of (1), (2), and (3) is optional and may not be used. In a specific case, one or more endogenous genes are knocked out or knocked down using CRISPR and guide RNAs for multiple genes. The process of generating the desired B cells also includes the specific duration of specific steps of the process (including one or more specific steps of the process).

[0007] The produced engineered B cells can be used for any purpose, including the treatment of medical conditions such as cancer, infectious diseases, and / or immune-related disorders. In some embodiments, those B cells are appropriately stored prior to use. The recipient individual of the engineered B cells can be autologous or allogeneic with respect to the origin of those cells. In some cases, the B cells produced by the methods of the present disclosure are used for therapeutic purposes after appropriate storage and may or may not be further modified during storage after production. For example, B cells may be engineered such that one or more endogenous genes in those cells are gene edited to enhance their viability, and then those B cells are stored, but prior to use, those B cells can be further modified to express a heterologous antigen receptor specific to the needs of the recipient individual (e.g., a receptor that targets an antigen on the cancer cells of that individual). In other cases, B cells may be engineered to express a heterologous antigen receptor (e.g., including those against antigens that are known cancer antigens), and then those B cells are stored, but prior to use, those B cells can be further modified to be gene edited to disrupt the expression of one or more endogenous genes. In this example, the heterologous antigen receptor may or may not be designed to target well-known cancer antigens or antigens present on various types of cancer cells.

[0008] In certain embodiments, the heterologous antigen receptor is a chimeric antigen receptor or a T cell receptor. The heterologous antigen receptor can target tumor-associated antigens. In specific cases, the heterologous antigen receptor targets an antigen selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99 and combinations thereof. Other antigens are listed elsewhere in this specification.

[0009] In certain embodiments, the endogenous gene whose expression is disrupted in the B cells is an inhibitory gene. In specific cases, the endogenous gene whose expression is disrupted in the B cells is NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, beta2-microglobulin, HLA, CD73, CD39 or combinations thereof.

[0010] In certain embodiments, the B cells are gene-edited and one or more compositions are delivered to the B cells by electroporation. When the B cells are electroporated, the electroporation is performed on a specific amount of B cells, e.g., about 200,000 to 1×1011 Individual B cells can be used. Electroporation can use about 200,000 to 2,000,000 cells. In specific cases, electroporation can use about 1,000,000 to 1×10 9 or more (10 10 、10 11 ) and any range of B cells derivable therefrom can be used.

[0011] In any method herein, any B cells that have been manipulated may be analyzed. For example, those cells may be analyzed by gene editing efficacy, functional assays, cytotoxicity assays, and / or in vivo activity. In specific embodiments, those cells are analyzed by flow cytometry, mass cytometry, RNA sequencing, PCR, or combinations thereof. Any of those cells can be preserved, such as by cryopreservation, for example, after a particular step of the process or after the final cell product has been made. An effective amount of B cells can be delivered to an individual in need thereof (e.g., an individual having cancer, an infection, and / or an immune-related disorder).

[0012] Embodiments of the present disclosure include a population of B cells made by any method included herein. Compositions containing such a population are contemplated, such as when the population of cells of the present disclosure is present in a pharmaceutically acceptable carrier.

[0013] Embodiments of the present disclosure include a method of treating an individual for a medical condition, the method including administering to the individual a therapeutically effective amount of B cells made by any method of the present disclosure. The individual may or may not have received one or more additional treatments for the medical condition and may be receiving or scheduled to receive such treatments.

[0014] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description, so it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only.

[0015] For a more complete understanding of the present disclosure, reference is made to the following description, which is to be construed in conjunction with the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0025] I. Definitions As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used with the word "comprising", the word "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more. Further, the terms "having", "including", "containing", and "comprising" are interchangeable, and those skilled in the art recognize that these terms are open-ended terms. In certain embodiments, aspects of the present disclosure may "consist essentially of" or "be composed of", for example, from one or more sequences of the present disclosure. Some embodiments of the present invention may consist of or consist essentially of one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein may be practiced with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As used herein, the terms "or" and "and / or" are utilized to combine multiple components or to describe them in an exclusive manner with respect to each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from the embodiments.

[0026] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives or unless the alternatives are shown to be mutually exclusive. However, the present disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" may mean at least a second, or more. The terms "about", "substantially", and "approximately" generally mean plus or minus 5% of the stated value.

[0027] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "an embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0028] "Immune disorder", "immune-related disorder", or "immune-mediated disorder" refers to a disorder in which the immune response plays an important role in the onset or progression of the disease. Immune-mediated disorders include autoimmune diseases, allograft rejection, graft-versus-host disease, and inflammatory and allergic diseases.

[0029] As used herein, the term "engineered" refers to an entity generated by human hand, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered article is synthetic, not found in nature, or composed of elements configured in a manner utilized in the present disclosure. With respect to cells, the cells can be engineered because the expression of one or more endogenous genes is reduced and / or because the cells express one or more heterologous genes (such as synthetic antigen receptors and / or cytokines). In this case, all manipulations are performed by human hand. With respect to antigen receptors, the antigen receptors may be considered engineered because they consist of multiple components that are genetically recombined to be so configured in the form of a fusion protein of components not found in nature.

[0030] As used herein, the term "large-scale" refers to those on the order of 10 10 or more, including NK cells in numbers such as 10 11 and up to 10 9 or more.

[0031] "Treatment" or management of a disease or condition refers to implementing a protocol, which may include administering to a patient one or more agents, as an effort to alleviate the signs or symptoms of the disease. Desirable effects of treatment include reducing the rate of progression of the disease, improving or alleviating the disease state, remission, or improving the prognosis. Alleviation can occur not only before the signs or symptoms of the disease or condition appear, but also after their appearance. Thus, "treating" or "managing" can include "preventing" or "precluding" a disease or undesirable condition. Further, "treating" or "treatment" does not require complete alleviation of signs or symptoms, and does not require a cure, and includes, in particular, protocols that give only a modest effect to a patient.

[0032] As used throughout this application, the terms "therapeutic benefit" or "therapeutically effective" refer to that which promotes or enhances the well-being of a subject with respect to the medical treatment of that condition. This includes, but is not limited to, a decrease in the frequency or severity of the signs or symptoms of the disease. For example, treatment of cancer may include, for example, a decrease in the size of a tumor, a decrease in the invasiveness of the tumor, a decrease in the growth rate of the cancer, a delay in the onset of the cancer, a delay in the onset of one or more symptoms of the cancer, or prevention of metastasis, a delay in the onset of metastasis. Treatment of cancer may also refer to prolonging the survival of a subject having cancer.

[0033] "Subject" and "patient" or "individual" mean either a human or non-human, such as a primate, mammal, and vertebrate.

[0034] As used herein, "mammal" is a suitable subject for the methods of the present disclosure. A mammal can be any member of the class Mammalia of higher vertebrates, including humans; characterized by reproduction, body hair, and mammary glands in the female that secrete milk for nourishing the young. Further, it is characteristic of mammals that they can maintain a constant body temperature even when the climate conditions change. Examples of mammals include humans, cats, dogs, cows, mice, rats, horses, goats, sheep, and chimpanzees. Mammals may be referred to as "patients" or "subjects" or "individuals".

[0035] The term "pharmaceutical or pharmaceutically acceptable" refers, where appropriate, to molecules and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients would be known to those of skill in the art in light of the present disclosure. Further, it will be understood that for administration to animals (e.g., humans), the preparations should meet the standards of sterility, pyrogenicity, general safety and purity required by the FDA's Biological Standards Division.

[0036] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, oxidizing agents, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, coloring agents, liquids and nutritional supplements, materials and combinations thereof known to those of skill in the art. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0037] As used herein, "disruption" of a gene means elimination or reduction of expression of one or more gene products encoded by the gene of interest in a cell, as compared to the level of expression of the gene product in the absence of disruption. Exemplary gene products include mRNA and protein products encoded by the gene. Disruption in some cases is transient or reversible, while in other cases it is permanent. In some cases, disruption results in a functional or full-length protein or mRNA, despite the fact that truncated or non-functional products may be produced. In some embodiments herein, in contrast to expression, gene activity or function is disrupted. Gene disruption is generally induced by artificial methods, i.e., by the addition or introduction of a compound, molecule, complex, or composition, and / or by disruption of the gene or nucleic acid associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene disruption techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense techniques such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in transient reduction of expression, and gene editing techniques, such as those that result in inactivation or disruption of a target gene by, for example, inducing cleavage and / or homologous recombination. Examples include insertions, mutations, and deletions. Disruption typically results in suppression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Exemplifications of such gene disruption include insertions, frameshift and missense mutations, deletions, knock-ins, and knockouts of the gene or part of the gene, including deletion of the entire gene. Such disruption can occur in the coding region, e.g., in one or more exons, such that no full-length product, functional product, or any product can be produced, for example, by insertion of a stop codon. Such disruption can also occur by disruption of the promoter or enhancer, or other regions that affect activation of transcription, so as to prevent transcription of the gene. Gene disruption includes gene targeting, such as inactivation of a target gene by homologous recombination.

[0038] As used herein, the term "heterologous" means derived from a cell type or species different from the recipient. Specifically, it refers to genes or proteins that are synthetic and / or not derived from B cells. This term also refers to synthetically derived genes or gene constructs. For example, cytokines are considered heterologous with respect to B cells even when they are naturally produced by B cells because they are synthetically derived, such as by recombinant means, which involves providing the B cells with a vector carrying the nucleic acid sequence encoding the cytokine.

[0039] The present disclosure relates to novel approaches for the large-scale expansion, CAR transduction, cytokine expression, and gene editing of B cells. In alternative embodiments, however, one or more of these events are not used in the generation of B cells. This approach enables the large-scale expansion of B cells derived from immortalized B cell lines (e.g., from EBV lymphoblastoid cell lines), B cell hybridomas, PBMCs, bone marrow, peripheral blood, umbilical cord blood, or stem cells (e.g., hematopoietic stem cells or induced pluripotent stem cells), and the expression of one or more cytokine genes as needed, and further enables them to be transduced to redirect their specificity, for example, towards tumor antigens. In some embodiments, the function of B cells is further improved by deleting genes involved in, for example, cell exhaustion and tumor-inducing dysfunction.

[0040] In certain embodiments, the present disclosure encompasses the combination of transduction of heterologous antigen receptors and deletion of single or multiple genes in human B cells, which contributes to the improvement of B cell functions including, but not limited to, improvement of antibody production, improvement of immunomodulatory functions, improvement of persistence, improvement of trafficking, or combinations thereof. In particular, a large-scale GMP-grade protocol enabling the generation of therapeutic B cells is disclosed. Embodiments of the present disclosure include the improvement of patient care using novel immunotherapy approaches that enhance the functions of the patient's own B cells or adoptively transferred B cells. II. Process

[0041] Embodiments of the present disclosure relate to a process for producing engineered B cells, particularly on a large scale. In specific embodiments, the process uses one or a combination of certain parameters to produce a particular type of engineered B cells, which parameters include a particular concentration of a reagent, a particular type of B cell (including effector or regulatory phenotypes), a particular duration for one or more steps, a particular type of cell modification mechanism, or a combination thereof. Optimal variables are described herein, but those skilled in the art will recognize that there are also variations of the process for producing a suitable amount of engineered B cells, and these are also included herein.

[0042] The above process generally relates to a sequence of steps. In specific embodiments, the sequence includes the expansion of B cells, the transduction or transfection of CD40L (also known as CD154), the manipulation of B cells in one, two or more aspects other than the transduction or transfection of CD40L, and the expansion of the engineered cells produced, followed by an optionally selected analysis step and / or an optionally selected administration step to an individual. In specific embodiments, the manipulation includes both (1) modifying the cells to express one or more heterologous proteins such as antigen receptors and / or cytokines, and (2) modifying the cells to reduce (knock down) or eliminate (knock out) the expression of one or more endogenous genes in the B cells. In some cases, (1) is performed before (2), while in other cases, (2) is performed before (1). Modifying the cells to reduce or eliminate expression can be done by any means, but in certain embodiments, the modification is performed by CRISPR.

