Method for producing CAR-T cells

Abstract

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

Description

Cross - reference to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 433,693, filed on December 19, 2022, the content of which is incorporated herein by reference in its entirety. Background of the Invention

[0002] The present invention generally relates to improved methods for producing engineered immune cells and populations thereof, including T cells expressing chimeric antigen receptors (CAR - T cells).

[0003] The production of engineered immune cells (such as CAR - T cells) typically involves immune cell activation, followed by viral transduction and expansion. However, certain methods for activating and / or expanding engineered immune cells or populations thereof (such as CAR - T cells) can lead to their progressive maturation and result in a population - related loss of potent immune cell subsets (e.g., naive T cells or stem cell memory T cells) in the population. This ultimately reduces the long - term therapeutic efficacy of using engineered immune cells produced by such methods. However, T cell activation can be particularly important for transduction efficiency (e.g., by certain viral vectors that require proliferation to be taken up by the virus). Thus, there remains a need for improved methods for producing engineered immune cells and populations thereof that increase, for example, the transduction efficiency of immune cells, the number of engineered immune cells produced (e.g., after expansion), and / or the number of potent immune cell subsets in the population. Summary of the Invention

[0004] The present disclosure particularly provides methods for producing engineered immune cell populations, methods for increasing the population of naive T cell or stem cell memory T cell subsets, engineered immune cells produced by such methods, compositions comprising an immune cell population and a cytokine, and methods for increasing the γδT cell population.

[0005] In one aspect, the present disclosure provides a method for producing an engineered immune cell population, the method comprising: (i) culturing an immune cell population, (ii) contacting the immune cell population with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence to provide an engineered immune cell population, and (iii) harvesting the engineered immune cell population, wherein step (i) and / or step (ii) are performed at least in part in the presence of (a) IL - 15 and (b) at least one of IL - 1b and IL - 12.

[0006] In some embodiments, step (i) is performed in the absence of a stimulant comprising a CD3 - binding domain and / or a TCR - binding domain.

[0007] In some embodiments, step (i) and / or step (ii) are performed at least in part in the presence of IL-15, IL-1β, and IL-12.

[0008] In some embodiments, the population of immune cells comprises T cells.

[0009] In some embodiments, the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR.

[0010] In some embodiments, the nucleic acid molecule is a viral vector. In some embodiments, the viral vector is a retroviral vector.

[0011] In some embodiments, step (i) and / or step (ii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

[0012] In one aspect, the present disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells, (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing a population of engineered immune cells, (iii) culturing the population of engineered immune cells resulting from step (ii), and (iv) harvesting the population of engineered immune cells for storage or administration, wherein step (i), step (ii), and / or step (iii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12.

[0013] In some embodiments, step (i) is performed in the absence of a stimulant comprising a CD3-binding domain and / or a TCR-binding domain.

[0014] In some embodiments, step (i), step (ii), and / or step (iii) are performed at least in part in the presence of IL-15, IL-1β, and IL-12

[0015] In some embodiments, the population of immune cells comprises T cells.

[0016] In some embodiments, the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR.

[0017] In some embodiments, the nucleic acid molecule is a viral vector. In some embodiments, the viral vector is a retroviral vector.

[0018] In some embodiments, step (iii) results in the expansion of the engineered immune cell population.

[0019] In some embodiments, step (i), step (ii), and / or step (iii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

[0020] In one aspect, the present disclosure provides a method of increasing the population of naive T cells or stem cell memory T cell subsets, which comprises contacting an immune cell population with (a) IL-15 and (b) IL-1β and / or IL-12.

[0021] In one aspect, the present disclosure provides a composition comprising an immune cell population and (a) IL-15 and (b) IL-1β and / or IL-12. In some embodiments, the composition comprises IL-15, IL-1β, and IL-12.

[0022] In some embodiments, the immune cell population comprises engineered immune cells.

[0023] In some embodiments, the engineered immune cell population expresses a chimeric antigen receptor (CAR).

[0024] In some embodiments, the immune cell population comprises T cells.

[0025] In one aspect, the present disclosure provides a method of increasing the γδ T cell population, which comprises contacting the γδ T cell population with IL-12. In some embodiments, the method further comprises contacting the γδ T cell population with IL-15 or IL-1β. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A -1C is a graph showing the results of Example 1. The cytokine mixture induced T cell aggregation. (1A) T cell morphology on day 2. (1B-1C) Time-lapse images and total aggregate target area of T cells from 0 to 96 hours after the cytokine mixture or IL-2 supplemented with TransACT™ (N = 8).

[0027] Figures 2A - 2B is a graph showing the results of Example 2. Genetically engineered T cells were prepared using the cytokine mixture. (2A) Microscopic image; (2B) mCherry-positive cells after manufacture.

[0028] Figures 3A - 3BA chart showing the results of Example 3. Potential cytokines were evaluated to remove individual cytokines from the cytokine mixture. (3A) Cell number, and (3B) mCherry-positive cells (N = 4, *p < 0.05). The p-value was calculated by one-way ANOVA and subsequent Tukey method.

[0029] Figure 4A -4F A chart showing the results of Example 4. By design of experiments (DOE) analysis, the key cytokines required for manufacturing genetically engineered T cells were determined. (4A) Culture conditions; (4B) Cell number; (4C) mCherry-positive cells; and (4D) Number of mCherry-positive cells after manufacturing. (4E) Correlation between predicted mCherry-positive cells (X-axis) and actual mCherry-positive cells (Y-axis) by Jackknife method; (4F) LogWorth of each source calculated by -log(p-value) of likelihood ratio test (N = 4).

[0030] Figures 5A - 5B A chart showing the results of Example 5. IL-1b, IL-12, and IL-15 promote the manufacturing of genetically engineered T cells. (5A) Cell number, and (5B) Number of mCherry-positive cells after manufacturing (N = 4, *p < 0.0002). The p-value was calculated by one-way ANOVA and subsequent Tukey method.

[0031] Figures 6A - 6B A chart showing the results of Example 6. When manufacturing CAR-T cells using IL-1b, IL-12, and IL-15, the naive / stem cell memory population was significantly higher compared to using IL-2 and TransACT™. (6A) CAR positivity rate, and (6B) Naive / stem cell memory population. The naive / stem cell memory population was determined as the CD45RA-positive and CCR7-positive population.

[0032] Figures 7A - 7B A chart showing the results of Example 7. IL-1b, IL-12, and IL-15 all slightly induced T cell proliferation. After culturing with IL-1b, IL-12, and IL-15, and IL-2 and TransACT™ respectively for 2 days, (7A) Cell number, and (7B) Cell cycle (N = 4, *p < 0.001). The p-value was calculated by one-way ANOVA and subsequent Tukey method.

[0033] Figures 8A - 8BChart showing the results of Example 8. IL-1b, IL-12, and IL-15 all slightly increased cell size. After culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively, (8A) cell diameter, (8B) FSC, and (C) SSC (N = 4, *p < 0.0005). p-values were calculated by one-way ANOVA and subsequent Tukey method.

[0034] Figure 9 Shows CD3 expression after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively.

[0035] Figures 10A - 10D Shows the expression of activation markers after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively. (10A) HLA-DR positive cells, (10B) CD25 positive cells, (10C) CD38 positive cells, and (10D) CD69 positive cells.

[0036] Figures 11A - 11D Shows the expression of senescence markers after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively. (11A) CTLA-4 positive cells, (11B) LAG-3 cells, (11C) PD-1 positive cells, and (11D) TIM-3 positive cells.

[0037] Figures 12A - 12C Shows the expression of exhaustion markers after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively. (12A) CD28 positive cells, (12B) CD57 positive cells, and (12C) KLRG1 positive cells.

[0038] Figures 13A - 13D Shows the amount of metabolites in the supernatant after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively. (13A) Glucose, (13B) Lactate, (13C) Glutamine, and (13D) NH4++.

[0039] Figure 14 Shows the results of the glucose uptake assay. The Y-axis shows the amount of glucose absorbed into the cells after treatment with the glucose uptake probe.

[0040] Figure 15 Shows the amount of Ca++ in the supernatant after culturing with IL-1b, IL-12, and IL-15, as well as IL-2 and TransACT™ for 2 days, respectively.

[0041] Figure 16 Shows the long-term expansion of CAR-T cells.

[0042] Figure 17 A graph showing the results of Example 12. IL-1b, IL-12, and IL-15 promoted the production of genetically engineered γδT cells.

[0043] Figures 18A - 18B Shows the co-culture assay of CAR-T cells. The cell number (Figure 18A) and killing activity (Figure 18B) after 24-hour co-culture were confirmed.

[0044] Figure 19 Shows the results of in vivo assays using xenograft models.

[0045] Figure 20 Shows the cell concentration of γδT cells cultured with IL-12 or IL-15. Detailed Description

[0046] It should be understood that certain aspects, modes, embodiments, variations, and features of the present method will be described below at different levels of detail in order to provide a substantial understanding of the present technology.

[0047] The present disclosure is not limited to the specific embodiments described in this application, which are intended to be illustrative of the various aspects of the present disclosure. All various embodiments of the present disclosure will not be described herein. Many modifications and variations can be made to the present disclosure without departing from its spirit and scope, which will be apparent to those skilled in the art. Methods and devices that are functionally equivalent to those described herein, in addition to the methods and devices listed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only to the terms of the appended claims and the full scope of equivalents to such claims.

[0048] In practicing the present technology, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology, and recombinant DNA are used. See, for example, 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., N.Y.)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 Edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins 10 eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir’s Handbook of Experimental Immunology.

