Methods for expanding and proliferating antigen-specific CAR-T cells, related compositions, and uses

Abstract

To provide methods for preparing and characterizing CAR-T cell cultures and preparations.SOLUTION: Provided herein are methods of generating allogeneic or autologous T cells that express a T cell receptor that specifically binds to an antigen (e.g., a virus antigen such as an EBV peptide) presented on a class I major histocompatibility complex (MHC) and a chimeric antigen receptor (CAR) that specifically binds to a target-cell antigen or cell surface marker (e.g., a cancer cell-associated antigen such as CD19).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 729,089, filed September 10, 2018, and U.S. Provisional Patent Application No. 62 / 896,707, filed September 6, 2019, which are incorporated in their entirety by reference. [Background technology]

[0002] Adoptive immunotherapy involves implanting or injecting disease-specific and / or engineered T cells, such as antigen-specific cytotoxic T cells (CTLs) and chimeric antigen receptor (CAR)-expressing T cells, into an individual with the aim of recognizing, targeting, and destroying disease-associated cells. Adoptive immunotherapy has become a promising approach for treating a number of diseases and disorders, including cancer, post-transplant lymphoproliferative disorders, infectious diseases (e.g., viral infections), and autoimmune diseases.

[0003] For example, exposure to the ubiquitous Epstein-Barr virus (EBV, also known as human herpesvirus 4) has been shown to predispose individuals to autoimmune diseases, including multiple sclerosis (MS), systemic autoimmune diseases (SAD), and inflammatory bowel disease (IBD), or to play a role in their pathogenesis. These conditions (i.e., MS, SAD, and IBD) result from an abnormal immune response to the body's own tissues. MS is characterized by the degradation of myelin, a protective lipid shell surrounding nerve fibers, by the body's own immune cells. SAD is a group of connective tissue diseases with diverse symptoms, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren's syndrome (SS). IBD is a group of inflammatory conditions of the colon and small intestine, including Crohn's disease, celiac disease, and ulcerative colitis. Recent studies have shown that individuals diagnosed with MS exhibit higher levels of EBV-related proteins in B cells aggregated in nerve tissue than healthy individuals.

[0004] The association between viral infection and the pathogenesis of multiple conditions (e.g., cancer and autoimmune diseases) has provided triggers for several groups to develop the production of allogeneic (e.g., see WO2017 / 203368 incorporated herein by reference) and autologous (e.g., see Pender et al., Multiple Sclerosis Journal. 2014; Vol. 20 (No. 11): pp. 1541-1544) antigen-specific T cells.

[0005] Immune surveillance by T cells plays a crucial role in the detection and elimination of a wide range of malignant cells (Gottschalk et al., 2005. Leuk Lymphoma. Vol. 46: pp. 1-10; Ochsenbein et al., 2002. Cancer Gene Ther. Vol. 9: pp. 1043-1010). The survival and spread of malignant cells are associated with their ability to evade recognition by CTLs (Bubenik et al., 2003. Oncol. Rep. Vol. 10: pp. 2005-2008; Rees et al., 1999. Cancer Immunol. Immunother. Vol. 48: pp. 374-381). In particular, antigen processing and presentation functions remain intact in EBV-related malignancies, such as Hodgkin lymphoma and nasopharyngeal carcinoma (Khanna et al., 1998. Cancer Res. 58: pp. 310-314; Lee et al., 1998. Blood Vol. 92: pp. 1020-1030). Recent studies suggest that immune surveillance (and subsequent responses) may be disrupted by the suppression of EBV-specific T cells mediated by regulatory T cells, leading to failure in the elimination of malignant cells. For example, upregulation of inhibitory immune checkpoint receptors, such as PD-1 and Tim-3, correlates with T cell dysfunction in patients with chronic HCV infection, and affects hepatitis C virus (HCV)-specific and HCV-nonspecific CD8 +This has been observed in T cells. Partial recovery of T cell proliferation and IFN-γ secretion can be achieved ex vivo by inhibiting the binding of PD-1 and Tim-3 to their respective ligands (i.e., B7-H1, also known as PD-L1, and galectin-9). Furthermore, recent reports have demonstrated that long-term administration of interferon therapy can lead to T cell "waste," a dysfunctional state characterized by decreased proliferative capacity, progressive loss of function, and persistent expression of inhibitory immune checkpoint receptors.

[0006] Chimeric antigen receptor (CAR)-T cells are an FDA-approved treatment for pediatric acute lymphoblastic leukemia and diffuse large cell lymphoma. In vivo proliferation and persistence of CAR-T cells are important factors in treatment efficacy. To this end, we hereby describe methods for generating and / or producing autologous and allogeneic antigen-specific T cells (e.g., T cells specifically sensitized to detect EBV-related antigens) that are engineered to express at least one functional chimeric antigen receptor directed to a selected disease-associated antigen. The generation of antigen-specific CTLs that also express one or more CARs provides a novel adoptive immunotherapy platform for the treatment of a wide range of diseases. Furthermore, central memory T cells (T) cm ) and stem cell-like memory T cells (T scm CAR-T cells exhibiting the phenotype promote proliferative capacity and sustained in vivo expansion after CAR T-cell injection (Kalos, 2018). Therefore, using enhanced culture and stimulation methods for T cells... cm and T scm Phenotypic enriched adoptive immunotherapy products (e.g., CAR-T cells) are advantageous for the in vivo potency, persistence, and efficacy of this type of therapy. Such enhancement of the T cell response to cancer and / or autoimmune-associated antigens by adoptive transplantation of engineered antigen-specific T cells (e.g., T cells expressing antigen-specific CARs) may offer an attractive alternative to current treatment strategies. [Overview of the Initiative]

[0007] Methods for generating allogeneic or autologous T cells expressing T cell receptors that specifically bind to antigens (e.g., viral antigens, e.g., EBV peptides) presented on class I major histocompatibility complexes (MHCs) and chimeric antigen receptors (CARs) that specifically bind to target cell antigens or cell surface markers (e.g., cancer cell-associated antigens, e.g., CD19) are provided herein. In some embodiments, antigen-specific T cells are generated by incubating a sample containing T cells (responder cells, e.g., PBMC samples or T cells isolated therefrom) with antigen-presenting cells (APCs, i.e., stimulator cells) that present antigens (e.g., viral peptides) on class I MHCs (e.g., class I MHCs encoded by HLA alleles present in the subject), thereby inducing the proliferation of antigen-specific responder T cells. Preferably, antigen-specific responder T cells are transduced with a viral vector containing a nucleic acid sequence encoding a CAR. Furthermore, a method for inducing ex vivo proliferation of a population of antigen-specific T cells expressing CAR is provided herein, comprising the steps of culturing an isolated population of T cells with antigen-presenting stimulator cells and transducing the resulting antigen-specific T cells with a viral vector containing a nucleic acid sequence encoding the CAR. In some embodiments, the transduced T cells are cultured with antigen-presenting stimulator cells to induce proliferation of antigen-specific CAR T cells. In certain other embodiments, the isolated T cells are transduced with a viral vector encoding the CAR before culturing with an APC. In further embodiments, the isolated T cells are cultured with an APC before and after transduction with a viral vector encoding the CAR.

[0008] In some embodiments, ex vivo methods for enriching central memory T cells are provided herein. In certain embodiments, such methods involve CD3 +Obtaining a sample of cells from a subject comprising T cells and said CD3 + comprising contacting the CD3 + T cells with antigen-presenting stimulator cells. In a preferred embodiment, the CD3 + T cells are isolated from the sample prior to contacting with the antigen-presenting stimulator cells by methods known in the art (e.g., positive selection of CD3 + cells from the sample and / or negative selection by depletion of unwanted cells or components from the sample). For example, without limitation, such methods include selection with anti-CD3 beads (e.g., magnetic beads), plastic adhesion, sedimentation, depletion of NK cells (e.g., using anti-CD56 beads) and / or combinations thereof. In some such embodiments, the CD3 + T cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR) before and / or after contact with the antigen-presenting stimulator cells. In some such embodiments, antigen-specific T cells expressing the CAR are cultured with the antigen-presenting stimulator cells.

[0009] In some embodiments, stimulator cells are prepared to present at least one viral peptide antigen by incubating the intended stimulator cells with a native / wild-type virus or with a viral vector encoding a viral peptide antigen. In some such embodiments, the viral peptide antigen is derived from viruses of a variety such as herpesviruses (e.g., EBV and CMV), papillomaviruses (e.g., HPV), adenoviruses, polyomaviruses (e.g., BKV, JCV, and Merkel cell viruses), retroviruses (e.g., HTLV-I, including lentiviruses such as HIV), picornaviruses (e.g., hepatitis A virus), hepadnaviruses (e.g., hepatitis B virus), hepaciviruses (e.g., hepatitis C virus), deltaviruses (e.g., hepatitis D virus), hepeviruses (e.g., hepatitis E virus), and oncoviruses. In certain preferred embodiments, stimulator cells are prepared to present an EBV peptide by incubating PBMCs with native / wild-type EBV. In other preferred embodiments, stimulator cells are prepared to present the EBV peptide by incubating PBMCs with a viral vector encoding the EBV peptide, thereby inducing the stimulator cells to present the EBV peptide. In some embodiments, the EBV peptide includes the LMP1 peptide or a fragment thereof, the LMP2A peptide or a fragment thereof, and / or the EBNA1 peptide or a fragment thereof. In some embodiments, the EBV peptide includes the sequences listed in Table 1. In some embodiments, the viral vector is a recombinant non-replicating adenovirus (e.g., AdE1-LMPpoly). In some embodiments, the stimulator cells may be B cells, antigen-presenting T cells, dendritic cells, or artificial antigen-presenting cells (e.g., cell lines expressing CD80, CD83, 41BB-L, and / or CD86, e.g., aK562 cells). Preferably, the antigen-presenting stimulator cells described herein also present a target cell antigen or cell surface marker against a CAR (e.g., CD19).In some embodiments, the stimulator cells are irradiated.

[0010] Furthermore, a method for identifying therapeutic preparations of CAR-T cells suitable for administration is provided herein. In some embodiments, a sample of a therapeutic preparation of CAR-T cells is obtained and the abundance of several T cell types and subtypes is evaluated. In some such embodiments, CD3 + CD4 + CD8 + CD62L + and / or CD45RO + The quantity of cells is evaluated.

[0011] In certain embodiments, a method for improving the proliferative capacity of T cells is provided herein, comprising the step of carrying out a method disclosed herein. In some embodiments, a method for improving the viability of CAR-expressing T cells is provided herein, comprising the step of carrying out a method disclosed herein. In certain embodiments, a T cell composition prepared by any one of the methods disclosed herein is provided herein. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows a flow diagram outlining the procedure and experimental groups for comparing CAR transduction of EBV-T cells derived from PBMCs or isolated T cells. [Figure 2] This figure shows the persistent central memory phenotype in antigen-stimulated T cells (EBV-CTLs). [Figure 3] This figure shows the growth curves of EBV-T cells derived from PBMCs and isolated T cells, transduced to express CAR. [Figure 4] This figure shows the enrichment of viable CAR+ cells derived from isolated T cells after blastosidine selection. [Figure 5] This figure shows a research design to investigate memory phenotypes in T cells after stimulation and CAR transduction. [Figure 6] This figure shows representative flow cytometry strategies for identifying CD3, CD4, CD8, CD62L, and CD45RO on CD19-CAR-T cells. [Figure 7] This figure shows representative dot plots of CD62L and CD45RO (gated to CD3+ cells) on CD19-CAR-T cells after stimulation with no stimulation, BLCL, soluble anti-CD3 / CD28, and bead-bound anti-CD3 / CD28. Naive PBMCs are also evaluated and serve as a reference for gateding to memory subsets; red rectangles represent CD62L expression on CD45RO+ cells. [Figure 8] This figure shows a representative dot plot of CD4 and CD8 expression (gated to CD3) on CD19-CAR T cells. [Figure 9] This figure shows that EBV-specific anti-CD19 CAR T cells exert potent, specific cytotoxicity. [Figure 10] This figure shows that EBV-specific anti-CD19 CAR T cells induce CD19-specific cytotoxicity. [Figure 11] This figure shows that while EBV-CTLs exhibit specific and potent HLA-independent cell lysis in both HLA-compatible (BLCL target) and HLA-incompatible (BLCL and Raji target) cells, they were only able to induce significant cell lysis in compatible BLCL target cells. [Figure 12] This figure shows conventionally generated anti-CD19 CAR T cells exhibiting enhanced alloreactive proliferation against EBV-specific anti-CD19 CAR T cells. [Figure 13]This figure shows the CellTrace® Violet dilution assay during co-culture with the indicated cell lines. Briefly, anti-CD19-CD28-CAR-EBV-CTLs induced cytolysis of CD19+ cell lines (i.e., BLCL, Raji, and NALM / 6; bottom-left), while preserving autologous and allogeneic targets lacking CD19 and EBV antigen expression (i.e., K562 and PHA blasts) and exhibiting significantly less alloreactivity to conventional CAR T cells (bottom-right, shaded). [Figure 14] This figure shows that the cytokine profiles of EBV antigen-specific anti-CD19 CAR T cells and conventionally generated anti-CD19 CAR T cells exhibit distinct responses. [Modes for carrying out the invention]

[0013] general The studies disclosed herein aim to determine the effects of various CAR-T cell stimuli to enrich memory T cell immunophenotypes in end-treatment products and to suit high-yield production processes. Standard anti-CD3 / CD28 bead-based stimulation is widely used in the art as a method for expanding T cells ex vivo or in vitro before transduction with CAR-encoding vectors. However, a process for producing antigen-stimulated T cells (e.g., virus-specific T cells) that also express one or more CARs is disclosed herein. Such a process involves CD3 + The process may include an initial T cell enrichment step in which T cells are enriched / purified from a more heterogeneous mixture of cells (e.g., from whole blood, from PBMCs, etc.), stimulated to recognize and respond to pre-selected antigens (e.g., viral antigens or other tumor / disease-associated antigens), and transduced with one or more CAR constructs, e.g., CD3 + Cell samples obtained from subjects containing T cells (e.g., PBMCs) are CD3 + T cells may also be enriched, as mentioned above. +T cells are brought into contact with antigen-presenting stimulator cells. Preferably, CD3 + T cells are isolated from the sample before contact with antigen-presenting stimulator cells. More preferably, the T cells and antigen-presenting stimulator cells (e.g., BLCL) originate from the same sample and are therefore HLA-matched. Such cell selection methods and techniques generally involve CD3 from the sample. + and / or CD19 + This includes positive selection of cells and / or negative selection by depletion of unwanted cells or components from the sample. For example, but not limited to, such methods include live cell sorting techniques (e.g., fluorescence-activated cell sorting), anti-CD3 and / or anti-CD19 beads (e.g., magnetic beads), plastic adhesion, NK cell depletion, selection by elutriation and / or combinations thereof. Before and / or after contact with antigen-presenting stimulator cells, the obtained CD3 + T cells can be transduced using viral vectors that encode chimeric antigen receptors (CARs).

[0014] The studies disclosed herein also provide a comparison of central and effector memory T cell subsets of anti-CD19 CAR-T cells derived from CTL stimulation in either CD3 / CD28 or BLCL co-cultures.

[0015] definition For convenience, the specific terms used in this specification, the examples, and the appended claims are set forth herein.

[0016] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, “one element” means one element or more than one element.

[0017] As used herein, the term “administer” means to provide a drug or composition to a subject, and includes, but is not limited to, administration by a healthcare professional and self-administration. Such drugs may include, for example, the peptides described herein, antigen-presenting cells provided herein, and / or CTLs provided herein.

[0018] The terms "binding" or "interacting" refer to an association that may be a stable association between two molecules, for example, between a TCR and a peptide / MHC, due to electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions under physiological conditions.

[0019] The terms “biological sample,” “tissue sample,” or simply “sample” refer, respectively, to a collection of cells obtained from the tissue of the subject. Sources of tissue samples may include solid tissue such as fresh, frozen, and / or preserved organs, tissue samples, biopsies, or aspirates; blood or any blood component; serum; blood; body fluids, such as cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; or cells from any point in time of the subject’s pregnancy or development.

[0020] As used herein, the term “cytokine” refers to any secreted polypeptide that affects cellular function and modulates intercellular interactions in immune, inflammatory, or hematopoietic responses. Cytokines include, but are not limited to, monokines and lymphokines, regardless of which cells produce them. For example, monokines are generally said to be produced and secreted by mononuclear cells such as macrophages and / or monocytes. However, many other cells, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes, also produce monokines. Lymphokines are generally said to be produced by lymphocytes. Examples of cytokines, though not limited to them, include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-15 (IL-15), tumor necrosis factor-alpha (TNFα), and tumor necrosis factor-beta (TNFβ).

[0021] The term “antibody fragment” refers to any derivative of an antibody smaller than its full length. In exemplary embodiments, an antibody fragment retains at least a substantial portion of the specific binding ability of the full-length antibody. Examples of antibody fragments, but not limited to, include Fab, Fab', F(ab')2, scFv, Fv, dsFv diabodies, Fc, and Fd fragments. Antibody fragments may be produced by any means. For example, an antibody fragment may be produced enzymatically or chemically by fragmentation of an intact antibody, by recombination from a gene encoding a partial antibody sequence, or by synthesis, either whole or partially. An antibody fragment may optionally be a single-chain antibody fragment. Alternatively, a fragment may contain multiple chains linked together, for example, by disulfide bonds. A fragment may also optionally be a multimolecular complex. Functional antibody fragments typically contain at least about 50 amino acids, and more typically, at least about 200 amino acids.

[0022] The term "antigen-binding site" refers to the region of an antibody that specifically binds to an epitope on an antigen.

[0023] The term "single-strand variable fragment" or "scFv" refers to an Fv fragment in which a heavy chain domain and a light chain domain are linked. One or more scFv fragments can be linked with other antibody fragments (e.g., constant domains of the heavy or light chain) to form an antibody construct having one or more antigen recognition sites.

[0024] The term "Fab fragment" refers to a fragment of an antibody containing an antigen-binding site, which is generated by the cleavage of an antibody with the enzyme papain, which cleaves the hinge region at the N-terminus of the inter-H chain disulfide bond, producing two Fab fragments from one antibody molecule.

[0025] The term "F(ab')2 fragment" refers to an antibody fragment containing two antigen-binding sites, which is generated by cleaving an antibody molecule with the enzyme pepsin, which cleaves the hinge region at the C-terminus of the inter-H chain disulfide bond.

[0026] The term "Fc fragment" refers to a fragment of an antibody that contains the constant domain of its heavy chain.

[0027] The term "Fv fragment" refers to a fragment of an antibody that contains the variable domains of its heavy and light chains.

[0028] The term "manipulated antibody" refers to a recombinant molecule comprising at least an antibody fragment that includes an antigen-binding site derived from the variable domains of the antibody's heavy and / or light chain, and optionally includes all or part of the variable and / or constant domains of an antibody derived from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM, and IgY).

