Methods of producing engineered immune cells

Abstract

The present disclosure provides improved methods of producing engineered T cells (e.g., CAR-T cells) that express exogenous genes. T cells expressing exogenous genes, as well as compositions comprising the T cells of the present technology, 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 and priority to U.S. Provisional Patent Application No. 63 / 344,255, filed May 20, 2022, which is incorporated herein by reference in its entirety for any and all purposes. Technical Field

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

[0003] In one aspect, the present disclosure provides a method for producing a T cell population expressing an exogenous gene product, the method comprising: (i) contacting the T cell population with a stimulatory agent, (ii) contacting the T cell population with a retroviral vector comprising a nucleic acid molecule encoding the exogenous gene product, thereby providing a T cell population expressing the exogenous gene product, and (iii) collecting the T cell population expressing the exogenous gene product for storage or administration, wherein: the T cell population expressing the exogenous gene product from step (iii) is not expanded, or is expanded by no more than 200% compared to the T cell population at the beginning of step (i), as assessed by the number of viable cells.

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

[0005] In some embodiments, the stimulatory agent comprises a CD3 binding domain.

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

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

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

[0009] In some embodiments, step (ii) is not initiated until step (i) is completed.

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

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

[0012] Figures 1A to 1C Shown is a comparison of CAR-T cell manufacturing methods in which the activation step was skipped (method 1), the transduction step was performed during the activation step (method 2), and the activation step was 48 hours followed by 1 day of transduction (method 3) (N=1). Figure 1A A scheme of the experimental design is shown. Figure 1B and Figure 1C Two experiments using Donor-3 and Donor-4 cells are shown, showing the relative cell numbers after expansion obtained using the large-scale method ( Figure 1B , the number of cells before expansion = 1) and the percentage of cells expressing CAR ( Figure 1C ) results.

[0013] FIG. 2A to FIG. 2C The in vitro characterization results from day 6 to day 8 during CAR-T cell manufacturing are shown (N=1). As the number of expansion days increases, both the proliferative capacity and pre-exhausted T cells (including stem cell memory T cells and central memory T cells) decrease. Figure 2A A scheme of the experimental design is shown. CAR-T cells were harvested on days 6 to 8. After harvesting, CAR-T cells were formulated and cryopreserved. Figure 2B The change in cell number during expansion is shown (cell number on day 3 = 1). Figure 2C The results of the proliferation capacity of CAR-T cells after thawing from day 6 to day 8 of cryopreservation are shown. The proliferation capacity was measured by "proliferation assay" (the number of cells before the proliferation assay = 1). Figure 2D to Figure 2G The changes in the percentage of each T cell phenotype of CAR-T cells from day 6 to day 8 are shown.

[0014] Figure 3 The results of in vivo efficacy studies using CAR-T cells on days 6, 7, and 8 are shown. In a xenograft model inoculated with HepG2, CAR-T cells manufactured with a shorter amplification cycle showed excellent anti-tumor efficacy. The X-axis represents the number of days after inoculation of the HepG2 cancer cell line. The Y-axis represents the tumor volume of the mouse. The data are presented as mean ± standard deviation, n=5.

[0015] FIG. 4A to FIG. 4I Results of in vitro characterization of CAR-T cells on days 3 and 7 are shown. Figure 4A A scheme showing the CAR-T cell manufacturing method. FIG. 4B to FIG. 4C (N=1) shows the percentage of cells expressing CAR ( Figure 4B ) and the copy number in the cell ( Figure 4C ) results. FIG. 4D to FIG. 4E (N=1) shows the changes in T cell phenotype in CD4+ and CD8+ T cells during manufacturing, respectively. FIG. 4D to FIG. 4E The legend is: Tn=naive T cells, Tscm=stem cell memory T cells, Tcm=central memory T cells, Tem=effector memory T cells, and Teff=effector T cells. Figures 4F to 4G The fold increase measured by the "expandability assay" is shown after thawing of CAR-T cells on days 3 and 7 of cryopreservation. CAR+CD4+ T cells ( Figure 4F ) or CAR+CD8+ T cells ( Figure 4G ) quantity. Figure 4H The assay scheme for the cytotoxicity assay using repeated antigen exposure is shown. Fig. 4I Luciferase activity in live target cells after four days of culture is shown. The luminescence ratio of co-culture wells to tumor-only wells was used to determine the percentage of luciferase activity. Data are presented as mean ± SD, n = 9 (Day 1), 6 (Day 2), 6 (Day 3), or 3 (Day 4). **p ≤ 0.01, ns; not significant, two-sided Student's t-test.

