Protein L for the activation and expansion of chimeric antigen receptor-modified immune cells

Abstract

Methods are provided herein for activating and expanding T cells. Further provided are methods for using T cells for therapy.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 733,291, filed Sep. 19, 2018, the entire disclosure of which is hereby incorporated by reference.

[0002] The present invention generally relates to the fields of immunology and medicine. More particularly, the present invention relates to methods and compositions of immune cells engineered to express chimeric antigen receptors.

Background Art

[0003] Adoptive cancer immunotherapy involves the transplantation of immune cells modified with chimeric antigen receptors (CARs) specific for tumor antigens, which can be produced by in vitro expansion culture of CAR-modified T cells (CAR-T). Currently, CAR-T cells are expanded in vitro using conventional methods with CD3 activating antibodies (Abs). In this method, all T cells are induced to proliferate regardless of CAR expression and not in a CAR-specific manner. This method can lead to "silencing" of CAR expression and function in CAR-T populations expanded with CD3.

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the case of CAR-T cells, they can be selectively expanded in culture having a tumor antigen specific for the CAR, either in isolated form or expressed by feeder cells, but the antigen culture is applicable only to specific CAR types. This approach is not general or very practical, especially for the production of therapeutic cells. Thus, similar to CD3 activation-based pan-T cell expansion cultures, the field of CAR-T cell therapy would benefit from a general pan-CAR-specific in vitro culture where CAR-T cells can be activated and expanded in a CAR-specific manner.

Means for Solving the Problems

[0005] In a first embodiment, the present disclosure provides an in vitro method for activating and / or expanding CAR-modified immune cells, comprising obtaining a starting population of CAR-modified immune cells and culturing the population of CAR-modified immune cells in the presence of protein L for a period sufficient to produce a population of CAR-modified immune cells that have been activated and / or expanded.

[0006] In certain embodiments, protein L is present at a concentration of 0.1 - 5 μg / cm 2 , for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 μg / cm 2 . In particular, protein L may be present at a concentration of 1 - 5 μg / mL, for example 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 μg / mL. In some embodiments, protein L is coated on the culture surface. For example, the culture surface can be a culture plate, a culture flask, a microcarrier, microparticles, hydrogel particles or a culture bag.

[0007] In some embodiments, the culture does not contain anti-CD3 antibody and / or antigen-specific target cells. In certain embodiments, the CAR-modified immune cells are T cells, NK cells, dendritic cells and / or macrophages. In certain embodiments, the T cells are CD8 + T cells, CD4 + T cells, αβ T cells or γδ T cells. In certain embodiments, the method further comprises selecting for CD8 + T cells.

[0008] In certain embodiments, the CAR-modified immune cells are allogeneic. In other embodiments, the CAR-modified immune cells are autologous. In specific embodiments, the CAR-modified immune cells are derived from pluripotent stem cells (PSCs). In certain embodiments, the PSC is an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is reprogrammed from blood cells or T cells. In specific embodiments, the iPSC is episomally reprogrammed. In some embodiments, the CAR-modified immune cells are derived from primary peripheral blood mononuclear cells (PBMCs) or primary hematopoietic stem cells. In certain embodiments, the iPSC differentiates into CD34 + progenitor cells through cytokine-induced differentiation. In certain embodiments, the iPSC differentiates into CD34 + progenitor cells through forward programming. For example, the CAR can include an antigen-binding domain selected from the group consisting of F(ab’)2, Fab’, Fab, Fv, and scFv. In some embodiments, the CAR includes a CD28 co-stimulation and a CD3ζ signaling domain.

[0009] In some embodiments, the culture surface is further coated with retronectin, fibronectin, or VCAM1. In certain embodiments, retronectin is added to the culture at a concentration of 0.1 - 1 μg / cm 2 , for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 or a concentration exceeding that. In further embodiments, the culture plate is further coated with the Notch ligand DLL4. In specific embodiments, DLL4 is added to the culture at a concentration of 0.1 - 1 μg / cm 2 , for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 or a concentration exceeding that.

[0010] In further embodiments, the culture further includes IL-2 and / or IL-15. In some embodiments, IL-12 and / or IL-15 are present at a concentration of 5 - 15 ng / mL, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ng / mL or a concentration exceeding that.

[0011] In certain embodiments, the culture is under hypoxic conditions. In some embodiments, the hypoxic conditions contain 5% oxygen.

[0012] In some embodiments, the sufficient period is 8 - 12 days, such as 8, 9, or 10 days. In some embodiments, the culture is in a medium containing SCF, TPO, FLT3L, and / or IL-7. In certain embodiments, SCF, TPO, FLT3L, and / or IL-7 are at a concentration of 50 ng / mL. In some embodiments, the medium further contains nicotinamide.

[0013] In certain embodiments, the method results in selective expansion culture of CAR-modified immune cells compared to non-CAR-modified immune cells. In some embodiments, at least 40% or 50% of the expanded population of CAR-modified immune cells are CAR-modified immune cells. In some embodiments, the expanded population of CAR-modified T cells is at least 25% CD3 + CD8 + and contains CAR-modified T cells. In certain embodiments, the expanded population of CAR-modified T cells contains cytotoxic activity that is 2 - 3 times higher compared to CAR-modified T cells expanded with anti-CD3. In some embodiments, the expanded population of CAR-modified T cells contains increased IFNγ and / or TNFα levels compared to CAR-modified T cells expanded with anti-CD3.

[0014] In another embodiment, a population of activated and / or expanded CAR-modified immune cells of the embodiments provided herein and their aspects is provided. Further provided herein is a pharmaceutical composition containing a population of CAR-modified immune cells of the embodiment and a pharmaceutically acceptable carrier.

[0015] The present disclosure further provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the expanded CAR-modified immune cells of the embodiment. In some embodiments, the CAR-modified immune cells are allogeneic. In other embodiments, the CAR-modified immune cells are autologous.

[0016] In a further aspect, the method further comprises administering at least a second therapeutic agent. In some aspects, the at least second therapeutic agent is a therapeutically effective amount of an immunomodulatory or immunosuppressive agent. In some aspects, the at least second therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, and immunotherapy. In certain aspects, the CAR-modified immune cells and / or the at least second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, or by direct injection or perfusion.

[0017] Use of a composition of activated and / or expanded CAR-modified immune cells of the embodiments provided herein for treating cancer in a subject in need thereof. In some aspects, the CAR-modified immune cells are allogeneic. In other aspects, the CAR-modified immune cells are autologous.

[0018] Furthermore, a composition comprising CAR-modified immune cells and Protein L is provided herein. In certain aspects, Protein L is present at a concentration of 0.1 - 5 μg / cm 2 , for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 μg / cm 2 . In particular, Protein L can be present at a concentration of 1 - 5 μg / mL, for example 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 μg / mL. In some aspects, Protein L is coated on the culture surface. For example, the culture surface can be a culture plate, a culture flask, a microcarrier, microparticles, hydrogel particles, or a culture bag.

[0019] In some aspects, the culture surface is further coated with retronectin, fibronectin, or VCAM1. In certain aspects, retronectin is present at a concentration of 0.1 - 1 μg / cm 2, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 or added to the culture at a concentration above that. In a further aspect, the culture plate is further coated with the Notch ligand DLL4. In a specific aspect, DLL4 is 0.1 - 1 μg / cm 2 , for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 or added to the culture at a concentration above that.

[0020] In some aspects, the culture does not contain anti - CD3 antibody and / or antigen - specific target cells. In a particular aspect, the CAR - modified immune cells are T cells, NK cells, dendritic cells and / or macrophages. In a particular aspect, the T cells are CD8 + T cells, CD4 + T cells, αβ T cells or γδ T cells. In a particular aspect, the method further includes selecting for CD8 + T cells.

[0021] In a particular aspect, the CAR - modified immune cells are allogeneic. In other aspects, the CAR - modified immune cells are autologous. In a specific aspect, the CAR - modified immune cells are derived from pluripotent stem cells (PSCs). In a particular aspect, the PSCs are induced pluripotent stem cells (iPSCs). In some aspects, the iPSCs are reprogrammed from blood cells or T cells. In a specific aspect, the iPSCs are episomally reprogrammed. In some aspects, the CAR - modified immune cells are derived from primary peripheral blood mononuclear cells (PBMCs) or primary hematopoietic stem cells. In a particular aspect, the iPSCs are differentiated into CD34 + progenitor cells through cytokine - induced differentiation. In a particular aspect, the iPSCs are differentiated into CD34 + progenitor cells through forward programming. For example, the CAR may comprise an antigen - binding domain selected from the group consisting of F(ab’)2, Fab’, Fab, Fv and scFv. In some aspects, the CAR comprises a CD28 co - stimulation and a CD3ζ signaling domain.

[0022] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various modifications and variations within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only.

[0023] The following drawings form a part of this specification and are included to further demonstrate specific aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

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Figure 1

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Figure 3

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Figure 4A - B

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Figure 5

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Figure 6

Mode for Carrying Out the Invention

[0030] Protein L is a bacterial protein with a unique specificity that binds to the light chain of an immunoglobulin containing the minimal antigen-binding portion - single-chain variable domain fragment (scFv) that constitutes the antigen-binding site of the CAR. It has been shown that protein L can bind to cell surface CAR (Zheng et al., 2012). Furthermore, Zheng et al. demonstrated the potential of protein L as a reagent for detecting CAR expression by flow cytometry.

