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

Abstract

Provided herein are methods for activating and expanding T cells. Further provided are methods of using the T cells for therapy.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 733,291, filed September 19, 2018, which is incorporated herein by reference in its entirety. background 1. Technical Field

[0003] The present invention relates generally to the fields of immunology and medicine. More specifically, the present invention relates to methods and compositions for immune cells engineered to express chimeric antigen receptors.

[0004] 2. Description of Related Technologies

[0005] Adoptive cancer immunotherapy involves delivering immune cells modified with chimeric antigen receptors (CARs) specific for tumor antigens, which can be generated by in vitro amplification of CAR-modified T cells (CAR-T). Currently, CAR-T cells are amplified in vitro using traditional methods using CD3-activating antibodies (Abs). In this method, all T cells are induced to proliferate regardless of whether CAR is expressed or not, and not in a CAR-specific manner. This method can result in "silencing" of CAR expression and function in CD3-expanded CAR-T populations.

[0006] Although CAR-T cells can be selectively expanded in culture using CAR-specific tumor antigens expressed in isolated form or by feeder cells, antigen culture is only applicable to specific CAR types. This approach is not universal or practical, especially for therapeutic cell production. Therefore, similar to the pan-T cell expansion culture based on CD3 activation, the field of CAR-T cell therapy would benefit from a universal pan-CAR-specific in vitro culture in which CAR-T cells can be activated and expanded in a CAR-specific manner. Summary of the Invention

[0007] In a first embodiment, the present invention provides a method for activating and / or expanding CAR-modified immune cells in vitro, 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 sufficient time to produce a population of activated and / or expanded CAR-modified immune cells.

[0008] In certain aspects, protein L is present at 0.1-5 μg / cm 2 , such as 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 2Specifically, protein L can be present at a concentration of 1-5 μg / mL, such as 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 μg / mL. In some aspects, protein L is coated on a culture surface. For example, the culture surface can be a culture plate, culture bottle, microcarrier, microparticle, hydrogel particle, or culture bag.

[0009] In some aspects, the culture does not contain anti-CD3 antibodies and / or antigen-specific target cells. In certain aspects, the CAR-modified immune cells are T cells, NK cells, dendritic cells, and / or macrophages. In specific aspects, the T cells are CD8 + T cells, CD4 + T cells, αβT cells or γδT cells. In some aspects, the method further comprises selecting CD8 + T cells.

[0010] In some aspects, the immune cells modified by CAR- are allogeneic. In other aspects, the immune cells modified by CAR- are autologous. In specific aspects, the immune cells modified by CAR- are derived from pluripotent stem cells (PSC). In specific aspects, PSC is an induced pluripotent stem cell (iPSC). In some aspects, iPSC is reprogrammed by blood cells or T cells. In specific aspects, iPSC is reprogrammed by epitopes. In some aspects, the immune cells modified by CAR- are derived from primary peripheral blood mononuclear cells (PBMC) or primary hematopoietic stem cells. In specific aspects, iPSC is differentiated into CD34 by cytokine-directed differentiation. + In a specific aspect, iPSCs are differentiated into CD34 + Progenitor cells. For example, CAR can include an antigen binding domain selected from the group consisting of F(ab')2, Fab', Fab, Fv and scFv. In some aspects, CAR includes a CD28 costimulatory domain and a CD3ζ signaling domain.

[0011] In some aspects, the culture surface is further coated with recombinant fibronectin, fibronectin or VCAM1. In specific aspects, recombinant fibronectin is coated at 0.1-1 μg / cm 2 , such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 In another aspect, the culture plate is further coated with Notch ligand DLL4. In a specific aspect, DLL4 is added to the culture at a concentration of 0.1-1 μg / cm 2 Such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2or higher concentrations were added to the culture.

[0012] In other aspects, the culture further comprises IL-2 and / or IL- 15. In some aspects, IL-12 and / or IL-15 are present at a concentration of 5-15 ng / mL, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ng / mL or more.

[0013] In certain aspects, the culture is under hypoxic conditions. In some aspects, the hypoxic conditions comprise 5% oxygen.

[0014] In some aspects, the sufficient time is 8-12 days, such as 8, 9, or 10 days. In some aspects, the culture is carried out in a culture medium containing SCF, TPO, FLT3L, and / or IL-7. In specific aspects, the concentration of SCF, TPO, FLT3L, and / or IL-7 is 50 ng / mL. In some aspects, the culture medium further contains nicotinamide.

[0015] In certain aspects, the method produces selective expansion of CAR-modified immune cells compared to non-CAR-modified immune cells. In some aspects, at least 40% or 50% of the expanded population of CAR-modified immune cells are CAR-modified immune cells. In some aspects, the expanded population of CAR-modified T cells comprises at least 25% CD3 + CD8 + CAR-modified T cells. In a specific aspect, the amplified population of CAR-modified T cells comprises 2-3 times higher cytotoxic activity than CAR-modified T cells amplified with anti-CD3. In some aspects, the amplified population of CAR-modified T cells comprises increased IFNγ and / or TNFα levels compared to CAR-modified T cells amplified with anti-CD3.

[0016] In another embodiment, there is provided herein a colony of activated and / or expanded CAR-modified immune cells of each embodiment and various aspects thereof. Further provided herein is a pharmaceutical composition comprising a colony of CAR-modified immune cells of the embodiment and a pharmaceutically acceptable carrier.

[0017] The present disclosure further provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of amplified CAR-modified immune cells of the embodiment. In some aspects, the CAR-modified immune cells are allogeneic. In other aspects, the CAR-modified immune cells are autologous.

[0018] In other aspects, the method further includes administering at least a second therapeutic agent. In some aspects, the at least second therapeutic agent is a therapeutically effective amount of an immunomodulator or immunosuppressant. In some aspects, the at least second therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy and immunotherapy. In specific aspects, the CAR-modified immune cells and / or the at least second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, or by direct injection or infusion.

[0019] The compositions of the activated and / or amplified CAR-modified immune cells of each embodiment provided herein are used to treat 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.

[0020] Further provided herein are compositions comprising CAR-modified immune cells and protein L. In certain aspects, protein L is administered at a concentration of 0.1-5 μg / cm 2 , such as 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 Specifically, protein L can be present at a concentration of 1-5 μg / mL, such as 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 μg / mL. In some aspects, protein L is coated on a culture surface. For example, the culture surface can be a culture plate, culture bottle, microcarrier, microparticle, hydrogel particle, or culture bag.

[0021] In some aspects, the culture surface is further coated with recombinant fibronectin, fibronectin or VCAM1. In specific aspects, recombinant fibronectin is coated at 0.1-1 μg / cm 2 , such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 In another aspect, the culture plate is further coated with Notch ligand DLL4. In a specific aspect, DLL4 is added to the culture at a concentration of 0.1-1 μg / cm 2 , such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / cm 2 or higher concentrations were added to the culture.

[0022] In some aspects, the culture does not contain anti-CD3 antibodies and / or antigen-specific target cells. In specific aspects, the CAR-modified immune cells are T cells, NK cells, dendritic cells and / or macrophages. In specific aspects, the T cells are CD8 + T cells, CD4 + In certain aspects, the method further comprises selecting CD8 + T cells.

[0023] In a specific 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 (PSC). In a specific aspect, PSC is an induced pluripotent stem cell (iPSC). In some aspects, the iPSC is reprogrammed by blood cells or T cells. In a specific aspect, iPSC is reprogrammed epitopically. In some aspects, the CAR-modified immune cells are derived from primary peripheral blood mononuclear cells (PBMC) or primary hematopoietic stem cells. In a specific aspect, the iPSC is differentiated into CD34 by cytokine-directed differentiation. + In a specific aspect, the iPSCs are differentiated into CD34 + For example, the CAR may include 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 costimulatory domain and a CD3ζ signaling domain.

[0024] Other objects, features and advantages of the present invention will become clear from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become clear to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0026] Figure 1 Protein L induces CAR-T cell expression in a dose-dependent manner. Figure 3 shows T cell fold expansion in the presence of Protein L alone or in combination with recombinant fibronectin and / or DLL4.

[0027] Figure 2: The combination of Protein L and recombinant fibronectin resulted in efficient CAR-T expansion compared to Protein L alone. Shown is flow cytometric analysis of CD8 and CD3 expression on T cells expanded in the presence of Protein L alone or in combination with recombinant fibronectin and / or DLL4.

[0028] Figure 3 : Comparison of T cell expansion using anti-CD3 monoclonal antibodies compared to protein L.

[0029] Figures 4A-4B :( Figure 4A ) and CD19 + Culture of P15 cells revealed inducible production of IFNγ, TNFα, granzyme B, sFasL, CCL3, CCL2, GM-CSF, IL2, and IL13. Comparison of cytokine production during T cell expansion using anti-CD3 monoclonal antibodies compared to protein L. ( Figure 4B ) CAR-dependent in vitro cytokine production induced by protein L.

[0030] Figure 5 : Anti-CD3 mAb and protein L-expanded CAR-T cells both showed anti-CD19 + Strong cytotoxic activity against P815, Daudi and Raji cells.

