Method for producing an engineered immune cell

Abstract

The present disclosure provides an improved method for producing engineered T cells (e.g., CAR-T cells) that express an exogenous gene. T cells that express the exogenous gene of the present technology, and compositions containing the same, are useful for treating various diseases, such as infections, autoimmune diseases, and tumors.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 344,255, filed May 20, 2022, which is hereby incorporated by reference in its entirety for any and all purposes.

[0002] The present technology generally relates to improved methods for producing engineered immune cells that express an exogenous gene, including T cells that express a chimeric antigen receptor (CAR - T cells).

Summary of the Invention

[0003] In one aspect, the present disclosure provides a method for producing a population of T cells that express an exogenous gene product, the method comprising: (i) contacting a population of T cells with a stimulant; (ii) contacting the population of T cells with a retroviral vector comprising a nucleic acid molecule encoding the exogenous gene product, thereby providing a population of T cells that express the exogenous gene product; and (iii) collecting the population of T cells that express the exogenous gene product for storage or administration, wherein the population of T cells that express the exogenous gene product from step (iii) is not increased or is increased by 200% or less, as evaluated by viable cell count, compared to the population of T cells at the start of step (i).

[0004] In some embodiments, the exogenous gene product is a chimeric antigen receptor (CAR).

[0005] In some embodiments, the stimulant comprises a CD3 - binding domain.

[0006] In some embodiments, the retroviral vector is a gammaretroviral vector. In some embodiments, the gammaretroviral vector is selected from a pMSGV vector, a pMSCV vector, a pSFG vector, or derivatives thereof.

[0007] In some embodiments, steps (i)-(iii) are performed in a single vessel.

[0008] In some embodiments, step (ii) is performed in the presence of a soluble additive of a cationic amphiphilic peptide.

[0009] In some embodiments, step (ii) is not initiated until after the completion of step (i).

[0010] In some embodiments, prior to step (i), the population of T cells is enriched for T cells expressing CD3, CD4, and / or CD8.

[0011] In some embodiments, step (i) is performed for between about 4 and about 96 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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[0013] To provide a substantial understanding of the present technology, it is understood that certain aspects, modes, embodiments, variations, and features of the present method are described below at various levels of detail.

[0014] The present disclosure is not limited to the perspective of the specific embodiments described in this application and is intended as a single illustration of the individual aspects of the present disclosure. Not all various embodiments of the present disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variations of the present disclosure can be made without departing from its spirit and scope. In addition to those listed herein, from the foregoing description, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims together with the full scope of equivalents to which such claims are entitled.

[0015] In carrying out this method, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology, and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Ausubel et al. eds. (2007) Current Protocols in Molecular Biology series, Methods in Enzymology (Academic Press, Inc., N.Y.) series, MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press), MacPherson et al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual, Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition, Gait ed. (1984) Oligonucleotide Synthesis, U.S. Patent No. 4,683,195, Hames and Higgins eds. (1984) Nucleic Acid Hybridization, Anderson (1999) Nucleic Acid Hybridization, Hames and Higgins eds. (1984) Transcription and Translation, Immobilized Cells and Enzymes (IRL Press (1986)), Perbal (1984) A Practical Guide to Molecular Cloning, Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory), Makrides ed.(2003) Gene Transfer and Expression in Mammalian Cells, Mayer and Walker eds., (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology should be referred to. Methods for detecting and measuring the level of polypeptide gene expression products (i.e., gene translation level) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999).).

[0016] The present technology provides an improved method for producing engineered immune cells that express exogenous genes for cell therapy, where the engineered immune cells include, but are not limited to, T cells that express a chimeric antigen receptor (CAR), T cells that express a T cell receptor (TCR), and T cells that express a synthetic T cell antigen receptor (STAR). In one embodiment, provided herein is a rapid CAR-T cell manufacturing process involving in vivo CAR-T expansion, i.e., a CAR-T cell manufacturing process that does not require ex vivo CAR-T expansion or requires limited ex vivo CAR-T expansion (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% or less of ex vivo expansion). In one embodiment, the improved CAR-T cell manufacturing process requires an activation step of about 1 to 2 days with stimulants such as anti-CD3 and anti-CD28 antibodies, and a transduction step of about 1 day (e.g., 20 to 28 hours) with a retroviral vector containing a nucleic acid molecule encoding the CAR. In one embodiment, the manufacturing process of the present technology produces CAR-T cells that exhibit improved in vitro and in vivo proliferation capabilities. The resulting CAR-T cells also exhibited improved efficacy (in vivo anti-tumor effect) and in vivo proliferation. Additionally, in one embodiment, the manufacturing process of the present technology enables an all-in-one process, i.e., in one embodiment, the manufacturing process can be performed in a single container.

[0017] Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0018] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0019] As used herein, the terms "about" or "approximately" mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within three standard deviations, or more than three standard deviations, according to the conventions in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within a magnitude of up to five-fold or two-fold of a value.

[0020] As used herein, the term "administration" of an agent to a subject includes any route by which the agent is introduced or delivered to the subject to effect the intended function. Administration can be effected by any suitable route, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes described herein. Administration includes self-administration and administration by another person.

[0021] As used herein, the term "activation" refers to the state of a T cell that has been sufficiently stimulated to induce cytokine production, a detectable effector function, and / or detectable cell proliferation.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, or can be an immunoreactive portion of an intact immunoglobulin. The antibodies of the present disclosure can exist in a variety of forms in which the antigen-binding portion of the antibody is expressed as part of a contiguous polypeptide chain, including, for example, single domain antibody fragments (sdAbs), single chain antibodies (scFvs), and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y., Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, Bird et al., 1988, Science 242:423-426).

[0023] As used herein, "antibody fragment" or "antigen-binding fragment" refers to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, sdAbs (V L or V H either), camelid V HHRefers to a multispecific antibody formed from a domain, scFv antibody, and antibody fragment. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a variable region of a light chain and at least one antibody fragment containing a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are continuously linked via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv may, for example, have variable regions V L and V H in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain V L -linker-V H or may contain V H -linker-V L The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences, e.g., two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues.

[0024] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule, which typically determines the class to which the antibody belongs.

[0025] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0026] As used herein, the term "synthetic antibody" refers to an antibody generated using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. This term also means an antibody generated by the synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein or an amino acid sequence specifying an antibody, and the DNA or amino acid sequence is to be construed as having been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0027] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can be accompanied by either antibody production, activation of certain immunologically competent cells, or both. One of ordinary skill in the art will understand that substantially any macromolecule, including virtually all proteins or peptides, can function as an antigen. Further, an antigen can be derived from recombinant DNA or genomic DNA. Thus, one of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by the full-length nucleotide sequence of a gene. The techniques herein include, but are not limited to, the use of partial nucleotide sequences of two or more genes, which are arranged in various combinations to encode polypeptides that elicit a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0028] As used herein, the term "self-antigen" means any self-antigen that is misrecognized as foreign by the immune system. Self-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins including cell surface receptors.

[0029] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens (auto-antigens). Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, celiac disease, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, ankylosing spondylitis, thyroiditis, autoimmune vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0030] As used herein, the term "self" means any material derived from the same individual that is later reintroduced into the individual. "Allogeneic" refers to a graft derived from a different animal of the same species. "Xenogeneic" refers to a graft derived from an animal of a different species.

[0031] As used herein, the term "tumor" or "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0032] As used herein, "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of the efficacy of a therapeutic agent for the treatment of a particular type of disease, a positive control (a composition known to exhibit a desirable therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are typically used.