[0031] Protein L is a bacterial protein that binds to immunoglobulins (Igs) through specific high-affinity interactions with the variable light chain without interfering with the antigen-binding site. Protein L can also bind to the light chain component within the single-chain variable fragment (scFv) that constitutes the antigen-binding domain of the CAR. Another unique feature of Protein L is its multi-domain structure with five identical Ig-binding sites in one molecule. Multivalent binding, and thus potential cross-linking of target molecules expressed on the cell surface, indicates that Protein L can function as a potent cell activator.

[0032] When bound to the scFv portion of the CAR, Protein L binds directly to the antigen-binding site without blocking it, while mimicking antigen binding and inducing CAR activation. In this way, it can be used as a general CAR activation reagent in cell culture applications involving CAR-modified cells, including functional analysis of CAR-induced cell responses and expansion culture of CAR-expressing cells for therapeutic applications.

[0033] Accordingly, in certain embodiments, the present disclosure provides methods and compositions comprising the use of Protein L for the activation and / or expansion culture of CAR-modified effector immune cells, such as CAR-T cells, CAR-NK cells, and CAR-macrophage cells. Thus, the method provides a general cell culture system for the activation and expansion culture of CAR-modified cells that is independent of their antigen specificity.

[0034] Using anti-human CD19 CAR-modified PSC-derived T cells, this study provides experimental evidence that immobilized Protein L induces a specific proliferative response within CAR-modified T cells (but not within unmodified T cells). While Retronectin (recombinant fibronectin fragment) supports the CAR-inducing function of Protein L, the Notch ligand DLL4 is CD19 +It has been found that the proliferative response and expansion culture of CAR-T cells can be further improved. When comparing CAR-T cells expanded in culture with anti-CD3 monoclonal antibody and protein L, superior CAR-mediated cytokine production, in vitro and in vivo antitumor activity were found in CAR-T cells expanded with protein L.

[0035] Accordingly, protein L was identified as a cell culture reagent for the specific activation, expansion culture, and analysis of CAR-modified cells in vitro. Since protein L is produced under animal-free conditions, it can be used as GMP-compliant culture suitable for therapeutic use. This method can be used for the development of PSC-derived CAR-T therapeutics. Specific uses include the selection of the optimal CAR conformation for PSC-derived T cells, quality control assays for CAR-T cells, and the expansion culture of CAR-T cells for preclinical animal studies.

[0036] I. Definitions As used herein, "essentially free of" is used herein to mean, from the perspective of a particular component, that the particular component intentionally formulated in the composition is absent and / or present only as a contaminant or in trace amounts. Thus, the total amount of the particular component obtained from any unintended contaminants of the composition is well below 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component cannot be detected by standard analytical methods.

[0037] As used herein, "a" or "an" herein can mean one or more. In this specification, when used in combination with the term "comprising" in the claims, the term "a" or "an" can mean one or more than one.

[0038] The use of the term "or" within the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives, or the alternatives are mutually exclusive even if the disclosure supports a definition that refers only to alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0039] The term "about" refers to ±5% of the recited value.

[0040] As used herein, a composition "substantially free of" a particular substance or material contains 30%, 20%, 15%, more preferably 10%, even more preferably 5% or most preferably 1% of that substance or material.

[0041] "Expression construct" or "expression cassette" means a nucleic acid molecule capable of inducing transcription. An expression construct includes at least one or more transcriptional regulatory elements (such as a promoter, enhancer or a functionally equivalent structure thereof) that induce gene expression in one or more desired cell types, tissues or organs. Additional elements such as transcription termination signals may also be included.

[0042] "Vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vehicle") refers to a macromolecule or complex of molecules that contains a polynucleotide to be delivered to a host cell, either in vitro or in vivo.

[0043] As a general type of vector, a "plasmid" is an extrachromosomal DNA molecule separated from chromosomal DNA and capable of replicating independently of chromosomal DNA. In certain cases, it is circular double-stranded.

[0044] As used herein, the terms "patient" or "subject" refer to a living mammal such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients include adults, juveniles, infants and fetuses.

[0045] The terms "tumor-associated antigen", "tumor antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the term refers to a protein, glycoprotein or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0046] An "epitope" is a site on an antigen that is recognized by an antibody, as determined by the specificity of the amino acid sequence. Two antibodies are said to bind to the same epitope if, when measured in a competitive binding assay, each competitively inhibits (blocks) the binding of the other to the antigen. Alternatively, two antibodies have the same epitope if most of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies are said to have overlapping epitopes if each partially inhibits the binding of the other to the antigen and / or if some of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0047] "Treating" or "treatment" of a disease or disorder refers to carrying out a protocol that may include administering one or more agents to a patient in an effort to reduce the signs or symptoms of the disease. Desirable therapeutic effects include reducing the rate of disease progression, remission or alleviation of the disorder and a favorable or improved prognosis. Alleviation can occur both before and after the signs or symptoms of the disease or disorder appear. Thus, "treating" or "treatment" can include "preventing" or "prevention" of a disease or undesirable condition. Further, "treating" or "treatment" includes protocols that do not require complete alleviation of signs or symptoms and do not require cure, specifically those having only a marginal effect on the patient.

[0048] As used herein, the term "effective" means sufficient to obtain the desired, expected or intended result when the term is used in the specification and / or claims. "Effective amount", "therapeutically effective amount" or "pharmaceutically effective amount", when used in connection with the treatment of a patient or subject with a compound, means that the amount of the compound, when administered to the subject or patient for treating or preventing a disease, is sufficient to effect such treatment or prevention of the disease.

[0049] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient who is experiencing or presenting the pathology or symptoms of the disease (e.g., stopping further development of the pathology and / or symptoms), (2) alleviating a disease in a subject or patient who is experiencing or presenting the pathology or symptoms of the disease (e.g., restoring the pathology and / or symptoms), and / or (3) effecting any measurable decrease in the disease or its symptoms in a subject or patient who is experiencing or presenting the pathology or symptoms of the disease.

[0050] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who is at risk of and / or susceptible to the disease but who has not yet experienced or presented any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the pathology or symptoms of a disease in a subject or patient who is at risk of and / or susceptible to the disease but who has not yet experienced or presented any or all of the pathology or symptoms of the disease.

[0051] The term "forward programming" refers to the programming of differentiated somatic cells that do not have pluripotency into multipotent or pluripotent cells different from them by the supply of one or more specific lineage-determining genes or gene products to the multipotent or pluripotent cells.

[0052] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable risk-benefit ratio.

[0053] "Pharmaceutically acceptable salts", as defined above, mean salts of the compounds disclosed herein that are pharmaceutically acceptable and have the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfates, aromatic sulfates, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, trimethylacetic acid and other organic acids. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be understood that the particular anions or cations forming part of any salt of the invention are not critical so long as the salt as a whole is pharmaceutically acceptable. Further examples of pharmaceutically acceptable salts, as well as methods for their preparation and use, are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0054] A "pharmaceutically acceptable carrier", "drug carrier" or simply "carrier" is a pharmaceutically acceptable substance formulated with an active ingredient drug involved in the conveyance, delivery and / or transport of a chemical drug. For example, drug carriers can be used to improve drug delivery and effectiveness, including sustained release techniques for modulating the bioavailability of a drug, reducing drug metabolism, and / or reducing drug toxicity. Some drug carriers can enhance the effectiveness of drug delivery to specific target sites. Examples of carriers include liposomes, microspheres (e.g., made from lactic acid-glycolic acid copolymers), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes and dendrimers.

[0055] The term "chimeric antigen receptor (CAR)", as used herein, refers to, for example, an artificial T cell receptor, a chimeric T cell receptor or a chimeric immune receptor, and may include a receptor modified to confer artificial specificity on a particular immune effector cell. CARs can be used to endow T cells with the specificity of monoclonal antibodies, thereby enabling the generation of large numbers of specific T cells for use, for example, in adoptive cell therapy. In specific embodiments, the CAR induces, for example, the specificity of cells for tumor-associated antigens. In some embodiments, the CAR includes an extracellular domain comprising a cell activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In certain aspects, the CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane domain and endodomain of CD3ζ. The specificity of other CAR designs can be derived from a ligand of the receptor (e.g., a peptide) or a pattern recognition receptor, such as Dectin. In certain cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In certain cases, the CAR includes domains for additional co-stimulation signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10, and / or OX40. Optionally, molecules including co-stimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally deplete T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR.

[0056] The term "culturing" refers to the in vitro maintenance, differentiation, and / or proliferation of cells in a suitable medium. "Enriched" means a composition that includes cells that are present in a greater percentage of total cells than when they are found in the tissues present within an organism.

[0057] An "anticancer" agent has the ability to negatively affect cancer cells / tumors in a subject, for example, by promoting the death of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting the immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject having cancer.

