Methods for Producing Engineered Immune Cells

Abstract

The present disclosure provides improved methods for producing engineered immune cells (e.g., CAR-T cells). The resulting engineered immune cells and compositions comprising them are useful for treating a variety of diseases, such as infections, autoimmune diseases, and tumors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 344,249, filed May 20, 2022, which is incorporated by reference in its entirety.

[0002] The present technology relates generally to improved methods for producing engineered immune cells, including T cells expressing chimeric antigen receptors (CAR-T cells). Summary of the Invention

[0003] In one aspect, the disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells; (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing a population of engineered immune cells; and (iii) harvesting the population of engineered immune cells, wherein step (i) and / or step (ii) are performed at least in part in the presence of dimethyl sulfoxide (DMSO).

[0004] In some embodiments, the population of immune cells comprises T cells and / or natural killer (NK) cells. In some embodiments, the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR.

[0005] In some embodiments, step (i) is performed in the presence of a stimulating agent. In some embodiments, the stimulating agent comprises a CD3 binding domain. In some embodiments, step (i) is performed in the presence of one or more cytokines.

[0006] In some embodiments, the nucleic acid molecule is a viral vector. In some embodiments, the viral vector is a retroviral vector.

[0007] In some embodiments, DMSO is present at a concentration of up to about 3% (v / v). In some embodiments, DMSO is present at a concentration of up to about 0.3% (v / v). In some embodiments, DMSO is present at a concentration ranging from about 0.001% (v / v) to about 0.03% (v / v). In some embodiments, DMSO is present at a concentration of about 0.01% (v / v).

[0008] In some embodiments, the method further comprises storing the population of engineered immune cells, hi some embodiments, the method further comprises administering at least some of the cells of the population of engineered immune cells to a subject in need thereof.

[0009] In another aspect, the disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells; (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing a population of engineered immune cells; (iii) culturing the population of engineered immune cells derived from step (ii); and (iv) harvesting the population of engineered immune cells for storage or administration, wherein step (i), step (ii), and / or step (iii) are performed at least in part in the presence of dimethyl sulfoxide (DMSO).

[0010] In another aspect, the disclosure provides a method of expanding a population of a subset of naive T cells or stem cell memory T cells, comprising contacting a population of immune cells with dimethylsulfoxide (DMSO).

[0011] In another aspect, the disclosure provides a method of increasing immune cell transduction efficiency comprising contacting a population of immune cells with dimethylsulfoxide (DMSO).

[0012] In yet another aspect, the disclosure provides a composition comprising a population of immune cells and dimethyl sulfoxide (DMSO), wherein the DMSO is present at a concentration ranging from about 0.01% (v / v) to less than 1% (v / v). [Brief description of the drawings]

[0013] [Figure 1] 1 shows the results of Example 1. In the activation step, 0.0037% to 0.3% DMSO was added to produce CAR-T cells expressing IL-7 and CCL19 genes. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Diagram 2] 1 shows the results of Example 2. In the activation step, 0.0037% to 0.3% DMSO was added to produce CAR-T cells. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Diagram 3] 1 shows the results of Example 3. In the gene transduction step, 0.0037% to 0.3% DMSO was added to produce CAR-T cells expressing IL-7 and CCL19 genes. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Figure 4]Figure 4 shows the results of Example 4. In the gene transduction process, 0.0037%-0.3% DMSO was added to produce CAR-T cells. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Diagram 5] 1 shows the results of Example 5. In the expansion step, 0.0037% to 0.3% DMSO was added to produce CAR-T cells expressing IL-7 and CCL19 genes. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Figure 6] 1 shows the results of Example 6. In the expansion step, 0.0037% to 0.3% DMSO was added to produce CAR-T cells. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Figure 7] 1 shows the results of Example 7. In the expansion step, 0.3% to 2.7% DMSO was added to produce CAR-T cells. (A) CAR positive rate, (B) CD4 positive naive T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive naive T cells, and (E) CD8 positive stem cell memory T cells were quantified by flow cytometry after DMSO supplementation (N=3, *p<0.05, **p<0.02). p values ​​were calculated by t-test. [Figure 8]A shows that DMSO suppresses CAR gene transduction in SK-Hep-1. B shows that DMSO improves CAR transduction efficiency. [Figure 9] A shows the scheme of the experiment using NK cells. DMSO was treated 2 days before the mCherry transduction step. In the pre-culture step before transduction, 0.01% to 0.1% DMSO was added to evaluate the effect of DMSO on the transduction of the mCherry gene into NK92 cells. B shows that DMSO treatment improved the transduction efficiency in NK92 cells. mCherry-positive cells were quantified by flow cytometry (B, N=3, *p<0.05, **p<0.02). p-values ​​were calculated by t-test. Detailed Description of the Invention

[0014] It will be understood that certain aspects, modes, embodiments, variations, and features of the present methods are described below at various levels of detail in order to provide an understanding of the present technology.

[0015] The present disclosure is not limited in terms of the specific embodiments described in this application, but is intended as a single illustration of each aspect 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 recited herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will become apparent to those skilled in the art from the foregoing description. 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, along with the full scope of equivalents to which such claims are entitled.

[0016] In carrying out the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology, and recombinant DNA are used. For example, see 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 series (Academic Press, Inc., NY), 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. Pat. No. 4,683,195, Hames and Higgins eds. (1984) Nucleic Acid Synthesis, 1999. 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.See, (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. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) 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)).

[0017] The present technology provides an improved method of producing engineered immune cells, e.g., CAR-T cells, for cell therapy. In particular, the improved method involves performing one or more of (1) pre-transduction activation or pre-culture (without stimulants), (2) transduction, and (3) optional ex vivo expansion of immune cells (e.g., T cells or NK cells) in the presence of dimethyl sulfoxide (DMSO). Prior to the present invention, DMSO was known to inhibit cell proliferation (Ogaki, et al., Sci Rep. 5:172297 (2015)) and / or gene transduction. Unexpectedly, the inventors of the present technology have found that the presence of DMSO in (1) pre-transduction activation or pre-culture, (2) transduction, and / or (3) optional ex vivo expansion steps significantly increases transduction efficiency. Additionally, in one embodiment, DMSO is used to stimulate the proliferation and differentiation of high-potency T cells, e.g., naive T cells, and / or stem cell memory T cells (T scm ) population.

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled 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 unless otherwise specified. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the disclosure.

[0019] 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.

[0020] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., on the limits of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice 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, particularly with respect to biological systems or processes, the term can mean within 5-fold or within 2-fold magnitude of a value.

[0021] As used herein, the term "administration" of an agent to a subject includes any route of introducing or delivering an agent to a subject to perform its intended function. Administration may be performed 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.

[0022] As used herein, the term "activated" refers to a state of T cells that has been sufficiently stimulated to induce cytokine production, detectable effector function, and / or detectable cell proliferation.

[0023] 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 from a recombinant source, or can be an immunoreactive portion of an intact immunoglobulin. The antibodies of the 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, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0024] 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 Camelidae V HHThe term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light and heavy chain variable regions are linked contiguously 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, an scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light and heavy chain variable regions are linked contiguously 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. L and V H The scFv may have a variable region, L -Linker-V H or 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., links 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.

[0025] 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 usually determines the class to which the antibody belongs.

[0026] 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.