[0029] The terms “specifically bind,” “specifically bind,” or “targeting,” as used herein when referring to polypeptides (including antibodies) or receptors, refer to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Therefore, a particular ligand or antibody “specifically binds” to its individual “targets” (e.g., an antibody specifically binds to an endothelial antigen) if, under specified conditions (e.g., immunoassay conditions in the case of an antibody), it does not bind in significant amounts to other proteins present in the sample, or to other proteins that the ligand or antibody may come into contact with in the organism. Generally, a first molecule that “specifically binds” to a second molecule will bind to that second molecule, and about 10 times that amount. 5 M -1 Larger (for example, 10) 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 and 10 12 M -1 It has an affinity constant (Ka) of 10⁻⁴ or greater. For example, in the case of a TCR's ability to bind to a peptide presented on an MHC (e.g., a class I MHC or a class II MHC), the TCR typically binds specifically to that peptide / MHC with an affinity of at least about 10⁻⁴ M or less, and binds with an affinity (as represented by KD) that is at least 10 times less, at least 100 times less, or at least 1000 times less than its affinity for binding to a given antigen / binding partner and to nonspecific and unrelated peptide / MHC complexes (e.g., those containing BSA peptides or casein peptides).

[0030] The term "epitope" refers to a protein determinant that can specifically bind to an antibody or TCR. Epitopes typically consist of chemically active surface groups of a molecule, such as amino acids or sugar side chains. A specific epitope can be defined by a particular sequence of amino acids to which an antibody can bind.

[0031] As used herein, the term “pharmaceutically acceptable” means a drug, compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit-to-risk ratio.

[0032] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle involved in transporting a drug from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, or material encapsulating a solvent. Each carrier must be compatible with the other components of the formulation and “acceptable” in the sense that it is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (1 (0) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffering agents, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Phenothermally hydrated substances; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffered solutions; (21) Polyesters, polycarbonates, and / or polyanhydrides; and (22) Other non-toxic, suitable substances used in pharmaceutical formulations.

[0033] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci(s) determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure, if present, may be made before or after the construction of the polymer. Polynucleotides may be further modified, for example, by conjugation with labeling components. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0034] As used herein, a therapeutic agent that “prevents” a condition means a compound that, when administered to a statistical sample before the onset of the disorder or condition, reduces the incidence of the disorder or condition in the treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0035] As used herein, “specific binding” refers to the ability of a TCR to bind to a peptide presented on an MHC (e.g., class I MHC or class II MHC). Typically, a TCR binds to at least about 10 -4 K below M DIt binds specifically to the peptide / MHC with affinity (K), and has an affinity (K) that is at least 10 times smaller, at least 100 times smaller, or at least 1000 times smaller than its affinity to binding to nonspecific and unrelated peptide / MHC complexes (e.g., those containing BSA peptide or casein peptide). D It binds to a predetermined antigen / binding partner (such as represented by ).

[0036] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0037] In certain embodiments, the agents of the present invention may be used by administration alone or in combination with another type of therapeutic agent. As used herein, the terms “combined administration” or “administered together” refer to any form of administration of two or more different therapeutic agents (e.g., compositions comprising CAR T and immune checkpoint inhibitors disclosed herein) in which a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include a synergistic effect between the two agents). For example, different therapeutic agents may be administered simultaneously or sequentially, either in the same formulation or in separate formulations. In some preferred embodiments, CAR T cells express further therapeutic agents (e.g., present on or secreted on the cell surface). In certain embodiments, different therapeutic agents (e.g., CAR T cells and immune checkpoint blocking molecules) may be administered to each other within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week. Therefore, those receiving such treatment may benefit from the combined effects of different medications.

[0038] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual being treated during the course of a clinicopathological condition. Desired effects of treatment include a reduction in the rate of progression, improvement or alleviation of a pathological condition, and remission or an improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0039] The term "vector" refers to a means by which nucleic acids can be replicated and / or transferred between organisms, cells, or cellular components. Examples of vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, and artificial chromosomes, which may or may not be able to replicate autonomously, or may be incorporated into the chromosomes of a host cell.

[0040] T cell enrichment To date, methods for producing CAR-T cells known in this art use crude, heterogeneous starting materials, such as PBMCs (see Sun et al., Journal for ImmunoTherapy of Cancer (2015) Vol. 3:5) or highly purified specific T cell types, namely CD4 + CD8 + The present invention has relied on either T cells or isolates of a specific proportion thereof (see Terakura et al. Blood (2011) Vol. 119:1 and Turtle et al. Sci Transl Med. (2016) Sep 7;8). However, the present invention disclosed herein provides an ex vivo method for enriching antigen-specific T cells to be used in the production of CAR-T cells. In particular, in some preferred embodiments, such a method extracts CD3 from a target. + The steps of obtaining a cell sample containing cells (e.g., PBMC) and the CD3 + The process includes the step of bringing cells into contact with antigen-presenting stimulator cells. Preferably, CD3 +Before contacting T cells with antigen-presenting stimulator cells, they are subjected to methods known in the art (e.g., CD3 from a sample). + The cells are isolated from the sample by positive selection of cells and / or negative selection by depletion of unwanted cells or components from the sample. For example, but not limited to, such methods include using fluorescence-activated cell sorting (FACS), depletion of NK cells using anti-CD3 beads (e.g., magnetic beads), plastic adhesives, anti-CD56, selection by elutriation and / or combinations thereof. + Sensitizing cells to a specific antigen, such as a viral antigen, can promote a central memory phenotype in the resulting antigen-specific T cell population. Such T cells can be transduced with a viral vector encoding a chimeric antigen receptor (CAR) before and / or after contact with antigen-presenting stimulator cells. In some such embodiments, CD3 expressing a CAR is used. + Antigen-specific T cells are cultured together with antigen-presenting stimulator cells.

[0041] The first CD3 disclosed herein + The concentration step provides a starting material that is considerably less heterogeneous than PBMC samples, but retains a certain level of heterogeneity relative to highly purified T cell fractions. I don't want to get bogged down in theory, but the first CD3 + By using the concentration step, the starting materials can work synergistically, increasing the viability and proliferation of antigen-specific T cells after contact with APC, improving the transduction efficiency of vectors expressing CARs in such antigen-specific T cells, and resulting in a higher percentage of T cells in the final therapeutic composition. CM A mixture of cells that can provide a favorable environment for cell growth (at least multiple effector T cell types, helper T cells (CD4) + T cells / T H cells), cytotoxic T cells (CD8 + T cells (CTLs), memory T cell type (i.e., central memory T cells (T) CM Cells), effector memory T cells (TEM T cells), tissue-resident memory T cells (T RM ) and virtual memory T cells (T VM cells)), regulatory T cells (T reg Cells, natural killer T cells (NKT cells), mucosa-associated invariant cells (MAIT cells), gamma delta T cells (γδ T cells), double-negative T cells (DNT), CD3 + It is assumed that the cells include B cells or any combination thereof. In some embodiments, further enrichment is performed after T cell stimulation with an antigen. For example, NK depletion (e.g., CD56 depletion) may be used before the subsequent antigen stimulation step (i.e., before the restimulation of enriched antigen-specific T cells with the antigen).

[0042] Stimulation and Transduction The production of T cells expressing T cell receptors that specifically bind to peptides presented on class I MHC requires T cell proliferation against defined antigens. In some embodiments, T cells also express CARs that specifically bind to disease-related peptides (e.g., tumor-related peptides, antigens, ligands, etc.).

[0043] Antigen delivery is performed using peripheral blood mononuclear cells (PBMCs) obtained from healthy donors or B-cell lymphoblastoid cells isolated therefrom (e.g., CD19). +Transduction can occur by viral infection of a BLCL sample with a native virus, or by transduction using a recombinant virus. Thus, the infected or transduced cells act as antigen-presenting cells and are called “stimulators.” In certain embodiments, stimulator cells also express peptides (i.e., antigens) on their cell surface that are recognized by CARs. Stimulator cells may endogenously express such CAR-targeted peptides, or they may be engineered to express such peptides. The viral vector used to transduce stimulators may be a recombinant non-replicating virus (e.g., adenovirus, e.g., AdE1-LMPpoly). Alternatively, the stimulator cells may be infected with a wild-type / native virus. A separate sample or culture (e.g., PBMCs from the same donor not used for transduction, samples of PBMCs from different healthy donors, or CD3 isolated therefrom) may be used. + The cells contain "responder" T cells, which are the active components of the therapy and express T cell receptors that specifically bind to peptide antigens presented on class I MHC. Responder T cells may be isolated from PBMCs by any suitable method, many of which are well known in the art. For example, "responder" T cells can be isolated from a sample before presentation to the stimulator fraction by methods known in the art, e.g., anti-CD3 beads (e.g., magnetic beads), plastic adhesives, elutriation and / or a combination thereof. Preferably, "stimulating" cells (e.g., PBMCs or BLCLs) are obtained from the same cell population (e.g., PBMC sample) as the "responder" cells, and as a result, they are also HLA-matched. For example, a PBMC sample obtained from a donor is separated into a "stimulating" cell fraction and a "responder" cell fraction, and the stimulator cells are presented to the CD3 + The cells may be concentrated, and responder cells may be transduced or infected to present a specific antigen on their cell surface. Optionally, the stimulator cell fraction may be treated with, for example, CD19, by a method known in the art prior to transduction / infection. +Cells may be enriched by selection. Therefore, antigen-presenting cells are T cells (e.g., CD3 + The antigen is presented to cells (which are concentrated with the stimulator fraction), thereby activating and inducing the proliferation of antigen-specific T cells. In some such embodiments, responder T cells (e.g., isolated T cells) are transduced with a CAR-encoding vector before antigen presentation by the stimulator fraction. In certain embodiments, responder T cells (e.g., isolated T cells) are transduced with a CAR-encoding vector after antigen presentation by the stimulator fraction.

[0044] Accordingly, methods for generating allogeneic or autologous T cells are provided herein that express a T cell receptor that expresses a chimeric antigen receptor (CAR) that specifically binds to an EBV peptide presented on a class I MHC and further expresses a chimeric antigen receptor (CAR) that binds to a selected target (e.g., CD19). In some embodiments, APCs are generated by viral infection of stimulator cells, for example, by a wild-type / natural EBV or adenovirus vector, for example, AdE1-LMPpoly. The AdE1-LMPpoly vector encodes a polyepitope of defined CTL epitopes derived from LMP1 and LMP2, which are fused with an EBNA1 sequence depleted of Gly-Ala repeats. The AdE1-LMPpoly vector is described, for example, in Smith et al., Cancer Research 72:1116 (2012); Duraiswamy et al., Cancer Research 64:1483-1489 (2004); and Smith et al., J. Immunol 117:4897-4906 (2006), each incorporated herein by reference. In some embodiments, stimulator cells are mixed with uninfected isolated T cells (responders) to present the EBV polyepitope to the T cells. In some embodiments, the isolated virus-specific T cells presented with the EBV polyepitope are activated and induced to proliferate.

[0045] T cells can be classified into one of three subtypes: naive or those that have undergone three major antigen changes: central memory T cells, effector memory T cells, and terminal effector T cells. CM Effector memory T cells (T) are commonly found in lymph nodes and peripheral circulation. This subtype expresses CD45RO, CC chemokine receptor type 7 (CCR7), and L-selectin (CD62L). EM These cells lack lymph node homing receptors and are therefore primarily found in peripheral circulation and tissues. EM The cells express CD45RO but lack expression of CCR7 and L-selectin. In certain embodiments, a method for identifying a therapeutic preparation of CAR-T cells as suitable for administration to a recipient is provided herein. In some embodiments, a sample of the therapeutic preparation of CAR-T cells is obtained and evaluated for different T cell subtypes. Preferably, the preparation is primarily CAR-expressing T cells. cm Cells and / or CD4 + Includes T cells.

[0046] In some embodiments, responder cells and stimulator cells are each derived from peripheral blood mononuclear cells (PBMCs). In some such embodiments, responder cells and stimulator cells are each derived from PBMCs from the same donor. In other embodiments, responder cells and stimulator cells are each derived from PBMCs from different donors. In some such embodiments, before contact with stimulator cells, responder cells are isolated and / or purified to be essentially devoid of T cells. In preferred embodiments, responder cells are isolated and / or CD3 + The cells are purified to be essentially the same. Most preferably, the responder cells are CD3 + Essentially derived from T cells.

[0047] Prior to presentation to responder cells (e.g., T cells), the stimulator cells may be infected with a native virus, such as EBV, thereby presenting viral antigens on their surface. In some embodiments, the stimulator cells are transduced with a viral vector, preferably an adenovirus vector containing a nucleic acid sequence encoding a herpesvirus antigen. In some such embodiments, the adenovirus vector is non-replicable. More preferably, the adenovirus vector contains one or more nucleic acid sequences encoding one or more EBV antigens. One or more EBV antigens may include an LMP1 peptide or fragment thereof, an LMP2A peptide or fragment thereof, and / or an EBNA1 peptide or fragment thereof. Most preferably, the adenovirus vector is AdE1-LMPpoly, encoding a polyepitope of defined CTL epitopes derived from LMP1 and LMP2 fused to an EBNA1 sequence with a Gly-Ala repeat depleted. In some embodiments, the stimulator cells are transduced with responder cells (e.g., uninfected PBMCs or CD33). + The cells are incubated with one or more cytokines before being cultured (i.e., presented to) cells that are concentrated with the antigen. Such stimulator cells may include B cells (e.g., BLCLs), antigen-presenting T cells, dendritic cells, artificial antigen-presenting cells, and / or aK562 cells. In a preferred embodiment, the stimulator cells are antigen-presenting BLCLs.

[0048] Antigen-specific T cells achieve activation and proliferation when presented with antigens by the stimulator fraction; however, such stimulator cells are undesirable in the final recovered CAR-T cell product. Furthermore, to minimize the risk of any viral recombination events in cell proliferation that could lead to the formation of competent viruses, stimulator cells are treated and / or modified prior to culture with responder T cells to inhibit proliferation, for example, by gamma irradiation or exposure to a drug, such as mitomycin C. For example, under such culture conditions, responder cells (e.g., T cells) are presented with peptide antigens by non-proliferating stimulator cells. In some such embodiments, the culture is maintained for at least 24 hours to at least 28 days before transduction with a CAR-encoding vector. In some embodiments, the culture is maintained for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 17 days, or at least 28 days before transduction with the CAR-encoding vector. Preferably, the culture is maintained for at least 2 days after antigen presentation by stimulator cells and before transduction with the CAR-encoding vector. Most preferably, the culture is maintained for at least 6 days after antigen presentation by stimulator cells and before transduction with the CAR-encoding vector. In further embodiments, the culture is maintained for at least 24 hours to at least 28 days after transduction with the CAR-encoding vector. In a particular manner, the culture is maintained for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 17 days, or at least 28 days after transduction with the vector encoding the CAR.In some embodiments, the culture is re-seeded and / or re-stimulated as needed. For example, responder T cells experience at least a first stimulation step (i.e., are presented with an antigen on an APC) and may be re-seeded and / or re-stimulated as needed (i.e., a second or more). The re-seeded and / or re-stimulated culture is maintained for at least 24 hours, at least 3 days, at least 9 days, at least 11 days, at least 14 days, at least 17 days, or at least 28 days before and / or after transduction with a CAR-encoding vector. In preferred embodiments, the responder cell culture is stimulated at least once before transduction with a CAR-encoding vector. More preferably, the responder culture is stimulated multiple times, with subsequent stimulation steps separated by any of 2 to 14 days. For example, a culture experiencing a first stimulation may experience a second stimulation (e.g., restimulation) 11 days after the first stimulation, and optionally, a third stimulation (e.g., restimulation) may be initiated 7 days after the second stimulation step. Cultures experiencing a stimulation step (e.g., restimulation) are maintained for at least 1 to 10 days before transduction with a CAR-encoding vector. Preferably, the culture is maintained for at least 2 days before transduction with a CAR-encoding vector. Most preferably, the culture is maintained for at least 6 days after antigen presentation by stimulator cells and before transduction with a CAR-encoding vector. Optionally, an NK depletion step may be used before stimulation. For example, CD56 from the culture immediately before stimulation with APC. + Cell depletion may occur (for example, when using anti-CD56 beads).

[0049] In certain embodiments, antigen-specific T cells may be frozen and stored for future thawing before and / or after transduction with a CAR-encoding vector, after at least a first presentation of the antigen to achieve activation and proliferation. For example, T cells expressing the antigen-specific CAR contemplated herein may be administered to a subject requiring it, or frozen for storage in a cell bank or repository for thawing at a later date. In some such embodiments, the thawed culture is restimulated at least one more time with the antigen (e.g., with antigen-presenting stimulator cells, e.g., BLCL, etc., as described herein) before and / or after transduction as described herein. In some embodiments, as described herein, the thawed culture is restimulated and / or reseeded as necessary, and the restimulated and / or reseeded culture is maintained for at least 24 hours, at least 2 days, at least 3 days, at least 9 days, at least 11 days, at least 14 days, at least 17 days, or at least 28 days before and / or after transduction with a CAR-encoding vector. Similarly, if the culture is re-stimulated multiple times, the subsequent stimulation steps are spaced apart by either 2 to 14 days, as described herein.

[0050] Activation and proliferation of antigen-specific T cells also require sufficient antigen presentation by stimulator cells. Therefore, the stimulation cultures intended herein (including, for example, restimulation cultures) include responder cells to stimulator cells in a known ratio. For example, the responder cell to stimulator cell ratio is about 0.1:1 to about 20:1. Preferably, the responder cell to stimulator cell ratio is about 0.2:1 to about 5:1. In some embodiments, the responder cell to stimulator cell ratio is approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some such embodiments, the responder cell to stimulator cell ratio is approximately 0.25:1, 0.43:1, or 4:1. Furthermore, if the culture is restimulated multiple times, each stimulus may include responder cells versus stimulator cells in the same or different ratios. For example, in a preferred embodiment, though not limited to, the initial stimulus may include a responder:stimulator ratio of approximately 0.43:1. Subsequent stimuli (e.g., restimulation) may include a responder:stimulator ratio of 0.25:1, and further stimuli (e.g., restimulation) may include a responder:stimulator ratio of 4:1.

[0051] Those skilled in the art will understand that cell growth in a culture can vary and is limited by the requirements for nutrients and oxygen, as well as by the accumulation of waste products, such as carbon dioxide and lactic acid. As such, those skilled in the art will be able to empirically determine an appropriate culture and (if necessary) reseeding schedule to achieve the T cells of the present invention. In some embodiments, the culture is maintained until a predetermined recovery date. Evaluation of the culture and determination of the presence or absence of active viruses are performed prior to and / or on the recovery date. Most preferably, the cultures and / or preparations disclosed herein are essentially free of active viruses at the time of CTL recovery.

[0052] Antigen peptide In certain embodiments, TCRs specifically bind to peptides (e.g., antigens) containing T cell epitopes presented on class I MHC, as well as to autoimmune disorders and cancers (e.g., MS, SAD, IBD and / or CD19 + Methods for generating allogeneic or autologous CAR-T cells expressing CARs that bind to selected targets, such as disease-related peptide targets, for treating B-cell malignancies, lymphomas, and leukemias are provided herein. + T cells suitable for CAR-T cell production are intended herein, which are generated by incubating a cell with antigen-presenting cells (APCs) that present one or more of the T cell epitopes described herein (e.g., APCs that present peptides described herein, including an EBV epitope on a class I MHC complex, e.g., EBV-infected or recombinantly transduced BLCL).