[0058] II. CAR-Modified Immune Cells Certain embodiments of the present disclosure relate to immune cells that express CAR. The immune cells can be T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + T cells, α-β T cells or γ-δ T cells), NK cells, invariant NK cells, NKT cells or stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem (iPSC) cells). In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils. Further provided herein are methods for generating and modifying immune cells and methods for using and administering the cells for adoptive cell therapy where the cells can be autologous or allogeneic. Thus, the immune cells can be used, for example, as an immunotherapy targeting cancer cells.

[0059] Methods are provided herein for activating and / or expanding culturing immune cells, such as CAR-modified immune cells, e.g., CAR-T cells, in the presence of Protein L. Protein L can be present in the culture alone or in combination with Retronectin and / or DLL4.

[0060] Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing treatment for a particular disease or condition. Immune cells can be collected from any location where they are present within the subject, such as, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly or stored for a period of time, for example, by freezing.

[0061] Immune cells can be concentrated / purified from any tissue in which they are present, such as, but not limited to, blood (including blood collected by a blood bank or cord blood bank), spleen, bone marrow, tissue removed and / or exposed during a surgical procedure, and tissue obtained via a biopsy procedure. The original tissue / organs from which the immune cells are concentrated, isolated, and / or purified can be isolated from both living and non-living subjects, where the non-living subject is an organ donor.

[0062] A. T cells In some embodiments, the immune cells are T cells. Some basic techniques for the induction, activation, and expansion culture of functional anti-tumor effector cells have been described in the last 20 years. These include autologous cells, such as tumor-infiltrating lymphocytes (TIL); autologous dendritic cells, lymphocytes, or T cell ligands, cells activated in vitro using beads or target cell membranes coated with artificial antigen-presenting cells (APC) or activating antibodies; allogeneic cells that naturally express an anti-host tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express a tumor-reactive TCR or chimeric TCR molecule with antibody-like tumor recognition ability known as a "T body". These techniques have led to a very large number of protocols for the preparation and immunization of T cells that can be used in the methods described herein.

[0063] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those isolated directly from a subject and / or those isolated and frozen from a subject. In some embodiments, the cells are one or more subsets of T cells or other cell types, such as the entire T cell population, CD4 + cells, CD8 + cells and their subpopulations, such as those defined by function, activation state, maturity, differentiation potential, expansion culture, recirculation, localization and / or persistence ability, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion characteristics and / or degree of differentiation. For the subject to be treated, the cells can be allogeneic and / or autologous. In some embodiments, the method includes isolating, preparing, treating, culturing and / or modifying the cells from a subject as described herein, and reintroducing them into the same patient, in the presence or absence of cryopreservation.

[0064] Among the subtypes and subpopulations of T cells (e.g., CD4 + and / or CD8 + T cells), there are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), central memory T (TC M ), effector memory T (T EM ) or highly differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, natural and adaptive regulatory T (T reg ) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / βT cells and δ / γT cells.

[0065] In some embodiments, one or more of the T cell populations are enriched or depleted for cells that are positive for a particular marker, such as a surface marker, or negative for a particular marker. Optionally, such a marker is absent or expressed at a relatively low level on a particular population of T cells (e.g., non-memory cells), but is present or expressed at a relatively higher level on a particular other population of T cells (e.g., memory cells).

[0066] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers (e.g., CD14) expressed on non-T cells, such as B cells, monocytes, or other white blood cells. In some aspects, CD4 + helper T cells and CD8 + cytotoxic T cells are separated, and a selection step for CD4 + or CD8 + is used. Such CD4 + populations and CD8 + populations can be further sorted into subpopulations by positive or negative selection for markers expressed on or relatively more highly expressed on one or more naive, memory, and / or effector T cell subpopulations.

[0067] In some embodiments, CD8 + T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (T CM ) cells is performed to enhance efficacy, for example, to improve long-term survival, expansion, and / or engraftment after administration, which is particularly robust in some aspects within such subpopulations.

[0068] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, for example, mechanically (e.g., by dissociating the tumor using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., using collagenase or deoxyribonuclease). The single cell suspension of the tumor enzymatic digest is cultured with interleukin-2 (IL-2).

[0069] The cultured T cells can be pooled and rapidly expanded. The rapid expansion results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) over a period of about 10 to about 14 days. More preferably, the rapid expansion results in an increase of at least about 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or more) over a period of about 10 to about 14 days.

[0070] Expansion culture can be achieved by any of several methods known in the art. For example, T cells can be rapidly expanded by non-specific T cell receptor stimulation in the presence of either feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (IL-2 is preferred). Non-specific T cell receptor stimulation can include about 30 ng / ml of OKT3, a murine monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cells can be optionally expanded by stimulation of peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of cancer that can be expressed from a vector (including the antigenic portion thereof, such as an epitope or cell), such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T cell growth factor, such as 300 IU / ml of IL-2 or IL-15 (IL-2 is preferred). In vitro-induced T cells are rapidly expanded by restimulation with the same antigen of cancer that is applied transiently onto antigen-presenting cells expressing HLA-A2. Alternatively, T cells can be restimulated, for example, with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0071] Autologous T cells can be modified to express a T cell growth factor that promotes the growth and activation of the autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rdSee, e.g., Alberts et al., Molecular Biology of the Cell, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, the modified autologous T cells express high levels of a T cell growth factor. T cell growth factor coding sequences, such as those of IL-12, are readily available in the art because their operable linkage to a T cell growth factor coding sequence, like a promoter, promotes high level expression.

[0072] In some embodiments, the T cells are activated and / or expanded in the presence of Protein L. Protein L can be added to the culture at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 μg / cm 2 of the culture. Protein L can be added to the culture immobilized on a surface or can be soluble in the medium. The culture surface can be a culture plate, a culture flask, a microcarrier, microparticles, hydrogel particles or a culture bag. Protein L can be added in combination with an extracellular matrix protein, such as retronectin or fibronectin.

[0073] B. Stem Cells In some embodiments, the CAR-modified immune cells of the disclosure can be or are derived from stem cells, such as induced pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs) or hematopoietic stem cells (HSCs).

[0074] With the exception of germ cells, erythrocytes, and platelets, any cell can be used as a starting point in iPSCs. For example, the cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. There are no restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can also be used as sources of somatic cells in the methods disclosed herein.

[0075] Somatic cells can be reprogrammed to generate iPS cells using methods known to those skilled in the art. Generally, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized, or Oct3 / 4, Sox2, Nanog, and Lin28 are utilized.

[0076] Once induced, the iPSCs can be cultured in a medium sufficient to maintain pluripotency. In certain embodiments, undefined conditions can be used; for example, pluripotent cells can be cultured on a fibroblast feeder cell or a medium exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, the cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division as the feeder cells. Alternatively, pluripotent cells can be cultured and maintained in an essentially undifferentiated state using a defined feeder-free culture system, such as TESR™ medium or E8™ / Essential 8™ medium.

[0077] C. Cytokine-Induced Differentiation Certain embodiments of the present disclosure relate to the differentiation of PSCs into HPCs. PSCs can be differentiated into HPCs by methods known in the art, for example, as described in U.S. Patent No. 8,372,642, which is incorporated herein by reference. In one method, a combination of BMP4, VEGF, Flt3 ligand, IL-3, and GM-CSF can be used to promote hematopoietic differentiation. In certain embodiments, continuous exposure of cell cultures to a first medium for preparing PSCs for differentiation, a second medium containing BMP4, VEGF, and FGF, followed by culture in a third medium containing Flt3 ligand, SCF, TPO, IL-3, and IL-6 can differentiate pluripotent cells into HPCs and hematopoietic cells. The second defined medium can also contain heparin. Furthermore, by adding FGF-2 (50 ng / ml) to the medium containing BMP4 and VEGF, the production efficiency of hematopoietic progenitor cells from pluripotent cells can be enhanced. Additionally, by adding a glycogen synthase kinase 3 (GSK3) inhibitor (e.g., CHIR99021, BIO, and SB-216763) to the first defined medium, the production of HPCs can be further enhanced.

[0078] Generally, the differentiation of pluripotent cells into hematopoietic progenitor cells can be performed using defined or undefined conditions. It will be understood that defined conditions are generally preferred in embodiments where the resulting cells are intended for administration to a human subject. Hematopoietic stem cells can be derived from pluripotent stem cells under defined conditions (e.g., using TeSR medium), and hematopoietic cells can be generated from embryoid bodies derived from pluripotent cells. In other embodiments, pluripotent cells can be co-cultured on OP9 cells or mouse embryonic fibroblasts and subsequently differentiated.

[0079] Pluripotent cells may be able to form embryoid bodies or aggregates as part of the differentiation process. The formation of "embryoid bodies" (EBs) or clusters of growing cells to induce differentiation generally involves in vitro aggregation of human pluripotent stem cells into EBs, enabling spontaneous and random differentiation into multiple tissue types representing the endoderm, ectoderm, and mesoderm origins of human pluripotent stem cells. Therefore, three-dimensional EBs can be used to generate some fractions of hematopoietic cells and endothelial cells.