[0031] Figure 6 : Tumor growth in mice treated with CAR-T cells up to 6 weeks after CAR-T injection. DETAILED DESCRIPTION

[0032] Protein L is a bacterial protein that has a unique specificity for binding to the light chain of an immunoglobulin, which includes a minimal antigen-binding portion - a single-chain variable domain fragment (scFv), which constitutes the antigen binding site of CAR. It has been shown that protein L can bind to cell surface CAR (Zheng et al., 2012). In addition, Zheng et al. demonstrated the potential of protein L as a reagent for detecting the expression of CAR by flow cytometry.

[0033] Protein L is a bacterial protein that binds to immunoglobulin (Ig) by specific high-affinity interactions with variable light chains without interfering with antigen binding sites. Protein L can also bind to the light chain components in single-chain variable fragments (scFv), which constitute the antigen binding domains of CAR. Another unique feature of protein L is its multi-domain structure, which has 5 identical Ig-binding sites in one molecule. Multivalent binding, and therefore potential cross-linking with target molecules expressed on the cell surface, shows that protein L can play a role as an effective cell activator.

[0034] Protein L, when bound to the scFv portion of the CAR, although not directly binding to and blocking the antigen binding site, can mimic antigen binding and induce CAR activation. In this way, it can be used as a universal CAR-activating reagent in cell culture applications using CAR-modified cells, including functional analysis of CAR-induced cellular responses and expansion of CAR-expressing cells for therapeutic applications.

[0035] Therefore, in certain embodiments, the present disclosure provides methods and compositions including the use of protein L for activating and / or amplifying CAR-modified effector immune cells (such as CAR-T cells, CAR-NK cells, and CAR-macrophages). Therefore, the methods of the present invention provide a universal cell culture system for activating and amplifying CAR-modified cells that is independent of its antigen specificity.

[0036] Using anti-human CD19 CAR-modified PSC-derived T cells, this study provides experimental evidence that immobilized protein L induces a specific proliferative response in CAR-modified T cells, but not in unmodified T cells. Recombinant fibronectin (recombinant fibronectin fragment) was found to support the CAR-inducible function of protein L, while the Notch ligand DLL4 could further enhance CD19 + Proliferative response and expansion of CAR-T cells. When CAR-T cells expanded in culture with anti-CD3 monoclonal antibodies and protein L were compared, protein L-expanded CAR-T cells showed superior CAR-mediated cytokine production, in vitro, and in vivo antitumor activity.

[0037] Therefore, Protein L was identified as a cell culture reagent for in vitro specific activation, expansion, and analysis of CAR-modified cells. Because Protein L is produced under animal-free conditions, Protein L can be used for GMP-compliant cultures suitable for therapeutic applications. This method can be used to develop PSC-derived CAR-T therapeutic products. Specific applications include selecting the optimal CAR construct for PSC-derived T cells, quality control assays for CAR-T cells, and expanding CAR-T cells for preclinical animal studies.

[0038] I. Definition

[0039] As used herein, "substantially free" with respect to a specific component means that the specific component is not purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specific component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the specific component cannot be detected using standard analytical methods.

[0040] As used in the specification herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one.

[0041] Unless explicitly stated to involve only alternatives or the alternatives are mutually exclusive, the term "or" used in the claims is used to mean "and / or," although the present disclosure supports definitions involving only alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0042] The term "about" means plus or minus 5% of the stated numerical value.

[0043] As used herein, a composition that is "substantially free" of a specified substance or material comprises 30%, 20%, 15%, more preferably 10%, even more preferably 5%, or most preferably 1% of the substance or material.

[0044] An "expression construct" or "expression cassette" refers to a nucleic acid molecule capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements (such as a promoter, enhancer, or functionally equivalent structures thereof) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements may also be included, such as transcription termination signals.

[0045] A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vector") refers to a macromolecule or complex of molecules that contains a polynucleotide to be delivered to a host cell in vitro or in vivo.

[0046] "Plasmid" is a common type of vector, which is an extrachromosomal DNA molecule separate from the chromosomal DNA, which can replicate independently of the chromosomal DNA. In some cases, it is circular and double-stranded.

[0047] As used herein, the term "patient" or "subject" refers to a living mammalian organism, 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 are adults, young adults, infants, and fetuses.

[0048] 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.

[0049] " epi-position " is the site on the antigen determined by the specificity of amino acid sequence that is recognized by antibody.As measured in competitive binding assay, if two antibodies competitively suppress (blocking) the combination of another antibody and antigen separately, then they are considered to be combined with identical epi-position.Alternatively, if most of the amino acid mutations that reduce or eliminate the combination of a kind of antibody in antigen reduce or eliminate the combination of another antibody, then these two antibodies have identical epi-position.If two antibodies partially suppress the combination of another antibody and antigen separately, and / or if some amino acid mutations that reduce or eliminate the combination of a kind of antibody reduce or eliminate the combination of another antibody, then these two antibodies are considered to have overlapping epi-position.

[0050] "Treatment" or "therapy" of a disease or condition refers to the implementation of a treatment regimen that may include administering one or more drugs to a patient in an effort to alleviate the signs or symptoms of the disease. Desirable effects of treatment include a reduction in the rate of disease progression, an improvement or palliation of the disease state, and a remission or improved prognosis. Relief can occur before signs or symptoms of the disease or condition appear, as well as after they appear. Thus, "treatment" or "therapy" can include "preventing" or "preventing" a disease or undesirable condition. In addition, "treatment" or "therapy" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes treatment regimens that have only a minimal effect on the patient.

[0051] The term "effective," as that term is used in the specification and / or claims, means sufficient to achieve a desired, expected, or intended result. "Effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or subject with a compound, means that the amount of the compound, when administered to a subject or patient for the treatment or prevention of a disease, is an amount sufficient to achieve such treatment or prevention of the disease.

[0052] "Treatment" or "treatment" includes (1) inhibiting a disease in a subject or patient who is experiencing or manifesting the pathology or symptoms of the disease (e.g., arresting further development of the pathology and / or symptoms), (2) alleviating the disease in a subject or patient who is experiencing or manifesting the pathology or symptoms of the disease (e.g., reversing the pathology and / or symptoms), and / or (3) achieving any measurable reduction in the disease or its symptoms in a subject or patient who is experiencing or manifesting the pathology or symptoms of the disease.

[0053] "Preventing" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not experience or display 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 may be at risk for and / or susceptible to the disease but who does not experience or display any or all of the pathology or symptoms of the disease.

[0054] The term "forward programming" refers to programming multipotent or pluripotent cells by providing them with one or more specific lineage-determining genes or gene products, rather than programming differentiated somatic cells without pluripotency.

[0055] As used generally herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0056] "Pharmaceutically acceptable salts" means salts of the compounds disclosed herein that are pharmaceutically acceptable as defined above and possess 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, and the like; or acid addition salts formed with organic acids such as 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 monocarboxylic and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid Pharmaceutically acceptable salts include, but are not limited to, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, enanthic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, 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 acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that can form base addition salts 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 will be appreciated that the specific anion or cation forming part of any salt of the invention is not important so long as the salt as a whole is pharmacologically acceptable. Further examples of pharmaceutically acceptable salts and their methods of preparation and use are found in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0057] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable substance formulated with an active ingredient drug that participates in carrying, delivering, and / or transporting a chemical agent. Drug carriers can be used to improve the delivery and effectiveness of drugs, including, for example, controlled-release technologies to modulate drug bioavailability, reduce drug metabolism, and / or minimize drug toxicity. Some drug carriers can increase the effectiveness of drug delivery to specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made from poly(lactic-co-glycolic acid) copolymers), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, viral particles, and dendrimers.

[0058] The term "chimeric antigen receptor (CAR)", as used herein, may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and encompasses a transformed receptor that transplants artificial specificity onto a specific immune effector cell. CAR can be used to confer the specificity of a monoclonal antibody to a T cell, thereby enabling the generation of a large number of specific T cells, for example, for adoptive cell therapy. In a specific embodiment, for example, CAR directs the specificity of cells to tumor-associated antigens. In some embodiments, CAR includes an intracellular activation domain, a transmembrane domain, and an extracellular domain including a tumor-associated antigen binding region. In a specific aspect, CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-ζ transmembrane domain and an intracellular domain. The specificity of other CAR designs can be derived from a ligand (e.g., a peptide) of a receptor or from a pattern recognition receptor, such as Dectin. In some cases, the interval between the antigen recognition domains can be changed to reduce activation-induced cell death. In some cases, CAR includes domains for other costimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some cases, molecules can be co-expressed with CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally eliminate T cells after the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0059] The term "culturing" refers to maintaining, differentiating and / or propagating cells in vitro in a suitable culture medium. "Enriched" means a composition comprising cells present at a greater percentage of total cells than is found in a tissue present in an organism.

[0060] An "anti-cancer" agent is capable of negatively affecting cancer cells / tumors in a subject, for example, by promoting the killing 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 blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0061] II. CAR-modified immune cells

[0062] Certain embodiments of the present disclosure relate to immune cells expressing 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, constant NK cells, NKT cells or stem cells (e.g., mesenchymal stem cells (MSC) or induced pluripotent stem cells (iPSC)). In some embodiments, the cells are monocytes or granulocytes, for example, myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils. Also provided herein are methods for producing and modifying immune cells and methods for using and administering the cells for adoptive cell therapy, in which case the cells can be autologous or allogeneic. Thus, the immune cells can be used as immunotherapy, such as for targeting cancer cells.