[0053] In some such embodiments, the peptide containing a T cell epitope as described herein includes epitopes derived from viruses, for example, but not limited to, herpesviruses (e.g., EBV and CMV), papillomaviruses (e.g., HPV), adenoviruses, polyomaviruses (e.g., BKV, JCV, and Merkel cell viruses), retroviruses (e.g., HTLV-I, including lentiviruses such as HIV), picornaviruses (e.g., hepatitis A virus), hepadnaviruses (e.g., hepatitis B virus), hepaciviruses (e.g., hepatitis C virus), deltaviruses (e.g., hepatitis D virus), and hepeviruses (e.g., hepatitis E virus). In some embodiments, the epitope is an HLA class I-restricted T cell epitope. In other embodiments, the epitope is an HLA class II-restricted T cell epitope.

[0054] The peptides provided herein may include sequences of any EBV viral protein (for example, sequences of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of any EBV protein). In some embodiments, the peptides provided herein include 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of an EBV viral protein.

[0055] The peptides provided herein may include a sequence of LMP1 (for example, a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of LMP1). In some embodiments, the peptides provided herein include 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of LMP1. An exemplary LMP1 amino acid sequence is provided below (SEQ ID NO: 1): JPEG0007882904000001.jpg83150JPEG0007882904000002.jpg8150

[0056] In some embodiments, the peptides provided herein include a sequence of LMP2A (for example, a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of LMP2A). In some embodiments, the peptides provided herein include 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of LMP2A. An example LMP2A amino acid sequence is provided below (SEQ ID NO: 2): JPEG0007882904000003.jpg118150

[0057] In some embodiments, the peptides provided herein include a sequence of EBNA1 (for example, a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of EBNA1). In some embodiments, the peptides provided herein include 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of EBNA1. An example EBNA1 amino acid sequence is provided below (SEQ ID NO: 3): JPEG0007882904000004.jpg15150JPEG0007882904000005.jpg43151

[0058] Preferably, the peptide contains the epitope sequences listed in Table 1.

[0059] [Table 1]

[0060] In certain embodiments, the peptides provided herein include peptides comprising epitopes comprising sequences homologous to any polyomavirus protein, e.g., BKV epitope, JCV epitope, MCV epitope, and / or CTLs that are recognized when presented on HLA. In certain embodiments, the epitopes described herein are useful for the prevention and / or treatment of polyomavirus infection (e.g., BKV, JCV, or MCV virus infection) and / or cancer (e.g., polyomavirus-associated cancer expressing the epitopes provided herein), and for the production of pharmaceuticals (e.g., CTLs and / or APCs) that are useful for the prevention and / or treatment of polyomavirus infection (e.g., BKV, JCV, or MCV virus infection) and / or cancer (e.g., polyomavirus-associated cancer expressing the epitopes provided herein). In some embodiments, the epitope is a hybrid epitope comprising amino acids derived from both the BKV epitope and the homogeneous JCV epitope, and / or amino acid variants found within different BKV or JCV epitopes. In some embodiments, the compositions and methods provided herein further comprise an MCV epitope. Exemplary peptides comprising the BKV epitope, JCV epitope, MCV epitope, and / or epitopes containing homologous sequences among the BKV, JCV, and / or MCV epitopes (e.g., hybrid epitopes) can be found in WO2018049165, the full text of which is incorporated herein.

[0061] In some embodiments, the peptides provided herein include peptides containing sequences of any cytomegalovirus protein, for example, epitopes recognized by cytotoxic T lymphocytes (CTLs) and useful in the prevention and / or treatment of CMV infection and / or cancer (e.g., cancer expressing the CMV epitope). Exemplary peptides containing CMV epitopes can be found in WO2017203370, WO2014059489, and WO2006056027, each of which is incorporated herein in full.

[0062] In some embodiments, the peptides provided herein include peptides containing sequences of any human papillomavirus protein, e.g., HPV epitopes that are recognized by cytotoxic T lymphocytes (CTLs) and are useful in the prevention and / or treatment of HPV infection and / or cancer (e.g., cancers expressing HPV epitopes) and / or precancerous lesions. Exemplary peptides containing HPV epitopes can be found in full in PCT / US2019 / 014727, which is incorporated herein.

[0063] In some embodiments, the peptide comprises epitopes derived from two or more viruses. In some embodiments, the peptide comprises epitopes derived from three or more viruses. In some embodiments, the peptide comprises epitopes derived from four or more viruses. In some embodiments, the peptide comprises epitopes derived from five or more viruses. For example, in some embodiments, the peptide comprises sequences derived from at least two, three, four, or five of JCV, BKV, MCV, EBV, CMV, and / or HPV.

[0064] In some embodiments, the peptides provided herein comprise two or more T cell epitopes (e.g., viral epitopes). In some embodiments, the peptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 T cell epitopes. For example, in some embodiments, the peptides provided herein comprise two or more T cell epitopes linked by a linker (e.g., a polypeptide linker). In some embodiments, the polypeptide or protein further comprises an amino acid sequence interposed between at least two of the multiple epitopes. In some embodiments, the interposed amino acid or amino acid sequence is a proteasome-free amino acid or amino acid sequence. Not limited to the proteasome-free amino acid or amino acid sequence, there are AD, K, or R, or include AD, K, or R. In some embodiments, the interposed amino acid or amino acid sequence is a TAP recognition motif. Typically, the TAP recognition motif may conform to the following formula: (R / N:I / Q:W / Y)n (wherein n is any integer ≥ 1). Examples of TAP recognition motifs that are not limited to this include RIW, RQW, NIW, and NQY. In some embodiments, the epitopes provided herein are linked or conjugated at the carboxyl terminus of each epitope by the proteasome free amino acid sequence and, optionally, by the TAP recognition motif.

[0065] In some embodiments, the peptide sequence includes the viral protein sequence with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications. As used herein, the term “conservative sequence modification” is intended to mean an amino acid modification that does not significantly affect or alter the interaction between the T cell receptor (TCR) and the peptide containing the amino acid sequence presented on the MHC. Such conservative modifications include amino acid substitutions, additions (e.g., addition of amino acids to the N-terminus or C-terminus of a peptide) and deletions (e.g., deletion of amino acids from the N-terminus or C-terminus of a peptide). A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues of the peptides described herein can be substituted with other amino acid residues from the same side chain family, and the modified peptides can be tested for TCR binding retention using methods known in the art. Modifications can be introduced into antibodies by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0066] In some embodiments, the peptides provided herein include sequences that are at least 80%, 85%, 90%, 95%, or 100% identical to a protein sequence (e.g., a sequence of a viral protein fragment). To determine the identity percentage of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). Next, the amino acid residues at the corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0067] Peptides may be chimeric or fusion peptides. As used herein, “chimeric peptide” or “fusion peptide” includes a peptide having a sequence provided herein that is linked to a separate peptide having a sequence that is not naturally linked. For example, the separate peptide may be fused to the N-terminus or C-terminus of the peptide provided herein either directly by a peptide bond or indirectly via a chemical linker. In some embodiments, the peptide provided herein is linked to another peptide containing a separate epitope. In some embodiments, the peptide provided herein is linked to a peptide containing an epitope associated with other viruses and / or infectious diseases. Chimeric or fusion peptides provided herein can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different peptide sequences are ligated in frame according to conventional techniques, for example, by using blunt-ended or stagger-ended ends for ligation, restriction enzyme digestion to provide suitable ends, attachment end completion as needed, alkaline phosphatase treatment to avoid undesirable linkages, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesis equipment. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that create complementary protrusions between two consecutive gene fragments, followed by annealing and re-amplification to generate a chimeric gene sequence (see, for example, *Current Protocols in Molecular Biology*, edited by Ausubel et al., John Wiley & Sons: 1992). Furthermore, numerous expression vectors encoding the fusion region are already commercially available.

[0068] The peptides provided herein can be isolated from cell or tissue sources by appropriate purification schemes using standard protein purification techniques, produced by recombinant DNA techniques, and / or chemically synthesized using standard peptide synthesis techniques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding the peptide(s) of the present invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for the expression of heterologous peptides in recombinant hosts, the chemical synthesis of peptides, and in vitro translation are well known in the art. Furthermore, the following are incorporated herein by reference: Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd edition, Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Vol. 152; Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken IM (1981) CRC Crit. Rev. Biochem. 11:255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, SBH (1988) Annu. Rev. This information is found in Biochem. 57:957, Offord, RE (1980) Semisynthetic Proteins, Wiley Publishing.

[0069] In certain embodiments, nucleic acid molecules encoding peptides described herein are provided herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is a viral vector containing the nucleic acid molecules described herein, for example, an adenovirus-based expression vector. In some embodiments, the vector provided herein encodes a plurality of epitopes (e.g., as polyepitopes) provided herein. In some embodiments, the vector provided herein encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 epitopes (e.g., epitopes provided in Table 1) provided herein.

[0070] In some embodiments, the vector is a viral vector (e.g., adenovirus, e.g., AdE1-LMPpoly). The AdE1-LMPpoly vector encodes a polyepitope of defined CTL epitopes derived from LMP1 and LMP2 fused to a Gly-Ala repeat-deficient EBNA1 sequence. AdE1-LMPpoly vectors are described, for example, by Smith et al., Cancer Research 72:1116 (2012), Duraiswamy et al., Cancer Research 64:1483~9 (2004), and Smith et al., J. Immunol 117:4897~4906 (2006), each of which is incorporated herein by reference.

[0071] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell and thereby replicate with the host genome. Furthermore, certain vectors can direct gene expression. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In some embodiments, nucleic acids functionally ligated to one or more regulatory sequences (e.g., promoters) in an expression vector are provided herein. In some embodiments, a cell transcribes the nucleic acid provided herein and thereby expresses the peptide described herein. The nucleic acid molecule may be integrated into the cell's genome, or it may be extrachromosomal.

[0072] In some embodiments, cells containing nucleic acids described herein (e.g., nucleic acids encoding peptides described herein) are provided herein. The cells may be, for example, prokaryotes, eukaryotes, mammals, birds, mice and / or humans. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are APCs (e.g., antigen-presenting T cells, dendritic cells, B cells, or aK562 cells). In the method, the nucleic acids described herein can be administered to cells in combination with a delivery reagent, for example, as nucleic acids without a delivery vehicle. In some embodiments, any nucleic acid delivery method known in the art may be used in the method described herein. Suitable delivery reagents include, but are not limited to, Mirus Transit TKO lipophilic reagents, lipofectin, lipofectamine, cellfectin, polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. In some embodiments of the method described herein, liposomes are used to deliver nucleic acids to cells or subjects. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and sterols, such as cholesterol. The selection of lipids is generally guided by considering factors such as the desired liposome size and the half-life of liposomes in the bloodstream. Various methods for preparing liposomes are known, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467, and U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369, the entire disclosure of which is incorporated herein by reference.

[0073] CAR-T cells This specification provides a method for treating cancer and autoimmune diseases (e.g., MS, SAD, IBD) by administering allogeneic or autologous CAR-T cells expressing a T cell receptor that specifically binds to an antigen (e.g., viral peptide antigen) presented on a major histocompatibility complex (MHC) molecule, and a chimeric antigen receptor molecule that specifically binds to a selected target peptide and has the ability to transmit subsequent activation signals via an immune receptor activation motif (ITAM) present in its cytoplasmic terminal.

[0074] In some embodiments, the antigen-specific T cells used to generate the CAR-T cells of the present invention are selected from a cell bank or library. Preferably, the MHC is class I MHC. In certain embodiments, the MHC is class II MHC and has an α-chain polypeptide which is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some such embodiments, the class II MHC has a β-chain polypeptide which is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. Such T cells as described herein (e.g., antigen-specific T cells and antigen-specific T cells expressing CARs) are stored in a cell library or bank before they are administered to a subject.

[0075] In some embodiments, the T cells used to generate the CAR-T cells of the present invention are multifunctional T cells, i.e., T cells capable of inducing multiple immune effector functions that provide a more effective immune response against a pathogen than cells that produce only a single immune effector (e.g., a single biomarker, e.g., cytokine or CD107a) do. Even low-multifunctional, monofunctional or "exhausted" T cells can dominate the immune response during chronic infection and thus negatively impact protection against virus-associated complications. Similarly, the CAR-T cells of the present invention are also multifunctional (e.g., exhibit, retain or have enhanced multifunctionality). In some embodiments, at least 50% of the T cells used to generate the CAR-T cells of the present invention are CD4 + These are T cells. In some such embodiments, the T cells are less than 50% CD4 + These are T cells. In further embodiments, the T cells are mainly CD4 + These are T cells. In some embodiments, at least 50% of the T cells used to generate the CAR-T cells of the present invention are CD8 + These are T cells. In some such embodiments, the T cells are less than 50% CD8 + These are T cells. In further embodiments, the T cells are mainly CD8 + These are T cells.

[0076] Furthermore, APCs that present peptides described herein (e.g., peptides containing T cell epitopes) are provided herein. In some embodiments, the APC is a B cell, an antigen-presenting T cell, a dendritic cell, or an artificial antigen-presenting cell (e.g., aK562 cell). In certain preferred embodiments, the APC is derived from lymphoblastoid cells, e.g., BLCL. Exemplary antigen-presenting BLCLs are described herein by reference in O'Reily et al., Immunol Res 2007 Vol. 38: pp. 237-250 and Koehne et al., Blood 2002 Vol. 99: pp. 1730-1740.

[0077] Dendritic cells for use in this process can be prepared by collecting PBMCs from a patient sample and attaching them to plastic. Generally, the monocyte population remains, and all other cells can be washed away. The adherent cell population is then differentiated with IL-4 and GM-CSF to produce monocyte-derived dendritic cells. These cells can be matured by adding IL-1β, IL-6, PGE-1 and TNF-α (which upregulate important costimulatory molecules on the surface of dendritic cells), and then transduced with one or more peptides provided herein.

[0078] In some embodiments, the APC is an artificial antigen-presenting cell, such as aK562 cells. In some embodiments, the artificial antigen-presenting cell is engineered to express CD80, CD83, 41BB-L, and / or CD86. An example of an artificial antigen-presenting cell, such as aK562 cells, is described in U.S. Patent Application Publication 2003 / 0147869, incorporated herein by reference.

[0079] In certain embodiments, a method is provided herein for producing an APC that presents one or more T cell epitopes described herein, the method comprising contacting the APC with a peptide containing the epitope and / or a nucleic acid encoding the epitope. In some embodiments, the APC is irradiated. Cells presenting the peptides described herein can be produced by standard techniques known in the art. For example, cells may be pulsed to promote peptide uptake. In some embodiments, cells are transfected with nucleic acids encoding the peptides provided herein. A method is provided herein for producing antigen-presenting cells (APCs), comprising the step of pulsing cells with the peptides described herein. An exemplary example of producing antigen-presenting cells can be found in WO2013088114, the full text of which is incorporated herein by reference.

[0080] In some embodiments, T cells (e.g., CD4 T cells and / or CD8 T cells) expressing a TCR (e.g., αβTCR or γδTCR) that recognizes the peptides described herein presented on an MHC are provided herein. In some embodiments, the T cells are CD8 T cells (CTLs) expressing a TCR that recognizes the peptides described herein presented on a class I MHC. In some embodiments, the T cells are CD4 T cells (helper T cells) that recognize the peptides described herein presented on a class II MHC.

[0081] In some embodiments, methods are provided herein for generating, activating, and / or inducing the proliferation of CAR-T cells (e.g., EBV-specific anti-CD19-CAR-T cells) that recognize one or more of the EBV epitopes described herein and also express a chimeric antigen receptor. In some embodiments, a culture (or sample thereof) containing T cells (i.e., isolated T cells) is incubated in the culture with APCs provided herein (e.g., APCs that present peptides containing EBV epitopes on a class I MHC complex, e.g., PBMCs transduced by the virus described herein). In some embodiments, the APCs are autologous to the subject from which the T cells are obtained. In some embodiments, the APCs are not autologous to the subject from which the T cells are obtained. In some embodiments, the sample containing T cells is incubated with APCs provided herein two or more times. In some embodiments, the T cells (and / or CAR-T cells) are incubated with APCs in the presence of at least one cytokine. In some embodiments, the cytokines are IL-2, IL-4, IL-7, and / or IL-15. Similarly, T cells and / or CAR-T cell cultures can be maintained in the presence of at least one cytokine, e.g., IL-2, IL-4, IL-7, and / or IL-15. Exemplary methods for inducing T cell proliferation using APCs are provided, for example, in U.S. Patent Publication 2015 / 0017723, incorporated herein by reference. In preferred embodiments, such antigen-specific (e.g., EBV-specific) T cells are transduced with a vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR), which typically includes an extracellular domain (also called an ectodomain), a transmembrane domain, and an intracellular signaling domain (also called an endodomain).

[0082] The extracellular domain allows CARs, when expressed on the surface of T cells, to direct T cell activity towards cells expressing targets recognized by the extracellular domain. By including a co-stimulatory domain, such as the 4-1BB(CD137) co-stimulatory domain, in series with CD3ζ within the intracellular domain, T cells can receive the co-stimulatory signal.

[0083] For illustrative purposes, first-generation CARs represent artificial receptors that, when expressed by T cells, allow them to retarget a given disease-associated antigen (e.g., tumor-associated antigen). Such CARs typically contain a single-strand variable fragment (scFv) derived from a target-specific antibody (e.g., tumor antigen) fused to a T cell receptor (TCR)-derived signaling domain, e.g., CD3ζ. Upon binding to the antigen, the CAR triggers phosphorylation of an immunoreceptor tyrosine-based activation motif (ITAMS), initiating a signaling cascade necessary for cell lysis, cytokine secretion, and proliferation, bypassing the endogenous antigen processing pathway and MHC constraints. Second-generation CAR designs include additional signaling domains, e.g., CD28 and / or 4-1BB, to enhance activation and co-stimulation. Second-generation CARs have been observed to induce greater IL-2 secretion, increase T cell proliferation and persistence, mediate greater tumor rejection, and extend T cell survival. Third-generation CARs are constructed by combining multiple signaling domains, such as CD3ζ-CD28-OC40 or CD3ζ-CD28-41BB, to enhance their efficacy with more potent cytokine production and cell death capabilities.

[0084] The ectodomains of CARs disclosed herein generally include a targeting domain, such as a ligand-binding domain or an antigen-recognition domain, that binds to a target antigen. In certain embodiments, the targeting domain is derived from innate T cell receptor (TCR) alpha and beta single-stranded ectodomains, as described herein. Preferably, such a targeting domain has a simple ectodomain (e.g., a CD4 ectodomain for recognizing HIV-infected cells). Alternatively, such an antigen-recognition domain includes an exogenous recognition component, such as a linked cytokine (these leads may lead to the recognition of cells having cytokine receptors). Generally, with respect to the methods disclosed herein, almost anything that binds with high affinity to a given target can be used as an antigen-recognition region. Thus, such a targeting domain is usually derived from an antibody. In some embodiments, the targeting domain is a functional antibody fragment or its derivative (e.g., scFv or Fab or any suitable antigen-binding fragment of an antibody). In preferred embodiments, the ectodomain includes a single-stranded variable fragment (scFv). In some such embodiments, the scFv is derived from a monoclonal antibody (mAb). In preferred embodiments, the antigen-specific binding domain (e.g., scFv) is fused to a transmembrane and / or signaling motif involved in lymphocyte activation, as disclosed in Sadelain M et al., Nat Rev Cancer 2003, Vol. 3: pp. 35-45, the full text of which is incorporated herein by reference. Optionally, a signal peptide (SP) may also be included, as a result the CAR can be glycosylated and immobilized on the cell membrane of immunoeffector cells. The affinity / specificity of the scFv is largely due to the heavy chain (V H ) and light chain (V L Driven by specific sequences within the complementarity-determining region (CDR) in each V. H and V L The sequence has three CDRs (CDR1, CDR2, CDR3).

