Method for preparing T cells for T cell therapy

Abstract

Provided herein are methods of preparing T cells for T cell therapy, comprising contacting a population of cells at a predetermined cell density with a concentration of anti-CD3 / CD28 nanomatrix and culturing the cells, thereby producing a T cell population comprising an increased proportion of at least one T cell subtype. In some embodiments, the method increases the proportion of stem memory T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 895,381, filed September 3, 2019, the contents of which are incorporated herein by reference in their entirety. [Technical field]

[0002] The present disclosure relates to methods for preparing one or more T cells for T cell therapy. In particular, the present disclosure relates to methods for increasing the proportion of at least one T cell subtype in a cell population by contacting the cell population having a predetermined cell density with a concentration of an anti-CD3 / CD28 nanomatrix selected to increase the proportion of the T cell subtype. [Background technology]

[0003] Human cancers, by their very nature, are composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens distinct from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells utilize various mechanisms to prevent immune cells, such as T lymphocytes and B lymphocytes, from successfully targeting cancer cells.

[0004] Human T cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in subjects (e.g., patients).Various techniques have been developed to enrich the natural T cells that can target tumor antigens, or the genetically modified T cells that specifically target known cancer antigens.These therapies have been demonstrated to have promising effects on tumor size and patient survival.

[0005] The transplantation of a mixed population of T cells is one factor that can prevent T cell therapy from reaching its full potential. In conventional T cell therapy, donor T cells are collected, optionally modified to target specific antigens (e.g., tumor cells) or selected for anti-tumor properties (e.g., tumor-infiltrating lymphocytes), expanded in vitro, and administered to a subject in need thereof. Typically, the resulting T cells contain a mixed population of largely mature cells, many of which are highly differentiated. As a result, the expected in vivo persistence of these cells can be limited, and the initially observed positive effects can be reversed over time as the tumor rebounds in the absence of the transplanted T cells. Therefore, there remains a need to increase the in vivo persistence of T cells for use in T cell therapy. Summary of the Invention

[0006] The present disclosure provides a method for producing a cell population comprising: 1) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting cell population in a volumetric ratio, wherein the starting cell population is at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6 and 2) culturing the cell population in culture medium, wherein the resulting T cell population comprises an increased proportion of stem memory T cells compared to the second T cell population, wherein the second T cell population at the same cell density is contacted with and cultured with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 1 volume of second starting cell population of 17.5 or more.

[0007] The present disclosure provides a method for producing a cell population comprising: 1) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting cell population in a volumetric ratio, wherein the starting cell population is at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6and 2) contacting the second starting cell population with anti-CD3 / CD28 nanomatrix at a cell density of 17.4 cells / ml and a volume ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 1 volume of starting cell population of 17.4 or less; and 2) culturing the cell population in culture medium, wherein the resulting T cell population comprises an increased proportion of stem memory T cells compared to the second T cell population, wherein the second starting cell population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 17.5 volumes of second starting cell population or greater, the method further comprising transducing and / or transfecting the starting cell population before, during, or after culturing the starting cell population with anti-CD3 / CD28 nanomatrix.

[0008] The present disclosure provides a method for treating PBMCs comprising: 1) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting PBMC population in a volumetric ratio, wherein the starting PBMC population comprises at least about 0.50 x 10 6 cells / ml ~ approx. 2.00x10 6 and 2) culturing the PBMC population in culture medium for 14 to 18 days, wherein the resulting PBMC population contains an increased proportion of stem memory T cells compared to the second PBMC population, wherein the second starting PBMC population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20 or 40 volumes of the second starting PBMC population, and cultured for the same number of days.

[0009] The present disclosure provides a method for identifying and identifying T cells comprising: 1) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting purified T cell population in a volumetric ratio, wherein the starting T cell population is at least about 0.80x10 6 cells / ml ~ approx. 1.60x10 6 and 2) culturing the purified T cell population in culture medium for 11 to 18 days, wherein the resulting purified T cell population contains an increased proportion of stem memory T cells compared to the second purified T cell population, wherein the second starting purified T cell population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20, 25, or 50 volumes of the second starting T cell population, and cultured for the same number of days.

[0010] The present disclosure further provides methods of treatment comprising administering a therapeutically effective amount of the resulting Tscm-enriched T cell population to a subject in need thereof. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a bar graph showing overall actual cell yields from days 1 to 18 for cells activated with Transact™ and relevant controls as shown against cell culture volume ratios.

[0012] [Figure 2] FIG. 2 is a bar graph showing actual cell yields from days 1 to 8 prior to cell culture in G-Rex® for cells activated with Transact™ and relevant controls shown against cell culture volume ratios.

[0013] [Figure 3]FIG. 3 is a bar graph showing actual cell yields from days 8 to 18 in G-Rex® for cells activated with Transact™ and relevant controls shown against cell culture volume ratios.