[0016] Figure 5A and Figure 5B Results of in vivo antitumor efficacy studies using day 3 and day 7 CAR-T cells in a murine xenograft model are shown. GSU cells expressing luciferase were subcutaneously inoculated into NSG mice. 2×10 5 and 5×10 5 Day 3 CAR-T cells, 2×10 5 and 1×10 6 On day 7, CAR-T cells or PBS were intravenously administered to mice. Figure 5A A graph summarizing in vivo data is shown. The X-axis represents the number of days after CAR-T administration. The Y-axis represents tumor growth measured by bioluminescence in mice. Data are presented as mean ± standard deviation, n=3 or 5. The significance of the PBS treatment group on day 12 was analyzed using the Bartlett test and Dunnett test (**p≤0.01; ***p≤0.001). Figure 5B Images of in vivo luminescence in anti-tumor efficacy studies are shown.

[0017] Fig. 6A and Figure 6B Results of in vivo antitumor efficacy studies using day 3 and day 7 CAR-T cells prepared with or without Prodigy are shown. GSU cells expressing luciferase were subcutaneously inoculated into NSG mice. 5×10 5 Day 3 CAR-T cells, 1×10 6On day 7, CAR-T cells or PBS were intravenously administered to mice. Fig. 6A A graph summarizing in vivo data is shown. The X-axis represents the number of days after CAR-T administration. The Y-axis represents tumor growth measured by bioluminescence in mice. Data are presented as mean ± standard deviation, n=3 or 5. The significance of the PBS treatment group on day 12 was analyzed using the Bartlett test and Dunnett test (**p≤0.01; ***p≤0.001). Figure 6B Images of in vivo luminescence in anti-tumor efficacy studies are shown.

[0018] 7A to 7F In vitro characterization results of non-armored CAR-T cells on days 3 and 7 are shown (N=1). Fig. 7A A scheme showing the CAR-T cell manufacturing method. FIG. 7B to FIG. 7C Shows the use of Fig. 7A The percentage of cells expressing CAR in the protocol ( Figure 7B ) and the copy number in the cell ( Figure 7C ) results. FIG. 7D to FIG. 7E Changes in T cell phenotype during manufacturing are shown in CD4+ and CD8+ T cells, respectively. FIG. 7D to FIG. 7E The legend is: Tn=naive T cells, Tscm=stem cell memory T cells, Tcm=central memory T cells, Tem=effector memory T cells, and Teff=effector T cells. Figure 7F The fold increase in the number of cells after 2 days of culture after thawing the cryopreserved CAR-T cells on days 3 and 7 is shown. The fold increase was calculated based on the ratio of the cell count after two days of culture to the initial cell count (determined by a nuclear counter).

[0019] Figure 8 Results of an in vivo antitumor efficacy study using day 3 and day 7 non-armored CAR-T cells in a murine xenograft model are shown. GSU cells were subcutaneously inoculated into NSG mice. Seven days after inoculation, 1×10 5 CAR-T cells or PBS were intravenously administered to mice on the 3rd and 7th days. The X-axis represents the number of days after CAR-T administration. The Y-axis represents tumor growth measured by a handheld Peira TM900 imaging device. Data are presented as mean ± standard deviation, n=5. The significance of the group on day 42 was analyzed using the Bartlett test and Dunnett test (ns, not significant; ***p<0.001).

[0020] 9A to 9E Shown are the in vitro characterization results of day 3 and day 7 CAR-T cells manufactured using CliniMACS Prodigy (N=1). Fig.9AThe scheme of the CAR-T cell manufacturing method is shown. CAR-T cells were manufactured using CliniMACS Prodigy (day 3 CAR-T cells). Day 3 CAR-T cells were further cultured in G-Rex bottles for 4 days (day 7 CAR-T cells). FIG. 9B to FIG. 9C The percentage of cells expressing CAR is shown ( Fig. 9B ) and the copy number in the cell ( Fig. 9C ) results. FIG. 9D to FIG. 9E Changes in T cell phenotype during manufacturing are shown in CD4+ and CD8+ T cells, respectively. FIG. 9D to FIG. 9E The legend is: Tn=naive T cells, Tscm=stem cell memory T cells, Tcm=central memory T cells, Tem=effector memory T cells, and Teff=effector T cells.