[0043] The first step of the above process may include the expansion of B cells that allows for an increase in the number of B cells towards the final modification. B cells may be obtained, for example, from a fresh source or a repository, or commercially. B cells may be of any type, but in specific embodiments, B cells are derived from peripheral blood mononuclear cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized B cell lines, B cell hybridomas, bone marrow, umbilical cord blood mononuclear cells, or mixtures thereof. In certain embodiments, the starting culture of B cells for the expansion step contains at least 5 million to 100 million cells, and in some cases, 1×10 9 、1×10 10 or 1×10 11 cells or more are used. Thus, in specific cases, the number of B cells to initiate this protocol is 5 million, 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, 50 million, 55 million, 60 million, 65 million, 70 million, 75 million, 80 million, 85 million, 90 million, 95 million, 100 million or more B cells. At any step of the above process, a range of cells containing 5 million to 100 million, 5 million to 90 million, 5 million to 75 million, 5 million to 50 million, 5 million to 25 million, 5 million to 10 million, 10 million to 100 million, 10 million to 90 million, 10 million to 75 million, 10 million to 60 million, 10 million to 50 million, 10 million to 25 million, 25 million to 100 million, 25 million to 90 million, 25 million to 75 million, 25 million to 50 million, 50 million to 100 million, 50 million to 90 million, 50 million to 75 million, 75 million to 100 million, 75 million to 90 million or 90 million to 100 million cells and any derivable range in between may be used.

[0044] 1. Expansion of B cells and transduction of CD40 ligand

[0045] In a specific embodiment, the protocol for generating genetically modified B cells using a vector (including a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector) is as follows:

[0046] On day 0, B cells can be obtained from a suitable source. In a specific case, peripheral blood mononuclear cells are isolated from a single buffy coat (or cord blood mononuclear cells are isolated from cord blood). The B cells may be negatively selected using CliniMACs immunomagnetic beads, but in certain embodiments, those cells are positively selected. The unlabeled B cells are collected, washed with CliniMACs buffer, and placed in complete SCGM (90% stem cell growth medium, 10% fetal bovine serum if not for GMP use, or 10% human AB serum if for GMP use) containing CpG (3 μg / ml), anti-IgM+IgG (10 μg / ml) and IL-4 (4 ng / ml) (or IL-2 or IL-10 or IL-6 depending on how the conditions are optimized to bias the B cells towards an effector or regulatory phenotype) at 0.5×10 6 / ml for stimulation. The B cells are incubated at 5% CO2, 37 °C for some cases for about 44 - 48 hours (or 30 - 55 hours, 35 - 50 hours, 40 - 50 hours, etc.).

[0047] Next, on the second day or about the second day, transduce the B cells with CD40 ligand (e.g., to support their survival and proliferation). To do so, as an example, prepare a RetroNectin transduction plate by incubating a non-tissue culture plate containing 1% RetroNectin diluted in PBS at 1 ml per well in an incubator at 37° C. for 5 hours. Next, aspirate the RetroNectin plate and add room temperature SCGM to the wells. Incubate the plate containing the medium for 10 minutes. Next, replace the medium with the retroviral supernatant and centrifuge at 2000 g at 32° C. for 2 hours. Next, replace the retroviral supernatant with fresh retroviral supernatant. Add a B cell suspension containing 0.5×10 6 cells per well together with IL-4 (4 ng / ml) and IL-21 (30 ng / ml) (or other cytokines as described elsewhere herein, with or without IL-7) or combinations thereof. Centrifuge the plate at 2000 g at 32° C. for 30 minutes and then incubate at 37° C., 5% CO2; 24 hours after transduction, 1 ml of SCGM together with IL-4 (4 ng / ml) and IL-21 (30 ng / ml) should be added to each well. On the fifth day, remove the CD40L-transduced B cells from the plate, centrifuge, and culture at 0.5×10 6 / ml in SCGM containing IL-4 (4 ng / ml) and IL-21 (30 ng / ml) or combinations thereof. At this point, the transduction efficiency of CD40L may be evaluated using flow cytometry. Continue expansion using SCGM / 10% serum + IL4 (4 ng / ml) and IL21 (30 ng / mL) (or other cytokines depending on the purpose of expansion) until collection can be performed.

[0048] 2. Expansion and gene editing of B cells

[0049] In this project, B cells expressing CD40L are subjected to gene editing to disrupt the expression of one or more endogenous genes in the B cells. In certain embodiments, the B cells are modified to express CD40L prior to gene editing and / or transfection with another heterologous molecule. In a specific embodiment, the B cells are expanded as needed prior to the gene editing step. Any method herein may or may not use expansion at any point during the process. Any method herein may or may not use transfection or transformation of CD40L into B cells at any point during the process.

[0050] In certain embodiments, the procedures for stimulation of B cells and transduction of CD40 ligand as detailed in "Expansion of B cells and transduction of CD40 ligand" can be followed. On about 4 to 5 days after CD40L transduction, the B cells are resuspended in SCGM (90% stem cell growth medium, 10% fetal bovine serum if not for GMP use, or 10% human AB serum if for GMP use) at 500,000 to 1,000,000 / mL in the presence of IL-4 (4 ng / mL) and IL-21 (30 ng / mL), and electroporation is performed using an example of the protocol described below. When editing one or two genes, electroporation of CRISPR-cas9 is performed 4 to 5 days after CD40L transduction. When targeting more than two genes, in at least some cases, after the first electroporation, the cells are allowed to rest for about 2 to 3 days, and then a second CRISPR+ electroporation with the desired gRNA is performed. (See below for details of the application of CRISPR Cas9). Depending on the number of genes to be disrupted, third, fourth and additional electroporation steps may be performed. In some cases, more than one gene is disrupted in the same electroporation step.

[0051] 3. Expansion of B cells, transduction of heterologous antigen receptor + / - electroporation

[0052] Follow the procedures for the stimulation of B cells and the transduction of CD40 ligand as detailed in "Expansion of B cells and transduction of CD40 ligand".

[0053] Chimeric antigen receptors can be transduced into B cells to redirect their specificity. Additionally, they may be transduced with chemokine receptors or homing receptors, cytokine genes, especially immunoglobulins, etc.

[0054] Prepare another RetroNectin transduction plate by incubating a non-tissue culture plate containing 1% RetroNectin diluted in PBS at 37 °C for 5 hours at 1 ml per well, about 2 - 3 days after CD40 ligand transduction (about day 4 - 5 from the start of culture). Then aspirate the RetroNectin plate at room temperature and add SCGM / serum to the wells. Incubate the plate containing the medium for 10 minutes. Then replace the medium with the retroviral supernatant and centrifuge at 2000 g, 32 °C for 2 hours. Next, replace the retroviral supernatant with fresh retroviral supernatant and add a B cell suspension containing 0.5×10 6 cells to each well. Centrifuge the plate at 2000 g, 32 °C for 30 minutes and then incubate at 37 °C, 5% CO2. Two days after transduction, check the transduction efficiency and continue expansion using SCGM / 10% serum + IL4 (4 ng / ml) and IL21 (30 ng / ml) until collection.

[0055] For the CRISPR-Cas9 gene editing step: When targeting 1 - 2 genes, electroporate the cells 2 - 3 days after CAR transduction (i.e., about day 6 - 7 from the start of culture). When targeting more than 2 genes, after the first electroporation, allow the cells to rest for about 2 - 3 days and then perform a second CRISPR-Cas9+ electroporation with the desired gRNA. (See below for details on the application of CRISPR Cas9).

[0056] The following provides one specific example of a CRISPR protocol, which may be extrapolated or optimized for other CRISPR protocols. Cas9 can be used in any CRISPR method used herein, although in some embodiments CpF1 is used instead. Pre-complexation of crRNA and electroporation (Lonza 4D) (for 5 million to 30 million B cells) Step 1: Prepare the crRNA+tracrRNA duplex [Table 1] The starting concentrations of crRNA and tracrRNA are 200 uM. The final concentration after mixing them at equimolar concentrations is 100 uM. a. Mix by pipetting and centrifuge. b. Incubate in a thermocycler at 95°C for 5 minutes. c. Cool to room temperature on the bench. Step 2: Combine the crRNA:tracrRNA duplex with Cas9 nuclease [Table 2] [Table 3] a. Mix by pipetting and centrifuge. b. Incubate the mixture at room temperature for 15 minutes. Step 3: Combine crRNA#1 and crRNA#2 from Step 3 [Table 4] [Table 5] Step 4: Perform electroporation a. Prepare a culture plate containing a medium containing IL-4 and IL21 (preferably antibiotic-free). b. Immediately before use, prepare 5E+106 cells (washed twice with PBS to remove FBS) and resuspend in 100 ul of Nucleofector solution for primary cells P3. c. Mix the cell suspension with RNP (final concentration of Cas9 is 4.6 uM and gRNA concentration is 4 uM), transfer to a nucleocuvette, and fix the lid in place. d. The electroporation program is EO-115, then add the cells to a culture plate and recover in a 37°C incubator. e. Maximum 5E+106-X units (Cat: AAF-1002X) (20 ul from the final product is sufficient. See above for final concentration). f. Maximum 30×10 6 To electroporate up to 1 ml of LV Kit L Unit (Cat.#: V4LC-2002) is used. g. Maximum 100×10 6 To electroporate 100 or more, 1 ml of LV Kit L Unit (Cat.#: AAF-1002L) is used. h. Divide the total number of cells by 5×10 6 and then multiply by the final amount of the RNP complex from step 3 to calculate the total amount of RNP complex required. i. Example: If the number of cells is 30×10 6 then 30×10 6 / 5×10 6 = 6, and 6×20 ul = 120 ul is used. j. Example: If the number of cells is 100×10 6 then 100×10 6 / 5×10 6 = 20, and 20×20 ul = 400 ul. CRISPR CAS9: Small-scale protocol (starting cell population of 250,000 to 3 million) 1. Pre-complexation and electroporation of sgRNA-Cas9 (Neon-Thermo Fisher) a. For each gene, one or two sgRNAs spanning the neighboring region were designed and used. For each gene, a reaction was carried out with 1.5 μg of cas9 (PNA Bio) and 500 ng of sgRNA (sum of all sgRNAs), and incubated on ice for 20 minutes. b. After 20 minutes, 250,000 B cells were added and resuspended in T - buffer * (included in the Neon Electroporation Kit, Invitrogen, the total volume containing the RNP complex and cells should be 14 μl), and electroporated using a 10 μl electroporation tip with the Neon Transfection System. c. The conditions for electroporation of B cells are 1600 V, 10 ms, and 3 pulses. Then, the cells were added to a culture plate containing medium and cytokines (IL4 and IL21) and allowed to recover in a 37°C incubator. 2. Pre - complexation and electroporation of crRNA (Neon - Thermo Fisher) Step 1: Prepare the crRNA + tracrRNA double - strand [Table 6] The initial concentrations of crRNA and tracrRNA are 200 μM. The final concentration after mixing them at equimolar concentrations is 44 μM. d. Mix with a pipette and centrifuge. e. Incubate in a thermocycler at 95°C for 5 minutes. f. Cool to room temperature on the bench. Step 2: Prepare the cas9 nuclease [Table 7] Step 3: Combine the crRNA:tracrRNA double - strand and Cas9 nuclease [Table 8] [Table 9] g. Mix with a pipette and centrifuge. h. Incubate the mixture at room temperature for 15 minutes. Step 4: Combine crRNA#1 and crRNA#2 from Step 3

Table 10

Table 11

[0057] Regarding the expansion step in the above protocol, in certain embodiments, the medium in which the expansion step is performed contains one or more agents that promote expansion, such as one or more cytokines. In a specific embodiment, the concentration of the cytokine in the medium is in the range of 100 - 300 units / mL (including 100, 125, 150, 175, 200, 225, 250, 275, or 300 units / mL) or 1 - 500 nM or any range derivable therefrom. The cytokine can be IL4, IL2, IL10, IL-21, or IL6 or a combination thereof, depending on the desired phenotype (effector or regulatory phenotype) of the B cells. Any expansion step can be performed at a specific temperature, for example, at about 35°C - 38°C including 35°C, 36°C, 37°C, or 38°C. Any expansion step can be performed at a specific oxygen level (e.g., 3% - 7% CO2). In a specific embodiment, the expansion step is performed with 3%, 4%, 5%, 6%, or 7% CO2. Any expansion step can continue over a specific time period, for example, over a specific number of days. In a specific embodiment, the expansion step continues for 4, 5, 6, 7 days or more, but in a specific case, the expansion step continues for 4 - 7, 4 - 6, 4 - 5, 5 - 6, 5 - 7, or 6 - 7 days. In certain embodiments, the medium in the expansion step may or may not be replaced during expansion, but in a specific embodiment, the medium is replaced on the third day after the start of the expansion step. The medium can be replaced to change to the same medium composition as described above or a different medium composition. In a specific case, the cells are centrifuged and resuspended in the same or a similar medium as described above (including a medium containing 100, 125, 150, 175, 200, 225, 250, 275, or 300 units / mL of cytokine). In a specific aspect, the expansion step is performed in a bioreactor, for example, a gas-permeable bioreactor, for example, within a G-Rex® 100M or G-Rex100®. In a certain aspect, the bioreactor is a gas-permeable bioreactor. In a specific aspect, the gas-permeable bioreactor is a G-Rex® 100M or G-Rex100®.In some embodiments, the stimulation in step (b) is performed in a specific volume of medium, e.g., 3 to 5 L of medium (e.g., 3, 3.5, 4, 4.5, or 5 L).