[0049] The production of engineered immune cells (e.g., CAR-T cells) generally involves immune cell activation, followed by viral transduction and expansion. However, certain methods for activating and / or expanding engineered immune cells or populations thereof (e.g., CAR-T cells) can lead to their progressive maturation and result in a population-related loss of a potent subset of immune cells in the population (e.g., naive T cells or stem cell memory T cells). This ultimately reduces the long-term efficacy of treatments using engineered immune cells produced by such methods. However, T cell activation can be important, particularly for transduction efficiency (e.g., by certain viral vectors that require proliferation to be taken up by the virus). Accordingly, there remains a need for improved methods of producing engineered immune cells and populations thereof that increase, for example, the transduction efficiency of immune cells, the number of engineered immune cells produced (e.g., after expansion), and / or the number of a potent subset of immune cells in the population.

[0050] Embodiments particularly relate to an improved method for producing engineered immune cells ( For example CAR-T cells) for cell therapy. Specifically, the improved method involves (i) culturing a population of immune cells, (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence to provide a population of engineered immune cells, and (iii) harvesting the population of engineered immune cells, wherein step (i) and / or step (ii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12. In some embodiments, step (i) is performed in the absence of an agonist comprising a CD3-binding domain (e.g., in the absence of immune cell activation, such as T cell activation). Unexpectedly, the inventors of the present technology have found that the presence of (a) IL-15 and (b) IL-1β and / or IL-12 at least in part during step (i) and / or step (ii) can significantly increase transduction efficiency, cell number, and / or the population of potent T cells produced (e.g., naive T cells, stem cell memory T cells). Definitions

[0051] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide general definitions of many of the terms used in this disclosure for those of ordinary skill in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings given to them below. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit this disclosure.

[0052] 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.

[0053] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" can mean within 3 or more 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, especially with respect to biological systems or processes, the term can mean within an order of magnitude of a value, within 5-fold, or within 2-fold.

[0054] As used herein, the term "administering" an agent to a subject includes any route by which the agent is introduced or delivered to the subject to perform its intended function. Administration can be carried out 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.

[0055] As used herein, the term "activated" refers to a state in which a T cell has been sufficiently stimulated to induce cytokine production, detectable effector function, and / or detectable cell proliferation.

[0056] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a whole immunoglobulin derived from a natural source or a recombinant source, or an immunoreactive portion of a whole immunoglobulin. The antibodies in the present disclosure can exist in various forms, where 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, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0057] 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 ), camelid V HH domains, scFv antibodies, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein that comprises at least one antibody fragment containing a light-chain variable region and at least one antibody fragment containing a heavy-chain variable region, wherein the light-chain variable region and the heavy-chain variable region are continuously linked via a short flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the whole antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have V L and V H variable regions in any order. For example, with respect to the N-terminus and C-terminus of the polypeptide, an scFv can comprise V L -linker-V H or can comprise V H -linker-V L . The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences (e.g., connecting 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.

[0058] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation and generally determines the class to which the antibody belongs.

[0059] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0060] As used herein, the term "synthetic antibody" refers to an antibody generated using recombinant DNA technology, such as, for example, an antibody expressed by a phage as described herein. The term should also be construed to mean an antibody generated 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 techniques available and well known in the art.

[0061] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. Such an immune response may involve the production of antibodies or the activation of specific immune competent cells or both. One of ordinary skill in the art will understand that any macromolecule (including substantially all proteins or peptides) can be used as an antigen. In addition, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" as used herein. In addition, one of ordinary skill in the art will understand that an antigen need not be encoded solely 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 a polypeptide that elicits a desired immune response. In addition, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is obvious that an antigen can be synthetic or can be derived from a biological sample. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell fluid or a biological fluid.

[0062] According to the present disclosure, the term "auto-antigen" means any self-antigen that is misrecognized by the immune system as foreign. Auto-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0063] As used herein, the term "autoimmune disease" is defined as a disorder caused by an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive reaction to self-antigens (autoantigens). 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 (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, autoimmune vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, etc.

[0064] As used herein, the term "autologous" is intended to refer to any material that is derived from the same individual and will later be re-introduced into that individual. "Allogeneic" refers to a graft derived from different animals of the same species. "Xenogeneic" refers to a graft derived from animals of different species.

[0065] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods shall mean excluding other elements that are of any significance to the composition or method. "Consisting of" shall mean excluding more than trace elements of other ingredients of the claimed composition and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure. Thus, it is contemplated that the methods and compositions may include additional steps and components (comprising), or include insignificant steps and compositions (consisting essentially of), or include only the method steps or compositions (consisting of).

[0066] 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 through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0067] 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's efficacy in treating a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (subjects or samples that do not receive therapy or receive a placebo) are typically employed.

[0068] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., dendritic cells, B cells, macrophages, monocytes, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing signals that mediate T cell responses, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to peptide-loaded MHC molecules. The T cell responses include, but are not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to: CD7, B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2, B7-H3, B7-H4, B7-H6, B7-H7 / HHLA2, BTLA, 4-1BBL, OX40L, PDCD6, VISTA (B7-H5, PD-1H), GITRL (TNFSF18), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD27 ligand (TNFSF7), CD28, CD28H (IGPR-1), CD30L, CD40, CD70, CD83, CTLA-4, HLA-G, MICA, MICB, HVEM, TIM-1 / KIM-1 / HAVCR, TIM-4, semaphorin 4A, galectin-9, butyrophilin-like molecules 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), nectin-2 (CD112), nectin-3, PVRIG, TIGIT, LILRA3 (CD85e), LILRA4 (CD85g, ILT7), LILRB3 (CD85a, ILT5), LILRB2 (CD85d, ILT4), LILRB1 (CD85j, ILT2), LILRB4 (CD85k, ILT3), B cell activating factor (BAFF) (BLyS, TNFSF13B), TL1A (TNFSF15), TNF-α, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind Toll-like receptors (TLRs), and ligands that specifically bind to B7-H3. Costimulatory ligands particularly also encompass antibodies that specifically bind to costimulatory molecules present on T cells.

[0069] As used herein, the term "costimulatory molecule" or "costimulatory domain" refers to the portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include: CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD40L, PD-1, PDL-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H3, CTLA-4, GITR (TNFRSF18), TIM-1, TIM-2, TIM-3, TIM-4, CD160, CD200, CD300a (LMIR1), CD300d (LMIR4), CLECL1 (DCAL-1), DAP12, Dectin-1 (CLEC7A), DPPIV (CD26), EphB6, integrin 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 CD83. Thus, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4-1BB, other costimulatory domains are also contemplated for use with the CARs described herein. Inclusion of one or more costimulatory signaling domains can enhance the efficacy and expansion of T cells expressing the CAR receptor. The intracellular signaling domain and the costimulatory signaling domain can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0070] As used herein, a "costimulatory signal" is 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.

[0071] "Disease" is a health state of an animal in which the animal cannot maintain homeostasis and in which, if the disease is not improved, the health state of the animal will continue to deteriorate. In contrast, a "disorder" of an animal is a health state in which the animal is able to maintain homeostasis, but in which the health state of the animal is not as good as when there is no disorder. If left untreated, a disorder does not necessarily lead to a further deterioration of the animal's health state.

[0072] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0073] As used herein, "endogenous" refers to any substance that is from within or produced within an organism, cell, tissue, or system.

[0074] As used herein, the term "exogenous" refers to any substance that is introduced from outside or produced outside an organism, cell, tissue, or system.

[0075] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter of the specific nucleotide sequence.

[0076] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0077] 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 the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acid.

[0078] As used herein, the term "immune cell" refers to any cell that plays a role in the immune response of a subject. Immune cells are 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 an immune cell that has been genetically modified. As used herein, the term "natural immune cell" refers to an immune cell that naturally exists in the immune system.

[0079] As used herein, the term "isolated" refers to a change or removal from a natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting substances in 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. A substantially purified cell also refers to a cell that has been separated from other cell types that are normally associated with it in its natural state of existence. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term only refers to a cell that has been separated from the cells that are naturally associated with it in a natural state. In some embodiments, cells are cultured in vitro. In other embodiments, cells are not cultured in vitro.

[0080] As used herein, the term "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 RNAs can include introns.

[0081] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence such that the heterologous nucleic acid sequence is expressed. 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 the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where two protein-coding regions are to be joined, are in the same reading frame.

[0082] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intracisternal, intrathecal or intrasternal injection, administration or infusion techniques.

[0083] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or its cells (whether in vitro or in situ) that are suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0084] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Additionally, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein may be used interchangeably. Those skilled in the art generally know that nucleic acid is a polynucleotide, 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 recombinant libraries or cell genomes, using common cloning techniques (such as PCR, etc.), and by synthetic methods.

[0085] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are commonly also referred to in the art as, for example, peptides, oligopeptides and oligomers, and long chains, which are generally referred to in the art as proteins, and there are various types of proteins. "Polypeptide" includes, for example, bioactive 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.

[0086] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cellular synthetic machinery or an introduced synthetic machinery, which is required to initiate the 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 the gene product in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, results in the production of the gene product in a cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0087] As used herein, a "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 linked gene. Regulatory regions include nucleotide sequences that confer inducible (i.e., requiring a substance or stimulus to increase transcription) expression of a gene. Gene expression can increase when an inducer is present or its concentration increases. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus reduces transcription). Gene expression can decrease when a repressor is present or its concentration increases. Regulatory regions are known to affect, regulate, or control many in vivo biological activities, including cell proliferation, cell growth and death, cell differentiation, and immune regulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in an increase or decrease in gene transcription.