[0014] [Figure 4] FIG. 4 is a graph plotting cell expansion folds versus days in process showing total cell expansion folds for Transact™ and relevant control activated cells shown against cell culture volume ratio.

[0015] [Figure 5] FIG. 5 is a bar graph and table showing fold expansion from day 0 to day 1, day 1 to day 4, day 4 to day 6, day 6 to day 8, and day 8 to day 18 for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0016] [Figure 6] FIG. 6 is a bar graph showing fold proliferation from days 0 to 1, 1 to 4, 4 to 6, and 6 to 8 prior to expansion in G-Rex® for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0017] [Figure 7] FIG. 7 is a bar graph showing the percentage of PBMC cell subsets on day 18 of the process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0018] [Figure 8] FIG. 8 is a bar graph showing the proportion of CD5+ T cell memory subsets (with CD45RO and CD62L markers) at day 18 of the process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0019] [Figure 9] FIG. 9 is a bar graph showing the percentage of CD4+ and CD8+ cell subsets on day 18 of the process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0020] [Figure 10-1] FIG. 10A is a bar graph showing the percentage of total CD5+ T cell subsets using CD45RA and CD62L markers at day 18 of the process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0021] [Figure 10-2] Figures 10B-10H are contour plots with CD62L on the vertical axis and CD45RA on the horizontal axis showing the percentage of CD5+ T cell subsets for cells activated with Transact™ and relevant controls as shown against cell culture volume ratios.

[0022] [Figure 11-1] FIG. 11A is a bar graph showing the percentage of total CD5+ T cell subsets using CD45RO and CD62L markers at day 18 of the process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0023] [Figure 11-2] Figures 11B-11H are contour plots with CD62L on the vertical axis and CD45RA on the horizontal axis showing the percentage of CD5+ T cell subsets for cells activated with Transact™ and relevant controls as shown against cell culture volume ratios.

[0024] [Figure 12]FIG. 12 is a bar graph showing the percentage of CAR+ cells and TCRα / β− cells at day 18 for cells activated with Transact™ and relevant controls as shown against cell culture volume ratios.

[0025] [Figure 13] FIG. 13A is a graph plotting percent cell viability versus days in process for cells activated with Transact™ and relevant controls as indicated against cell culture volume ratios.

[0026] FIG. 13B is a graph plotting cell diameter versus days in process for cells activated with Transact™ and relevant controls shown against cell culture volume ratio.

[0027] [Figure 14] Figures 14A and 14B are graphs plotting the optimal T cell proliferation achieved at TransAct™ dilutions of 1:10 and 1:15. FMC63-41BB-CD3ζ anti-CD19 T cells from two different donors and UT control T cells were stimulated with either T Cell Activation / Proliferation Kit beads (Miltenyi Biotec) or T Cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at the indicated bead:cell or volume:volume ratios, respectively. Total T cells were counted 9 and 14 days after activation (day 0), and fold expansion was calculated. 561 = donor number; 658 = donor number; UT = untransduced T cells; M = T Cell Activation / Proliferation Kit; T = TransAct™; FMC = FMC63-41BB-CD3ζ anti-CD19 CAR T cells; D9 = day 9; D14 = day 14.

[0028] [Figure 15]Figures 15A and 15B are bar graphs showing that TransAct™ dilution does not alter the percentage of CAR+ T cells at the end of the manufacturing process. FMC63-41BB-CD3ζ anti-CD19 T cells from two different donors were stimulated with either T Cell Activation / Proliferation Kit beads (Miltenyi Biotec) or T Cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at the indicated bead:cell or volume:volume ratios, respectively. The percentage of CAR+ T cells at day 14 was determined by flow cytometry. 561 = donor number; 658 = donor number; Miltenyi = T Cell Activation / Proliferation Kit; T = TransAct™; D14 = day 14; CAR = chimeric antigen receptor.

[0029] [Figure 16] Figures 16A and 16B are bar graphs showing that low concentrations of TransAct™ increase the percentage of CD4+ CAR T cells in the final product. FMC63-41BB-CD3ζ anti-CD19 T cells and UT control T cells from two different donors were stimulated with either T Cell Activation / Proliferation Kit beads (Miltenyi Biotec) or T Cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at the indicated bead:cell or volume:volume ratios, respectively. The percentages of CD4+ and CD8+ T cells at day 14 were determined by flow cytometry. 561 = donor number; 658 = donor number; UT = untransduced T cells; M = T Cell Activation / Proliferation Kit; T = TransAct™; FMC = FMC63-41BB-CD3ζ anti-CD19 CAR T cells; D14 = day 14.