[0027] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term also refers to an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, and it should be understood that the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0028] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunologically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present technology includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, which nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled 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 synthesized or derived from a biological sample. Such biological samples may include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0029] The term "autoantigen" refers, according to the present disclosure, to any self-antigen that is incorrectly recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0030] 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 a self-antigen (auto-antigen). Examples of autoimmune diseases include, but are not limited to, Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, celiac disease, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, ankylosing spondylitis, thyroiditis, autoimmune vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

[0031] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is subsequently 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.

[0032] As used herein, the term "tumor" or "cancer" is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0033] As used herein, a "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 treating a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are typically used.

[0034] "Costimulatory ligand," as that term is used herein, includes a molecule on an antigen-presenting cell (e.g., dendritic cell, B cell, macrophage, monocyte, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, 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, for example, by binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory 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 costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), C D27 ligand (TNFSF7), CD28, CD28H (IGPR-1), CD30L, CD40, CD70, CD83, CTLA-4, HLA-G, MICA, MICB, HVEM, TIM-1 / KIM-1 / HAVCR, TIM-4, semaphorin 4A, galectin-9, BTN1A1 (butyrophilin), BTN2A1, BTN2A2 (butyrophilin 2A2), BTN3A1 / 2, BTN3A2, BTN3A3, BTNL2 / butyrophilin Butyrophilins such as Lin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, and BTNL10, 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), and LILR Examples of such antibodies include agonists or antibodies that bind to B2 (CD85d, ILT4), LILRB1 (CD85j, ILT2), LILRB4 (CD85k, ILT3), B cell activating factor (BAFF) (BLyS, TNFSF13B), TL1A (TNFSF15), TNF-alpha, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, 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.

[0035] As used herein, the term "costimulatory molecule" or "costimulatory domain" refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an 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 activating 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. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains can enhance the potency and proliferation of T cells expressing a CAR receptor. The intracellular signaling and costimulatory signaling domains can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0036] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0037] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than if the disorder were not present. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

[0038] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0039] As used herein, "endogenous" refers to any material that is produced from or within an organism, cell, tissue, or system.

[0040] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue, or system.

[0041] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0042] 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. The nucleic acid may be derived from another organism or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0043] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The term "substantially homologous" or "substantially identical" refers to 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 (e.g., wild-type or naturally occurring sequence) used for comparison. In some embodiments, substantially homologous or substantially identical polypeptides contain one or more amino acid substitutions, insertions, or deletions compared to the sequence used for comparison. In some embodiments, substantially homologous or substantially identical polypeptides contain one or more unnatural amino acids or amino acid analogs, including D-amino acids and retroinverso amino acids, to replace the homologous sequence.

[0044] As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acid.

[0045] As used herein, the term "immune cell" refers to any cell that plays a role in a subject's immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; 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 "native immune cell" refers to an immune cell that occurs naturally in the immune system.

[0046] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of the natural state is "isolated." An isolated nucleic acid or protein may be present in a substantially purified form or may be present in a non-native environment, such as, for example, a host cell. As used herein, a "purified" or "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in a naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in a natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0047] As used herein, the term "modulating" means mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses disrupting and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0048] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs can contain introns.

[0049] The term "operably linked" refers to the functional link 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 when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, in the same reading frame to join two protein coding regions.

[0050] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to an abnormal level of expression of a tumor antigen in cells from a disease area, such as a solid tumor, in a particular tissue or organ of a patient, compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0051] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intracisternal, intrathecal, or intrasternal injection, administration, or infusion techniques.

[0052] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof that are amenable to the methods described herein, either in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0053] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, any nucleic acid sequence obtained by any means available in the art and synthetic means, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques such as PCR.

[0054] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein sequence or a peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0055] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell, or introduced synthetic machinery, 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 the production of a 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 the production of a gene product in a cell substantially only if 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 encoding or specifying a gene product, causes the production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0056] As used herein, a "regulatory sequence" or "regulatory region" of a nucleic acid molecule refers to a cis-acting nucleotide sequence that positively or negatively influences the expression of an operably linked gene. A regulatory region includes a sequence of nucleotides that confers inducible (i.e., requires a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentrations, gene expression can be increased. A regulatory region also includes a sequence that confers repression of gene expression (i.e., a substance or stimulus reduces transcription). When a repressor is present or at increased concentrations, gene expression can be reduced. Regulatory regions are known to affect, regulate, or control many in vivo biological activities, including cell proliferation, cell growth and death, cell differentiation, and immune regulation. Regulatory regions typically bind one or more trans-acting proteins, resulting in either increased or decreased transcription of a gene.

[0057] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically located 5' of the translation start site. Promoters are usually located within 1 Kb of the translation start site, but can be located farther away, including, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to 10 Kb. Enhancers are known to affect gene expression when located 5' or 3' of the gene, or when located in or part of an exon or intron. Enhancers can also function at significant distances from the gene, for example, about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more. Regulatory regions, in addition to promoter regions, also include, but are not limited to, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to allow in-frame translation of the mRNA, as well as multigene or polycistronic messages to provide proper polyadenylation of the transcript and stop codons of the gene of interest, leader sequences, and fusion partner sequences, internal ribosome binding site (IRES) elements, and may optionally be included in the expression vector.

[0058] As used herein, the term "sample" refers to a clinical sample obtained from a subject. In certain embodiments, the 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 fluid (BAL), bronchial lavage fluid (BW), whole blood, body fluid, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0059] As used herein, the term "secreted" in reference to a polypeptide refers to a polypeptide that is released from the cell via the secretory pathway through the endoplasmic reticulum, the Golgi apparatus, and vesicles that transiently fuse with the cytoplasmic membrane and release the protein outside the cell. Small molecules, such as drugs, can also be secreted by diffusion through the membrane to the outside of the cell.

[0060] As used herein with respect to an antibody, the term "specifically binds" refers to 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 that antigen from one or more species. However, such cross-species reactivity in itself 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 an antibody of a different allelic form. However, such cross-reactivity in itself does not change the classification of the antibody as specific. In some cases, the term "specific binding" or "specifically binding" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species, e.g., an antibody recognizes and binds to a particular protein structure rather than a protein in general. If 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 an antibody will reduce the amount of labeled A bound to the antibody. As used herein, the terms "specifically binding," "specifically binding," or "specific for" a particular molecule (e.g., an antigen) refer to, for example, a binding affinity of about 10 -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 K of M d This can be represented by having:

[0061] As used herein, the term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation may mediate changes in expression of certain molecules, such as downregulation of TGFβ and / or rearrangement of cytoskeletal structure.

[0062] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0063] As used herein, a "stimulatory ligand" or "stimulatory agent" refers to 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. Stimulatory agents are well known in the art and include, among others, MHC class I molecules loaded with 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 concanavalin A (ConA).

[0064] The term "therapeutically effective amount" refers to an amount of a compound of interest that will elicit the biological or medical response of a tissue, system, or subject that is desired 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 or alleviate to some extent one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0065] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0066] 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.

[0067] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration route is the same or different.More specifically, sequential use refers to the full administration of one of the active ingredients before the administration of the other or before the administration of the other is started.Thus, one of the active ingredients can be administered for several minutes, hours, or days before the administration of the other active ingredient.In this case, there is no simultaneous treatment.

[0068] As used herein, the term "T cells" 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 memory T cells, effector memory T cells (e.g., T EMThese include T cells (T cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells (NKT), mucosal-associated invariant T cells, αβ T cells, double-negative T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. In certain embodiments, CAR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype. In some embodiments, CAR-T cells are any immune cells derived from pluripotent stem cells (e.g., induced pluripotent stem (iPS) cells).