[0080] EBs can be formed using the following protocol. Undifferentiated iPSCs adapted to feeder-free growth on MATRIGEL™-coated plates can be harvested at a culture density for about 8 - 10 minutes using 0.5 M EDTA treatment at room temperature. The EDTA is aspirated after incubation, and EBs can be formed by collecting the cells in SFD medium containing a rock inhibitor or brevistatin. The medium can be changed the next day to EB1 differentiation medium containing different cytokine formulations. The cells are seeded at a density of 250,000 - 500,000 cells / ml to promote aggregate formation.

[0081] To promote aggregate formation, the cells are transferred to a low-attachment plate and have 0.05% N2 and B-27 without RA supplement, 200 mM 1-glutamine, 0.05 mg / ml ascorbic acid-2-phosphate magnesium salt (Asc 2-P) (WAKO), and 4.5×10 -4It can be incubated overnight in serum-free differentiation (SFD) medium consisting of 75% IMDM (Gibco) and 25% Ham's modified F12 (Cellgro) supplemented with MTG. The next day, the cells can be collected from each well and centrifuged. Next, the cells can be resuspended in "EB differentiation medium" consisting of SFD basal medium supplemented with approximately 50 ng / ml of bone morphogenetic protein (BMP4), approximately 50 ng / ml of vascular endothelial growth factor (VEGF), and 50 ng / ml of zbFGF for the first 4 days of differentiation. The cells are fed half every 48 hours. On day 5 of differentiation, the medium is replaced with a second medium consisting of SFD medium supplemented with 50 ng / ml of stem cell factor (SCF), approximately 50 ng / ml of Flt-3 ligand (Flt-3L), 50 ng / ml of interleukin-6 (IL-6), 50 ng / ml of interleukin-3 (IL-3), and 50 ng / ml of thrombopoietin (TPO). The cells are fed half with fresh differentiation medium every 48 hours. The medium change is performed by centrifuging the differentiation culture at 300 g for 5 minutes, aspirating half of the volume from the differentiation culture, and replenishing it with fresh medium. In certain embodiments, the EB differentiation medium can contain BMP4 (e.g., approximately 50 ng / ml), VEGF (e.g., approximately 50 ng / ml), and optionally FGF-2 (e.g., approximately 25 - 75 ng / ml or approximately 50 ng / ml). The supernatant can be aspirated and replaced with fresh differentiation medium. Alternatively, the cells can be fed half with fresh medium every 2 days. The cells can be collected at different time points during the differentiation process.

[0082] HPC can be cultured from pluripotent stem cells using defined media. Using defined media, pluripotent cells are differentiated into hematopoietic CD34 + Methods for differentiating into hematopoietic stem cells are described, for example, in U.S. Patent Application Publication No. 12 / 715,136, which is hereby incorporated by reference in its entirety. It is expected that these methods can be used in conjunction with the present disclosure.

[0083] For example, hematopoietic CD34 +To induce differentiation, a defined medium can be used. The defined medium can contain growth factors BMP4, VEGF, Flt3 ligand, IL-3 and / or GMCSF. The pluripotent cells are cultured in a first defined medium containing BMP4, VEGF and optionally FGF-2, and subsequently can be cultured in a second medium containing either (Flt3 ligand, IL-3 and GMCSF) or (Flt3 ligand, IL-3, IL-6 and TPO). The first and second media can also contain one or more of SCF, IL-6, G-CSF, EPO, FGF-2 and / or TPO. Substantially hypoxic conditions (e.g., less than 20% O2) can further promote hematopoietic or endothelial differentiation.

[0084] The cells can be substantially individualized via mechanical or enzymatic means (e.g., using trypsin or TrypLE™). A ROCK inhibitor (e.g., H1152 or Y-27632) can also be included in the medium. It is expected that these procedures can be automated, for example, using robotic automation.

[0085] In certain embodiments, substantially hypoxic conditions can be used to promote the differentiation of pluripotent cells into hematopoietic progenitor cells. As would be understood by one of ordinary skill in the art, an atmospheric oxygen content of less than about 20.8% would be considered hypoxic. Human cells in culture can be grown under atmospheric conditions having a reduced oxygen content compared to the outside air. This relative hypoxia can be achieved by reducing the atmospheric oxygen exposed to the medium. Embryonic cells typically occur in vivo under hypoxic conditions where the atmospheric oxygen is generally about 1% to about 6% and the carbon dioxide is at ambient levels. Without wishing to be bound by theory, it is predicted that hypoxic conditions can reproduce aspects of certain embryonic developmental conditions. As shown in the examples below, in certain embodiments, hypoxic conditions can be used to promote further differentiation of pluripotent cells, such as iPSCs or hESCs, into more differentiated cell types, such as HPCs.

[0086] To promote the differentiation of pluripotent cells into hematopoietic progenitor cells, the following hypoxic conditions can be used. In certain embodiments, to promote the differentiation into hematopoietic progenitor cells, an atmospheric oxygen content of less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3%, about 2% or about 1% can be used. In certain embodiments, the hypoxic atmosphere contains about 5% oxygen gas.

[0087] In certain embodiments, to promote the differentiation into hematopoietic progenitor cells, a dissolved oxygen content of less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, about 25%, about 20%, about 15%, about 10% or about 5% can be used. In certain embodiments, the dissolved oxygen content level is about 25% oxygen.

[0088] Irrespective of the specific medium being used in the expansion culture of any given hematopoietic progenitor cell, the medium used is preferably supplemented with at least one cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more typically 10 ng / mL to 100 ng / mL. Suitable cytokines include, but are not limited to, c-kit ligand (KL) (also referred to as stem cell factor (StI), mast cell growth factor (MGF) and stem cell factor (SCF)), IL-6, G-CSF, IL-3, GM-CSF, IL-1α, IL-11, MIP-1α, LIF, c-mpl ligand / TPO and flk2 / flk3 ligand (Flt2L or Flt3L). In particular, the culture will comprise at least one of SCF, Flt3L and TPO. More specifically, the culture will comprise SCF, Flt3L and TPO.

[0089] In some embodiments, the HPCs exhibit disrupted methyl-CpG binding protein 2 (MeCP2) and are cultured under conditions to promote myeloid or lymphoid differentiation. In some aspects, the HPCs express a non-functional MeCP2 that essentially does not have binding to methylated DNA. In certain aspects, the HPCs do not express MeCP2 at levels sufficient to confer MeCP2 DNA binding activity. In certain aspects, MeCP2 is non-functional due to a cleavage or mutation in the MeCP2 gene. In some aspects, obtaining HPCs that exhibit disrupted MeCP2 includes contacting the HPCs with an siRNA, shRNA, or small molecule inhibitor of MeCP2.

[0090] D. Forward programming Certain embodiments of the present disclosure provide HPCs by forward programming of CAR-PSCs through the expression of a combination of programming genes important for hematopoietic cell differentiation / function. In one method, the PSCs are modified to express at least three hematopoietic precursor programming genes, such as ETS genes (e.g., ETV2 or ERG), hematopoietic development genes (e.g., GATA2), and homeobox genes (e.g., HOXA9), as described in International Application PCT / US Patent Application Publication No. 2016 / 057893, which is hereby incorporated by reference in its entirety. In certain aspects, the ETV2 / ERG, GATA2, and HOXA9 genes are co-expressed by a single vector, such as an inducible PiggyBac vector, using a bidirectional Tight promoter transfected into the CAR-PSCs.

[0091] Furthermore, the EGH-CAR-PSCs can be further modified to express additional genes for long-term engraftment ability. Exemplary genes include HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, and / or RBAK. For example, the PSCs can be transfected with one or more vectors to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, and HOXA4.

[0092] Preferably, the ETV2 / GAT2 / HOXA9 gene is expressed for a period sufficient for the forward program of PSCs into hematopoietic progenitor cells. Thus, the programming gene of the hematopoietic progenitor can be under the control of an inducible promoter. Thus, the expression of the programming gene of the hematopoietic progenitor can be induced in PSCs for a period sufficient for the forward program for multi-lineage hematopoietic progenitor cells. That period can be from about 1 to about 20 days, such as about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days. Alternatively, the programming gene of the hematopoietic progenitor can be introduced into PSCs by an episomal vector. Thus, the programming gene of the hematopoietic progenitor can be transiently expressed in PSCs.

[0093] E. antigen The CARs provided herein can have any antigen specificity useful in the treatment of a disease or disorder. Among the antigens targeted by the genetically modified antigen receptor are those expressed in the context of a disease, condition, or cell type targeted for treatment via adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders such as cancer and tumors, such as hematologic cancers, cancers of the immune system such as lymphoma, leukemia, and / or myeloma, such as B, T, and myeloid leukemia, lymphoma, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition, such as a tumor or pathogenic cell, compared to normal or non-targeted cells or tissues, and is an antigen associated with autoimmunity or alloimmune abnormalities or a pathogen-specific antigen. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0094] Any suitable antigen may be found for use in the methods. Exemplary antigens include, but are not limited to, antigen molecules from infectious pathogens, auto / self antigens, tumor / cancer-associated antigens, and tumor neoantigens. In certain aspects, antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA.

