Methods of producing engineered immune cells

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 344,249, filed on May 20, 2022, under 35 U.S.C. 119(e), the entire contents of which are incorporated herein by reference. Technical Field

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

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

[0004] In some embodiments, immune cell group comprises T cell and / or natural killer (NK) cell.In some embodiments, heterologous amino acid sequence comprises chimeric antigen receptor (CAR), thereby providing the engineered immune cell group expressing CAR.

[0005] In some embodiments, step (i) is performed in the presence of a stimulatory agent. In some embodiments, the stimulatory 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 in a concentration of up to about 3% (vol / vol). In some embodiments, DMSO is present in a concentration of up to about 0.3% (vol / vol). In some embodiments, DMSO is present in a concentration ranging from about 0.001% (vol / vol) to about 0.03% (vol / vol). In some embodiments, DMSO is present in a concentration of about 0.01% (vol / vol).

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

[0009] In another aspect, the present disclosure provides a method for producing an engineered immune cell population, the method comprising: (i) culturing the immune cell population, (ii) contacting the immune cell population with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing an engineered immune cell population, (iii) culturing the engineered immune cell population derived from step (ii), and (iv) harvesting the engineered immune cell population 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 present disclosure provides a method of increasing a subset population of naive T cells or stem cell memory T cells, comprising contacting the population of immune cells with dimethyl sulfoxide (DMSO).

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

[0012] In yet another aspect, the present disclosure provides a composition comprising an immune cell population and dimethyl sulfoxide (DMSO), wherein the DMSO is present at a concentration ranging from about 0.01% (vol / vol) to less than 1% (vol / vol). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A- Figure 1 E is a chart showing the results of Example 1. In the activation step, 0.0037%-0.3% DMSO is added to produce CAR-T cells expressing IL-7 and CCL19 genes. After supplementing DMSO, (A) CAR positive rate, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. P value is calculated by t test.

[0014] Figure 2 It is a chart showing the results of Example 2. In the activation step, 0.0037%-0.3% DMSO is added to produce CAR-T cells. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0015] Figure 3It is a chart showing the results of Example 3. In the gene transduction step, 0.0037%-0.3% DMSO is added to produce CAR-T cells expressing IL-7 and CCL19 genes. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0016] Figure 4 It is a chart showing the results of Example 4. In the gene transduction step, 0.0037%-0.3% DMSO is added to produce CAR-T cells. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0017] Figure 5 It is a chart showing the results of Example 5. In the amplification step, 0.0037%-0.3% DMSO is added to produce CAR-T cells expressing IL-7 and CCL19 genes. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0018] Figure 6 It is a chart showing the results of Example 6. In the amplification step, 0.0037%-0.3% DMSO is added to produce CAR-T cells. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0019] Figure 7It is a chart showing the results of Example 7. In the amplification step, 0.3%-2.7% DMSO is added to produce CAR-T cells. After supplementing DMSO, (A) CAR positivity, (B) CD4 positive unsensitized T cells, (C) CD4 positive stem cell memory T cells, (D) CD8 positive unsensitized T cells and (E) CD8 positive stem cell memory T cells (N=3, *p<0.05, **p<0.02) are quantified by flow cytometry. The p value is calculated by t test.

[0020] Figure 8 A shows that DMSO inhibits CAR gene transduction in SK-Hep-1. Figure 8 B shows that DMSO increased CAR transduction efficiency.

[0021] Fig. 9 A shows the experimental scheme using NK cells. DMSO was used for 2 days before the mCherry transduction step. 0.01%-0.1% DMSO was added in the pre-culture step before transduction to evaluate the effect of DMSO on mCherry gene transduction into NK92 cells. Fig. 9 B shows that DMSO treatment improves the transduction efficiency of 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

[0022] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present methods will be described below in varying levels of detail in order to provide an understanding of the present technology.

[0023] The present disclosure is not limited to the specific embodiments described in this application, which are intended to be single illustrations of individual aspects of the present disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and changes may be made to the present disclosure without departing from its spirit and scope, which will be apparent to those skilled in the art. In addition to those methods and devices listed herein, functionally equivalent methods and devices within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and changes are intended to fall within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims and the full scope of equivalents enjoyed by such claims.

[0024] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., NY) series; MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press of 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 ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; edited by Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (ColdSpring Harbor Laboratory); edited by Makrides (2003) Gene Transfer and Expression inMammalian 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 the level 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 quantitative techniques. (See also, Strachan & Read, Human Molecular Genetics, 2nd Edition (John Wiley and Sons, Inc., NY, 1999)). ;

[0025] The present technology provides an improved method for producing engineered immune cells (e.g., CAR-T cells) for cell therapy. Specifically, the improved method involves performing one or more of the following on immune cells (e.g., T cells or NK cells) in the presence of dimethyl sulfoxide (DMSO): (1) activation or pre-culture before transduction (without stimulants); (2) transduction; and (3) optional ex vivo expansion. Prior to the present invention, DMSO was known to inhibit cell proliferation (see Ogaki et al., SciRep. 5: 172297 (2015)) and / or gene transduction. Unexpectedly, the inventors of the present technology found that the presence of DMSO in (1) activation or pre-culture before transduction, (2) transduction and / or (3) optional ex vivo expansion steps significantly improved transduction efficiency. In addition, in one embodiment, DMSO increases the population of efficient T cells, e.g., unsensitized T cells; and / or stem cell memory T cells (T scm ). definition

[0026] Unless otherwise defined, all technical terms and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. The following references provide general definitions of many terms used in the present disclosure for those of ordinary skill in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (ed.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms have the meanings assigned to them below. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure.

[0027] 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.

[0028] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 3 or more than 3 standard deviations. Alternatively, "about" can mean 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 an order of magnitude, within 5-fold, or within 2-fold of a value.

[0029] As used herein, the term "administering" an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be performed by any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes as described herein. Administration includes self-administration and administration by another person.

[0030] As used herein, the term "activated" refers to a state in which T cells have been sufficiently stimulated to induce cytokine production, detectable effector function, and / or detectable cell proliferation.

[0031] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be complete immunoglobulins derived from natural or recombinant sources, or may be immunoreactive portions of complete immunoglobulins. Antibodies disclosed herein may exist in a variety of forms, wherein the antigen-binding portion of the antibody is expressed as part of a continuous polypeptide chain, including, for example, single domain antibody fragments (sdAb), single chain antibodies (scFv), 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).

[0032] As used herein, "antibody fragment" or "antigen-binding fragment" refers to Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, sdAb (V L or V H ), Camelidae V HH Domains, scFv antibodies, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected via a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have V in any order. L and V H The variable region, for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a V L -Connector-V H Or it can contain V H -Connector-V L The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences (e.g., connects 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.

[0033] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformation, and generally determines the class to which the antibody belongs.

[0034] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) light chains and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0035] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technology available and well known in the art.

[0036] The term "antigen" or "Ag" as used herein is defined as a molecule that causes an immune response. This immune response may involve the production of antibodies or the activation of specific immune-competent cells or both. It will be understood by the technician that any macromolecule (including almost all proteins or peptides) can be used as an antigen. In addition, antigens can be derived from recombinant DNA or genomic DNA. It will be understood by the technician that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein (which causes an immune response) is therefore encoded as the term "antigen" used herein. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present technology includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that cause a desired immune response. In addition, it will be understood by the technician that antigens do not need to be encoded by "genes" at all. It is obvious that antigens can be synthesized or can be derived from biological samples. This biological sample can include but is not limited to tissue samples, tumor samples, cell fluids or biological fluids.

[0037] According to the present disclosure, the term "auto-antigen" means any self-antigen that is mistakenly 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.

[0038] As used herein, the term "autoimmune disease" is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens (self-antigens). Examples of autoimmune diseases include, but are not limited to, Addision's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Celiac disease, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, and gravis), pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, autoimmune vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, etc.

[0039] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into the individual. "Allogeneic" refers to a transplant derived from a different animal of the same species. "Xenogeneic" refers to a transplant derived from an animal of a different species.

[0040] The term "tumor" or "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0041] As used herein, a "control" is an alternative sample used for comparison purposes in an experiment. A control can be "positive" or "negative." For example, when the purpose of an experiment is to determine the relevance of a therapeutic agent for the efficacy of 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 the therapy or receives a placebo) are typically employed.