[0069] As used herein, "treating" or "treatment" encompasses the treatment of a disease or disorder as described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., preventing its onset, (ii) relieving the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, 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 either direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or mitigating the disease state, and remission, or improving prognosis.

[0070] As used herein, a "vector" is a replicable nucleic acid that can express one or more heterologous proteins when the vector is transformed into a suitable host cell. Reference to a vector typically includes such vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced by restriction digestion and ligation. Reference to a vector also includes such vectors that contain a nucleic acid encoding a polypeptide. A vector is used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. A vector typically remains episomal, but can be designed to affect integration of a gene or a portion thereof into a chromosome of the genome. Vectors can include viral vectors. Viral vectors are engineered viruses that are operably linked to an exogenous gene to transfer the exogenous gene into a cell (as a vehicle or shuttle).

[0071] The viral vector of this technology can be a retroviral vector.One advantage that retroviral vector offers is the ability to convert their single-stranded RNA genome into double-stranded DNA molecule, and stably integrate double-stranded DNA molecule into target cell genome.Therefore, retroviral vector can be used to permanently modify host cell nuclear genome.

[0072] The retroviral vectors of the present technology may be derived from any member of the Retroviridae family, such as Spumaviruses or Fomieviruses (e.g., human and simian viruses), Betaretroviruses (e.g., MMTV), Gammaretroviruses (e.g., MLV), Alpharetroviruses (e.g., ALV), Deltaretroviruses (e.g., BLV and HTLV-1), Lentiviruses (e.g., HIV1), and Epsilonretroviruses (e.g., WDSV and WEHV1 / 2), or derivatives thereof.

[0073] To construct an expression vector containing a nucleic acid encoding any of the polypeptides provided herein, any method known to those of skill in the art for inserting heterologous nucleic acid sequences into a vector (e.g., a retroviral vector) can be used.

[0074] Chimeric antigen receptors (CARs) CARs are engineered receptors that include extracellular and intracellular domains. The extracellular domain includes an antigen-binding portion. In some embodiments, the extracellular domain also includes a hinge domain. In some embodiments, the intracellular domain or otherwise the cytoplasmic domain includes a CD3 zeta chain and / or a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient response of lymphocytes to antigens.

[0075] A linker or spacer domain may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "spacer domain" generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to either the extracellular or cytoplasmic domain of a polypeptide chain. A spacer domain may be composed of up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0076] Antigen-binding portion.

[0077] 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 the target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen moiety domain in the CAR of the presently disclosed subject matter include those associated with viral, bacterial, and parasitic infections (e.g., pathogen antigens), autoimmune diseases (e.g., autoantigens), and cancer cells (e.g., tumor-specific or tumor-associated antigens).

[0078] In one embodiment, the CAR of the presently disclosed subject matter can be engineered to target a tumor antigen of interest by engineering a desired antigen-binding moiety that specifically binds to an antigen on a tumor cell. A tumor antigen can be a protein produced by a tumor cell that elicits an immune response, e.g., a T-cell mediated immune response. The selection of the antigen-binding moiety of the presently disclosed subject matter will depend on the particular type of cancer to be 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 These include hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, 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.

[0079] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve 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 prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates as target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0080] The type of tumor antigen referred to in the subject matter of the present disclosure can also be tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is usually present at very low levels on normal cells, but is expressed at much higher levels on tumor cells.

[0081] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique 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, CA 15-3\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 an antigen 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, and the like.

[0082] Depending on the desired antigen to be targeted, the CAR of the present disclosure can be engineered to include an appropriate antigen-binding portion that is specific to 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 CAR of the present technology.

[0083] Transmembrane domain.

[0084] Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein in order to minimize interaction with other members of the receptor complex.

[0085] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present technology may be derived from (i.e., may include at least the transmembrane region(s) thereof) the α, β, or ζ chain of the T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0086] Cytoplasmic domain.

[0087] The cytoplasmic domain or otherwise intracellular signaling domain of the CAR of the subject disclosure is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized 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 specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases 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 the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0088] Examples of intracellular signaling domains for use in the CARs of the presently disclosed subject matter include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capability.

[0089] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: 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 costimulatory signals (secondary cytoplasmic signaling sequences).

[0090] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0091] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the presently disclosed subject matter 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 in the CAR of the presently disclosed subject matter comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0092] In some embodiments, the cytoplasmic domain of the CAR can be designed to include a CD3ζ signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present technology. For example, the cytoplasmic domain of the CAR can include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. 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 a ligand that specifically binds to CD83.

[0093] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the presently disclosed subject matter can be linked together in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0094] 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.

[0095] Methods for Producing Engineered Immune Cells of the Present Technology In one aspect, the disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells; (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide encoding a heterologous polypeptide, thereby providing a population of engineered immune cells (the transduction step); and (iii) harvesting the population of engineered immune cells, wherein step (i) and / or step (ii) are performed at least in part in the presence of dimethyl sulfoxide (DMSO).

[0096] In some embodiments, the heterologous polypeptide comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR (e.g., CAR-T cells).

[0097] The engineered immune cells of the subject matter of the present disclosure can be cells of lymphoid or myeloid lineages. The myeloid lineage can include monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. The lymphoid lineage, including B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of lymphoid lineage include T cells, natural killer (NK) cells, fetal stem cells, and pluripotent stem cells (e.g., those that can be differentiated from lymphocytes). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The subject T cells of this disclosure can be any type of T cell, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. In certain embodiments, CAR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype. In some embodiments, the engineered immune cell is any immune cell derived from a pluripotent stem cell (e.g., an induced pluripotent stem (iPS) cell). Regardless of the type of cell, in this disclosure, CAR-T can encompass any immune cell that expresses a CAR.

[0098] In some embodiments, the population of immune cells comprises T cells and / or natural killer (NK) cells. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during natural immune responses. NK cells do not require prior activation to carry out cytotoxic effects on target cells.

[0099] The population of immune cells of the present technology 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 infection site, ascites, pleural effusion, spleen tissue, and tumor. In certain embodiments of the present technology, any immune cell available in the art can be used. In certain embodiments of the present technology, the population of immune cells can be obtained from a unit of blood collected from a subject using various techniques known to those skilled in the art, for example, apheresis. In some embodiments, the population of immune cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes.

[0100] 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 with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents conjugated to or used in conjunction with the mAb, including, but not limited to, complement and cytotoxins; and panning, elution, or any other convenient technique using antibodies attached to a solid matrix, e.g., a plate, chip.

[0101] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., multiple color channels, low-angle and obtuse-angle light scatter detection channels, impedance channels, and fluorescence-activated cell sorting (FACS).

[0102] In some embodiments, CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Specific subpopulations of immune 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 immune cells can be enriched for T cells expressing CD4 and / or CD8. These selection techniques are well known to those skilled in the art. Non-limiting examples include CD4 + The cells may be enriched by negative selection by treating the mixture of cells with a monoclonal antibody cocktail containing antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, regulatory T cells may be depleted by anti-CD25 conjugated beads.

[0103] In some embodiments, the immune cells may be frozen after a washing step prior to activation (step (i)). The freezing and subsequent thawing step may provide a more homogenous product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Freezing solutions and parameters are known in the art. In certain embodiments, the cryopreserved cells may be thawed, washed, and allowed to rest at room temperature for about 1 hour prior to step (i).