[0095] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma cancer, ovarian cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4; 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. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate acid phosphatase, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0096] Other tumor-related antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, the tumor antigen can be a self-peptide hormone, for example, the full-length gonadotropin-releasing hormone of a short 10-amino acid long peptide, which is useful in the treatment of many cancers.

[0097] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as the expression of HER-2 / neu. The tumor-associated antigens of interest include lineage-specific tumor antigens such as melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related protein. Exemplary tumor-associated antigens include, but are not limited to, p53, 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, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, 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, β-catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250, 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) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2).Nuclear factor κB (NF-κB), Notch receptors (e.g., Notch1-4), 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-α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), 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, RGsS, 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 tumor antigens derived from an idiotype or comprising any one or more of them.

[0098] Antigens include epitope regions or epitope peptides derived from genes mutated within tumor cells or genes transcribed at different levels within tumor cells compared to normal cells, such as the telomerase enzyme, survivin, mesothelin, mutant ras, rearrangement of bcr / abl, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and abnormally expressed intron sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangement of immunoglobulin genes that generate idiotypes unique to multiple myeloma and B-cell lymphoma; tumor antigens including epitope regions or epitope peptides derived from the processes of oncogenic viruses, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated cancer fetal proteins with tumor-selective expression, such as cancer fetal antigen and alpha-fetoprotein.

[0099] In other embodiments, the antigen is obtained or derived from pathogenic microorganisms or opportunistic pathogenic microorganisms (also referred to herein as infectious microorganisms), such as viruses, fungi, parasites, and bacteria. In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0100] Exemplary pathogenic organisms for which the use in the methods described herein for an antigen is contemplated 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, Staphylococcus species, such as Methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, such as Streptococcus pneumoniae. As will be understood by those skilled in the art, the proteins and nucleotide sequences encoding proteins from these and other pathogenic microorganisms for use as an antigen as described herein can be identified in publications and public databases such as GENBANK®, Swiss-Prot®, and TrEMBL®.

[0101] An antigen derived from human immunodeficiency virus (HIV) includes 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.

[0102] Examples of antigens derived from herpes simplex virus (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes mainly encodes proteins that form virion particles. Such proteins include five proteins from (UL) that form the viral capsid: UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins being considered for use as antigens herein include the proteins of ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD). The HSV genome contains at least 74 genes, each of which encodes a protein potentially usable as an antigen.

[0103] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during the immediate-early and early phases of 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 from a cluster of genes from UL128 - UL150, and envelope glycoproteins B (gB), gH, gN, and pp150. As would be understood by one of ordinary skill in the art, CMV proteins for use as antigens as described herein can be identified in public databases such as GENBANK®, Swiss-Prot®, and TrEMBL®.

[0104] Antigens derived from Epstein - Barr virus (EBV) being considered for use in certain embodiments include the EBV lytic proteins gp350 and gp110, EBV proteins produced during latent cycle infection, such as 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.

[0105] Antigens derived from respiratory syncytial virus (RSV) under consideration for use in this specification include any one of 11 proteins encoded by the RSV genome or antigenic fragments thereof: 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 regulation), RNA polymerase, and phosphorylated protein P.

[0106] Antigens derived from vesicular stomatitis virus (VSV) under consideration for use include any one of 5 major proteins encoded by the VSV genome and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphorylated protein (P), and matrix protein (M).

[0107] Antigens derived from influenza virus under consideration 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.

[0108] Exemplary viral antigens include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoprotein, core or non-structural protein), herpesvirus polypeptides (including herpes simplex virus or varicella-zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptide), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0109] In certain embodiments, the antigen can be a bacterial antigen. In certain embodiments, the bacterial antigen of interest can be a secreted polypeptide. In other certain embodiments, the bacterial antigen includes an antigen having one or more portions of a polypeptide exposed on the outer cell surface of the bacteria.

[0110] Antigens from Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA) under consideration for use, include virulence regulators such as the Agr system, Sar and Sae, Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), Srr system and TRAP. Other Staphylococcus proteins that can function as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes (N315 and Mu50) in two strains of Staphylococcus aureus have been sequenced and are publicly available, for example, in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As would be understood by those skilled in the art, Staphylococcus proteins for use as antigens can also be identified in other public databases such as GENBANK®, Swiss-Prot® and TrEMBL®.

[0111] Antigens 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. The complete genome sequence of virulent strains of Streptococcus pneumoniae has 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®. Proteins of particular interest as antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of pneumococci.

[0112] Examples of bacterial antigens that can be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., Borrelia burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., Haemophilus influenzaeinfluenza type b outer membrane protein), Helicobacter polypeptide, Klebsiella polypeptide, L-form bacteria polypeptide, Leptospira polypeptide, Listeria polypeptide, Mycobacteria polypeptide, Mycoplasma polypeptide, Neisseria polypeptide, Neorickettsia polypeptide, Nocardia polypeptide, Pasteurella polypeptide, Peptococcus polypeptide, Peptostreptococcus ococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides) (see discussion herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., S. pyogenes M protein), group B streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis F1 and V antigens).

[0113] Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides,Examples include Trichosporon polypeptides and Xylohypha polypeptides.

[0114] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides,Haemonchus polypeptide, Lagochilascaris polypeptide, Loa polypeptide, Mansonella polypeptide, Muellerius polypeptide, Nanophyetus polypeptide, Necator polypeptide, Nematodirus polypeptide, Oesophagostomum polypeptide, Onchocerca polypeptide, Opisthorchis polypeptide, Ostertagia polypeptide, Parafilaria polypeptide, Paragonimus polypeptide, Parascaris polypeptide, Physaloptera polypeptide, Protostrongylus polypeptide, Setaria polypeptide, Spirocerca polypeptide, Spirometra polypeptide, Stephanofilaria polypeptide, Strongyloides polypeptide, Strongylus polypeptide, Thelazia polypeptide, Toxascaris polypeptide, Toxocara polypeptide, Trichinella polypeptide, Trichostrongylus polypeptide, Trichuris polypeptide, Uncinaria polypeptide and Wuchereria polypeptide (for example, around the sporozoite of Plasmodium falciparum (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver-stage antigen 1 (PfLSA1 c-term) and transport protein 1 (PfExp-1), Pneumocystis polypeptide, Sarcocystis polypeptide, Schistosoma polypeptide,Examples include Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0115] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) derived from fleas; ticks, such as Ixodes and Ornithodoros; flies, such as midges, mosquitoes, horse flies, black flies, buffalo gnats, sand flies, stable flies, biting flies, tsetse flies, blow flies, and bot flies; ants; spiders, lice; mites; and true bugs, such as bed bugs and boxelder bugs.

[0116] F. Chimeric antigen receptors In some embodiments, the CAR has an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, similar to a TCR.

[0117] In some embodiments, the chimeric antigen receptor comprises a) an intracellular signaling domain, b) a hinge and transmembrane domain, and c) an extracellular domain comprising an antigen-binding region.

[0118] In some embodiments, the modified antigen receptor comprises a chimeric antigen receptor (CAR) including an activating or stimulatory CAR, a co-stimulatory CAR (see WO 2014 / 055668 pamphlet) and / or an inhibitory CAR (see Fedorov et al., 2013). Generally, in some aspects, the CAR comprises an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components via a linker and / or transmembrane domain. Such molecules typically recapitulate or approximate signals through the native antigen receptor, signals through such receptors in combination with co-stimulatory receptors and / or signals through co-stimulatory receptors alone.

[0119] Certain embodiments of the present disclosure relate to the use of a nucleic acid encoding an antigen-specific CAR polypeptide, comprising a humanized CAR (hCAR) that is humanized to reduce 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 the complementarity determining regions of a monoclonal antibody, the variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0120] The human CAR nucleic acid can be considered a human gene used to enhance cellular immunotherapy in human patients. In specific embodiments, the invention comprises a full-length CAR cDNA or coding region. The antigen-binding region or domain can be, for example, the V of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody as described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. H chain and V LIt may contain fragments of the lock. The fragments can also be several different antigen-binding domains of human antigen-specific antibodies. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage in human intracellular expression.

[0121] The construct can be multimeric, such as, for example, a bispecific antibody or a multimer. The multimer is most easily formed by cross-pairing of the variable portions of the light and heavy chains into a bispecific antibody. Since the hinge portion of the construct is entirely deleted, there can be multiple options such as maintaining the first cysteine, substitution with proline instead of serine, and cleavage 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, either the CH2 or CH3 domain from human immunoglobulin, can be used. The hinge, CH2, and CH3 regions of human immunoglobulin that have been modified to improve dimerization can also be used. Even only the hinge portion of the immunoglobulin can be used. A portion of CD8α can also be used.

[0122] In some embodiments, the CAR nucleic acid contains sequences encoding other co-stimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Examples of other co-stimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137).

[0123] In some embodiments, a chimeric antigen receptor (CAR) is constructed that has specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, such as a cancer marker and / or an antigen intended to induce a hyporesponse, such as an antigen expressed in a normal or non-diseased cell type. Thus, the CAR typically includes, in its extracellular portion, one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains or portions or one or more antibody variable domains and / or antibody molecules. In some embodiments, the CAR includes one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (V H ) chain and variable light (V L ) chain of a monoclonal antibody (mAb).