[0063] Provided herein are methods of activating and / or expanding the immune cells to selectively activate and / or expand CAR-modified immune cells (such as CAR-T cells) in the presence of protein L. Protein L can be present in the culture alone or in combination with recombinant fibronectin and / or DLL4.

[0064] The immune cells can be isolated from a subject, particularly a human subject. The immune cells can be obtained from a subject of interest, such as a subject suspected of having a specific disease or condition, a subject suspected of being susceptible to a specific disease or condition, or a subject undergoing therapy for a specific disease or condition. Immune cells can be collected from any part of the subject in which they reside, including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly, or they can be stored for a period of time, such as by freezing.

[0065] Immune cells can be enriched / purified from any tissue in which they reside, including, but not limited to, blood (including blood collected through a blood bank or umbilical cord blood bank), spleen, bone marrow, tissue removed and / or exposed during a surgical procedure, and tissue obtained via a biopsy procedure. The tissue / organ used to enrich, isolate and / or purify immune cells can be isolated from both living and non-living subjects, wherein the non-living subject is an organ donor.

[0066] AT cells

[0067] In some embodiments, immune cells are T cells. Several basic methods for deriving, activating and amplifying functional anti-tumor effector cells have been documented over the past two decades. These methods include: autologous cells, such as tumor infiltrating lymphocytes (TILs); T cells activated in vitro using autologous DCs, lymphocytes, artificial antigen presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells separated by means of captured target cell membranes; allogeneic cells naturally expressing anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells of chimeric TCR molecules called "T-antibodies" that are genetically reprogrammed or "redirected" to express tumor-reactive TCRs or display antibody-like tumor recognition capabilities. These methods have produced a large number of protocols for T cell preparation and immunization, which can be used in the methods described herein.

[0068] In some embodiments, the T cells are derived from blood, bone marrow, lymph nodes, 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 from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as a full T cell population, CD4 + cells, CD8 + Cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, type of antigen receptor, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. In some embodiments, the method comprises isolating cells from the subject, preparing, processing, culturing, and / or modifying them as described herein, and reintroducing them into the same patient with or without cryopreservation.

[0069] In T cell subtypes and subsets (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 subsets, such as stem cell memory T (TSC M ), central memory T (TC M ), effect memory T(T EM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and acquired regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0070] In some embodiments, one or more T cell populations are enriched or depleted of cells that are positive for a specific marker (such as a surface marker), or cells that are negative for a specific marker. In some cases, such markers are absent or expressed at relatively low levels on a specific T cell population (e.g., non-memory cells) but present or expressed at relatively high levels on specific other T cell populations (e.g., memory cells).

[0071] In some embodiments, T cells are isolated from PBMC samples by negatively selecting for markers expressed on non-T cells (such as B cells, monocytes, or other white blood cells), such as CD14. + or CD8 + Select steps to isolate CD4 + Helper and CD8 + Cytotoxic T cells. Such CD4 + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers that are expressed or expressed at relatively high levels on one or more naive, memory and / or effector T cell subpopulations.

[0072] In some embodiments, the CD8 + The T cells are further enriched or depleted of naive T cells, central memory T cells, effector memory T cells, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subsets. In some embodiments, central memory T (T CM ) cells to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly potent in such subpopulations.

[0073] 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 (using, for example, gentleMACS TMDissociator, Miltenyi Biotec, Auburn, Calif., for tumor disintegration) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests were incubated in the presence of interleukin-2 (IL-2).

[0074] The T cells of cultivation can be brought together and rapidly expanded. Rapid expansion increases the number of antigen-specific T cells by at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90- or 100-fold, or higher) within the period of about 10 to about 14 days. More preferably, rapid expansion increases at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-fold, or higher) within the period of about 10 to about 14 days.

[0075] Expansion can be achieved by any of a number of methods known in the art. For example, T cells can be rapidly expanded using nonspecific T-cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15), preferably IL-2. Nonspecific T-cell receptor stimulation can include about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Raritan, NJ). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of the cancer (including antigenic portions thereof, such as one or more epitopes, or cells) in the presence of T-cell growth factors, such as 300 IU / ml IL-2 or IL-15 (preferably IL-2), which can optionally be expressed by a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptide. The in vitro induced T-cells are rapidly expanded by restimulation with the same antigen of the cancer pulsed on antigen presenting cells expressing HLA-A2. Alternatively, the T-cells can be rapidly expanded, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2 + Allogeneic lymphocytes and IL-2 restimulation.

[0076] Autologous T cells can be modified to express T cell growth factors, which promote the growth and activation of 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, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In a specific aspect, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences, such as the coding sequence of IL-12, are readily available in the art, and promoters are also readily available in the art, and the operably connected promoter to the T cell growth factor coding sequence promotes high-level expression.

[0077] In some embodiments, T cells are activated and / or expanded in the presence of protein L. Protein L can be at 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 Protein L can be added to the culture at a concentration of 1:1. Protein L can be added to the culture, immobilized on a surface, or dissolved in the culture medium. The culture surface is a culture plate, culture bottle, microcarrier, microparticle, hydrogel particle, or culture bag. Protein L can be added in combination with an extracellular matrix protein, such as recombinant fibronectin or fibronectin.

[0078] B. Stem cells

[0079] In some embodiments, the CAR-modified immune cells of the present disclosure can be stem cells, or be derived from stem cells, such as induced pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs).

[0080] Any cell, except germ cells, red blood cells, and platelets, can be used as the starting point for iPSCs. For example, the cell type may be a keratinocyte, a fibroblast, a hematopoietic cell, a mesenchymal cell, a hepatocyte, or a gastric cell. There is no limitation on the degree of cell differentiation or the age of the animal from which the cells are collected; in the methods disclosed herein, even undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as somatic cell sources.

[0081] Somatic cells can be reprogrammed to produce iPS cells using methods known to those skilled in the art. Pluripotent stem cells are typically generated from somatic cells using nuclear reprogramming factors. 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, or Oct3 / 4, Sox2, Nanog, and Lin28 are utilized.

[0082] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. In certain embodiments, non-limiting conditions can be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or on a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts (as feeder cells) that have been treated with radiation or antibiotics to terminate cell division. Alternatively, a defined culture system that does not require feeder cells (such as TESR) can be used. TM culture medium or E8 TM / Essential 8 TM culture medium), culturing and maintaining pluripotent cells in a largely undifferentiated state.

[0083] C. Cytokine-directed differentiation

[0084] Certain embodiments of the present disclosure relate to differentiating PSC into HPC. By methods known in the art, such as the method described in U.S. Patent No. 8,372,642, PSC can be differentiated into HPC, and U.S. Patent No. 8,372,642 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, cell culture is sequentially exposed to a first culture medium to prepare PSC for differentiation, a second culture medium including BMP4, VEGF and FGF, and then cultured in a third culture medium including Flt3 ligand, SCF, TPO, IL-3 and IL-6, pluripotent cells can be differentiated into HPC and hematopoietic cells. The culture medium determined by the second component may also include heparin. In addition, including FGF-2 (50ng / ml) in the culture medium containing BMP4 and VEGF can enhance the efficiency of generating hematopoietic precursor cells from pluripotent cells. In addition, inclusion of glycogen synthase kinase 3 (GSK3) inhibitors (eg, CHIR99021, BIO, and SB-216763) in the first defined medium can further enhance HPC yield.

[0085] Typically, pluripotent cells can be differentiated into hematopoietic precursor cells using defined or undefined conditions. It will be appreciated that in embodiments where the resulting cells are expected to be administered to a human subject, defined conditions are typically preferred. Hematopoietic stem cells can be derived from pluripotent stem cells under defined conditions (e.g., using TeSR culture medium), and hematopoietic cells can be produced 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.

[0086] As part of the differentiation process, pluripotent cells can be made to form embryoid bodies or aggregates. For inducing differentiation, the formation of "embryoid bodies" (EBs) or growth cell clusters generally involves human pluripotent stem cells gathering into EBs in vitro and enabling human pluripotent stem cells to spontaneously and randomly differentiate into a variety of tissue types representing endoderm, ectoderm, and mesoderm origin. Therefore, three-dimensional EBs can be used to produce a portion of hematopoietic cells and endothelial cells.

[0087] EBs can be formed using the following protocol. Undifferentiated iPSCs can be harvested at confluence by treating with 0.5M EDTA for approximately 8-10 minutes at room temperature. Undifferentiated iPSCs are suitable for MATRIGEL®. TMGrow cells on coated plates without a feeder layer. After incubation, aspirate the EDTA and harvest the cells to form EBs in SFD medium containing rock inhibitor or blebbistatin. The next day, change the medium to EB1 differentiation medium containing various cytokine preparations. Plate cells at a density of 250,000 to 500,000 cells per ml to promote aggregate formation.