[0058] On about day 2 after the start of B cell expansion, the expanded B cells can be subjected to modification by transduction of CD40L. On about days 2 to 3 after CD40L transduction (days 4 to 5 after the start of culture), one or more heterologous genes (e.g., one or more heterologous antigen receptors, one or more chemokine receptors, one or more homing receptors, one or more cytokine genes and / or one or more immunoglobulins) can be transduced or transfected into the cells.

[0059] The modification after transduction or transfection of CD40L can be transducing or transfecting the B cells to express one or more heterologous antigen receptors, but in some cases, the modification after transduction or transfection of CD40L is disruption of the expression of one or more endogenous genes of the B cells. When the modification is transducing or transfecting the B cells to express a heterologous antigen receptor, the subsequent modification can be disruption of the expression of one or more endogenous genes of that B cell. When the modification is disrupting the expression of one or more endogenous genes of the B cells, the subsequent modification is transducing or transfecting that B cell to express a heterologous antigen receptor.

[0060] In a specific embodiment, the expanded B cells are transduced or transfected to have a heterologous antigen receptor gene before being gene edited to disrupt the expression of one or more endogenous genes. In certain embodiments, a specific vector containing an expression construct encoding one or more chimeric antigen receptors, one or more T cell receptors or combinations thereof is transduced or transfected into the cells. The vector can be of any kind including at least nanoparticles, plasmids, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors, etc. Vectors that enable those B cells to express multiple heterologous proteins (e.g., heterologous antigen receptors, suicide genes, and one or more cytokines selected from the group consisting of IL-4, IL-10, IL-7, IL-2, IL-15, IL-12, IL-18, IL-21 and combinations thereof) can be transfected or transduced into the B cells. The genes of multiple heterologous proteins may be present on the same vector, but in some cases, they are present on multiple vectors. Once those cells are transduced or transfected, they may be tested for the expression of heterologous proteins, which can be done 1, 2, 3 days or later after transfection / transduction. When testing an aliquot from a population of modified (transduced or transfected) B cells, the test may be performed before further modification, such as before gene editing of the B cells, or not.

[0061] In some embodiments, within about 6, 7, 8, 9, 10, 11, or 12 days after the start of expansion of B cells (including modified B cells), they can be subjected to a gene editing method. The gene editing step may or may not be performed within 1, 2, or 3 days or later after the transfection / transduction step. Gene editing of the modified B cells may be performed by any suitable method, but in certain embodiments, gene editing of B cells (including modified B cells) is performed by the CRISPR method. Thus, in certain embodiments, the modified cells are exposed to a suitable amount of Cas9 and guide RNA. If the expression of more than one gene in the B cells is disrupted, there may be a population of guide RNAs that includes one or more sequence-specific guide RNAs for each gene to be edited. In certain embodiments, the modified B cells are subjected to two or more different electroporation steps that are temporally separated by 1, 2, 3 days or more. In some cases, the first electroporation step includes targeting one or more genes, and the second electroporation step includes targeting one or more genes that are different genes from those of the first electroporation step. In some cases, there are consecutive electroporation steps that include 3, 4, 5 or more additional electroporation steps beyond two electroporation steps. In any case where multiple electroporation steps are used, the next electroporation may or may not be performed only after a certain period of time has elapsed, such as after 1, 2, 3, 4 days or more have passed since the previous electroporation step.

[0062] In certain embodiments, the B cells first undergo a gene editing step and then a transduction or transfection step for having a heterologous antigen receptor gene.

[0063] After the gene editing and transformation / transduction of the above B cells, those B cells may or may not be subjected to a second expansion step to increase the number of genetically edited modified B cells. In a specific embodiment, the second or subsequent expansion step in the process may be substantially the same as the first expansion step in the process, but in an alternative embodiment, the second or subsequent expansion step is different from the first expansion step, such as having a different medium, different compounds added externally, different culture / expansion times, different expansion flasks (e.g., GREX or WAVE as some examples), or a combination thereof. In a specific case, the second or subsequent expansion step includes culturing the genetically edited modified B cells with one or more specific cytokines.

[0064] At any step during the above process, the above cells can be used, analyzed, stored, etc. In a specific embodiment, those cells are analyzed for functions, cytotoxicity, in vivo activity, etc. For example, those cells can be analyzed for (1) the ability of a heterologous antigen receptor to bind to a target antigen; (2) the expression of the edited gene or the loss of its expression to confirm knockdown or knockout of expression; or (3) both. In a specific case, those cells are subjected to, for example, mass spectrometry and / or RNA sequencing.

[0065] In a specific embodiment, the produced B cells are stored, such as by cryopreservation. For example, the B cells can be cryopreserved for use by the individual from whom the starting B cells were obtained, or the B cells can be cryopreserved for use by an individual different from the individual from whom the starting B cells were obtained. III. Heterologous Antigen Receptor

[0066] The B cells of the present disclosure can be genetically engineered to express one or more heterologous antigen receptors (e.g., engineered TCRs, CARs, chimeric cytokine receptors, chemokine receptors, combinations thereof, etc.). Those heterologous antigen receptors are synthetically made by human hands. In certain embodiments, those B cells are modified to express one or more CARs and / or TCRs having antigen specificity for cancer antigens. Multiple CARs and / or TCRs (e.g., for different antigens) can be added to those B cells. In some aspects, those immune cells are engineered to express a CAR or TCR by knocking in the CAR or TCR at a specific locus, such as by using CRISPR.

[0067] The above B cells are edited particularly using CRISPR, although alternative suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, those cells can be transduced to express a TCR having antigen specificity for cancer antigens using the transduction methods described in Heemskerk et al., 2008 and Johnson et al., 2009. In some embodiments, those cells include one or more nucleic acids introduced via genetic manipulation that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, those nucleic acids are heterologous, i.e., they do not normally exist in a certain cell or a sample obtained from that cell, e.g., nucleic acids obtained from another organism or cell, e.g., nucleic acids not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, those nucleic acids do not occur naturally, e.g., nucleic acids not found in nature (e.g., chimeric).

[0068] In some embodiments, the B cells are modified to express a chimeric antigen receptor (CAR) comprising one or more extracellular antigen recognition domains that specifically bind to an antigen. In some embodiments, the antigen is a protein expressed on the cell surface (including on the surface of cancer cells). In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen (e.g., a peptide antigen of an intracellular protein) that is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, similar to a TCR.

[0069] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering those receptors and methods for introducing them into cells include, for example, those described in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patents Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, genetically engineered antigen receptors include CARs such as those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668Al. A. Chimeric Antigen Receptor

[0070] In some embodiments, the CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising one or more antigen-binding regions. When the extracellular domain comprises two or more antigen-binding regions, the two or more antigens are different antigens, which in some cases comprise different antigens expressed on the same cell surface or on the same type of cell.

[0071] In some embodiments, engineered antigen receptors include CARs such as activating or stimulatory CARs, co-stimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally comprise an extracellular antigen (or ligand) binding domain linked, in some embodiments via a linker and / or transmembrane domain, to one or more intracellular signaling components. Such molecules typically mimic or emulate signals via natural antigen receptors, signals via such receptors together with co-stimulatory receptors, and / or signals via co-stimulatory receptors alone.

[0072] Certain embodiments of the present disclosure relate to the use of a nucleic acid comprising a nucleic acid encoding an antigen-specific CAR polypeptide (including a humanized CAR (hCAR) for reducing immunogenicity) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope comprising a space shared between one or more antigens. In certain embodiments, the binding region can comprise complementarity-determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen-binding fragments thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0073] The human CAR nucleic acid is contemplated to be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the invention includes the full-length cDNA or coding region of the CAR. Its antigen-binding region or domain is the V H chain and the V L chain fragments (e.g., those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference). The fragment can also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0074] The configuration can be multimeric (e.g., diabody or multimer). The multimer is most likely formed by cross-pairing of the variable portions of the light and heavy chains in the diabody. There can be multiple options for the hinge portion of the construct, ranging from complete deletion, maintaining the first cysteine, being a proline substitution instead of a serine substitution, to being cleaved up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains, for example, the CH2 domain or CH3 domain of human immunoglobulin, can be used. The hinge, CH2, and CH3 regions of human immunoglobulin modified to improve dimerization can also be used. Only the hinge portion of the immunoglobulin can be used. A portion of CD8 alpha can also be used.

[0075] In some embodiments, the CAR nucleic acid includes sequences encoding other co-stimulatory receptor domains, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Specific co-stimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to the primary signal induced by CD3ζ, additional signals provided by human co-stimulatory receptors inserted into the human CAR are important for the complete activation of B cells and can help with in vivo persistence and improved therapeutic success of adoptive immunotherapy.

[0076] In some embodiments, the CAR is engineered to have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and / or an antigen intended to induce an attenuated response (e.g., an antigen expressed on a normal cell type or a non-affected cell type). Thus, the CAR typically includes, in its extracellular portion, one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, antigen-binding domains, or antigen-binding portions) or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR includes the antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0077] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain including the antigen-binding region) includes a domain that binds to a tumor-associated antigen or, in some cases, a pathogen-specific antigen. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as dectin-1. The tumor-associated antigen can be of any type as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like. In certain embodiments, the CAR can be co-expressed with a cytokine to improve persistence in cases where the amount of the tumor-associated antigen is low. In such cases, the cytokine is not part of the CAR protein. For example, the CAR can be co-expressed with one or more cytokines (e.g., IL-4, IL-10, IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or combinations thereof).

[0078] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA origin, cDNA origin, or can be synthesized (e.g., via PCR), or can be a combination thereof. Since introns have been found to stabilize mRNA, depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof. It may also be further beneficial to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0079] It is contemplated that the chimeric construct can be introduced into immune cells either as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor that is included in a plasmid expression vector in an orientation appropriate for expression.

[0080] In some embodiments, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric construct into immune cells. Vectors suitable for use according to the methods of the present disclosure are non-replicative vectors in immune cells. A number of virus-based vectors are known (e.g., vectors based on HIV, SV40, EBV, HSV, or BPV), and the copy number of the virus maintained intracellularly is low enough to maintain the viability of the cell.

[0081] In some embodiments, the component that specifically binds to the antigen or the antigen-specific recognition component is linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0082] The transmembrane domain is, in some embodiments, of natural or synthetic origin. When the origin is natural, the domain is, in some aspects, derived from any membrane-bound or transmembrane protein. The transmembrane region includes transmembrane regions derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (i.e., at least including the transmembrane regions of those molecules). Alternatively, the transmembrane domain is, in some embodiments, a synthetic transmembrane domain. In some aspects, the synthetic transmembrane domain mainly includes hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0083] In certain embodiments, the platform technologies disclosed herein for genetically modifying immune cells such as B cells include: (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) a CAR that signals through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) a CAR having an extracellular domain of variable length that connects an antigen recognition domain to the cell surface, and in some cases, (iv) a CAR + Examples include artificial antigen-presenting cells (aAPCs) derived from K562 (Singh et al., 2008; Singh et al., 2011) that can robustly and numerically expand immune cells. B. T cell receptor (TCR)

[0084] In some embodiments, the genetically engineered antigen receptor includes a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. A "T cell receptor" or "TCR" is a molecule that includes a variable alpha chain and a variable beta chain (also known as TCRα and TCRβ, respectively) or a variable gamma chain and a variable delta chain (also known as TCRγ and TCRδ, respectively), and that can specifically bind to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0085] Generally, TCRs that exist as αβ and γδ types are generally similar in structure, but the T cells that express them can differ in anatomical location or function. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are usually involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules on their surface. In some embodiments, the TCR can also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of the TCR can have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass its functional TCR fragments. This term also encompasses intact or full-length TCRs, including αβ or γδ type TCRs.

[0086] Thus, for the purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., the specific antigen peptide bound in an MHC molecule, i.e., the antigen-binding portion of the TCR that binds to an MHC-peptide complex). The "antigen-binding portion" or antigen-binding fragment of a TCR, which can be used interchangeably, refers to a molecule that contains only a part of the structural domain of the TCR but binds to the antigen (e.g., MHC-peptide complex) to which the complete TCR binds. In some cases, the antigen-binding portion includes the variable domains of the TCR (e.g., the variable alpha and variable beta chains of the TCR) sufficient to form a binding site for binding to a specific MHC-peptide complex, and generally, each chain contains three complementarity-determining regions, for example.