[0042] As used herein, the term "co-stimulatory ligand" includes molecules on antigen presenting cells (e.g., dendritic cells, B cells, macrophages, monocytes, etc.) that specifically bind to cognate co-stimulatory molecules on T cells, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to the MHC molecule loaded with the peptide. Costimulatory ligands may 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), CD27 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 (Semaphorin 4A), galectin-9, butyrophilin-like proteins such as BTN1A1 (butyrophilin), BTN2A1, BTN2A2 (butyrophilin 2A2), BTN3A1 / 2, BTN3A2, BTN3A3, BTNL2 / butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10, CD277 / BTN3A1, LAIR1, LAIR2, CD96, CD155 / PVR, CRTAM, DNAM-1 (CD226), integrin-2 (CD112), integrin-3, PVRIG, TIGIT, LILRA3 (CD85e), LILRA4 (CD85g, ILT7), LILRB3 (CD85a, ILT5), LILRB2 (CD85d, ILT4), LILRB1 (CD85j, ILT2), LILRB4 (CD85k, ILT3), B cell activating factor (BAFF) (BLyS, TNFSF13B), TL1A (TNFSF15), TNF-α, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll-like receptors (TLRs), and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules present on T cells.

[0043] As used herein, the term "costimulatory molecule" or "costimulatory domain" refers to the portion of a CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the efficient activation and function of T lymphocytes after binding to an antigen. Examples of such co-stimulatory 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 α4β1, integrin α4β7 / LPAM-1, LAG-3, TSLP R, B cell activating factor receptor (BAFF R) (TNFRSF13C), DR3 (TNFRSF25), lymphotoxin-α (TNF-β), 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 a ligand that specifically binds to CD83. Therefore, although the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-1BB, other costimulatory domains are also considered for use with the CAR described herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and amplification of T cells expressing CAR receptors. The intracellular signaling domain and the costimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0044] As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0045] A "disease" is a health state in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health state will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is not as good as it would be if the disorder were not present. A disorder will not necessarily result in a further deterioration in the animal's health state if left untreated.

[0046] As used herein, an "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0047] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0048] As used herein, the term "exogenous" refers to any substance introduced from outside of an organism, cell, tissue, or system or produced outside of an organism, cell, tissue, or system.

[0049] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0050] As used herein, the term "heterologous nucleic acid molecule or heterologous polypeptide" refers to a nucleic acid molecule (e.g., cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0051] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a certain position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a certain position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at this position. "Substantially homologous" or "substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least about 50% or higher homology or identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% homology or identity with a sequence for comparison (e.g., a wild type or native sequence) at the amino acid level or nucleic acid level. In some embodiments, substantially homologous or substantially identical polypeptides contain one or more amino acid substitutions, insertions or deletions relative to the sequence 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) in place of the homologous sequence.

[0052] As used herein, a "host cell" is a cell that is used to receive, maintain, propagate, 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.

[0053] As used herein, the term "immune cell" refers to any cell that plays a role in a subject's immune response. Immune cells are derived from hematopoiesis 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 a genetically modified immune cell. As used herein, the term "natural immune cell" refers to an immune cell that occurs naturally in the immune system.

[0054] "Isolated" means to change or be separated from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting substances of its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment (such as, for example, a host cell). As used herein, a "purified" or "substantially purified" cell is a cell that is substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that are usually associated with them in the naturally occurring state of the substantially purified cells. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term refers only to cells that have been separated from cells that are naturally associated with cells in a natural state. In some embodiments, cells are cultured in vitro. In other embodiments, cells are not cultured in vitro.

[0055] As used herein, the term "modulate" means mediating a detectable increase or decrease in a subject's response level compared to the subject's response level in the absence of the treatment or compound and / or compared to the response level of an otherwise identical but untreated subject. The term encompasses perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0056] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0057] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting 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 in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. In general, operably linked DNA sequences are continuous and in the same reading frame when two protein coding regions need to be linked.

[0058] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to mean that within a particular tissue or organ of a patient, the expression level of the tumor antigen in cells in a diseased area (such as a solid tumor) is abnormal relative to the expression level in normal cells in 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.

[0059] "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intracisternal, intrathecal or intrasternal injection, application or infusion techniques.

[0060] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof (whether in vitro or in situ) suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0061] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. It is generally known to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any available means in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from a recombinant library or cell genome, using common cloning techniques (such as PCR, etc.), and by synthetic methods.

[0062] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence or a peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are generally referred to in the art as proteins, and there are many types of proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0063] The term "promoter" as used herein is defined as a DNA sequence recognized by a cellular synthetic machinery or an introduced synthetic machinery that is required for initiating 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, results in 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, results in the production of a gene product in a cell essentially only when there is an inducer corresponding to the promoter in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a gene encoding or specified polynucleotide, results in the production of a gene product in a cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0064] As used herein, "regulatory sequence" or "regulatory region" of a nucleic acid molecule means a cis-acting nucleotide sequence that has a positive or negative effect on the expression of an operably connected gene. The regulatory region includes a nucleotide sequence that imparts inducible (i.e., requires a substance or stimulant to increase transcription) expression of a gene. When an inducer is present or the concentration increases, gene expression can increase. The regulatory region also includes a sequence that imparts gene expression inhibition (i.e., a certain substance or stimulant will reduce transcription). When an inhibitor is present or the concentration increases, gene expression can decrease. Known regulatory regions can affect, regulate or control many biological activities in vivo, including cell proliferation, cell growth and death, cell differentiation and immunomodulation. The regulatory region is usually combined with one or more trans-acting proteins, which results in an increase or decrease in gene transcription.

[0065] The specific example of gene regulatory region is promoter and enhancer.Promoter is positioned at transcription start site or translation start site around, usually positioned at the sequence of 5' of translation start site.Promoter is usually positioned at 1 Kb of translation start site, but can be positioned at more distant position, for example, positioned at 2 Kb, 3 Kb, 4 Kb, 5 Kb or more distant of translation start site, at most and including 10 Kb.Known enhancer can affect gene expression when positioned at 5' or 3' of gene or when positioned in exon or intron or as part of exon or intron.Enhancer can also play a role at the position far away from gene, for example, about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more distant from gene. In addition to the promoter region, regulatory regions 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, stop codons, leader and fusion partner sequences, internal ribosome binding site (IRES) elements for creating polygenic or polycistronic messages, polyadenylation signals to provide proper polyadenylation of the transcript of the gene of interest and stop codons, and may optionally be included in an expression vector.

[0066] 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 a tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchoalveolar lavage fluid (BAL), bronchial washings (BW), whole blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0067] As used herein, the term "secretion" with respect to a polypeptide means a polypeptide released by a cell via the secretory pathway, through the endoplasmic reticulum, the Golgi apparatus, and as a vesicle that temporarily fuses on the plasma membrane of the cell, thereby releasing the protein to the outside of the cell. Small molecules, such as drugs, can also be secreted by diffusing across the membrane to the outside of the cell.

[0068] As used herein, the term "specific binding" about antibodies means that the antibody recognizes a specific antigen, but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to the antigen from one or more species. However, this cross-species reactivity itself does not change the classification of the antibody as a specific antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, this cross-reactivity itself does not change the classification of the antibody as a specific antibody. In some cases, the term "specific binding" or "specifically binding" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than a general protein. If the antibody is specific to epitope "A", then in the reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. As used herein, the terms "specifically bind", "bind specifically to", or "have specificity" for a particular molecule (e.g., an antigen) can be expressed, for example, as the K of a molecule to which it binds. d 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 M.

[0069] As used herein, the term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction via a TCR / CD3 complex). Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGFβ and / or reorganization of cytoskeletal structure, etc.

[0070] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0071] As used herein, "stimulatory ligand" or "stimulatory agent" means a ligand that, when present on antigen presenting cells (e.g., dendritic cells, B cells, macrophages, monocytes, 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 of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulators are well known in the art and particularly encompass peptide-loaded MHC class I molecules, 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).

[0072] The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit a biological or medical response in a tissue, system, or subject that is sought by a researcher, veterinarian, medical doctor, or other clinician. The term "therapeutically effective amount" includes an amount of the compound that, when administered, is sufficient to prevent the development of the condition or disease being treated or to alleviate to some extent one or more signs or symptoms of the condition 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 to be treated.