[0088] To promote aggregate formation, cells were transferred to low-adhesion culture plates and incubated overnight in serum-free differentiation (SFD) medium consisting of 75% IMDM (Gibco), 25% Ham's modified F12 (Cellgro), supplemented with 0.05% N2 and B-27 without RA supplement, 200 mM l-glutamine, 0.05 mg / ml ascorbic acid-2-phosphate magnesium salt (Asc2-P) (WAKO), and 4.5 x 10 -4 MTG. The next day, cells can be collected from each well and centrifuged. Then, in the first four days of differentiation, the cells can be resuspended in "EB differentiation medium", which is composed of SFD basal medium supplemented with about 50ng / ml bone morphogenetic factor (BMP4), about 50ng / ml vascular endothelial growth factor (VEGF) and 50ng / ml zb FGF. Half of the cell culture fluid is replaced every 48 hours. On the fifth day of differentiation, the culture medium is replaced with a second culture medium, which comprises SFD culture medium, which is wherein supplemented with 50ng / ml stem cell factor (SCF), about 50ng / ml Flt-3 ligand (Flt-3L), 50ng / ml interleukin-6 (IL-6), 50ng / ml interleukin-3 (IL-3), and 50ng / ml thrombopoietin (TPO). Half of the cell culture fluid is replaced with fresh differentiation medium every 48 hours. The culture medium is replaced by centrifuging the differentiation culture at 300g for 5 minutes and drawing half the volume from the differentiation culture and supplementing with fresh culture medium. In certain embodiments, EB differentiation medium may include about BMP4 (e.g., about 50 ng / ml), VEGF (e.g., about 50 ng / ml), and optionally FGF-2 (e.g., about 25-75 ng / ml or about 50 ng / ml). The supernatant may be aspirated and replaced with fresh differentiation medium. Alternatively, half of the cell culture medium may be replaced with fresh medium every two days. Cells may be harvested at various time points during the differentiation process.

[0089] HPCs can be cultured from pluripotent stem cells using defined media. Pluripotent cells can be differentiated into hematopoietic CD34 +Stem cell methods are described, for example, in U.S. Application 12 / 715,136, which is incorporated by reference in its entirety. It is contemplated that these methods can be used with the present disclosure.

[0090] For example, defined culture medium can be used to induce hematopoietic CD34 + Differentiation. The defined medium may comprise the growth factors BMP4, VEGF, Flt3 ligand, IL-3 and / or GMCSF. Pluripotent cells may be cultured in a first defined medium comprising BMP4, VEGF and optionally FGF-2, and then cultured in a second medium comprising (Flt3 ligand, IL-3 and GMCSF) or (Flt3 ligand, IL-3, IL-6 and TPO). The first and second culture media may also comprise one or more of SCF, IL-6, G-CSF, EPO, FGF-2 and / or TPO. Substantially hypoxic conditions (e.g., less than 20% O2) may further promote hematopoietic or endothelial differentiation.

[0091] The enzymes can be cleaved mechanically or enzymatically (e.g., using trypsin or TrypLE TM ) to substantially individualize the cells. ROCK inhibitors (e.g., H1152 or Y-27632) may also be included in the culture medium. It is contemplated that these methods can be automated using, for example, robotic automation.

[0092] In certain embodiments, the condition of hypoxia can be used to promote pluripotent cells to differentiate into hematopoietic progenitor cells. As will be appreciated by those skilled in the art, the atmosphere oxygen content lower than about 20.8% will be considered as hypoxia. Compared with ambient air, the human cells in the culture medium can grow under the atmospheric conditions that oxygen content reduces. This relative hypoxia can be achieved by reducing the atmospheric oxygen that is exposed to culture medium. Embryonic cells grow in vivo conventionally under the condition that oxygen reduces, and are generally about 1% to about 6% atmospheric oxygen, and carbon dioxide is in ambient level. Do not wish to be bound by theory, expection hypoxia condition can simulate the aspect of specific embryo development condition. As shown in the following examples, hypoxia condition can be used in certain embodiments to promote pluripotent cells (such as iPSC or hESC) to be differentiated into more highly differentiated cell types (such as HPC) in addition.

[0093] The following hypoxic conditions can be used to promote that pluripotent cells are differentiated into hematopoietic progenitor cells.In certain embodiments, it is possible to use an atmosphere oxygen content 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% to promote differentiation into hematopoietic progenitor cells.In certain embodiments, hypoxic atmosphere comprises about 5% oxygen.

[0094] In certain embodiments, a dissolved oxygen level 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 to promote differentiation into hematopoietic precursor cells. In certain embodiments, the dissolved oxygen level is about 25% oxygen.

[0095] Regardless of which specific culture medium is used in any given hematopoietic progenitor cell expansion, the culture 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 known as steel 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 include at least one of SCF, Flt3L, and TPO. More particularly, the culture will include SCF, Flt3L, and TPO.

[0096] In some embodiments, HPCs exhibit disruption of methyl-CpG binding protein 2 (MeCP2) and are cultured under conditions that promote myeloid or lymphoid differentiation. In some aspects, the HPCs express non-functional MeCP2 that does not substantially bind to methylated DNA. In certain aspects, the HPCs do not express MeCP2 at levels sufficient to affect MeCP2 DNA binding activity. In specific aspects, MeCP2 is non-functional due to truncation or mutation in the MeCP2 gene. In some aspects, obtaining HPCs that exhibit disruption of MeCP2 comprises contacting the HPCs with siRNA, shRNA, or a small molecule inhibitor of MeCP2.

[0097] D. Forward Programming

[0098] Certain embodiments of the present disclosure provide HPC by forward programming CAR-PSC, which is performed by expressing a combination of programming genes important for hematopoietic cell differentiation / function. In one method, PSC is modified to express at least three hematopoietic precursor programming genes, such as ETS genes (e.g., ETC2 or ERG), hematopoietic development genes (e.g., GATA2) and homeobox genes (e.g., HOXA9), such as described in PCT / US2016 / 057893, which are incorporated herein by reference in their entirety. In a particular aspect, ETV2 / ERG, GATA2 and HOXA9 genes are co-expressed by a vector (such as an inducible PiggyBac vector), which uses a bidirectional Tight promoter and is transfected into CAR-PSC.

[0099] In addition, EGH-CAR-PSCs can be further modified to express additional genes for long-term engraftment potential. Exemplary genes include HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, and / or RBAK. For example, PSCs can be transfected with one or more vectors to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, and HOXA4.

[0100] Preferably, only the ETV2 / GAT2 / HOXA9 gene expression is sufficient to forward program the PSC into hematopoietic precursor cells for a period of time. Therefore, the hematopoietic precursor programming gene can be under the control of an inducible promoter. In this way, the hematopoietic precursor programming gene expression can be induced in the PSC for a period of time sufficient to forward program into multi-lineage hematopoietic precursor cells. This period of time can be about 1 day to about 20 days, such as about 3, 4, 5, 6, 7, 8, 9 or 10 days. Alternatively, the hematopoietic precursor programming gene can be introduced into the PSC via an episomal vector. In this way, the hematopoietic precursor programming gene can be transiently expressed in the PSC.

[0101] E. Antigen

[0102] CAR provided herein can have antigen specificity useful in the treatment of disease or illness.The antigen targeted by genetically engineered antigen receptors is to be expressed in the case of the disease, condition or cell type targeted by adoptive cell therapy.Disease and the condition are hyperplasia, tumor and malignant diseases and conditions, including cancer and tumor, including blood cancer, immune system cancer, such as lymphoma, leukemia and / or myeloma, such as B, T and myeloid leukemia, lymphoma and multiple myeloma.In some embodiments, compared with normal or untargeted cells or tissues, antigen is selectively expressed or overexpressed on disease or condition cells (such as tumors or pathogenic cells), and the antigen is related to autoimmune disease or alloimmune disease, or is a pathogen-specific antigen.In other embodiments, antigen is expressed on normal cells and / or expressed on transformed cells.

[0103] Any suitable antigen can be used in this method. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, self / autoantigens, tumor / cancer-associated antigens, and neoplastic antigens. In specific aspects, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA.

[0104] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3 and MAGE4; PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a variety of tumor types (such as melanoma, lung cancer, sarcoma and bladder cancer). Tumor-associated antigens for prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphatase, NKX3.1 and prostate six transmembrane epithelial antigen (STEAP).

[0105] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone, a short peptide of 10 amino acids long, which can be used to treat many cancers.

[0106] Tumor antigen includes the tumor antigen that is derived from cancer, and the feature of described cancer is that tumor-associated antigen expresses, such as HER-2 / neu expresses.Interested tumor-associated antigen includes pedigree specific tumor antigen, such as melanocyte-melanoma pedigree antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-associated protein.Exemplary tumor-associated antigen includes but is not limited to be derived from or comprise following any one or more tumor antigen: p53, Ras, c-Myc, cytoplasmic serine / threonine kinase (such as A-Raf, B-Raf and C-Raf, cyclin dependent kinase), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, G AGE-6, GAGE-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, phosphatidylinositol 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, ELF2 M, 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 signaling protein 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 its 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 breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucose GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, 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 idiotype. ,

[0107] Antigens may include epitope regions or epitope peptides derived from genes that are mutated in tumor cells or that are transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosamine transferase-V; clonal rearrangements of immunoglobulin genes that produce unique idiotypes in myeloma and B-cell lymphoma; tumor antigens, including epitope regions or epitope peptides derived from oncogenic viral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0108] In other embodiments, antigens are 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.