[0030] [Figure 17]Figures 17A-17D are bar graphs showing that 1:10 and 1:15 TransAct™ dilutions result in a decrease in the percentage of CD25+ CAR T cells in the final product. FMC63-41BB-CD3ζ anti-CD19 T cells and UT control T cells from two different donors were stimulated with either T Cell Activation / Proliferation Kit beads (Miltenyi Biotec) or T Cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at the indicated bead:cell or volume:volume ratios, respectively. The percentage of CD25+ and 4-1BB+ T cells at day 14 was determined by flow cytometry. 561 = donor number; 658 = donor number; UT = untransduced T cells; M = T Cell Activation / Proliferation Kit; T = TransAct™; FMC = FMC63-41BB-CD3ζ anti-CD19 CAR T cells; D14 = day 14.

[0031] [Figure 18] Figures 18A-18D are bar graphs showing that TransAct™ dilutions of 1:10 and 1:15 preserve TSCM cells in both CD4+ and CD8+ CAR T cells, and TCM cells in CD8+ CAR T cells. FMC63-41BB-CD3ζ anti-CD19 T cells from two different donors and UT control T cells were stimulated with either T Cell Activation / Expansion Kit beads (Miltenyi Biotec) or T Cell TransAct™ Polymer Nanomatrix (Miltenyi Biotec) at the indicated bead:cell or volume:volume ratios, respectively. The percentages of TSCM and TCM cells on day 14 were determined by flow cytometry. 561 = donor number; 658 = donor number; UT = untransduced T cells; M = T cell activation / expansion kit; T = TransAct™; FMC = FMC63-41BB-CD3ζ anti-CD19 CAR T cells; D14 = day 14; TEFF = effector T cells; TEM = effector memory T cells; TCM = central memory T cells; TSCM = stem cell memory T cells. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present disclosure relates to methods for preparing T cells for use in T cell therapy. In particular, the present disclosure relates to methods for preparing immature, less differentiated T cells in a T cell population (e.g., stem and memory T cells (hereinafter "T SCM The present invention relates to enhancing the proportion of specific T cell subtypes, including immature and less differentiated T cells. By enriching for immature and less differentiated T cells, the persistence potential of T cells may be increased once administered to a subject, e.g., a patient. As a result, enriched populations of immature T cells are more likely to produce a durable anti-tumor effect than populations of T cells at mixed stages of differentiation.

[0033] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly defined herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this disclosure.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0035] Units, prefixes, and symbols are designated in the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety. Accordingly, the terms defined below are more fully defined by reference to the specification in its entirety.

[0036] As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or enumerated members.

[0037] The terms "about" or "essentially comprising" refer to a value or composition within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "essentially comprising" can mean within one or more standard deviations. Alternatively, "about" or "essentially comprising" can mean a range of up to 10% (i.e., + / - 10%). For example, "about 3 mg" can include any number between 2.7 mg and 3.3 mg (about 10%). Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range for that particular value or composition.

[0038] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate.

[0039] As used herein, the term "and / or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] Wherever embodiments are described herein with the term "comprising," it is understood that otherwise similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0041] The term "activation" or "activated" refers to a state of immune cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division. T cell activation can be characterized by increased T cell expression of one or more biomarkers, including, but not limited to, CD57, PD1, CD107a, CD25, CD137, CD69, and / or CD71.

[0042] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for T cells prepared by the methods disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0043] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. Generally, antibodies may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains, CH1, CH2 CH3, and / or CH4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0044] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are known in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-chain Abs, including camelid antibodies. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and the context suggests otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain Abs.

[0045] An "antigen-binding molecule" or "antibody fragment" refers to any portion of an antibody that is less than the entire antibody. An antigen-binding molecule may contain antigenic complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.

[0046] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, engineered autologous cell therapy (eACT™) involves the collection of lymphocytes from a donor, e.g., a patient, which are then engineered to express, e.g., a CAR construct, and then administered back to the same donor, e.g., patient.

[0047] The term "allogeneic" refers to any material derived from one individual that is subsequently introduced into another individual of the same species, for example, allogeneic T cell transplantation or therapy.

[0048] The term "anti-CD3 / 28 nanomatrix" refers to, for example, TransAct™ nanomatrix from Miltenyi Biotec, Inc. (Auburn, CA). TM Refers to a nanometer-scale matrix containing antibodies and / or fragments thereof that bind to CD3 and CD28, provided as a T cell reagent (see, for example, Catalog No. 200-076-202 MACS GMP T Cell Transact-CRR, Catalog No. 170-076-156 MACS GMP T Cell Transact for Research use).

[0049] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that can invade neighboring tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors of various stages. In certain embodiments, the cancer or tumor is at stage 0, e.g., the cancer or tumor is very early in development and has not metastasized. In some embodiments, the cancer or tumor is at stage I, e.g., the cancer or tumor is relatively small, has not spread to nearby tissues, and has not metastasized. In other embodiments, the cancer or tumor is at stage II or stage III, e.g., the cancer or tumor is larger than stage 0 or stage I and has grown into neighboring tissues but has not metastasized, except possibly to lymph nodes. In other embodiments, the cancer or tumor is at stage IV, e.g., the cancer or tumor has metastasized. Stage IV may also be referred to as advanced cancer or metastatic cancer.