[0087] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity-determining regions (CDRs) similar to immunoglobulins, thereby resulting in antigen recognition, determining peptide specificity by forming the binding site of the TCR molecule, and determining peptide specificity. Usually, similar to immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the major CDR involved in the recognition of processed antigens, whereas the CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigen peptide, while the CDR1 of the beta chain interacts with the C-terminal part of that peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the beta chain may include an additional hypervariable (HV4) region.

[0088] In some embodiments, the TCR chains include constant domains. For example, similar to immunoglobulins, the extracellular part of the TCR chains (e.g., the alpha chain, beta chain) is two immunoglobulin domains, the variable domain at the N-terminus (e.g., V aor Vp; typically, amino acids 1 - 116 based on Kabat numbering of Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed. (amino acids 1 - 116), and one constant domain adjacent to the cell membrane (e.g., the a - chain constant domain or C a , typically, amino acids 117 - 259 based on Kabat, the β - chain constant domain or Cp, typically, amino acids 117 - 295). For example, in some cases, the extracellular portion of the TCR formed by those two chains includes two membrane - proximal constant domains and two membrane - distal variable domains containing CDRs. The constant domains of the TCR domains include short connecting sequences where cysteine residues form disulfide bonds, thereby forming a linkage between those two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α - chain and β - chain so that the TCR contains two disulfide bonds in the constant domain.

[0089] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, due to its structure, the TCR can associate with other molecules such as CD3. For example, a TCR containing a constant domain together with a transmembrane region can anchor the protein to the cell membrane and associate with an invariant subunit of the CD3 signaling apparatus or complex.

[0090] In general, CD3 is a multi-protein complex that can have three different chains (γ, δ, and ε) and the ζ chain in mammals. For example, in mammals, this complex can include the CD3γ chain, the CD3δ chain, two CD3ε chains, and the homodimeric CD3ζ chain. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, which is a property that allows these chains to associate with the positively charged T cell receptor chains. Each intracellular tail of the CD3γ chain, CD3δ chain, and CD3ε chain contains a single conserved motif known as an immunoreceptor activation tyrosine motif or ITAM, whereas each CD3ζ chain contains three. In general, ITAM is involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in the propagation of signals from the TCR to the cell. The CD3 chains and the ζ chain together with the TCR form a complex known as the T cell receptor complex.

[0091] In some embodiments, the TCR can be a heterodimer of two chains α and β (or γ and δ if desired) or a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α chain and β chain or γ chain and δ chain) linked by, for example, disulfide bonds. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, the nucleic acid encoding the TCR can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological origin, such as a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from a patient and the TCR can be isolated. In some embodiments, the T cells can be cultured T cell hybridomas or clones. In some embodiments, the TCR clone against the target antigen is a clone produced in a transgenic mouse engineered with human immune system genes (e.g., the human leukocyte antigen system, i.e., HLA). See, for example, tumor antigens (e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate the TCR against the target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or its antigen-binding portion can be synthetically made based on knowledge of the TCR sequence. C. Antigen-presenting cells

[0092] Antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, are identified by the expression of specific MHC molecules. APCs internalize antigens and re-express a portion of those antigens on the outer membrane of their cell membranes, together with MHC molecules. MHC is a large gene complex containing multiple loci. The MHC loci encode two major classes of MHC membrane molecules, called class I and class II MHC. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, and T cell cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. MHC is called the HLA complex in humans and the H-2 complex in mice.

[0093] In some cases, aAPCs are useful in preparing the therapeutic compositions and cell therapy products of the above embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0094] The aAPC system may include at least one exogenous co-stimulatory molecule. Any suitable number and combination of co-stimulatory molecules can be used. The co-stimulatory molecules can be selected from co-stimulatory molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules include, for example, carbohydrate-binding glycoproteins such as selectins that promote cell-cell contact or cell-matrix contact, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecule (ICAM). Exemplary adhesion molecules include LFA-3 and ICAM such as ICAM-1. Methods, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary co-stimulatory molecules and adhesion molecules, including co-stimulatory molecules and adhesion molecules, are illustrated, for example, in US Pat. Nos. 6,225,042, 6,355,479, and 6,362,001. D. Antigen

[0095] Antigens targeted by genetically engineered antigen receptors include antigens expressed in the context of a disease, condition, or cell type targeted via adoptive cell therapy. Those diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including blood cancers, cancers of the immune system (e.g., lymphomas, leukemias, and / or myelomas, e.g., B, T, and myeloid leukemias, lymphomas, and multiple myelomas). In some embodiments, the antigen is selectively expressed or overexpressed on the cells of that disease or condition, e.g., on tumor cells or pathogenic cells, compared to normal cells or normal tissues or non-targeted cells or non-targeted tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0096] Any suitable antigen can be targeted in the present method. The antigen may be associated with certain cancer cells, but in some cases, it may not be associated with non-cancerous cells. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, auto / self-antigens, tumor-associated antigens / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain embodiments, antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In certain embodiments, antigens for the above two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. The sequences of these antigens are known in the art, for example, the following in the GenBank® database: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_029383.1), HER2 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0097] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, gastric cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY-ESO1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types such as melanoma, lung cancer, sarcoma and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate acid phosphate, NKX3.1 and six-transmembrane epithelial antigen of the prostate (STEAP).

[0098] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Further, the tumor antigen can be a self-peptide hormone useful in the treatment of many cancers, for example, the full-length gonadotropin-releasing hormone (GnRH), which is a short 10-amino acid long peptide.

[0099] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens such as the expression of HER-2 / neu. Tumor-associated antigens of interest include lineage-specific tumor antigens such as the melanocyte-melanoma lineage antigens MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related protein.

[0100] Exemplary cancer antigens include CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, beta-catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (particularly, EGFRvIII), platelet-derived growth factor receptor (PDGFR),Vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-κB), Notch receptors (e.g., Notch1-4), NY-ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1 and LRRN1 are mentioned.,

[0101] Antigens can include epitope regions or epitope peptides derived from genes mutated in tumor cells or genes transcribed at different levels in tumor cells compared to normal cells (e.g., epitope regions or epitope peptides of abnormally expressed intron sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V); clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitope regions or epitope peptides derived from the processes of oncoviruses (e.g., human papillomavirus proteins E6 and E7); Epstein-Barr virus protein LMP2; non-mutated tumor fetal proteins selectively expressed in tumors (e.g., carcinoembryonic antigen and alpha-fetoprotein).

[0102] In other embodiments, the antigen is obtained from or derived from pathogenic microorganisms or opportunistic pathogenic microorganisms (also referred to herein as infectious microorganisms) (e.g., viruses, fungi, parasites, and bacteria). In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0103] Exemplary pathogenic organisms having antigens contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), and streptococcal species including Streptococcus pneumoniae. As will be understood by those skilled in the art, proteins derived from these and other pathogenic microorganisms for use as antigens as described herein, as well as nucleotide sequences encoding those proteins, can be identified in publications and public databases such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0104] Antigens derived from human immunodeficiency virus (HIV) include any of the HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0105] Antigens derived from herpes simplex virus (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. Late group genes mainly encode proteins that form virion particles. Such proteins include five proteins that form the viral capsid: (UL) UL6, UL18, UL35, UL38, as well as the major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each of which may encode a protein that can be used as an antigen.

[0106] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed early and immediate early to viral replication, glycoproteins I and III, capsid proteins, coat proteins, the lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products of the gene cluster of UL128 - UL150 (Rykman, et al., 2006), envelope glycoproteins B (gB), gH, gN, as well as pp150. As will be understood by those skilled in the art, CMV proteins for use as antigens described herein can be identified in public databases such as GENBANK®, SWISS - PROT®, and TREMBL® (see, for example, Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0107] Antigens derived from Epstein - Barr virus (EBV) contemplated for use in certain embodiments include EBV lytic proteins gp350 and gp110, Epstein - Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA - leader protein (EBNA-LP), and latent membrane proteins (LMP)-1, LMP-2A, and LMP-2B, which are EBV proteins produced during the latent infection cycle (see, for example, Lockey et al., 2008).

[0108] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any one of the 11 proteins encoded by the RSV genome or their antigenic fragments: NS1, NS2, N (nucleocapsid protein), M (matrix protein), SH, G, and F (viral coat protein), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription control), RNA polymerase, and phosphoprotein P.

[0109] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins encoded by the VSV genome and their antigenic fragments: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, for example, Rieder et al., 1999).

[0110] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1 - F2, and PB2.

[0111] Exemplary viral antigens include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (e.g., hepatitis B core or surface antigens, hepatitis C virus E1 or E2 glycoproteins, core or non-structural proteins), herpesvirus polypeptides (including glycoproteins of herpes simplex virus or varicella-zoster virus), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin polypeptides and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptides), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides, but are not limited thereto.

[0112] In certain embodiments, the antigen can be a bacterial antigen. In certain embodiments, the bacterial antigen of interest can be a secreted polypeptide. In certain other embodiments, bacterial antigens include antigens having a portion of a polypeptide exposed on the outer surface of the bacterial cell.

[0113] Antigens derived from species of the genus Staphylococcus, including methicillin-resistant Staphylococcus aureus (MRSA), for which use is contemplated, include virulence regulators such as the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP. Other Staphylococcus proteins that may serve as antigens include Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two strains of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by those skilled in the art, Staphylococcus proteins for use as antigens may also be identified in other public databases such as GenBank®, Swiss-Prot® and TrEMBL®.

[0114] Antigens derived from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, e.g., Zysk et al., 2000). The complete genomic sequences of virulent strains of Streptococcus pneumoniae have been sequenced, and as will be understood by those skilled in the art, S. pneumoniae proteins for use herein may also be identified in other public databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Particularly interesting proteins as antigens according to the present disclosure include pathogenic factors and proteins predicted to be exposed on the surface of pneumococci (see, e.g., Frolet et al., 2010).

[0115] Examples of bacterial antigens that can be used as antigens include polypeptides of the genus Actinomyces, polypeptides of the genus Bacillus, polypeptides of the genus Bacteroides, polypeptides of the genus Bordetella, polypeptides of the genus Bartonella, polypeptides of the genus Borrelia (e.g., B. burgdorferi OspA), polypeptides of the genus Brucella, polypeptides of the genus Campylobacter, polypeptides of the genus Capnocytophaga, polypeptides of the genus Chlamydia, polypeptides of the genus Corynebacterium, polypeptides of the genus Coxiella, polypeptides of the genus Dermatophilus, polypeptides of the genus Enterococcus, polypeptides of the genus Ehrlichia, polypeptides of the genus Escherichia, polypeptides of the genus Francisella, polypeptides of the genus Fusobacterium, polypeptides of the genus Hemobartonella, polypeptides of the genus Haemophilus (e.g., H. influenzae type b outer membrane protein), polypeptides of the genus Helicobacter, polypeptides of the genus Klebsiella, polypeptides of L-form bacteria, polypeptides of the genus Leptospira, polypeptides of the genus Listeria, polypeptides of the genus Mycobacterium, polypeptides of the genus Mycoplasma, polypeptides of the genus Neisseria, polypeptides of the genus Neorickettsia, polypeptides of the genus Nocardia, polypeptides of the genus Pasteurella, polypeptides of the genus Peptococcus, polypeptides of the genus Peptostreptococcus, polypeptides of pneumococcus (i.e., polypeptides of S. pneumoniae), polypeptides of the genus Proteus, polypeptides of the genus Pseudomonas, polypeptides of the genus Rickettsia, polypeptides of the genus Rochalimaea, polypeptides of the genus Salmonella, polypeptides of the genus Shigella, polypeptides of the genus Staphylococcus, polypeptides of group A streptococcus (e.g., M protein of S. pyogenes), polypeptides of group B streptococcus (S. agalactiae), polypeptides of the genus Treponema, and polypeptides of the genus Yersinia (e.g., F1 and V antigens of Y. pestis), but are not limited thereto.

[0116] Examples of fungal antigens include, but are not limited to, polypeptides of Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Coccidioides, Conidiobolus, Cryptococcus, Curvularia, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Microsporum, Moniliera, Mortierella, Pichia, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Phytophthora, Rhinosporidium, Scopulariopsis, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha.