[0109] Exemplary pathogenic microorganisms whose antigens are contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza viruses A, B, and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenoviruses, Staphylococcus species including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species including Streptococcus pneumoniae. As will be appreciated by those skilled in the art, proteins from these and other pathogenic microorganisms and nucleotide sequences encoding these proteins for use as antigens described herein can be found in publications and in public databases such as and ) was identified.

[0110] Antigens derived from human immunodeficiency virus (HIV) include HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0111] Antigens derived from herpes simplex viruses (e.g., HSV 1 and HSV2) include, but are not limited to, proteins expressed by HSV late genes. Late group genes primarily encode proteins that form virions. Such proteins include 5 proteins (UL) that form the viral capsid: UL6, UL18, UL35, UL38, and major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome includes at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

[0112] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed in the very early and early stages of viral replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the gene cluster of UL128-UL150, envelope glycoprotein B (gB), gH, gN and pp150. As will be appreciated by those skilled in the art, CMV proteins useful as antigens described herein can be identified in public databases such as and

[0113] Antigens derived from Epstein-Barr virus (EBV) contemplated for use in certain embodiments include the EBV lytic proteins gp350 and gp110, and EBV proteins produced during latent infection include Epstein-Ban nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B.

[0114] Antigens derived from respiratory syncytial virus (RSV) contemplated for use in the present invention include any of the 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 proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulator), RNA polymerase, and phosphoprotein P.

[0115] Antigens contemplated for use from vesicular stomatitis virus (VSV) include any of the five major proteins encoded by the VSV genome, and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M).

[0116] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0117] Exemplary viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoprotein, core or nonstructural protein), herpesvirus polypeptides (including herpes simplex virus or varicella-zoster virus glycoprotein), 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 polypeptides), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0118] 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 comprises an antigen having a portion or multiple portions of a polypeptide exposed on the outer surface of the bacterium.

[0119] Antigens from Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), contemplated for use include virulence regulators such as the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and TRAP. Other Staphylococcal proteins that may serve as antigens include the Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA, and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two S. aureus species (N315 and Mu50) have been sequenced and are publicly available, for example, at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by the skilled artisan, staphylococcal proteins used as antigens may also be found in other public databases such as and ) was identified.

[0120] Antigens derived from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and fimbriae 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 strains of Streptococcus pneumoniae has been sequenced, and as will be appreciated by those skilled in the art, Streptococcus pneumoniae proteins used herein may also be identified in other public databases, such as and Proteins of particular interest for use as antigens according to the present disclosure include virulence factors and proteins expected to be exposed on the surface of pneumococci.

[0121] 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., B. 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, Bartonella polypeptides, Haemophilus polypeptides (e.g., Haemophilus influenzae type b outer membrane protein), Helicobacter polypeptides , Klebsiella polypeptides, L-form bacterial polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacterium polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcal polypeptides (i.e., S. pneumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rocalima polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., Streptococcus pyogenes M protein), Group B Streptococcus (Streptococcus agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Yersinia pestis F1 and V antigens).

[0122] 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, Conid iobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides , Moniliella polypeptide, Mortierella polypeptide, Mucor polypeptide, Paecilomyces polypeptide, Penicillium polypeptide, Philalemonium polypeptide, Phialophora polypeptide, Prototheca polypeptide, Pseudallescheria polypeptide, Pseudomi crodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides and Xylohypha polypeptides.

[0123] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Bagnacles polypeptides, Benoysia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalosporin polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentaflagellate polypeptides, Plasmodium polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilus polypeptides, Catstrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Diplocachus polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Dipylidium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Ascaris polypeptides, Loa filaria polypeptides, peptides, Mansonella polypeptides, Muellera polypeptides, dwarf trematode polypeptides, flat nematode polypeptides, nematode polypeptides, nodular nematode polypeptides, onchocerca polypeptides, opisthorchis polypeptides, gastropoda polypeptides, parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, blister nematode polypeptides, Protocircle polypeptides, abdominal filarial nematode (Setaria) polypeptides, Spirocera polypeptides, Spirocera polypeptides, Crown filaria polypeptides, Strongyloides polypeptides, Strongyloides polypeptides, sucking nematode polypeptides, Toxocara polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Whipworm polypeptides, Uncinaria polypeptides and Wuchereria polypeptides. (e.g., Plasmodium falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-terminus), and exportin 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0124] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and stink bugs, such as bed bugs and triatomine bugs.

[0125] F. Chimeric Antigen Receptor

[0126] In some embodiments, CAR contains an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, 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, just like a TCR.

[0127] 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.

[0128] In some embodiments, the modified antigen receptor includes a chimeric antigen receptor (CAR), which includes activating or stimulating CAR, costimulatory CAR (see WO2014 / 055668), and / or inhibitory CAR (iCAR, see Fedorov et al., 2013). CAR typically comprises an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components, which is connected in some aspects by a linker and / or a transmembrane domain. Such molecules typically simulate or approximate the signal of a natural antigen receptor, the signal of a combination of this receptor and a costimulatory receptor and / or the signal of a costimulatory receptor alone.

[0129] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides, including CAR (hCAR) that has been humanized to reduce immunogenicity, which includes an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, CAR can recognize an epitope comprising a shared space between one or more antigens. In certain embodiments, the binding region may include the complementary determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or its antigen binding fragment. In another embodiment, specificity is derived from a peptide (e.g., cytokine) bound to a receptor.

[0130] It is contemplated that human CAR nucleic acids may be human genes for enhancing cellular immunotherapy in human patients. In specific embodiments, the present invention includes full-length CAR cDNA or coding regions. The antigen binding region or domain may comprise a V domain of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody. H and V L In one embodiment, the fragment is a fragment of a human antigen-specific antibody, such as a fragment of a human antigen-specific antibody, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. The fragment can also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage expressed in human cells.

[0131] The arrangement can be multimeric, such as a diabody or a multimer. Multimers are most likely formed by cross-pairing the variable portions of the light and heavy chains into diabodies. The hinge portion of the construct can have a variety of options, from complete deletion to retaining the first cysteine, to proline substitution instead of serine substitution, until being truncated to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can achieve this goal. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2 and CH3 regions of a modified human immunoglobulin can also be used to improve dimerization. Only the hinge portion of an immunoglobulin can also be used. Parts of CD8α can also be used.

[0132] In some embodiments, CAR nucleic acid includes sequences encoding other costimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains.Other costimulatory receptors include but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137).

[0133] In some embodiments, CAR is constructed to be specific for a specific antigen (or marker or ligand), such as an antigen (e.g., cancer marker) expressed in a specific cell type to be targeted by adoptive therapy, and / or an antigen (such as an antigen expressed on a normal or non-disease cell type) intended to induce a weakened response. Therefore, CAR typically comprises 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 its extracellular portion. In some embodiments, CAR includes one or more antigen binding portions of an antibody molecule, such as a variable heavy chain (V) derived from a monoclonal antibody (mAb). H ) and variable light chain (V L ) of a single-chain antibody fragment (scFv).

[0134] 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 an antigen binding region) comprises a tumor-associated antigen or a 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 kind, 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 antigens, melanoma-associated antigens, mutated p53, mutated ras, etc. In certain embodiments, CAR can be co-expressed with cytokines to improve persistence when there is a small amount of tumor-associated antigens. For example, CAR can be co-expressed with IL-15.

[0135] The sequence encoding the open reading frame of the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR) or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA. Furthermore, it may be further advantageous to use endogenous or exogenous noncoding regions to stabilize mRNA.

[0136] Can expect that chimeric construct can be introduced into immune cell in the form of naked DNA or suitable vector.Using naked DNA by the method for electroporation stable transfection cell is known in the art.Naked DNA generally refers to the DNA of the encoding chimeric receptor that is contained in the plasmid expression vector with correct expression direction.

[0137] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the methods of the present disclosure are non-replicative in immune cells. A large number of viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0138] In some aspects, antigen-specific binding or recognition components are connected to one or more transmembrane and intracellular signaling domains. In some embodiments, CAR includes a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of these domains to the transmembrane domains of the same or different surface membrane proteins, minimizing the interaction with other members of the receptor complex.

[0139] In some embodiments, membrane spaning domain is derived from natural or synthetic source.If source is natural, then this domain is derived from any membrane-bound protein or membrane spaning protein in some aspects.Membrane spaning region includes those derived from (i.e. at least including its membrane spaning region) following: α, β or ζ chain of T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS / CD278, and GITR / CD357 molecule. Alternatively, in some embodiments, membrane spaning domain is synthetic. In some aspects, synthetic membrane spaning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, phenylalanine, tryptophan and valine triplet will be found at each end of synthetic membrane spaning domain.