[0050] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, inhibition of tumor growth, a reduction in tumor cell count, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors.Anti-tumor effect can also refer to the prevention of tumor development, for example, a vaccine.

[0051] As used herein, the term "progression-free survival," which may be abbreviated as PFS, refers to the time from the date of treatment to the date of disease progression according to the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.

[0052] "Disease progression" is assessed by measuring malignant lesions on radiographs or by other methods.

[0053] As used herein, "duration of response," which may be abbreviated as DOR, refers to the period from a subject's first objective response to the date of confirmed disease progression by the modified IWG Response Criteria for Malignant Lymphoma, or the date of death.

[0054] The term "overall survival," which may be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0055] As used herein, "cytokine" refers to a non-antibody protein that can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to mediate an immune response. In some embodiments, one or more cytokines are released in response to T cell therapy. In certain embodiments, the cytokines secreted in response to T cell therapy can be an indication of effective T cell therapy.

[0056] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of T cells or DC cells produced by the method and used alone or in combination with another therapeutic agent to protect a subject from developing a disease or promote disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of defects or disorders resulting from disease. The ability of T cells or DC cells to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, for example, by assaying the activity of agents in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or in in vitro assays.

[0057] As used herein, the term "lymphocyte" may include natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that represents a major component of the innate immune system. NK cells reject tumor- and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). Their T cell receptors (TCRs) differentiate them from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells.

[0058] There are several types of T cells: helper T cells (e.g., CD4 + cells, effector T EFF cells), cytotoxic T cells (TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8 + T cells, also known as killer T cells), memory T cells ((i) stem memory T SCM The cells, like naive cells, express CD45RO - , CCR7 + , CD45RA + , CD62L + (L-selectin), CD27+ , CD28 + , and IL-7Rα + (ii) central memory T cells, which also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1 and exhibit many functional properties characteristic of memory cells; CM The cells express L-selectin and CCR7 + and CD45RO + , which secrete IL-2 but not IFNβ or IL-4, and (iii) effector memory T EM However, these cells do not express L-selectin or CCR7, but do express CD45RO and produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4 + CD25 + Regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. T cells found within tumors are called "tumor-infiltrating lymphocytes" or "TILs."

[0059] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells become CD4 + or CD8 + In their naive state, T cells express L-selectin (CD62L + ), IL-7 receptor-α (IL-7R-α), and CD132, but not CD25, CD44, CD69, and CD45RO. As used herein, "immature" also refers to T cells that exhibit phenotypic characteristics of either naive or immature T cells, e.g., T SCM Cells or T CM For example, immature T cells express L-selectin (CD62L + ), IL-7R-α, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, and LFA-1. Naive or immature T cells are T EM Cells and TEFF These can be contrasted with terminally differentiated effector T cells, such as T cells.

[0060] As referred to herein, "T cell function" refers to the normal characteristics of healthy T cells. In some embodiments, T cell function includes T cell proliferation. In some embodiments, T cell function includes T cell activity. In some embodiments, T cell function includes cytolytic activity.

[0061] As used herein, "cell proliferation" refers to the ability of T cells to grow in number through cell division. Proliferation can be measured, for example, by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation can occur in vitro, for example, in T cell culture, or in vivo, for example, after administration of T cell therapy.

[0062] As used herein, "T cell activity" refers to any activity common to healthy T cells. In some embodiments, T cell function includes cytokine production. In certain embodiments, T cell activity includes production of one or more cytokines selected from interferon gamma (IFNγ), tissue necrosis factor alpha (TNFα), and both.

[0063] As used herein, "cytolytic activity" or "cytotoxicity" refers to the ability of a T cell to destroy a target cell. In some embodiments, the target cell is a cancer cell, e.g., a tumor cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cell expresses a target antigen.

[0064] The terms "genetically engineered," "gene editing," or "engineered" refer to methods of modifying a cell's genome, including, but not limited to, deleting a coding or non-coding region, or portion thereof, or inserting a coding region, or portion thereof. In some embodiments, the cell to be modified is a lymphocyte, e.g., a T cell, which can be obtained from either a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the cell's genome.

[0065] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils), and soluble macromolecules (including Abs, cytokines, and complement) produced by either these cells or the liver, that result in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0066] The term "immunotherapy" refers to the treatment of a subject suffering from or at risk of suffering from or relapsing from a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), engineered allogeneic cell therapy, and allogeneic T cell transplantation. However, those skilled in the art will recognize that the methods of preparing T cells disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, and International Publication No. WO2008 / 081035.