[0073] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0074] As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

[0075] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, with the same or different routes of administration. More specifically, sequential use refers to the complete administration of one of the active ingredients before the start of administration of one or more active ingredients. Thus, one of the active ingredients may be administered a few minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no simultaneous treatment.

[0076] As used herein, the term "T cell" includes naive T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, and antigen-specific T cells. For more specific examples, the T cells of the subject matter disclosed in the present invention include, but are not limited to: CD4 + T cells, CD8 +T cells, helper T cells, cytotoxic T cells, central memory T cells, stem cell memory T cells, effector memory T cells (e.g., T EM Cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells (NKT), mucosal-associated constant T cells, αβ T cells, double negative T cells and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are subsets of T lymphocytes that can induce the death of infected somatic cells or tumor cells. In certain embodiments, CAR-expressing T cells express Foxp3 to achieve and maintain T regulatory phenotypes. In some embodiments, CAR-T cells are any immune cells derived from pluripotent stem cells (e.g., induced pluripotent stem (iPS) cells).

[0077] As used herein, "treating" or "treatment" covers the treatment of a disease or condition described herein in a subject (such as a human), and includes: (i) inhibiting the disease or condition, i.e., preventing its development; (ii) alleviating the disease or condition, i.e., causing regression of the condition; (iii) slowing the progression of the condition; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or condition. The therapeutic effects of treatment include, but are not limited to, inhibiting the recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0078] As used herein, "vector" is a reproducible nucleic acid that can express one or more heterologous proteins when the vector is transformed into an appropriate host cell. The vectors mentioned include those vectors in which the nucleic acid encoding a polypeptide or its fragment can be introduced, usually by restrictive digestion and connection. The vectors mentioned also include those vectors containing nucleic acids encoding polypeptides. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell to amplify the nucleic acid or express / display the polypeptide encoded by the nucleic acid. The vector usually maintains an additional type, but can be designed to achieve integration of a gene or part thereof into the chromosome of the genome. The vector can include a viral vector. A viral vector is an engineered virus that is operably connected to an exogenous gene to transfer the exogenous gene (as a vehicle or transport) to the cell.

[0079] The viral vector of the present technology can be a retroviral vector. An advantage provided by retroviral vectors is that they can convert their single-stranded RNA genome into a double-stranded DNA molecule that is stably integrated into the target cell genome. Therefore, retroviral vectors can be used to permanently modify the host cell nuclear genome.

[0080] The retroviral vector of the present technology can be derived from any member of the Retroviridae family, such as Spumavirus or Fomie virus (e.g., human and monkey virus), beta retrovirus (e.g., MMTV), gamma retrovirus (e.g., MLV), alpha retrovirus (e.g., ALV), delta retrovirus (e.g., BLV and HTLV-1), lentivirus (e.g., HIV 1) and epsilon retrovirus (e.g., WDSV and WEHV1 / 2) or their derivatives.

[0081] Any method known to those skilled in the art for inserting a heterologous nucleic acid sequence into a vector (eg, a retroviral vector) can be used to construct an expression vector containing a nucleic acid encoding any of the polypeptides provided herein. Chimeric Antigen Receptor (CAR)

[0082] CAR is an engineered receptor comprising an extracellular domain and an intracellular domain. The extracellular domain comprises an antigen binding portion. In some embodiments, the extracellular domain further comprises a hinge domain. In some embodiments, the intracellular domain or cytoplasmic domain comprises a CD3ζ chain and / or a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of a CAR comprising an intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for the efficient response of lymphocytes to antigens in addition to antigen receptors or their ligands.

[0083] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint or spacer domain can be incorporated. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide, which is used to connect the transmembrane domain to the extracellular domain or cytoplasmic domain in the polypeptide chain. The spacer domain may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

[0084] Antigen binding part

[0085] The selection of antigen binding moiety depends on the type and quantity of the ligand defining the target cell surface. For example, antigen binding domains can be selected to identify ligands, which act as cell surface markers on target cells associated with a specific disease state. Therefore, examples of cell surface markers of ligands of antigen moiety domains in the CAR of the subject matter disclosed in the present invention include cell surface markers related to viruses, bacteria and parasites (e.g., pathogen antigens), autoimmune diseases (e.g., self-antigens) and cancer cells (e.g., tumor-specific antigens or tumor-associated antigens).

[0086] In one embodiment, the CAR of the subject matter disclosed in the present invention can be engineered to target tumor antigens of interest by engineering the desired antigen binding portion that specifically binds to the antigen on the tumor cell. Tumor antigens may be proteins produced by tumor cells that cause an immune response (e.g., a T cell-mediated immune response). The selection of the antigen binding portion of the subject matter disclosed in the present invention will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2 (ephrinB2), CD22, insulin growth factor (IGF)-1, IGF-II, IGF-I receptor, CA125, CA19-9, MUC-1, WT-1, glypican 3 (GPC3) and mesothelin.

[0087] In one embodiment, tumor antigens include one or more antigen cancer epitopes associated with malignant tumors. Malignant tumors express a variety of proteins that can be used as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to transformation-related molecule groups, such as oncogenes HER-2 / Neu / ErbB-2. Another target antigen group is onco-fetal antigen (onco-fetal antigen), such as carcinoembryonic antigen (carcinoembryonic antigen, CEA). In B cell lymphoma, tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen unique to a single tumor. B cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens of B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but the success rate is limited.

[0088] The types of tumor antigens referred to in the subject matter disclosed in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but rather are also expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. Expression of antigens on tumors may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0089] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA19-9, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, phosphatidylinositol glycan 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 antigens targeted by the antigen binding portion of CAR include but are not limited to: cMet, CD 19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, cMet, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc.

[0090] Depending on the desired antigen to be targeted, the CAR of the subject matter disclosed herein can be engineered to include an appropriate antigen binding portion specific for the desired antigen target. For example, if CD19 is a desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding portion incorporated into the CAR of the present technology.

[0091] Transmembrane domain.

[0092] About transmembrane domain, CAR can be designed to include transmembrane domain fused with the extracellular domain of CAR.In one embodiment, a transmembrane domain naturally associated with a domain in CAR is used.In some cases, a transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0093] Transmembrane domain can be derived from natural origin or synthetic origin.When the source is natural, the domain can be derived from any membrane binding or transmembrane protein.The particularly useful transmembrane region in the present technology can be derived from (that is, at least one or more transmembrane regions comprising the following items) α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or derived from immunoglobulin, such as IgG4.Alternatively, the transmembrane domain can be synthetic, in which case it will mainly include hydrophobic residues, such as leucine and valine.Preferably, there are triplets of phenylalanine, tryptophan and valine at each end of the synthetic transmembrane domain.Optionally, short oligopeptides or polypeptide linkers (preferably between 2 and 10 amino acids in length) can form a connection between the transmembrane domain of CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0094] Cytoplasmic domain.

[0095] The cytoplasmic domain or intracellular signaling domain of the CAR of the subject matter disclosed in the present invention is responsible for activating at least one normal effector function of the immune cell where the CAR is located. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity (including the secretion of cytokines). Therefore, the term "intracellular signaling domain" refers to a part of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, it is not necessary to use the entire chain in many cases. In the case of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain, as long as the truncated portion transduces the effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0096] Examples of intracellular signaling domains for use in the CARs of the presently disclosed subject matter include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0097] It is known that the signal generated by the TCR alone is insufficient to fully activate T cells, and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different categories of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0098] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0099] Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in the subject matter disclosed herein include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferably, the cytoplasmic signaling molecule in the CAR of the subject matter disclosed herein includes a cytoplasmic signaling sequence derived from CD3ζ.

[0100] In some embodiments, the cytoplasmic domain of CAR can be designed to include the CD3ζ signaling domain itself or to include it and any other desired cytoplasmic domain useful in the context of CAR of the present technology. For example, the cytoplasmic domain of CAR may include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of a CAR that includes an intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for the 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 ligands that specifically bind to CD83, etc.

[0101] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR of the subject matter disclosed in the present invention can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0102] 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 and the signaling domains of CD28 and 4-1BB. Methods for producing engineered immune cells of the present technology

[0103] In one aspect, the present disclosure provides a method for producing an engineered immune cell population, the method comprising: (i) culturing the immune cell population, (ii) contacting the immune cell population with a nucleic acid molecule comprising nucleotides encoding a heterologous polypeptide, thereby providing an engineered immune cell population (transduction step), and (iii) harvesting the engineered immune cell population, wherein step (i) and / or step (ii) is performed at least in part in the presence of dimethyl sulfoxide (DMSO).