[0140] III. How to use

[0141] In some embodiments, the present disclosure provides a method for immunotherapy, which includes administering an effective amount of immune cells expressing CAR of the present disclosure. In one embodiment, a medical disease or condition is treated by transferring a group of immune cells that elicit an immune response. In certain embodiments of the present disclosure, cancer or infection is treated by transferring a group of immune cells that elicit an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, which includes administering an effective amount of antigen-specific cell therapy to the individual. This method may be applicable to the treatment of immune diseases, solid cancers, blood cancers, and viral infections.

[0142] In a specific embodiment, a method of treating a cancer patient is provided by administering immune cells, such as NK cells and / or T cells, that express a CAR provided herein that has an antigen binding domain specific for an antigen expressed by the cancer.

[0143] The tumors to which the therapeutic methods of the present invention are applicable include any malignant cell type, such as those found in solid tumors or hematologic tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. The example of exemplary hematologic tumors includes myeloid tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. More examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0144] The cancer may specifically be of the following histological types, although not limited thereto: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; combined hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; cervical adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant testicular blastoma; sertoli cell carcinoma cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo maligna; acral lentigo melanoma; nodular melanoma; malignant melanoma within giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii); choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma;Primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; small lymphocytic malignant lymphoma; large cell diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; B-cell lymphoma; low-grade / follicular non-Hodgkin's 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 cells NHL; bulky NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0145] Specific embodiments relate to methods for treating leukemia. Leukemia is a cancer of the blood or bone marrow characterized by an abnormal proliferation (multiplication) of blood cells, typically white blood cells (leukocytes). It is part of a larger group of diseases known as hematologic neoplasms. Leukemia is a broad term encompassing several diseases. Clinically and pathologically, leukemia is classified into acute and chronic forms.

[0146] In certain embodiments of the present disclosure, immune cells are delivered to individuals in need, such as individuals suffering from cancer or infection. The cells then enhance the immune system of the individual to attack each cancer or pathogenic cell. In some cases, one or more doses of immune cells are provided to the individual. In the case of providing two or more doses of immune cells to the individual, the duration between administrations should be sufficient to allow for propagation time in the individual, and in specific embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7 or more days.

[0147] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject suffers from an autoimmune disease. Non-limiting examples of autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal glands, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-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, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Fuzzy's disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (such as minimal change disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, essential agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome syndrome), generalized myotonia syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject can also suffer from an allergic condition such as asthma.

[0148] A therapeutically effective amount of immune cells can be administered by a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0149] The therapeutically effective amount of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in the treated subject. For example, this can be the number of immune cells necessary to inhibit progression, or cause regression of an autoimmune or alloimmune disease, or to alleviate the symptoms (such as pain and inflammation) caused by an autoimmune disease. It can be the amount necessary to alleviate symptoms associated with inflammation (such as pain, edema, and elevated body temperature). It can also be the amount necessary to reduce or prevent transplant organ rejection.

[0150] The immune cell population can be administered according to a treatment regimen consistent with the disease, such as a single or several doses administered over one to several days to improve the disease state, or periodic doses administered over an extended period of time to inhibit disease progression and prevent disease recurrence. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease or condition and should be determined based on 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 the disease, and the mode of administration. In some embodiments, a therapeutic dose for a human subject may be at least 3.8×10 4 , at least 3.8×10 5 , at least 3.8×10 6 , at least 3.8×10 7 , at least 3.8×10 8 , at least 3.8×10 9 or at least 3.8×10 10 Immune cells / m 2 In certain embodiments, the therapeutic dose for human subjects is about 3.8×10 9 to about 3.8×10 10 Immune cells / m 2 In other embodiments, the therapeutically effective amount of immune cells may be about 5×10 6 cells to about 7.5×10 cells per kilogram of body weight 8 The number of cells varies from one to another, such as about 2×10 per kilogram of body weight. 7 cells to approximately 5×10 8 cells, or about 5×10 per kilogram of body weight 7 cells to approximately 2×10 8 The exact number of immune cells is readily determined by one skilled in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be inferred from dose-response curves obtained in vitro or in animal model test systems.

[0151] Immune cells can be co-administered with one or more other therapeutic agents to treat immune-mediated disorders. Combination therapy can include, but is 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), immunoscavengers (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids (e.g., dexamethasone or prednisone)), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone)) or nonsteroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-β), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or tolerogenic agents may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, Treosulfan, busulfan); irradiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Depending on the desired effect, such additional agents may be administered before, during, or after the administration of immune cells. Such administration of cells and agents may be performed by the same route or by different routes and may be administered at the same site or at different sites.

[0152] B. Pharmaceutical Compositions

[0153] Also provided herein are pharmaceutical compositions and formulations comprising immune cells (eg, T cells or NK cells) and a pharmaceutically acceptable carrier.

[0154] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (such as an antibody or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the receptor 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 (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polyols. peptides; 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, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, for example, sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0155] C. Combination therapy

[0156] In certain embodiments, the compositions and methods of the present embodiment relate to a combination of an immune cell population and at least one additional therapy. Additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing therapies. Additional therapy can be the form of adjuvant or neoadjuvant therapy.

[0157] In some embodiments, additional therapy is to administer a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, additional therapy is to administer a side effect limiter (for example, an agent that expects to reduce the incidence and / or severity of the side effects of treatment, such as an antiemetic). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation. In some embodiments, additional therapy is therapy targeting PBK / AKT / mTOR pathways, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and / or chemopreventive agents. Additional therapy can be one or more chemotherapeutics as known in the art.

[0158] Immune cell therapy can be relative to additional cancer therapy, such as before, during, after immune checkpoint therapy, or with various combinations.The application can be carried out at intervals ranging from a few minutes to a few days to a few weeks. In the embodiment in which immune cell therapy and additional therapeutic agents are provided to the patient individually, it is generally ensured that the time between each delivery is not too long apart, so that the two compounds can still play a favorable comprehensive effect to the patient. In this case, it is considered that antibody therapy and anticancer therapy can be provided to the patient, both of which are within about 12-24h or 72h, more specifically, within about 6-12h. In some cases, significantly extending the time period for treatment can be desirable, between each administration, a few days (2, 3, 4, 5, 6 or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks).

[0159] Various combinations can be used. For the following example, CAR immune cell therapy is "A" and anti-cancer therapy is "B":

[0160] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0161] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0162] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy.

[0163] 1. Chemotherapy

[0164] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are categorized by their intracellular activity, e.g., whether they affect the cell cycle and at which stage of the cell cycle. Alternatively, an agent can be characterized based on its ability to directly crosslink DNA, to insert into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0165] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquinone, metodepa, and uredepa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trihydroxymethylmelamine; acetogenins (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesine, carzelesin, and biszelesin); cryptophycins (particularly cryptophycin); 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolidinone hydrochloride, melphalan, neomuscarin, phenylephrine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics , such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma and calicheamicin omega; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins; and neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores, aclarubicin, dactinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin , esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zoloft, and doxyrubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;Androgens, such as kalutestosterone, drostanolone propionate, epithioandrostol, melastane, and testolactone; antiadrenal agents, such as mitotane and trilostane; folic acid supplements, such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclocycline; diazocine; elfomithine; elliptonium acetate; epothilone; etoposide; Lu; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; diamine nitrazol; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; cysoflavone; spirogermanium; tenuisporic acid; triamin quinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, etc.) A), bacitracin A and serpentin); urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; piperobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel and the docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinums; etoposide (VP-16); ifosfamide; mitoxantrone; vinblastine Neoral; vinorelbine; vinorelbine; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans-platinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0166] 2. Radiotherapy

[0167] Other factors that cause DNA damage and have been widely used include the directed delivery of generally known gamma-rays, X-rays and / or radioisotopes to tumor cells. Other forms of DNA damaging factors are also considered, such as microwaves, proton beam irradiation and UV-irradiation. It is most likely that all of these factors cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dosage range of X-rays ranges from a daily dose of 50-200 roentgens for an extended period of time (3-4 weeks) to a single dose of 2000-6000 roentgens. The dosage range of radioisotopes varies greatly, depending on the half-life of the isotope, the intensity and type of the radiation emitted, and the uptake by tumor cells.

[0168] 3. Immunotherapy

[0169] The skilled artisan will appreciate that additional immunotherapeutic drugs may be combined or coupled with the methods of the described 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 In some embodiments, the present invention relates to a method for treating a tumor cell by binding to a target antigen. The method comprises the steps of: firstly, binding to a target antigen of a tumor cell, and secondly, binding to a target antigen of a tumor cell, and then ...

[0170] Antibody-drug conjugates (ADCs) include monoclonal antibodies (MAbs) covalently linked to cell-killing drugs and can be used in combination therapies. This approach combines the high specificity of MAbs for their antigenic targets with highly potent cytotoxic drugs, forming "armed" Mabs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, leading to reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include (brentuximab vedotin) and (Trastuzumab emtansine conjugate or T-DM1).

[0171] In one aspect of immunotherapy, tumor cells must carry some markers that can be suitable for targeting, that is, these markers are not present on most other cells. There are many tumor markers, and in the case of the present embodiment, any one of these can be suitable for targeting. 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 anticancer effects with immunostimulatory effects. 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.