[0085] In some embodiments, the targeting domain is derived from a native antibody, such as a monoclonal antibody. In some cases, the antibody is human. In some cases, the antibody has undergone modifications to make it less immunogenic when administered to humans. For example, the modifications may include one or more techniques selected from chimerization, humanization, CDR transplantation, deimmunization, and mutations of framework amino acids to correspond to the closest human germline sequence. In further embodiments, the targeting domain is a ligand-based targeting domain, in which, for example, scFv disclosed herein is replaced with a tumor marker ligand. Thus, a CAR expressing the ligand of the IL13 receptor (IL13R) makes it possible to redirect T cells to IL13R expressed on glioblastoma.

[0086] Furthermore, a bispecific CAR capable of binding to at least two molecular targets (e.g., cell-specific markers and tumor antigens) is disclosed. Also disclosed are CARs designed to work only with another CAR that binds to a different antigen, e.g., a cancer-associated antigen. For example, in these embodiments, the endodomain of the first CAR contains only a signaling domain (SD) or a co-stimulatory signaling region (CSR), but not both. The second CAR provides a deficiency signal when activated. For example, if the disclosed CAR contains an SD but not a CSR, an immune effector cell containing this CAR is activated only when another CAR (or T cell) containing a CSR binds to its respective antigen. Similarly, if the disclosed CAR contains a CSR but not an SD, an immune effector cell containing this CAR is activated only when another CAR (or T cell) containing an SD binds to its respective antigen.

[0087] Exemplary antigens include, but are not limited to, tumor antigens, i.e., proteins produced by tumor cells that induce an immune response, particularly a T cell-mediated immune response. Further antigen-binding domains may be antibodies or natural ligands of the tumor antigen. The selection of further antigen-binding domains varies depending on the specific type of cancer being treated. Tumor antigens are well known in this art, and examples include glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, TIM3, cyclin Bl, lectin-reactive AFP, Fos-associated antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESl, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, and polysialic acid. acid), PLAC1, RUL, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, HSP70, HSP27, mutation (mut) hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivorbin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAGE-A2, MAGE-C1, MAGE-C2, Annexin-A2, MAD-CT-1, MAD-CT-2, Melan A / MART 1, XAGE1, ELF2M, ERG (TMPRSS2) Examples include ETS fusion genes, NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnn B2, CD20, CD22, CD24, CD30, TIM3, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, FlT3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In certain preferred embodiments, the tumor antigen is selected from folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, TIM3, BCMA, GD2, CLL-1, CA-IX, MUCl, HER2, and any combination thereof.

[0088] Further, non-limiting, examples of tumor antigens include: differentiation antigens, e.g., tyrosinase, TRP-1, TRP-2 and tumor-specific multiseries antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutant tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include SCCA, GP73, FC-GP73, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA 19-9, CA 72-4, and CAM. 17.1, NuMa, K-ras, Beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, Alpha-fetoprotein, Beta-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASL, SDCCAG1 6. Examples include TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, TAG72, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0089] The transmembrane domain (TD) connects the ectodomain (i.e., the extracellular domain) to the endodomain (i.e., the intracellular domain) and, when expressed by a cell, resides within the cell membrane. The transmembrane domain can originate from either a natural or synthetic source. If the source is natural, the domain can originate from any membrane-binding or transmembrane protein. For example, the transmembrane region includes the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, and IL7R. α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, C The transmembrane domains may be derived from D96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp (i.e., including at least one of their transmembrane domains). Alternatively, the transmembrane domain may be synthetic, in which case it mainly comprises hydrophobic residues, such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.For example, short oligo- or polypeptide linkers with a length of 2 to 10 amino acids can form a link between the transmembrane domain and the endoplasmic domain of a CAR.

[0090] The endodomain transmits an activation signal to immune effector cells after antigen recognition, activating at least one of the normal effector functions of the immune effector cells. In certain embodiments, the effector function of the T cell may be, for example, cytolytic activity including cytokine secretion or helper activity. Generally, the endodomain may contain an intracellular signaling domain (ISD) and, optionally, a co-stimulatory signaling region (CSR). The endodomain may contain an ISD of a T cell receptor (TCR) and, optionally, a co-receptor. The entire intracellular signaling domain may be used, but the entire chain does not need to be used. As long as a cleaved portion of the intracellular signaling domain is used, such a cleaved portion may be used in place of the intact chain as long as it transmits the effector function signal. A "signaling domain (SD)," for example, an ISD, generally modulates the primary activation of the TCR complex acting in a stimulative manner and contains a cytoplasmic signaling sequence consisting of a signaling motif known as an immune receptor tyrosine-based activation motif (ITAM). The signaling cascade is activated when the ITAM is phosphorylated. The term “costimulatory signaling region (CSR)” refers to intracellular signaling domains derived from costimulatory protein receptors, such as CD28, 41BB, and ICOS, which can enhance T cell activation by T cell receptors. In some embodiments, the endodomain contains either SD or CSR, but not both. In these embodiments, immunoeffector cells containing the disclosed CAR are activated only when another CAR (or T cell receptor) containing the deficiency domain also binds to its respective antigen. Examples of ITAM-containing cytoplasmic signaling sequences include those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0091] In certain embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3ζ) (TCR zeta, GenBank accession number BAG36664.1). The T cell surface glycoprotein CD3 zeta (CD3ζ) chain is also known as the T cell receptor T3 zeta chain or CD247 (differentiation antigen group 247), and in humans, it is a protein encoded by the CD247 gene. The intracellular tail of the CD3 molecule contains a single ITAM, which is essential for the signaling ability of the TCR. The intracellular tail of the ζ chain (CD3ζ) contains three ITAMs. In some embodiments, the ζ chain is a mutant ζ chain. For example, the mutant ζ chain includes a mutation in at least one ITAM, e.g., a point mutation, to render the ITAM nonfunctional. In some such embodiments, either or both of the membrane-proximal ITAM (ITAM1) and / or membrane-distal ITAM (the third ITAM at the C-terminus, ITAM3) are nonfunctional. In further embodiments, two membrane-proximal ITAMS (ITAM1 and ITAM2) or two membrane-distal ITAMS (ITAM2 and ITAM3) are nonfunctional. In even further embodiments, only ITAM2 is nonfunctional. In some embodiments, the mutant ζ chain contains a deletion (e.g., truncation) mutation, resulting in the absence of at least one ITAM. In some such embodiments, the ζ chain is missing either a membrane-proximal ITAM (ITAM1), a membrane-distal ITAM (ITAM3), or both. In other embodiments, the ζ chain is missing either one of the two membrane-proximal ITAMS (ITAM1 and ITAM2) or two membrane-distal ITAMS (ITAM2 and ITAM3). In even further embodiments, the ζ chain is missing ITAM2. Methods for producing the mutant CD3ζ are known to those skilled in the art (Bridgeman JS et al., Clin Exp Immunol. February 2014; Vol. 175 (No. 2): pp. 258-257 and WO2019 / 133969, incorporated herein by reference). CD3ζ-mediated apoptosis can be reduced by removing at least one ITAM from the introduced CAR. Alternatively, the size of the introduced CAR can be reduced without loss of function by removing at least one ITAM.

[0092] In some embodiments, the CAR includes a hinge sequence. The hinge sequence is a short sequence of amino acids that promotes antibody mobility (see, e.g., Woof et al., Nat. Rev. Immunol., vol. 4(2): pp. 89-99 (2004)). The hinge sequence may be located between the antigen recognition portion (e.g., anti-CD19, -CD20, -CD22, or -CLEC4 scFv) and the transmembrane domain. The hinge sequence may be derived from or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0093] In a preferred embodiment, the disclosed CAR is defined by the following formula: SP-TD-HG-TM-CSR-SD, or SP-TD-HG-TM-SD-CSR [In the formula, "SP" represents an optional signal peptide, "TD" stands for Targeted Domain, "HG" represents an optional hinge domain (spacer domain), "TM" stands for transmembrane domain, "CSR" represents one or more co-stimulatory signaling regions. "SD" stands for signal transduction domain, The hyphen "-" represents a peptide bond or linker.

[0094] In some embodiments, the CAR contains one signaling domain. In other embodiments, the CAR contains one or more signaling domains (including a co-stimulatory signaling domain). The one or more signaling domains may be polypeptides selected from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137 (41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40 and their mutants. For example, the endodomain of the CAR may be designed to contain a CD3ζ signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in relation to the CAR of the present invention. Alternatively, the cytoplasmic domain of the CAR may contain a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the portion of a co-stimulatory molecule that includes the intracellular domain, known as the CAR. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, NKG2D, and their mutants. Therefore, while CARs are primarily exemplified together with CD28 as a co-stimulatory signaling element, other co-stimulatory elements and their mutants may be used alone or in combination with other co-stimulatory signaling elements. For example, in some such embodiments, the preferred CAR co-stimulatory signaling domain is the CD28 mutant domain known as "Mut06," as described in WO2019 / 010383.

[0095] In some embodiments, the CAR has one or more transmembrane domains, which may be repeats of the same transmembrane domain or different transmembrane domains.

[0096] In some embodiments, the CAR is a polychain CAR, as described in WO2015 / 039523, which is incorporated by reference for this teaching. The polychain CAR may contain distinct extracellular ligand-binding and signaling domains within different transmembrane polypeptides. The signaling domains can be designed to assemble near the membrane, thereby forming a flexible structure similar to that of the intrinsic receptor and conferring optimal signaling. For example, the polychain CAR may contain portions of the FCERI alpha chain and portions of the FCERI beta chain, resulting in the FCERI chains spontaneously dimerizing together to form the CAR.

[0097] Further CAR constructs are described, for example, in Fresnak AD et al., Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. August 23, 2016; Vol. 16 (No. 9): pp. 566-561, the full text of which is incorporated by reference for teaching these CAR models.

[0098] In certain embodiments, a CAR may be, for example (but not limited to), a TRUCK, a universal CAR, a self-driving CAR, an armored CAR, a self-destruct CAR, a conditional CAR, a marked CAR, a TenCAR, a dual CAR, or an sCAR.

[0099] TRUCK (T cells redirected to universal cytokine death) co-express chimeric antigen receptors (CARs) and antitumor cytokines. Cytokine expression can be constitutive or induced by T cell activation. Localized production of pro-inflammatory cytokines targeted by CAR specificity can recruit endogenous immune cells to tumor sites and enhance the antitumor response.

[0100] Universal allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0101] Self-driven CARs co-express CARs and chemokine receptors that bind to tumor ligands, thereby enhancing tumor homing.

[0102] In certain embodiments disclosed herein, the present invention utilizes immune checkpoint inhibition / blockade strategies. Immune checkpoint therapies target key regulators of the immune system that stimulate or inhibit the immune response. Such immune checkpoints may be utilized in disease states (e.g., by tumors) to evade attack by the immune system. Checkpoint inhibitor studies have described the activity of PD-1 inhibitor therapy (El-Khoueiry, Sangro et al., 2017), and the FDA approved nivolumab for the second-line treatment of HCC with an objective response rate of 20%. CAR T cells engineered to be resistant to immunosuppression (armored CARs) can be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD-1)). Exemplary "knockdown" and "knockout" techniques include, but are not limited to, RNA interference (RNAi) (e.g., asRNA, miRNA, shRNA, siRNA, etc.) and CRISPR interference (CRISPRi) (e.g., CRISPR-Cas9). In certain embodiments, CAR T cells are engineered to express a checkpoint molecule in a dominant-negative form. In some such embodiments, the extracellular ligand-binding domain (i.e., ectodomain) of the immune checkpoint molecule is fused to the transmembrane to compete for ligand binding. For example, the extracellular ligand-binding domain of PD-1 may be fused to the CD8 transmembrane domain, thus competing for a PD-1 ligand derived from the target cell. In some embodiments, CAR T cells are engineered to express an immune checkpoint switch receptor to utilize an inhibitory immune checkpoint ligand present on the target cell. In such embodiments, the extracellular ligand-binding domain of the immune checkpoint molecule is fused to a signaling, stimulating, and / or co-stimulating domain. For example, the extracellular ligand-binding domain of PD-1 may be fused to the CD28 domain, thus providing CD28 co-stimulation while blocking PD-1 signaling. In a further embodiment, CAR T cells are aptamers or monoclonal cells that block immune checkpoint signaling. They may be administered together with a nal antibody. In some such embodiments, CAR T cells (e.g., CAR T cell therapy) are administered in combination with a PD-1 blockade method, such as a PD-1 / PD-L1 antagonistic aptamer or an anti-PD-1 / PD-L1 antibody. In preferred embodiments, CAR T cells and PD-1 pathway blocking antibodies are administered together. In further embodiments, CAR T cells are engineered to express, or express and secrete, immune checkpoint blocking antibodies, such as anti-PD-1 or anti-PD-L1 or fragments thereof. In even further embodiments, CAR T cells are administered with a vector (e.g., an engineered virus) expressing an immune checkpoint blocking molecule as described herein.

[0103] Self-destructive CARs can be designed to encode CARs using RNA delivered by electroporation. Alternatively, T cell-induced apoptosis can be achieved based on more recently described systems of human caspase-9 activation in genetically modified lymphocytes using ganciclovir conjugated with thymidine kinase or small molecule dimerizing agents.

[0104] Conditioned CAR T cells are inactive or switched "off" by default until small molecules are added to complete the "circuit" (e.g., molecular pathway), enabling the complete transmission of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells can be engineered to express adapter-specific receptors that have affinity for a subsequently administered secondary antibody against a target antigen.

[0105] Marked CAR T cells express tumor epitopes to which CAR and existing monoclonal antibody substances bind. In settings of unacceptable adverse effects, administration of monoclonal antibodies eliminates CAR T cells and alleviates symptoms without additional off-tumor effects.

[0106] Tandem CAR (TanCAR) T cells express a single CAR consisting of two linked single-stranded variable fragments (scFv) with different affinities, fused to the intracellular costimulatory domain(s) and CD3ζ domain. TanCAR T cell activation is achieved only when the target cell simultaneously expresses both targets.

[0107] Dual-CAR T cells express two distinct CARs with different ligand-binding targets. In some, but not limited to, one CAR may contain only the CD3ζ domain, while the other CAR contains only the co-stimulatory domain(s). In some such embodiments, dual-CAR T cells are activated when both targets are expressed in a tumor.

[0108] Safety CARs (sCARs) consist of extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing standard CARs possess the standard CAR target but are activated only when they encounter target cells lacking the sCAR target.

[0109] Also disclosed are polynucleotides and polynucleotide vectors encoding target-specific CARs that enable the expression of the CARs in disclosed immune effector cells (e.g., T cells).

[0110] The disclosed CARs and nucleic acid sequences encoding their regions can be obtained using recombinant methods known in the art, for example, by screening libraries obtained from cells expressing the gene using standard techniques, by extracting the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it. Alternatively, they can be produced by synthesis rather than cloning the gene of interest.

[0111] The expression of CAR-encoding nucleic acids is typically achieved by operably ligating the CAR polypeptide-encoding nucleic acid to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, start sequences, and promoters useful for regulating the expression of a desired nucleic acid sequence.

[0112] The disclosed nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly suitable vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0113] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses (e.g., gamma retroviruses), adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a functional origin of replication, a promoter sequence, a useful restriction endonuclease site, and one or more selectable markers in at least one organism. In some embodiments, the polynucleotide vector is a lentiviral or retroviral vector. Preferably, the polynucleotide vector is a gamma retrovirus vector.

[0114] Several virus-based systems have been developed for gene delivery into mammalian cells. For example, retroviruses provide a useful platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo.

[0115] One example of a suitable promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, but are not limited to, the monkey virus 40 (SV40) early promoter, the MND (myeloprenovatory sarcoma virus) promoter, the mouse mammary cancer virus (MMTV), the human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus very early promoter, the Roussarcoma virus promoter, and human gene promoters, including, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. The promoter may also be an inducible promoter. Examples of inductive promoters, though not limited to them, include the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0116] Further promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although it has recently been discovered that some promoters contain similarly functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, resulting in the preservation of promoter function even when elements are inverted or moved relative to each other.

[0117] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and whose expression is indicated by several readily detectable characteristics, such as enzymatic activity, which encodes a polypeptide. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes include those encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, a construct having the smallest 5' flanking region exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated to the reporter gene and used to evaluate the substance's ability to modulate promoter-driven transcription.

[0118] Methods for introducing and expressing genes in cells are well known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0119] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0120] The use of DNA and RNA vectors are biological methods for introducing target polynucleotides into host cells. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells.

[0121] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo include liposomes (e.g., artificial membrane vesicles).

[0122] When a non-viral delivery system is used, the exemplary delivery vehicle is a liposome. In another embodiment, nucleic acids may associate with lipids. Nucleic acids associated with lipids may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome by linking molecules that associate with both the liposome and oligonucleotides, encapsulated within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "broken" structure. They may also be readily dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring lipids or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") is available from Sigma, ST. Louis, Mo; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0123] choice To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector to be introduced into cells (e.g., antigen-specific T cells) may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be held on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and reporter gene may be flanked with appropriate regulatory sequences to enable expression in host cells. In some embodiments, the CAR-encoding vector includes a lentiviral vector. In some preferred embodiments, the CAR-encoding vector includes a gamma-retroviral vector. Most preferably, the CAR-encoding vector includes a nucleic acid sequence encoding a selectable marker or physical tag that enables the selection of only cells expressing the CAR(s) encoded by the vector. Useful selectable markers include, but are not limited to, the expression of antibiotic resistance genes, reporter genes, or physical tags. Examples of physical tags include, but are not limited to, terminally cleaved cell surface peptides (e.g., cell surface receptors lacking intracellular signaling domains), which can be selected with appropriate antibodies using methods known in the art. In some embodiments, the vector encoding the CAR includes an antibiotic resistance gene, e.g., a nucleic acid sequence encoding bor blastosidine resistance. In some such embodiments, CAR T cells (e.g., antigen-specific CAR T cells) are cultured in the presence of a selectable marker (e.g., blastosidine), thereby enabling the selection and expansion of T cells expressing the CAR described herein.

[0124] Treatment method In some embodiments, the methods provided herein relate to treating cancer and / or autoimmune disorders in a subject by administering autologous or allogeneic CAR-T cells, as provided herein, to the subject.