[0104] The population of immune cells may be collected at any time necessary for subsequent activation, transduction, expansion, formulation, and used in cell therapy for any disease or condition that would benefit from immune cell therapy. In one embodiment, a blood sample or apheresis may be taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis may be taken from a generally healthy subject who is at risk of developing a disease, but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the sample may be collected from a patient immediately after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, the cells may be isolated from a blood sample or apheresis from a subject before, during, or after any relevant treatment modality, including, but not limited to, treatment with agents such as antiviral agents, chemotherapy, radiation, immunotherapy (e.g., checkpoint inhibitors), or immunosuppressants.

[0105] In some embodiments of the present technology, a population of immune cells can be obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of the obtained immune cells (e.g., T cells) immediately after treatment during the period when the patient would normally be recovering from the treatment can be optimal or improved for ex vivo manipulation (e.g., activation, transduction, or expansion).

[0106] Immune cell pre-culture or immune cell activation

[0107] In some other embodiments, immune cells (e.g., T cells) are activated by contacting the immune cells with a stimulatory agent. In some embodiments, the stimulatory agent may include an agent that stimulates a CD3 / TCR complex-associated signal and / or a ligand that stimulates a costimulatory molecule on the surface of the immune cell. In some embodiments, the stimulatory agent includes any of the costimulatory ligands disclosed herein, including but not limited to a CD3 binding domain, a CD28 binding domain, a CD134 binding domain, and / or a CD137 binding domain. In some embodiments, the stimulatory agent includes a CD3 binding domain and / or a CD28 binding domain. In some embodiments, the stimulatory agent 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.

[0108] In certain embodiments, the stimulatory agent comprises an anti-CD3 antibody and an anti-CD28 antibody. Each of the anti-CD3 antibody and the anti-CD28 antibody can be independently 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).

[0109] In one embodiment, anti-CD3 and anti-CD28 antibodies are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." In one embodiment, the molar ratio of anti-CD3 to anti-CD28 antibodies ranges from 100:1 to 1:100 and all integer values ​​therebetween. The ratio of beads to cells 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 cells. One skilled in the art can readily appreciate that any cell concentration can be used. For example, in one embodiment, the cell concentration may be about 10 to 15 million cells / ml, about 15 to 20 million cells / ml, about 20 to 25 million cells / ml, about 25 to 30 million cells / ml, about 30 to 35 million cells / ml, about 35 to 40 million cells / ml, about 40 to 45 million cells / ml, about 45 to 50 million cells / ml, about 50 to 55 million cells / ml, about 55 to 60 million cells / ml, about 60 to 65 million cells / ml, about 65 to 70 million cells / ml, about 70 million cells / ml, about 80 million cells / ml, about 85 million cells / ml, about 86 million cells / ml, about 87 million cells / ml, about 88 million cells / ml, about 89 million cells / ml, about 90 million cells / ml, about 91 million cells / ml, about 92 million cells / ml, about 93 million cells / ml, about 94 million cells / ml, about 95 million cells / ml, about 96 million cells / ml, about 97 million cells / ml, about 98 million cells / ml, about 99 million cells / ml, about 100 million cells / ml, about 105 million cells / ml, about 106 million cells / ml, about 107 million cells / ml, about 108 million cells / ml, about 109 million cells / ml, about 110 million cells / ml, about 111 million cells / ml, about 112 million cells / ml, about 113 million cells / ml, about 114 million cells / ml, about 115 million cells / ml, about 116 million cells / ml, about 117 million cells / ml, about 118 million cells / ml, about 119 million cells / ml, about 120 million cells / ml, about 121 million cells / ml, about 122 million cells / ml, Concentrations of about 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 may be used.

[0110] In certain embodiments, it may be desirable to significantly increase the concentration of cells to ensure maximum contact between cells and particles.In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest.For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have weak CD28 expression.

[0111] In other embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of immune cells (e.g., T cells) and surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express a large amount of the desired antigen that is bound to the particles. For example, CD4 expressing higher levels of CD28 may be used. + T cells were identified as CD8 + They can be captured more efficiently than T cells.

[0112] In one embodiment, immune cells (e.g., T cells) may be contacted with the stimulatory agents (e.g., anti-CD3 and anti-CD28 antibodies) for a period of about 3 hours to about 14 days, or any integer value in between. In some embodiments, the contacting is for about 4 to about 96 hours, e.g., 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 The incubation time can be 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, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, or about 96 hours. In some embodiments, the immune cells (e.g., T cells) may be contacted with the stimulatory agent for about 4-60 hours. In some embodiments, the immune cells (e.g., T cells) may be contacted with the stimulatory agent for about 4-48 hours. In some embodiments, the immune cells (e.g., T cells) may be contacted with the stimulatory agent for about 12-48 hours. In some embodiments, the immune cells (e.g., T cells) may be contacted with the stimulatory agent for about 24-48 hours. The beads and cells may then be separated and the cells may then be washed and collected for transduction.

[0113] In some embodiments, immune cells (e.g., T cells, NK cells) are cultured with DMSO without stimulants prior to the transduction step (pre-incubation before transduction).

[0114] Transduction process

[0115] In some embodiments, the transduction step, i.e., contacting a population of immune cells (e.g., T cells) with a nucleic acid molecule (e.g., a viral vector) that includes a nucleotide encoding a heterologous amino acid sequence, is not initiated until after completion of the activation step, i.e., contacting a population of immune cells (e.g., T cells) with a stimulatory agent. For example, immune cells (e.g., T cells) from the activation step can be washed and collected for transduction.

[0116] The nucleic acid molecule that comprises the nucleotide that codes for the heterologous amino acid sequence can be based on any RNA or DNA vector known in the art.The method of introducing nucleic acid molecule into host cell is known to those skilled in the art.For example, nucleic acid molecule can be transferred into host cell by physical, chemical or biological means.

[0117] Physical methods for introducing nucleic acid molecules into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.

[0118] Biological methods for introducing nucleic acid molecules of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0119] Chemical means for introducing nucleic acid molecules into host cells include macromolecule complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0120] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. For the introduction of nucleic acid molecules into host cells (in vitro, ex vivo, or in vivo), the use of lipid formulations is contemplated. In another embodiment, the nucleic acid molecule can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of the liposome, can be interspersed within the lipid bilayer of the liposome, can be attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, can be entrapped in the liposome, can be complexed with the liposome, can be dispersed in a lipid-containing solution, can be mixed with lipid, can be combined with lipid, can be contained as a suspension in lipid, or can be otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist as micelles or in a bilayer structure with a "collapsed" structure. They may also simply be scattered in the solution and form aggregates that are not uniform in size or shape.Lipids are fatty substances and can be naturally occurring lipids or synthetic lipids.For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0121] Lipids suitable for use may be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") may be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") may be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") may be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol may be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vehicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having a vesicular structure with a phospholipid bilayer membrane and an aqueous medium inside. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before a closed structure is formed, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also included. For example, lipids may assume a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Lipofectamine nucleic acid complexes are also contemplated.

[0122] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0123] Retroviral vectors are particularly well developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No. 5,399,346). In some embodiments, for the initial genetic modification of immune cells (e.g., T cells) to produce engineered immune cells (e.g., CAR-T cells), a retroviral vector containing a nucleotide molecule encoding a heterologous amino acid sequence is 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. For the subsequent genetic modification of cells to provide cells containing an antigen-presenting complex that includes at least two costimulatory ligands, retroviral gene transfer (transduction) has proven to be effective as well. The combination of a retroviral vector with a suitable packaging line is also suitable, in which case the capsid protein will function to infect human cells. A variety of 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 envelopes, and any others known in the art, are also suitable.