[0033] "Co-stimulatory ligand", as the term is used herein, specifically binds to cognate co-stimulatory molecules on T cells, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of, for example, a peptide-loaded MHC molecule to the TCR / CD3 complex. Co-stimulatory ligands include molecules on antigen-presenting cells (e.g., dendritic cells, B cells, macrophages, monocytes, etc.). Examples of co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2, B7-H3, B7-H4, B7-H6, B7-H7 / HHLA2, BTLA, 4-1BBL, OX40L, PDCD6, VISTA (B7-H5, PD-1H), GITRL (TNFSF18), inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD27 ligand (TNFSF7), CD28, CD28H (IGPR-1), CD30L, CD40, CD70, CD83, CTLA-4, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, TIM-1 / KIM-1 / HAVCR, TIM-4, semaphorin 4A, galectin-9, BTN1A1 (butyrophilin), BTN2A1, BTN2A2 (butyrophilin 2A2), BTN3A1 / 2, BTN3A2, BTN3A3, BTNL2 / butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10 and other butyrophilins, CD277 / BTN3A1, LAIR1, LAIR2, CD96, CD155 / PVR, CRTAM, DNAM-1 (CD226), nectin-2 (CD112), nectin-3, PVRIG, TIGIT, LILRA3 (CD85e), LILRA4 (CD85g, ILT7), LILRB3 (CD85a, ILT5), LILRB2 (CD85d, ILT4), LILRB1 (CD85j, ILT2), LILRB4 (CD85k, ILT3), B cell-activating factor (BAFF) (BLyS, TNFSF13B), TL1A (TNFSF15), TNF-alpha, agonists or antibodies that bind to Toll-like receptors (TLRs), and ligands that specifically bind to B7-H3.Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on T cells.

[0034] As used herein, the terms "costimulatory molecule" or "costimulatory domain" refer to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that, upon binding to an antigen, provides a second signal required for efficient activation and function of T lymphocytes. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD40L, PD-1, PDL-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H3, CTLA-4, GITR (TNFRSF18), TIM-1, TIM-2, TIM-3, TIM-4, CD160, CD200, CD300a (LMIR1), CD300d (LMIR4), CLECL1 (DCAL-1), DAP12, dectin-1 (CLEC7A), DPPIV (CD26), EphB6, integrin alpha4beta1, integrin alpha4beta7 / LPAM-1, LAG-3, TSLP R, B cell activation factor receptor (BAFF R) (TNFRSF13C), DR3 (TNFRSF25), lymphotoxin-alpha (TNF-beta), RELT (TNFRSF19L), TACI (TNFRSF13B), TNFR2 (TNFRSF1B), 2B4 (CD244, SLAMF4), BLAME (SLAMF8), CD2, CD2F-10 (SLAMF9), CD48 (SLAMF2), CD58 (LFA-3), CD84 (SLAMF5), CD229 (SLAMF3), CRACC (SLAMF7), NTB-A (SLAMF6), SLAM (CD150), and ligands that specifically bind to CD83. Accordingly, the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-1BB, although other costimulatory domains are contemplated for use with the CARs described herein. By including one or more costimulatory signaling domains, the efficacy and proliferation of T cells expressing the CAR receptor can be enhanced. The intracellular signaling and costimulatory signaling domains can be tandemly linked in any order at the carboxyl terminus of the transmembrane domain.

[0035] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0036] "Disease" is the health state of an animal where the animal is unable to maintain homeostasis and, if the disease is not improved, the health of the animal continues to deteriorate. In contrast, a "disorder" in an animal is a health state where the animal is able to maintain homeostasis, but the health state of the animal is less favorable than it would be in the absence of the disorder. By leaving it untreated, a disorder does not necessarily cause a further decrease in the health state of the animal.

[0037] As used herein, the term "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0038] As used herein, "endogenous" refers to any material produced from or within a living organism, cell, tissue, or system.

[0039] As used herein, the term "exogenous" refers to any material introduced from or produced outside of a living organism, cell, tissue, or system.

[0040] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0041] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid can be derived from another organism or can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0042] "Identical" refers to sequence similarity or identity between two polypeptides or two nucleic acid molecules. When the positions in both of the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, when the positions in each of two DNA molecules are occupied by adenine, those molecules are identical at that position. The terms "substantially identical" or "substantially the same" mean a polypeptide or nucleic acid molecule that exhibits at least 50% or more homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison (e.g., wild-type or native sequence). In some embodiments, a substantially identical, or substantially the same, polypeptide contains one or more amino acid substitutions, insertions, or deletions compared to the sequence used for comparison. In some embodiments, a substantially identical, or substantially the same, polypeptide contains one or more unnatural amino acids or amino acid analogs, including D-amino acids and retroinverso amino acids, to replace the homologous sequence.

[0043] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acid.

[0044] As used herein, the term "immune cell" refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term "engineered immune cell" refers to an immune cell that has been genetically modified. As used herein, the term "innate immune cell" refers to an immune cell that occurs naturally in the immune system.

[0045] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting materials is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell. As used herein, a "purified" or "substantially purified" cell is a cell that essentially does not contain other cell types. A substantially purified cell also refers to a cell that is separated from other cell types that normally associate in its natural state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that are separated from the cells with which they naturally associate in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0046] As used herein, the term "modulating" means mediating a detectable increase or decrease in the level of response in a subject as compared to the level of response in the subject in the absence of treatment or compound and / or as compared to the level of response in an otherwise identical but untreated subject. This term encompasses disrupting and / or affecting natural signals or responses such that they mediate a beneficial therapeutic response in a subject, preferably a human.

[0047] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate versions of each other and include all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0048] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, are in the same reading frame to join two protein-coding regions.

[0049] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in cells from a disease area such as a solid tumor in a particular tissue or organ of a patient as compared to the level of expression in normal cells from that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0050] Examples of "parenteral" administration of an immunogenic composition include subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intracavitary, intrathecal, or intrasternal injection, administration, or infusion techniques.

[0051] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or its cells that can receive the methods described herein in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is human.

[0052] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. One of ordinary skill in the art has the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtainable by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques such as PCR, and synthetic means.

[0053] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to, for example, both short chains, which are generally referred to in the art as peptides, oligopeptides, and oligomers, and long chains, which are generally referred to in the art as proteins, and there are many types. Examples of "polypeptides" include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0054] As used herein, the term "promoter" is defined as a DNA sequence recognized by a cellular synthetic machinery or an introduced synthetic machinery that is required to initiate specific transcription of a polynucleotide sequence. A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes production of the gene product in a cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0055] As used herein, a "regulatory sequence" or "regulatory region" of a nucleic acid molecule means a cis-acting nucleotide sequence that positively or negatively affects the expression of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. Gene expression can be increased when an inducer is present or at increased concentration. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). Gene expression can be decreased when a repressor is present or at increased concentration. Regulatory regions are known to affect, modulate, or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation, and immune regulation. Regulatory regions typically bind to one or more trans-acting proteins and result in either an increase or a decrease in transcription of a gene.