[0104] In some embodiments, the heterologous polypeptide comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells expressing CAR (e.g., CAR-T cells).

[0105] The engineered immune cells of the subject matter disclosed in the present invention can be cells of lymphoid lineage or bone marrow lineage. Bone marrow lineage can include monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils and granulocytes. Lymphoid lineage includes B cells, T cells and natural killer (NK) cells, which provide the production of antibodies, the regulation of cellular immune system, the detection of foreign substances in blood, the detection of host foreign cells, etc. Non-limiting examples of the immune cells of lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells and pluripotent stem cells (for example, those cells from which lymphoid cells can be differentiated). T cells can be lymphocytes matured in the thymus, and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the subject matter disclosed in the present invention can be any type of T cells, including but not limited to: helper T 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 called suppressor T cells), natural killer T cells, mucosal-associated constant T cells and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells 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 cell type, in the present disclosure, CAR-T can encompass any immune cell expressing CAR.

[0106] In some embodiments, the immune cell population comprises T cells and / or natural killer (NK) cells. Natural killer (NK) cells can be lymphocytes, which are part of cell-mediated immunity and play a role during the innate immune response. NK cells do not need to be pre-activated to exert their cytotoxic effects on target cells.

[0107] The immune cell group 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 of 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, immune cell group can be obtained from blood units collected from subjects using various techniques known to technicians (e.g., apheresis). In some embodiments, immune cell group can be separated from peripheral blood lymphocytes by lysing red blood cells and depleting mononuclear cells.

[0108] Separation procedures include, but are not limited to, density gradient centrifugation (e.g., using PERCOLL ® gradient); counter-flow centrifugal elutriation; resetting; coupling to particles that modify cell density; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents linked to or used in conjunction with mAbs, including but not limited to complement and cytotoxins; and panning, elutriation, or any other convenient technique using antibodies attached to a solid matrix (e.g., a plate, a chip).

[0109] Techniques used for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low-angle blunt light scatter detection channels, impedance channels, and Fluorescence-Activated Cell Sorting (FACS).

[0110] In some embodiments, specific immune cell subsets, such as CD3 + T cells, CD28 + T cells, CD4 + T cells, CD8 + T cells, CD45RA + T cells and CD45RO + T cells. In some embodiments, prior to step (i), the immune cell population may be enriched for T cells expressing CD4 and / or CD8. These selection techniques are well known to those skilled in the art. As a non-limiting example, CD4 can be enriched by negative selection by treating the cell mixture with a mixture of monoclonal antibodies including antibodies to CD14, CD20, CD 11b, CD 16, HLA-DR, and CD8. + In certain embodiments, regulatory T cells can be depleted by anti-CD25 conjugated beads.

[0111] In some embodiments, the immune cells may be frozen after the washing steps prior to activation (step (i)). The freezing and subsequent thawing steps may provide a more uniform product by removing granulocytes and to a certain extent monocytes from the cell population. After the washing steps 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 and washed prior to step (i) and allowed to stand at room temperature for about one hour.

[0112] Immune cell populations can be collected at any time point required for subsequent activation, transduction, amplification, and preparation, and used for cell therapy of any disease or illness that benefits from immune cell therapy. In one embodiment, a blood sample or a single blood component can be obtained from a generally healthy subject. In certain embodiments, a blood sample or a single blood component is obtained from a generally healthy subject with a risk of illness but not yet ill, and then the cells of interest are separated and frozen for later use. In certain embodiments, samples can be collected from a patient shortly after being diagnosed with a specific disease as described herein but before any treatment. In a further embodiment, cells can be separated from a blood sample or a single blood component of a subject before, during, or after any relevant treatment modality, and the treatment includes but is not limited to treatment with agents such as antiviral agents, chemotherapy, radiotherapy, immunotherapy (e.g., checkpoint inhibitors) or immunosuppressants.

[0113] In some embodiments of the present technology, immune cell populations can be obtained directly from patients after treatment. In this regard, it has been observed that following certain cancer treatments (particularly treatments using drugs that damage the immune system), the quality of the immune cells (e.g., T cells) obtained may be optimal or improved for ex vivo manipulation (e.g., activation, transduction, or expansion) shortly after treatment, while the patient is typically recovering from treatment.

[0114] Pre-culture of immune cells or activation of immune cells

[0115] In some other embodiments, immune cells (e.g., T cells) are activated by contacting immune cells with stimulants. In some embodiments, the stimulant may include an agent that stimulates CD3 / TCR complex-related signals and / or a ligand that stimulates co-stimulatory molecules on the surface of immune cells. In some embodiments, the stimulant includes any co-stimulatory ligand disclosed herein, including but not limited to CD3 binding domains, CD28 binding domains, CD134 binding domains, and / or CD137 binding domains. In some embodiments, the stimulant includes a CD3 binding domain and / or a CD28 binding domain. In some embodiments, the stimulant includes an anti-CD3 antibody and / or an anti-CD28 antibody. Examples of anti-CD28 antibodies include but are not limited to 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France). Examples of anti-CD3 antibodies include but are not limited to OKT3, 145–2C11, 17A2, UCHT1, and SK7.

[0116] In certain embodiments, the stimulating agent comprises an anti-CD3 antibody and an anti-CD28 antibody. The anti-CD3 antibody and the anti-CD28 antibody can each be independently present in a solution or coupled to a surface. When both are coupled to a surface, the anti-CD3 antibody and the anti-CD28 can be coupled to the same surface (i.e., "cis" formation) or a separate surface (i.e., "trans" formation).

[0117] In one embodiment, the anti-CD3 antibody and the anti-CD28 antibody are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". In one embodiment, the molar ratio of the anti-CD3 antibody to the anti-CD28 antibody ranges from 100:1 to 1:100 and all integer values ​​therebetween. 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 and the size and type of the cells. One of ordinary skill in the art will readily appreciate that any cell concentration can be used. For example, in one embodiment, about 10 million to 15 million cells / ml, about 15 million to 20 million cells / ml, about 20 million to 25 million cells / ml, about 25 million to 30 million cells / ml, about 30 million to 35 million cells / ml, about 35 million to 40 million cells / ml, about 40 million to 45 million cells / ml, about 45 million to 50 million cells / ml, about 50 million to 55 million cells / ml, about 55 million to 60 million cells / ml, about 60 million to 65 million cells / ml, about 65 million to 70 million cells / ml, about 70 million to 75 million cells / ml, about 75 million to 80 million cells / ml, about 70 million to 8 ... In some embodiments, the present invention relates to a concentration of about 70 million cells / ml, about 70 million to 75 million cells / ml, about 75 million to 80 million cells / ml, about 80 million to 85 million cells / ml, about 85 million to 90 million cells / ml, about 90 million to 95 million cells / ml, about 95 million to 100 million cells / ml, about 100 million to 125 million cells / ml, about 125 million to 150 million cells / ml, about 150 million to 200 million cells / ml, about 200 million to 500 million cells / ml, about 500 million to 1 billion cells / ml, or about 1 billion to 2 billion cells / ml.

[0118] In certain embodiments, it may be desirable to significantly increase the cell concentration 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 targeted antigen of interest. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0119] 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 surfaces (e.g., particles such as beads), interactions between particles and cells are minimized. This selects cells that express a greater amount of the desired antigen to bind to the particles. For example, at dilute concentrations, CD4+ cells that express higher levels of CD28 may be more likely to bind to the surface. + T cells may be more sensitive than CD8 + T cells are captured more efficiently.

[0120] In one embodiment, the immune cells (eg, T cells) can be contacted with the stimulatory agents (eg, anti-CD3 antibodies and anti-CD28 antibodies) for about 3 hours to about 14 days, or any integer number of hours therebetween. In some embodiments, the contact time can be from about 4 hours to about 96 hours, for example, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours 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, 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, immune cells (e.g., T cells) can be contacted with stimulants for about 4 hours to 60 hours. In some embodiments, immune cells (e.g., T cells) can be contacted with stimulants for about 4 hours to 48 hours. In some embodiments, immune cells (e.g., T cells) can be contacted with stimulants for about 12 hours to 48 hours. In some embodiments, immune cells (e.g., T cells) can be contacted with stimulants for about 24 hours to 48 hours. Subsequently, beads and cells can be separated, and then cells can be washed and collected for transduction.