[0125] The methods of this disclosure rely to some extent on the principle of T cell constraint to a specific target antigen. For example, products and preparations containing donor-derived CTLs targeted against antigens expressed by a virus, e.g., Epstein-Barr virus (EBV), can be induced in targeted antigen-presenting (e.g., stimulator) cell lines containing EBV-transformed B lymphoblasts (BLCLs). With respect to the disclosed virus antigen-specific CAR T cell preparations (e.g., anti-CD19-CD28-CAR-EBV-CTLs), the target cells and / or recipients of the CAR-T products produced by the methods provided herein may be autologous or allogeneic. In the case of allogeneic targets, the donor's human leukocyte antigen (HLA) identity must be known and matched to the HLA identity of the target (i.e., the cultured cell line and / or recipient).

[0126] Preparations of CAR T cells may contain a mixture of antigen-specific and non-specific CTLs. Therefore, to quantify alloreactive CTLs (e.g., antigen-specific CTLs with or without CARs), a reaction to HLA-incompatible targets is induced. Ideally, cell lysis should be minimal or below a predetermined threshold. Furthermore, the level of CTLs capable of expansion or expressing CARs can be quantified by limiting dilution assays and cytotoxic dilutions against allogeneic targets.

[0127] The target cells for the assays disclosed herein are typically selected for their homogeneous phenotype and availability in large quantities. In certain embodiments, the target cell line is an antigen-presenting cell (APC). In some such embodiments, the target cell line is a B cell, an antigen-presenting T cell, a dendritic cell, or an artificial antigen-presenting cell (e.g., aK562 cell). In certain embodiments, the target cell line comprises peripheral blood mononuclear cells (PBMCs). In some such embodiments, the target cell line is a lymphoblast. In certain preferred embodiments, the target cell line is transformed by a virus. In preferred embodiments, the target cell line comprises phytohemagglutinin-stimulated peripheral blood lymphocytes (PHA blasts / PHAbs) and B lymphoblast-like cell lines (BLCLs) transformed by Epstein-Barr virus. As illustrated herein for the evaluation of antigen-specific CTLs expressing CAR, BLCLs have the advantage of being large cells with high viability and the ability to hold a substantial lactate dehydrogenase (LDH) and / or large intracellular chromium (Cr) reservoirs, which makes them favorable for cytotoxicity assessment by Cr or LDH release assays. However, in the case of EBV antigen, BLCLs (e.g., those used as APCs) are EBV antigen-positive, which allows for EBV antigen-specific cross-presentation to the T cell receptor (TCR), which is specific to incompatible HLA alleles. PHA blasts have the advantage of being EBV antigen-negative and cannot cross-present EBV antigen to the TCR. Nevertheless, PHA blasts are smaller and generally more fragile cells. This results in smaller reservoir and reporter permeability, ultimately providing a higher possibility of false-positive cytotoxicity results. Therefore, BLCLs and PHA blasts can be used as target pairs derived from the same donor. In some such embodiments, the target cells are derived from promising recipients of the allogeneic CAR T cell preparations described herein, in order to guide the appropriate selection of the CAR T cell preparation to be administered.

[0128] In certain embodiments, where the preparation induces lysis in a plurality of target cell lines above a predetermined threshold, the method further includes quantifying in the preparation the frequency of expandable CAR-T CTLs capable of lysing the target cell lines, and the sample is identified as not being clinically alloreactive when it contains expandable CAR-T CTLs at a level below the predetermined threshold. In certain embodiments, prior to quantifying the frequency of cytolytic expandable CAR-T CTLs, the preparation induces more than 15% lysis in each of an allogeneic PHA blast cell line and an allogeneic cell line.

[0129] In some embodiments, quantifying the frequency of expandable CAR-T CTLs in the preparation includes, for example, evaluating cytolytic function after a period of CAR-T CTL expansion by performing limiting dilution analysis. In further embodiments, evaluating cytolytic function includes performing a detection method, such as 51 chromium (e.g.,

[0130] In some embodiments, the target cell line includes allogeneic cells. In some such embodiments, the target cells carry human leukocyte antigen (HLA) alleles that are not compatible with the HLA alleles to which the CAR-T CTLs of the preparation are restricted. In other such embodiments, the target cells carry HLA alleles that are partially compatible with the HLA alleles to which the CAR-T CTLs of the preparation are restricted. In still other such embodiments, the target cells carry HLA alleles that are compatible with the HLA alleles to which the CAR-T CTLs of the preparation are restricted. In a preferred embodiment, the CAR-T CTLs are restricted to the HLA alleles of the target cells encoding MHC class I proteins.

[0131] In other embodiments, the target cell line includes autologous cells. In some such embodiments, the CAR-T CTL preparation is identified as suitable for use against target cells by confirming the ability of the preparation to lyse two or more autologous target cell lines above, below, or exceeding a given threshold.

[0132] In certain embodiments, autologous or allogeneic CAR-T cells include central memory T cells (T CM cells), for example, at least 60%, 70%, 80% of the cells are Tcm cells. In some embodiments, autologous or allogeneic CAR-T cells have a central memory T cell to effector memory cell (T CM :T EM ) ratio of at least 1:1 to at least 3:1, for example, at least 1:1, 1.4:1, 2.5:1, or 3:1. In some such embodiments, autologous or allogeneic CAR-T cells are primarily CD4 + T cells. In some embodiments, autologous or allogeneic CAR-T cells include at least 80% CD4 + CAR-T cells and at least 15% CD8 + CAR-T cells). In some embodiments, autologous or allogeneic CAR-T cells have a CD4 + T cell to CD8 + T cell ratio of at least 1:1 to at least 3:1, for example, at least 1:1, 1.4:1, 2.5:1, or 3:1. In some of the methods described herein, allogenic T cells are selected from a cell bank (e.g., a bank of pre-generated third-party donors of epitope-specific T cells).

[0133] In some embodiments, any autoimmune disease can be treated using the methods provided herein. Examples of autoimmune diseases include, for example, glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjögren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Still's disease, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, polyarteritis nodosa, rheumatic fever, vitiligo, Behçet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, aseptic disease, pemphigus, autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, autoimmune achlorhydria, celiac disease, and cuscinosis. Examples include G's syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, lymphopenia, complex connective tissue disease, bullous pemphigoid, pemphigus vulgaris, pernicious anemia, lens-induced uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt syndrome, focal scleroderma (or Crest syndrome), sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroidopathy, type B insulin resistance, type I diabetes mellitus, ulcerative colitis, and Wegener's granulomatosis.

[0134] In some embodiments, the methods provided herein are used to treat MS. In some embodiments, MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS.

[0135] In some embodiments, the methods provided herein are used to treat SAD. For example, in certain embodiments, the methods provided herein are used to treat rheumatoid arthritis, systemic lupus erythematosus, and / or Sjögren's syndrome.

[0136] In some embodiments, the methods provided herein are used to treat IBD. For example, in certain embodiments, the methods provided herein are used to treat Crohn's disease (focal bowel disease, e.g., inactive and active forms), celiac disease (e.g., inactive and active forms), and / or ulcerative colitis (e.g., inactive and active forms). In some embodiments, the methods provided herein are used to treat irritable bowel syndrome, microscopic colitis, lymphocytic plasmacytic colitis, celiac disease, collagen colitis, lymphocytic colitis, eosinophilic colitis, uncertain colitis, infectious colitis (viral, bacterial or protozoal, e.g., amoebic colitis) (e.g., Clostridium dificile colitis), pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behçet's disease, sarcoidosis, scleroderma, IBD-related dysplasia, dysplasia-associated masses or lesions, and / or primary sclerosing cholangitis.

[0137] In some embodiments, a method for treating cancer in a subject by administering the therapeutic CAR-T cell preparation described herein is provided herein.

[0138] In some embodiments, the methods provided herein can be used to treat any cancer. For example, in some embodiments, the methods and CAR-T cells described herein may be used to treat any cancerous or precancerous tumor. In some embodiments, cancer includes solid tumors. In some embodiments, cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancer cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer can be, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilosacarcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrin-producing tumor; malignant; cholangiocarcinoma; hepatocellular carcinoma; combination of hepatocellular carcinoma and cholangiocarcinoma; cord-like adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma; familial colonic polyposis; solid tumor; carcinoid tumor; malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; pigmentophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma Cancer, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary adenocarcinoma and follicular adenocarcinoma, non-encapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrioid carcinoma, cutaneous adnexal carcinoma, apocrine adenocarcinoma, sebaceous gland carcinoma, cerumen adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous Gonadal carcinoma, signet ring cell carcinoma, invasive 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 capsular cell tumor, malignant granulosa cell tumor, malignant androgenic cell tumor (malignantRoblastoma, Sertoli cell tumor, malignant Leydig cell tumor, malignant lipid cell tumor, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, angioglobulosarcoma, malignant melanoma, achromatic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevi, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed Tumors, Müllerian mixed tumors, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymal tumor, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, undifferentiated germ cell tumor, fetal carcinoma, malignant teratoma, malignant ovarian goiter, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangioectocytoma, lymphangiosarcoma, osteosarcoma, paraosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic odontosarcoma Malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pineal glandoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioblastoma, primitive neuroectodermal tumor, cerebellar sarcoma, ganglioblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant schwannoma, malignant granuloma, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, lateral granuloma, Small lymphocytic lymphoma, diffuse large cell lymphoma, follicular lymphoma, mycosis fungoides, other designated non-Hodgkin lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative bowel disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myelosarcoma, and hairy cell leukemia. In some such preferred embodiments, the compositions and methods provided herein are for B-cell malignancies including chronic lymphocytic leukemia (CLL), ALL, and numerous non-Hodgkin lymphomas (e.g., CD19 + It can be used to treat malignant tumors.

[0139] In some embodiments, the methods provided herein are used to treat EBV-related cancers. In some embodiments, the EBV-related cancer is an EBV-related non-pectorisic tumor (NPC). In some embodiments, the EBV-related cancer is a post-transplant lymphoproliferative disorder (PTLD), an NK / T-cell lymphoma, an EBV-positive gastric cancer, or an EBV-positive leiomyosarcoma.

[0140] In some embodiments, the subject is exposed to a virus (e.g., EBV) such that viral particles are detectable in the subject's blood. In some embodiments, the method further includes measuring the viral load in the subject (e.g., before or after administering peptide-specific CAR-T cells to the subject). Determining the viral load in the subject can be a good prognostic marker for the effectiveness of immunotherapy. In some embodiments, the selection of CAR-T cells further includes determining the number of viral DNA copies in the subject (e.g., in a tissue or blood sample). In some embodiments, the viral load is measured two or more times. The actual dosage levels of the active ingredient in the pharmaceutical compositions provided herein can be varied to achieve an amount, composition, and mode of administration of the active ingredient that is effective in achieving a desired therapeutic response for a particular patient without toxicity to the patient.

[0141] The selected dosage level depends on various factors, including the activity of the specific drug used, the route of administration, the time of administration, the rate of elimination or metabolism of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific compound used, the age, sex, weight, condition, overall health status, and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0142] In some embodiments, the method described herein includes the step of selecting allogeneic T cells from a cell bank (e.g., a pre-generated third-party donor-derived bank of epitope-specific T cells). In some embodiments, T cells are selected because they express a class I MHC-bound TCR encoded by an HLA allele present in the subject. In some embodiments, T cells are selected if the T cells and the subject share at least two (e.g., at least three, at least four, at least five, at least six) HLA alleles and the T cells are bound by the shared HLA alleles. In some embodiments, the method includes the step of testing the TCR repertoire of pre-generated third-party donor-derived epitope-specific T cells (i.e., allogeneic T cells) by flow cytometry. In some embodiments, epitope-specific T cells are detected using a tetramer assay, ELISA assay, Western blot assay, fluorescence microscopy assay, Edman degradation assay and / or mass spectrometry assay (e.g., protein sequencing). In some embodiments, the TCR repertoire is analyzed using nucleic acid probes, nucleic acid amplification assays, and / or sequencing assays.

[0143] In some embodiments, compositions comprising T cells and / or APCs (e.g., therapeutic compositions) provided herein are used to treat and / or prevent autoimmune diseases in subjects by administering an effective amount of the composition to the subject. In some embodiments, methods for treating autoimmune disorders using compositions (e.g., pharmaceutical compositions such as compositions comprising allogeneic CTLs) are provided herein. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) CTLs provided herein.

[0144] [Examples] [Example 1] Comparison of CAR transduction of EBV CTLs derived from PBMCs and isolated T cells. Frozen PBMCs from healthy donors were thawed and removed into RPMI medium. The cells were divided into two fractions; one-third of the cells (stimulators) were infected with AdE1-LMPpoly adenovirus at 37°C for 1 hour. The stimulators were then washed twice, resuspended in RPMI / AB serum culture medium, and irradiated with 2500 cGy (2500 rads). The remaining two-thirds of the cells (responders) were transferred to RPMI / AB serum medium and kept at 37°C until they could be mixed with the stimulators (e.g., PBMC-derived BLCL), or CD3 + T cells were used to isolate them, and then they were cultured with stimulator PBMCs (see Figure 1).

[0145] PBMC activation Day 0, 9 x 10 6 Irradiated stimulator PBMC cells and 2.1 × 10⁶ cells 7 6 wells (10 cm) with individual responder PBMC cells 2 In GRex culture plates, culture was started and returned to tissue culture incubation at 37°C. Optionally, on days 9 and 10, CD56 was extracted from the cell culture before transduction. + This depleted the NK (natural killer) cells.

[0146] Isolated CD3 + T cell activation On day 0, T cells were isolated (i.e., enriched) from PBMCs by means known in the art (e.g., viable FACS or anti-CD3 coated magnetic beads). Six wells (10 cm³) containing medium with 20 U / ml IL2 were used. 2 T cells were started in a GRex culture plate at a ratio of 1 T cell / 4 stimulator PBMC cells and returned to tissue culture incubation at 37°C. On days 9 and 10, the cell cultures were transduced to express CAR (as described above, NK cell depletion before transduction was optional). The starting material was sensitized to a viral antigen, e.g., EBV (e.g., CD3). +After enrichment, the resulting population exhibits an increased percentage of central memory phenotypic T cells (see Figure 2, left). These central memory T cells are advantageously persistent over time, while markers of T cell depletion (PD1 and CTLA4) remain low in virus-sensitized cells (Figure 2, right).

[0147] CAR trait introduction On days 9 and 10, isolated T cell and PBMC cultures were transduced with recombinant viruses encoding anti-CD19 CAR (and blastosidin selection marker). Briefly, retronectin (0.5 mL 10 μg / mL) was added to each well of a 24-well non-tissue culture treatment plate for each stimulation condition, and incubated at room temperature for 2 hours. The retronectin was then removed by aspirate and replaced with 0.5 mL of blocking buffer in each well, and the plate was incubated at room temperature for 30 minutes. The plate was then washed with wash buffer. The viruses encoding anti-CD19 CAR were thawed at room temperature and added to the plates. The plates were wrapped in paraffin film and centrifuged at 32°C and 2000 × g for 2 hours. The viral supernatant was aspirated, and 1 ml of the prepared cell suspension (0.5 × 10⁶ cells / mL resuspended in YH5 medium) from each stimulation group was added. The plates were centrifuged at 32°C and 1000×g for 15 minutes, then incubated overnight at 37°C and 5% CO2. Cells from each stimulus condition were then removed and pooled for counting. The cells were measured at 0.5–1.0 × 10⁶. 6 The cells were resuspended in fresh YH5 medium at a concentration of cells / mL.

[0148] On day 11, each stimulated state (i.e., of isolated T cells or responder PBMCs) was returned to the culture along with the irradiated stimulator PBMCs. Samples were taken for fluorescence-activated cell sorting (FACS) on days 15, 23, and 27, respectively (e.g., CD3 + scFV + (For the cells). Drug selection (blastosidine) was induced on day 19.

[0149] result EBV-CTLs derived from isolated T cells demonstrate improved viability and proliferative capacity. CD3 + The concentrated starting material yielded more than 10 times the yield compared to conventional expanded growth and transduction conditions, demonstrating significantly improved viability and proliferative capacity (see Figure 3). The efficiency of downstream CAR vector transduction varies depending on proliferative capacity. Therefore, higher viability and proliferative capacity in antigen-stimulated T cells results in improved downstream CAR transduction efficiency. Compared to transduction in crude PBMC cultures, the initial CD3 + CAR derived from the concentration step + EBV CTLs demonstrated a significant enhancement of downstream CAR transduction efficiency after BLCL stimulation and also showed greater cell viability depending on blastosidine selection (see Figure 4).

[0150] [Example 2] Evaluation of memory T cell phenotype in anti-CD19-CAR-EBV-CTL f cells after BLCL stimulation. Standard anti-CD3 / CD28 bead-based stimulation is widely used in this field as a method for expanding T cell proliferation in vitro before transduction with CAR vectors. The following experiments were conducted to determine the effects of various CAR-T cell stimulations suitable for high-yield production processes of memory T cell immunophenotypes for end-product therapeutics.

[0151] Study design and procedure Isolated CD3 + EBV antigen-specific T cells were stimulated and expanded for 3 days under four different culture conditions; 1. Unstimulated, 2. Soluble anti-CD3 / CD28, 3. Anti-CD3 / CD28 magnetic beads, and 4. Using BLCL cells.

[0152] After 3 days of stimulation, each culture was transduced with a viral vector encoding a chimeric antigen receptor (CAR), and then returned to a 7-day incubation in standard (YH medium) cultures to allow for the expansion and proliferation of transduced T cells. The cells were then harvested and their phenotypic analysis was performed by cell staining and fluorescence-activated cell sorting (FACS), as outlined in Figure 5.

[0153] Cell lines and culture conditions CD3 + EBV antigen-specific T cells were removed from liquid nitrogen storage, thawed, and 2 × 10⁶ cells were stored. 6 The cells were suspended in YH medium at a concentration of cells / mL with 100 IU / mL of IL-2. Using 2 mL of suspension per well, the suspension was plated into a 24-well plate and incubated overnight. After this overnight recovery, the cells were counted and, as shown in Figure 5 (see also Table 2), 1 × 10⁶ cells were obtained. 6 6 × 10⁶ cells per group at a concentration of individual cells / mL 6 The individual cells were divided into four different treatment groups.

[0154] [Table 2]

[0155] Cells were stimulated with anti-CD3 / CD28 DynaBeads in a 1:1 cell-to-bead ratio. Before direct addition to the cell suspension, a sufficient volume of beads was removed and washed with DPBS. The stimulated cultures were then plated in 2 mL of YH5 medium with 100 IU / mL IL-2 per well in a 24-well plate as described above, and incubated for 3 days.

[0156] Stimulation with soluble anti-CD3 / CD28 was performed by direct addition of CD3 / CD28 ImmunoCulT® reagent at a concentration of 25 μl per 1 mL (i.e., 50 μl per well in a 24-well plate, as described above), and incubated for 3 days.

[0157] Stimulation was performed using EBV-transformed lymphoblastoid B cell lines (BLCLs) at a ratio of 4 BLCLs to 1 EBV antigen-specific T cell. Briefly, CD3+, EBV antigen-specific T cells were removed from liquid nitrogen storage, thawed, suspended in YH medium, and allowed to recover. For preparation for stimulation culture, EBV-BLCLs were irradiated with a total dose of 90 Gy (9000 rads). EBV-BLCL antigen-specific T cells were incubated in YH5 medium with 100 IU / mL IL-2 in the same 24-well plates as above for 3 days.