[0117] Examples of protozoan parasite antigens include, but are not limited to, polypeptides of Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, and Plasmodium. Examples of helminth parasite antigens include, but are not limited to, polypeptides of Acantocheilonema, Aelurostrongylus, Ancylostoma, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyma, Dipetalonema, Diphyllobothrium, Dipylidium, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Nodularia, Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris, Phyllodistomum,Polypeptides of Protostrongylus, polypeptides of Setaria, polypeptides of Spirocerca, polypeptides of Spirometra, polypeptides of Stephanofilaria, polypeptides of Strongyloides, polypeptides of Strongylus, polypeptides of Terrazia, polypeptides of Toxascaris, polypeptides of Toxocara, polypeptides of Trichinella, polypeptides of Trichomuris, polypeptides of Trichuris, polypeptides of Uncinaria, and polypeptides of Wuchereria (e.g., the circumsporozoite polypeptide of P. falciparum (PfCSP)), sporozoite surface protein 2 (PfSSP2), the carboxyl terminus of liver state antigen 1 (PfLSA1 c-terminus), and exported protein 1 (PfExp-1), polypeptides of Pneumocystis, polypeptides of Sarcocystis, polypeptides of Schistosoma, polypeptides of Theileria, polypeptides of Toxoplasma, and polypeptides of Trypanosoma, but not limited to these.

[0118] Examples of ectoparasite antigens include fleas; ticks including Ixodes and Dermacentor; flies such as midges, mosquitoes, black flies, horse flies, stable flies, horn flies, biting midges, tsetse flies, house flies, flies causing myiasis, and small biting gnats; ants; spiders, lice; mites; and hemipteran insects such as bedbugs and assassin bugs, polypeptides (including antigens and allergens). However, it is not limited to these. E. Suicide gene

[0119] In some cases, any cell of the present disclosure is modified to produce one or more agents other than, for example, heterologous cytokines, engineered receptors, etc. In a specific embodiment, a cell such as a B cell is engineered to have one or more suicide genes, and the term "suicide gene" as used herein is defined as a gene in which, when a prodrug is administered, the gene product is changed into a compound that kills the host cell. In some cases, B cell therapy may be subject to the use of any type of one or more suicide genes when an individual undergoing B cell therapy and / or an individual who has undergone B cell therapy exhibits one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, anaphylaxis / allergic reactions, and / or on-target / off-tumor toxicity (by way of example)), or when there is a risk of having one or more symptoms (including when imminent). The use of suicide genes can be part of a protocol planned for treatment or can be used only when the need to use them is recognized. In some cases, cell therapy is terminated by using an agent that targets the suicide gene or its gene product because it is no longer needed.

[0120] Examples of suicide genes include engineered non-secretory (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009, which is hereby incorporated by reference in its entirety), and these polypeptides can be targeted by the delivery of antibodies that bind to the TNF-alpha mutants. Examples of suicide gene / prodrug combinations that can be used include herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytarabine. The so-called suicide gene, purine nucleoside phosphorylase from E. coli, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET) and thymidine phosphorylase (TP) as examples. F. Delivery Methods

[0121] One of ordinary skill in the art would be fully capable of constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) to express the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., those derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including their replicable, replication-deficient, and gutless forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors, but are not limited thereto.

[0122] In a specific embodiment, the vector is a multicistronic vector as described in PCT / US19 / 62014, which is incorporated herein by reference in its entirety. In such a case, a single vector can encode a CAR or TCR (the expression construct of which can be configured in a modular format to allow for interchange of parts of the CAR or TCR), a suicide gene, and one or more cytokines. 1. Viral vectors

[0123] Viral vectors encoding antigen receptors can be provided in certain embodiments of the present disclosure. When producing recombinant viral vectors, genes that are not essential are usually replaced with genes or coding sequences of heterologous (or non-native) proteins. A viral vector is a type of expression construct that utilizes viral sequences to introduce nucleic acids and optionally proteins into cells. Due to the ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into the host cell's genome and stably and efficiently express viral genes, these viruses have become attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain embodiments of the present invention are described below.

[0124] Lentiviruses are complex retroviruses and contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0125] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, recombinant lentiviruses capable of infecting non-dividing cells (where suitable host cells are transfected with two or more vectors having packaging functions, namely gag, pol, and env, and rev and tat) are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. a. Regulatory element

[0126] Expression cassettes included in vectors useful in the present disclosure include, in particular, a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence (in the 5' to 3' direction). Promoters and enhancers that control the transcription of genes encoding proteins are composed of multiple genetic elements. Cellular machinery can collect and integrate the regulatory information carried by each element, thereby enabling different genes to exhibit different patterns of transcriptional control, often in complex patterns. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters. b. Promoter / Enhancer

[0127] The expression constructs provided herein include a promoter that drives the expression of an antigen receptor. A promoter generally includes a sequence that functions to specify the position of the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, the TATA box is absent and discontinuous elements that overlap the start site itself help to fix the location of initiation. Additional promoter elements control the frequency of transcription initiation. Usually, these are located in the region 30 - 110 bp upstream of the start site, but some promoters have been shown to also contain functional elements downstream of the start site. To place a coding sequence "under the control of" a promoter, the 5' end of the transcription start site of the transcription reading frame is placed "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0128] The spacing between promoter elements is often flexible, and the function of the promoter is conserved even when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be widened by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may be thought to function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0129] A promoter can be a promoter that is naturally associated with a nucleic acid sequence, such as one that may be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter can be referred to as an "endogenous" promoter. Similarly, an enhancer can be an enhancer that is naturally associated with a nucleic acid sequence and is located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not naturally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not naturally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers from other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that do not "naturally occur," i.e., contain various elements of different transcriptional control regions and / or mutations that alter expression. For example, among the most commonly used promoters in the construction of recombinant DNA are the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, recombinant cloning and / or PCR TMUsing nucleic acid amplification techniques, including but not limited to, a sequence can be generated in relation to the compositions disclosed herein. Additionally, regulatory sequences that direct transcription and / or expression of sequences within organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.) are also contemplated to be used similarly.

[0130] Of course, it is important to use promoters and / or enhancers that effectively direct the expression of DNA segments in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of using combinations of promoters, enhancers, and cell types for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter useful under appropriate conditions that directs high-level expression of the introduced DNA segment (e.g., a promoter beneficial in the large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0131] Furthermore, any combination of promoters / enhancers (e.g., according to the Eukaryotic Promoter Data Base EPDB via the world wide web at epd.isb-sib.ch / ) can also be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters when an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0132] Non-limiting examples of promoters include early viral promoters or late viral promoters (e.g., SV40 early or late promoter, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic cell promoters (e.g., beta-actin promoter, GADPH promoter, metallothionein promoter); and tandem response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter near minimal TATA box (tre)). It is also possible to use the human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC45007). In certain embodiments, the promoter is a CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however, any other promoter useful for driving the expression of a therapeutic gene can also be applied to the practice of the present disclosure.

[0133] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase the activity of a promoter, which act in cis and regardless of their orientation, and have the ability to act over relatively long distances (up to several kilobases away from the target promoter). However, since an enhancer can also function proximally to a given promoter, the function of an enhancer is not necessarily limited to such long distances. c. Initiation signal and linked expression

[0134] For efficient translation of the coding sequence, specific initiation signals can also be used in the expression constructs provided by the present disclosure. These signals include the ATG start codon or adjacent sequences. In some cases, exogenous translation control signals (including the ATG start codon) may need to be provided. One of ordinary skill in the art will be able to readily determine this and provide the necessary signals. It is well known that for ensuring translation of the entire insert, the start codon must be "in-frame" with the reading frame of the desired coding sequence. The exogenous translation control signals and start codons can be either natural or synthetic. By including appropriate transcriptional enhancer elements, the expression efficiency can be enhanced.

[0135] In certain embodiments, the use of an internal ribosome entry site (IRES) element is employed to generate a multi-gene message, i.e., a polycistronic message. The IRES element can bypass the ribosome scanning model of 5'-methylated cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the Picornaviridae family (poliovirus and encephalomyocarditis), as well as IRESs from mammalian messages, have been reported. The IRES element can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to generate a polycistronic message. Thanks to the IRES element, each open reading frame can be accessible to ribosomes for efficient translation. It is also possible to efficiently express multiple genes using a single promoter / enhancer to transcribe a single message.

[0136] Furthermore, certain 2A sequence elements can be used to effect linked or co-expression of genes within the constructs provided by the present disclosure. For example, cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A). d. Replication origin

[0137] To propagate the vector in a host cell, the vector can include one or more replication initiation sites (often referred to as “ori”), e.g., a specific nucleic acid sequence at which replication is initiated, such as the oriP of EBV as described above or a nucleic acid sequence corresponding to a genetically engineered oriP that may have a similar or enhanced function in programming. Alternatively, the origin of replication of other viruses that replicate episomally as described above, or an autonomous replication sequence (ARS), can be used. e. Selection markers and screenable markers

[0138] In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers confer an identifiable change to the cell that allows cells containing the expression vector to be readily identified. Generally, a selection marker is a marker that confers a property that allows for selection. A positive selection marker is a marker that allows for selection by virtue of the presence of the marker, and a negative selection marker is a marker whose presence prevents selection. Examples of positive selection markers are drug resistance markers.

[0139] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype allowing discrimination of transformants based on the execution of a condition, other types of markers are contemplated, including screenable markers such as GFP, based on colorimetric analysis. Alternatively, enzymes that can be screened as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT), can be utilized. One of ordinary skill in the art will likely also be aware of methods of using immunological markers, perhaps in conjunction with FACS analysis. The marker used is thought not to be important as long as it can be co-expressed with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those of ordinary skill in the art. 2. Other nucleic acid delivery methods

[0140] In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods are considered in the present disclosure as additional methods of recombinant gene delivery to a given host cell.

[0141] The introduction of nucleic acids such as DNA or RNA into immune cells of the present disclosure may use any method suitable for nucleic acid delivery for transforming cells as described herein or known to those skilled in the art. Such methods include direct delivery of DNA (e.g., ex vivo transfection, injection (including microinjection)); electroporation; calcium phosphate precipitation; use of polyethylene glycol following DEAE-dextran; direct sonoporation; liposome-mediated transfection and receptor-mediated transfection; microprojectile bombardment; agitation with silicon carbide fibers; Agrobacterium-mediated transformation; drying / inhibition-mediated DNA uptake, and any combination of such methods, but are not limited thereto. By applying techniques such as these, organelles, cells, tissues or organisms can be stably or transiently transformed. IV. Gene Editing and CRISPR

[0142] The B cell production process of the present disclosure may include gene editing of B cells. In some cases, the gene editing is performed in B cells expressing one or more heterologous antigen receptors, while in other cases, the gene editing is performed in B cells not expressing heterologous antigen receptors. In certain embodiments, the gene-edited B cells are expanded B cells.

[0143] In certain cases, one or more endogenous genes of the above B cells are modified, such as being disrupted in expression, where the expression is partially or completely reduced. In specific cases, one or more genes are knocked down or knocked out using the processes of the present disclosure. In specific cases, multiple genes are knocked down or knocked out in the same step or in multiple steps. The genes to be edited in B cells can be of any type, but in specific embodiments, those genes are genes whose gene products inhibit the activity and / or proliferation of B cells. In specific cases, the genes edited in B cells enable the B cells to function more effectively in the tumor microenvironment. In specific cases, those genes are one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, beta2-microglobulin, HLA, CD73 and CD39. In a specific embodiment, the TGFBR2 gene is knocked out or knocked down in B cells.

[0144] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as alterations via RNA-guided endonucleases (RGENs). For example, the alterations can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In some aspects, CpF1 can be used in place of Cas9. Generally, the term "CRISPR system" collectively refers to CRISPR-associated ("Cas") genes (including sequences encoding Cas genes), tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" in the context of endogenous CRISPR systems and partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts derived from the CRISPR locus, as well as transcripts and other elements that participate in or direct the expression of such sequences and transcripts and their activities.

[0145] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA sequence specifically, and a Cas protein (e.g., Cas9) having nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II or type III CRISPR system, and can be derived, for example, from a particular organism (e.g., Streptococcus pyogenes) including an endogenous CRISPR system.

[0146] In some embodiments, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and a given tracrRNA) are introduced into a cell. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to its target site, e.g., a gene, by complementary base pairing. The target site can 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 is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence. Usually, a "target sequence" generally refers to a sequence that is designed such that the guide sequence has complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.

[0147] The CRISPR system can induce disruptions or modifications as discussed herein following a double-strand break (DSB) at the target site. In other embodiments, a Cas9 variant considered a "nickase" is used to introduce a single-strand nick at the target site. For example, to improve specificity, pairs of nickases can be used, each of which is guided by a different pair of gRNAs that target the sequence, and when the nicks are introduced simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or transcriptional activator to affect gene expression.