[0056] Specific examples of gene regulatory regions are promoters and enhancers. A promoter is a sequence located around the transcription or translation start site, typically located 5' of the translation start site. A promoter is usually located within 1 Kb from the translation start site, but can be located further away, for example, including 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to 10 Kb. An enhancer is known to affect gene expression when located 5' or 3' of a gene, or in an exon or intron, or in a part thereof. An enhancer can also function at a significant distance from a gene, for example, at a distance of about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more. Also, regulatory regions include, in addition to the promoter region, but not limited to, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of a gene to allow in-frame translation of the mRNA, and multiple genes or polycistronic messages for providing appropriate polyadenylation of the transcript of the gene of interest and stop codons, stop codons for generating polyadenylation signals, leader sequences, and fusion partner sequences, internal ribosome entry site (IRES) elements, and can optionally be included in an expression vector.

[0057] As used herein, the term "sample" refers to a clinical sample obtained from a subject. In certain embodiments, a sample is obtained from a biological source (i.e., a "biological sample") such as tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchoalveolar lavage (BAL), bronchial wash (BW), whole blood, body fluid, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0058] As used herein, the term "secreted" in reference to a polypeptide means a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum and Golgi apparatus, and means a vesicle that transiently fuses with the cytoplasmic membrane and releases the protein outside the cell. Small molecules such as drugs can also be secreted by diffusion across the membrane to the outside of the cell.

[0059] As used herein with respect to antibodies, the term "specifically binds" means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity per se does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to antibodies of different allelic forms. However, such cross-reactivity per se does not change the classification of the antibody as specific. In some instances, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a particular structure on the chemical species (e.g., an antigenic determinant or epitope), e.g., an antibody generally recognizes and binds to a specific protein structure rather than a protein. When an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody reduces the amount of labeled A bound to the antibody. As used herein, the terms "specifically binds to", "specifically binds", or "is specific for" a particular molecule (e.g., an antigen) mean, for example, that the molecule has a K -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M of K d that can be exhibited by having.

[0060] As used herein, the term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation may mediate changes in the expression of certain molecules such as downregulation of TGFβ and / or reorganization of the cytoskeletal structure.

[0061] When used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0062] As used herein, the term "stimulatory ligand" or "stimulator" means a ligand that, when present on an antigen-presenting cell (e.g., dendritic cell, B cell, macrophage, monocyte, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulators are well known in the art and include, inter alia, peptides, CD3 binding domains (e.g., anti-CD3 antibodies), CD28 binding domains (e.g., superagonist anti-CD28 antibodies), CD2 binding domains (e.g., superagonist anti-CD2 antibodies), and MHC class I molecules loaded with concanavalin A (ConA).

[0063] The term "therapeutically effective amount" refers to the amount of a subject compound that will elicit a biological or medical response of a tissue, system, or subject that is sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the onset of, or to alleviate to some extent, one or more of the symptoms or manifestations of the disorder or disease being treated. A therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0064] As used herein, the terms "transfected", "transformed", or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected", "transformed", or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes primary subject cells and their progeny.

[0065] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes.

[0066] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration routes may be the same or different. More specifically, sequential use refers to the complete administration of one of the active ingredients before the other administration or before the other administration is initiated. Thus, it is possible to administer one of the active ingredients over minutes, hours, or days before administering the other active ingredient. In this case, there is no co-therapy.

[0067] As used herein, the term "T cell" includes naive T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, and antigen-specific T cells. As a more specific example, the T cells of the subject matter of the present disclosure include, but are not limited to, CD4 + T cells, CD8 + T cells, T helper cells, cytotoxic T cells, central memory T cells, stem cell-like memory T cells (stem cell memory T cells or stem-like memory T cells), effector memory T cells (e.g., T EMCells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells (NKT), mucosa-associated invariant T cells, αβ T cells, double-negative T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. In certain embodiments, the CAR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype. In some embodiments, the CAR-T cells are any immune cells derived from pluripotent stem cells (e.g., induced pluripotent stem (iPS) cells).

[0068] As used herein, "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject such as a human, and includes (i) inhibiting the disease or disorder, i.e., preventing its onset, (ii) reducing the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, reducing, or slowing the progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, but are not limited to, inhibiting recurrence of the disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission, or improving the prognosis.

[0069] As used herein, a "vector" is a replicable nucleic acid that, when the vector is transformed into a suitable host cell, is capable of expressing one or more heterologous proteins. References to a vector typically include such vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, typically by restriction digestion and ligation. References to a vector also include such vectors that contain a nucleic acid encoding a polypeptide. Vectors are used to introduce into host cells a nucleic acid encoding a polypeptide, for amplification of the nucleic acid or for expression / presentation of the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to affect the integration of a gene or a portion thereof into the genomic chromosome. Examples of vectors include viral vectors. A viral vector is an engineered virus that is operably linked to an exogenous gene for transferring the exogenous gene (as a vehicle or shuttle) into cells.

[0070] The viral vectors of the present technology can be retroviral vectors. One advantage provided by retroviral vectors is their ability to transduce their single-stranded RNA genomes into double-stranded DNA molecules and stably integrate the double-stranded DNA molecules into the target cell genome. Thus, retroviral vectors can be used to permanently modify the host cell nuclear genome.

[0071] The retroviral vectors of the present technology can be derived from any member of the retroviridae family, such as spumavirus or Fomie virus (e.g., human and simian viruses), betaretrovirus (e.g., MMTV), gammaretrovirus (e.g., MLV), alpharetrovirus (e.g., ALV), deltaretrovirus (e.g., BLV and HTLV-1), lentivirus (e.g., HIV1), and epsilonretrovirus (e.g., WDSV and WEHV1 / 2), or derivatives thereof.

[0072] To construct an expression vector containing a nucleic acid encoding any of the polypeptides provided herein, any method known to those skilled in the art for inserting a heterologous nucleic acid sequence into a vector (e.g., a retroviral vector) can be used.

[0073] CAR-T cell The CAR-T cells provided herein are T cells engineered to express at least one chimeric antigen receptor (CAR). A CAR is an engineered receptor that includes extracellular and intracellular domains. The extracellular domain includes an antigen-binding portion. In some embodiments, the extracellular domain also includes a hinge domain. The intracellular domain, or otherwise the cytoplasmic domain, includes a CD3ζ chain and / or a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to an antigen.

[0074] A linker or spacer domain can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligo or polypeptide that functions to link a transmembrane domain to either the extracellular domain or the cytoplasmic domain of a polypeptide chain. The spacer domain can be composed of up to 300 amino acids, preferably 10 - 100 amino acids, most preferably 25 - 50 amino acids.

[0075] Antigen-binding portion.

[0076] The selection of the antigen-binding portion depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen portion domain in the CARs of the subject matter of the present disclosure include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0077] In one embodiment, the CARs of the subject matter of the present disclosure can be engineered to target a desired tumor antigen by engineering an antigen-binding portion that specifically binds to an antigen on tumor cells. The tumor antigen can be a protein produced by tumor cells that elicits an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding portion of the subject matter of the present disclosure will likely depend on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, CA125, CA19-9, MUC-1, WT-1, glypican 3 (GPC3), and mesothelin.

[0078] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules such as the cancer gene HER-2 / Neu / ErbB-2. Yet another group of target antigens are cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates as target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0079] The types of tumor antigens referred to in the subject matter of this disclosure can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur in other cells in the body. TAA-related antigens are not unique to tumor cells; instead, they are expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of the antigen on the tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0080] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; fetal antigens overexpressed such as CEA; oncogenes overexpressed and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; specific tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, cMet, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA19-9, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, glypican 3 (GPC3), HTgp-175, M344, MA-50, mesothelin, MG7-Ag, MOV18, MUC-1, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, and WT-1. In one embodiment, the antigen-binding portion of the CAR targets antigens including, but not limited to, cMet, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc.