[0088] Specific examples of gene regulatory regions are promoters and enhancers. A promoter is a sequence that is located around the transcription start site or the translation start site, usually at the 5' of the translation start site. A promoter is typically within 1 Kb of the translation start site, but can be located further away, such as at 2 Kb, 3 Kb, 4 Kb, 5 Kb, or further from the translation start site, up to and including 10 Kb. Enhancers are known to affect gene expression when located at the 5' or 3' of a gene or when located in an exon or intron or as part of an exon or intron. Enhancers can also act at positions quite far from a gene, such as about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb, or further from the gene. In addition to the promoter region, regulatory regions include, but are not limited to: sequences that facilitate translation, splicing signals of introns, maintenance of the correct reading frame of a gene to allow in-frame translation of mRNA, stop codons, leader sequences, and fusion partner sequences, internal ribosome entry site (IRES) elements for creating polygenic or polycistronic messages, polyadenylation signals that provide appropriate polyadenylation of transcripts of the gene of interest and stop codons, and can optionally be included in an expression vector.

[0089] As used herein, the term "sample" refers to a clinical sample obtained from a subject. In certain embodiments, the sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchoalveolar lavage fluid (BAL), bronchial wash (BW), whole blood, body fluid, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0090] As used herein, the term "secretion" with respect to a polypeptide means the release of the polypeptide from a cell via the secretory pathway, through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses with the cytoplasmic membrane to release the protein extracellularly. Small molecules, such as drugs, can also be secreted by diffusing across the membrane to the extracellular side.

[0091] As used herein, the term "specifically binds" with respect to an antibody means that the antibody recognizes a particular antigen, but essentially does not recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity by 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 allelic forms of the antigen. Nevertheless, such cross-reactivity by itself does not change the classification of the antibody as a specific antibody. In some cases, the term "specifically binds" or "binds specifically" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a specific structure (e.g., an 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 an antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody. As used herein, the terms "specifically binds", "binds specifically to", or "is specific for" a particular molecule (e.g., an antigen) can be manifested, for example, by the molecule having a K d of about 10 −4 M, 10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M, 10 −10 M, 10 −11 M or 10 −12 M.

[0092] As used herein, the term "stimulate" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events (such as, but not limited to, signal transduction via the TCR / CD3 complex). Stimulation can mediate alterations in the expression of certain molecules, such as downregulation of TGFβ and / or reorganization of the cytoskeletal structure, etc.

[0093] 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.

[0094] As used herein, "stimulatory ligand" or "stimulator" means a ligand that, when present on an antigen-presenting cell (e.g., dendritic cell, B cell, macrophage, monocyte, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response of the T cell, the response including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulators are well known in the art and particularly encompass TCR-binding domains (e.g., peptide-loaded MHC class I molecules), CD3-binding domains (e.g., anti-CD3 antibodies), mannose receptor family-binding domains (e.g., anti-CD206 antibodies, anti-mannose-6-phosphate receptor (M6PR) antibodies), CD28-binding domains (e.g., superagonist anti-CD28 antibodies), CD2-binding domains (e.g., superagonist anti-CD2 antibodies), CD27-binding domains (e.g., superagonist anti-CD27 antibodies), CD30-binding domains (e.g., superagonist anti-CD30 antibodies), CD40L-binding domains (e.g., superagonist anti-CD40L antibodies), CD226-binding domains (e.g., superagonist anti-CD226 antibodies), 4-1BB-binding domains (e.g., superagonist anti-4-1BB antibodies), OX40-binding domains (e.g., superagonist anti-OX40 antibodies), and concanavalin A (ConA).

[0095] The term "therapeutically effective amount" refers to the amount of a subject compound that will elicit a biological or medical response of a tissue, system, or subject as sought by a researcher, veterinarian, medical doctor, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of the treated disorder or disease or to alleviate to some extent one or more signs or symptoms of the treated disorder or disease. A 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.

[0096] As used herein, the terms "transfection" or "transformation" or "transduction" or "introduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" or "introduced" cell is a cell that has been transfected, transformed, transduced, or introduced with exogenous nucleic acid. The cells include primary subject cells and their progeny.

[0097] As used herein, the term "co-therapy" refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes.

[0098] As used herein, the term "sequential" treatment usage refers to the administration of at least two active ingredients at different times, by the same or different routes of administration. More specifically, sequential usage means that one of the active ingredients is administered completely before the start of the administration of one or more active ingredients. Thus, one of the active ingredients can be administered within minutes, hours, or days before the administration of one or more other active ingredients. There is no concurrent treatment in this case.

[0099] As used herein, the term "T cell" includes naive T cells, memory T cells, activated T cells, anergic T cells, tolerogenic 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, T helper cells, cytotoxic T cells, central memory T cells, stem cell memory T cells, effector memory T cells (e.g., T EM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells (NKT), mucosa-associated invariant T cells, αβ T cells, double-negative T cells, and γδ ("γδ") T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. In certain embodiments, the T cells expressing CAR express Foxp3 to achieve and maintain a T regulatory phenotype. In some embodiments, CAR-T cells are any immune cells derived from pluripotent stem cells (e.g., induced pluripotent stem (iPS) cells).

[0100] "Treating" or "treatment" as used herein encompasses the treatment of a disease or disorder described herein in a subject (such as a human) and includes: (i) inhibiting the disease or disorder, i.e., preventing its development; (ii) alleviating the disease or disorder, i.e., causing the symptoms to subside; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. The therapeutic effects of treatment include, but are not limited to, inhibiting disease recurrence, 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 the prognosis.

[0101] As used herein, a "vector" is a replicable nucleic acid that, when transformed into a suitable host cell, can express one or more heterologous proteins. Vectors mentioned include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced by restriction digestion and ligation. Vectors mentioned also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells to amplify the nucleic acids or to express / display polypeptides encoded by the nucleic acids. Vectors generally remain episomal, but can be designed to achieve integration of a gene or part thereof into the chromosomes of the genome. Vectors can include viral vectors. A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or delivery means) the foreign gene into cells.

[0102] The viral vectors of the present technology can be retroviral vectors. One advantage provided by retroviral vectors is that they are capable of converting their single-stranded RNA genomes into double-stranded DNA molecules that stably integrate into the genome of the target cell. Thus, retroviral vectors can be used to permanently modify the host cell nuclear genome.

[0103] The retroviral vectors of the present technology can be derived from any member of the Retroviridae family, such as Spumavirus or Fomie virus (e.g., human and simian viruses), betaretrovirus (e.g., MMTV), gammaretrovirus (e.g., MLV), alpharetrovirus (e.g., ALV), deltaretrovirus (e.g., BLV and HTLV-1), lentivirus (e.g., HIV 1) and epsilonretrovirus (e.g., WDSV and WEHV1 / 2) or derivatives thereof.

[0104] Any method known to those skilled in the art for inserting a heterologous nucleic acid sequence into a vector (e.g., a retroviral vector) can be used to construct an expression vector containing a nucleic acid encoding any polypeptide provided herein. Chimeric antigen receptor (CAR)

[0105] A CAR is an engineered receptor that contains an extracellular domain and an intracellular domain. The extracellular domain contains an antigen-binding portion. In some embodiments, the extracellular domain also contains a hinge domain. In some embodiments, the intracellular domain or cytoplasmic domain contains a CD3ζ chain and / or a co-stimulatory signaling region. A co-stimulatory signaling region refers to a part of the CAR that contains the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule required for an efficient response of lymphocytes to an antigen in addition to an antigen receptor or its ligand.

[0106] Between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR, a linker or spacer domain can be incorporated. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to an extracellular or cytoplasmic domain in a polypeptide chain. The spacer domain can comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Antigen-binding portion

[0107] The selection of the antigen-binding portion depends on the type and amount of ligand that defines the target cell surface. For example, an antigen-binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for the antigen portion domain in the CARs of the subject matter disclosed herein include those associated with viral, bacterial, and parasitic infections (e.g., pathogen antigens), autoimmune diseases (e.g., autoantigens), and cancer cells (e.g., tumor-specific antigens or tumor-associated antigens).

[0108] In one embodiment, the CARs of the subject matter disclosed herein can be engineered to target a tumor antigen of interest by engineering a desired antigen-binding portion that specifically binds to an antigen on a tumor cell. A tumor antigen can be a protein produced by a tumor cell that elicits an immune response (e.g., a T cell-mediated immune response). The selection of the antigen-binding portion of the subject matter disclosed herein will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example: glioma-associated antigens, carcinoembryonic antigen (CEA), β-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-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostasin, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-1, IGF-II, IGF-I receptor, CA125, CA19-9, MUC-1, WT-1, glypican 3 (GPC3), and mesothelin.

[0109] In one embodiment, the tumor antigen comprises one or more cancer epitopes of antigens associated with a malignant tumor. 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 prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is the onco-fetal antigen such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes the true tumor-specific immunoglobulin antigen unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in 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 with limited success rates.

[0110] The types of tumor antigens referred to in the subject matter disclosed in the present invention can also be tumor-specific antigens (TSA) or tumor-associated antigens (TAA). TSA are unique to tumor cells and are not present on other cells in the body. TAA-related antigens are not unique to tumor cells; rather, under conditions where an immune tolerance state against the antigen cannot be induced, TAA-related antigens are also expressed on normal cells. The expression of tumor antigens can occur under conditions that enable the immune system to respond to the antigen. TAA 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 are expressed at much higher levels on tumor cells.

[0111] 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 resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include: TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, cMet, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-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 the 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.