[0124] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain comprising the antigen-binding region) comprises a tumor-associated antigen or pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors, such as dectin 1. Tumor-associated antigens can be of any type as long as they are expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD319 (CS1), 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 when the tumor-associated antigen is present in low amounts. For example, the CAR can be co-expressed with IL-15.

[0125] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or combinations thereof. Since introns have been found to stabilize mRNA, it may be desirable to use cDNA or combinations thereof, depending on the size of the genomic DNA and the number of introns. Additionally, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0126] The chimeric construct is considered to be introducible into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. Naked DNA generally refers to DNA encoding a chimeric receptor contained within a plasmid expression vector in the appropriate orientation for expression.

[0127] Alternatively, 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 within immune cells. When the copy number of the virus maintained within the cell is low enough to maintain cell viability, a number of virus-based vectors are known, such as vectors based on HIV, SV40, EBV, HSV, or BPV. <S

[0128] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. Optionally, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domains of the same or different surface membrane proteins of such domains and to minimize interaction with other members of the receptor complex.

[0129] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. When the source is natural, in some aspects the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, and GITR / CD357 molecules (i.e., including at least their transmembrane regions). Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, a triple of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0130] III. Methods of Use In some embodiments, the present disclosure provides a method for immunotherapy, the method comprising administering an effective amount of immune cells expressing the CAR of the present disclosure. In one embodiment, a medical disease or disorder is treated by transplantation of an immune cell population that induces an immune response. In certain embodiments of the present disclosure, cancer or an infection is treated by transplantation of an immune cell population that induces an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, 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 viral infections.

[0131] In certain embodiments, provided is a method for treating a cancer patient by administering immune cells expressing the CAR provided herein that have an antigen-binding domain specific for an antigen expressed by the cancer, such as NK cells and / or T cells.

[0132] Tumors for which the present treatment methods are useful include those found in any malignant cell type, such as solid tumors or hematological tumors. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovaries, 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 may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, gastric 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 or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0133] Cancer specifically includes, but is not limited to, the following histological types: tumor, malignant; cancer; cancer, undifferentiated; giant cell cancer and spindle cell cancer; small cell cancer; papillary cancer; squamous cell cancer; lymphoepithelial cancer; basal cell cancer; hair matrix cancer; transitional cell cancer; papillary transitional cell cancer; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; small columnar adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid cancer; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic cancer; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic cancer; clear cell adenocarcinoma; granular cell cancer; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing cancer; adrenocortical carcinoma; endometrial cancer; skin appendage cancer; apocrine adenocarcinoma; sebaceous gland cancer; ceruminous gland cancer; mucoepidermoid cancer; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell cancer; invasive ductal carcinoma; medullary cancer; lobular cancer; inflammatory cancer; Paget's disease, breast; acinar cell cancer; adenosquamous cancer; adenocarcinoma associated with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; capsular cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell cancer; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficially spreading melanoma; malignant lentigo melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant congenital 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 cell tumor; embryonal cancer tumor; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; vascular endothelial tumor, malignant; Kaposi sarcoma; hemangioendothelioma, malignant; lymphangiosarcoma; osteosarcoma; parosteal 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; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Anaplastic glioma; Anaplastic glioblastoma; Undifferentiated neuroectodermal; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulocytic sarcoma, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; 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; Large tumor lesion NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myelogenous leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastoid leukemia; Myelosarcoma; Hairy cell leukemia; Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); And chronic myelogenous leukemia.

[0134] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by the abnormal proliferation (production by proliferation) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases referred to as hematological neoplasms. Leukemia is a broad term encompassing a spectrum of diseases. Leukemia is clinically and pathologically divided into its acute and chronic forms.

[0135] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, such as an individual having cancer or an infection. The cells then enhance the individual's immune system to attack each cancer or pathogenic cell. Optionally, one or more doses of immune cells are provided to the individual. When two or more doses of immune cells are provided to the individual, the duration between administrations needs to be sufficient to allow for growth in the individual over time. In a specific embodiment, the duration between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0136] 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 syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue 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, Ménière's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, nephrotic syndrome (such as 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 (such as polyarteritis nodosa, Takayasu arteritis, temporal arteritis / giant cell arteritis or herpes zoster vasculitis), vitiligo and Wegener's granulomatosis. Thus, some examples of autoimmune diseases treatable 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.

[0137] A therapeutically effective amount of immune cells can be administered by several routes, such as parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal or intra-articular injection or infusion.

[0138] The therapeutically effective amount of immune 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 bring about regression of an autoimmune or alloimmune disease, or the ability to reduce symptoms caused by an autoimmune disease, such as pain and inflammation. It can be the amount necessary to reduce symptoms associated with inflammation, such as pain, swelling, and increased temperature. It can also be the amount necessary to reduce or prevent rejection of a transplanted organ.

[0139] The immune cell population can be administered in a treatment regimen that is tailored to the disease, such as a single or multiple doses over one day to several days to achieve remission of the condition, or regular doses over an extended period to inhibit disease progression and prevent disease recurrence. The exact dose utilized in the formulation will also depend on the route of administration and the severity of the disease or disorder and will need to be determined according to the judgment of the practitioner and the circumstances of each patient. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dosage range that may be considered useful in the treatment of human subjects is at least 3.8×10 4 cells, at least 3.8×10 5 cells, at least 3.8×10 6 cells, at least 3.8×10 7 cells, at least 3.8×10 8 cells, at least 3.8×10 9 cells or at least 3.8×10 10 immune cells / m 2 . 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 immune cells / m 2 . In further embodiments, the therapeutically effective amount of immune cells is from about 5×10 6 cells / kg body weight to about 7.5×10 8 cells / kg body weight, such as 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 immune cells can be readily determined by one of ordinary skill in the art based on the age, weight, gender, and physiological state of the subject. The effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0140] The immune cells can be administered in combination with one or more other therapeutic agents for the treatment of immune-mediated disorders. The combination therapies include, but are not limited to, one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune depletion 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, such as hydrocortisone, dexamethasone, or prednisone, or non-steroidal anti-inflammatory agents, such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin 10 or transforming growth factor β), hormones (e.g., estrogen), or vaccines. Further, immunosuppressive or immunomodulatory agents, including but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., those recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceuticals can be administered before, during, or after the administration of the immune cells, depending on the desired effect. This administration of the cells and agents can be by the same route or different routes, and at the same site or different sites.

[0141] B. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising immune cells (e.g., T cells or NK cells) and a pharmaceutically acceptable carrier.

[0142] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (such as an antibody or polypeptide) having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 22 nd edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin, etc.; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0143] C. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment include an immune cell population combined with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., breast tumor excision and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0144] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an anti-nausea agent, etc.). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapeutic agent targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more of the chemotherapeutic agents known in the art.

[0145] Immune cell therapy can be administered before, during, after, or in various combinations with additional cancer therapies, such as immune checkpoint therapy. The administration can be at intervals ranging from simultaneous to several minutes, days, and weeks. In embodiments where immune cell therapy is provided to a patient separately from an additional therapeutic agent, generally, a significant period is ensured not to be interrupted between each delivery time point so that the two compounds can further exert an advantageously combined effect on the patient. In such cases, it is considered that antibody therapy and anti-cancer therapy can be provided to the patient within about 12 hours to about 24 hours or within about 72 hours of each other, more particularly within about 6 to 12 hours of each other. Depending on the situation, when several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration, it may be desirable to significantly extend the treatment period.

[0146] Various combinations can be utilized. The following examples: 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 In the following, the CAR immune cell therapy is "A", and the anti-cancer therapy is "B".

[0147] In the administration of any compound or therapeutic agent of the present embodiment to a patient, in consideration of the toxicity of the drug (if any), the general protocol for the administration of such a compound will be followed. Thus, in some embodiments, a step of monitoring the toxicity that may result from the combination therapy is provided.

[0148] 1. Chemotherapy A variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of agents to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified, for example, by their mode of activity within cells, such as whether they affect the cell cycle and at which stage they do so. Alternatively, drugs can be characterized based on their ability to induce chromosomal and mitotic abnormalities, such as by directly cross-linking to DNA, intercalating into DNA, or affecting nucleic acid synthesis.