[0158] CAR trait introduction For transduction preparation, a 24-well plate was prepared as follows: 0.5 mL of retronectin (10 μg / mL) was added to each well of a 24-well non-tissue culture treatment plate and incubated at room temperature for 2 hours. The retronectin was then removed and replaced with 0.5 mL of blocking buffer and incubated at room temperature for 30 minutes. The blocking buffer was then removed and each well was washed with at least 1 mL of washing buffer. The plate was kept at 4°C with the washing buffer until ready for use.

[0159] Lentiviral vectors encoding CAR (containing either the 4-1BB or CD28 signaling domain) were thawed at room temperature and added to each well of retronectin-coated plates to achieve MOI (Moritomoor of Infection) or 15 viral particles / cell (see Table 2). The plates were then wrapped in paraffin film and centrifuged at 32°C and 2000 × g for 2 hours. During this time, cells obtained from each stimulation group were counted and added to YH5 medium in a total volume of 0.5 × 10⁶ cells. 6 The cells were resuspended at a concentration of cells / mL. After centrifugation, the viral supernatant was aspirated from each well and replaced with 1 mL of the prepared cell suspension, including the control well. The plates were centrifuged at 1000 × g for 15 minutes at 32°C, and then incubated overnight at 37°C in 5% CO2.

[0160] [Table 3]

[0161] Cells obtained from each stimulation group were pooled and counted, and the number of cells was reduced to 0.5–1.0 × 10⁶ per 1 mL of fresh YH5 medium. 6 The cells were resuspended at their original concentration and then returned to incubation at 37°C, 5% CO2 for 2 days. Pooling and resuspension were repeated every 2 days until day 7 (recovery). Once recovered, the cells were labeled (see Table 3), and a flow cytometry gating strategy was applied to identify CD3, CD4, CD8, CD62L, and CD45RO in CAR-expressing T cells (see Figure 6). Briefly, the target cell signals were first identified by size and granularity (i.e., forward scattering (FSC) versus side scattering (SSC)). Live cells were then identified based on staining with a viability dye (e.g., Live / Dead® BV510). Live single cells were then gated based on pulse area versus pulse height (FSC-A versus FSC-H). Individual CD3 cells were then identified using a fluorescently labeled antibody. + T cells and their subsequent subpopulations (e.g., CD4) + CD8 + We were able to identify central memory T cells.

[0162] result

[0163] [Table 4]

[0164] Flow cytometry evaluation showed that CD62L was superior to soluble CD3 / CD28 or bead stimulation. + / CD45RO + As assessed by cells, transduced CD19-CAR-T cells stimulated with EBV-BLCL were shown to exhibit a higher percentage of the central memory T cell subset (see Figure 7). CD19-CAR-T cells stimulated with soluble or bead-bound CD3 / CD28 showed a higher percentage of CD62L compared to BLCL-stimulated cells. - / CD45RO+ As assessed by the cells, they had a higher percentage of the effector memory T cell subset (see Table 4).

[0165] [Table 5]

[0166] CD4 and CD8 expression were also evaluated in CD19-CAR-T cells. + In the group, CD4 was stimulated more when stimulated with BLCL compared to CD3 / CD28 stimulation. + There was a higher percentage of cells (see Figure 8 and Table 5).

[0167] These data demonstrate that stimulation of CAR-T cell cultures in BLCL expressing CAR-targeting antigens leads to a shift to a dominant central memory phenotype when compared to results from CD3 / CD28 stimulation with beads-bound antibodies or soluble antibodies. This study strongly supports the potential for improving the quality of CAR-T cell cultures (such as those represented by antigen-presenting BLCL) utilizing antigen-positive APC stimulation compared to the quality predicted when using standard anti-CD3 / CD28 beads or other cell-free modes of stimulation.

[0168] [Example 3] Anti-CD19-CAR-EBV-CTLs exhibit potent and specific cytotoxicity. Similarly, a lentiviral vector encoding CAR (containing either the 4-1BB or CD28 signaling domain) is subjected to EBV-BLCL-stimulated CD3 + It was used for the transduction of antigen-specific T cells.

[0169] In short, virus-specific T cells expressing CAR were prepared by the following method.

[0170] Day 0: Unfreeze the PBMC sample and CD3 +Cells were enriched (e.g., by viable FACS or anti-CD3 coated magnetic beads). Then these CD3 + T cells were stimulated by culturing them with EBV antigen-presenting BLCL as described herein. • Eleven days after stimulation (day 11), the culture was depleted of NK cells (e.g., using anti-CD56 beads), and fresh EBV antigen-presenting BLCLs were re-stimulated for 7 days at a responder / stimulator ratio of 1:4. On day 18, the cultures were stimulated for the third time over two days with a responder / stimulator ratio of 4:1. On day 20, transduction was initiated with a viral vector encoding a chimeric antigen receptor (CAR), and the cells were returned to incubation in standard (YH medium) culture to allow for the expansion and proliferation of transduced virus-specific T cells. Optionally, an NK cell depletion step may be applied immediately before transduction. By day 25, CAR expression could be evaluated (e.g., by FACS analysis), and virus-specific T cells expressing CAR (effectors) could be added to the target culture for cytotoxicity assays. Optionally, cells could be frozen on day 25 and then thawed on day 28 for cytotoxicity testing.

[0171] Cytotoxicity, as measured by LDH release, was observed 4 hours after co-culturing EBV-CAR19-CAR T cells or control EBV CTLs at the indicated E:T ratio (see Figures 9 and 10). EBV-CD19-CAR T cells are CD19 + (NALM6 and Raji) cells and EBV + / CD19 +This demonstrates HLA-independent CD19-specific cytotoxicity with low allocytotoxicity, as observed by the specific death of HLA-matched and HLA-incompatible BLCL cells (see Figures 9 A and B, and Figure 10). In contrast, K562 cells, as well as HLA-matched and HLA-incompatible PHA blasts, all lack EBV and CD19 antigens and are not killed (see Figures 9 A and C, and Figure 10). Furthermore, EBV-sensitized anti-CD19 CAR T cells are not killed by all CD19 + It is cytotoxic to cell lines and has little off-target cytotoxicity; that is, it is cytotoxic or nonexistent to cells lacking both CD19 and EBV expression. When observed for 3 days after effector addition (i.e., addition of EBV-CTL or EBV-CD19 CAR T cells), specific and potent HLA-independent cytolysis was observed in targeted cells. Cytolysis was induced in both HLA-matched (BLCL target) and HLA-incompatible (BLCL and Raji target) cells. However, EBV-CTL was only able to induce significant cytolysis in compatible BLCL target cells (see Figure 11).

[0172] Compared to conventionally generated CAR T cells (i.e., without enrichment of the starting T cell culture or antigen stimulation), antigen-specific CAR T cells (e.g., anti-CD19-CD28-CAR-EBV-CTL) exhibit comparable, if not superior, cytotoxicity (see Figure 12). However, anti-CD19-CD28-CAR-EBV-CTL appears to be less alloreactive. The proliferative capacity of EBV-specific CD19-CAR T cells was observed by the CellTrace® Violet dilution assay during co-culture with the indicated cell lines (see Figure 13). Anti-CD19-CD28-CAR-EBV-CTLs retained the ability to kill B lymphoblastoid cell lines (BLCLs) (Figure 13, bottom left), but preserved autologous and allogeneic PHA blast cell targets lacking CD19 and EBV antigen expression, and exhibited significantly less alloreactivity to conventional CAR T cells (Figure 13, bottom right, shaded). Furthermore, compared to conventionally generated CAR T cells, anti-CD19-CD28-CAR-EBV-CTLs exhibited a distinct cytokine profile characterized by increased IFNγ production (see Figure 14).

[0173] Additional Embodiments The following are a number of specific embodiments of the present invention disclosed herein. These embodiments are illustrative and for illustrative purposes only. The present invention is not limited to these embodiments, but it will be understood that they encompass all such forms and combinations thereof that fall within the scope of the above disclosure.

[0174] Embodiment 1. A method for producing a preparation containing antigen-specific T cells expressing a chimeric antigen receptor (CAR), (i) CD3 + A step in which a cell culture is prepared, and the culture contains T cells and antigen-presenting stimulator cells. (ii) Transducing the T cells of the culture with a viral vector containing a nucleic acid sequence encoding a CAR, (iii) culturing the transduced T cells to allow proliferation of antigen-specific T cells expressing the CAR, and (iv) recovering the antigen-specific T cells expressing the CAR A method comprising the above steps.

[0175] Embodiment 2. A method for inducing ex vivo proliferation of a population of antigen-specific T cells expressing a CAR, comprising: (i) preparing a culture of CD3 + cells, wherein the culture contains T cells and antigen-presenting stimulator cells, (ii) transducing the T cells with a viral vector containing a nucleic acid sequence encoding the CAR, (iii) culturing the population of transduced T cells to expand antigen-specific T cells expressing the CAR, and (iv) recovering the antigen-specific T cells expressing the CAR for treatment A method comprising the above steps.

[0176] Embodiment 3. The method according to Embodiment 1 or 2, further comprising culturing the transduced T cells in step (iii) together with antigen-presenting stimulator cells.

[0177] Embodiment 4. The method according to any one of Embodiments 1 to 3, further comprising incubating the culture of step (i), (ii), (iii) or any combination thereof with one or more cytokines.

[0178] [[ID=3E]]Embodiment 5. The method according to any one of Embodiments 1 to 4, further comprising incubating the stimulator cells with one or more cytokines before culturing them with CD3 + cells.

[0179] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the stimulator cells are irradiated antigen-presenting stimulator cells.

[0180] Embodiment 7. CD3 + The method according to any one of Embodiments 1 to 6, wherein the cells include a sample of peripheral blood mononuclear cells (PBMCs) depleted of red blood cells, platelets, monocytes, and granulocytes.

[0181] Embodiment 8. CD3 + Cells, CD3 + The method according to any one of embodiments 1 to 7, comprising a sample of PBMCs from which lymphocytes are positively selected.

[0182] Embodiment 9. CD3 + Cells include effector T cells and T helper cells (T H cells), cytotoxic T cells (CTLs), memory T cell types, regulatory T cells (T reg The method according to Embodiment 8, comprising a plurality of cell types including any one of the following: cells, natural killer T cells (NKT cells), mucosa-associated invariant cells (MAIT cells), gamma delta T cells (γδ T cells), double-negative T cells (DNTs), CD3+ B cells, or any combination thereof.

[0183] Embodiment 10. CD3 + The method according to any one of Embodiments 1 to 9, wherein the cells and the stimulator cells are each derived from the same donor.

[0184] Embodiment 11. CD3 + The method according to any one of embodiments 1 to 9, wherein the cells are derived from a donor different from the stimulator cells.

[0185] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the stimulator cells include lymphoblastoid cells, B cells, antigen-presenting T cells, dendritic cells, artificial antigen-presenting cells and / or K562 cells.

[0186] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein the stimulator cells include lymphoblastoid B cells (BLCLs).

[0187] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein stimulator cells are positively selected from a donor sample.

[0188] Embodiment 15. Positively selected cells are CD19 + The method according to Embodiment 14, wherein the cells are cells.

[0189] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein stimulator cells are infected with a natural and / or wild-type virus containing at least one immunogenic peptide antigen including a T cell epitope.

[0190] Embodiment 17. The method according to any one of Embodiments 1 to 15, wherein the stimulator cells express a viral vector comprising a nucleic acid sequence encoding at least one immunogenic peptide antigen containing a T cell epitope.

[0191] Embodiment 18. The method according to Embodiment 16 or 17, wherein the immunogenic peptide antigen is derived from oncovirus.

[0192] Embodiment 19. The method according to any one of Embodiments 16 to 18, wherein the immunogenic peptide antigen is derived from a herpesvirus, papillomavirus, adenovirus, polyomavirus, or retrovirus.

[0193] Embodiment 20. The method according to any one of Embodiments 16 to 19, wherein the immunogenic peptide antigen is derived from Epstein-Barr virus (EBV), cytomegalovirus (CMV), human papillomavirus (HPV), BK virus (BKV), John Cunningham virus (JCV), Merkel cell virus (MCV), human T lymphotropic virus (HTLV), or human immunodeficiency virus (HIV).

[0194] Embodiment 21. The method according to any one of Embodiments 16 to 18, wherein the immunogenic peptide antigen is derived from a virus known to cause viral hepatitis.

[0195] Embodiment 22. The method according to embodiment 17, wherein the viral vector is replication-incompetent.

[0196] Embodiment 23. The method according to any one of embodiments 17 to 22, wherein the viral vector comprises a nucleic acid sequence encoding one or more immunogenic peptide antigens.

[0197] Embodiment 24. The method according to any one of embodiments 17 to 23, wherein the viral vector is an adenoviral vector.

[0198] Embodiment 25. The method according to embodiment 24, wherein the adenoviral vector comprises a nucleic acid sequence encoding one or more EBV antigens.

[0199] Embodiment 26. The method according to embodiment 24 or 25, wherein the adenoviral vector is AdE1-LMPpoly.

[0200] Embodiment 27. The method according to any one of embodiments 1 to 26, wherein the stimulator cells present more than one immunogenic peptide antigen comprising a T cell epitope.

[0201] Embodiment 28. The method according to any one of embodiments 1 to 27, wherein the stimulator cells express one or more EBV antigens.

[0202] Embodiment 29. The method according to embodiment 28, wherein the one or more EBV antigens comprise an LMP1 peptide or a fragment thereof, an LMP2A peptide or a fragment thereof, and / or an EBNA1 peptide or a fragment thereof.

[0203] Embodiment 30. The method according to any one of embodiments 1 to 29, wherein the stimulator cells endogenously express an antigen or ligand targeted by a CAR.

[0204] Embodiment 31. The method according to any one of Embodiments 1 to 29, wherein the stimulator cells express a vector containing a nucleic acid sequence encoding an antigen or ligand targeted by CAR.

[0205] Embodiment 32. The method according to Embodiment 30 or 31, wherein the CAR antigen or ligand is CD19.

[0206] Embodiment 33. For future trait introduction, CD3 + The method according to any one of Embodiments 1 to 32, optionally comprising the step of freezing and storing a cell culture.

[0207] Embodiment 34. Before transduction, CD3 + The method according to Embodiment 33, wherein the cell culture is thawed and re-cultured at least once with stimulator cells.

[0208] Embodiment 35. Prior to transduction, CD3 is administered to the culture for at least 2 to at least 28 days. + The method according to any one of embodiments 1 to 34, comprising the step of maintaining the cells.

[0209] Embodiment 36. Before transduction, CD3 is administered to the culture for at least two days. + The method according to embodiment 35, comprising the step of maintaining the cells.

[0210] Embodiment 37. Before transduction, CD3 is administered to the culture for at least 9 days. + The method according to embodiment 35, comprising the step of maintaining the cells.

[0211] Embodiment 38. Before transduction, CD3 is administered to the culture for at least 20 days. + The method according to embodiment 35, comprising the step of maintaining the cells.

[0212] Embodiment 39. Before transduction, CD3 is administered to the culture for at least 28 days. +The method according to embodiment 35, comprising the step of maintaining the cells.

[0213] Embodiment 40. The method according to any one of Embodiments 1 to 39, further comprising the step of maintaining the transduced T cells in the culture for at least 2 to at least 17 days after transduction.

[0214] Embodiment 41. The method according to Embodiment 40, further comprising the step of maintaining the transduced T cells in the culture for at least two days after transduction.

[0215] Embodiment 42. The method according to Embodiment 40, further comprising the step of maintaining the transduced T cells in the culture for at least 7 days after transduction.

[0216] Embodiment 43. The method according to Embodiment 40, further comprising the step of maintaining the transduced T cells in the culture for at least 17 days after transduction.

[0217] Embodiment 44. The CD3 of the culture + The method according to any one of embodiments 37 to 39, further comprising the step of restoring the cells with the stimulator cells at least once.

[0218] Embodiment 45. The CD3 of the culture + The method according to any one of embodiments 37 to 39, further comprising the step of restoring the cells with the stimulator cells at least twice.

[0219] Embodiment 46. The method according to Embodiment 44 or 45, wherein the re-culturing step is initiated at least every 2 to 14 days.

[0220] Embodiment 47. The method according to any one of Embodiments 44 to 46, wherein the first re-culturing step is initiated 11 days after the preparation of the culture.

[0221] Embodiment 48. The method according to any one of Embodiments 44 to 47, wherein the second re-culturing step is initiated 7 days after the first re-culturing step.

[0222] Embodiment 49. The method according to any one of Embodiments 1 to 48, wherein the viral vector containing the nucleic acid sequence encoding CAR is a lentiviral vector.

[0223] Embodiment 50. The method according to Embodiment 49, wherein the viral vector encoding CAR is a gamma retroviral vector.

[0224] Embodiment 51. The method according to Embodiment 49 or 50, wherein the viral vector is non-replicable.

[0225] Embodiment 52. The method according to Embodiment 51, wherein the nucleic acid sequence encoding the CAR comprises one or more signaling domains.

[0226] Embodiment 53. The method according to Embodiment 52, wherein one or more signaling domains include a CD28 signaling domain, a 4-1BB signaling domain and / or a CD3 signaling domain, or a fragment thereof.

[0227] Embodiment 54. The method according to Embodiment 53, wherein the CD3 signaling domain is CD3ζ.

[0228] Embodiment 55. The method according to any one of Embodiments 49 to 54, wherein a viral vector comprising a nucleic acid sequence encoding a CAR further comprises a nucleic acid sequence encoding a selectable marker.

[0229] Embodiment 56. The method of Embodiment 55, wherein the viral vector comprising a nucleic acid sequence encoding CAR further comprises a nucleic acid sequence encoding antibiotic resistance.

[0230] Embodiment 57. The method according to Embodiment 56, wherein the antibiotic is blastocydin.

[0231] Embodiment 58. T cells expressing the recovered CAR, CD3 + The method according to any one of embodiments 1 to 57, wherein the cell is a T cell.

[0232] Embodiment 59. The method of Embodiment 58, optionally comprising the step of freezing and storing CAR-expressing T cells recovered in a cell bank for the preparation of an adoptive immunotherapy composition at a later date.

[0233] Embodiment 60. The method according to Embodiment 59, wherein CAR-expressing T cells are thawed and re-cultured at least once with stimulator cells before preparing the adoptive immunotherapy composition.

[0234] Embodiment 61. T cells expressing the recovered CAR become central memory T cells (T CM T cells) and effector memory T cells (T EM The method according to any one of embodiments 58 to 60, comprising each of the cells.

[0235] Embodiment 62. T cells expressing the recovered CAR are mainly T CM The method according to any one of embodiments 58 to 60, wherein the cells are cells.

[0236] Embodiment 63. T CM Cell vs. T EM The method according to Embodiment 61, wherein the cell ratio is greater than 1:1.

[0237] Embodiment 64. T CM Cell vs. T EM The method according to Embodiment 63, wherein the cell ratio is at least 1.4:1.

[0238] Embodiment 65. T CM Cell vs. T EM The method according to Embodiment 63, wherein the cell ratio is at least 2.5:1.