[0021] Fig.10 Results are shown for the percentage of CAR-expressing cells produced with CliniMACS Prodigy with or without Vectofusin-1 during transduction (N=1). DETAILED DESCRIPTION

[0022] It should 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 a substantial 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 a single illustration of various aspects of the present disclosure. All various embodiments of the present disclosure will not be described herein. Many modifications and changes can 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 (Cold Spring HarborLaboratory); Makrides editor (2003) Gene Transfer and Expression in Mammalian Cells;Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring 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 expressing exogenous genes for use in cell therapy, wherein the engineered immune cells include but are not limited to T cells expressing chimeric antigen receptors (CARs), T cells expressing T cell receptors (TCRs), and T cells expressing synthetic T cell antigen receptors (STARs). In one embodiment, the present invention provides a rapid CAR-T cell manufacturing method with in vivo CAR-T expansion, that is, a CAR-T cell manufacturing method that does not require or only requires limited ex vivo CAR-T expansion (e.g., no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or 200% ex vivo expansion). In one embodiment, the improved CAR-T cell manufacturing method requires an activation step of about 1 day to 2 days, the activation step involves a stimulant, such as anti-CD3 and anti-CD28 antibodies, and a transduction step of about 1 day (e.g., 20 hours to 28 hours), the transduction step involves a retroviral vector comprising a nucleic acid molecule encoding CAR. In one embodiment, the CAR-T cells produced by the manufacturing method of the present technology show improved in vitro and in vivo amplification capabilities. The resulting CAR-T cells also show improved efficacy (in vivo anti-tumor effect) and in vivo proliferation. Additionally, in one embodiment, the manufacturing method of the present technology realizes an integrated method, that is, in one embodiment, the manufacturing method can be performed in a single container. 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 generated, synthesized, or can be derived from biological samples. This biological sample can include but is not limited to tissue samples, tumor samples, cells 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, Addison'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, 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 and to be 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, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, TIM-1 / KIM-1 / HAVCR, TIM-4, Semaphorin 4A, Galectin-9, butyrophilin 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), integrins-2 (CD112), integrins-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, 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 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 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. Such a nucleic acid may be from another organism, or it may 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 them. "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 to initiate specific transcription of a polynucleotide sequence. A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, 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 an inducer corresponding to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a 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 a 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 an antibody 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" may 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-like memory T cells (stem cell memory T cells or stem cell-like 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 regulatory T cell 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 remains an additional type, but can be designed to achieve integration of a gene or part thereof into the chromosome of the genome. The vector includes 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 transduce their single-stranded RNA genome into double-stranded DNA molecules, thereby stably integrating 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. CAR-T cells

[0082] The CAR-T cells provided herein are T cells engineered to express at least one chimeric antigen receptor (CAR). 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. The intracellular domain or other cytoplasmic domains comprise 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 for connecting a transmembrane domain to an extracellular domain or a cytoplasmic domain in a 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 moieties depends on the type and quantity of ligands defining the target cell surface. For example, antigen binding domains may be selected to identify ligands that act as cell surface markers associated with a specific disease state on target cells. Therefore, examples of cell surface markers that can act as ligands for antigen moiety domains in the CAR of the subject matter disclosed herein include cell surface markers associated with viruses, bacteria and parasitic infections, autoimmune diseases and cancer cells.

[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 (particularly 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 invention.

[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-bound protein or transmembrane protein.The particularly useful transmembrane region in the present invention can be derived from (i.e., at least comprising the following transmembrane region) 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 is a triplet of phenylalanine, tryptophan and valine at each end of the synthetic transmembrane domain.Optionally, a short oligopeptide or polypeptide linker (preferably a length of 2 to 10 amino acids) can be connected 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 the CAR of the present invention. 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.

[0103] In the present disclosure, CAR-T cells may encompass any immune cells expressing CAR.

[0104] The CAR-T cells of the present technology can be administered to a subject in need (e.g., a human subject) to treat any disease, including but not limited to infection, autoimmune disease, or tumor. Methods for producing engineered immune cells of the present technology

[0105] In one aspect, the present disclosure provides a method for producing a T cell population expressing an exogenous gene product, the method comprising (i) contacting a T cell population with a stimulator, (ii) contacting a T cell population with a retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product, thereby providing a T cell population expressing an exogenous gene product, and (iii) collecting a T cell population expressing an exogenous gene product for storage or administration, wherein: the T cell population expressing the exogenous gene product from step (iii) is not expanded, or is expanded by no more than 200% compared to the T cell population at the beginning of step (i), as assessed by the number of viable cells. In some embodiments, the exogenous gene product is a chimeric antigen receptor (CAR).

[0106] Step (i): Activation

[0107] Prior to step (i), T cell populations can be obtained from any source known in the art, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, tumor, T cells derived from pluripotent stem cells and T cells derived from direct differentiation. In certain embodiments of the present technology, any T cell line available in the art can be used. In certain embodiments of the present technology, T cells can be obtained from blood units collected from subjects using various techniques known to technicians (e.g., apheresis). In some embodiments, prior to step (i), T cell populations can be separated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes.