[0149] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (especially bradycidin and bradycidinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, nobemycin, phenesterine, prednimustine, trophosphamide and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enzyin antibiotics chromophore, actinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardifilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, tubercidin, ubenimex, dinostatin and zorubicin; antimetabolites, for example methotrexate and 5-fluorouracil (5-FU); folic acid analogues, for example denopterin, pteropterin and trimetrexate; purine analogues, for example fludarabine, 6-mercaptopurine, thiampurine and thioguanine; pyrimidine analogues, for example ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine and floxuridine; androgens, for example calusterone, drostanolone propionate, epitostanol, mepitiostane and testolactone; antiadrenal substances, for example mitotane and trilostane; folic acid supplements, for example folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, for example maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rizoxin; schizophyllan;Spirogyrnium; tenuazonic acid; triazicon; 2,2’,2”-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, such as paclitaxel and docetaxel · gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; zeloda; ibandronic acid; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum and pharmaceutically acceptable salts, acids or derivatives of any of the above are included.;

[0150] 2. Radiation therapy Other elements that cause DNA damage and are widely used generally include direct delivery of γ-rays, those known as X-rays and / or radioactive isotopes to tumor cells. Other forms of DNA damage elements, such as microwaves, proton beam irradiation and UV irradiation, are also considered. All of these elements are very likely to affect extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. The dose in X-rays ranges from a daily dose of 50 to 200 roentgens over a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range in radioactive isotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by neoplastic cells.;

[0151] 3. Immunotherapy 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 embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example of such. An immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone can serve as an effector of treatment, or it can recruit other cells to actually affect cell death. 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 carrying surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0152] 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 MAbs for their antigen targets with highly potent cytotoxic drugs to yield "armed" MAbs that deliver the payload (drug) to tumor cells having abundant levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0153] In one aspect of immunotherapy, tumor cells need to be adaptable for targeting, i.e., have some markers that are not present in most other cells. There are numerous 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 the anti-cancer effect with an immunostimulatory effect. There are also immunostimulatory molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0154] Examples of immunotherapeutic agents 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 utilized in conjunction with the antibody therapy described herein.

[0155] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints upregulate or downregulate signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte 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, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0156] The immune checkpoint inhibitor can be an agent such as a small molecule, a recombinant form of a ligand or receptor, or particularly an antibody, e.g., a human antibody. Known inhibitors of immune checkpoint proteins or their analogs are used, particularly those that can be chimerized, humanized, or the human form of the antibody can be used. As will be understood by those skilled in the art, alternative and / or equivalent names may be used for the specific antibodies described in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is recognized that pembrolizumab is also known under the alternative and equivalent names MK-3475 and lambrolizumab.

[0157] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 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.

[0158] 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 the extracellular or PD-1 binding portion of PDL1 or PDL2 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® (registered trademark), is an anti-PD-1 antibody that can be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® (registered trademark) and SCH-900475, is an exemplary anti-PD-! 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.

[0159] [[ID=?]] 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 that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells through the T cell receptor and CD28 results in increased expression of CTLA-4, an inhibitory receptor on the B7 molecule.

[0160] 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.

[0161] An anti-human CTLA-4 antibody (or V derived therefrom) suitable for use in the method H and / or V LThe (domain) can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies known 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 and light chain CDRs or 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 with and / or binds to the same epitope on CTLA-4 as the above antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibody (e.g., at least about 90%, 95% or 99% variable region identity with ipilimumab).

[0162] 4. Surgery Approximately 60% of people with cancer will undergo some type of surgery, including prophylactic, diagnostic or staging, curative and palliative surgery. Curative surgery includes excisions in which all or part of the cancerous tissue is physically removed, excised and / or destroyed, and can be combined with other treatments, such as the treatments of this embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery and microsurgery (Mohs surgery).

[0163] When part or all of the cancer cells, tissue or tumor are excised, cavities can form in the body. The treatment can be achieved by perfusion, direct injection or local application of additional anti-cancer therapy to the area. Such treatments 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 in various doses.

[0164] 5. Other Agents To improve the therapeutic effect of the treatment, it is contemplated that other agents may be used in combination with specific aspects of the present embodiment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell division arrest agents and differentiating agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing factors, or other biological agents. Enhancement of intercellular signaling by increasing the number of gap junctions will enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell division arrest agent or differentiating agent may be used in combination with specific aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are thought to improve the effectiveness of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. Further, to improve treatment effectiveness, other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, are contemplated to be combinable with specific aspects of the present embodiment.

[0165] IV. Product or Kit Products or kits containing immune cells are also provided herein. The product or kit may further include an accompanying document containing instructions for using the immune cells for treating or delaying the progression of cancer in an individual or enhancing the immune function of an individual having cancer. Any of the 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 can be formed from various materials such as glass, plastic (such as polyvinyl chloride or polyolefin) or alloy (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation and the label thereon, or in relation thereto, the container may display instructions for use. The product or kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes and accompanying documents with instructions for use. In some embodiments, the product further includes one or more other agents (such as chemotherapeutic agents and anti-neoplastic agents). Containers suitable for one or more agents include, for example, bottles, vials, bags and syringes.

Examples

[0166] To demonstrate preferred embodiments of the present invention, the following examples are included. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that have been found by the inventors to function well in the practice of the present invention and thus can be considered to constitute preferred modes in its practice. However, those skilled in the art will understand that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed and still obtain similar or analogous results without departing from the spirit and scope of the present invention.

[0167] Example 1 - Activation of Protein L and Expansion Culture of CAR-T Cells Protein L induces the expansion culture of CAR-T cells: E11 pluripotent stem cells (TiPSCs) reprogrammed from peripheral blood T cells using an episomal vector were modified for the constitutive expression of a second-generation anti-human CD19 chimeric antigen receptor (CAR) consisting of a human CD19-binding scFv domain derived from the FMC63 monoclonal antibody, CD28 co-stimulation, and CD3ζ signaling domains.

[0168] Non-modified and CAR-modified E11 TiPSCs were differentiated into T / NK CD34 + progenitor cells through cytokine-induced differentiation. The isolated CD34+ progenitor cells were further differentiated into CD3+ T cells in a T cell differentiation medium (TCDM) consisting of StemSpan SFEM (Stem Cell Technologies) supplemented with magnesium ascorbate phosphate (0.25 mM), nicotinamide (2 mM), GlutaMax (Gibco), and cytokines (SCF, TPO, FLT3L, IL7; 50 ng / ml each) on a DLL4 / retronectin-coated plate in a low-oxygen culture for 2 weeks.

[0169] CAR-T cells were cultured on plates coated with different concentrations of Protein L (Pierce; 0, 0.1, 0.5, 2.5 μg / cm + in the presence or absence of IL2 and IL15 (both 10 ng / mL) in TCDM at a density of 10,000 CD3 2 T cells / cm 2 ) and human DLL4-Fc (Acro Biosystems; 0.5 μg / cm 2 ). The cultures were maintained under low-oxygen (5% O2) conditions and harvested after 10 days. All harvested cells were counted, the percentage of CD3 2 cells was determined, and the T cell expansion fold was calculated (output / input T cell number). +

[0170] Protein L induced the expansion culture of CAR-T cells in a dose-dependent manner, while Retronectin enhanced the proliferative response (Figure 1). DLL4 is a complementary T cell growth factor that further significantly improved the protein L-dependent expansion culture of CAR-T cells. In parallel, since normoxic cultures showed a decrease in the expansion amount in the test variants, a hypoxic environment was used to achieve efficient CAR-T cell expansion culture.

[0171] DLL4 promotes the expansion culture of CD8+ CAR-T: The expression of CD8α was determined in CAR-T cells collected from the 10-day expansion culture with protein L combined with Retronectin and DLL4-Fc (all used at 0.5 μg / cm 2 ) (Figure 2).

[0172] The combination of protein L and Retronectin resulted in an efficient CAR-T expansion culture compared to protein L alone. However, despite a much higher expansion rate, the expression of CD8 remained low. In contrast, when DLL4 was added, there was an additional approximately 25% improvement in the CAR-T expansion culture rate, along with the expression of CD8α in a significant proportion of CD3 + T cells (Figure 2).

[0173] Expansion culture of CAR-specific T cells induced by protein L: The specificity of CAR-induced T cell expansion culture in protein L cultures was verified with CAR-modified vs. unmodified T cells seeded for expansion culture in both CD3-induced pan-T cells (control) and protein L-induced expansion cultures. CAR-modified and unmodified E11-derived T cells were cultured on plates coated with protein L (0.5 μg / cm 2 ) or Retronectin and DLL4-Fc (both in the respective culture variants at 0.5 μg / cm 2 ) along with anti-CD3 monoclonal antibody (OKT3 clone; 0.5 μg / cm 2 ). In TCDM supplemented with IL2 and IL15 (both at 10 ng / ml), 10,000 CD3 + T cells / cm 2Cells were seeded at a density of. After 8 days, the cells were collected, counted, the percentage of CD3 + was determined, and the T cell expansion ratio was calculated (output / input T cell number) (Figure 3).

[0174] In contrast to CD3-activated cultures in which both T cells and CAR-T cells were expanded with similar efficiency, exclusive expansion cultures of CAR-T cells were observed in protein L cultures. The efficiency of CAR-T cell expansion cultures in anti-CD3 and protein L cultures was equivalent. When the expression of CAR in the expanded CAR-T cells was determined by protein L staining, CAR + T cells were found at a significantly higher percentage, and positive selection of CAR + T cells in protein L cultures was shown.