[0067] T cells for immunotherapy can be obtained from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from donors. The donor can be a subject, for example, a subject who needs anti-cancer treatment, or a healthy donor. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. T cells can also be obtained from the artificial thymic organoid (ATO) cell culture system, which replicates the human thymic environment and supports efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0068] The term "engineered autologous cell therapy," also known as adoptive cell transfer and abbreviated as "eACT™," is a process in which a patient's own T cells are collected and then genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, one or more chimeric antigen receptors (CARs), or one or more T cell receptors (TCRs), and combinations thereof. CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling moiety containing a costimulatory domain and an activation domain. Costimulatory domains include, for example, CD28, CTLA4, CD16, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), inducible T cell costimulator (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14;TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, N Kp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof;The activation domain can be derived from CD3, for example, CD3 zeta, epsilon, delta, gamma, etc. In certain embodiments, CARs are designed to have two, three, four, or more costimulatory domains. CAR scFvs can be designed to target CD19, a transmembrane protein expressed by cells of the B-cell lineage, including all normal B cells and B-cell malignancies, including, but not limited to, NHL, CLL, and non-T-cell ALL. Examples of CAR+T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties.

[0069] As used herein, a "patient" includes any human suffering from a disease, including cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers herein to a subject whose cells are being obtained for further in vitro manipulation. The donor subject may be a cancer patient to be treated with a population of cells produced by the methods described herein (i.e., an autologous donor), or may be an individual who provides a lymphocyte sample that will be used to treat a different individual or cancer patient upon production of a population of cells produced by the methods described herein (i.e., an allogeneic donor). A subject who receives cells prepared by the present methods may be referred to as a "recipient subject."

[0070] As used herein, "stimulation" refers to a primary response elicited by the binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an artificial antigen-presenting cell (aAPC), a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. As used herein, "activated" or "active" refers to stimulated T cells. Activated T cells may be characterized by the expression of one or more markers selected from the group consisting of CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

[0071] The term "exogenous" refers to any substance that comes from an outside source.

[0072] As used herein, the term "persistence" refers to the ability of, for example, one or more transplanted T cells or their progeny (e.g., differentiated or mature T cells) administered to a subject to remain in the subject at detectable levels for a period of time. As used herein, increasing the persistence of one or more transplanted T cells or their progeny (e.g., differentiated or mature T cells) refers to increasing the time that the transplanted T cells are detectable in the subject after administration. For example, the in vivo persistence of one or more transplanted T cells can be increased by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. Additionally, the in vivo persistence of one or more transplanted T cells can be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to one or more transplanted T cells not prepared by the methods disclosed herein.

[0073] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change below an original value. "Reduce" and "decrease" are relative terms, requiring a comparison of a pre-measurement and post-measurement. "Reduce" and "decrease" include complete depletion. In some embodiments, the terms "reduce" and "decrease" include a comparison of T cell effects between T cells prepared by the methods of the present disclosure (e.g., strong activation) and T cells without this preparation.

[0074] As used herein, the term "modulating" T cell maturation refers to using any of the interventions described herein to affect the maturation, e.g., differentiation, of one or more T cells. In some embodiments, "modulating" refers to delaying or inhibiting T cell maturation. In other embodiments, "modulating" refers to accelerating or promoting T cell maturation. In particular, as used herein, "delaying or inhibiting T cell maturation" refers to maintaining one or more T cells in an immature or undifferentiated state. For example, "delaying or inhibiting T cell maturation" refers to maintaining one or more T cells in an immature or undifferentiated state. EM or T EFF "Delaying or inhibiting T cell maturation" may also refer to maintaining T cells in a naive or TCM state, as opposed to progressing to a naive or TCM state. Also, "delaying or inhibiting T cell maturation" may refer to maintaining immature or undifferentiated T cells (e.g., naive T cells and / or T cells) within a mixed population of T cells. CM The state of T cells (e.g., mature or immature) can be determined, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of T cells. For example, L-selectin (CD62L + The presence of one or more markers selected from the group consisting of: IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof can be indicative of less mature, less differentiated T cells.

[0075] "Treatment of a subject" or "treating" a subject refers to any type of intervention or process performed, or the administration of one or more T cells prepared using the present disclosure, with the intent to reverse, alleviate, ameliorate, inhibit, delay, or prevent the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0076] Various aspects of the disclosure are described in further detail in the following subsections. Method for preparing immune cells

[0077] The present disclosure relates to methods for preparing immune cells (e.g., lymphocytes or dendritic cells) for use in cell therapy. It has been found that certain in vitro engineered cells (e.g., CAR T cells, T cells, or dendritic cells) may be less effective when administered to patients after in vitro manipulation. Without intending to be bound by any particular theory, it is noted that one reason is that lymphocytes may be prematurely differentiated in vitro before being administered to patients. In certain embodiments, the present disclosure reveals a method for expanding the number of immature, less differentiated cells in a T cell population by using strong activating conditions.