[0108] Separation procedures include, but are not limited to, density gradient centrifugation (e.g., using PERCOLL ® gradients); counter-flow centrifugal elutriation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents conjugated or used in conjunction with mAbs, including but not limited to complement and cytotoxins; and panning, elutriation, or any other convenient technique with antibodies attached to a solid matrix (e.g., plate, 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 T 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 T cell population may be enriched for T cells expressing CD3, CD4 and / or CD8. These selection techniques are well known to those skilled in the art. As a non-limiting example, CD4 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, before step (i), T cells can be frozen after the washing step, or can be frozen without washing steps. Freezing and subsequent thawing steps can provide a more uniform product by removing the granulocytes and a certain degree of monocytes in the cell population. After the washing steps of removing plasma and platelets, the cells can be suspended in a freezing solution. Freezing solutions and parameters are known in the art. In certain embodiments, the cells can be thawed and washed before step (i) and allowed to stand at room temperature for about one hour.

[0112] T cell sources can be collected at any necessary time point for subsequent activation, transduction, preparation, and for T cell therapy for any disease or illness that can benefit from T 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, a population of T cells can be obtained directly from a patient after treatment. In this regard, it has been observed that following certain cancer treatments (particularly treatments with drugs that compromise the immune system), the quality of the T cells obtained may be optimal or improved for ex vivo manipulation (e.g., activation and engineering) shortly after treatment, while the patient is typically recovering from treatment.

[0114] In any of the above embodiments, the T cell is activated or stimulated by a stimulator. The stimulator may include an agent that stimulates CD3 / TCR complex-related signals and / or a ligand that stimulates co-stimulatory molecules on the surface of T cells. In some embodiments, the stimulator includes a CD3 binding domain, a CD28 binding domain, a CD134 binding domain, and / or a CD137 binding domain. In some embodiments, the stimulator includes a CD3 binding domain and / or a CD28 binding domain. In some embodiments, the stimulator 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.

[0115] 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).

[0116] 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 some embodiments, the molar ratio of the anti-CD3 antibody to the anti-CD28 antibody ranges from 100:1 to 1:100 and all integer values ​​therebetween. In some embodiments, the 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. In some embodiments, the stimulator is MACS GMP T-Cell TransACT (Miltenyi Biotec) ("TransAct"). In some embodiments, the ratio of T cell suspension to TransAct is about 17:1. One of ordinary skill in the art can 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 10,000 to 70 million cells / ml, about 70 million to 75 million cells / ml, about 75 million to 80 million cells / ml, about 80 million to 85 million cells / ml, about 85 million to 90 million cells / ml, about 90 million to 95 million cells / ml, about 95 million to 100 million cells / ml, about 100 million to 125 million cells / ml, about 125 million to 150 million cells / ml, about 150 million to 200 million cells / ml, about 200 million to 500 million cells / ml, about 500 million to 1 billion cells / ml, or about 1 billion to 2 billion cells / ml.

[0117] 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.

[0118] In other embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles, such as beads), interactions between particles and cells are minimized. This selects for cells that express large amounts of the desired antigen that binds to the particles. For example, at dilute concentrations, CD4+ cells that express high 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.

[0119] In one embodiment, the T cells can be contacted with the stimulatory agent (e.g., anti-CD3 antibody and anti-CD28 antibody) for about 4 to about 96 hours, such as about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours 8 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, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 hours, about 111 hours, about 112 hours, about 113 hours, about 114 hours, about 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, about 120 hours, about 121 hours, about 122 hours, about 123 hours, about 124 hours In some embodiments, the T cells may be contacted with the stimulating agent for about 4 hours to 48 hours. In some embodiments, the T cells may be contacted with the stimulating agent for about 12 hours to 48 hours. In some embodiments, the T cells may be contacted with the stimulating agent for about 24 hours to 48 hours. Subsequently, the beads and cells may be separated, and the cells may then be washed and collected for transduction.

[0120] Step (ii): Transduction

[0121] In any embodiment, step (ii) (contacting the T cell population with a retroviral vector) is not initiated until step (i) (contacting the T cell population with a stimulating agent) is completed. For example, the T cells from step (i) can be washed and collected for use in step (ii).