[0112] Based on the desired antigen to be targeted, the CARs of the subject matter disclosed herein can be engineered to include an appropriate antigen-binding portion that is specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding portion incorporated into the CARs of the present technology. Transmembrane domain

[0113] Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0114] The transmembrane domain can be derived from a natural source or a synthetic source. In the case where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present technology can be derived from (i.e., at least include one or more transmembrane regions of the following) the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or from immunoglobulins such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker (preferably having a length between 2 and 10 amino acids) can form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. Cytoplasmic domain

[0115] The cytoplasmic domain or intracellular signaling domain of the CAR of the subject matter disclosed by the present invention is responsible for activating at least one normal effector function of the immune cell in which the CAR is located. The term "effector function" refers to the specialized function of a cell. For example, the effector functions of T cells can be cytolytic activity or helper activity (including the secretion of cytokines). Thus, the term "intracellular signaling domain" refers to the part of a protein that transduces the signal of the effector function and directs the cell to perform the specialized function. Although usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. In the case of using a truncated portion of the intracellular signaling domain, such truncated portion can be used in place of the full chain as long as the truncated portion transduces the signal of the effector function. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the signal of the effector function.

[0116] Examples of intracellular signaling domains for the CAR of the subject matter disclosed by the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors, which cooperate to initiate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capacity.

[0117] It is known that the signal generated solely through the TCR is not sufficient to fully activate T cells, and a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two different classes 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 a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0118] The primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are called immunoreceptor tyrosine activation motifs or ITAMs.

[0119] Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in the subject matter disclosed by the present invention 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 by the present invention contains a cytoplasmic signaling sequence derived from CD3ζ.

[0120] In some embodiments, the cytoplasmic domain of the CAR can be designed to include the CD3ζ signaling domain itself or a combination thereof with any other desired cytoplasmic domain useful in the context of the CARs of the present technology. For example, the cytoplasmic domain of the CAR can include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the part of the CAR that contains the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc.

[0121] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CARs of the subject matter disclosed herein can be linked to each other in a random or specified order. Optionally, short oligopeptide or polypeptide linkers (preferably between 2 and 10 amino acids in length) can form the linkage. A glycine-serine dyad provides a particularly suitable linker.

[0122] 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. Method for producing engineered immune cells of the present technology

[0123] In one aspect, the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells (culturing step), (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence to provide a population of engineered immune cells (transduction step), and (iii) harvesting the population of engineered immune cells, wherein step (i) and / or step (ii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12.

[0124] In some embodiments, step (i) and / or step (ii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

[0125] In some embodiments, the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing a population of engineered immune cells that express the CAR (e.g., CAR-T cells).

[0126] The engineered immune cells of the disclosed subject matter can be T cells. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the disclosed subject matter can be any type of T cell, including but not limited to naive T cells, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell memory T cells (or stem-like memory T cells) and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, γδ T cells, and αβ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. The engineered immune cells can also be any T cells (iPS-derived T cells) derived from pluripotent stem cells (e.g., induced pluripotent stem (iPS) cells).

[0127] In some embodiments, the T cells are αβ T cells, γδ T cells, or iPS-derived T cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the T cells are γδ T cells. In some embodiments, the T cells are iPS-derived T cells.

[0128] The population of immune cells of the present technology can be obtained from any source known in the art, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present technology, the population of immune cells can be obtained from a blood unit collected from a subject using various techniques known to those skilled in the art (e.g., apheresis). In some embodiments, the population of immune cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes.

[0129] Isolation procedures include but are not limited to density gradient centrifugation (e.g., using a PERCOLL ® gradient); countercurrent centrifugal elutriation; resetting; coupling with particles that modify cell density; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents conjugated or used in combination with mAbs, including but not limited to complement and cytotoxins; and panning, elutriation, or any other convenient technique using antibodies attached to a solid matrix (e.g., plates, chips).

[0130] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of complexity, such as multiple color channels, low-angle blunt light scatter detection channels, impedance channels, and fluorescence-activated cell sorting (FACS).

[0131] In some embodiments, specific immune cell subsets, such as αβ T cells or γδ T cells, can be further separated by positive or negative selection techniques (e.g., using selection techniques well known to those skilled in the art).

[0132] In some embodiments, prior to step (i), T cells expressing CD4 and / or CD8 in the immune cell population can be enriched. These selection techniques are well known to those skilled in the art. By way of a non-limiting example, CD4 cells can be enriched by negative selection by treating the cell mixture with a monoclonal antibody mixture comprising antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. + In certain embodiments, regulatory T cells can be depleted by anti-CD25 conjugated beads.

[0133] In some embodiments, optionally after a washing step, the immune cells can be frozen (e.g., before culturing, after harvesting). The freezing and subsequent thawing steps can provide a more uniform product by removing granulocytes and to some extent monocytes from the cell population. After a washing step (e.g., 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, cryopreserved cells can be thawed and washed and allowed to sit at room temperature for about one hour prior to culturing (e.g., step (i) as described herein).

[0134] An immune cell population can be collected at any time point desired for subsequent activation, transduction, expansion, and / or formulation and used for cell therapy of any disease and / or disorder that can benefit from immune cell therapy. In some embodiments, a blood sample or apheresis component can be obtained from a generally healthy subject. In some embodiments, a blood sample or apheresis component is obtained from a generally healthy subject at risk of developing a disease but not yet having developed the disease, and the cells of interest are isolated and cryopreserved for later use. In some embodiments, a sample can be collected from a patient shortly after being diagnosed with a specific disease as described herein but prior to any treatment. In another embodiment, cells can be isolated from a blood sample or apheresis component of a subject before, during, or after any relevant treatment modality, the treatment including but not limited to treatment with an agent such as an antiviral agent, chemotherapy, radiation therapy, immunotherapy (e.g., checkpoint inhibitors), or immunosuppressants.

[0135] In some embodiments of the present technology, an immune cell population can be obtained directly from a patient after treatment. In this regard, it has been observed that after certain cancer treatments (especially those using drugs that damage the immune system), shortly after treatment, during the period when the patient is typically recovering from the treatment, the quality of the immune cells obtained (e.g., T cells) can be optimal or improved for ex vivo manipulation (e.g., transduction, expansion). Culturing step

[0136] The method of producing the engineered immune cell population described herein can include a culturing step of culturing an immune cell population. In some embodiments, the culturing step is carried out at least in part in the presence of IL-12 and / or IL-15. In some embodiments, the culturing step is carried out at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12. In some embodiments, the culturing step is carried out at least in part in the presence of IL-12 and IL-15. In some embodiments, the culturing step is carried out at least in part in the presence of IL-12 and IL-1β. In some embodiments, the culturing step is carried out at least in part in the presence of IL-15, IL-1β, and IL-12.

[0137] In some embodiments, the culturing step is carried out for about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 12 hours to about 72 hours, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 72 hours, about 24 hours to about 60 hours, or about 24 hours to about 48 hours. In some embodiments, the culturing step is carried out for about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours. In some embodiments, the culturing step is carried out for about 12 hours. In some embodiments, the culturing step is carried out for about 24 hours. In some embodiments, the culturing step is carried out for about 48 hours. In some embodiments, the culturing step is carried out for about 72 hours.

[0138] In some embodiments, the culturing step is carried out at about 30 °C to about 40 °C. In some embodiments, the culturing step is carried out at about 30 °C. In some embodiments, the culturing step is carried out at about 32 °C. In some embodiments, the culturing step is carried out at about 35 °C. In some embodiments, the culturing step is carried out at about 37 °C. In some embodiments, the culturing step is carried out at about 39 °C.

[0139] The production of engineered immune cells (e.g., CAR-T cells) generally involves immune cell activation. However, many methods for activating engineered immune cells (such as CAR-T cells) can lead to their gradual maturation and result in the associated loss of populations of potent immune cell subsets (e.g., naive T cells, stem cell memory T cells). This ultimately reduces the long-term therapeutic efficacy of using engineered immune cells. Immune cells (such as T cells) can be activated by contacting the immune cells with a stimulant. Thus, the methods of the present disclosure can be carried out in the absence of a stimulant (e.g., a stimulant comprising a CD3 binding domain). In some embodiments, step (i) (e.g., the culturing step) of the methods described herein is carried out in the absence of a stimulant comprising a CD3 binding domain. Transduction step

[0140] Methods for producing the engineered immune cell populations described herein can include a transduction step of contacting an immune cell population (e.g., T cells) with a nucleic acid molecule (e.g., a viral vector) comprising a nucleotide sequence encoding a heterologous amino acid sequence. In some embodiments, the transduction step is performed at least in part in the presence of IL-12 and / or IL-15. In some embodiments, the transduction step is performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12. In some embodiments, the transduction step is performed at least in part in the presence of IL-12 and IL-15. In some embodiments, the transduction step is performed at least in part in the presence of IL-12 and IL-1β. In some embodiments, the transduction step is performed at least in part in the presence of IL-15, IL-1β, and IL-12.

[0141] The nucleic acid molecule comprising a nucleotide encoding a heterologous amino acid sequence can be based on any RNA or DNA vector known in the art. Methods for introducing nucleic acid molecules into host cells are known to those of skill in the art. For example, nucleic acid molecules can be transferred into host cells by physical, chemical, or biological methods.

[0142] Physical methods for introducing nucleic acid molecules into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.

[0143] Biological methods for introducing nucleic acid molecules of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus type I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362. In some embodiments, the nucleic acid molecule is a viral vector (e.g., a retroviral vector). In some embodiments, the nucleic acid molecule is a retroviral vector.

[0144] Chemical means for introducing nucleic acid molecules into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0145] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid formulations are considered for introducing nucleic acid molecules into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid molecules can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped within liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, included as a suspension within lipids, included within micelles or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles or with a "collapsed" structure. They can also be merely dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be natural lipids or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm and the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0146] Suitable lipids for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform / methanol can be stored at a temperature of about -20°C. Since chloroform evaporates more readily than methanol, chloroform is used as the sole solvent. "Liposome" is a general term that encompasses a variety of single and multi-layer lipid vehicles formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in an excess of aqueous solution, they spontaneously form. The lipid components undergo self-rearrangement before forming a closed structure and enclose water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicular structures in solution are also encompassed. For example, lipids can assume a micellar structure or exist only in the form of non-uniform aggregates of lipid molecules. Liposome transfection amine (lipofectamine)-nucleic acid complexes are also considered.