[0124] Possible methods of transduction also include direct co-culture of producer cells with immune cells (e.g., T cells), for example by the method of Bregni, et al., Blood 80:1418-1422 (1992), or culture with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, for example by the method of Xu, et al., Exp. Hemat. 22:223-230 (1994), and Hughes, et al., J. Clin. Invest. 89:1817 (1992). In some embodiments, contacting the population of immune cells (e.g., T cells) with the retroviral vector is performed in the presence of a soluble additive of a cationic amphipathic peptide, e.g., vectofusin-1.

[0125] In some embodiments, the retroviral vector expressing CAR can be an oncoretroviral vector, a gammaretroviral vector, a lentiviral 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 a pMSGV vector, a pMSCV vector, a pSFG vector, or any two or more combinations thereof.

[0126] In one embodiment, contacting a population of immune cells (e.g., T cells) with a nucleic acid molecule (e.g., a retroviral vector) comprising a nucleotide molecule encoding a heterologous amino acid sequence (e.g., a CAR or a fluorescent protein) can be performed for about 1 to about 72 hours, e.g., 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 The incubation period may be 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 immune cells may be contacted with a nucleic acid molecule (e.g., a retroviral vector) comprising a heterologous amino acid sequence (e.g., a CAR) for about 16 to 28 hours, e.g., 24 hours.

[0127] In some embodiments, a nucleic acid molecule (e.g., a retroviral vector) that includes a nucleotide molecule encoding a heterologous amino acid sequence (e.g., a CAR) can include a nucleotide molecule encoding IL-7 and / or CCL19. In some embodiments, the nucleotide molecule encoding the heterologous amino acid sequence (e.g., a CAR) and the nucleotide molecule encoding IL-7 and / or CCL19 can be on the same nucleic acid molecule. In some embodiments, the nucleotide molecule encoding the heterologous amino acid sequence (e.g., a CAR) and the nucleotide molecule encoding IL-7 and / or CCL19 can be on separate nucleic acid molecules.

[0128] Suitable conditions for immune cell culture (e.g., during activation and / or transduction steps) include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo15, (Lonza)) that may contain factors necessary for viability and / or proliferation, including, but not limited to, 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 cytokine or additive for the growth of cells known to one of skill in the art. In some embodiments, immune cells (e.g., during activation and / or transduction steps) were cultured in the presence of IL-2.

[0129] Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, IMDM, Advanced DMEM / F12, X-Vivo10, supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of hormones, and / or a sufficient amount of cytokine(s) for T cell growth and / or proliferation. 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, ImmunoCultTM -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 Examples of suitable media include serum-free expansion medium, and OptiPEAK T Lymphocyte XPR. The target cells are maintained in the 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 ) is maintained below

[0130] In any of the above embodiments, DMSO may be present in an amount up to about 3% (v / v), e.g., about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), about 1.0% (v / v), about 1.1% (v / v), about 1.2% (v / v), about 1.3% (v / v), about 1.4% (v / v), or about 1.5% (v / v). In some embodiments, the soluble soluble soluble material is present at a concentration of about 1.5% (v / v), about 1.6% (v / v), about 1.7% (v / v), about 1.8% (v / v), about 1.9% (v / v), about 2.0% (v / v), about 2.1% (v / v), about 2.2% (v / v), about 2.3% (v / v), about 2.4% (v / v), about 2.5% (v / v), about 2.6% (v / v), about 2.7% (v / v), about 2.8% (v / v), about 2.9% (v / v), or about 3.0% (v / v).

[0131] In any of the above embodiments, DMSO may be present in an amount up to about 0.3% (v / v), e.g., about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01% (v / v), about 0.02% (v / v), about 0.03% (v / v), about 0.04% (v / v), about 0.05% (v / v), about 0.06% (v / v), about 0.07% (v / v), about 0.08% (v / v), about 0.09% (v / v), about 0.10% (v / v), about 0.11% (v / v), or the like.

[0033] The compound is present at a concentration of about 0.12% (v / v), about 0.13% (v / v), about 0.14% (v / v), about 0.15% (v / v), about 0.16% (v / v), about 0.17% (v / v), about 0.18% (v / v), about 0.19% (v / v), about 0.20% (v / v), about 0.21% (v / v), about 0.22% (v / v), about 0.23% (v / v), about 0.24% (v / v), about 0.25% (v / v), about 0.26% (v / v), about 0.27% (v / v), about 0.28% (v / v), about 0.29% (v / v), or about 0.30% (v / v). In some embodiments, DMSO is present at a concentration of about 0.01% (v / v), about 0.02% (v / v), or about 0.03% (v / v).

[0132] In some embodiments, the presence of DMSO in the activation and / or transduction steps significantly increases the transduction efficiency, e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, 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%, about 95% to about 100%, or more, as compared to a control without DMSO. Transduction efficiency can be measured by methods known in the art, including but not limited to, methods using FACS, PCR, or image analysis.

[0133] In some embodiments, the presence of DMSO in the activation and / or transduction steps significantly increases the population of high potent T cells by, for example, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, 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%, about 95% to about 100% or more, as compared to a control without DMSO. In some embodiments, highly pharmacologically active T cells include, but are not limited to, naive T cells and / or stem cell-like memory T cells (T scm ). The immunophenotype of T cells may be measured by methods known in the art, including, but not limited to, methods using FACS, PCR, or image analysis. In some embodiments, the T cell phenotype may be measured using an anti-CD4 antibody (e.g., clone SK3, catalog number 344604, BioLegend), an anti-CD8 antibody (e.g., clone SK1, catalog number 344710, BioLegend), an anti-CCR7 antibody (e.g., clone G043H7, catalog number 353204, BioLegend), an anti-CD45RA antibody (e.g., clone L48, catalog number 337167, BD Biosciences), an anti-CD27 antibody (e.g., clone O323, catalog number 302836, BioLegend), and an anti-CD95 antibody (e.g., clone DX2, catalog number 305612, BioLegend). CCR7 / CD45RA negative cells were defined as effector memory T cells, CCR7 positive CD45RA negative cells were defined as central memory T cells, CCR7 negative CD45RA positive cells were defined as effector T cells, CCR7 / CD45RA / CD27 / CD95 positive cells were defined as stem cell memory T cells, and the other CCR7 / CD45RA positive cells were defined as naive T cells.

[0134] Storage / Formulation / Administration

[0135] The engineered immune cells (e.g., CAR-T cells) from the transduction process can be harvested for storage, formulation, and / or administration according to protocols well known in the art. Thus, in some embodiments, the methods of the present technology can further include storing the population of engineered immune cells and / or administering at least a portion of the cells of the population of engineered immune cells to a subject in need thereof.