[0239] Embodiment 66. T CM Cell vs. T EMThe method according to Embodiment 63, wherein the cell ratio is at least 3:1.

[0240] Embodiment 67. T cells expressing the recovered CAR, CD4 + and CD8 + The method according to any one of embodiments 58 to 60, comprising each of the T cells.

[0241] Embodiment 68. T cells expressing the recovered CAR mainly CD4 + The method according to any one of embodiments 58 to 60, wherein the cell is a T cell.

[0242] Embodiment 69. CD4 + T cells vs. CD8 + The method according to embodiment 68, wherein the ratio of T cells is greater than 1:1.

[0243] Embodiment 70. CD4 + T cells vs. CD8 + The method according to embodiment 69, wherein the ratio of T cells is at least 1.4:1.

[0244] Embodiment 71. CD4 + T cells vs. CD8 + The method according to embodiment 69, wherein the ratio of T cells is at least 2.5:1.

[0245] Embodiment 72. CD4 + T cells vs. CD8 + The method according to embodiment 69, wherein the ratio of T cells is at least 3:1.

[0246] Embodiment 73. At least 60% of the T cells expressing the recovered CAR are T CM The method according to any one of embodiments 62 to 72, wherein the cell is a cell.

[0247] Embodiment 74. An ex vivo method for enriching T cells expressing antigen-specific CARs, (i) Obtain a cell sample from the subject, and if the sample is CD3 + Steps including T cells, (ii) From the above sample, CD3 + The cells were isolated, and the CD3 + Cells are brought into contact with antigen-presenting stimulus cells, thereby allowing antigen-specific CD3 + A step to selectively enhance the proliferation of T cells. (iii) CD3 + CD3 is a viral vector that encodes a chimeric antigen receptor (CAR) that is transduced into cells to express an antigen-specific CAR. + A step of providing a population of T cells, and (iv) Optionally, CD3 expressing CAR + A population of T cells is cultured ex vivo in a culture medium together with antigen-presenting stimulator cells to produce CD3 cells that express antigen-specific CARs. + A step to selectively enhance T cell proliferation. Methods that include...

[0248] Embodiment 75. The method according to Embodiment 74, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

[0249] Embodiment 76. Isolated CD3 + The method according to Embodiment 74 or 75, wherein the cells and antigen-presenting stimulator cells are derived from the same subject, respectively.

[0250] Embodiment 77. Isolated CD3 + The method according to any one of embodiments 74 to 76, wherein the cells are derived from a different subject than the antigen-presenting stimulator cells.

[0251] Embodiment 78. The method according to any one of Embodiments 74 to 77, wherein the antigen-presenting stimulator cells include lymphoblastoid cells, B cells, antigen-presenting T cells, dendritic cells, artificial antigen-presenting cells and / or K562 cells.

[0252] Embodiment 79. The method according to any one of Embodiments 74 to 79, wherein the antigen-presenting stimulator cells are BLCLs.

[0253] Embodiment 80. Antigen-presenting stimulator cells are CD19 + The method according to any one of embodiments 74 to 79.

[0254] Embodiment 81. The method according to any one of Embodiments 74 to 80, wherein antigen-presenting stimulator cells express one or more EBV antigens.

[0255] Embodiment 82. The method according to Embodiment 81, wherein one or more EBV antigens comprise an LMP1 peptide or a fragment thereof, an LMP2A peptide or a fragment thereof, and / or an EBNA1 peptide or a fragment thereof.

[0256] Embodiment 83. Before transduction, CD3 + The method according to any one of embodiments 74 to 82, comprising the step of maintaining T cells for at least 3 to at least 28 days after contact with the antigen-presenting stimulator cells.

[0257] Embodiment 84. Before transduction, CD3 + The method according to Embodiment 83, comprising the step of maintaining T cells for at least 3 days after contact with the antigen-presenting stimulator cells.

[0258] Embodiment 85. Before transduction, CD3 + The method according to Embodiment 83, comprising the step of maintaining T cells for at least 6 days after contact with the antigen-presenting stimulator cells.

[0259] Embodiment 86. Before transduction, CD3 + The method according to Embodiment 83, comprising the step of maintaining T cells for at least 9 days after contact with the antigen-presenting stimulator cells.

[0260] Embodiment 87. Before transduction, CD3 + The method according to Embodiment 83, comprising the step of maintaining T cells for at least 28 days after contact with the antigen-presenting stimulator cells.

[0261] Embodiment 88. The isolated CD3 + The method according to any one of embodiments 83 to 87, further comprising the step of bringing T cells into contact with antigen-presenting stimulator cells at least once again.

[0262] Embodiment 89. The isolated CD3 + The method according to any one of embodiments 83 to 88, further comprising the step of bringing T cells into contact with antigen-presenting stimulator cells at least twice again.

[0263] Embodiment 90. The isolated CD3 + The method according to embodiment 88 or 89, wherein the step of re-contacting T cells with antigen-presenting stimulator cells is initiated at least every 2 to 14 days.

[0264] Embodiment 91. The isolated CD3 + The method according to any one of embodiments 88 to 90, wherein the first step of re-contacting T cells with antigen-presenting stimulator cells is initiated after 11 days.

[0265] Embodiment 92. The method according to any one of embodiments 88 to 91, wherein the second re-contact step is initiated 7 days after the first re-contact step with the isolated CD3+ T cell antigen-presenting stimulator cells.

[0266] Embodiment 93. CD3 expressing antigen-specific CAR + The method according to any one of embodiments 74 to 92, wherein the culture that selectively enhances T cell proliferation includes antigen-presenting stimulator cells.

[0267] Embodiment 94. In a culture medium that selectively enhances the proliferation of T cells expressing antigen-specific CARs after transduction, CD3 cells expressing antigen-specific CARs are introduced. + The method according to Embodiment 93, comprising the step of maintaining T cells for at least two days.

[0268] Embodiment 95. In a culture medium that selectively enhances the proliferation of T cells expressing antigen-specific CARs after transduction, CD3 cells expressing antigen-specific CARs are introduced. + The method according to embodiment 94, comprising the step of maintaining T cells for at least 7 days.

[0269] Embodiment 96. In a culture medium that selectively enhances the proliferation of T cells expressing antigen-specific CARs after transduction, CD3 cells expressing antigen-specific CARs are introduced. + The method according to Embodiment 94, comprising the step of maintaining T cells for at least 17 days.

[0270] Embodiment 97. The method according to any one of Embodiments 74 to 96, wherein the viral vector encoding CAR is a lentiviral vector.

[0271] Embodiment 98. The method according to Embodiment 97, wherein the viral vector encoding CAR is a gamma retroviral vector.

[0272] Embodiment 99. The method according to either Embodiment 97 or 98, wherein the viral vector encoding CAR is non-replicable.

[0273] Embodiment 100. The method according to Embodiment 99, wherein the nucleic acid sequence encoding the CAR comprises one or more signaling domains.

[0274] Embodiment 101. The method according to Embodiment 100, wherein one or more signaling domains include a CD28 signaling domain, a 4-1BB signaling domain and / or a CD3 signaling domain, or a fragment thereof.

[0275] Embodiment 102. The method according to Embodiment 101, wherein the CD3 signaling domain is CD3ζ or a fragment thereof.

[0276] Embodiment 103. The method according to any one of Embodiments 97 to 102, wherein a viral vector comprising a nucleic acid sequence encoding a CAR further comprises a nucleic acid sequence encoding a selectable marker.

[0277] Embodiment 104. The method according to any one of Embodiments 97 to 102, wherein a viral vector comprising a nucleic acid sequence encoding CAR further comprises a nucleic acid sequence encoding antibiotic resistance.

[0278] Embodiment 105. CD3 expressing antigen-specific CAR + The method according to any one of embodiments 74 to 104, wherein the culture that selectively enhances T cell proliferation further comprises an antibiotic.

[0279] Embodiment 106. The method of Embodiment 105, wherein the antibiotic is blastocydin.

[0280] Embodiment 107. A method for preparing an adoptive immunotherapy composition comprising any of the embodiments described in Embodiments 74 to 106, wherein the composition expresses an antigen-specific CAR in CD3 + Central memory T cells (T cm A method that mainly includes ).

[0281] Embodiment 108. At least 60% of the adoptive immunotherapy composition is a CD3 expressing antigen-specific CARs. + T cm The method according to Embodiment 107, including the method described above.

[0282] Embodiment 109. CD3 expressing antigen-specific CARs + T cm However, mainly CD4 + The method according to either embodiment 107 or 108, wherein the cell is a T cell.

[0283] Embodiment 110. The method according to any one of Embodiments 1 to 109, wherein the CAR includes a targeting domain that binds to CD19.

[0284] Embodiment 111. The method according to Embodiment 110, wherein the CAR comprises an anti-CD19 single-stranded variable fragment (ScFv).

[0285] Embodiment 112. A method for improving the proliferative capacity of T cells, comprising the step of carrying out the method according to any one of Embodiments 1 to 111.

[0286] Embodiment 113. A method for improving the transduction efficiency of T cells, comprising the step of carrying out the method according to any one of Embodiments 1 to 112.

[0287] Embodiment 114. A method for improving the viability of CAR-expressing T cells, comprising the step of carrying out the method described in any one of Embodiments 1 to 113.

[0288] Embodiment 115. A T cell composition prepared by the method described in any one of Embodiments 1 to 114.

[0289] Embodiment 116. A T cell preparation comprising antigen-specific T cells expressing recombinant chimeric antigen receptors (CARs), wherein at least 60% of the CAR-expressing T cells are T cm T cell preparations, which are cells.

[0290] Embodiment 117. T expressing CAR cm Cells mainly CD4 + A T cell preparation according to Embodiment 116, wherein the T cells are T cells.

[0291] Embedding by reference All publications, patents, patent applications, and sequence accession numbers described herein are incorporated herein by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In case of any conflict, this application shall prevail, including the definitions herein.

[0292] Equivalents Those skilled in the art will recognize, or can verify by ordinary experiment alone, numerous equivalents of the specific embodiments of the present invention described herein. Such equivalents shall be encompassed by the following claims. The present invention includes, for example, the following embodiments: [Embodiment 1] A method for producing a composition comprising cytotoxic T cells expressing a chimeric antigen receptor (CAR) that is specific to binding to a first antigen and specific to binding to a second antigen, (i) CD3 + A culture of concentrated T cells was prepared, and the CD3 + A step in which T cells are stimulated to recognize and bind to the first antigen via T cell receptors, (ii) CD3 of the culture + A step of transducing T cells with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, (iii) CD3 with CAR introduced + A step of culturing T cells to enable proliferation of first antigen-specific cytotoxic T cells expressing CAR, and (iv) Step of collecting the first antigen-specific cytotoxic T cells expressing CAR. Methods that include... [Embodiment 2] A method for preparing an adoptive immunotherapy composition comprising cytotoxic T cells expressing a chimeric antigen receptor (CAR) that is specific to binding to a first antigen and specific to binding to a second antigen, (i) CD3 + A culture of concentrated T cells was prepared, and the CD3 + A step in which T cells are stimulated to recognize and bind to the first antigen via T cell receptors, (ii) CD3 of the culture + A step of transducing T cells with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, (iii) CD3 with CAR introduced +A step of culturing T cells to enable proliferation of first antigen-specific cytotoxic T cells expressing CAR, and (iv) Recovering first antigen-specific cytotoxic T cells expressing CAR, thereby providing the adoptive immunotherapy composition. Methods that include... [Embodiment 3] A method for inducing the proliferation of a population of cytotoxic T cells expressing a chimeric antigen receptor (CAR) that is specific to binding to a first antigen and specific to binding to a second antigen, (i) CD3 + A culture of concentrated T cells was prepared, and the CD3 + A step in which T cells are stimulated to recognize and bind to the first antigen via T cell receptors, (ii) CD3 of the culture + The steps include transducing T cells with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, and (iii) CD3 with CAR introduced + A step to culture T cells and enable the proliferation of the first antigen-specific cytotoxic T cells expressing CAR. Methods that include... [Embodiment 4] The method according to any one of Embodiments 1 to 3, further comprising the step of incubating the culture from step (i), (ii), (iii) or any combination thereof with one or more cytokines. [Embodiment 5] CD3 + The method according to any one of Embodiments 1 to 4, wherein the concentrated cell culture includes a sample of peripheral blood mononuclear cells (PBMCs) depleted of erythrocytes, platelets, monocytes, and granulocytes. [Embodiment 6] CD3 + Regarding the cells, the concentrated cell culture was found to contain CD3 from the PBMC sample. + The method according to any one of Embodiments 1 to 5, prepared by a process including positive selection of cells. [Embodiment 7] CD3 stimulated to recognize and bind to the first antigen via the T cell receptor +The step of preparing a culture of cells enriched with T cells is CD3 + The method according to any one of Embodiments 1 to 6, comprising the step of co-culturing a population of cell responders together with stimulator cells that present the first antigen on their cell surface. [Embodiment 8] The method according to Embodiment 7, wherein the co-culturing step is initiated with a responder cell:stimulator cell ratio of 1:4. [Embodiment 9] CD3 stimulated to recognize and bind to the first antigen via the T cell receptor + The method according to either Embodiment 7 or 8, wherein the prepared culture of cells enriched with T cells is optionally re-cultured with stimulator cells that present the first antigen on their surface before CAR transduction. [Embodiment 10] The method according to Embodiment 9, wherein the first re-culture step is initiated at least 7 days after the start of the co-culture step. [Embodiment 11] The method according to any one of Embodiments 7 to 10, wherein the responder and stimulater cells are derived from the same donor. [Embodiment 12] The method according to any one of Embodiments 7 to 11, wherein the stimulator cells are gamma-irradiated antigen-presenting stimulator cells. [Embodiment 13] The method according to any one of Embodiments 7 to 12, wherein the stimulator cells include lymphoblastoid B cells (BLCLs). [Embodiment 14] The method according to any one of Embodiments 7 to 13, wherein stimulator cells are infected with a natural and / or wild-type virus comprising at least one immunogenic peptide antigen including a T cell epitope. [Embodiment 15] The method according to any one of Embodiments 7 to 14, wherein the stimulator cells are engineered to express at least one immunogenic peptide antigen containing a T cell epitope. [Embodiment 16] The method according to any one of Embodiments 1 to 15, wherein the first antigen is a viral antigen. [Embodiment 17] The method according to Embodiment 16, wherein the viral antigen is Epstein-Barr virus (EBV), cytomegalovirus (CMV), human papillomavirus (HPV), BK virus (BKV), John Cunningham virus (JCV), Merkel cell virus (MCV), human T lymphotropic virus (HTLV), or human immunodeficiency virus (HIV) antigen. [Embodiment 18] The method according to Embodiment 16 or 17, wherein the viral antigen comprises EBV LMP1 peptide or a fragment thereof, EBV LMP2A peptide or a fragment thereof, or EBV EBNA1 peptide or a fragment thereof. [Embodiment 19] The method according to any one of Embodiments 7 to 15, wherein the stimulator cells further express the second antigen. [Embodiment 20] The method according to any one of Embodiments 1 to 19, wherein the second antigen is a CD19 antigen, a CD20 antigen, or a mesothelin antigen. [Embodiment 21] For future trait introduction, CD3 + The method according to any one of Embodiments 1 to 20, optionally comprising the step of freezing and storing a cell culture enriched with T cells. [Embodiment 22] The method according to any one of Embodiments 1 to 21, optionally comprising the step of freezing and storing a culture of first antigen-specific cytotoxic T cells expressing CAR for future use. [Embodiment 23] The method according to any one of Embodiments 1 to 22, optionally comprising the step of freezing and storing first antigen-specific cytotoxic T cells expressing the recovered CAR for future administration to patients in need. [Embodiment 24] Before introducing CAR, CD3 + The method according to any one of Embodiments 1 to 23, comprising the step of maintaining a cell culture enriched with T cells for at least 2 to at least 28 days. [Embodiment 25] Before introducing CAR, CD3 + The method according to any one of Embodiments 1 to 24, comprising the step of maintaining a cell culture enriched with T cells for at least two days. [Embodiment 26] Before introducing CAR, CD3 + The method according to any one of Embodiments 1 to 25, comprising the step of maintaining a cell culture enriched with T cells for at least 9 days. [Embodiment 27] Before introducing CAR, CD3 + The method according to any one of Embodiments 1 to 26, comprising the step of maintaining a cell culture enriched with T cells for at least 20 days. [Embodiment 28] Before introducing CAR, CD3 + The method according to any one of Embodiments 1 to 27, comprising the step of maintaining a cell culture enriched with T cells for at least 28 days. [Embodiment 29] Before the recovery step, CD3 that has been transduced with CAR + The method according to any one of Embodiments 1 to 28, comprising the step of culturing T cells for at least 2 to at least 17 days. [Embodiment 30] Before the recovery step, CD3 that has been transduced with CAR + The method according to any one of embodiments 1 to 29, comprising the step of culturing T cells for at least two days. [Embodiment 31] Before the recovery step, CD3 that has been transduced with CAR + The method according to any one of embodiments 1 to 30, comprising the step of culturing T cells for at least 7 days. [Embodiment 32] Before the recovery step, CD3 that has been transduced with CAR + The method according to any one of embodiments 1 to 31, comprising the step of culturing T cells for at least 17 days. [Embodiment 33] CD3 transduced with CAR + The method according to any one of Embodiments 1 to 32, optionally comprising the step of co-culturing T cells with stimulator cells expressing the first antigen at least once. [Embodiment 34] The method according to any one of Embodiments 1 to 33, wherein the viral vector containing the nucleic acid sequence encoding CAR is a lentivirus or a retroviral vector. [Embodiment 35] The method according to any one of Embodiments 1 to 34, wherein the viral vector is non-replicable. [Embodiment 36] The method according to any one of Embodiments 1 to 35, wherein the CAR comprises one or more signal transduction domains. [Embodiment 37] The method according to Embodiment 36, wherein one or more signaling domains include a CD28 signaling domain, a 4-1BB signaling domain, a CD3 signaling domain, or a variant or fragment thereof. [Embodiment 38] The method according to Embodiment 37, wherein the CD3 signaling domain is CD3ζ. [Embodiment 39] The method according to Embodiment 36, wherein the CAR includes a variant CD3ζ domain lacking at least one functional ITAM region. [Embodiment 40] The method according to Embodiment 36, wherein CAR includes a Mut06 domain. [Embodiment 41] The method according to any one of Embodiments 1 to 40, wherein the viral vector comprising a nucleic acid sequence encoding a CAR further comprises a nucleic acid sequence encoding a selectable marker. [Embodiment 42] The method according to Embodiment 41, wherein a selectable marker confers antibiotic resistance. [Embodiment 43] The method according to Embodiment 42, wherein the antibiotic is blastocydin. [Embodiment 44] The first antigen-specific cytotoxic T cells expressing the recovered CAR are central memory T cells (T CM T cells) and effector memory T cells (T EM The method according to Embodiment 1 or 2, comprising each of the cells. [Embodiment 45] The first antigen-specific cytotoxic T cells expressing the recovered CAR are mainly T CM The method according to Embodiment 44, wherein the cells are cells. [Embodiment 46]T CM Cell vs. T EM The method according to embodiment 44 or 45, wherein the cell ratio is greater than 1:1. [Embodiment 47]T CM Cell vs. T EMThe method according to any one of embodiments 44 to 46, wherein the cell ratio is at least 1.4:1. [Embodiment 48]T CM Cell vs. T EM The method according to any one of embodiments 44 to 47, wherein the cell ratio is at least 2.5:1. [Embodiment 49]T CM Cell vs. T EM The method according to any one of embodiments 44 to 48, wherein the cell ratio is at least 3:1. [Embodiment 50] The first antigen-specific cytotoxic T cells expressing the recovered CAR are CD4 + and CD8 + The method according to either Embodiment 1 or 2, comprising each of the T cells. [Embodiment 51] The first antigen-specific cytotoxic T cells expressing the recovered CAR mainly CD4 + The method according to Embodiment 50, wherein the cells are T cells. [Embodiment 52] An ex vivo method for generating a composition comprising cytotoxic T cells expressing a chimeric antigen receptor (CAR) that is specific to binding to a first antigen and specific to binding to a second antigen, (i) From the target, CD3 + Steps to obtain a sample of cells containing T cells, (ii) The cell sample is CD3 + A step of enriching T cells and providing a concentrated sample of cells. (iii) A step of providing a sample of cells stimulated by the first antigen by bringing the concentrated sample of the cells into contact with antigen-presenting stimulator cells that present the first antigen on their cell surface. (iv) Transducing a sample of cells stimulated by the first antigen with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, (v) A step of culturing cells transduced with CAR to enable the proliferation of a first antigen-specific cytotoxic T cell expressing CAR, and (vi) A step of recovering a first antigen-specific cellular composition expressing CAR. Methods that include... [Embodiment 53] An ex vivo method for generating a composition comprising cytotoxic T cells expressing a chimeric antigen receptor (CAR) that is specific to binding to a first antigen and specific to binding to a second antigen, (i) From the target, CD3 + Steps to obtain a sample of cells containing T cells, (ii) The cell sample is CD3 + A step of enriching T cells and providing a concentrated sample of cells. (iii) Transducing the concentrated cell sample with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, (iv) The step of bringing cells transduced with CAR into contact with antigen-presenting stimulator cells that present the first antigen on their cell surface, and (v) A step of recovering a first antigen-specific cellular composition expressing CAR. Methods that include... [Embodiment 54] A method for improving the proliferative capacity of T cells, comprising the step of carrying out the method described in any one of Embodiments 1 to 53. [Embodiment 55] A method for improving the transduction efficiency of T cells, comprising the step of carrying out the method described in any one of Embodiments 1 to 54. [Embodiment 56] A method for improving the viability of CAR-expressing T cells, comprising the step of carrying out the method described in any one of Embodiments 1 to 55. [Embodiment 57] A method for treating a disorder associated with the expression of a peptide antigen in a patient in need thereof, comprising the step of administering to the patient a first antigen-specific cytotoxic T cell expressing a CAR obtained by the method of any one of Embodiments 1 to 56. [Embodiment 58] A T cell composition prepared by the method described in any one of Embodiments 1 to 57.