[0122] For the initial T cell genetic modification of the T cell expressing the exogenous gene product (e.g., CAR) in step (ii), retroviral vectors are generally used for transduction.For example, the polynucleotide encoding CAR can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat sequence, or from an alternative internal promoter.In order to carry out subsequent genetic modification to cells to provide cells comprising antigen presentation complexes (the antigen presentation complexes include at least two costimulatory ligands), retroviral gene transfer (transduction) is also proven to be effective.The combination of retroviral vectors and appropriate packaging cell lines is also suitable, in which 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.

[0123] Possible transduction methods also include directly co-culturing T cells with production cells, for example, by Bregni et al., Blood 80: 1418-1422 (1992) methods, or cultured 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) methods. In some embodiments, in the presence of a soluble additive of a cationic amphipathic peptide (e.g., Vectofusin-1), a T cell group is contacted with a retroviral vector comprising a nucleic acid molecule encoding CAR. In some embodiments, in the presence of a fibronectin-like peptide (e.g., Retronectin), a T cell group is contacted with a retroviral vector comprising a nucleic acid molecule encoding CAR.

[0124] In some embodiments, the retroviral vector expressing the CAR disclosed in the present invention can be an oncoretroviral vector, a γ retroviral 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 derivative thereof.

[0125] 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. Pat. No. 5,399,346). Other viral vectors that can be used include, for example, adenovirus and adeno-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr Virus.

[0126] In one embodiment, a population of T cells can be contacted with a retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product (e.g., a CAR) 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, about 72 hours, about 73 hours, about 74 hours Hour, 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, T cells can be contacted with a retroviral vector comprising a nucleic acid molecule encoding a CAR for about 1 hour to 28 hours. In some embodiments, T cells can be contacted with a retroviral vector comprising a nucleic acid molecule encoding an exogenous gene product (e.g., CAR) for about 16 hours to 28 hours, such as 24 hours.

[0127] Conditions suitable for culturing T cells in step (i) and / or step (ii) 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 additives for cell growth known to those skilled in the art. 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, to which amino acids, sodium pyruvate and vitamins are added, 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 grow and / or expand T cells. 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% CO2).

[0128] Step (iii): Storage, formulation and / or administration

[0129] According to protocols well known in the art, the engineered T cells expressing the exogenous gene product (e.g., CAR-T cells) from step (ii) are optionally washed and collected for storage, preparation and / or administration in step (iii). Specifically, current techniques for producing engineered T cells (e.g., CAR-T cells) do not require or only require limited ex vivo expansion. In some embodiments, the engineered T cells (e.g., CAR-T cells) group from step (iii) is not expanded, or the T cell group at the beginning of step (i) is expanded by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or 200%, such as assessed by viable cell number. Any method for assessing viable cell number known in the art can be used, including but not limited to hemocytometer, automatic cell counter and any known cell viability and cytotoxicity determination. These methods may be based on PCR, spectrophotometer, pH meter, ATP measurement, weight measurement or metabolite analyzer.

[0130] In some embodiments, steps (i) to (iii) are all performed in a single container. Suitable containers for the methods of producing engineered T cells (e.g., CAR-T cells) of the present technology include, but are not limited to, G-REX® bioreactors, CliniMACS Prodigy®, Xuri TM Cell expansion system, WAVE bioreactor TM , Coccon® Platform, PBS Bioreactors, Ambr Bioreactors, Biostat Bioreactors, Cell Factory Systems, CellSTAK as well as cell culture bags, dishes, plates and flasks.

[0131] Engineered T cells (e.g., CAR-T cells) can be prepared for administration or long-term storage. For long-term storage, the engineered T cells (e.g., CAR-T cells) of step (ii) can be cryopreserved. Methods for cryopreservation are well known to those skilled in the art. For example, engineered T cells (e.g., CAR-T cells) can be suspended in a cell cryopreservation solution containing a cryoprotectant (e.g., DMSO) and a protein (e.g., human serum albumin), a polymer and / or a sugar, 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 can affect the quality of engineered T 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.

[0132] Preparation

[0133] Engineered T cells (e.g., CAR-T cells) and compositions of engineered T cells comprising 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 a 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. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions may include a carrier, which may 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.

[0134] 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.

[0135] 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 T cells (e.g., CAR-T cells) of the subject matter disclosed in the present invention.

[0136] 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.

[0137] 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 definite route of administration and the property of a particular dosage form (for example, liquid dosage form) (for example, whether the composition will be formulated into a solution, a suspension, a gel or another liquid form, such as a sustained release form or a liquid-filled form).

[0138] 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 T 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 and the documents cited herein.