[0078] In one embodiment, the present disclosure provides a method for identifying immature, less differentiated cells in a population of collected T cells (e.g., stem cell memory T cells; T SCM The methods described herein relate to methods for increasing the proportion of CD8+ T cells. Accordingly, the methods described herein can be used in cell therapy (e.g., T cell therapy) to increase the in vivo persistence of transplanted T cells or DC cells or their progeny. Furthermore, in some embodiments, the invention provides that the resulting T cells exhibit increased proliferation in vitro. Furthermore, in some embodiments, the invention provides that the resulting T cells exhibit an increased proportion of CD8+ T cells.

[0079] In another embodiment, the invention includes a method of enriching the proportion of immature, less differentiated cells in a T cell population by contacting one or more cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population, wherein the T cell population is at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6Further preparation of T cells is described elsewhere herein.

[0080] The present disclosure provides a method for identifying and characterizing T cells in a sample, the method comprising: (a) contacting a cell population comprising one or more T cells in vitro with a higher concentration of anti-CD3 / 28 nanomatrix; (b) increasing the number of immature and less differentiated T cells in the sample, e.g., T SCM Thus, in another embodiment, the present invention provides a method for producing stem cell-like CD4 T cells, comprising culturing one or more T cells in a medium comprising a ratio of 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, to 17 or fewer volumes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population. + T cells or CD8 + A method for generating T cells is provided, wherein the T cell population is about 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6 In other embodiments, the disclosure provides a method for treating T cells comprising contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes), wherein the T cell population is at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6 and (c) expanding one or more T cells, wherein the cell density of the sample is 100%. + CD45RA + / CD62L + T cells and / or CD8 + CD45RO - / CD62L +

[0010] Provided are methods for enriching a population of T cells. Producing increased concentrations of immature and undifferentiated T cells or DC cells can increase the in vivo persistence of the cells upon transplantation into a subject in need of cell therapy (e.g., T cell therapy or DC cell therapy). Accordingly, in another embodiment, the present disclosure provides a method for enriching a population of T cells by (i) contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population prior to administration to the subject, wherein the T cell population is approximately 0.2x10 6 cells / ml ~ approx. 5.8x10 6

[0013] Provided is a method for extending the in vivo persistence of one or more T cells in adoptive cell therapy, comprising contacting the one or more transferred T cells with a cell density of 1000 to 15000 cells / ml; wherein the in vivo persistence is extended compared to one or more transferred T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, to greater than 15 volumes (e.g., 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 25, 30, or 40 volumes) of the T cell population.

[0081] The methods disclosed herein include modulating, e.g., enriching, a T cell population for immature, less differentiated cells in vitro. The delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, enrichment of a cell population for immature, less differentiated T cells or DC cells can be measured by detecting the presence of one or more biomarkers. The presence of one or more biomarkers can be detected by any method known in the art, including, but not limited to, immunohistochemistry and / or fluorescence-activated cell sorting (FACS). In some embodiments, the one or more biomarkers are L-selectin (CD62L), L-cell proliferation (IL-1), L-cell denaturation (IL-2), L-cell proliferation (IL-3), L-cell proliferation (IL-4), L-cell proliferation (IL-5), L-cell proliferation (IL-6), L-cell proliferation (IL-7), L-cell proliferation (IL-8), L-cell proliferation (IL-9), L-cell proliferation (IL-10), L-cell proliferation (IL-11), L-cell proliferation (IL-12), L-cell proliferation (IL-13), L-cell proliferation (IL-14), L-cell proliferation (IL-15), L-cell proliferation (IL-16), L-cell proliferation (IL-17), L-cell proliferation (IL-18), L-cell proliferation (IL-19 ... +), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof. In certain embodiments, enrichment of the cell population for immature, less differentiated T cells is achieved by L-selectin (CD62L + ), CD45RA, and CD45RO. Those skilled in the art will appreciate that while the present methods can increase the relative proportion of immature and less differentiated T cells or DC cells in a population of collected cells, some mature and differentiated cells may still be present. Consequently, enrichment of a cell population for immature and less differentiated T cells can be achieved by contacting one or more cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population, where the T cell population is approximately 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6This can be measured by calculating the total percentage of immature and undifferentiated cells in the cell population before and after contacting, including the cell density in cells / ml. The methods disclosed herein increase the percentage of immature and undifferentiated T cells in a T cell population. In certain embodiments, one or more T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) comprise at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% immature or undifferentiated T cells. In other embodiments, the T cell population is 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes), wherein the T cell population is about 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6 One or more T cells contacted with one volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population comprising a cell density of 1000 cells / ml, comprise at least about 10% to at least about 90%, at least about 20% to at least about 80%, at least about 30% to at least about 70%, at least about 40% to at least about 60%, at least about 10% to at least about 50%, at least about 20%, at least about 40%, at least about 35% to at least about 45%, at least about 20% to at least about 60%, or at least about 50% to at least about 90% immature or undifferentiated T cells. In certain embodiments, the immature or undifferentiated T cells include naive T cells and / or central memory T cells. SCM It is a cell.