[0136] In some embodiments, the engineered immune cells (e.g., CAR-T cells) may be formulated for long-term storage. In some embodiments, the engineered immune cells (e.g., CAR-T cells) may be cryopreserved. Methods for cryopreservation are well known to those of skill in the art. For example, the engineered immune cells (e.g., CAR-T cells) may be suspended in a cell cryopreservation solution containing a cryoprotectant (e.g., dimethyl sulfoxide) and human serum albumin, and subjected to freezing at -80°C for one day, and the cryopreserved cells may be further stored in liquid nitrogen (LN) (e.g., below -150°C). Many factors in cryopreservation may affect the quality of the engineered immune cells (e.g., CAR-T cells) and therefore the outcome of the cell therapy. These factors include (1) formulation and introduction of the freezing medium, (2) cooling rate, (3) storage conditions, (4) thawing conditions, and (5) post-thaw processing. Optimization of these factors to achieve the desired outcome of the cell therapy is within the level of one of skill in the art.

[0137] Formulation

[0138] The engineered immune cells (e.g., CAR-T cells) of the present technology and compositions comprising the same can be conveniently provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, particularly 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 include a carrier, which can be a solvent or dispersion medium, for example, containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0139] Sterile injectable solutions can be prepared by incorporating the subject compositions of the present disclosure in the required amount of a suitable solvent with various amounts of other ingredients, if necessary. Such compositions can be in admixture with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, and the like. 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 buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, dyes, and the like, depending on the desired route of administration and preparation. Standard textbooks, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted in order to prepare suitable preparations without undue experimentation.

[0140] Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with the engineered immune cells (e.g., CAR-T cells) of the subject matter of the present disclosure.

[0141] The composition may be isotonic, i.e., have the same osmotic pressure as blood and tears. The desired isotonicity of the composition of the presently disclosed subject matter may be achieved using sodium chloride or other pharma- ceutically 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.

[0142] If desired, pharma- ceutically acceptable thickening agents 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, etc. The concentration of the thickening agent can depend on the selected drug. The important point is to use an amount that will achieve the selected viscosity. Obviously, the selection of suitable carriers and other additives will depend on the appropriate route of administration and the nature of a particular dosage form, for example, a liquid dosage form (e.g., whether the composition is formulated into a solution, suspension, gel, or another liquid form such as a time-release form or liquid-filled form).

[0143] One of 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 immune cells (e.g., CAR-T cells) described in the subject matter of the present disclosure. This does not present a problem to one of skill in the art of chemical and pharmaceutical principles from this disclosure and the documents cited herein, or can be easily circumvented by reference to standard textbooks or by simple experimentation (without undue experimentation).

[0144] One consideration regarding the therapeutic use of the engineered immune cells (e.g., CAR-T cells) of the subject of the present disclosure is the amount of cells required to achieve optimal effect. The amount of cells administered will vary for the subject being treated. In certain embodiments, about 10 2 ~about 10 12 pieces, about 10 3 ~about 10 11 pieces, about 10 4 ~about 10 10 pieces, about 10 5 ~about 10 9 Pieces, or about 10 6 ~about 10 8 The engineered immune cells (e.g., CAR-T cells) of the presently disclosed subject matter are administered to a subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least 1×10 8 pieces, approximately 2×10 8 pieces, about 3 x 10 8 pieces, about 4×10 8 pieces, about 5×10 8 pieces, about 1×10 9 pieces, about 5×10 9 pieces, about 1×10 10 pieces, about 5×10 10 pieces, about 1×10 11 pieces, about 5×10 11 pieces, about 1×10 12One or more of the engineered immune cells (e.g., CAR-T cells) of the subject of the present disclosure are administered to a human subject. The exact amount that will be considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage amount can be easily ascertained by those skilled in the art from this disclosure and knowledge in the art. In general, the engineered immune cells (e.g., CAR-T cells) are administered at a dose that is non-toxic or tolerated by the patient.

[0145] One of skill in the art can readily determine the amount of cells, and optional additives, vehicles, and / or carriers, in the compositions administered in the methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or agent(s)) in phosphate buffered saline are present in an amount of about 0.001% to about 50% by weight solution, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001% to about 5% by weight, about 0.0001% to about 1% by weight, about 0.0001% to about 0.05% by weight, about 0.001% to about 20% by weight, about 0.01% to about 10% by weight, or about 0.05% to about 5% by weight. For any composition administered to animals or humans, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice, as well as the dosage of the composition(s), the concentration of the components in the composition, and the time of administration of the composition(s) to induce a suitable response. Such determinations do not require undue experimentation from the knowledge of those skilled in the art, this disclosure, and documents cited herein. And the time of sequential administration can be ascertained without undue experimentation.

[0146] Administration

[0147] The engineered immune cells (e.g., CAR-T cells) of the subject of the present disclosure can be provided systemically or directly to a subject to treat various diseases, including, but not limited to, infection, autoimmune disease, or tumors. In certain embodiments, the engineered immune cells (e.g., CAR-T cells) are directly injected into an organ of interest. Additionally or alternatively, the engineered immune cells (e.g., CAR-T cells) are provided indirectly to an organ of interest, for example, by administration into the circulatory system or by administration into a tissue of interest. Growth and differentiation agents can be provided before, during, or after administration of the cells and compositions to increase the production of engineered immune cells (e.g., CAR-T cells) in vitro or in vivo.

[0148] The engineered immune cells (e.g., CAR-T cells) of the subject disclosure can be administered systemically or locally in any physiologically acceptable vehicle, usually intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus). In certain embodiments, at least 1×10 5 cells may be administered, ultimately resulting in a total of 1 x 10 10 In certain embodiments, the number of 6A single cell may be administered. The cell population containing engineered immune cells (e.g., CAR-T cells) may include a population of purified cells. One skilled in the art can easily determine the percentage of engineered immune 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 containing engineered immune cells (e.g., CAR-T cells) may 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 easily adjusted by one skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered immune cells (e.g., CAR-T cells) may be introduced by injection, catheter, etc. If desired, factors may also be included, including, but not limited to, interleukins, e.g., 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, interferons, e.g., gamma-interferon.

[0149] In certain embodiments, the compositions of the presently disclosed subject matter include pharmaceutical compositions comprising engineered immune cells (e.g., CAR-T cells) and a pharma- ceutically acceptable carrier. Administration can be autologous or non-autologous. For example, engineered immune cells (e.g., CAR-T cells) and compositions comprising same can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood (e.g., from in vivo, ex vivo, or in vitro) derived from immune cells of the presently disclosed subject matter or their progeny can be administered via catheter administration, systemic injection, local injection, local injection, including intravenous injection, or parenteral administration. When the pharmaceutical compositions of the presently disclosed subject matter are administered, they can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0150] In another aspect, the disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) contacting a population of immune cells with a stimulant (an activation step); (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing a population of engineered immune cells (a transduction step); (iii) culturing the population of engineered immune cells derived from step (ii) (an ex vivo expansion step); and (iv) harvesting the population of engineered immune cells for storage or administration, wherein steps (i), (ii), and / or (iii) are performed at least in part in the presence of dimethyl sulfoxide (DMSO).

[0151] In some embodiments, in the ex vivo expansion step, the engineered immune cells may be cultured for about 3 hours to about 21 days, or any integer value of time units therebetween. Also, several cycles of stimulation may be desirable, such that the culture time of the engineered immune cells may be 60 days or more. In some embodiments, the population of engineered immune cells derived from step (ii) may be cultured for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. Suitable conditions for ex vivo expansion T cell culture are essentially the same as those discussed above for the activation and / or transduction steps.

[0152] In some embodiments, the presence of DMSO in the activation, transduction, and / or ex vivo expansion steps significantly increases the transduction efficiency, e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, 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%, about 95% to about 100%, or more, as compared to a control without DMSO.