[0121] In some embodiments, prior to the transduction step (pre-transduction pre-culture), immune cells (eg, T cells, NK cells) are cultured with DMSO without a stimulatory agent.

[0122] Transduction steps

[0123] In some embodiments, the transduction step, i.e., contacting the immune cell population (e.g., T cells) with a nucleic acid molecule (e.g., a viral vector) comprising nucleotides encoding a heterologous amino acid sequence, is not initiated until after the activation step (i.e., contacting the immune cell population (e.g., T cells) with a stimulating agent) is completed. For example, the immune cells (e.g., T cells) from the activation step can be washed and collected for transduction.

[0124] The nucleic acid molecule comprising the nucleotides encoding the heterologous amino acid sequence can be based on any RNA vector or DNA vector known in the art. The method of introducing the nucleic acid molecule into the host cell is known to those skilled in the art. For example, the nucleic acid molecule can be transferred to the host cell by physical, chemical or biological methods.

[0125] Physical methods for introducing nucleic acid molecules into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.

[0126] 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 methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from slow viruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0127] Chemical means for introducing nucleic acid molecules into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0128] In the case of utilizing non-viral delivery systems, exemplary delivery vehicles are liposomes. Consider using lipid formulations for introducing nucleic acid molecules into host cells (in vitro, in vitro or in vivo). In another aspect, nucleic acid molecules can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes via connecting molecules associated with liposomes and oligonucleotides, wrapped in liposomes, compounded with liposomes, dispersed in solutions containing lipids, mixed with lipids, combined with lipids, included in lipids as suspensions, included in micelles or compounded with micelles, or otherwise associated with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any specific structure in solution. For example, they can be present in a double-layer structure, as micelles or have a "collapse" structure. They can also be only dispersed in solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring lipid or a synthetic lipid. For example, lipids include naturally occurring fat droplets in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0129] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, Mo.; dihexadecyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform / methanol can be stored at a temperature of about -20°C. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of monolayer and multilayer lipid vehicles formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and encapsulate water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids may present micellar structures or exist only in the form of non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0130] No matter what method is used to introduce exogenous nucleic acid into host cells, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as, for example, by immunological means (ELISA and Western blotting) or by assays described herein to detect the presence or absence of specific peptides to identify agents falling within the scope of the present invention.

[0131] Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Patent No. 5,399,346). In some embodiments, in order to perform initial genetic modification on immune cells (e.g., T cells) to produce engineered immune cells (e.g., CAR-T cells), a retroviral vector comprising a nucleotide molecule encoding a heterologous amino acid sequence is used for transduction. For example, the polynucleotide encoding CAR can be cloned into a retroviral vector, and expression can be driven by its endogenous promoter, a retroviral long terminal repeat sequence, or an alternative internal promoter. In order to perform subsequent genetic modification on cells to provide cells comprising an antigen presentation complex (the antigen presentation complex comprises at least two costimulatory ligands), retroviral gene transfer (transduction) is also proven to be effective. The combination of a retroviral vector and an appropriate packaging cell line is also suitable, in which the capsid protein will have the function of infecting human cells. Various cell lines are known for producing amphotropic viruses, 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 are also suitable, for example, particles pseudotyped with VSVG, RD114 or GALV envelopes, and any other particles known in the art.

[0132] Possible transduction methods also include direct co-culturing of immune cells (e.g., T cells) with production cells, for example, by the method of Bregni et al., Blood 80: 1418-1422 (1992), or culturing with viral supernatant alone or with concentrated vector stock solutions (containing or not containing appropriate growth factors and polycations), for example, by Xu et al., Exp. Hemat. 22: 223-230 (1994); and Hughes et al., J. Clin. Invest. 89: 1817 (1992). In some embodiments, a population of immune cells (e.g., T cells) is contacted with a retroviral vector in the presence of a soluble additive of a cationic amphiphilic peptide (e.g., Vectofusin-1).

[0133] In some embodiments, the retroviral vector expressing CAR can be an oncoretroviral vector, a γ retroviral vector, a lentiviral vector, or a spumaretroviral vector. In some embodiments, the retroviral vector can be a γ retroviral vector. In some embodiments, the γ retroviral vector is selected from a pMSGV vector, a pMSCV vector, a pSFG vector, or a combination of any two or more thereof.

[0134] In one embodiment, a population of immune cells (e.g., T cells) is contacted 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), and the contact can be performed for about 1 hour to about 72 hours, such as about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours , about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours. In some embodiments, the immune cell can 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 hours to 28 hours, e.g., 24 hours.

[0135] In some embodiments, a nucleic acid molecule (e.g., a retroviral vector) comprising a nucleotide molecule encoding a heterologous amino acid sequence (e.g., CAR) may include a nucleotide molecule encoding IL-7 and / or CCL19. In some embodiments, a nucleotide molecule encoding a heterologous amino acid sequence (e.g., CAR) and a nucleotide molecule encoding IL-7 and / or CCL19 may be located on the same nucleic acid molecule. In some embodiments, a nucleotide molecule encoding a heterologous amino acid sequence (e.g., CAR) and a nucleotide molecule encoding IL-7 and / or CCL19 may be located on a separate nucleic acid molecule.

[0136] Conditions suitable for culturing immune cells (e.g., culturing immune cells in activation and / or transduction steps) include an appropriate culture medium (e.g., Minimal Essential Media or RPMI medium 1640 or X-vivo 15, (Lonza)), which may contain factors necessary for viability and / or proliferation, including but not limited to serum (e.g., fetal bovine serum 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 cell growth known to those skilled in the art. In some embodiments, immune cells are cultured in the presence of IL-2 (e.g., in activation and / or transduction steps).

[0137] Other additives for cell growth include, but are not limited to, surfactants, plasma protein preparations (plasmanates), and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, IMDM, Advanced DMEM / F12, X-Vivo 10 TM 、X-Vivo 15 TM 、X-Vivo 20 TM 、TheraPEAK TM X-Vivo 10, TheraPEAK TM X-Vivo 15 TM 、TheraPEAK TM X-Vivo 20 TM , CTS TM Optimizer TM T cell expansion SFM, CTS Optmizer Pro serum-free medium, 4Cell Nutri-T medium, LymphoONE TM Xeno-free medium for T cell expansion, ImmunoCult TM -XF T Cell Expansion Medium, ExCellerate Human T Cell Expansion Medium, Stemline T Cell Expansion Medium, CAR T Cell Medium, TexMACS TM Culture medium, Corning serum-free lymphocyte culture medium, Corning 88-581-CM culture medium, CellGenix T cell culture medium, SmarT TM T cell expansion medium, StemSpan TMSerum-free Expansion Medium and OptiPEAK T Lymphocyte XPR, supplemented with amino acids, sodium pyruvate, and vitamins, are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in an amount sufficient to allow T cell growth and / or expansion. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., room temperature or 37°C) and atmosphere (e.g., air plus 5% CO 2 ).

[0138] In any of the above embodiments, DMSO is present in a concentration of up to about 3% (v / v), for example, 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), 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% (vol / vol), about 2.3% (vol / vol), about 2.4% (vol / vol), about 2.5% (vol / vol), about 2.6% (vol / vol), about 2.7% (vol / vol), about 2.8% (vol / vol), about 2.9% (vol / vol) or about 3.0% (vol / vol).

[0139] In any of the above embodiments, DMSO is present at a concentration of 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), about 0.12% (v / v), about 0.13% (v / v), about 0.14% (v / v), about In some embodiments, DMSO is present at a concentration of about 0.01% (vol / vol), about 0.02% (vol / vol), or about 0.03% (vol / vol).

[0140] In some embodiments, the presence of DMSO in the activation and / or transduction step significantly increases 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, 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.