[0172] Examples of immunotherapeutic drugs include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be employed in conjunction with the antibody therapies described herein.

[0173] In some embodiments, immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints turn up signals (e.g., co-stimulatory molecules) or turn down signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockers include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuation protein (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 inhibitor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target PD-1 axis and / or CTLA-4.

[0174] Immune checkpoint inhibitors can be drugs such as small molecules, recombinant forms of ligands or receptors, or specifically, antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or their analogs can be used, and in particular chimeric, humanized or human forms of antibodies can be used. As known to professionals, alternative and / or equivalent names can be used for some antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, alternative and equivalent names of lambrolizumab are also known to be MK-3475 and pembrolizumab.

[0175] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, 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.

[0176] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an 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 is an anti-PD-1 antibody that can be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, and SCH-900475, are exemplary anti-PD-1 antibodies. 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 soluble PDL2-Fc fusion receptor.

[0177] 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 Genbank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is found on the surface of T cells and acts as a "close" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells, which transmits inhibitory signals to T cells. CTLA4 is similar to T-cell co-stimulatory protein CD28, and both molecules bind CD80 and CD86 on antigen-presenting cells, also known as B7-1 and B7-2, respectively. 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 its function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4 (inhibitory receptor for B7 molecules).

[0178] 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.

[0179] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and ) 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 the above-mentioned antibodies for binding to the same epitope on CTLA-4 and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0180] 4. Surgery

[0181] Approximately 60% of cancer patients will undergo the same type of surgery, including preventive surgery, diagnostic surgery or staging surgery, radical surgery and palliative surgery. Radical surgery includes resection in which all or part of the cancerous tissue is physically removed, cut and / or destroyed, and can be combined with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microscope-controlled surgery (Mohr's surgery).

[0182] After removal of some or all of the cancerous cells, tissues, or tumors, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or topical application of an additional anticancer 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 administered at varying doses.

[0183] 5. Other medicines

[0184] It is contemplated that other agents may be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy of treatment. These additional agents include agents that influence the upregulation of cell surface receptors and the connection of GAPs, cytostatics and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing the number of GAP connections by increasing intracellular signaling will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell groups. In other embodiments, cytostatics or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of treatment. It is contemplated that cell adhesion inhibitors are used to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (such as antibody c225) may be used in combination with certain aspects of this embodiment to improve therapeutic efficacy.

[0185] IV. Articles or Kits

[0186] Also provided herein are products or kits comprising immune cells. The products or kits may further include a package insert including instructions for use of the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of a cancer patient. Any of the antigen-specific immune cells described herein may be included in the products or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastics (such as polyvinyl chloride or polyolefins), or metal alloys (such as stainless steel or nickel-based alloys). In some embodiments, the container holds a formulation, and a label on or associated with the container may indicate instructions for use. The products or kits may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the products further include one or more other agents (e.g., chemotherapeutic agents, and antitumor drugs). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0187] V. Examples

[0188] The following examples are included to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, in light of this disclosure, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0189] Example 1 - Protein L activation and expansion of CAR-T cells

[0190] Protein L-induced CAR-T cell expansion: E11 pluripotent stem cells reprogrammed from peripheral blood T cells (TiPSCs) using an episomal vector were modified to constitutively express a second-generation anti-human CD19 chimeric antigen receptor (CAR), which consists of a human CD19-binding scFv domain derived from the FMC63 monoclonal antibody, a CD28 costimulatory domain, and a CD3ζ signaling domain.

[0191] Differentiation of unmodified and CAR-modified E11 TiPSCs into T / NK CD34 T cells by cytokine-directed differentiation + Progenitor cells. The isolated CD34 + Progenitor cells were further differentiated into CD3+ T cells in 2 weeks of hypoxic culture on DLL4 / recombinant fibronectin-coated plates in T cell differentiation medium (TCDM) consisting of StemSpan SFEM (Stem Cell Technologies) supplemented with magnesium ascorbyl phosphate (0.25 mM), nicotinamide (2 mM), GlutaMax (Gibco), and cytokines (SCF, TPO, FLT3L, IL7; 50 ng / ml each).

[0192] CAR-T cells were injected with 10,000 CD3 + T cells / cm 2 The density of the coated cells was different with different concentrations of protein L (Pierce; 0, 0.1, 0.5, 2.5 μg / cm 2 ) with or without recombinant fibronectin (Takara; 0.5 μg / cm 2 ) and human DLL4-Fc (Acro Biosystems; 0.5 μg / cm 2 The cells were cultured on plates supplemented with IL-2 and IL-15 (10 ng / mL each) in TCDM. The cultures were maintained under hypoxic conditions (5% O2) and harvested after 10 days. All harvested cells were counted and CD3 + The percentage of T cells was determined and the T cell expansion fold (output / input T cell number) was calculated.

[0193] Protein L induced the expansion of CAR-T cells in a dose-dependent manner; however, recombinant fibronectin enhanced the proliferative response ( Figure 1DLL4 is a supplemental T cell growth factor that also significantly enhances protein L-dependent expansion of CAR-T cells. A hypoxic environment was used to achieve efficient CAR-T cell expansion, as parallel normoxic cultures showed poor expansion yields in the tested variants.

[0194] DLL4 promotes CD8+ CAR-T expansion: In the combination of protein L, recombinant fibronectin and DLL4-Fc (all at 0.5 μg / cm 2 CD8α expression was measured in CAR-T cells harvested on day 10 of the expansion culture ( Figure 2 ).

[0195] Compared with protein L alone, the combination of protein L and recombinant fibronectin produced efficient CAR-T cell expansion. However, despite the much higher expansion rate, CD8 expression was still very low. In contrast, when DLL4 was added, not only was the CAR-T cell expansion rate increased by more than 25%, but a significant proportion of CD3 + T cells acquired CD8α expression ( Figure 2 ).

[0196] Protein L-induced CAR-specific T cell expansion: The specificity of CAR-induced T cell expansion in protein L culture was verified by comparing CAR-modified T cells with unmodified T cells. Both cells were plated and expanded in CD3-induced pan-T cell culture (control) and protein L-induced expansion culture. CAR-modified and unmodified E11-derived T cells were cultured in the presence of anti-CD3 monoclonal antibody (OKT3 clone; 0.5 μg / cm 2 ) or protein L (0.5 μg / cm 2 )-coated plates, containing recombinant fibronectin and DLL4-Fc (0.5 μg / cm 2 ). Cells were expressed as 10,000 CD3 + T cells / cm 2 Cells were seeded at high density in TCDM supplemented with IL2 and IL15 (10 ng / ml each). After 8 days, cells were collected, counted, and CD3 + The percentage of T cell expansion was calculated (output / input T cell number) ( Figure 3 ).

[0197] In contrast to CD3-activated cultures, in which both T cells and CAR-T cells proliferated with similar efficiency, exclusive expansion of CAR-T cells was observed in protein L cultures. The efficiency of CAR-T cell expansion was comparable in anti-CD3 and protein L cultures. When CAR expression in expanded CAR-T cells was determined by protein L staining, a significantly higher proportion of CAR was found in the protein L-expanded CAR-T population. + T cells, showing that CAR + Positive selection of T cells.

[0198] Cytokine production in protein L-expanded CAR-T cells: CAR-T cells expanded for 8 days in culture with anti-CD3 mAb and protein L were incubated with untransfected P815 cells (control) and P815 cells transfected with human CD19 CAR antigen. CAR-T effector cells (E) and P815 target (T) cells were expressed at 10 5 and 2x10 5 1:2 E / T ratio) was added to the co-culture. Supernatants were collected after 24 hours for cytokine analysis using the LegendPlex multiplex flow cytometry assay (BioLegend).

[0199] Although CAR-T co-culture with untransfected P815 cells showed no significant target cell-induced cytokine production, + Culture of P815 cells was shown to induce the production of IFNγ, TNFα, granzyme B, sFasL, CCL3, CCL2, GM-CSF, IL2, and IL13; thus, a panel of cytokines was identified in response to CAR activation. The cytokine secretion profile was similar to that of CAR-T cells expanded in culture with anti-CD3 mAb or protein-L, although higher IFNγ and TNFα levels were detected in protein-L-expanded CAR-T cells ( Figure 4A ).

[0200] Protein L induces CAR-dependent cytokine production in vitro: Cytokine production induced by CAR activation was evaluated by incubating CAR-T cells with protein L adsorbed on plastic. E11 PSC-derived CAR-T cells were added to plastic-coated plates with protein L and recombinant fibronectin (both 0.5 μg / cm 2 ), or recombinant fibronectin alone (control). Cells were seeded at a density of 20,000 cells / well in 0.5 mL TCDM. After 24 hours of incubation, supernatants were collected for cytokine analysis using LegendPlex multiplex flow cytometry (BioLegend).

[0201] In wells coated with recombinant fibronectin alone, CAR-T cells produced granzyme B (GzmB), CCL3, and low levels of GM-CSF and IL13 during a 24-hour incubation period. When CAR-T cells were incubated in wells coated with protein L, induction of TNFα, IP10, IFNγ, sFasL, and IL6 was detected, indicating that protein L induced a CAR-dependent cytokine production response ( Figure 4B Treatment of CAR-modified immune cells with protein L can be used to evaluate CAR-dependent functional responses in vitro, including cytokine production.