[0139] One consideration for therapeutic use of engineered T 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 104 to about 10 10 About 10 5 to about 10 9 or about 10 6 to about 10 8 The engineered T cells (e.g., CAR-T cells) of the subject matter of the present disclosure may be administered in even smaller quantities to increase the potency of the cells. In some embodiments, at least about 1 × 10 8 pcs, about 2 × 10 8 pcs, about 3 × 10 8 pcs, about 4 × 10 8 pcs, about 5 × 10 8 pcs, about 1 × 10 9 pcs, about 5 × 10 9 pcs, about 1 × 10 10 pcs, about 5 × 10 10 pcs, about 1 × 10 11 pcs, about 5 × 10 11 pcs, about 1 × 10 12 The engineered T cells (e.g., CAR-T cells) of one or more of the themes disclosed in the present invention. The precise determination of how much can be considered an effective dose can be based on individual factors of each subject, including their body shape, age, sex, weight, and the disease of a particular subject. Those skilled in the art can easily determine the dosage based on the present disclosure and the knowledge in the art. In general, engineered T cells (e.g., CAR-T cells) are administered at a dose that is non-toxic or tolerable to the patient.

[0140] 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.

[0141] Application

[0142] The engineered T cells (e.g., CAR-T cells) of the subject disclosed in the present invention can be provided to a subject systemically or directly to treat infection, autoimmune disease or tumor. In certain embodiments, engineered T cells (e.g., CAR-T cells) are directly injected into an organ of interest. Additionally or alternatively, engineered T 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 or compositions to increase cell production in vitro or in vivo.

[0143] The engineered T 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 6Cells. The cell group comprising engineered T cells (e.g., CAR-T cells) may include purified cell groups. Those skilled in the art can easily determine the percentage of engineered T 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 T 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 T 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.

[0144] In certain embodiments, the composition of the subject disclosed in the present invention includes a pharmaceutical composition comprising engineered T cells (e.g., CAR-T cells) and a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous (allogeneic). For example, engineered T cells (e.g., CAR-T cells) and compositions comprising the engineered T cells can be obtained from a subject and applied to the same subject or different compatible subjects. The peripheral blood-derived T 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.

[0145] Some embodiments of the present disclosure are further described below by examples. The examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure in any way. Therefore, the present disclosure is not limited to the specific embodiments described herein by examples. Example General experimental methods

[0146] Culture medium

[0147] 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 a basal cell culture medium. 20 IU / mL or 40 IU / mL of MACS GMP IL-2 (Miltenyi Biotec) was added to the basal cell culture medium.

[0148] SK-HEP-1-Luc and HepG2 cell culture medium: MEM, L-Gln (+) (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.), 1% 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 WakoPure Chemical Industries, Ltd.).

[0149] GSU-Luc cell culture medium: RPMI1640 (ThermoFisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.) and 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0150] Production of CAR-T cells

[0151] After thawing Leukopak (Hemacare), CD4+ and CD8+ cells were enriched by CliniMACS Prodigy (Miltenyi Biotech). The enriched cells were diluted to less than or equal to 2.0 × 10 in CAR-T cell culture medium. 6Cells / mL (pre-production raw material). Cell suspension: MACS GMP T-Cell TransACT (MiltenyiBiotec) = 17.5:1 was inoculated in a culture bag and cultured for about 48 hours (cell activation step). The activated cells were diluted in culture medium using a LOVO cell processing system (Fresenius Kabi) or a centrifuge 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 transduction process). 6 cells / cm 2 Inoculate culture flasks (G-Rex, Wilson Wolf) and optionally culture for 3 to 7 days to produce CAR-T cells. After culture, CAR-T cells are cryopreserved in Cryostor CS-10 freezing medium (BioLifeSolutions) as final products. The CAR gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the IL-7 gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 3, and the CCL19 gene used has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 4. See Table 1.

[0152] CAR-T cell production using CliniMACS Prodigy

[0153] After thawing Leukopak (Hemacare), CD4+ and CD8+ cells were enriched in CliniMACS Prodigy (Miltenyi Biotech). After enrichment, cells were diluted in CAR-T cell culture medium with MACS GMP T-Cell TransACT (Miltenyi Biotech) and cultured for about 48 hours before retroviral transduction with or without MCAS GMP Vectofusin-1 (Miltenyi Biotech) for 1 day in CliniMACS Prodigy. On day 3, transduced cells were recovered from CliniMACS Prodigy and then cultured at less than 2.2×10 6 cells / cm 2Inoculate in culture flasks (G-Rex, Wilson Wolf) and optionally culture for 3 to 7 days to produce CAR-T cells (final product). After culture, CAR-T cells are cryopreserved in CryoStor CS10 freezing medium (BioLife Solutions) as the final product.