[0141] In some embodiments, the presence of DMSO in the activation and / or transduction step significantly increases the population of efficient T cells, 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, compared to a control without DMSO. In some embodiments, efficient T cells include, but are not limited to, naive T cells and / or stem cell-like memory T cells (T cells). scm ). The immunophenotype of T cells can 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 can be measured using anti-CD4 antibodies (e.g., clone SK3, catalog number 344604, BioLegend), anti-CD8 antibodies (e.g., clone SK1, catalog number 344710, BioLegend), anti-CCR7 antibodies (e.g., clone G043H7, catalog number 353204, BioLegend), anti-CD45RA antibodies (e.g., clone L48, catalog number 337167, BD Biosciences), anti-CD27 antibodies (e.g., clone O323, catalog number 302836, BioLegend) and anti-CD95 antibodies (e.g., clone DX2, catalog number 305612, BioLegend). CCR7 / CD45RA-negative cells are effector memory T cells, CCR7-positive CD45RA-negative cells are central memory T cells, CCR7-negative CD45RA-positive cells are effector T cells, CCR7 / CD45RA / CD27 / CD95-positive cells are defined as stem cell memory T cells, and CCR7 / CD45RA-positive cells other than them are defined as naive T cells.

[0142] Storage / Preparation / Administration

[0143] The engineered immune cells (e.g., CAR-T cells) from the transduction step can be harvested for storage, preparation and / or administration according to protocols well known in the art. Therefore, in some embodiments, the method of the present technology may further include storing engineered immune cell populations, and / or administering at least some cells in the engineered immune cell populations to subjects in need.

[0144] In some embodiments, engineered immune cells (e.g., CAR-T cells) can be prepared for long-term storage. In some embodiments, engineered immune cells (e.g., CAR-T cells) can be cryopreserved. Methods for cryopreservation are well known to those skilled in the art. For example, engineered immune cells (e.g., CAR-T cells) can be suspended in a cell cryopreservation solution containing a cryoprotectant (e.g., dimethyl sulfoxide) and human serum albumin and frozen at -80°C for 1 day; cryopreserved cells can be further stored in liquid nitrogen (LN) (e.g., < -150°C). Many factors in cryopreservation may affect the quality of engineered immune cells (e.g., CAR-T cells), thereby affecting the results of cell therapy. These factors include (1) preparation and introduction of freezing medium, (2) cooling rate, (3) storage conditions, (4) thawing conditions, and (5) post-thawing treatment. Optimizing these factors to achieve the desired results of cell therapy is within the level of ordinary technicians in the art.

[0145] preparation

[0146] Engineered immune cells (e.g., CAR-T cells) and compositions comprising engineered immune cells of the present technology can be conveniently provided as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to selected pH.Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are slightly more convenient to apply, especially by injection. In another aspect, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a suitable mixture thereof.

[0147] Sterile injectable solutions can be prepared as needed by incorporating the compositions of the disclosed subject matter into the appropriate solvent of the desired amount and other components of various amounts. Such compositions can be mixed with suitable carriers, diluents or excipients (such as sterile water, normal saline, glucose, dextrose, etc.). Compositions can also be lyophilized. According to route of administration and desired preparations, compositions can contain auxiliary substances, such as wetting agents, dispersants or emulsifiers (for example, methylcellulose), pH buffers, gelling agents or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc. Can refer to standard texts to prepare suitable preparations without excessive experiments, described standard texts such as "REMINGTON'S PHARMACEUTICALSCIENCE", the 17th edition, 1985, which is incorporated herein by reference.

[0148] Various additives for enhancing the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.) can be used to ensure that microbial action is prevented. The absorption of injectable drug forms can be extended by using agents that delay absorption (e.g., aluminum monostearate and gelatin). However, according to the subject matter disclosed in the present invention, any vehicle, diluent or additive used must be compatible with the engineered immune cells (e.g., CAR-T cells) of the subject matter disclosed in the present invention.

[0149] The compositions can be isotonic, that is, they can have the same osmotic pressure as blood and tears. Sodium chloride or other pharmaceutically acceptable agents (such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes) can be used to achieve the desired isotonicity of the compositions of the presently disclosed subject matter. Sodium chloride is particularly suitable for buffers containing sodium ions.

[0150] If desired, pharmaceutically acceptable thickeners can be used to maintain the viscosity of the composition at a selected level. Methylcellulose can be used because it is easily available and economical, and easy to operate. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer (carbomer) etc. The concentration of the thickener can depend on the selected agent. Emphasis is on the use of the amount that will achieve the selected viscosity. Obviously, the selection of suitable carriers and other additives will depend on the properties of definite route of administration and specific dosage form, for example, liquid dosage form (for example, whether the composition is to be formulated into solution, suspension, gel or another liquid form, such as sustained release form or liquid filled form).

[0151] It will be appreciated by those skilled in the art that the components of the composition should be selected to be chemically inert and will not affect the viability or efficacy of engineered immune cells (e.g., CAR-T cells) as described in the subject matter disclosed in the present invention. This will not be a problem for those skilled in the art of chemical and pharmaceutical principles, or the problem can be easily avoided by reference to standard texts or by simple experiments (not involving excessive experiments) according to the present disclosure or the documents cited herein.

[0152] One consideration for therapeutic use of engineered immune cells (e.g., CAR-T cells) of the disclosed subject matter is the amount of cells required to achieve optimal effect. The amount of cells to be administered will vary depending on the subject being treated. In certain embodiments, about 10 2 to about 10 12 , about 10 3 to about 10 11 , about 10 4to about 10 10 , about 10 5 to about 10 9 or about 10 6 to about 10 8 The engineered immune cells (e.g., CAR-T cells) of the subject matter of the present disclosure may be administered in even smaller quantities to provide more effective cells. In some embodiments, at least about 1 × 10 8 , about 2 × 10 8 , about 3 × 10 8 , about 4 × 10 8 , about 5 × 10 8 , about 1 × 10 9 , about 5 × 10 9 , about 1 × 10 10 , about 5 × 10 10 , about 1 × 10 11 , about 5 × 10 11 , about 1 × 10 12 Or more engineered immune cells (e.g., CAR-T cells) of the subject disclosed in the present invention. The accurate determination of the effective dose can be based on the individual factors of each subject, including its body shape, age, sex, weight and the disease of the specific subject. Those skilled in the art can easily determine the dosage according to the disclosure and the knowledge in the art. In general, engineered immune cells (e.g., CAR-T cells) are administered in a dose that is non-toxic or tolerable to the patient.

[0153] Technicians can easily determine the amount of cells and optional additives, vehicles and / or carriers in the composition to be administered in the method for the theme disclosed by the present invention.Usually, any additive (except active cells and / or agent) is present in phosphate buffered saline solution with an amount of about 0.001 % by weight to about 50 % by weight, and active ingredient exists with the magnitude of about microgram to milligram, such as about 0.0001 % by weight to about 5 % by weight, about 0.0001 % by weight to about 1 % by weight, about 0.0001 % by weight to about 0.05 % by weight, about 0.001 % by weight to about 20 % by weight, about 0.01 % by weight to about 10 % by weight or about 0.05 % by weight to about 5 % by weight.For any composition to be applied to an animal or a person, and for any specific method of application, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (for example, rodents, such as mice); and the dosage of the composition, the concentration of the component therein and the time of applying the composition, so as to cause a suitable reaction. Such determinations can be made without undue experimentation, based on the knowledge of those skilled in the art, this disclosure, and the documents cited herein. Also, the timing of sequential administration can be determined without undue experimentation.

[0154] Application

[0155] The engineered immune cells (e.g., CAR-T cells) of the subject disclosed in the present invention can be provided to subjects systemically or directly to treat various diseases, including but not limited to infection, autoimmune diseases or tumors. In certain embodiments, engineered immune cells (e.g., CAR-T cells) are directly injected into an organ of interest. Additionally or alternatively, engineered immune cells (e.g., CAR-T cells) are indirectly provided to an organ of interest, for example, by administration to the circulatory system or to a tissue of interest. Amplifiers and differentiation agents can be provided before, during, or after the administration of cells and compositions to increase the production of engineered immune cells (e.g., CAR-T cells) in vitro or in vivo.