[0081] Depending on the desired antigen to be targeted, the CARs of the subject matter of the present disclosure can be engineered to include an appropriate antigen-binding portion that is specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding portion for incorporation into the CARs of the present invention.

[0082] Transmembrane domain.

[0083] With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains within the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such a domain to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0084] The transmembrane domain can be derived from either a natural source or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful in the present invention can be derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or immunoglobulins such as IgG4 (i.e., can include at least their transmembrane region(s)). Alternatively, the transmembrane domain can be synthetic, in which case it will primarily include hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length, can form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0085] Cytoplasmic domain.

[0086] The cytoplasmic domain of the CAR of the subject matter of the present disclosure or, in other cases, the intracellular signaling domain, is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is located. The term "effector function" refers to a special function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a special function. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of an intact chain as long as it transmits the effector function signal. Thus, the term intracellular signaling domain means any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0087] Examples of intracellular signaling domains for use in the CAR of the subject matter of the present disclosure include the cytoplasmic sequence of the T cell receptor (TCR), and co-receptors that act in concert to initiate signaling following antigen receptor engagement, and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0088] Signals generated through the TCR alone are known to be insufficient for complete activation of T cells, and secondary or co-stimulatory signals are also required. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: namely, sequences that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0089] The primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either in a stimulatory or inhibitory manner. A primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM.

[0090] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in the context of the present disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule within the CAR of the present disclosure includes a cytoplasmic signaling sequence derived from CD3ζ.

[0091] In some embodiments, the cytoplasmic domain of the CAR can be designed to include the CD3ζ signaling domain, either by itself or in combination with any other desirable cytoplasmic domain(s) useful in the context of the CARs of the present invention. For example, the cytoplasmic domain of the CAR can include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0092] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked to each other in a random or specified order. Optionally, short oligos or polypeptide linkers, preferably 2 to 10 amino acids in length, may form the linkages. Glycine-serine doublets provide particularly suitable linkers.

[0093] In one embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3ζ and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3ζ and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3ζ, as well as the signaling domains of CD28 and 4-1BB.

[0094] In the present disclosure, CAR-T cells can include any immune cells that express a CAR.

[0095] The CAR-T cells of the present technology can be administered to a subject (e.g., a human subject) in need of treatment for any disease, including but not limited to infection, autoimmune disease, or tumor.

[0096] Method for producing engineered immune cells of the present technology In one aspect, the present disclosure provides a method of producing a population of T cells that express an exogenous gene, the method comprising: (i) contacting a population of T cells with a stimulant; (ii) contacting the population of T cells with a retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product, thereby providing a population of T cells that express the exogenous gene product; and (iii) collecting the population of T cells that express the exogenous gene product for storage or administration, wherein the population of T cells that express the exogenous gene product from step (iii) is not increased or is increased by 200% or less, as evaluated by viable cell count, compared to the population of T cells at the start of step (i). In some embodiments, the exogenous gene product is a chimeric antigen receptor (CAR).

[0097] Step (i): Activation

[0098] Prior to step (i), the population of T cells can be obtained from any source known in the art, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, tumor, T cells derived from pluripotent stem cells, and T cells derived from direct differentiation. In certain embodiments of the present technology, any T cell line available in the art can be used. In certain embodiments of the present technology, T cells can be obtained from units of blood collected from a subject using various techniques known to those skilled in the art, such as apheresis. In some embodiments, the population of T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes prior to step (i).

[0099] Procedures for separation include, but are not limited to, density gradient centrifugation (e.g., using a PERCOLL® gradient); counterflow centrifugal elutriation; resetting; binding to particles that modify cell density; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents conjugated to or used in combination with mAbs, including but not limited to complement and cytotoxins; and panning, elution, or any other convenient technique using an antibody adhered to a solid matrix, such as a plate or chip.

[0100] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low-angle and obtuse-angle light scatter detection channels, impedance channels, and fluorescence-activated cell sorting (FACS).

[0101] In some embodiments, CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Certain subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. In some embodiments, prior to step (i), the population of T cells can be enriched for T cells expressing CD3, CD4, and / or CD8. These selection techniques are well known to those skilled in the art. As a non-limiting example, CD4 + cells can be enriched by negative selection by treating the cell mixture with a monoclonal antibody cocktail containing antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, regulatory T cells can be depleted by anti-CD25 conjugated beads.

[0102] In some embodiments, the T cells can be frozen after the washing step or without a washing step prior to step (i). The freezing and subsequent thawing steps can provide a more homogeneous product by removing granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. The freezing solution and parameters are known in the art. In certain embodiments, the cryopreserved cells can be thawed, washed, and allowed to rest at room temperature for about 1 hour prior to step (i).

[0103] The source of T cells can be collected at any point in time necessary for use in T cell therapy for any disease or condition that would benefit from subsequent activation, transduction, formulation, and T cell therapy. In one embodiment, a blood sample or apheresis can be obtained from a generally healthy subject. In certain embodiments, a blood sample or apheresis can be obtained from a generally healthy subject at risk of developing a disease but who has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the sample can be collected from a patient immediately following diagnosis of a particular disease described herein but prior to any treatment. In a further embodiment, the cells can be isolated from a blood sample or apheresis from a subject before, during, or after any related treatment modality, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunotherapy (e.g., checkpoint inhibitors), or immunosuppressive agents.

[0104] In some embodiments of the present technology, T cells can be obtained from a patient immediately following treatment. In this regard, it has been observed that following certain cancer treatments, particularly those with drugs that damage the immune system, the quality of the obtained T cells immediately following treatment, during a period in which the patient would normally be recovering from the treatment, can be optimal or improved for ex vivo manipulation (e.g., activation and engineering).

[0105] In any of the above embodiments, the T cells are activated or stimulated by a stimulant. The stimulant may include an agent that stimulates CD3 / TCR complex-related signals and / or a ligand that stimulates co-stimulatory molecules on the surface of T cells. In some embodiments, the stimulant includes a CD3 binding domain, a CD28 binding domain, a CD134 binding domain, and / or a CD137 binding domain. In some embodiments, the stimulant includes a CD3 binding domain and / or a CD28 binding domain. In some embodiments, the stimulant includes an anti-CD3 antibody and / or an anti-CD28 antibody. Examples of anti-CD28 antibodies include, but are not limited to, 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France). Examples of anti-CD3 antibodies include, but are not limited to, OKT3, 145-2C11, 17A2, UCHT1, and SK7.

[0106] In certain embodiments, the stimulant includes an anti-CD3 antibody and an anti-CD28 antibody. Each of the anti-CD3 antibody and the anti-CD28 antibody can independently be in solution or bound to a surface. When both are bound to a surface, the anti-CD3 antibody and the anti-CD28 can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration).