[0154] Determination of T cell transduction rate and immunophenotype using flow cytometry

[0155] CAR targeting antigen was used to measure the CAR introduction rate introduced into T cells on 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. 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.

[0156] Proliferation assay

[0157] The frozen CAR-T cells were thawed and cultured in CAR-T cell culture medium for 3 days. After culture, the cells were counted.

[0158] Amplification assay

[0159] The frozen CAR-T cells were thawed and cultured in CAR-T cell culture medium without IL-2 for 7 days. After culture, the cells were counted.

[0160] Cytotoxicity assay

[0161] CAR-T cells (effector cells) and SK-HEP-1 cells expressing luciferase were inoculated in a cell culture plate containing SK-HFP-1 medium at a ratio of effector cells: target cells = 5: 1 (0.1M: 20K per well). After 24 hours of incubation, the effector cells were collected and counted with an NC-200 instrument. In addition, the luciferase activity derived from the target cells was measured to determine the target cell killing rate. All collected effector cells were transferred to a new cell culture plate. Before inoculating the effector cells, 20K cells / well of target cells were re-inoculated into a new plate. After 24 hours of re-inoculation, the cell number and luciferase activity were re-measured. These steps were repeated at 24 hours, 48 ​​hours, 72 hours, and 96 hours, and the effector cell number and cytotoxic activity were measured.

[0162] In vivo experiments

[0163] GSU-Luc cells or HepG2 were subcutaneously inoculated into NSG mice (Charles River Japan). Seven days after inoculation, CAR-T cells or PBS were intravenously administered to mice. In order to analyze tumor growth by measuring the luciferase activity of GSU-Luc cells, D-luciferin (Promega) was injected intraperitoneally, and luminescence was measured by IVIS imaging (Summit Pharmaceutical International). To determine the tumor volume of HepG2, a caliper was used. Example 1: The activation process is crucial for CAR-T cell manufacturing

[0164] CAR-T cells expressing IL-7 gene and CCL19 gene were produced as described above (using CAR as described in SEQ ID NO: 1). CAR-T manufacturing methods were compared in two experiments using donor-3 and donor-4, respectively, in which the activation step was skipped (method 1), the transduction step was performed during the activation step (method 2), and the transduction method was performed 1 day after 48 hours of activation (method 3) ( Figure 1A ). CAR-T cells manufactured as described in method 1 could not be produced at all. However, CAR-T cells manufactured by both method 2 and method 3 could be produced without any problems ( Figure 1B and Figure 1C ). Example 2: In vitro characterization of CAR-T cells during the expansion step

[0165] CAR-T cells expressing IL-7 gene and CCL19 gene were produced as described above (using CAR as described in SEQ ID NO: 1). CAR-T cells were harvested on day 6 to day 8 ( Figure 2A During the manufacturing period, the number of cells increases day by day ( Figure 2B). CAR-T cells from day 6 to day 8 were used to analyze cell proliferation capacity and T cell phenotype ( Figure 2C to Figure 2G ). As the number of expansion days increased, both the proliferative capacity and pre-exhausted cells (including stem cell memory T cells and central memory T cells) decreased. These results indicate that the cell quality of CAR-T cells (including proliferative capacity and the percentage of each T cell phenotype) changed from day 6 to day 8. Example 3: In vivo study of CAR-T cells from day 6 to day 8

[0166] Seven days after HepG2 was inoculated into NSG mice, the CAR-T cells produced in Example 2 were administered. In the xenograft model inoculated with HepG2, the CAR-T cells produced with a shorter expansion cycle showed excellent anti-tumor efficacy ( Figure 3 ). Example 4: In vitro characterization of CAR-T cells on days 3 and 7

[0167] CAR-T cells expressing IL-7 and CCL19 genes were produced as described above (using the CAR described in SEQ ID NO: 2). For the preparation of day 3 cells, the expansion step was skipped ( Figure 4A CAR protein expression in day 3 cells measured by flow cytometry was lower than that in day 7 cells ( Figure 4B In another aspect, CAR mRNA expression in day 3 cells measured as copy number by qPCR is the same as that in day 7 cells ( Figure 4C ). This indicates that the transduction efficiency did not change between day 3 cells and day 7 cells. A shift in T cell phenotype during CAR-T manufacturing was observed. Stem cell memory T cells peaked between day 2 and day 3 ( Figure 4D and Figure 4E Compared with day 7 cells, day 3 cells showed higher expansibility ( Figures 4F to 4G ) and cytotoxicity ( FIG. 4H to FIG. 4I ). Example 5: In vivo study of CAR-T cells on days 3 and 7