[0153] In some embodiments, the presence of DMSO in the activation, transduction, and / or ex vivo expansion steps significantly increases the population of highly pharmacologically active T cells by, for example, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, 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%, about 95% to about 100% or more compared to a control without DMSO. In some embodiments, highly pharmacologically active T cells include, but are not limited to, naive T cells and / or stem cell-like memory T cells (T scm ) are mentioned. EXAMPLES

[0154] General Experimental Method Culture medium

[0155] CAR-T cell culture medium: Basal cell culture medium was prepared by adding 2.6% OpTmizer Expansion Basal Supplement (Thermo Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific), 1% streptomycin, and 2% CTS Immune Cell SR (Thermo Fisher Scientific) to OpTmizer CTS T-Cell Expansion basal medium (Thermo Fisher Scientific), which was supplemented with 20 IU / mL or 40 IU / mL MACS GMP IL-2 (Miltenyi Biotec).

[0156] SK-HEP-1 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.).

[0157] NK92 cell culture medium: RPMI, L-Gln(+) (Thermo Fisher Scientific) was prepared by adding 20% ​​FBS (Biosera Co., Ltd.).

[0158] Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Ltd.) was added to each medium to obtain the indicated levels and used.

[0159] CAR-T cell production

[0160] After thawing Leukopak (Hemacare), 2.0 × 10 cells were cultured in CAR-T cell culture medium. 6 The cells were diluted to 6.07 × 10 cells / mL or less (pre-produced 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 culture medium using a LOVO Cell processing system (Fresenius Kabi) or a centrifuge, and cultured at 6.07 × 10 cells / mL or less (pre-produced raw material). 5 cells / cm 2The cells were inoculated at less than 2.2 × 10 cells per well and cultured until the next day (gene transfer process). 6 cells / cm 2 The cells were seeded at less than 100 μg / ml and cultured for 3 to 7 days to produce CAR-T cells (final product). The CAR gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1, the IL-7 gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2, and the CCL19 gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 3 (the same applies to the SK-HEP-1 cells below). Please refer to Table 1 for the aforementioned sequences.

[0161] Transduction of CAR into SK-HEP-1 cells

[0162] SK-HEP-1 cells (ATCC) were cultured at 2.0 × 10 6 The cells were diluted to 6.07 × 10 cells / mL or less and plated on a culture plate coated with RetroNectin (Takara Bio Co., Ltd.) and a retrovirus carrying the CAR gene. 5 cells / cm 2 The CAR gene was introduced into SK-HEP-1 cells by seeding at less than 100 μg / mL and culturing for 4 days.

[0163] Introducing mCherry into NK92 cells

[0164] NK92 cells (ATCC) were cultured at 2.0 × 10 6 Dilute to 6.07 × 10 cells / mL or less with or without DMSO. 5 cells / cm 2 The NK92 cells were then cultured with RetroNectin (Takara Bio Co., Ltd.) and a retrovirus carrying the mCherry gene, and cultured for 2 days to introduce the mCherry gene into the NK92 cells.

[0165] Determination of T cell transduction rate and immunophenotype using flow cytometry

[0166] The rate of CAR transduction into T cells was determined using CAR-targeted antigens on a BD FACSCanto II flow cytometer (BD Biosciences). The immunophenotype of T cells was measured using anti-CD4 (clone SK3, catalog no. 344604, BioLegend), anti-CD8 (clone SK1, catalog no. 344710, BioLegend), anti-CCR7 (clone G043H7, catalog no. 353204, BioLegend), anti-CD45RA (clone L48, catalog no. 337167, BD Biosciences), anti-CD27 (clone O323, catalog no. 302836, BioLegend), and anti-CD95 (clone DX2, catalog no. 305612, BioLegend) antibodies. CCR7 / CD45RA / CD27 / CD95 positive cells in the CD4+ or CD8+ T cell population were used as stem cell memory T cells, and the remaining CCR7 / CD45RA positive cells were used as naive T cells.

[0167] Example 1: CAR-T production with the addition of DMSO in the activation step (Armored) CAR-T cells expressing IL-7 and CCL19 genes were produced as described above. 0.0037% to 0.3% DMSO was added during the production in the transduction step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 1A). The CAR positive rate was significantly increased in the 0.0037%, 0.011%, 0.033%, and 0.1% DMSO groups compared to the group without DMSO.

[0168] In addition, naive T cells and stem cell memory T cells positive for CD4 or CD8 were also measured (Figure 1B-Figure 1E). CD4 positive naive T cells and CD4 positive stem cell memory T cells were significantly increased in the 0.011%, 0.033%, and 0.1% DMSO supplemented groups compared to the group without DMSO supplementation (Figure 1B-Figure 1C). CD8 positive naive T cells and CD8 positive stem cell memory T cells were significantly increased in the 0.0037%, 0.011%, 0.033%, and 0.1% DMSO supplemented groups compared to the group without DMSO supplementation (Figure 1D-Figure 1E).

[0169] Example 2: CAR-T production with DMSO added to the activation step (Unarmored) CAR-T cells were produced as described above. 0.0037% to 0.3% DMSO was added during their production in the transduction step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 2A). The CAR-positive rate was significantly increased in the 0.0037%, 0.011%, 0.033%, 0.1%, and 0.3% DMSO groups compared to the group without DMSO.

[0170] In addition, naive T cells and stem cell memory T cells positive for CD4 or CD8 were also measured (Figure 2B-Figure 2E). CD4 positive stem cell memory T cells were significantly increased in the 0.0037%, 0.011%, 0.033%, 0.1%, and 0.3% DMSO supplemented groups compared to the non-DMSO supplemented group (Figure 2C). CD8 positive stem cell memory T cells were also significantly increased in the 0.1% and 0.3% DMSO supplemented groups compared to the non-DMSO supplemented group (Figure 2E).

[0171] Example 3: CAR-T Production with Addition of DMSO to the Gene Transduction Step (Armored) CAR-T cells expressing IL-7 and CCL19 genes were produced as described above. 0.0037% to 0.3% DMSO was added during the production in the transduction step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 3A). The CAR positive rate was significantly increased in the 0.0037%, 0.011%, 0.033%, and 0.1% DMSO groups compared to the group without DMSO.

[0172] In addition, naive T cells and stem cell memory T cells positive for CD4 or CD8 were also measured (Figure 3B-Figure 3E). CD4 positive naive T cells and CD4 positive stem cell memory T cells were significantly increased in the 0.011%, 0.033%, 0.1%, and 0.3% DMSO supplemented groups compared to the group without DMSO supplementation (Figure 3B-Figure 3C). CD8 positive naive T cells were increased by 0.011% and 0.033% DMSO supplementation compared to the group without DMSO supplementation, and CD8 positive stem cell memory T cells were increased by 0.0037%, 0.011%, 0.033%, and 0.1% DMSO supplementation compared to the group without DMSO supplementation (Figure 3D-Figure 3E).

[0173] Example 4: CAR-T production with the addition of DMSO to the gene transduction process (Unarmored) CAR-T cells were produced as described above. 0.0037% to 0.3% DMSO was added during their production in the transduction step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 4A). The CAR positive rate was increased in the 0.0037%, 0.011%, and 0.033% DMSO groups compared to the group without DMSO.

[0174] In addition, naive T cells and stem cell memory T cells positive for CD4 or CD8 were also measured (Figure 4B-E). CD4 positive naive T cells were increased in the 0.011% and 0.033% DMSO treatment groups compared to the group without DMSO treatment (Figure 4B). CD8 positive stem cell memory T cells were increased in the 0.0037%, 0.011%, 0.033%, 0.01%, and 0.3% DMSO supplemented groups compared to the group without DMSO supplementation (Figure 4E).