[0107] In one embodiment, the anti-CD3 antibody and the anti-CD28 antibody are immobilized on beads either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". In some embodiments, the molar ratio of the anti-CD3 antibody to the anti-CD28 antibody ranges from 100:1 to 1:100 and all integer values therebetween. In some embodiments, the bead-to-cell ratio can range from 1:500 to 500:1 and any integer value therebetween. The optimal ratio will vary depending on the particle size as well as the size and type of the cells. In some embodiments, the stimulant is MACS GMP T-Cell TransACT (Miltenyi Biotec) ("TransAct"). In some embodiments, the ratio of the T cell suspension to TransAct is about 17:1. One of ordinary skill in the art can readily understand that any cell concentration can be used. For example, in one embodiment, a concentration of about 10 million to 15 million cells / ml, about 15 million to 20 million cells / ml, about 20 million to 25 million cells / ml, about 25 million to 30 million cells / ml, about 30 million to 35 million cells / ml, about 35 million to 40 million cells / ml, about 40 million to 45 million cells / ml, about 45 million to 50 million cells / ml, about 50 million to 55 million cells / ml, about 55 million to 60 million cells / ml, about 60 million to 65 million cells / ml, about 65 million to 70 million cells / ml, about 70 million to 75 million cells / ml, about 75 million to 80 million cells / ml, about 80 million to 85 million cells / ml, about 85 million to 90 million cells / ml, about 90 million to 95 million cells / ml, about 95 million to 100 million cells / ml, about 100 million to 125 million cells / ml, about 125 million to 150 million cells / ml, about 150 million to 200 million cells / ml, about 200 million to 500 million cells / ml, about 500 million cells / ml to 1 billion cells / ml, or about 1 billion cells / ml to 2 billion cells / ml can be used.

[0108] In certain embodiments, it may be desirable to significantly increase the concentration of cells to ensure maximum contact between the cells and the particles. Additionally, the use of high cell concentrations enables more efficient capture of cells that may weakly express the target antigen of interest. For example, using high concentrations of cells enables more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0109] In other embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), the interaction between the particles and the cells is minimized. This results in the selection of cells that express a large amount of the desired antigen bound to the particles. For example, CD4 + T cells at a diluted concentration can be captured more efficiently than CD8 + T cells.

[0110] In one embodiment, the T cells may be contacted with a stimulant (e.g., anti-CD3 antibody and anti-CD28 antibody) for about 4 to about 96 hours, such as about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, or about 96 hours. In some embodiments, the T cells may be contacted with the stimulant for about 4 to 48 hours. In some embodiments, the T cells may be contacted with the stimulant for about 12 to 48 hours. In some embodiments, the T cells may be contacted with the stimulant for about 24 to 48 hours. The beads and cells can then be separated, and then the cells can be washed and collected for transduction.

[0111] Step (ii): Transduction

[0112] In any embodiment, step (ii) (contacting the population of T cells with a retroviral vector) is not initiated until after the completion of step (i) (contacting the population of T cells with a stimulant). For example, the T cells from step (i) can be washed and collected for step (ii).

[0113] For the initial genetic modification of T cells to produce T cells that express an exogenous gene product (e.g., CAR) in engineering (ii), generally, retroviral vectors are used for transduction. For example, a polynucleotide encoding a CAR can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, the retroviral long terminal repeat, or an alternative internal promoter. Retroviral gene transfer (transduction) has also proven to be equally effective for the subsequent genetic modification of cells to provide cells that contain an antigen-presenting complex comprising at least two costimulatory ligands. A combination of a retroviral vector and an appropriate packaging strain is also suitable, in which case the capsid protein will function to infect human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al., Mol. Cell. Biol. 5:431-437 (1985)), PA317 (Miller, et al., Mol. Cell. Biol. 6:2895-2902 (1986)), and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelope, and any others known in the art are also suitable.

[0114] Also, as methods for transduction, for example, direct co-culture of producer cells and T cells by the method of Bregni, et al., Blood 80:1418-1422 (1992), or, for example, culture with viral supernatant alone, or culture with a concentrated vector stock, with or without appropriate growth factors and polycations, by the methods of Xu, et al., Exp. Hemat. 22:223-230 (1994), and Hughes, et al., J. Clin. Invest. 89:1817 (1992) can be mentioned. In some embodiments, contacting a population of T cells with a retroviral vector containing a nucleic acid molecule encoding a CAR is carried out in the presence of a soluble additive of a cationic amphiphilic peptide, for example, Vectofusin-1. In some embodiments, contacting a population of T cells with a retroviral vector containing a nucleic acid molecule encoding a CAR is carried out in the presence of a fibronectin-like peptide (for example, RetroNectin).

[0115] In some embodiments, the retroviral vector expressing the CAR of the present disclosure can be an oncoretroviral vector, a gammaretroviral vector, or a spumaretroviral vector. In some embodiments, the retroviral vector can be a gammaretroviral vector. In some embodiments, the gammaretroviral vector is selected from the pMSGV vector, the pMSCV vector, the pSFG vector, or derivatives thereof.

[0116] Retroviral vectors are particularly well-developed and are used in the clinical setting (Rosenberg et al., N. Engl. J. Med 323:370 (1990), Anderson et al., U.S. Patent No. 5,399,346). Other viral vectors that can be used include, for example, adenovirus, and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpesviruses such as Epstein-Barr virus.

[0117] In one embodiment, contacting a population of T cells with a retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product (e.g., a CAR) can be performed for about 1 to about 72 hours, such as about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours. In some embodiments, the T cells may be contacted with the retroviral vector comprising a nucleic acid molecule encoding a CAR for about 1 to 28 hours. In some embodiments, the T cells may be contacted with the retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product (e.g., a CAR) for about 16 to 28 hours, such as 24 hours.

[0118] Suitable conditions for T cell culture in step (i) and / or step (ii) include, but are not limited to, a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo15, (Lonza)) containing factors necessary for viability and / or proliferation, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFNγ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ, and TNFα, or any other additive for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, Plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may be supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or a sufficient amount of cytokine(s) for T cell growth and / or proliferation. RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, IMDM, Advanced DMEM / F12, X-Vivo10 TM 、X-Vivo15 TM 、X-Vivo20 TM 、TheraPEAK TM X-Vivo10, TheraPEAK TM X-Vivo15 TM 、TheraPEAK TM X-Vivo20 TM 、CTS TM Optimizer TM T Cell Expansion SFM, CTS Optmizer Pro Serum Free Medium, 4Cell Nutri-T Medium, LymphoONE TM T-Cell Expansion Xeno-Free Medium, ImmunoCult TM-XF T Cell Expansion Medium, ExCellerate Human T Cell Expansion Medium, Stemline T Cell Expansion Medium, CAR T-Cell Medium, TexMACS TM Medium, Corning Lymphocyte Serum-free Medium, Corning 88-581-CM Medium, CellGenix T Cell Medium, SmarT TM T cell Expansion Medium, StemSpan TM Serum-Free Expansion Medium, and OptiPEAK T Lymphocyte XPR can be mentioned. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., room temperature or 37°C) and atmosphere (e.g., air + 5% CO 2 ).

[0119] Step (iii): Storage, formulation, and / or administration

[0120] According to protocols well-known in the art, in step (iii), the engineered T cells (e.g., CAR-T cells) expressing the exogenous gene product from step (ii) are optionally washed and collected for storage, formulation, and / or administration. In particular, the technology for producing engineered T cells (e.g., CAR-T cells) does not require ex vivo expansion or requires limited ex vivo expansion. In some embodiments, the population of engineered T cells (e.g., CAR-T cells) from step (iii) is not expanded or is expanded by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% as evaluated by viable cell count compared to the population of T cells at the start of step (i). Any method known in the art for evaluating viable cell count can be used, including but not limited to a hemocytometer, an automated cell counter, and any known cell viability and cytotoxicity assays. These methods can be based on PCR, a spectrophotometer, a pH meter, ATP measurement, gravimetry, or a metabolite analyzer.