[0148] The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination to a targeted locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0149] Typically, in the context of an endogenous CRISPR system, cleavage of one or both strands occurs 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) by the formation of a CRISPR complex (including a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins). A tracr sequence that can include all or part of the wild-type tracr sequence (e.g., about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more of the wild-type tracr sequence or more than about 20 nucleotides, more than about 26 nucleotides, more than about 32 nucleotides, more than about 45 nucleotides, more than about 48 nucleotides, more than about 54 nucleotides, more than about 63 nucleotides, more than about 67 nucleotides, more than about 85 nucleotides or more) or consists of them can also form part of the CRISPR complex by hybridization along at least part of the tracr sequence to all or part of a tracr mate sequence operably linked to the guide sequence. The tracr sequence has sufficient complementarity (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) to hybridize and participate in the formation of the CRISPR complex.

[0150] One or more vectors that drive the expression of those elements of the CRISPR system can be introduced into a cell such that the formation of the CRISPR complex is directed at one or more target sites by the expression of one or more elements of the CRISPR system. Additionally, the components can be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the 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 can be combined in a single vector, and one or more additional vectors provide any components of the CRISPR system not included in the first vector. The vector can include 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 of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0151] The vector can include a regulatory element operably linked to an enzyme coding sequence encoding 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, homologs thereof or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database under accession number Q99ZW2.

[0152] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumoniae). In some embodiments, CpF1 can be used in place of Cas9. The CRISPR enzyme can direct cleavage of one or both strands at the position of the target sequence, such as within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated compared to the corresponding wild-type enzyme, and the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that each target the sense and antisense strands of its DNA target. This combination can be used to nick both strands and to induce NHEJ or HDR.

[0153] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be a cell of a particular organism (e.g., a mammal including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate) or a cell derived from such an organism. Generally, codon optimization refers to the process of modifying a nucleic acid sequence so that its expression is enhanced in a target host cell by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit specific biases for certain codons of a particular amino acid. Codon bias (differences in codon usage frequencies among organisms) often correlates with the translation efficiency of messenger RNA (mRNA), and the translation efficiency is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The fact that the selected tRNA is dominant intracellularly usually reflects that it is the codon most frequently used in peptide synthesis. Thus, based on codon optimization, a gene can be adapted for optimal gene expression in a given organism.

[0154] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to hybridize to a target polynucleotide sequence and sufficient complementarity to direct sequence-specific binding of a CRISPR complex to that 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 more than about 50%, more than about 60%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, more than about 99% or more when optimally aligned using a suitable alignment algorithm.

[0155] Optimal alignment can be determined using any suitable algorithm for aligning the sequences. Non-limiting 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).

[0156] The CRISPR enzyme can be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include 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, but are not limited thereto. Non-limiting 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 glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), but are not limited thereto. The CRISPR enzyme can be fused to a gene sequence encoding a protein or a fragment of a protein that binds to a DNA molecule or to other cellular molecules (including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions). Additional domains that can form part of the fusion protein including the CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, which is incorporated herein by reference. V. Treatment Methods

[0157] In some embodiments, the B cells produced by the methods of the present disclosure are used in a method of treating an individual in need thereof. Embodiments of the present disclosure include, by way of example, methods of treating an individual for cancer, any type of infectious disease, and any immune disorder. The individual may use the treatment methods of the present disclosure as a first treatment, or after (or together with) another treatment. In some embodiments, the method of immunotherapy can be tailored to the needs of an individual with cancer based on the type and / or stage of the cancer, and in at least some cases, the immunotherapy can be modified for the individual during the course of treatment.

[0158] In specific instances, examples of treatment methods are as follows: (1) adoptive cell therapy using the produced B cells (expanded ex vivo or expressing a CAR or TCR) to treat cancer patients having any type of hematologic malignancy, (2) adoptive cell therapy using the produced B cells (expanded ex vivo or expressing a CAR and / or TCR) to treat cancer patients having any type of solid cancer, (3) adoptive cell therapy using the produced B cells (expanded ex vivo or expressing a CAR and / or TCR) to treat patients having an infectious disease and / or an immune disorder.

[0159] In some embodiments, the present disclosure provides a method for immunotherapy comprising administering an effective amount of B cells produced by the methods of the present disclosure. In one embodiment, in at least certain cases, a medical disease or disorder is treated by engraftment of a B cell population that elicits an immune response, produced by the methods herein. In certain embodiments of the present disclosure, cancer or an infectious disease is treated by delivery of one or more B cell populations that elicits an immune response, produced by the methods of the present disclosure. Methods are provided herein for treating an individual's cancer or delaying the progression of cancer, the method comprising administering to the individual an effective amount of antigen-specific cell therapy. The method can be applied to the treatment of immune disorders, solid cancers, blood cancers, and / or viral infections.

[0160] The tumors for which the present treatment method is useful include any malignant cell type, such as cell types found in solid tumors or hematological tumors. Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers that can be treated using the methods provided herein include lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer), peritoneal cancer, gastric cancer or stomach 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 or renal carcinoma, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma, but are not limited thereto.

[0161] Cancer can specifically be, but is not limited to, cancers of the following tissue types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma-cholangiocarcinoma; cord adenocarcinoma; adenoid cystic carcinoma; adenomatous polyp-in situ adenocarcinoma; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary-follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficially spreading melanoma; malignant lentigo melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal tumor; fetal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; angioendothelioma, malignant; Kaposi sarcoma; hemangioepithelioma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontogenic sarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; anaplastic glioma; oligodendroblastoma; primitive neuroectodermal;Cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neuroepithelioma; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell type, diffuse; malignant lymphoma, follicular; fungating polyposis; other specified non-Hodgkin lymphoma; 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 non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myelogenous leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); and chronic myelogenous leukemia.

[0162] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by mitotic division) of blood cells (usually white blood cells (leukocytes)). Leukemia is part of a broad group of diseases known as blood cancers. Leukemia is a broad term encompassing a wide variety of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

[0163] In certain embodiments of the present disclosure, B cells are delivered to an individual in need thereof (e.g., an individual having cancer or an infectious disease). The cells then enhance the individual's immune system to attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with B cells more than once. When the individual is provided with B cells more than once, the time between administrations should be sufficient for the cells to propagate in the individual. In a specific embodiment, the time between administrations is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hours or more, 1, 2, 3, 4, 5, 6, 7 days or more, or 1, 2, 3, 4 weeks, or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. And any range derived therefrom may be used. The successive dosages may or may not be the same amount as each other. In some cases, the successive dosages decrease over time or increase over time.

[0164] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes or immune-mediated diabetes, myasthenia gravis, nephrotic syndrome (e.g., minimal change group, focal segmental glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu arteritis, temporal arteritis / giant cell arteritis or herpes dermatitis vasculitis), vitiligo as well as Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis or psoriasis. The subject may also have an allergic disorder such as asthma.

[0165] In yet another embodiment, the subject is a recipient of an organ or stem cell to be transplanted, and immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a complication that can occur with any transplant using or involving stem cells from a related or unrelated donor. There are two types of GVHD, acute and chronic. Acute GVHD appears within the first three months after transplantation. Symptoms of acute GVHD include a reddish rash on the hands and feet, which may spread with peeling or blistering of the skin and may become more severe. Acute GVHD can also affect the stomach and intestines, in which case muscle spasms, nausea, and diarrhea are observed. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops three months or more after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD can also affect the mucous glands of the eyes, the salivary glands of the mouth, and the glands that line the stomach wall and intestines. Any of the immune cell populations disclosed herein can be used. Examples of organs to be transplanted include organ grafts such as kidneys, livers, skin, pancreas, lungs, and / or hearts, or cell grafts such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft can be a composite graft such as facial tissue. The immune cells can be administered before, at the same time as, or after transplantation. In some embodiments, the immune cells are administered before transplantation, for example, at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month before transplantation. In one non-limiting specific example, administration of a therapeutically effective amount of immune cells is performed 3 to 5 days before transplantation.

[0166] In some embodiments, the subject may be administered a non-myeloablative lymphodepleting chemotherapy prior to the immune cell therapy. The non-myeloablative lymphodepleting chemotherapy can be any suitable such therapy that can be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy can include, for example, the administration of cyclophosphamide and fludarabine, particularly in the case of melanoma where the cancer can be metastatic. Exemplary routes of administration of cyclophosphamide and fludarabine are intravenous. Similarly, any suitable doses of cyclophosphamide and fludarabine can be administered. In certain embodiments, approximately 60 mg / kg of cyclophosphamide is administered for 2 days, followed by approximately 25 mg / m 2 of fludarabine administered for 5 days.

[0167] In certain embodiments, one or more growth factors that promote B cell growth and activation are administered to the subject either concomitantly with or subsequent to the B cells. The growth factor can be any suitable growth factor that promotes NK cell growth and activation. Examples of suitable immune cell growth factors include IL-2, IL-4, IL-10, IL-7, IL-12, IL-15, IL-18, or IL-21, which can be used alone or in various combinations (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-4, and IL-10, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2).

[0168] A therapeutically effective amount of the generated NK cells can be administered by several routes including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, intratumoral, intrathecal, intraventricular, reservoir, intra-articular injection or infusion.

[0169] The therapeutically effective amount of the engineered B cells for use in adoptive cell therapy is the amount that achieves the desired effect in the subject being treated. For example, this can be the amount of immune cells necessary to inhibit progression, or the amount of B cells necessary to regress an autoimmune or alloimmune disease, or an amount that can alleviate symptoms caused by an autoimmune disease, such as pain and inflammation. This can be an amount necessary to alleviate symptoms associated with inflammation, such as pain, swelling, and elevated body temperature. This can also be an amount necessary to reduce or prevent rejection of a transplanted organ.

[0170] The engineered B cell population can be administered, for the purpose of restoring a disease state, in a treatment regimen consistent with the disease, e.g., once or several times over a period of 1 day to several days, or for the purpose of inhibiting disease progression and preventing disease recurrence, by periodic administration over a long period. The exact dosage used in the formulation also depends on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the physician and the circumstances of each patient. The therapeutically effective dosage of B cells depends on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dosage that can be used in the treatment of human subjects is at least 3.8×10 4 at least 3.8×10 5 at least 3.8×10 6 at least 3.8×10 7 at least 3.8×10 8 at least 3.8×10 9 or at least 3.8×10 10 B cells / m 2 and varies. In certain embodiments, the dosage used in the treatment of human subjects is from about 3.8×10 9 to about 3.8×10 10 B cells / m 2 and varies. In further embodiments, the therapeutically effective amount of B cells is from about 5×10 6 cells / kg body weight to about 7.5×10 8 cells / kg body weight, e.g., from about 2×10 7 cells to about 5×108 cells / kg body weight or about 5×10 7 cells to about 2×10 8 cells / kg body weight and may vary. The exact amount of B cells will be readily determined by one of ordinary skill in the art based on the age, weight, sex and physiological condition of the subject. The effective amount can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0171] The above B cells can be administered in combination with one or more other therapeutic agents for treating immune-mediated disorders. Examples of combination therapies include one or more antibacterial agents (e.g., antibiotics, antiviral agents, and antifungal agents), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (e.g., azathioprine or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone) or non-steroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium)), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines, but are not limited thereto. Further, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., CD3, CD4, CD40, CD154, CD45, IVIG, or an antibody that recognizes B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or immunosuppressive or immunotolerogenic agents including, but not limited to, chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceuticals can be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the above cells and agents can be by the same route or different routes, and at the same site or different sites. A. Pharmaceutical composition

[0172] Also provided herein are pharmaceutical compositions and formulations comprising B cells made by the processes encompassed herein and a pharmaceutically acceptable carrier.

[0173] Pharmaceutical compositions and formulations as described herein comprise an active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity and one or more freely selected pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 22 ndBy mixing with (edition, 2012), it can be prepared in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and such carriers include buffering agents (e.g., phosphoric acid, citric acid and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenolic alcohol, butyl alcohol or benzyl alcohol; alkyl parabens (e.g., methyl paraben or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (e.g., EDTA); saccharides (e.g., sucrose, mannitol, trehalose or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complex); and / or nonionic surfactants (e.g., polyethylene glycol (PEG)), but are not limited thereto. Exemplary pharmaceutically acceptable carriers in the present specification further include interstitial drug dispersants, such as neutral and active soluble hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGP containing rHuPH20 and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinase. B. Combination Therapy

[0174] In certain embodiments, the compositions and methods of the present embodiments include a B cell population that is used in combination with at least one additional treatment. The additional treatment can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy (in addition to those included herein), bone marrow transplantation, nano-therapy, monoclonal antibody therapy, or a combination of the foregoing. The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0175] In some cancer embodiments, the additional treatment is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is the administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of the side effects of a treatment, such as an anti-nausea agent, etc.). In some embodiments, the additional treatment is radiotherapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiotherapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a treatment that targets the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional treatment can be one or more of the chemotherapy agents known in the art.