[0156] The engineered immune cells (e.g., CAR-T cells) of the presently disclosed subject matter can be administered systemically or regionally in any physiologically acceptable vehicle, typically intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they can also be introduced into the bone or other convenient sites (e.g., thymus) where the cells can find appropriate sites of regeneration and differentiation. In certain embodiments, at least 1 × 10 5 cells, eventually reaching 1 × 10 10 In some embodiments, at least 1 × 10 6 Cells. The cell group comprising engineered immune cells (e.g., CAR-T cells) may include purified cell groups. Those skilled in the art can easily determine the percentage of engineered immune cells (e.g., CAR-T cells) in a cell group using various well-known methods (such as fluorescence activated cell sorting (FACS)). The purity range of the cell group comprising 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%. Those skilled in the art can easily adjust the dosage (e.g., a reduction in purity may require an increase in dosage). Engineered immune cells (e.g., CAR-T cells) may be introduced by injection, catheter, etc. If desired, factors including, but not limited to, interleukins, e.g., IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21 and others, colony stimulating factors, such as G-CSF, M-CSF and GM-CSF, interferons, e.g., gamma-interferon, may also be included.

[0157] In certain embodiments, the composition of the subject disclosed in the present invention includes a pharmaceutical composition comprising an engineered immune cell (e.g., CAR-T cell) and a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous. For example, engineered immune cells (e.g., CAR-T cells) and compositions comprising the engineered immune cells can be obtained from a subject and applied to the same subject or different compatible subjects. The peripheral blood-derived immune cells or their offspring (e.g., in vivo, ex vivo or in vitro derived) of the subject disclosed in the present invention can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When the pharmaceutical composition of the subject disclosed in the present invention is administered, it can be formulated into an injectable form (solution, suspension, emulsion) of a unit dose.

[0158] In another aspect, the present disclosure provides a method for producing an engineered immune cell population, the method comprising: (i) contacting the immune cell population with a stimulatory agent (activation step), (ii) contacting the immune cell population with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing an engineered immune cell population (transduction step), (iii) culturing the engineered immune cell population derived from step (ii) (ex vivo expansion step), and (iv) harvesting the engineered immune cell population 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).

[0159] In some embodiments, for the ex vivo expansion step, the engineered immune cells can be cultured for about 3 hours to about 21 days or any hour integer value therebetween. It may also be desirable to have several stimulation cycles so that the culture time of the engineered immune cells can be 60 days or longer. In some embodiments, the engineered immune cell population derived from step (ii) can 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. The conditions suitable for the T cell culture of ex vivo expansion are substantially the same as discussed above for the activation step and / or the transduction step.

[0160] In some embodiments, the presence of DMSO in the activation, transduction and / or ex vivo expansion steps significantly increases 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, compared to a control without DMSO.

[0161] In some embodiments, the presence of DMSO in the activation, transduction and / or ex vivo expansion steps significantly increases the population of efficient T cells, 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, compared to a control without DMSO. In some embodiments, efficient T cells include, but are not limited to, naive T cells and / or stem cell-like memory T cells (T cells). scm ). Example General experimental methods

[0162] Culture medium

[0163] CAR-T cell culture medium: 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) were added to OpTmizer CTS T Cell Expansion Basal Medium (ThermoFisher Scientific) to prepare the basal cell culture medium. 20 IU / mL or 40 IU / mL of MACSGMP IL-2 (Miltenyi Biotec) was added thereto.

[0164] SK-HEP-1 cell culture medium: MEM, L-Gln (+) (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.), 1% nonessential 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 PureChemical Industries, Ltd.).

[0165] NK92 cell culture medium: RPMI, L-Gln(+) (Thermo Fisher Scientific) was prepared by adding 20% ​​FBS (Biosera Co., Ltd.).

[0166] Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Ltd) was added to the corresponding culture medium to obtain the indicated levels and used.

[0167] Production of CAR-T cells

[0168] After thawing in Leukopak (Hemacare), cells were diluted to less than or equal to 2.0 × 10 6 Cells / mL (pre-production raw material). Cell suspension: MACS GMP T-Cell TransACT (Miltenyi Biotec) = 17.5:1 was inoculated into the culture bag and cultured for about 48 hours (cell activation step). The activated cells were diluted in the culture medium using the LOVO cell processing system (Fresenius Kabi) or a centrifuge to a concentration of less than 6.07×10 5 cells / cm 2 The cells were inoculated in a culture bag pre-coated with retronectin® (Takara Bio Co., Ltd.) and retrovirus introduced with CAR gene or CAR gene, IL-7 gene, and CCL19 gene and cultured until the next day (gene transfer process). 6 cells / cm 2Inoculate culture flasks (G-REX, Wilson Wolf) and culture for 3-7 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 following SK-HEP-1 cells are also applicable). The aforementioned sequences are shown in Table 1.

[0169] Introduction of CAR into SK-HEP-1 cells

[0170] SK-HEP-1 cells (ATCC) were diluted with SK-HEP-1 cell culture medium to a concentration of 2.0 × 10 6 cells / mL or lower, less than 6.07 × 10 5 cells / cm 2 The CAR gene was introduced into SK-HEP-1 cells by seeding on a culture plate coated with retronectin (Takara Bio Co., Ltd.) and the retrovirus into which the CAR gene was introduced and culturing for 4 days.

[0171] Introduction of mCherry into NK92 cells

[0172] NK92 cells (ATCC) were diluted with NK92 cell culture medium to a concentration of 2.0 × 10 6 cells / mL or lower, less than 6.07 × 10 5 cells / cm 2 The cells were plated on culture plates containing or not containing DMSO for 2 days. After that, NK92 cells were cultured on retronectin (Takara Bio Co., Ltd.) and retrovirus into which the mCherry gene was introduced, and cultured for 2 days to introduce the mCherry gene into NK92 cells.

[0173] Determination of T cell transduction rate and immunophenotype using flow cytometry

[0174] The rate of CAR incorporation into T cells was measured using the CAR targeting antigen on a BD FACSCanto II flow cytometer (BD Biosciences). Anti-CD4 antibody (clone SK3, catalog number 344604, BioLegend), anti-CD8 antibody (clone SK1, catalog number 344710, BioLegend), anti-CCR7 antibody (clone G043H7, catalog number 353204, BioLegend), anti-CD45RA antibody (clone L48, catalog number 337167, BD Biosciences), anti-CD27 antibody (clone O323, catalog number 302836, BioLegend) and anti-CD95 antibody (clone DX2, catalog number 305612, BioLegend) were used to measure the immunophenotype of T cells, and CCR7 / CD45RA / CD27 / CD95-positive cells in the CD4-positive T cell population or the CD8-positive T cell population were used as stem cell memory T cells, and CCR7 / CD45RA-positive cells other than these were used as naive T cells. Example 1: CAR-T production with addition of DMSO to the activation step (Armor)

[0175] CAR-T cells expressing IL-7 gene and CCL19 gene were produced as described above. During its production, 0.0037% to 0.3% DMSO was added in the transduction step. After production, the introduction rate of CAR gene was measured by flow cytometry ( Figure 1 A). Compared with the no DMSO group, the CAR positive rates in the 0.0037% DMSO group, 0.011% DMSO group, 0.033% DMSO group, and 0.1% DMSO group were significantly increased.

[0176] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 1 B- Figure 1 E). Compared with the non-DMSO supplemented group, the CD4-positive naive T cells and CD4-positive stem cell memory T cells in the 0.011% DMSO supplemented group, the 0.033% DMSO supplemented group, and the 0.1% DMSO supplemented group increased significantly ( Figure 1 B- Figure 1 C). Compared with the non-DMSO supplemented group, the CD8-positive naive T cells and CD8-positive stem cell memory T cells in the 0.0037% DMSO supplemented group, the 0.011% DMSO supplemented group, the 0.033% DMSO supplemented group, and the 0.1% DMSO supplemented group increased significantly ( Figure 1 D- Figure 1 E). Example 2: CAR-T production with addition of DMSO to the activation step (non-armored)

[0177] CAR-T cells were produced as described above. During their production, 0.0037% to 0.3% DMSO was added during the transduction step. After production, the introduction rate of the CAR gene was measured by flow cytometry ( Figure 2 A). Compared with the no DMSO group, the CAR positive rates in the 0.0037% DMSO group, 0.011% DMSO group, 0.033% DMSO group, 0.1% DMSO group, and 0.3% DMSO group were significantly increased.