[0175] Cytokine production in CAR-T cells expanded with protein L: CAR-T cells expanded for 8 days in anti-CD3 mAb cultures and protein L cultures were incubated with non-transfected P815 cells (control) and P815 cells transfected with the human CD19 CAR antigen. CAR-T effector (E) and P815 target (T) cells were added to the co-cultures at 10 5 cells / ml and 2×10 5 cells / ml (1:2 E / T ratio). After 24 hours, supernatants were collected for cytokine analysis by LegendPlex multiplex flow cytometry assay (BioLegend).

[0176] CAR-T co-cultures with non-transfected P815 cells did not show significant target cell-induced cytokine production, while CD19 +Cultures with P815 cells showed induced production of IFNγ, TNFα, granzyme B, sFasL, CCL3, CCL2, GM-CSF, IL2 and IL13, thus defining a set of cytokines produced in response to CAR activation. Cytokine secretion profiles were similar in CAR-T cells expanded in either anti-CD3 mAb cultures or protein L cultures, although higher levels of IFNγ and TNFα were detected in CAR-T cells expanded with protein L (Figure 4A).

[0177] Protein L-induced CAR-dependent in vitro cytokine production: Cytokine production induced by CAR activation was evaluated by incubating CAR-T cells with plastic-adsorbed protein L. E11 PSC-derived CAR-T cells were added to wells coated with protein L and retronectin (both at 0.5 μg / cm 2 ) or retronectin alone (control). Cells were seeded at a density of 20,000 cells / well in 0.5 mL of TCDM. After 24 hours of incubation, supernatants were collected for cytokine analysis by LegendPlex multiplex flow cytometry assay (BioLegend).

[0178] CAR-T cells produced granzyme B (GzmB), CCL3 and low levels of GM-CSF and IL13 during 24-hour incubation in wells coated with retronectin alone. When CAR-T cells were incubated in wells coated with protein L, induction of TNFα, IP10, IFNγ, sFasL and IL6 was detected, thus indicating a CAR-dependent cytokine production response induced by protein L (Figure 4B). Treatment of CAR-modified immune cells with protein L can be used to evaluate in vitro CAR-dependent functional responses, including cytokine production.

[0179] Cytotoxic activity of CAR-T cells expanded with Protein L: The cytotoxic function of CAR-T cells expanded for 8 days in anti-CD3 monoclonal antibody cultures and Protein L cultures was evaluated using an in vitro cytotoxicity assay against non-transfected P815 cells expressing luciferase (control), P815 cells transfected with the human CD19 CAR antigen, CD19+ B-cell lymphoma Daudi and Raji (Figure 5). CAR-T effector (E) cells were incubated with tumor target (T) cells at an E / T ratio of 1:2 for 24 hours, and luciferase activity in the culture lysate was quantified using the Steady-Glo luciferase assay reagent (Promega). The absolute number of target cells in the 24-hour cytotoxic culture was determined by calibration against serial dilutions of each target cell standard. Cytotoxicity was expressed as the absolute number of target cells lysed by a single effector cell and calculated by the formula: (T control - T experiment) / E, where "experiment" and "control" are the target cell cultures in the presence and absence of effector cells, respectively; T and E are the absolute numbers of target and effector cells, respectively. Furthermore, the cytotoxic function of CAR-T cells expanded in Protein L cultures was verified by real-time GFP+ cell counting using the Incucyte S3 live cell analysis system (Essen Biosciences) against Raji cells expressing GFP.

[0180] Minimal cytotoxicity was detected in cultures with CD19 CAR antigen-negative P815 cells, while both anti-CD3 monoclonal antibody- and Protein L-expanded CAR-T cells showed strong cytotoxic activity against CD19 + P815, Daudi, and Raji cells. In particular, CAR-T cells expanded with Protein L showed approximately 2- to 3-fold higher cytotoxic activity than CAR-T cells expanded in anti-CD3 monoclonal antibody cultures. The cytolytic activity of CAR-T cells expanded with Protein L was also confirmed in cultures with GFP + Raji cells, where GFP +A significant decrease in viable cell count was detected only in the cultures with CAR-T cells, but not in the cultures with non-modified T cells.

[0181] Anti-tumor ability of CAR-T cells expanded with Protein L: 8-week-old NSG mice were intraperitoneally (ip) injected with Daudi cells expressing luciferase. Four days later, when tumor cell inoculation was confirmed by control imaging, the mice were divided into a control (tumor only) and two experimental groups treated with CAR-T cells expanded in either anti-CD3 monoclonal antibody cultures or Protein L cultures. CAR-T cells were injected twice at 2-day intervals. IL2 and IL15 cytokines were injected (ip) into all mice during CAR-T injection and for an additional week. Tumor growth was monitored weekly by bioluminescence in vivo imaging. Anesthetized mice injected (ip) with In Vivo-Glo luciferin (Promega) were analyzed using a Pearl Trilogy in vivo imager (LiCor) within 15 minutes after luciferin injection. Images of mice in the designated groups are shown (left panel). Tumor growth was evaluated in vivo by quantification of bioluminescence signal intensity (BLI) using Image Studio software (LiCor). The graph shows the mean ± STD-BLI values over time for each group.

[0182] In mice treated with CAR-T cells, tumor growth was significantly suppressed over 6 weeks after CAR-T injection (Figure 6). Consistent with the in vitro cytotoxic ability, the in vivo anti-tumor effect was higher in CAR-T cells expanded in Protein L cultures.

[0183] All of the methods disclosed and claimed in this specification can be constructed and implemented without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or series of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related agents can be replaced with the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the scope of the spirit, scope and concept of the present invention as defined by the appended claims. References The following references are specifically incorporated herein by reference to the extent that they present exemplary procedural or other details supplementing the description herein. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994 International Patent Application Number International Application PCT / US Patent Application Publication No. 2016 / 057893 Remington’s Pharmaceutical Sciences 22 nd edition, 2012. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., 2001. US Patent Application Publication No. 12 / 715,136 US Patent No. 8,372,642 Zheng et al, J Transl Med, 10:29, 2012.

Claims

1. An in vitro method for activating and / or expanding CAR-modified immune cells selected from T cells and / or NK cells, comprising: (a) obtaining a starting population of CAR-modified immune cells derived from induced pluripotent stem cells (iPSCs) selected from T cells and / or NK cells; and (b) culturing the starting population of the iPSC-derived CAR-modified immune cells on a culture surface coated with (i) protein L and (ii) RetroNectin, which is a recombinant fibronectin fragment, for a period sufficient to generate a population of activated and / or expanded CAR-modified immune cells. The in vitro method as described above.

2. The method according to claim 1, wherein the culture surface is a culture plate, a culture flask, a microcarrier, microparticles, hydrogel particles, or a culture bag.

3. The method according to claim 1, wherein the culturing is performed in the absence of anti-CD3 antibody and / or antigen-specific target cells.

4. The method according to claim 1, wherein the T cells are CD8+ T cells, CD4+ T cells, αβ T cells, or γδ T cells.

5. The method according to claim 4, further comprising selecting for CD8+ T cells.

6. The method according to claim 1, wherein the iPSCs are iPSCs reprogrammed from blood cells.

7. In step (a), the induced pluripotent stem cells (iPSCs) are induced into CAR-modified immune cells and differentiated into CD34+ progenitor cells through cytokine-induced differentiation or forward programming to obtain a starting population of CAR-modified immune cells. The method according to claim 1.

8. The RetroNectin is coated on the culture surface at a concentration of 0.1 to 1 μg / cm 2 or 0.5 μg / cm 2 The method according to claim 1, wherein the coating is performed at the concentration of.

9. The method according to claim 1, wherein the culture surface is further coated with the Notch ligand DLL4.

10. The DLL4 is at a concentration of 0.1 to 1 μg / cm 2 or 0.5 μg / cm 2 The method according to claim 9, wherein the concentration is such.

11. The method according to claim 1, wherein the culturing is performed in the presence of IL-2 and / or IL-15.

12. The method according to claim 11, wherein the IL-2 and / or IL-15 are present at a concentration of 5-15 ng / mL.

13. The method according to claim 1, wherein the culturing is under hypoxic conditions.

14. The method according to claim 1, wherein the sufficient period is 8-12 days.

15. The method according to claim 1, wherein the culturing is in a medium containing SCF, TPO, FLT3L, and / or IL-7.

16. The method according to claim 15, wherein the SCF, TPO, FLT3L and / or IL-7 is at a concentration of 50 ng / mL.

17. The method according to claim 15, wherein the medium further contains nicotinamide.

18. The method according to claim 1, which results in selective expansion of CAR-modified immune cells compared to non-CAR-modified immune cells.

19. The method according to claim 18, wherein at least 40% or 50% of the expanded population of the CAR-modified immune cells are CAR-modified immune cells.

20. The method according to claim 1, wherein the expanded population of the CAR-modified T cells contains at least 25% CD3+CD8+ CAR-modified T cells.

21. The method according to claim 1, wherein the expanded population of the CAR-modified T cells has a cytotoxic activity 2 to 3 times higher, increased IFNγ and / or TNFα levels compared to CAR-modified T cells expanded with anti-CD3.

22. The method according to claim 1, wherein the CAR-modified immune cells are T cells.

23. The method according to claim 1, wherein the CAR-modified immune cells are NK cells.

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