[0176] The B cell therapy of the present disclosure can be administered before, during, after, or in various combinations with further cancer treatments such as immune checkpoint therapy. Those administrations can be performed at intervals ranging from simultaneous to several minutes, days, or weeks. In embodiments where the immune cell therapy is provided to the patient separately from the further therapeutic agent, it is common to ensure that a significant period does not elapse between each delivery time so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy can be provided to the patient within about 12 to 24 or within about 6 to 12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period if several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) have elapsed between each administration.

[0177] Various combinations can be used. In the following examples, the immune cell therapy is "A" and the anti-cancer therapy is "B": A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0178] The administration of any compound or treatment of the present embodiment to a patient follows the general protocol for administering such a compound, taking into account its toxicity to the agent. Thus, in some embodiments, there is a step of monitoring the toxicity that may result from the combination therapy. 1. Chemotherapy

[0179] A variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the treatment of cancer using drugs. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell, for example, by whether they affect the cell cycle and at which stage they affect the cell cycle. Alternatively, the agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0180] Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa and uredopa); ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine); acetogenins (particularly, bratexacin and bratexanone); camptothecin (including topotecan, a synthetic analog); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including KW-2189 and CB1-TM1, synthetic analogs); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma1I and calicheamicin omegaI1)); dynemicin (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and neocarzinostatin chromophore and related pigment proteins, engomycin antibiotic chromophore, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, daunorubicin, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, pteropterin and trimethoprim); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine and thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine and floxuridine); androgens (e.g., calusterone, drostanolone propionate, epithiostanol, mepitiostane and testolactone); anti-adrenals (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;Bisantrene; Edatraxate; Defofamine; Demecortin; Diazicon; Elformithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids (e.g., Maytansine and Ansamitocins); Mitoguazone; Mitoxantrone; Mopidanmol; Nitralin; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2’,2”-Trichlorotriethylamine; Trichothecins (especially, T-2 toxin, Verrucarin A, Roridin A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Taxoids, e.g., Paclitaxel and Docetaxel Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes (e.g., Cisplatin, Oxaliplatin and Carboplatin); Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids (e.g., Retinoic acid); Capecitabine;Examples include carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, a farnesyl-protein transferase inhibitor, transplatinum, and any pharmaceutically acceptable salts, acids or derivatives of the above.; 2. Radiation therapy

[0181] Other widely used agents that cause DNA damage include γ-rays, X-rays and / or those commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation and UV irradiation are also contemplated. It is most likely that all of these agents will cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosomal assembly and maintenance. The range of X-ray doses is from a daily dose of 50-200 roentgens over a long period (3-4 weeks) to a single dose of 2000-6000 roentgens. The range of radioisotope doses varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumorous cells. 3. Immunotherapy

[0182] One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the above embodiments. In the context of treating cancer, immunotherapeutic agents typically rely on using immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can function alone as an effector of treatment or recruit other cells to actually affect cell killing. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector can be a lymphocyte having surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0183] Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to a cell-killing drug and can be used in combination therapies. This approach combines the high specificity of the MAb for the antigen target with a very potent cytotoxic drug to result in an "armed" MAb that delivers the payload (drug) to tumor cells having abundant levels of the antigen. Also, the targeted delivery of the drug reduces toxicity and improves the therapeutic index since exposure to normal tissues is minimized. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0184] In one aspect of immunotherapy, the tumor cells must have some marker that is susceptible to targeting, i.e., some marker that is not present on most other cells. There are many tumor markers, and any of these 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, erb B, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect with an immune-stimulating effect. There are also immune-stimulating molecules such as cytokines like IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines like MIP-1, MCP-1, IL-8, and growth factors like FLT3 ligand.

[0185] Examples of immunotherapy include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy such as interferons α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be used in combination with the antibody therapy described herein.

[0186] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. An immune checkpoint either enhances a signal (e.g., a costimulatory molecule) or weakens a signal. Inhibitory immune checkpoints that can be targeted by blocking the immune checkpoint include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4.

[0187] The immune checkpoint inhibitor can be a drug such as a small molecule, a recombinant ligand or receptor, or in particular, an antibody such as a human antibody. Known inhibitors of the immune checkpoint protein or its analog can be used, in particular, chimeric, humanized or human antibodies can be used. As known to those skilled in the art, alternative and / or equivalent names can be used for certain antibodies described in the present disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that pembrolizumab is also known by the alternative and equivalent names MK-3475 and lambrolizumab.

[0188] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits PDL1 from binding to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits PDL2 from binding to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide.

[0189] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 or a PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and OPDIVO®, is an anti-PD-1 antibody that can be used. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA® and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0190] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. CTLA4 resembles the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important for the function of those cells. Activation of T cells via the T cell receptor and CD28 results in high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0191] 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide.

[0192] An anti-human CTLA-4 antibody (or VH and / or VL domains derived therefrom) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101 and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy chain CDRs and light chain CDRs or heavy chain VRs and light chain VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the above-described antibody and / or binds to the same epitope on CTLA-4 as the above-described antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (e.g., at least about 90%, 95% or 99% variable region identity to ipilimumab). 4. Surgery

[0193] Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic surgery or staging surgery, curative surgery, and palliative surgery. Curative surgery includes excisions that physically remove, excise and / or destroy all or part of the cancerous tissue and can be used in combination with other treatments (e.g., the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies). Tumor excision refers to physically removing at least part of the tumor. Surgical treatments include, in addition to tumor excision, laser surgery, cryosurgery, electrocautery and microsurgery (Mohs surgery).

[0194] When removing part or all of a cancerous cell, tissue, or tumor, a cavity can form in the body. The treatment can be achieved by perfusion, direct injection, or topical application of that area by further anti-cancer therapy. 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 can also be treatments with various dosages. 5. Other agents

[0195] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell division inhibitors and differentiating agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intercellular signaling by increasing the number of gap junctions can enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell division inhibitor or differentiating agent may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effectiveness of the treatment. An inhibitor of cell adhesion is contemplated to improve the effectiveness of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents (e.g., antibody c225) that enhance the sensitivity of hyperproliferative cells to apoptosis may be used in combination with certain aspects of the present embodiment to improve the effectiveness of the treatment. VI. Product or kit

[0196] Also provided herein are products or kits comprising B cells. Products or kits are provided that comprise engineered B cells and / or one or more reagents for making the same. The B cells can be of any origin, the B cells can be made by the methods encompassed herein, or the kit can include reagents for making such engineered B cells. In some embodiments, the B cells are already modified to express CD40L and can be further modified, such as being gene edited and / or expressing one or more heterologous antigen receptors, and can be provided in the kit. In specific embodiments, the B cells are already modified to express one or more heterologous antigen receptors and / or to be gene edited and can be further modified and can be provided in the kit.

[0197] In specific embodiments, one or more reagents for making the above B cells (e.g., reagents that target a specific gene, reagents that include one or more heterologous antigen receptors (or one or more reagents for making a heterologous antigen receptor), a transfection vector or transduction vector or expression construct for CD40L, or combinations thereof) are provided in the kit. In general embodiments, those reagents can include nucleic acids, such as DNA or RNA, proteins, media, buffers, salts, cofactors, and the like. In specific cases, the kit includes one or more CRISPR-related reagents that include reagents for targeting a desired specific gene.

[0198] The above-mentioned product or kit may further include a package insert containing instructions for using immune cells to treat cancer in an individual, delay the progression of cancer, or enhance the immune function of an individual having cancer. Any antigen-specific immune cells described herein may be included in the product or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from various materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloy (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation and the label, which is attached to or associated with the container, and the usage method may be indicated on the container. The product or kit may further include other materials desirable from a commercial and user perspective, such materials including other buffers, diluents, filters, needles, syringes, and a package insert containing instructions for use. In some embodiments, the product further includes one or more other agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0199] When the components of the above kit are provided as one and / or more than one liquid solution, the liquid solution may be an aqueous solution, and a sterilized aqueous solution is particularly contemplated. The above composition may also be formulated into a syringeable composition or any form suitable for administration such as intravenous administration. In that case, the container means may itself be a syringe, pipette, and / or other such device, from which the formulation may be applied to an infected area of the body, injected into an animal, and / or applied to and / or mixed with other components of the kit.

Examples

[0200] The following examples are included to demonstrate preferred embodiments of the present invention. The techniques disclosed in the following examples are those that the inventors have found to function well in the practice of the present invention and should be recognized by those skilled in the art as being a preferred form for its implementation. However, those skilled in the art will recognize that, in view of the present disclosure, many changes can be made in the specific embodiments disclosed, and such changes will still result in the same or similar results without departing from the spirit and scope of the present invention. Example 1 Large-scale CRISPR / Cas9-mediated manipulation strategy for primary B cells

[0201] This example includes an example of a protocol for large-scale manipulation of B cells, including editing by CRISPR / Cas9. As an example, FIG. 1 shows one protocol for first stimulating peripheral blood (PB) B cells with CpG, anti-BCR (antibody against the B cell receptor (IgG + iIM) and cytokines (IL-2, IL-4, IL-7, IL-10, IFN-gamma (separately or in combination)) for 48 hours. Then, they are transduced with a CD40L construct via a vector such as a retroviral vector. In some cases, they are transduced with CD40L and IL-21. The IL-21 may or may not be membrane-bound. In any case, IL-21 and CD40L can be expressed in one bicistronic construct or from different constructs. In some cases, the transduced cells are expanded, such as in the presence of one or more cytokines. Then, the B cells may be used or stored as needed. In some cases, the B cells are subjected to functional studies, mass cytometry, and / or RNA sequencing.

[0202] In one example, the transduction efficiency of primary B cells with CD40L versus CD40L-IL21 is measured. PBMCs are isolated from peripheral blood using Ficoll density centrifugation, and B cells are negatively selected using magnetic selection. B cells are activated for 48 hours using CpG and anti-IgM / IgG. The transduction efficiency of B cells transduced with CD40L via a retroviral vector is measured in Figure 2A, and the transduction efficiency of B cells transduced with CD40L-IL21 via a retroviral vector is shown in Figure 2B. After culturing for a specific period using different cytokine combinations, the growth rate of the transduced B cells is measured. Figure 2D shows the suppression of T cell cytokine secretion by B cells. Allogeneic T cells are isolated from cryopreserved PBMC samples and activated with anti-CD3 / CD28 microbeads. They are cultured with B cells at various ratios for 48 hours, and then intracellular staining is performed to evaluate the production levels of IFNγ, TNFα, and IL-2 in the T cells. B cells were able to decrease the cytokine production levels of T cells in a dose-dependent manner. Functional assays were performed 5, 7, and 9 days after transduction of CD40L-IL21 into B cells, and it was found that if the B cells remained in culture, they lost their inhibitory capacity (Figure 2E).

[0203] Figure 3A provides another example of a protocol that combines transduction of CD40L and transduction of a CAR into primary B cells derived from peripheral blood. First, B cells are stimulated in vitro with CpG, anti-BCR, and cytokines (IL-2, IL-4, IL-7, IL-10, IFN-gamma (separately or in combination)) for 48 hours. Then, they are transduced with CD40L via a retroviral vector and supplemented with IL-21 and IL-4. Two days after CD40L transduction, the B cells are re-transduced with a CAR construct (as a mere example, CD19 or CD5) via a retroviral vector. The B cells are maintained in a culture medium that includes IL-4 and IL-21 in at least some cases. In some cases, the cells are cultured until a sufficient number is available for functional studies, mass cytometry (CytoF), and / or RNA sequencing. Figure 3B provides the transduction efficiency of CD40L-B cells that were transduced with CAR CD19 or CAR CD5 a second time. The second transduction does not reduce the expression of the first CD40L transduction.

[0204] Figure 4A shows another example of a protocol that combines CD40L transduction and CRISPR Cas9 gene editing into primary B cells derived from peripheral blood. First, PB B cells are stimulated in vitro with CpG, anti-BCR, and cytokines (IL-2, IL-4, IL-7, IL-10, IFN-gamma (separately or in combination)) for 48 hours. Then, they are transduced with CD40L via a retroviral vector and supplemented with IL-21 and IL-4. Two days after CD40L transduction, CRISPR-cas9 gene editing is performed using ribonucleoprotein complexes (RNPs). For example, the B cells can be maintained in a culture medium until a sufficient number is available for functional studies, mass cytometry, and RNA sequencing as needed. The knockout efficiency of the CD86, CD95, IL10R, and PD-1 genes is provided in Figure 4B.