[0178] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 2 B- Figure 2 E). Compared with the non-DMSO supplemented group, the CD4 positive stem cell memory T cells in the 0.0037% DMSO supplemented group, the 0.011% DMSO supplemented group, the 0.033% DMSO supplemented group, the 0.1% DMSO supplemented group, and the 0.3% DMSO supplemented group increased significantly ( Figure 2 C). CD8-positive stem cell memory T cells also increased significantly in the 0.1% DMSO-supplemented group and the 0.3% DMSO-supplemented group compared to the non-DMSO-supplemented group ( Figure 2 E). Example 3: CAR-T production with addition of DMSO during gene transduction step (Armor)

[0179] CAR-T cells expressing IL-7 gene and CCL19 gene were produced as described above. During its production, 0.0037% to 0.3% DMSO was added in the transduction step. After production, the introduction rate of CAR gene was measured by flow cytometry ( Figure 3 A). Compared with the no DMSO group, the CAR positive rates in the 0.0037% DMSO group, 0.011% DMSO group, 0.033% DMSO group, and 0.1% DMSO group were significantly increased.

[0180] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 3 B- Figure 3 E). Compared with the non-DMSO supplemented group, the CD4-positive naive T cells and CD4-positive stem cell memory T cells in the 0.011% DMSO supplemented group, the 0.033% DMSO supplemented group, the 0.1% DMSO supplemented group, and the 0.3% DMSO supplemented group increased significantly ( Figure 3 B- Figure 3C). Compared with no DMSO supplementation, 0.011% DMSO supplementation and 0.033% DMSO supplementation increased CD8-positive naive T cells, and compared with no DMSO supplementation, 0.0037% DMSO supplementation, 0.011% DMSO supplementation, 0.033% DMSO supplementation, and 0.1% DMSO supplementation increased CD8-positive stem cell memory T cells ( Figure 3 D- Figure 3 E). Example 4: CAR-T production with addition of DMSO during gene transduction step (non-armored)

[0181] CAR-T cells were produced as described above. During their production, 0.0037% to 0.3% DMSO was added during the transduction step. After production, the introduction rate of the CAR gene was measured by flow cytometry ( Figure 4 A). Compared with the no DMSO group, the CAR positive rate increased in the 0.0037% DMSO group, the 0.011% DMSO group, and the 0.033% DMSO group.

[0182] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 4 B- Figure 4 E). Compared with the group without DMSO treatment, the CD4-positive naive T cells in the 0.011% treatment group and the 0.033% treatment group increased ( Figure 4 B). Compared with the non-DMSO supplemented group, the CD8-positive stem cell memory T cells in the 0.0037% DMSO supplemented group, the 0.011% DMSO supplemented group, the 0.033% DMSO supplemented group, the 0.01% DMSO supplemented group, and the 0.3% DMSO supplemented group increased ( Figure 4 E). Example 5: CAR-T preparation with addition of DMSO during the amplification step (Armor)

[0183] CAR-T cells expressing IL-7 and CCL19 genes were produced as described above. During its production, 0.0037% to 0.3% DMSO was added in the ex vivo expansion step. After production, the introduction rate of the CAR gene was measured by flow cytometry ( Figure 5 A).

[0184] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 5 B- Figure 5 E). Compared with the DMSO non-supplemented group, the CD8 positive stem cell memory T cells in the 0.3% DMSO supplemented group increased significantly ( Figure 5 E). Example 6: CAR-T preparation with addition of DMSO during the expansion step (non-armored)

[0185] CAR-T cells were produced as described above. During their production, 0.0037% to 0.3% DMSO was added in the ex vivo expansion step. After production, the introduction rate of the CAR gene was measured by flow cytometry ( Figure 6 A).

[0186] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells ( Figure 6 B- Figure 6 E). Compared with the group without DMSO supplementation, the number of CD4-positive naive T cells and CD4-positive stem cell memory T cells in the group with 0.3% DMSO supplementation increased ( Figure 6 B- Figure 6 C). Example 7: CAR-T preparation with high concentration of DMSO added during the amplification step (non-armored)

[0187] CAR-T cells were produced as described above. During their production, 0.0037% to 0.3% DMSO was added in the ex vivo expansion step. After production, the introduction rate of the CAR gene was measured by flow cytometry ( Figure 7 A).

[0188] In addition, CD4-positive or CD8-positive naive T cells and stem cell memory T cells were measured. Compared with the no DMSO group, the CD4-positive or CD8-positive naive T cells and stem cell memory T cells in the 0.6%-2.7% DMSO group increased ( Figure 7 B- Figure 7 E). Example 8: DMSO inhibits CAR gene transduction in SK-Hep-1 but improves CAR-T transduction efficiency

[0189] CAR-T cells were produced as described above. SK-Hep-1 cells expressing CAR were produced as discussed above. During its production, 0.1% DMSO was added in the transduction step. After production, the introduction rate of the CAR gene was measured by flow cytometry.

[0190] like Figure 8 A- Figure 8 As shown in B, DMSO inhibited CAR gene transduction in SK-Hep-1 ( Figure 8 A) but significantly improved CAR transduction efficiency ( Figure 8 B). Example 9: mCheery gene transduction of NK cells using DMSO

[0191] mCherry-positive NK92 cells were produced as described above. During their production, 0.01% to 0.1% DMSO was added during the pre-culture step before transduction. Fig. 9 A. After production, the incorporation rate of the mCherry gene was measured by flow cytometry. Fig. 9 As shown in B, 0.03% DMSO and 0.1% DMSO significantly improved the mCherry transduction efficiency. Table 1

[0192] The present technology is not limited to the specific embodiments described in the application, and the embodiments are intended to be a single description of the individual aspects of the present technology. Many modifications and changes can be made to the present technology without departing from its spirit and scope, as is apparent to those skilled in the art. In addition to those methods and equipment listed herein, the functionally equivalent methods and equipment within the scope of the present technology will be apparent to those skilled in the art according to the foregoing description. Such modifications and changes are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to ad hoc methods, reagents, compounds, compositions or biological systems, which can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive.

[0193] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0194] It will be understood by those skilled in the art that for any and all purposes, particularly in providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as fully describing and capable of decomposing the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. It will also be understood by those skilled in the art that all languages, such as "at most", "at least", "greater than", "less than", etc., all include the enumerated numbers and refer to the ranges that can be subsequently decomposed into sub-ranges as discussed above. Finally, it will be understood by those skilled in the art that a range includes each individual member. Therefore, for example, a group having 1-3 units refers to a group having 1, 2, or 3 units. Similarly, a group having 1-5 units refers to a group having 1, 2, 3, 4, or 5 units, etc.

[0195] All patents, patent applications, provisional applications, and publications mentioned or cited herein 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. A method for producing an engineered immune cell population, the method comprising: (i) Cultivating immune cell populations, (ii) contacting the immune cell population with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing the engineered immune cell population, and (iii) harvesting the engineered immune cell population, wherein step (i) and / or step (ii) is at least partially carried out in the presence of dimethyl sulfoxide (DMSO).

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

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

4. The method of any one of claims 1 to 3, wherein step (i) is performed in the presence of a stimulating agent.

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

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

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

8. The method of any one of claims 1 to 7, further comprising administering at least some of the engineered immune cell population 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% (vol / vol).

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

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 of 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. A method for producing an engineered immune cell population, the method comprising: (i) Cultivating immune cell populations, (ii) contacting the immune cell population with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing the engineered immune cell population, (iii) culturing the engineered immune cell population derived from step (ii), and (iv) harvesting the engineered immune cell population for storage or administration, wherein step (i), step (ii) and / or step (iii) are at least partially carried out in the presence of dimethyl sulfoxide (DMSO).

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

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

18. The method of any one of claims 15 to 17, wherein step (i) is performed in the presence of a stimulating agent.

19. 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 immune cell population.

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

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

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

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

26. The method of any one of claims 15 to 22, wherein DMSO is present at a concentration of 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. The method of any one of claims 15 to 27, wherein step (i) and / or step (ii) is performed at least in part in the presence of dimethyl sulfoxide (DMSO), wherein the presence of DMSO increases the transduction efficiency and / or the subset of naive T cells or stem cell memory T cells.

29. A method of increasing a subset population of naive T cells or stem cell memory T cells comprising contacting the immune cell population with dimethyl sulfoxide (DMSO).

30. A method for increasing the transduction efficiency of immune cells, comprising contacting a population of immune cells with dimethyl sulfoxide (DMSO).

31. 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% (vol / vol) to less than 1% (vol / vol).

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

33. A composition as described in claim 32, wherein the engineered immune cell population expresses a chimeric antigen receptor (CAR).

34. The composition of any one of claims 32, wherein the immune cell population comprises T cells and / or natural killer (NK) cells.

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