[0121] In some embodiments, steps (i)-(iii) are all performed in a single container. Suitable containers for the method of producing the engineered T cells (e.g., CAR-T cells) of the technology include, but are not limited to, G-REX® bioreactors, CliniMACS Prodigy®, Xuri TM Cell Expansion systems, WAVE Bioreactors TM , Coccon® platforms, PBS bioreactors, Ambr bioreactors, Biostat bioreactors, Cell Factory systems, CellSTAKs and cell culture bags, dishes, well plates, and flasks.

[0122] The engineered T cells (e.g., CAR-T cells) can be formulated for administration or long-term storage. For long-term storage, the engineered T cells (e.g., CAR-T cells) from step (ii) can be cryopreserved. Methods for cryopreservation are well known to those skilled in the art. For example, the engineered T cells (e.g., CAR-T cells) can be suspended in a cell cryopreservation solution containing a cryoprotectant (e.g., DMSO) and a protein (e.g., human serum albumin), a polymer, and / or a sugar, can be frozen at -80°C for one day, and the cryopreserved cells can be further stored in liquid nitrogen (LN) (e.g., below -150°C). Many factors in cryopreservation affect the quality of the engineered T cells (e.g., CAR-T cells) and thus the outcome of cell therapy. These factors include: (1) the formulation and introduction of the cryopreservation medium, (2) the cooling rate, (3) the storage conditions, (4) the thawing conditions, and (5) the post-thaw treatment. Optimization of these factors to achieve a desirable outcome of cell therapy is within the level of those skilled in the art.

[0123] Formulation

[0124] The engineered T cells (e.g., CAR-T cells) of the present technology and compositions containing the same can be conveniently provided as a sterile liquid preparation, e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient, especially for administration by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, physiological saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0125] Sterile injectable solutions can be prepared, if necessary, by incorporating the compositions of the subject matter of the present disclosure into a required amount of a suitable solvent containing various amounts of other ingredients. Such compositions can be admixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, dextrose, etc. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc., depending on the desired route of administration and the preparation. To prepare suitable preparations without undue experimentation, reference may be made to standard textbooks such as "REMINGTON’S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0126] Various additives can be added to enhance the stability and sterility of the compositions, including antibacterial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Sustained absorption of injectable pharmaceutical forms can be brought about by the use of agents that retard absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used should be compatible with the engineered T cells (e.g., CAR-T cells) of the subject matter of the present disclosure.

[0127] The compositions can be isotonic, i.e., can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the subject matter of the present disclosure can be achieved using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly suitable for buffers containing sodium ions.

[0128] If desired, a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level. Methylcellulose can be used because it is readily and economically available and easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the selected agent. The important point is to use an amount that will achieve the selected viscosity. Clearly, the selection of a suitable carrier and other additives will depend on the proper route of administration and the nature of the particular dosage form, for example, whether the composition is formulated into a solution, suspension, gel, or other liquid form such as a time-release or liquid-filled form.

[0129] One of ordinary skill in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or efficacy of the engineered T cells (e.g., CAR-T cells) described in the subject matter of the present disclosure. This can be readily avoided from the present disclosure and the documents cited herein, without presenting a problem to one of ordinary skill in the chemical and pharmaceutical arts, by referring to standard textbooks, or by simple experiments (without undue experimentation).

[0130] One consideration regarding the therapeutic use of the engineered T cells (e.g., CAR-T cells) of the subject matter of the present disclosure is the amount of cells necessary to achieve an optimal effect. The amount of cells administered will vary for the subject being treated. In certain embodiments, about 10 2 ~ about 10 12 cells, about 10 3 ~ about 10 11 cells, about 10 4 ~ about 10 10 cells, about 10 5 ~ about 10 9 cells, or about 10 6 ~ about 10 8Engineered T cells (e.g., CAR-T cells) of the present disclosure are administered to a subject. More effective cells can be administered in even fewer numbers. In some embodiments, at least 1×10 8 cells, about 2×10 8 cells, about 3×10 8 cells, about 4×10 8 cells, about 5×10 8 cells, about 1×10 9 cells, about 5×10 9 cells, about 1×10 10 cells, about 5×10 10 cells, about 1×10 11 cells, about 5×10 11 cells, about 1×10 12 cells, or more engineered T cells (e.g., CAR-T cells) of the present disclosure are administered to a human subject. The precise determination of the amount that would be considered an effective dose can be based on individual factors for each subject, including the size, age, gender, weight, and condition of the particular subject. Dosages can be readily ascertained by one of ordinary skill in the art from the present disclosure and the knowledge in the art. Generally, engineered T cells (e.g., CAR-T cells) are administered at a dosage that is non-toxic or tolerable to the patient.

[0131] One skilled in the art can readily determine the amounts of cells, as well as optional additives, vehicles, and / or carriers, in the compositions administered in the methods of the subject disclosure. Typically, any additives (in addition to the active cell(s) and / or agent(s)) in phosphate buffered saline are present in amounts of about 0.001 wt% to about 50 wt% solution, and the active ingredient is present in microgram to milligram ranges such as about 0.0001 wt% to about 5 wt%, about 0.0001 wt% to about 1 wt%, about 0.0001 wt% to about 0.05 wt%, about 0.001 wt% to about 20 wt%, about 0.01 wt% to about 10 wt%, or about 0.05 wt% to about 5 wt%. For any composition administered to an animal or human and any particular method of administration, toxicity should be determined, for example, by determining the lethal dose (LD) and LD50 in a suitable animal model such as rodents like mice, as well as the dosage of the composition(s), the concentration of the components in the composition, and the timing of administration of the composition(s), whereby a suitable response is elicited. Such determinations do not require undue experimentation from the knowledge of one skilled in the art, the present disclosure, and the documents cited herein. And the duration of continuous administration can be confirmed without undue experimentation.

[0132] Administration

[0133] The engineered T cells (e.g., CAR-T cells) of the subject disclosure can be provided systemically or directly to a subject to treat an infection, autoimmune disease, or tumor. In certain embodiments, the engineered T cells (e.g., CAR-T cells) are injected directly into the organ of interest. Additionally or alternatively, the engineered T cells (e.g., CAR-T cells) are provided indirectly to the organ of interest, for example, by administration into the circulatory system or by administration to the tissue of interest. Mitogens and differentiating agents can be provided before, during, or after administration of the cells and compositions to increase the production of T cells in vitro or in vivo.

[0134] The engineered T cells (e.g., CAR-T cells) of the subject matter of the present disclosure can be administered systemically or locally, usually intravascularly, intraperitoneally, intrathecally, or intrapleurally, in any physiologically acceptable vehicle, but they can also be introduced into bone or other convenient sites where the cells can find an appropriate site (e.g., thymus) for regeneration and differentiation. In certain embodiments, at least 1×10 5 cells can be administered, ultimately reaching 1×10 10 or more. In certain embodiments, at least 1×10 6 cells can be administered. A cell population comprising engineered T cells (e.g., CAR-T cells) can comprise a purified cell population. One skilled in the art can readily determine the percentage of engineered T cells (e.g., CAR-T cells) in a cell population using various well-known methods such as fluorescence-activated cell sorting (FACS). The range of purity in a cell population comprising engineered T cells (e.g., CAR-T cells) can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95 to about 100%. The dosage can be readily adjusted by one skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered T cells (e.g., CAR-T cells) can be introduced by injection, catheter, etc. If desired, but not limited to, factors can also include interleukins such as IL-2, IL-3, IL6, IL-11, IL-7, IL-12, IL-15, IL-21, and other interleukins, colony-stimulating factors such as G-, M-, and GM-CSF, and interferons such as γ-interferon.