[0205] A further protocol combining CD40L transduction and CRISPR Cas9 gene editing into primary B cells derived from peripheral blood is shown in Figure 5A. First, PB B cells are stimulated in vitro with CpG, anti-BCR and cytokines (IL-2, IL-4, IL-7, IL-10, IFN-gamma (separately or in combination)) for 48 hours. Then, they are transduced with CD40L via a retroviral vector and supplemented with IL-21 and IL-4. Two days after CD40L transduction, CRISPR-cas9 gene editing using ribonucleoprotein complexes (RNPs) is performed. The B cells can be maintained in culture medium until a sufficient number is available for functional studies, mass cytometry and RNA sequencing as needed. As shown in Figure 5B, this experimental plan enables successful performance of separate double or triple gene knockouts in a single session.

[0206] Figure 6 shows a specific embodiment of large-scale CRISPR cas9 gene editing of primary B cells transduced with CD40L. First, PB B cells are stimulated in vitro with CpG, anti-BCR and cytokines (IL-2, IL-4, IL-7, IL-10, IFN-gamma (separately or in combination)) for 48 hours. Then, they are transduced with CD40L via a retroviral vector and supplemented with IL-21 and IL-4. Two days after CD40L transduction, CRISPR-cas9 gene editing using ribonucleoprotein complexes (RNPs) is performed. The B cells are maintained in culture medium until a sufficient number is available for functional studies, mass cytometry and / or RNA sequencing as needed.

[0207] A comparison of the electroporation efficiency and knockout efficiency of CRISPR-Cas9 gene editing settings at small and large scales is provided in Figure 7. As shown in Figure 7A, the electroporation efficiency is over 95% in both small-scale and large-scale CRISPR-edited B cells. Figure 7B demonstrates that the knockout efficiency of CD47 remains high in both small-scale and large-scale settings.

[0208] Figure 8 shows the expression levels of miR-155 in B cells after CRISPR-Cas9-mediated gene knockout. To find the maximum efficiency, various combinations of gRNAs were used. The combination of 2 and 5 gRNAs achieved the most efficient knockout.

[0209] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps of the methods described herein, or the order of the steps, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be used in place of the agents described herein as long as the same or similar results are achieved. All such similar substitutes and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

**Claim 1** An in vitro method for producing engineered B cells, comprising: (a) stimulating B cells obtained from peripheral blood mononuclear cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized B cell lines, B cell hybridomas, bone marrow, and / or umbilical cord blood mononuclear cells over a first period, said stimulation comprising exposing the B cells to one or more suitable cytokines, thereby producing stimulated B cells; (b) transducing the stimulated B cells with CD40 ligand to produce CD40L-positive B cells, and numerically expanding the CD40L-positive B cells over a second period; and (c1) and (d1), or (c2) and (d2), one of the following: (c1) delivering an effective amount of Cas9 or Cpf1 and one or more guide RNAs to the expanded CD40L-positive B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited B cells; and (d1) transducing or transfecting the gene-edited B cells of (c1) with a vector encoding a heterologous antigen receptor to produce gene-edited modified B cells; or (c2) transducing or transfecting the expanded CD40L-positive B cells with a vector encoding a heterologous antigen receptor to produce modified B cells; and (d2) delivering an effective amount of Cas9 or Cpf1 and one or more guide RNAs to the modified B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited modified B cells, wherein in (b), transduction further comprises transducing the stimulated B cells with IL-21, and / or expansion occurs in the presence of IL-21. **Claim 2** The method of claim 1, wherein the gene-edited modified B cells are produced from the steps of (c1) and (d1). **Claim 3** The method of claim 1, wherein the gene-edited modified B cells are produced from the steps of (c2) and (d2). **Claim 4** The method of claim 1, wherein the steps of (c1) and (d2) are each further defined as two or more delivery steps. **Claim 5** The method according to claim 4, wherein the first delivery step comprises delivering a guide RNA that targets one or more genes, and the second delivery step comprises delivering a guide RNA that targets one or more genes different from the one or more genes in the first delivery step.

6. The method according to claim 5, wherein the time between the first delivery step and the second delivery step is at least 2 days ± 5%.

7. The method according to claim 5, wherein the time between the first delivery step and the second delivery step is 2 ± 5% to 3 ± 5% days.

8. The method according to any one of claims 1 to 7, wherein the first period is 30 to 55 hours or 44 ± 5% to 48 ± 5% hours.

9. The method according to any one of claims 1 to 8, wherein the second period is 4 ± 5% to 5 ± 5% days after transduction.

10. The method according to any one of claims 1 to 9, wherein the expansion in (b) is performed in the presence of IL-4, IL-10, IL-21, IL-2, CpG, anti-BCR, or a combination thereof.

11. The method according to any one of claims 1 to 10, wherein the step of (c1) or (d2) is performed in the presence of IL-4 and / or IL-21.

12. The method according to any one of claims 1 to 11, wherein the delivery step is a step by electroporation.

13. Electroporation using 200,000 ± 5% to 1 × 10 ９ ± 5% B cells, the method according to claim 12.

14. The method according to claim 12, wherein electroporation uses 200,000 ± 5% to 2,000,000 ± 5% cells.

15. Electroporation using from 1,000,000 ± 5% to 1 × 10 ９ ± 5% B cells, according to claim 12

16. The method according to any one of claims 1 to 15, wherein the concentration of the guide RNA in the electroporation step is 3, 4, or 5 μM.

17. The method according to any one of claims 1 to 16, wherein the concentration of CpF1 or Cas9 nuclease in the electroporation step is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 μM.

18. The method according to any one of claims 1 to 17, wherein the cytokine is IL-2, IL-4, IL-6, IL-10, or a combination thereof.

19. The method according to claim 18, wherein the concentration of the cytokine is 100, 125, 150, 175, 200, 225, 250, 275 or 300 units / ml or 1 to 500 nM.

20. The method according to any one of claims 1 to 19, wherein the heterologous antigen receptor is a chimeric antigen receptor, a T cell receptor, a chemokine receptor, or a homing receptor.

21. The method according to any one of claims 1 to 20, wherein one or more cytokine genes and / or one or more immunoglobulins are transduced or transfected into the stimulated B cells, expanded CD40L-positive B cells, gene-edited B cells, or gene-edited modified B cells.

22. The method according to any one of claims 1 to 21, wherein the heterologous antigen receptor targets a cancer antigen.

23. The heterologous antigen receptor is CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinase, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7A, GAGE-7B, GAGE-8, NA88-A, MC1R, MDA-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase, TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1), TACSTD2, receptor tyrosine kinase, epidermal growth factor receptor (EGFR), EGFRvIII, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2,The method according to any one of claims 1 to 22, which targets an antigen selected from the group consisting of cytoplasmic tyrosine kinase, integrin-linked kinase (ILK), signal transducer and activator of transcription 3, STAT5 and STAT6, HIF-1, HIF-2, nuclear factor-kappa B (NF-κB), Notch receptor NY-ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), PSMA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI), CAIX, STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, TMPRSS2 ETS fusion gene, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1 and combinations thereof.

24. The method according to any one of claims 1 to 23, wherein the gene whose expression is disrupted in the B cells is an inhibitory gene.

25. The method according to claim 24, wherein the inhibitory gene is selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, beta2-microglobulin, HLA, CD73, CD39, and combinations thereof.

26. The method according to any one of claims 1 to 25, wherein the cells are expanded in the presence of IL-4 and / or IL-21 after the method.

27. The method according to any one of claims 1 to 26, wherein any of the cells are analyzed.

28. The method according to claim 27, wherein the cells are analyzed by one or more functional assays, cytotoxicity assays, and / or in vivo activity.

29. The method according to claim 27 or 28, wherein the cells are analyzed by flow cytometry, mass cytometry, RNA sequencing, CytoF, or combinations thereof.

30. The method according to any one of claims 1 to 29, which preserves any of said cells.

31. The method according to any one of claims 1 to 30, which cryopreserves any of said cells.

32. A population of B cells produced by the following method: An in vitro method for producing engineered B cells, (a) A step of stimulating B cells obtained from peripheral blood mononuclear cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized B cell lines, B cell hybridomas, bone marrow and / or umbilical cord blood mononuclear cells over a first period, said stimulation comprising exposing to one or more appropriate cytokines, thereby producing stimulated B cells; (b) Transducing the stimulated B cells with CD40 ligand to produce CD40L-positive B cells, and numerically expanding the CD40L-positive B cells over a second period; and (c1) and (d1), or one of (c2) and (d2): (c1) Delivering an effective amount of Cas9 or CpF1 and one or more guide RNAs to the expanded CD40L-positive B cells to disrupt the expression of one or more genes in said B cells, thereby producing gene-edited B cells; and (d1) Transducing or transfecting the gene-edited B cells of (c1) with a vector encoding a heterologous antigen receptor to produce gene-edited modified B cells; or (c2) Transducing or transfecting the expanded CD40L-positive B cells with a vector encoding a heterologous antigen receptor to produce modified B cells; and (d2) Delivering an effective amount of Cas9 or CpF1 and one or more guide RNAs to the modified B cells to disrupt the expression of one or more genes in said B cells, thereby producing gene-edited modified B cells, wherein in (b), transduction further comprises transducing the stimulated B cells with IL-21, and / or expansion occurs in the presence of IL-21.

33. A composition comprising the population according to claim 32.

34. The composition according to claim 33, wherein said population is contained in a pharmaceutically acceptable carrier.

35. A composition for treating an individual for a medical condition, comprising a therapeutically effective amount of B cells produced by the following method: An in vitro method for producing engineered B cells, comprising: (a) stimulating B cells obtained from peripheral blood mononuclear cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized B cell lines, B cell hybridomas, bone marrow and / or umbilical cord blood mononuclear cells for a first period, said stimulation comprising exposing the B cells to one or more appropriate cytokines, thereby producing stimulated B cells; (b) transducing the stimulated B cells with CD40 ligand to produce CD40L-positive B cells, and numerically expanding the CD40L-positive B cells for a second period; and (c1) and (d1), or one of (c2) and (d2): (c1) delivering an effective amount of Cas9 or Cpf1 and one or more guide RNAs to the expanded CD40L-positive B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited B cells; and (d1) transducing or transfecting the gene-edited B cells of (c1) with a vector encoding a heterologous antigen receptor to produce gene-edited modified B cells; or (c2) transducing or transfecting the expanded CD40L-positive B cells with a vector encoding a heterologous antigen receptor to produce modified B cells; and (d2) delivering an effective amount of Cas9 or Cpf1 and one or more guide RNAs to the modified B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited modified B cells, wherein in (b), transduction further comprises transducing the stimulated B cells with IL-21, and / or expansion occurs in the presence of IL-21.

36. The composition according to claim 35, wherein the medical condition is cancer.

37. The composition according to claim 36, wherein the cancer includes hematological malignancies or solid tumors.

38. The composition according to claim 35, wherein the medical condition is an infectious disease and / or an immune-related disorder.

39. The composition according to any one of claims 35 to 38, wherein the B cells are administered to the individual one or more times.

40. The composition according to claim 39, wherein when the B cells are administered to the individual multiple times, the time between administrations includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours.

41. The composition according to claim 39, wherein when the B cells are administered to the individual multiple times, the time between administrations is 1, 2, 3, 4, 5, 6 or 7 days.

42. The composition according to claim 39, wherein when the B cells are administered to the individual multiple times, the time between administrations includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.

43. The composition according to any one of claims 35 to 42, wherein an effective amount of one or more additional treatments for the medical condition is administered to the individual.

44. The composition according to claim 43, wherein the additional treatment is administered to the individual before, during and / or after administration of the B cells.

45. An in vitro method of producing engineered B cells, (a) stimulating B cells obtained from peripheral blood mononuclear cells, hematopoietic stem cells, induced pluripotent stem cells, immortalized B cell lines, B cell hybridomas, bone marrow and / or umbilical cord blood mononuclear cells over a first period, said stimulation comprising exposing to one or more appropriate cytokines, thereby producing stimulated B cells; (b) transducing the stimulated B cells with CD40 ligand to produce CD40L-positive B cells and numerically expanding the CD40L-positive B cells over a second period; and (c1) and (d1), or one of (c2) and (d2): (c1) delivering an effective amount of Cas9 or CpF1 and one or more guide RNAs to the expanded CD40L-positive B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited B cells; and (d1) transducing or transfecting the gene-edited B cells of (c1) with a vector encoding a heterologous antigen receptor to produce gene-edited modified B cells; or (c2) transducing or transfecting the expanded CD40L-positive B cells with a vector encoding a heterologous antigen receptor to produce modified B cells; and (d2) delivering an effective amount of Cas9 or CpF1 and one or more guide RNAs to the modified B cells to disrupt the expression of one or more genes in the B cells, thereby producing gene-edited modified B cells, wherein, in (b), transduction further comprises transducing the stimulated B cells with IL-21 and / or expansion occurs in the presence of IL-21, a kit comprising the B cells produced by the method and / or one or more reagents for producing the B cells.

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