[0147] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence of a specific peptide by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents falling within the scope of the present invention.

[0148] Retroviral vectors have been particularly well developed and have been used in a clinical setting (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Patent No. 5,399,346). In some embodiments, to perform an initial genetic modification of immune cells (e.g., T cells) to produce engineered immune cells (e.g., CAR-T cells), transduction is carried out using a retroviral vector comprising a nucleotide molecule encoding a heterologous amino acid sequence. For example, a polynucleotide encoding a CAR can be cloned into a retroviral vector, and expression can be driven by its endogenous promoter, the retroviral long terminal repeat, or an alternative internal promoter. To perform a subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) has also proven effective. A combination of a retroviral vector and a suitable packaging cell line is also suitable, wherein the capsid protein will have the function of infecting human cells. There are various cell lines 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, e.g., particles pseudotyped with VSVG, RD114, or GALV envelopes and any other particles known in the art.

[0149] Possible transduction methods also include direct co - culture of immune cells (e.g., T cells) with producer cells, e.g., by the method of Bregni et al., Blood 80: 1418 - 1422 (1992), or culture only with viral supernatant or with concentrated vector stock solution (with or without appropriate growth factors and polycations), e.g., by the methods of Xu et al., Exp. Hemat. 22:223 - 230 (1994); and Hughes et al., J. Clin. Invest. 89: 1817 (1992). In some embodiments, a population of immune cells (e.g., T cells) is contacted with a retroviral vector in the presence of a soluble additive of a cationic amphiphilic peptide (e.g., Vectofusin - 1).

[0150] In some embodiments, the retroviral vector expressing the CAR can be an oncoretroviral vector, a gamma - retroviral vector, a lentiviral vector, or a spumaretroviral vector. In some embodiments, the retroviral vector can be a gamma - retroviral vector. In some embodiments, the gamma - retroviral vector is selected from the pMSGV vector, the pMSCV vector, the pSFG vector, or a combination of any two or more thereof.

[0151] In one embodiment, an immune cell population (e.g., T cells) is contacted with a nucleic acid molecule (e.g., a retroviral vector) comprising a nucleotide molecule encoding a heterologous amino acid sequence (e.g., a CAR or a fluorescent protein). The contacting can be carried out for about 1 hour to about 72 hours, such as about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours. In some embodiments, the immune cells can be contacted with a nucleic acid molecule (e.g., a retroviral vector) comprising a heterologous amino acid sequence (e.g., a CAR) for about 16 hours to 28 hours, such as 24 hours. Culture conditions

[0152] Conditions suitable for immune cell culture (e.g., immune cell culture in the culturing and / or transduction steps) include a suitable 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), certain cytokines, growth factors, or additives known to those skilled in the art for cell growth.

[0153] Other additives for cell growth include but are not limited to surfactants, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The culture medium can 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 T cell expansion xenogeneic-free medium, ImmunoCult TM -XF T cell expansion medium, ExCellerate human T cell expansion medium, Stemline T cell expansion medium, CAR T cell medium, TexMACS TM Medium, Corning lymphocyte serum-free medium, Corning 88-581-CM medium, CellGenix T cell medium, SmarT TM T cell expansion medium, StemSpan TM Serum-free expansion medium and OptiPEAK T lymphocyte XPR (e.g., L-glutamine), supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma, e.g., CTS™ Immune Cell SR) or a defined set of hormones, and / or cytokines in an amount sufficient to allow T cell growth and / or expansion, and / or antibiotics (e.g., streptomycin) that can be used in the culture method. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., room temperature or 37 °C) and atmosphere (e.g., air plus 5% CO2).

[0154] In any of the above embodiments, there is (a) IL-15 and (b) IL-1b and / or IL-12. In any of the above embodiments, IL-15, IL-1b, and IL-12 are present. In any of the above embodiments, IL-12 and / or IL-15 is present. In any of the above embodiments, IL-12 and IL-15 are present. In any of the above embodiments, IL-12 and IL-1b are present.

[0155] In any of the above embodiments, IL-15 is present at the following concentrations: about 1 to about 100 μg / mL, about 1 to about 90 μg / mL, about 1 to about 80 μg / mL, about 1 to about 70 μg / mL, about 1 to about 60 μg / mL, about 1 to about 50 μg / mL, about 5 to about 100 μg / mL, about 5 to about 90 μg / mL, about 5 to about 80 μg / mL, about 5 to about 70 μg / mL, about 5 to about 60 μg / mL, about 5 to about 50 μg / mL, about 10 to about 100 μg / mL, about 10 to about 90 μg / mL, about 10 to about 80 μg / mL, about 10 to about 70 μg / mL, about 10 to about 60 μg / mL or about 10 to about 50 μg / mL. In any of the above embodiments, IL-15 is present at the following concentrations: about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL or about 50 μg / mL. In any of the above embodiments, IL-15 is present at a concentration of about 5 μg / mL. In any of the above embodiments, IL-15 is present at a concentration of about 10 μg / mL. In any of the above embodiments, IL-15 is present at a concentration of about 25 μg / mL. In any of the above embodiments, IL-15 is present at a concentration of about 50 μg / mL.

[0156] In any of the above embodiments, IL-1β is present at the following concentrations: about 1 to about 100 μg / mL, about 1 to about 90 μg / mL, about 1 to about 80 μg / mL, about 1 to about 70 μg / mL, about 1 to about 60 μg / mL, about 1 to about 50 μg / mL, about 5 to about 100 μg / mL, about 5 to about 90 μg / mL, about 5 to about 80 μg / mL, about 5 to about 70 μg / mL, about 5 to about 60 μg / mL, about 5 to about 50 μg / mL, about 10 to about 100 μg / mL, about 10 to about 90 μg / mL, about 10 to about 80 μg / mL, about 10 to about 70 μg / mL, about 10 to about 60 μg / mL, or about 10 to about 50 μg / mL. In any of the above embodiments, IL-1β is present at the following concentrations: about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, or about 50 μg / mL. In any of the above embodiments, IL-1β is present at a concentration of about 5 μg / mL. In any of the above embodiments, IL-1β is present at a concentration of about 10 μg / mL. In any of the above embodiments, IL-1β is present at a concentration of about 25 μg / mL. In any of the above embodiments, IL-1β is present at a concentration of about 50 μg / mL.

[0157] In any of the above embodiments, IL-12 is present at the following concentrations: about 1 to about 100 μg / mL, about 1 to about 90 μg / mL, about 1 to about 80 μg / mL, about 1 to about 70 μg / mL, about 1 to about 60 μg / mL, about 1 to about 50 μg / mL, about 5 to about 100 μg / mL, about 5 to about 90 μg / mL, about 5 to about 80 μg / mL, about 5 to about 70 μg / mL, about 5 to about 60 μg / mL, about 5 to about 50 μg / mL, about 10 to about 100 μg / mL, about 10 to about 90 μg / mL, about 10 to about 80 μg / mL, about 10 to about 70 μg / mL, about 10 to about 60 μg / mL or about 10 to about 50 μg / mL. In any of the above embodiments, IL-12 is present at the following concentrations: about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL or about 50 μg / mL. In any of the above embodiments, IL-12 is present at a concentration of about 5 μg / mL. In any of the above embodiments, IL-12 is present at a concentration of about 10 μg / mL. In any of the above embodiments, IL-12 is present at a concentration of about 25 μg / mL. In any of the above embodiments, IL-12 is present at a concentration of about 50 μg / mL.

[0158] In some embodiments, compared to a control (e.g., the culturing and / or transduction step in the absence of IL-15 and IL-1β and / or IL-12), the presence of (a) IL-15 and (b) IL-1β and / or IL-12 in the culturing and / or transduction step significantly increases the transduction efficiency, e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100% or more.

[0159] The transduction efficiency can be measured by methods known in the art, including but not limited to methods using FACS, PCR or image analysis.

[0160] In some embodiments, compared to a control (e.g., a comparable method of producing an engineered immune cell population such as activation using IL-2 + TransAct™), the presence of (a) IL15 and (b) IL-1β and / or IL-12 in the culture and / or transduction step significantly increases the highly efficient T cell population (e.g., naive T cells or stem cell memory T cells), e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100% or more. In some embodiments, the highly efficient T cells include, but are not limited to, naive T cells and / or stem cell memory T cells.

[0161] The immunophenotype of T cells can be measured by methods known in the art, including but not limited to methods using FACS, PCR, or image analysis. In some embodiments, the T cell phenotype can be measured using anti-CD4 antibody (e.g., clone SK3, catalog number 344604, BioLegend), anti-CD8 antibody (e.g., clone SK1, catalog number 344710, BioLegend), anti-CCR7 antibody (e.g., clone G043H7, catalog number 353204, BioLegend), anti-CD45RA antibody (e.g., clone L48, catalog number 337167, BD Biosciences), anti-CD27 antibody (e.g., clone O323, catalog number 302836, BioLegend), and anti-CD95 antibody (e.g., clone DX2, catalog number 305612, BioLegend). CCR7 / CD45RA negative cells are effector memory T cells, CCR7 positive CD45RA negative cells are central memory T cells, CCR7 negative CD45RA positive cells are effector T cells, CCR7 / CD45RA / CD27 / CD95 positive cells are defined as stem cell memory T cells, and CCR7 / CD45RA positive cells other than them are defined as naive T cells.

[0162] In some embodiments, compared to a control (e.g., a culturing and / or transduction step in the absence of IL-15 and IL-1β and / or IL-12), the presence of (a) IL15 and (b) IL-1β and / or IL-12 in the culturing and / or transduction step significantly increases the number of engineered immune cells produced (cell number), e.g., by about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 5 to about 10-fold, about 6 to about -10-fold or more. The cell number can be measured by methods known in the art, including but not limited to methods using a hemocytometer and / or an automated cell counter.