[0082] In another embodiment, the enrichment of the cell population for immature, less differentiated T cells or DC cells is 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population, wherein the T cell population is about 0.2x10 6 cells / ml ~ approx. 5.8x10 6 by calculating the total percentage of immature and less differentiated cells in a cell population contacted with one volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, relative to a T cell population, including cell density in cells / ml, and comparing it to the percentage of immature, less differentiated T cells or DC cells in a reference cell population contacted with one volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, relative to a T cell population of greater than 17 volumes, wherein the reference T cell population is approximately 0.2x10 6 cells / ml ~ approx. 5.8x10 6In certain embodiments, one or more T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, relative to 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of a T cell population contain at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% more immature or undifferentiated T cells, e.g., Tscm cells, than the reference T cell population. In certain embodiments, one or more T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of a T cell population are 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x more immature or non-differentiated T cells, e.g., T cells, than the reference T cell population. SCM Contains cells.

[0083] The present disclosure provides methods for enriching the proportion of immature, less differentiated cells in a T cell population by contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population, wherein the T cell population is at least about 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6The cell density includes cells / ml. One or more T cells or DC cells can be contacted with the anti-CD3 / 28 nanomatrix via any means known in the art. For example, the anti-CD3 / 28 nanomatrix can be added to the culture medium used to culture one or more T cells or DC cells.

[0084] One or more T cells of the present disclosure can be administered to a subject for use in T cell therapy. Thus, one or more T cells can be collected from a subject in need of T cell therapy or from a donor. Once collected, the one or more T cells can be treated for any suitable period of time before being administered to a subject. During this time, for any period between collection of the T cells from the donor and administration to the subject, the one or more T cells can be contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes), where the T cell population is approximately 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6For example, one or more T cells can be cultured in contact with, e.g., in the presence of, 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of a T cell population for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days. In some embodiments, one or more T cells are contacted with (e.g., cultured in the presence of) 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population for about 1 day to about 18 days, about 1 day to about 14 days, about 1 day to about 10 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, or about 2 days to about 6 days. In one particular embodiment, one or more T cells are contacted with, e.g., cultured in the presence of, 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell from the day the T cells are collected (e.g., day 0) until the day the T cells are administered to the subject.In another embodiment, the T cells are contacted with, e.g., cultured in the presence of, 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population from day 0 until administration, from day 1 until administration, from day 2 until administration, from day 3 until administration, from day 4 until administration, from day 5 until administration, or from day 6 until administration. In some embodiments, the one or more T cells are washed to remove the anti-CD3 / 28 nanomatrix prior to administration.

[0085] The methods described herein can further include enriching a population of lymphocytes obtained from a donor. Enrichment of a population of lymphocytes, such as one or more T cells, can be achieved by any suitable separation method, including, but not limited to, the use of a separation medium (e.g., FICOLL® PAQUE, ROSETTESEP™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X), or similar), cell size, shape, or density separation by filtration or elution, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation. Additionally, enrichment of the T cell population can be achieved using a Pan T Isolation Kit, Human (Miltenyi Biotec, Auburn, CA, #130-096-535), CD4 / CD8 Selection Kit (Miltenyi Biotec, Auburn, CA, #130-122-352 StraightFrom® Leukopak® CD4 / CD8 MicroBead Kit, #130-030-401 CliniMACS CD4 MicroBeads, and #130-030-801 CliniMACS CD8 MicroBeads and CD3 MicroBead Kit).

[0086] The methods described herein can further include stimulating the population of lymphocytes with one or more T cell stimulatory agents to produce a population of activated T cells under suitable conditions. The lymphocytes, e.g., T cells or PBMCs, can be fresh or frozen, and can be derived, for example, from peripheral blood or umbilical cord blood. Any combination of one or more suitable T cell stimulatory agents may be used to generate a population of activated T cells, including, but not limited to, antibodies or functional fragments thereof that target T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or functional fragments thereof), or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands for T cell stimulatory or costimulatory molecules, or anti-CD3 / 28 nanomatrix, e.g., TransAct™ T cell reagent (Miltenyi Biotec, Auburn, CA).

[0087] Suitable conditions for stimulating the lymphocyte populations described herein may include a temperature, a length of time, and / or in the presence of a level of CO2. In certain embodiments, the temperature for stimulation is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In certain embodiments, the temperature for stimulation is about 34-38°C. In certain embodiments, the temperature for stimulation is about 35-37°C. In certain embodiments, the temperature for stimulation is about 36-38°C. In certain embodiments, the temperature for stimulation is about 36-37°C or about 37°C.