[0175] Example 5: CAR-T preparation with DMSO added to the expansion step (Armored) CAR-T cells expressing IL-7 and CCL19 genes were produced as described above. 0.0037% to 0.3% DMSO was added during the production in the ex vivo amplification step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 5A).

[0176] In addition, CD4 or CD8 positive naive T cells and stem cell memory T cells were also measured (Figure 5B-Figure 5E). CD8 positive stem cell memory T cells were significantly increased in the 0.3% DMSO supplemented group compared to the non-DMSO supplemented group (Figure 5E).

[0177] Example 6: CAR-T preparation with DMSO added to the expansion process (Unarmored) CAR-T cells were produced as described above. 0.0037% to 0.3% DMSO was added during their production in the ex vivo amplification step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 6A).

[0178] In addition, CD4 or CD8 positive naive T cells and stem cell memory T cells were also measured (Figures 6B to 6E). CD4 positive naive T cells and CD4 positive stem cell memory T cells were increased in the 0.3% DMSO supplemented group compared to the non-DMSO supplemented group (Figures 6B to 6C).

[0179] Example 7: CAR-T preparation with high concentration DMSO added to the expansion process (Unarmored) CAR-T cells were produced as described above. 0.3% to 2.7% DMSO was added during their production in the ex vivo amplification step. After production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 7A).

[0180] In addition, CD4 or CD8 positive naive T cells and stem cell memory T cells were also measured. CD4 or CD8 positive naive T cells and stem cell memory T cells were increased in the 0.6-2.7% DMSO group compared to the no DMSO group (Figures 7B-7E).

[0181] Example 8: DMSO suppresses CAR gene transduction in SK-Hep-1 but improves CAR-T transduction efficiency CAR-T cells were produced as described above. SK-Hep-1 cells expressing CAR were produced as discussed above. 0.1% DMSO was added during the transduction step during its production. After production, the transduction rate of CAR gene was measured by flow cytometry.

[0182] As shown in Figures 8A-B, DMSO suppresses CAR gene transduction in SK-Hep-1 (Figure 8A) but significantly improves CAR transduction efficiency (Figure 8B).

[0183] Example 9: NK cell mCheery gene transduction using DMSO mCherry-positive NK92 cells were produced as described above. 0.01% to 0.1% DMSO was added during the production in a pre-culture step before transduction. See Figure 9A. After production, the transduction rate of the mCherry gene was measured by flow cytometry. As shown in Figure 9B, 0.03% and 0.1% DMSO significantly improve the mCherry transduction efficiency.

[0184] [Table 1]

[0185] The present technology is not limited in terms of the specific embodiment described in this application, but is intended as a single illustration of each aspect of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present disclosure. The present technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which can of course vary, and it is understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0186] In addition, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or any subgroups of the Markush group.

[0187] As will be understood by those of skill in the art, for any and all purposes, especially in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges, as well as combinations of subranges thereof. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into three lower thirds, middle thirds, upper thirds, etc. Also, as will be understood by those of skill in the art, all language such as "up to," "at least," "greater than," "less than," etc. refers to a range that includes the recited numbers and can then be broken down into subranges as discussed above. Finally, as will be understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0188] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent they do not contradict the explicit teachings of this specification.

Claims

1. 1. A method for producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells; (ii) contacting said population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing said population of engineered immune cells; (iii) harvesting the population of engineered immune cells; Step (i) and / or step (ii) are carried out at least in part in the presence of dimethylsulfoxide (DMSO). method.

2. 2. The method of claim 1, wherein the population of immune cells comprises T cells and / or natural killer (NK) cells.

3. 3. The method of claim 1 or 2, wherein the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR.

4. The method according to any one of claims 1 to 3, wherein step (i) is carried out in the presence of a stimulant.

5. The method of claim 4, wherein the stimulatory agent comprises a CD3 binding domain.

6. The method according to any one of claims 1 to 5, wherein step (i) is carried out in the presence of one or more cytokines.

7. The method of any one of claims 1 to 6, further comprising storing the engineered population of immune cells.

8. 8. The method of any one of claims 1 to 7, further comprising administering at least a portion of the cells of the population of engineered immune cells to a subject in need thereof.

9. The method of any one of claims 1 to 8, wherein DMSO is present at a concentration of up to about 3% (v / v).

10. The method of any one of claims 1 to 9, wherein DMSO is present at a concentration of up to about 0.3% (v / v).

11. The method of any one of claims 1 to 10, wherein DMSO is present at a concentration ranging from about 0.001% (v / v) to about 0.03% (v / v).

12. The method of any one of claims 1 to 11, wherein DMSO is present at a concentration of about 0.01% (v / v).

13. The method according to any one of claims 1 to 12, wherein the nucleic acid molecule is a viral vector.

14. The method of claim 13 , wherein the viral vector is a retroviral vector.

15. 1. A method for producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells; (ii) contacting said population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing said population of engineered immune cells; (iii) culturing the population of engineered immune cells resulting from step (ii); and (iv) harvesting the population of engineered immune cells for storage or administration; The method, wherein step (i), step (ii), and / or step (iii) are carried out at least in part in the presence of dimethylsulfoxide (DMSO).

16. 16. The method of claim 15, wherein the population of immune cells comprises T cells and / or natural killer (NK) cells.

17. 17. The method of claim 15 or 16, wherein the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR.

18. The method according to any one of claims 15 to 17, wherein step (i) is carried out in the presence of a stimulant.

19. 20. The method of claim 19, wherein the stimulatory agent comprises a CD3 binding domain.

20. The method of any one of claims 15 to 19, further comprising (v) storing the engineered population of immune cells.

21. 21. The method of any one of claims 15 to 20, further comprising administering at least some of the cells of the population of engineered immune cells to a subject in need thereof.

22. The method of any one of claims 15 to 21, wherein step (iii) results in the expansion of the population of engineered immune cells.

23. 23. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration of up to about 3% (v / v).

24. 23. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration ranging from about 1% to about 3%.

25. 23. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration of up to about 0.3% (v / v).

26. 23. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration ranging from about 0.001% (v / v) to about 0.03% (v / v).

27. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration of about 0.01% (v / v).

28. 28. The method of any one of claims 15 to 27, wherein step (i) and / or step (ii) is at least partially performed in the presence of dimethylsulfoxide (DMSO), wherein the presence of DMSO increases transduction efficiency and / or subsets of naive or stem cell memory T cells.

29. A method of expanding a population of a subset of naive T cells or stem cell memory T cells, comprising contacting a population of immune cells with dimethylsulfoxide (DMSO).

30. 1. A method of increasing immune cell transduction efficiency, comprising contacting a population of immune cells with dimethylsulfoxide (DMSO).

31. A composition comprising a population of immune cells and dimethylsulfoxide (DMSO), wherein the DMSO is present at a concentration ranging from about 0.01% (v / v) to less than 1% (v / v).

32. 32. The composition of claim 31 , wherein the population of immune cells comprises engineered immune cells.

33. 33. The composition of claim 32, wherein the engineered population of immune cells expresses a chimeric antigen receptor (CAR).

34. 33. The composition of claim 32, wherein the population of immune cells comprises T cells and / or natural killer (NK) cells.

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