[0163] In some embodiments, compared to a control (e.g., a culturing and / or transduction step in the absence of one or more of IL-12, IL-15, and IL-1β), the presence of one or more of IL-12, IL-15, and IL-1β in the culturing and / or transduction step significantly increases the number of γδ T cells produced (e.g., engineered γδ T cells), e.g., by about 2 to about 15-fold, about 3 to about 15-fold, about 4 to about 15-fold, about 5 to about 15-fold, about 6 to about -15-fold or more. The cell number can be measured by methods known in the art, including but not limited to methods using a hemocytometer and / or an automated cell counter. Storage / Formulation / Administration

[0164] Engineered immune cells (e.g., CAR-T cells) from the transduction step can be harvested according to well-known protocols in the art for storage, formulation, and / or administration. Thus, in some embodiments, the methods of the present technology can further include storing a population of engineered immune cells and / or administering at least some of the cells from the population of engineered immune cells to a subject in need thereof.

[0165] In some embodiments, engineered immune cells (e.g., CAR-T cells) can be formulated for long-term storage. In some embodiments, engineered immune cells (e.g., CAR-T cells) can be cryopreserved. Methods for cryopreservation are well known to those skilled in the art. For example, engineered immune cells (e.g., CAR-T cells) can be suspended in a cell cryopreservation solution containing a cryoprotectant (e.g., dimethyl sulfoxide) and human serum albumin and frozen at -80°C for 1 day; the cryopreserved cells can be further stored in liquid nitrogen (LN) (e.g., < -150°C). Many factors in cryopreservation can affect the quality of engineered immune cells (e.g., CAR-T cells), thereby affecting the outcome of cell therapy. These factors include, for example, (1) formulation and introduction of the cryopreservation medium, (2) cooling rate, (3) storage conditions, (4) thawing conditions, and (5) post-thaw handling. Optimizing such factors to achieve the desired outcome of cell therapy is within the level of ordinary skill in the art. Formulate

[0166] Engineered immune cells (e.g., CAR-T cells) and compositions containing the engineered immune cells of the present technology can be conveniently provided as sterile liquid preparations, such as, 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 somewhat more convenient to administer, 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 particular tissue. The liquid or viscous composition can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0167] Sterile injectable solutions can be prepared as needed by incorporating the compositions of the subject matter disclosed herein into a desired amount of a suitable solvent and various amounts of other ingredients. Such compositions can be mixed with suitable carriers, diluents, or excipients, such as sterile water, saline, dextrose, dextran, etc. The compositions can also be lyophilized. Depending on the route of administration and the desired preparation, the compositions can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling agents, or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared with reference to standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985, which is incorporated herein by reference without undue experimentation.

[0168] Additives that enhance the stability and sterility of various compositions can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of microbial action can be ensured by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.). Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption (such as aluminum monostearate and gelatin). However, according to the subject matter disclosed in the present invention, any vehicle, diluent, or additive used must be compatible with the engineered immune cells (such as CAR-T cells) of the subject matter disclosed in the present invention.

[0169] The compositions can be isotonic, i.e., 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 subject matter disclosed in the present invention. Sodium chloride is particularly suitable for buffers containing sodium ions.

[0170] If desired, pharmaceutically acceptable thickening agents can be used to maintain the viscosity of the composition at a selected level. Methylcellulose can be used because it is readily available, economical, and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The concentration of the thickening agent can depend on the selected agent. The key is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage forms (e.g., whether the composition is to be formulated as a solution, suspension, gel, or another liquid form, such as a sustained-release form or a liquid-filled form).

[0171] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and that they will not affect the viability or efficacy of the engineered immune cells (such as CAR-T cells) as described in the subject matter disclosed in the present invention. This will not pose any problem for those skilled in the field of chemical and pharmaceutical principles, or the problem can be easily avoided according to the present disclosure or the documents cited herein by referring to standard texts or through simple experiments (without involving undue experimentation).

[0172] One consideration regarding the therapeutic use of the engineered immune cells (such as CAR-T cells) of the subject matter disclosed in the present invention is the amount of cells required to achieve optimal effects. The amount of cells to be administered will vary depending on the subject being treated. In certain embodiments, about 10 2 to about 10 12 、about 10 3 to about 10 11 、about 10 4from about 0 to about 10 10 about 10 5 to about 10 9 or about 10 6 to about 10 8 engineered immune cells (e.g., CAR-T cells) of the subject matter disclosed herein. Even fewer numbers of more effective cells can be administered. In some embodiments, at least about 1 × 10 8 about 2 × 10 8 about 3 × 10 8 about 4 × 10 8 about 5 × 10 8 about 1 × 10 9 about 5 × 10 9 about 1 × 10 10 about 5 × 10 10 about 1 × 10 11 about 5 × 10 11 about 1 × 10 12 or more engineered immune cells (e.g., CAR-T cells) of the subject matter disclosed herein. The precise determination of an effective dose can be based on the individual factors of each subject, including their body size, age, gender, weight, and the affliction of the particular subject. One of ordinary skill in the art can readily determine the dose based on the present disclosure and knowledge in the art. Generally, engineered immune cells (e.g., CAR-T cells) are administered at a dose that is non-toxic or tolerable to the patient.

[0173] One of ordinary skill in the art can readily determine the amount of cells and optional additives, vehicles, and / or carriers in the composition to be administered in the methods of the subject matter disclosed herein. Generally, any additive (other than the active cells and / or agents) is present in a phosphate buffered saline solution in an amount from about 0.001 wt% to about 50 wt%, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt%, or from about 0.05 wt% to about 5 wt%. For any composition to be administered to an animal or a human, and for any particular method of administration, the toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent, such as a mouse); and the dose of the composition, the concentration of the components therein, and the time of administration of the composition to elicit a suitable response. Such determinations do not require undue experimentation according to the knowledge of one of ordinary skill in the art, the present disclosure, and the documents cited herein. Also, the time of sequential administration can be determined without undue experimentation. Administration

[0174] Engineered immune cells (e.g., CAR-T cells) of the subject matter disclosed herein can be administered systemically or directly to a subject to treat a variety of diseases, including but not limited to infections, autoimmune diseases, or tumors. In certain embodiments, the engineered immune cells (e.g., CAR-T cells) are directly injected into the organ of interest. Additionally or alternatively, the engineered immune cells (e.g., CAR-T cells) are indirectly provided to the organ of interest, e.g., by administration to the circulatory system or administration to the tissue of interest. Expansion agents and differentiation agents can be provided before, during, or after administration of the cells and compositions to increase the production of engineered immune cells (e.g., CAR-T cells) in vitro or in vivo.

[0175] Engineered immune cells (e.g., CAR-T cells) of the subject matter disclosed herein can be administered systemically or regionally in any physiologically acceptable vehicle, typically intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they can also be introduced into bone or other convenient sites (e.g., the thymus) where the cells can find appropriate sites for regeneration and differentiation. In certain embodiments, at least 1 × 10 5 cells can be administered, ultimately reaching 1 × 10 10 cells or more. In certain embodiments, at least 1 × 10 6 cells can be administered. A cell population comprising engineered immune cells (e.g., CAR-T cells) can comprise a purified cell population. One of ordinary skill in the art can readily determine the percentage of engineered immune cells (e.g., CAR-T cells) in a cell population using a variety of well-known methods such as fluorescence-activated cell sorting (FACS). The purity of a cell population comprising engineered immune cells (e.g., CAR-T cells) can range from 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%. One of ordinary skill in the art can readily adjust the dose (e.g., a decrease in purity may require an increase in dose). Engineered immune cells (e.g., CAR-T cells) can be introduced by injection, catheter, etc. If desired, the following factors can also be included, 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 other interleukins, colony-stimulating factors such as G-CSF, M-CSF, and GM-CSF, interferons, e.g., gamma-interferon.

[0176] In certain embodiments, the compositions of the subject matter disclosed herein comprise a pharmaceutical composition comprising engineered immune cells (e.g., CAR-T cells) and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, the engineered immune cells (e.g., CAR-T cells) and the compositions comprising the engineered immune cells can be obtained from a subject and administered to the same subject or a different compatible subject. The peripherally blood-derived immune cells or progeny thereof (e.g., derived in vivo, ex vivo or in vitro) of the subject matter disclosed herein can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When administering the pharmaceutical composition of the subject matter disclosed herein, it can be formulated into a unit dose injectable form (solution, suspension, emulsion).

[0177] In another aspect, the present disclosure provides a method of producing a population of engineered immune cells, the method comprising: (i) culturing a population of immune cells (culturing step), (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence to provide a population of engineered immune cells (transduction step), (iii) culturing the population of engineered immune cells derived from step (ii) (ex vivo expansion step), and (iv) harvesting the population of engineered immune cells for storage or administration, wherein step (i), step (ii) and / or step (iii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12.

[0178] In some embodiments, step (i) is performed in the absence of a stimulant (e.g., a stimulant comprising a CD3 binding domain).

[0179] In some embodiments, step (i), step (ii) and / or step (iii) are performed at least in part in the presence of IL-15, IL-1β and IL-12.

[0180] In some embodiments, step (i), step (ii) and / or step (iii) are performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

[0181] In some embodiments, step (iii) results in the expansion of the population of engineered immune cells.

[0182] In some embodiments, for the ex vivo expansion step, the engineered immune cells can be cultured for about 3 hours to about 21 days or any whole integer value of hours in between. It may also be desirable to have several stimulation cycles such that the culture time of the engineered immune cells can be 60 days or longer. In some embodiments, the population of engineered immune cells derived from step (ii) can be cultured for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. The conditions for T cell culture suitable for ex vivo expansion are substantially the same as those discussed above for the culture step and / or transduction step.