[0135] In certain embodiments, the compositions of the subject matter of the present disclosure include pharmaceutical compositions comprising engineered T cells (e.g., CAR-T cells) and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic (allogeneic). For example, engineered T cells (e.g., CAR-T cells) and compositions containing them can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood derived from the T cells of the subject matter of the present disclosure or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered via catheter administration, systemic injection, local injection, including local injection by intravenous injection, or parenteral administration. When administering the pharmaceutical compositions of the subject matter of the present disclosure, they can be formulated in unit dosage injectable forms (solutions, suspensions, emulsions).

[0136] Some embodiments of the present disclosure are further described below through examples. The examples are intended to be illustrative and do not mean to limit the scope of the present disclosure in any way. Therefore, the present disclosure is not limited to the specific embodiments described through the examples herein.

Examples

[0137] General experimental methods Medium CAR-T cell culture medium: 2.6% OpTmizer Expansion Basal Supplement (Thermo Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific), and 1% streptomycin, 2% CTS Immune Cell SR (Thermo Fisher Scientific) were added to OpTmizer CTS T-Cell Expansion basal medium (Thermo Fisher Scientific) to prepare a basal cell culture medium. 20 IU / mL or 40 IU / mL of MACS GMP IL-2 (Miltenyi Biotec) was added to this basal cell culture medium.

[0138] SK-HEP-1-Luc and HepG2 cell culture medium: MEM, L-Gln(+) (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.), 1% non-essential amino acids (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0139] GSU-Luc cell culture medium: RPMI1640 (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.) and 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0140] Production of CAR-T cells

[0141] After thawing Leukopak (Hemacare), CD4+ and CD8+ cells were concentrated by CliniMACS Prodigy (Miltenyi Biotech). The concentrated cells were diluted to 2.0×10 6 cells / mL or less (pre-production raw material). Cell suspension: MACS GMP T-Cell TransACT (Miltenyi Biotec) = 17.5:1 was seeded in a culture bag and cultured for about 48 hours (cell activation step). The activated cells were diluted in the culture medium using a LOVO Cell processing system (Fresenius Kabi) or a centrifuge, and in a culture bag pre-coated with RetroNectin® (Takara Bio Co., Ltd.) and a retrovirus introducing the CAR gene or the CAR gene, IL-7 gene, and CCL19 gene, at 6.07×10 5 cells / cm2 Seeded at less than [quantity] and cultured until the next day (gene transduction process). Culture bottles (G-Rex, Wilson Wolf) were seeded at less than 2.2×10 6 cells / cm 2 less than [quantity], and optionally cultured for 3 - 7 days to produce CAR-T cells. After culture, the final product, CAR-T cells, were cryopreserved in Cryostor CS-10 Freezing Media (BioLife Solutions). The CAR gene used had a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, the IL-7 gene used had a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3, and the CCL19 gene used had a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. See Table 1.

[0142] Production of CAR-T cells using CliniMACS Prodigy

[0143] After thawing Leukopak (Hemacare), CD4+ and CD8+ cells were enriched in CliniMACS Prodigy (Miltenyi Biotech). After enrichment, the cells were diluted in CAR-T cell culture medium using MACS GMP T-Cell TransACT (Miltenyi Biotech) and cultured for approximately 48 hours, followed by retroviral transduction with or without MCAS GMP Vectofusin-1 (Miltenyi Biotech), and the transduction was carried out in CliniMACS Prodigy for 1 day. On day 3, the transduced cells were recovered from CliniMACS Prodigy, and then culture bottles (G-Rex, Wilson Wolf) were seeded at less than 2.2×10 6 cells / cm 2 less than [quantity], and optionally cultured for 3 - 7 days to produce CAR-T cells (final product). After culture, the final product, CAR-T cells, were cryopreserved in CryoStor CS10 Freezing Media (BioLife Solutions).

[0144] Determination of Transduction Efficiency and Immunophenotype of T Cells Using Flow Cytometry

[0145] The CAR transduction efficiency into T cells was determined using a CAR-targeted antigen on a BD FACSCanto II flow cytometer (BD Biosciences). The immunophenotype of T cells was measured using anti-CD4 antibody (clone SK3, catalog number 344604, BioLegend), anti-CD8 antibody (clone SK1, catalog number 344710, BioLegend), anti-CCR7 antibody (clone G043H7, catalog number 353204, BioLegend), anti-CD45RA antibody (clone L48, catalog number 337167, BD Biosciences), anti-CD27 antibody (clone O323, catalog number 302836, BioLegend), and anti-CD95 antibody (clone DX2, catalog number 305612, BioLegend). CCR7 / CD45RA-negative cells were effector memory T cells, CCR7-positive CD45RA-negative cells were central memory T cells, CCR7-negative CD45RA-positive cells were effector T cells, CCR7 / CD45RA / CD27 / CD95-positive cells were defined as stem cell memory T cells, and CCR7 / CD45RA-positive cells other than these were defined as naive T cells.

[0146] Proliferation Assay

[0147] Frozen CAR-T cells were thawed and cultured in CAR-T cell culture medium for 3 days. After culture, cell counting was performed.

[0148] Proliferative Capacity Assay

[0149] Frozen CAR-T cells were thawed and cultured in CAR-T cell culture medium without IL-2 for 7 days. After culture, cell counting was performed.

[0150] Cytotoxicity Assay

[0151] CAR-T cells (effector cells) and luciferase-expressing SK-HEP-1 cells were seeded into a cell culture plate containing SK-HEP-1 culture medium at an effector:target ratio of 5:1 (0.1M: 20K per well). After 24 hours of incubation, the effector cells were collected and counted using an NC-200 instrument. In addition, the luciferase activity derived from the target cells was measured to determine the target cell killing rate. All the collected effector cells were transferred to a new cell culture plate. Target cells at 20K cells / well were reseeded into the new plate, and then the effector cells were seeded. Twenty-four hours after reseeding, the cell number and luciferase activity were measured again. These steps were repeated at 24 hours, 48 hours, 72 hours, and 96 hours to measure the effector cell number and cytotoxic activity.

[0152] In vivo experiment

[0153] GSU-Luc cells or HepG2 were subcutaneously inoculated into NSG mice (Charles River Japan). Seven days after inoculation, CAR-T cells or PBS were intravenously administered to the mice. To analyze tumor growth by measuring the luciferase activity of GSU-Luc cells, D-luciferin (Promega) was intraperitoneally injected, followed by measuring the luminescence by IVIS imaging (Summit Pharmaceutical International). A caliper was used to determine the tumor volume of HepG2.

[0154] Example 1: An activation process is essential for CAR-T cell production As described above, CAR-T cells expressing the IL-7 gene and the CCL19 gene were produced (using the CAR described as SEQ ID NO: 1). This was compared with the CAR-T manufacturing process in two experiments using Donor 3 and Donor 4 respectively, where the activation step (Process No. 1) was skipped, the transduction step was carried out during the activation step (Process No. 2), and a 1-day transduction process (Process No. 3) was carried out following 48 hours of activation (Figure 1A). CAR-T cells produced as Process No. 1 could not be produced at all. However, CAR-T cells produced by both Process No. 2 and Process No. 3 could be produced without any problems (Figure 1B and Figure 1C).