[0293] [Sequence List] SEQUENCE LISTING <110> ATARA BIOTHERAPEUTICS, INC. <120> METHODS FOR EXPANDING ANTIGEN-SPECIFIC CAR-T CELLS, COMPOSITIONS AND USES RELATED THERETO <130> PA24-421 <150> US 62 / 729,089 <151> 2018-09-10 <150> US 62 / 896,707 <151> 2019-09-06 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 376 <212> PRT <213> Human gammaherpesvirus 4 <400> 1 Met Asp Leu Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Tyr Ile Ala Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Ile Met Ser Asn Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ala Phe Ala Leu Met Leu Val Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Leu Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Ile Trp Val Tyr Phe Leu Glu 115 120 125 Ile Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Ile Leu Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Ser Asn His Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 His Leu Leu Val Ser Gly Ala Gly Asp Ala Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asp Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Asn Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Arg Gly Pro 325 330 335 Pro Ser Met Thr Asp Gly Gly Gly Gly Asp Pro His Leu Pro Thr Leu 340 345 350 Leu Leu Gly Thr Ser Gly Ser Gly Gly Asp Asp Asp Asp Pro His Gly 355 360 365 Pro Val Gln Leu Ser Tyr Tyr Asp 370 375 <210> 2 <211> 497 <212> PRT <213> Human gammaherpesvirus 4 <400> 2 Met Gly Ser Leu Glu Met Val Pro Met Gly Ala Gly Pro Pro Ser Pro 1 5 10 15 Gly Gly Asp Pro Asp Gly Asp Asp Gly Gly Asn Asn Ser Gln Tyr Pro 20 25 30 Ser Ala Ser Gly Ser Asp Gly Asn Thr Pro Thr Pro Pro Asn Asp Glu 35 40 45 Glu Arg Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu Asp Leu Asp 50 55 60 Trp Gly Asn Gly Asp Arg His Ser Asp Tyr Gln Pro Leu Gly Asn Gln 65 70 75 80 Asp Pro Ser Leu Tyr Leu Gly Leu Gln His Asp Gly Asn Asp Gly Leu 85 90 95 Pro Pro Pro Pro Tyr Ser Pro Arg Asp Asp Ser Ser Gln His Ile Tyr 100 105 110 Glu Glu Ala Gly Arg Gly Ser Met Asn Pro Val Cys Leu Pro Val Ile 115 120 125 Val Ala Pro Tyr Leu Phe Trp Leu Ala Ala Ile Ala Ala Ser Cys Phe 130 135 140 Thr Ala Ser Val Ser Thr Val Val Thr Ala Thr Gly Leu Ala Leu Ser 145 150 155 160 Leu Leu Leu Ala Ala Val Ala Ser Ser Tyr Ala Ala Ala Gln Arg 165 170 175 Lys Leu Leu Thr Pro Val Thr Val Leu Thr Ala Val Val Thr Phe Phe 180 185 190 Ala Ile Cys Leu Thr Trp Arg Ile Glu Asp Pro Pro Phe Asn Ser Leu 195 200 205 Leu Phe Ala Leu Leu Ala Ala Ala Gly Gly Leu Gln Gly Ile Tyr Val 210 215 220 Leu Val Met Leu Val Leu Leu Ile Leu Ala Tyr Arg Arg Arg Trp Arg 225 230 235 240 Arg Leu Thr Val Cys Gly Gly Ile Met Phe Leu Ala Cys Val Leu Val 245 250 255 Leu Ile Val Asp Ala Val Leu Gln Leu Ser Pro Leu Leu Gly Ala Val 260 265 270 Thr Val Val Ser Met Thr Leu Leu Leu Leu Ala Phe Val Leu Trp Leu 275 280 285 Ser Ser Pro Gly Gly Leu Gly Thr Leu Gly Ala Ala Leu Leu Thr Leu 290 295 300 Ala Ala Ala Leu Ala Leu Leu Ala Ser Leu Ile Leu Gly Thr Leu Asn 305 310 315 320 Leu Thr Thr Met Phe Leu Leu Met Leu Leu Trp Thr Leu Val Val Leu 325 330 335 Leu Ile Cys Ser Ser Cys Ser Ser Cys Pro Leu Thr Lys Ile Leu Leu 340 345 350 Ala Arg Leu Phe Leu Tyr Ala Leu Ala Leu Leu Leu Leu Ala Ser Ala 355 360 365 Leu Ile Ala Gly Gly Ser Ile Leu Gln Thr Asn Phe Lys Ser Leu Ser 370 375 380 Ser Thr Glu Phe Ile Pro Asn Leu Phe Cys Met Leu Leu Leu Ile Val 385 390 395 400 Ala Gly Ile Leu Phe Ile Leu Ala Ile Leu Thr Glu Trp Gly Ser Gly 405 410 415 Asn Arg Thr Tyr Gly Pro Val Phe Met Cys Leu Gly Gly Leu Leu Thr 420 425 430 Met Val Ala Gly Ala Val Trp Leu Thr Val Met Thr Asn Thr Leu Leu 435 440 445 Ser Ala Trp Ile Leu Thr Ala Gly Phe Leu Ile Phe Leu Ile Gly Phe 450 455 460 Ala Leu Phe Gly Val Ile Arg Cys Cys Arg Tyr Cys Cys Tyr Tyr Cys 465 470 475 480 Leu Thr Leu Glu Ser Glu Glu Arg Pro Pro Thr Pro Tyr Arg Asn Thr 485 490 495 Val <210> 3 <211> 238 <212> PRT <213> Human gammaherpesvirus 4 <400> 3 Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu Tyr Leu Gln 1 5 10 15 Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly Ala Ile Glu 20 25 30 Gln Gly Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr Gly Pro Arg 35 40 45 Gly Gln Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys 50 55 60 His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn Ile Ala Glu 65 70 75 80 Gly Leu Arg Val Leu Leu Ala Arg Ser His Val Glu Arg Thr Thr Glu 85 90 95 Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly Ser Lys Thr 100 105 110 Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala Ile Pro Gln Cys 115 120 125 Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala Pro Gly Pro 130 135 140 Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys Tyr Phe Met 145 150 155 160 Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys Asp Ala Ile 165 170 175 Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn Ile Lys Val 180 185 190 Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro Trp Phe Pro 195 200 205 Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly Asp Asp Gly 210 215 220 Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln Glu 225 230 235 <210> 4 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 4 Cys Leu Gly Gly Leu Leu Thr Met Val 1 5 <210> 5 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 5 Phe Leu Tyr Ala Leu Ala Leu Leu Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 6 Tyr Leu Gln Gln Asn Trp Trp Thr Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 7 Tyr Leu Leu Glu Met Leu Trp Arg Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 8 Ala Leu Leu Val Leu Tyr Ser Phe Ala 1 5 <210> 9 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 9 Leu Leu Ser Ala Trp Ile Leu Thr Ala 1 5 <210> 10 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 10 Leu Thr Ala Gly Phe Leu Ile Phe Leu 1 5 <210> 11 <211> 11 <212> PRT <213> Human gammaherpesvirus 4 <400> 11 Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys Ile 1 5 10 <210> 12 <211> 8 <212> PRT <213> Human gammaherpesvirus 4 <400> 12 Pro Tyr Leu Phe Trp Leu Ala Ala 1 5 <210> 13 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 13 Thr Tyr Gly Pro Val Phe Met Cys Leu 1 5 <210> 14 <211> 10 <212> PRT <213> Human gammaherpesvirus 4 <400> 14 Val Put Ser Asn Thr Leu Leu Ser Ala Trp 1 5 10 <210> 15 <211> 8 <212> PRT <213> Human gammaherpesvirus 4 <400> 15 Cys Pro Leu Ser Lys Ile Leu Leu 1 5 <210> 16 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 16 Arg Arg Arg Trp Arg Arg Leu Thr Val 1 5 <210> 17 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 17 Ile Glu Asp Pro Pro Phe Asn Ser Leu 1 5 <210> 18 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 18 Ile Ala Leu Tyr Leu Gln Gln Asn Trp 1 5 <210> 19 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 19 Met Ser Asn Thr Leu Leu Ser Ala Trp 1 5 <210> 20 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 20 Val Leu Lys Asp Ala Ile Lys Asp Leu 1 5 <210> 21 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 21 Arg Pro Gln Lys Arg Pro Ser Cys Ile 1 5 <210> 22 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 22 Ile Pro Gln Cys Arg Leu Thr Pro Leu 1 5 <210> 23 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 23 Tyr Asn Leu Arg Arg Gly Thr Ala Leu 1 5 <210> 24 <211> 11 <212> PRT <213> Human gammaherpesvirus 4 <400> 24 His Pro Val Gly Glu Ala Asp Tyr Phe Glu Tyr 1 5 10 <210> 25 <211> 9 <212> PRT <213> Human gammaherpesvirus 4 <400> 25 Leu Ser Arg Leu Pro Phe Gly Met Ala 1 5 <210> 26 <211> 10 <212> PRT <213> Human gammaherpesvirus 4 <400> 26 Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu 1 5 10

Claims

1. A method for producing a composition comprising cytotoxic T cells (CTLs) including (a) a T cell receptor (TCR) that binds to a first antigen, and (b) a chimeric antigen receptor (CAR) that binds to a second antigen, (i) A step of preparing a cell culture containing human peripheral blood mononuclear cells (PBMCs), (ii) The step of co-culturing the culture containing PBMCs on day 0 with human lymphoblastoid B cells (BLCLs) that have been manipulated to express the first antigen, (iii) Maintaining the culture containing PBMC and BLCL for at least 10 days to induce selective proliferation of CTLs containing TCRs that bind to the first antigen, (iv) Transducing the CTL of the culture with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, thereby providing a CTL containing (a) a TCR that binds to the first antigen and (b) a CAR that binds to the second antigen. (v) A first antigen-specific CTL expressing CAR is co-cultured with BLCL for at least two days to enable proliferation of such CTLs, and (vi) A step of recovering a composition comprising (a) a TCR that binds to the first antigen and (b) a CTL that contains a CAR that binds to the second antigen. A method that includes depleting CD56+ cells from the cell culture before step (iv).

2. A method for preparing an adoptive immunotherapy composition comprising cytotoxic T cells (CTLs) including (a) a T cell receptor (TCR) that binds to a first antigen, and (b) a chimeric antigen receptor (CAR) that binds to a second antigen, (i) A step of preparing a cell culture containing human peripheral blood mononuclear cells (PBMCs), (ii) The step of co-culturing the culture containing PBMCs with human lymphoblastoid B cells (BLCLs) that have been manipulated to express the first antigen, (iii) Maintaining the culture containing PBMC and BLCL for at least 10 days to induce selective proliferation of CTLs containing TCRs that bind to the first antigen, (iv) Transducing the CTL of the culture with a viral vector containing a nucleic acid sequence encoding a CAR that binds to the second antigen, thereby providing a CTL containing (a) a TCR that binds to the first antigen and (b) a CAR that binds to the second antigen. (v) A first antigen-specific CTL expressing CAR is co-cultured with BLCL for at least two days to enable proliferation of such CTLs, and (vi) A step of recovering a composition comprising (a) a TCR that binds to the first antigen and (b) a CTL that binds to the second antigen, thereby providing the adoptive immunotherapy composition. A method that includes depleting CD56+ cells from the cell culture before step (iv).

3. The method according to claim 1 or 2, further comprising the step of incubating any culture from step (i), (ii), (iii), (v) or any combination thereof with one or more cytokines.

4. The method according to claim 3, wherein one or more cytokines include IL-2.

5. The method according to any one of claims 1 to 4, wherein the simultaneous culture in step (ii) is carried out with a PBMC:BLCL ratio of 20 or less:

1.

6. The method according to any one of claims 1 to 5, wherein the transduction in step (iv) is performed 1 to 3 days after the co-culturing in step (ii).

7. The method according to any one of claims 1 to 6, further comprising step (x) of resterung, after step (v) and before step (vi).

8. The method according to any one of claims 1 to 7, wherein step (x) comprises re-culturing a first antigen-specific CTL expressing the CAR together with a BLCL manipulated to express the first antigen, 5 to 8 days after step (v) of transduction.

9. The method according to claim 8, wherein the re-culturing step (x) is carried out with a CTL:BLCL ratio of 0.2:

1.

10. Immediately before step (x) of re-culturing, CD56 ＋ The method according to any one of claims 7 to 9, wherein the cells are depleted.

11. Step (vi) is, (a) Re-culture the first antigen-specific CTL expressing the CAR together with BLCL manipulated to express the first antigen, 1 to 8 days after the transduction step (iv), and (b) At least 14 days after the transduction step (iv), the first antigen-specific CTL expressing the CAR is re-cultured together with BLCL manipulated to express the first antigen. The method according to claim 1 or 2, including the method described in claim 1 or 2.

12. The method according to claim 11, wherein the re-culturing step (a) is carried out with a CTL:BLCL ratio of 0.2:1, and the re-culturing step (b) is carried out with a CTL:BLCL ratio of 4:

1.

13. The method according to claim 11 or 12, wherein the first re-culture step is initiated at least 7 days after the start of the co-culture step.

14. The method according to any one of claims 1 to 13, wherein the PBMC and BLCL are derived from the same donor.

15. The method according to any one of claims 1 to 14, wherein the BLCL is gamma-irradiated before co-culturing with the PBMC.

16. The method according to any one of claims 1 to 15, wherein BLCL is infected with a virus comprising at least one immunogenic peptide antigen, the at least one immunogenic peptide antigen comprising the first antigen.

17. The method according to any one of claims 1 to 16, wherein the first antigen is Epstein-Barr virus (EBV) antigen, cytomegalovirus (CMV) antigen, human papillomavirus (HPV) antigen, BK virus (BKV) antigen, John Cunningham virus (JCV) antigen, Merkel cell virus (MCV) antigen, human T lymphotropic virus (HTLV) antigen, or human immunodeficiency virus (HIV) antigen.

18. The method according to claim 17, wherein the viral antigen comprises EBV LMP1 peptide or a fragment thereof, EBV LMP2A peptide or a fragment thereof, or EBV EBNA1 peptide or a fragment thereof.

19. The method according to any one of claims 1 to 18, optionally comprising the step of freezing and storing first antigen-specific cytotoxic T cells expressing the recovered CAR for future administration to patients in need.

20. The method according to any one of claims 1 to 19, wherein the viral vector comprising a nucleic acid sequence encoding a CAR is a lentivirus or a retroviral vector.

21. The method according to any one of claims 1 to 20, wherein the viral vector is incapable of replication.

22. The method according to any one of claims 1 to 21, wherein the CAR comprises one or more signaling domains.

23. The method according to claim 22, wherein one or more signaling domains include a CD28 signaling domain, a 4-1BB signaling domain, a CD3 signaling domain, or a variant or fragment thereof.

24. The method according to claim 23, wherein the CD3 signaling domain is CD3ζ.

25. The method according to claim 23, wherein the CAR comprises a variant CD3ζ domain lacking at least one functional ITAM region.

26. The method according to claim 22, wherein the CAR comprises the Mut06 domain.

27. The method according to any one of claims 1 to 26, wherein a viral vector comprising a nucleic acid sequence encoding a CAR further comprises a nucleic acid sequence encoding a selectable marker.

28. The method according to claim 27, wherein a selectable marker confers antibiotic resistance.

29. The method according to claim 28, wherein the antibiotic is blastosidine.

30. A method for improving the proliferative capacity of antigen-specific CTLs expressing CAR, comprising the step of carrying out the method according to any one of claims 1 to 29.

31. A method for improving the transduction efficiency of antigen-specific CTLs, comprising the step of carrying out the method according to any one of claims 1 to 30.

32. A method for improving the viability of antigen-specific CTLs expressing CAR, comprising the step of carrying out the method according to any one of claims 1 to 31.

33. Use of a first antigen-specific CTL expressing a CAR obtained by any one of claims 1 to 32 in the manufacture of a pharmaceutical product for treating a disorder associated with the expression of a peptide antigen in a patient in need thereof.

34. A composition comprising a first antigen-specific CTL expressing a CAR obtained by any one of claims 1 to 32, for use in the treatment of a patient in need of a peptide antigen expression-related disorder.

35. A T cell composition prepared by the method described in any one of claims 1 to 32.

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