[0183] In another aspect, the present disclosure provides a method of increasing the population of naive T cells or stem cell memory T cell subsets, which comprises contacting a population of immune cells with (a) IL-15 and (b) IL-1β and / or IL-12.

[0184] In some embodiments, compared to a control (e.g., the culture, transduction, and / or ex vivo expansion steps in the absence of IL-15 and IL-1β and / or IL-12), the presence of (a) IL-15 and (b) IL-1β and / or IL-12 in the culture, transduction, and / or ex vivo expansion steps significantly increases the transduction efficiency, e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100% or more.

[0185] In some embodiments, compared to a control (e.g., a comparable method of producing an engineered immune cell population, such as activation using IL-2 + TransAct™), the presence of (a) IL-15 and (b) IL-1β and / or IL-12 in the culturing, transducing, and / or ex vivo expansion steps significantly increases the population of highly efficient T cells, e.g., by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100% or more. In some embodiments, the highly efficient T cells include, but are not limited to, naive T cells and / or stem cell memory T cells.

[0186] In some embodiments, compared to a control (e.g., culturing and / or transducing steps in the absence of IL-15 and IL-1β and / or IL-12), the presence of (a) IL-15 and (b) IL-1β and / or IL-12 in the culturing and / or transducing steps significantly increases the number of engineered immune cells produced (cell number), e.g., by about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 5 to about 10-fold, about 6 to about 10-fold or more. The cell number can be measured by methods known in the art, including but not limited to methods using a hemocytometer and / or an automated cell counter.

[0187] In another aspect, the present disclosure provides a method of increasing a population of γδ T cells, which comprises contacting a population of γδ T cells with IL-12 and / or IL-15. In some embodiments, the γδ T cells are contacted with IL-12. In some embodiments, the γδ T cells are contacted with IL-15. In some embodiments, the γδ T cells are contacted with IL-12 and IL-15. In some embodiments, the γδ T cells are contacted with IL-12 and IL-1β. In some embodiments, the γδ T cells are contacted with IL-15 and IL-1β. In some embodiments, the γδ T cells are contacted with IL-12, IL-15, and IL-1β.

[0188] In some embodiments, the presence of one or more of IL-12, IL-15, and IL-1β together with γδ T cells significantly increases the number of γδ T cells produced, e.g., by about 2 to about 15-fold, about 3 to about 15-fold, about 4 to about 15-fold, about 5 to about 15-fold, about 6 to about 15-fold, or more, compared to a control (e.g., not contacting γδ T cells with one or more of IL-12, IL-15, and IL-1β). The cell number can be measured by methods known in the art, including but not limited to methods using a hemocytometer and / or an automated cell counter. Example General experimental methods

[0189] The following materials and methods were used in the following examples.

[0190] Media: 2.6% OpTmizer Expansion Base 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 Base Medium (Thermo Fisher Scientific) to prepare the basal cell medium. SK-Hep Medium: MEM, L-Gln (+) (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.), 1% non-essential amino acids (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd.). GSU-Luc Cell Medium: RPMI 1640 (Thermo Fisher Scientific) was prepared by adding 10% FBS (Biosera Co., Ltd.) and 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0191] Cytokines: MACS GMP® recombinant human IL-1β (Miltenyi Biotec.), MACS GMP® recombinant human IL-2 (Miltenyi Biotec.), MACS GMP® recombinant human IL-3 (Miltenyi Biotec.), MACS GMP® recombinant human IL-4 (Miltenyi Biotec.), MACS GMP® recombinant human IL-6 (Miltenyi Biotec.), MACS GMP® recombinant human IL-7 (Miltenyi Biotec.), MACS GMP® recombinant human IL-12 (Miltenyi Biotec.), MACS GMP® recombinant human IL-15 (Miltenyi Biotec.), and MACS GMP® recombinant human IL-21 (Miltenyi Biotec.) were used. The cytokine mixture included all the cytokines described above.

[0192] Production of genetically engineered T cells: After thawing Leukopak (Hemacare) or γδ T cells (Hemacare), the cells were diluted in basal medium to less than or equal to 4.0×10 6 cells / mL. Cell suspension: MACS GMP T-Cell TransACT™ (Miltenyi Biotec) was inoculated into a culture bag at a ratio of 17.5:1 and cultured for 2 days (activation step). The activated cells were diluted in basal medium using a LOVO cell processing system (Fresenius Kabi) or a centrifuge, and then at 6.07×10 5 cells / cm 26 cells / cm 2 were seeded into culture flasks (G-REX, Wilson Wolf) and cultured for 4 days or longer to generate T cells expressing the mCherry gene or T cells expressing the CAR gene.

[0193] Flow cytometry was used to determine the transduction rate and immunophenotype of T cells: Zombie-NIR fixable viability dye (BioLegend) was used to remove dead cells in the samples. The transduction rates of the mCherry gene and the CAR gene into T cells were determined on a BD FACSCanto II flow cytometer (BD Biosciences). Anti-CAR antibodies were used to measure CAR transduction. Anti-CD4 antibodies (BioLegend), anti-CD8 antibodies (BioLegend), anti-CCR7 antibodies (BioLegend), anti-CD45RA antibodies (BD Biosciences), anti-CD27 antibodies (BioLegend), and anti-CD95 antibodies (BioLegend) were used to measure the immunophenotype of T cells, and CCR7 / CD45RA / CD27 / CD95-positive cells in the CD4-positive T cell population or CD8-positive T cell population were used as stem cell memory T cells, and CCR7 / CD45RA-positive cells other than these were used as naive T cells.

[0194] Flow cytometry was used to determine the expression of several surface markers on T cells: The expression levels of several surface markers on T cells were determined using anti-CD3 antibodies (BioLegend), anti-HLA-DR antibodies (BioLegend), anti-CD25 antibodies (BD Biosciences), anti-CD38 antibodies (BioLegend), anti-CD69 antibodies (BioLegend), anti-CD152 (CTLA4) antibodies (BD Biosciences), anti-CD223 (LAG3) antibodies (BioLegend), anti-CD279 (PD1) antibodies (Thermo), anti-CD366 (TIM3) antibodies (BD Biosciences), anti-CD28 antibodies (BioLegend), anti-CD57 antibodies (Miltenyi Biotech), and anti-KLRG1 antibodies (BioLegend) on a BD FACSCanto II flow cytometer. Forward scatter (FSC) and side scatter (SSC) were also evaluated on the BD FACSCanto II flow cytometer.

[0195] Cell cycle analysis: The cell cycle was calculated using NucleoCounter® NC-3000 (Cheomometec) according to the instruction manual.

[0196] Metabolite analysis: The supernatant after the activation step was collected and analyzed using BioProfile® FLEX2 (NovaBiomedical) according to the instruction manual.

[0197] Glucose uptake assay: Glucose uptake was measured using a Glucose Uptake Assay Kit - Green (Dojindo) according to the instruction manual.

[0198] Co - culture assay: CAR - T cells (effector cells) and luciferase - expressing SK - HEP - 1 cells were seeded in a cell culture plate with SK - HFP - 1 medium at a ratio of effector cells: target cells = 1:1 (0.1M:0.1M per well). After incubation for 24 hours, the effector cells were collected and counted using an NC - 200 instrument. In addition, the luciferase activity derived from the target cells was measured to determine the target cell killing rate.

[0199] In vivo experiment: GSU - Luc cells were subcutaneously inoculated into NSG mice (Charles River Japan). Seven days after inoculation, CAR - T cells or PBS were intravenously administered to the mice. To measure the tumor volume, calipers were used. Example 1: The cytokine mixture slightly induced T - cell aggregation

[0200] T cells were cultured without cytokines, with a cytokine mixture (containing IL - 1b, IL - 2, IL - 3, IL - 4, IL - 6, IL - 7, IL - 12, IL - 15, and IL - 21), or with IL - 2 and TransACT™. On day 2, no T - cell aggregation was observed when T cells were cultured without cytokines, but T - cell aggregation was observed when T cells were cultured with the cytokine mixture or with IL - 2 and TransACT™ ( Figure 1A ). The size of the aggregates was smaller when T cells were cultured with the cytokine mixture compared to when cultured with IL - 2 and TransACT™ (Figures 1B and 1C). Example 2: Production of genetically engineered T cells in the presence of a cytokine mixture added to the medium

[0201] Genetically engineered T cells were produced as described above (Figure 2A). After production, the introduction rate of the mCherry gene was measured by flow cytometry (Figure 2B). mCherry - positive cells were detected in the cytokine mixture group, although the positivity of these cells was lower compared to the IL - 2 and TransACT™ group. Example 3: IL-1b, IL-3, IL-7, IL-12, and IL-15 are candidates for generating genetically engineered T cells

[0202] Individual cytokines were removed from the cytokine mixture to elucidate which cytokine triggered the generation of genetically engineered T cells. Removal of IL-7 or IL-15 significantly reduced the cell number compared to the cytokine mixture (Figure 3A). In addition, removal of IL-1b, IL-3, or IL-12 decreased the ratio of mCherry-positive cells. On the other hand, removal of IL-4 significantly induced the mCherry transduction efficiency (Figure 3B). Given the above results, IL-1b, IL-3, IL-7, IL-12, and IL-15 are candidates for the production of genetically engineered T cells. Example 4: DOE analysis

[0203] Using JMP ® Version 15.0.0 (SAS Institute Inc.) statistically evaluated the effects of the candidate cytokines selected from Example 3 by design of experiments (DoE) analysis.