[0155] Example 2: In vitro characterization of CAR-T cells during the proliferation process As described above, CAR-T cells expressing the IL-7 gene and the CCL19 gene were produced (using the CAR described as SEQ ID NO: 1). The CAR-T cells were collected on days 6 to 8 (Figure 2A). The cell number increased daily during production (Figure 2B). The cell proliferation ability and T cell phenotype were analyzed using CAR-T cells from days 6 to 8 (Figure 2C to Figure 2G). As the number of proliferation days progresses, both the proliferation ability and the pre-exhausted T cells including stem cell memory T cells and central memory T cells decrease. These results suggested changes in the quality of the cells, including both the proliferation ability of CAR-T cells from days 6 to 8 and the percentage of each T cell phenotype.

[0156] Example 3: In Vivo Study Using CAR-T Cells from Days 6 to 8 Seven days after inoculating NSG mice with HepG2, the CAR-T cells produced in Example 2 were administered. CAR-T cells produced with a shorter proliferation period showed excellent antitumor efficacy in the HepG2-inoculated xenograft model (Figure 3).

[0157] Example 4: In vitro characterization of CAR-T cells on day 3 and day 7 As described above, CAR-T cells expressing the IL-7 gene and the CCL19 gene were produced (using the CAR described as SEQ ID NO: 2). In the preparation of cells on day 3, the proliferation step was skipped (Figure 4A). CAR protein expression measured by flow cytometry in cells on day 3 was lower than that in cells on day 7 (Figure 4B). On the other hand, the expression of CAR mRNA measured by qPCR as the copy number in cells on day 3 was the same as that in cells on day 7 (Figure 4C). These indicated that the transduction efficiency did not change between cells on day 3 and cells on day 7. A shift in the T cell phenotype during CAR-T production was observed. From day 2 to day 3, the stem cell memory T cells reached their peak (Figures 4D and 4E). Cells on day 3 showed higher proliferative capacity (Figures 4F - 4G) and cytotoxic ability (Figures 4H - 4I) compared to cells on day 7.

[0158] Example 5: In vivo study using CAR-T cells on day 3 and day 7 Seven days after inoculation of GSU-LUC into NSG mice, the CAR-T cells produced in Example 4 were administered. Cells on day 3 at 0.2M and 0.5M (produced without an ex vivo expansion step) showed in vivo efficacy. However, cells on day 7 at 0.2M and 1M did not show efficacy (Figures 5A and 5B).

[0159] Example 6: In vivo study using CAR-T cells on day 3 and day 7 produced in a single container by CliniMACS Prodigy CAR-T cells expressing the IL-7 gene and the CCL19 gene were produced (using the CAR described as SEQ ID NO: 2) with or without using CliniMACS prodigy. The efficacy of CAR-T cells produced in a single container using CliniMACS Prodigy showed a strong tendency compared to CAR-T cells produced without using CliniMACS Prodigy (Figures 6A and 6B).

[0160] Example 7: In vitro characterization of CAR-T cells (unarmed) on day 3 and day 7 As described above, unarmed CAR-T cells (that do not express exogenous cytokine or chemokine genes) were produced (using the CAR described as SEQ ID NO: 2). In the preparation of cells on day 3, the proliferation step was skipped (Figure 7A). CAR protein expression measured by flow cytometry in cells on day 3 was lower than that in cells on day 7 (Figure 7B). On the other hand, the expression of CAR mRNA on day 3 measured by qPCR as the copy number was the same as that in cells on day 7 (Figure 7C). These results indicated that the transduction efficiency did not change between cells on day 3 and cells on day 7. The T cell phenotypes of cells on day 3 and cells on day 7 were observed. Cells on day 3 showed a higher stem cell memory T cell (Tscm) population compared to cells on day 7 (Figures 7D to 7E). In addition, cells on day 3 showed a higher proliferative capacity compared to cells on day 7 (Figure 7F).

[0161] Example 8: In vivo study using CAR-T cells (unarmed) on day 3 and day 7 GSU cells were subcutaneously inoculated into NSG mice. Seven days after inoculation, either 1 × 10 5 cells of unarmed CAR-T cells produced according to Example 7 on day 3 and day 7, or PBS, was intravenously administered to the mice. Unarmed CAR-T cells on day 3 (produced without an ex vivo proliferation step) showed in vivo efficacy, while unarmed CAR-T cells on day 7 did not show in vivo efficacy (Figure 8).

[0162] Example 9: In vitro characterization of CAR-T cells on day 3 and day 7 produced using CliniMACS Prodigy CAR-T cells expressing the IL-7 gene and the CCL19 gene were described in paragraph

[0163] Produced using CliniMACS Prodigy according to the method described in " Production of CAR-T Cells Using CliniMACS Prodigy". A CAR containing the amino acid sequence of SEQ ID NO: 2 was used. Figure 9A shows a scheme of the CAR-T cell manufacturing process. CAR-T cells were manufactured using CliniMACS Prodigy (CAR-T cells on day 3). The CAR-T cells on day 3 were further cultured in a G-Rex culture bottle for 4 more days (CAR-T cells on day 7). Figures 9B to 9C show the results of the percentage of CAR-expressing cells (Figure 9B) and the copy number within the cells (Figure 9C). Figures 9D to 9E show the changes in the T cell phenotypes during production in CD4+ and CD8+ T cells, respectively. The cells on day 3 showed a higher central memory T cell and stem cell memory T cell population compared to the cells on day 7.

[0164] Example 10: CAR-T cells on day 3 and day 7 produced using CliniMACS Prodigy with or without Vectofusin-1 in the transduction process CAR-T cells expressing the IL-7 gene and the CCL19 gene were produced using CliniMACS prodigy, with or without Vectofusin-1 in the transduction step. A CAR containing the amino acid sequence of SEQ ID NO: 2 was used. Figure 10 shows the results of the percentage of CAR-expressing cells produced with or without Vectofusion-1.

[0165] One of ordinary skill in the art will recognize, or be able to confirm, many equivalents to the specific embodiments of the present disclosure described herein using only routine experiments. Such equivalents are intended to be encompassed by the following claims.

[0166] [Table 1]

Claims

1. A method for producing a population of T cells that express an exogenous gene product, comprising: (i) contacting a population of T cells with a stimulant; (ii) contacting the population of T cells with a retroviral vector comprising a nucleic acid molecule encoding the exogenous gene product, thereby providing a population of T cells that express the exogenous gene; (iii) collecting the population of T cells that express the exogenous gene product for storage or administration; and the population of T cells that express the exogenous gene product from step (iii) is not expanded or is expanded by 200% or less, as evaluated by viable cell count, compared to the population of T cells at the start of step (i). A method.

2. The method according to claim 1, wherein the exogenous gene product is a chimeric antigen receptor (CAR).

3. The method according to claim 1, wherein the stimulant comprises a CD3-binding domain.

4. The method according to claim 1, wherein the retroviral vector is a gammaretroviral vector.

5. The method according to claim 3, wherein the gammaretroviral vector is selected from a pMSG vector, a pMSCV vector, a pSFG vector, or a derivative of a pMSG vector, a pMSCV vector, or a pSFG vector.

6. The method according to claim 1, wherein steps (i)-(iii) are performed in a single container.

7. The method according to claim 1, wherein step (ii) is performed in the presence of a soluble additive of a cationic amphiphilic peptide.

8. The method according to claim 1, wherein step (ii) is not initiated until after completion of step (i).

9. The method according to claim 1, wherein prior to step (i), the population of T cells is enriched for T cells that express CD3, CD4, and / or CD8.

10. The method according to claim 1, wherein step (i) is performed for about 4 to about 96 hours.

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