[0202] Cytotoxic activity in protein L-expanded CAR-T cells: luciferase-expressing non-transfected P815 cells (control), P815 cells transfected with human CD19 CAR antigen, CD19 + In vitro cytotoxicity assay to evaluate the cytotoxic function of CAR-T cells expanded for 8 days in culture with anti-CD3 monoclonal antibody and protein L in B-cell lymphoma Daudi and Raji Figure 5 ). CAR-T effector (E) cells were incubated with tumor target (T) cells at a 1:2 E / T ratio for 24 hours, and luciferase activity in culture lysates was quantified by 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 individual target cell standards. Cytotoxicity was expressed as the absolute number of target cells lysed by a single effector cell and calculated by the following formula: (T control – T experimental / E, where “experimental” and “control” – target cell cultures with and without effector cells, respectively; T and E – the absolute number of target cells and effector cells, respectively. In addition, the cytotoxic function of CAR-T cells expanded in protein L culture was verified by real-time GFP+ cell counting against GFP-expressing Raji cells using the Incucyte S3 live cell analysis system (Essen Biosciences).

[0203] Although minimal cytotoxicity was detected in culture with CD19 CAR antigen-negative P815 cells, both anti-CD3 mAb and protein L-expanded CAR-T cells showed anti-CD19 + Strong cytotoxic activity against P815, Daudi, and Raji cells. Notably, protein L-expanded CAR-T cells showed ~2-3x higher cytotoxic activity than CAR-T cells expanded in culture with anti-CD3 monoclonal antibody. + The cytolytic activity of protein L-expanded CAR-T cells was also confirmed in culture with Raji cells, where GFP was detected only in culture with CAR-T cells.+ The significant decrease in viable cell counts was not seen in cultures with unmodified T cells.

[0204] Protein L-amplified CAR-T cells' anti-tumor potential: 8-week-old NSG mice were injected intraperitoneally (ip) with Daudi cells expressing luciferase. After 4 days, when tumor cell inoculation was confirmed by control imaging, the mice were divided into a control group (tumor only) and two experimental groups treated with CAR-T cells amplified in anti-CD3 monoclonal antibody culture or protein L culture. CAR-T cells were injected twice at intervals of 2 days. During CAR-T injection and another week, all mice were injected (ip) with IL2 and IL15 cytokines. Tumor progression was monitored weekly by bioluminescent in vivo imaging. Anesthetized mice injected with In Vivo-Glo luciferin (Promega) were analyzed using 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 in vivo was assessed by quantifying bioluminescent signal intensity (BLI) using Image Studio software (LiCor). The graph shows the mean ± STDBLI values ​​in each group over time.

[0205] It was observed that tumor growth was significantly inhibited in mice treated with CAR-T cells until 6 weeks after CAR-T injection ( Figure 6 ). Based on the in vitro cytotoxic potential, the anti-tumor effect of CAR-T cells expanded in protein L culture was higher.

[0206] In light of the present disclosure, all methods disclosed and claimed herein can be made and performed without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods and steps described herein, or the order of steps of the methods described herein, can be varied without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain reagents that are chemically and physiologically related can be substituted for the reagents described herein while obtaining the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0207] References

[0208] The following references, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0209] Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, NY, 1994

[0210] International Patent Application No. PCT / US2016 / 057893

[0211] Remington's Pharmaceutical Sciences 22nd Edition, 2012.

[0212] Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001.

[0213] U.S. Patent Application No. 12 / 715,136

[0214] U.S. Patent No. 8,372,642

[0215] Zheng et al., J Transl Med, 10:29, 2012.

Claims

1. A method for activating and / or expanding CAR-modified T cells or NK cells in vitro, the method comprising: (a) Obtaining a starting population of iPSC-derived CAR-modified T cells or NK cells; as well as (b) culturing the population of iPSC-derived CAR-modified T cells or NK cells in the presence of protein L and recombinant fibronectin (retronectin) for a sufficient time to generate a population of activated and / or expanded CAR-modified T cells or NK cells. The method according to claim 1 , wherein the protein L is coated on the culture surface.

3. The method of claim 2, wherein the culture surface is a culture plate, a culture flask, a microcarrier, a microparticle, a hydrogel particle or a culture bag.

4. The method according to claim 2, wherein the culturing is performed in the absence of anti-CD3 antibodies and / or antigen-specific target cells.

5. The method of claim 1, wherein the T cells are CD8 + T cells, CD4 + T cells, αβ T cells, or γδ T cells.

6. The method according to claim 5, wherein the method further comprises selecting CD8 + T cells.

7. The method of claim 1, wherein the CAR-modified T cells or NK cells are allogeneic.

8. The method of claim 1, wherein the CAR-modified T cells or NK cells are autologous.

9. The method of claim 1, wherein the iPSCs are reprogrammed from blood cells.

10. The method of claim 1, wherein the iPSCs are reprogrammed from T cells. The method of claim 1 , wherein the iPSCs are episomal reprogrammed.

12. The method according to any one of claims 1 to 11, wherein the iPSCs are differentiated into CD34 + Progenitor cells.

13. The method according to any one of claims 1 to 11, wherein the iPSCs are differentiated into CD34 + Progenitor cells.

14. The method of claim 1, wherein the CAR comprises an antigen binding domain selected from the group consisting of F(ab')2, Fab', Fab, Fv, and scFv.

15. The method of claim 1, wherein the CAR comprises a CD28 co-stimulatory domain and a CD3 zeta signaling domain.

16. The method of claim 2, wherein the culture surface is further coated with recombinant fibronectin.

17. The method according to claim 16, wherein the concentration of the recombinant fibronectin is 0.1-1 μg / cm 2 .

18. The method according to claim 16, wherein the concentration of the recombinant fibronectin is 0.5 μg / cm 2 .

19. The method of claim 2 or 16, wherein the culture surface is further coated with Notch ligand DLL4.

20. The method of claim 19, wherein the concentration of DLL4 is 0.1-1 μg / cm 2 .

21. The method of claim 19, wherein the concentration of DLL4 is 0.5 μg / cm 2 .

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

23. The method of claim 22, wherein the IL-12 and / or IL-15 is present at a concentration of 5-15 ng / mL.

24. The method of claim 22, wherein the IL-12 and / or IL-15 is present at a concentration of 10 ng / mL.

25. The method of claim 1, wherein the culturing is under hypoxic conditions.

26. The method of claim 25, wherein the hypoxic conditions comprise 5% oxygen.

27. The method of claim 1, wherein the sufficient time is 8-12 days.

28. The method of claim 27, wherein the sufficient time is 8, 9 or 10 days.

29. The method of claim 1, wherein the culturing is performed in a culture medium comprising SCF, TPO, FLT3L and / or IL-7.

30. The method of claim 29, wherein the concentration of SCF, TPO, FLT3L and / or IL-7 is 50 ng / mL.

31. The method of claim 29, wherein the culture medium further comprises nicotinamide.

32. The method of claim 1, wherein the method results in selective expansion of CAR-modified T cells or NK cells compared to non-CAR-modified T cells or NK cells.

33. The method of claim 32, wherein at least 40% or 50% of the expanded population of CAR-modified T cells or NK cells are CAR-modified T cells or NK cells.

34. The method of claim 1, wherein the expanded population of CAR-modified T cells comprises at least 25% CD3 + CD8 + CAR-modified T cells.

35. The method of claim 1, wherein the expanded population of CAR-modified T cells comprises 2-3 times higher cytotoxic activity compared to anti-CD3 expanded CAR-modified T cells.

36. The method of claim 1, wherein the expanded population of CAR-modified T cells comprises increased levels of IFNγ and / or TNFα compared to anti-CD3 expanded CAR-modified T cells.

37. A population of activated and / or expanded CAR-modified T cells or NK cells prepared according to the method of any one of claims 1-36.

38. A pharmaceutical composition comprising a population of activated and / or expanded CAR-modified T cells or NK cells according to claim 37 and a pharmaceutically acceptable carrier.

39. Use of the population of activated and / or expanded CAR-modified T cells or NK cells of claim 37 in the preparation of a medicament for treating cancer in a subject.

40. The use according to claim 39, wherein the activated and / or expanded CAR-modified T cells or NK cells are allogeneic.

41. The use according to claim 39, wherein the activated and / or expanded CAR-modified T cells or NK cells are autologous.

42. The use according to claim 39, wherein the activated and / or expanded CAR-modified T cells or NK cells are administered locally.

43. The use of claim 39, wherein the activated and / or expanded CAR-modified T cells or NK cells are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, by direct injection or by infusion.

44. Use of the pharmaceutical composition of claim 38 in the preparation of a medicament for treating cancer in a subject in need thereof.

45. A composition comprising CAR-modified T cells or NK cells and protein L and recombinant fibronectin.

46. ​​The composition of claim 45, wherein the protein L is coated on a culture surface.

47. The composition of claim 45, wherein the composition comprises protein L and recombinant fibronectin, and the protein L and recombinant fibronectin are coated on a culture surface.

Images