[0204] The sources, levels, and responses were defined as follows. Based on these components, an experiment was designed ( Figure 4A ). - Sources: IL-1b, IL-3, IL-7, IL-12, and IL-15 - Levels: Their concentrations were 0, 10, and 50 μg / mL. - Response: mCherry-positive cells after culture. The mCherry-positive cells were calculated by multiplying the cell number and the mCherry transduction rate ( Figures 4B - 4D ). The predicted mCherry-positive cells were calculated by the Jackknife method, and its p-value was 0.0074, so this experiment worked well (Figure 4E). Given the above results, IL-1b, IL-12, and IL-15 had a positive effect on the number of mCherry-positive cells (Figure 4F). Example 5: IL-1b, IL-12, and IL-15 synergistically promote multiple genetically engineered T cells

[0205] mCherry-positive T cells were generated by using a combination of IL-1β, IL-12, and IL-15. IL-15 synergistically promoted cell numbers and mCherry-positive cells with IL-1β or IL-12. In addition, the mCherry-positive cells were increased when cells were generated with IL-1β, IL-12, and IL-15 compared to when cells were generated with IL-15 and IL-1β or IL-12 (Figures 5A and 5B). Example 6: IL-1β, IL-12, and IL-15 induce naive / stem cell memory enriched CAR-T cells

[0206] CAR-T cells were generated using IL-2 and TransACT™ or IL-1β, IL-12, and IL-15. After CAR-T cell production, the transduction rate of the CAR gene was measured by flow cytometry (Figure 6A).

[0207] In addition, the T cell phenotype was measured as described above (Figure 6B). The CD45RA-positive and CCR7-positive naive / stem cell memory T cells were increased in the IL-1β, IL-12, and IL-15 group compared to the IL-2 and TransACT™ group (Figure 6B). Example 7: IL-1β, IL-12, and IL-15 slightly increase cell proliferation and cell size

[0208] T cells were cultured with IL-2 and TransACT™ and IL-1β, IL-12, and IL-15 for 2 days. After culture, cell numbers, cell cycle, and cell diameter were measured by NC-3000 ( Figures 7A - 7B and 8A).

[0209] In addition, FSC and SSC were measured by flow cytometry (Figure 8B). Cell proliferation and cell size were increased in the IL-1β, IL-12, and IL-15 group compared to the no additive group. However, these were decreased in the IL-1β, IL-12, and IL-15 group compared to the IL-2 and TransACT™ group. Example 8: IL-1β, IL-12, IL-15 do not cause inhibition of CD3 expression.

[0210] T cells were cultured with IL-2 and TransACT™ and IL-1β, IL-12, and IL-15 for 2 days. After culture, CD3 expression was measured by flow cytometry. CD3 expression was not decreased in the IL-1β, IL-12, and IL-15 group. Example 9: Expression of cell surface markers.

[0211] Although the CD3 expression of IL-2- and TransACT™-stimulated T cells was downregulated, the expression of their cells stimulated by cytokines did not change( Figure 9 ). This means that cytokine stimulation does not occur via CD3ζ signaling, because cell surface CD3 molecules will be internalized by TCR-CD3 stimulation.

[0212] The expression levels of several well-known surface markers that are functional for T cells were evaluated. HLA-DR, CD25, CD38, and CD69 are known as activation markers( Figures 10A - 10D ), CTLA4, LAG3, PD1, and TIM3 are known as exhaustion markers( Figures 11A - 11D ), and CD28, CD57, and KLRG1 are known as senescence markers( Figures 12A - 12C ). Comparing cytokine-stimulated cells with IL-2- and TransACT™-stimulated cells, similar expression of activation markers and senescence markers in both types of cells was observed, but the exhaustion markers on cytokine-stimulated cells were lower than those on IL-2- and TransACT™-stimulated cells. Example 10: IL-1b, IL-12, and IL-15 do not promote glycolysis and glutaminolysis

[0213] T cells were cultured with IL-2 and TransACT™, as well as IL-1b, IL-12, and IL-15 for 2 days. After culturing, glucose, lactose, glutamine, NH4+, and Ca++ in the supernatant were measured by BioProfile FLEX2( Figures 13A - 13D and 15 ). In addition, a glucose uptake test was also performed( Figure 14 ). As a result, compared with the group without additives, glycolysis and glutaminolysis were promoted in the IL-1b, IL-12, and IL-15 groups. However, compared with the IL-1b, IL-12, and IL-15 groups, these were further increased in the IL-2 and TransACT™ groups. Example 11; Long-term expansion of CAR-T cells

[0214] CAR-T cells were generated using IL-2 and TransACT™ or IL-1b, IL-12, and IL15. After production, the CAR-T cells were cultured with IL-2 or IL-1b, IL-12, and IL-15. Compared with the IL-2 and TransACT™ groups, the number of cells increased after long-term culture in the IL-1b, IL-12, and IL-15 groups( Figure 16 ). Example 12: IL-1b, IL-12, and IL-15 promoted genetically engineered γδ T cells.

[0215] mCherry-positive γδ T cells were generated by using a combination of IL-1β, IL-12, and IL-15. IL-1β at 50 ng / mL, IL-12 at 10 or 50 ng / mL, and IL-15 at 10 or 50 ng / mL promoted mCherry-positive γδ T cells ( Figure 17 ). Example 13: CAR-T cell co-culture experiment.

[0216] Three different donor-derived CAR-T cells generated by IL-2 and TransAct™ or IL-1β, IL-12, and IL-15 were co-cultured with the tumor cell line SK-Hep-1 cells for 1 day to confirm their potency. In terms of cell proliferation of CAR-T cells just after co-culture, CAR-T cells generated by IL-1β, IL-12, and IL-15 increased more than CAR-T cells generated by IL-2 and TransAct™ (Figure 18A). Additionally, CAR-T cells generated by IL-1β, IL-12, and IL-15 showed higher killing activity compared to CAR-T cells generated by IL-2 and TransAct™ (Figure 18B). These results indicate that CAR-T cells generated by IL-1β, IL-12, and IL-15 have a more effective phenotype. Example 14: In vivo study using CAR-T cells generated by IL-1β, IL-12, and IL-15

[0217] Seven days after inoculating tumor cells into NSG mice, CAR-T cells generated by using IL-1β, IL-12, and IL-15 were administered. The CAR-T cells showed in vivo efficacy ( Figure 19 ). Example 15: Expansion of γδ T cells using IL-12 or IL-15.

[0218] γδ T cells (Hemacare) were thawed and the cells were diluted to less than or equal to 4.0×10 6 cells / mL. The cells were cultured with cytokine-containing medium for 7 days. γδ T cells cultured with IL-12 or IL-15 without using TransAct™ showed cell proliferation ( Figure 20 ).

Claims

1. A method for producing an engineered population of immune cells, the method comprising: (i) culturing a population of immune cells, (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing the engineered population of immune cells, and (iii) harvesting the engineered population of immune cells, wherein step (i) and / or step (ii) is performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12.

2. The method according to any one of claim 1, wherein step (i) is performed in the absence of a stimulant comprising a CD3-binding domain and / or a TCR-binding domain.

3. The method according to claim 1, wherein step (i) and / or step (ii) is performed at least in part in the presence of IL-15, IL-1β and IL-12.

4. The method according to claim 1, wherein the population of immune cells comprises T cells.

5. The method according to claim 1, wherein the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing the engineered population of immune cells expressing the CAR.

6. The method according to claim 1, wherein the nucleic acid molecule is a viral vector.

7. The method according to claim 6, wherein the viral vector is a retroviral vector.

8. The method according to claim 1, wherein step (i) and / or step (ii) is performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

9. A method for producing an engineered population of immune cells, the method comprising: (i) culturing a population of immune cells, (ii) contacting the population of immune cells with a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous amino acid sequence, thereby providing the engineered population of immune cells, (iii) culturing the engineered population of immune cells derived from step (ii), and (iv) harvesting the engineered population of immune cells for storage or administration, wherein step (i), step (ii) and / or step (iii) is performed at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12.

10. The method according to claim 9, wherein step (i) is performed in the absence of a stimulant comprising a CD3-binding domain and / or a TCR-binding domain.

11. The method according to claim 9, wherein step (i), step (ii) and / or step (iii) is performed at least in part in the presence of IL-15, IL-1β and IL-12.

12. The method according to claim 9, wherein the population of immune cells comprises T cells.

13. The method according to claim 9, wherein the heterologous amino acid sequence comprises a chimeric antigen receptor (CAR), thereby providing the engineered population of immune cells expressing the CAR.

14. The method according to claim 9, wherein the nucleic acid molecule is a viral vector.

15. The method according to claim 14, wherein the viral vector is a retroviral vector.

16. The method according to claim 9, wherein step (iii) results in the expansion of the engineered immune cell population.

17. The method according to claim 9, wherein step (i), step (ii) and / or step (iii) are carried out at least in part in the presence of (a) IL-15 and (b) IL-1β and / or IL-12, wherein the presence of (a) IL-15 and (b) IL-1β and / or IL-12 increases the naive T cell or stem cell memory T cell subset.

18. A method of increasing the population of naive T cells or stem cell memory T cell subsets, comprising contacting an immune cell population with (a) IL-15 and (b) IL-1β and / or IL-12.

19. A composition comprising an immune cell population and (a) IL-15 and (b) IL-1β and / or IL-12.

20. The composition according to claim 19, wherein the composition comprises IL-15, IL-1β and IL-12.

21. The composition according to claim 19, wherein the immune cell population comprises engineered immune cells.

22. The composition according to claim 21, wherein the engineered immune cell population expresses a chimeric antigen receptor (CAR).

23. The composition according to claim 19, wherein the immune cell population comprises T cells.

24. A method of increasing the population of γδ T cells, comprising contacting a γδ T cell population with IL-12.

25. The method according to claim 24, wherein the method further comprises contacting the γδ T cell population with IL-15 or IL-1β.

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