[0168] CAR-T cells produced in Example 4 were administered 7 days after GSU-LUC inoculation into NSG mice. Day 3 cells at 0.2 M and 0.5 M (produced without an ex vivo expansion step) showed in vivo efficacy. However, day 7 cells at 0.2 M and 1 M did not show in vivo efficacy ( Figure 5A and Figure 5B ). Example 6: In vivo studies of day 3 and day 7 CAR-T cells manufactured in a single vessel by CliniMACS Prodigy

[0169] CAR-T cells expressing IL-7 gene and CCL19 gene were produced with or without CliniMACS Prodigy (using CAR as described in SEQ ID NO: 2). The efficacy of CAR-T cells produced in a single container using CliniMACS Prodigy showed an enhanced trend compared with CAR-T cells produced without CliniMACS Prodigy ( Fig. 6A and Figure 6B ). Example 7: In vitro characterization of CAR-T cells (non-armored) on days 3 and 7

[0170] Non-armored CAR-T cells (not expressing exogenous cytokine or chemokine genes) were produced as described above (using the CAR described in SEQ ID NO: 2). For preparation of day 3 cells, the expansion step was skipped ( Fig. 7A CAR protein expression in day 3 cells measured by flow cytometry was lower than that in day 7 cells ( Figure 7B In another aspect, the expression of CAR mRNA in cells at day 3 measured as copy number by qPCR is the same as that in cells at day 7 ( Figure 7C ). These results indicate that there was no change in transduction efficiency between day 3 cells and day 7 cells. The T cell phenotypes of day 3 cells and day 7 cells were observed. Day 3 cells showed a higher population of stem cell memory T cells (Tscm) compared to day 7 cells ( FIG. 7D to FIG. 7E In addition, day 3 cells showed higher expansibility compared to day 7 cells ( Figure 7F ). Example 8: In vivo study of CAR-T cells (non-armored) on days 3 and 7

[0171] GSU cells were subcutaneously inoculated into NSG mice. Seven days after inoculation, 1×10 5 Day 3 and day 7 non-armored CAR-T cells manufactured according to Example 7 or PBS were intravenously administered to mice. Day 3 non-armored CAR-T cells (produced without an ex vivo expansion step) showed in vivo efficacy, but day 7 non-armored CAR-T cells did not show in vivo efficacy ( Figure 8 ). Example 9: In vitro characterization of day 3 and day 7 CAR-T cells produced using CliniMACS Prodigy

[0172] According to the method described in paragraph

[00150] "Production of CAR-T cells using CliniMACS Prodigy", CAR-T cells expressing IL-7 gene and CCL19 gene are produced using CliniMACS Prodigy. A CAR comprising the amino acid sequence of SEQ ID NO: 2 is used. Fig.9A The scheme of the CAR-T cell manufacturing method is shown. CAR-T cells were manufactured using CliniMACS Prodigy (day 3 CAR-T cells). Day 3 CAR-T cells were further cultured in G-Rex culture bottles for 4 days (day 7 CAR-T cells). FIG. 9B to FIG. 9C The percentage of cells expressing CAR is shown ( Fig. 9B ) and the copy number in the cell ( Fig. 9C ) results. FIG. 9D to FIG. 9E The changes in T cell phenotype during manufacturing are shown in CD4+ and CD8+ T cells, respectively. Day 3 cells showed higher populations of central memory T cells and stem cell memory T cells compared to day 7 cells. Example 10: Day 3 and Day 7 CAR-T cells manufactured using CliniMACS Prodigy with or without Vectofusin-1 during transduction

[0173] During transduction, CAR-T cells expressing IL-7 gene and CCL19 gene were produced using CliniMACS Prodigy with or without Vectofusin-1. CAR comprising the amino acid sequence of SEQ ID NO: 2 was used. Fig.10 Results showing the percentage of CAR-expressing cells produced with or without Vectofusion-1.

[0174] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing a T cell population expressing an exogenous gene product, the method comprising: (i) contacting the T cell population with a stimulatory agent, (ii) contacting the T cell population with a retroviral vector comprising a nucleic acid molecule encoding the exogenous gene product, thereby providing a T cell population expressing the exogenous gene, and (iii) collecting the T cell population expressing the exogenous gene product for storage or administration, in: The population of T cells expressing the exogenous gene product from step (iii) is not expanded, or is expanded by no more than 200% compared to the population of T cells at the beginning of step (i), as assessed by viable cell count.

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

3. The method of claim 1, wherein the stimulatory agent comprises a CD3 binding domain.

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

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

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

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

8. The method of claim 1, wherein step (ii) is not initiated until step (i) is completed.

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

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

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