[0088] Another condition for stimulating the lymphocyte population described herein may include the time for stimulation. In some embodiments, the time for stimulation is about 0 to 72 hours. In some embodiments, the time for stimulation is about 0 to 24 hours, about 24 to 72 hours, about 24 to 36 hours, about 30 to 42 hours, about 36 to 48 hours, about 40 to 52 hours, about 42 to 54 hours, about 44 to 56 hours, about 46 to 58 hours, about 48 to 60 hours, about 54 to 66 hours, or about 60 to 72 hours. In a specific embodiment, the time for stimulation is about 48 hours or at least about 48 hours. In other embodiments, the time for stimulation is about 44 to 52 hours. In certain embodiments, the time for stimulation is about 40 to 44 hours, about 40 to 48 hours, about 40 to 52 hours, or about 40 to 56 hours. In one embodiment, the time for stimulation is about 0 to 24 hours, and the lymphocyte population is fresh.

[0089] Other conditions for stimulating lymphocyte populations described herein may include a CO2 level. In some embodiments, the CO2 level for stimulation is about 1.0-10% CO2. In some embodiments, the CO2 level for stimulation is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for stimulation is about 3-7% CO2. In another embodiment, the CO2 level for stimulation is about 4-6% CO2. In yet another embodiment, the CO2 level for stimulation is about 4.5-5.5% CO2. In one specific embodiment, the CO2 level for stimulation is about 5% CO2.

[0090] The conditions for stimulating the population of lymphocytes can include, in any combination, a temperature, a length of time for stimulation, and / or in the presence of a level of CO. For example, stimulating the population of lymphocytes can include stimulating the population of lymphocytes with one or more T cell stimulatory agents at a temperature of about 36-38°C, for about 44-52 hours, in the presence of a CO level of about 4.5-5.5% CO.

[0091] The lymphocyte concentration useful in the methods herein is about 0.5 to 10.0 x 10 6 In certain embodiments, the lymphocyte concentration may be about 1.0-2.0 x 10 cells / mL. 6 cells / mL, approximately 1.0~3.0×10 6 cells / mL, approximately 1.0~4.0×10 6 cells / mL, approximately 1.0~5.0×10 6 cells / mL, approximately 1.0~6.0×10 6 cells / mL, approximately 1.0~7.0×10 6 cells / mL, approximately 1.0~8.0×10 6 cells / mL, 1.0~9.0x10 6 cells / mL, or approximately 1.0–10.0 × 10 6 In certain embodiments, the lymphocyte concentration is about 1.0-2.0 x 10 cells / mL. 6 cells / mL or 2.0-3.0x10 6 In certain embodiments, the lymphocyte concentration is about 1.0-1.2 x 10 cells / mL. 6 cells / mL, approximately 1.0~1.4×10 6 cells / mL, approximately 1.0~1.6×10 6 cells / mL, approximately 1.0~1.8×10 6 cells / mL, or approximately 1.0–2.0 × 10 6 In certain embodiments, the lymphocyte concentration is about 2.1-3.0 x 10 cells / mL. 6 cells / mL, approximately 2.1~3.0x10 6 cells / mL, approximately 2.1~3.0x10 6 cells / mL, approximately 2.4~3x10 6 cells / mL, approximately 2.5~3.0x10 6 cells / mL, approximately 2.6~3.0x10 6 cells / mL, approximately 2.7~3.0x10 6 cells / mL, 2.8~3.0x10 6 cells / mL, or 2.8-3.0 x 10 6 In certain embodiments, the lymphocyte concentration is at least about 0.2 x 10 cells / mL. 6 cells / mL, 0.3×10 6cells / mL, 0.4×10 6 cells / mL, 0.5×10 6 cells / mL, 0.6×10 6 cells / mL, 0.7×10 6 cells / mL, 0.8×10 6 cells / mL, 0.9×10 6 cells / mL, 1.0×10 6 cells / mL, at least approximately 1.1 x 10 6 cells / mL, at least approximately 1.2 x 10 6 cells / mL, at least approximately 1.3 x 10 6 cells / mL, at least approximately 1.4 x 10 6 cells / mL, at least approximately 1.5 x 10 6 cells / mL, at least approximately 1.6 × 10 6 At least approximately 1.7 × 10 cells / mL 6 cells / mL, at least approximately 1.8 x 10 6 At least approximately 1.9 × 10 cells / mL 6 At least approximately 2.0 × 10 cells / mL 6 cells / mL, at least approximately 4.0 x 10 6 cells / mL, at least approximately 6.0 × 10 6 cells / mL, at least approximately 8.0 × 10 6 , or at least about 10.0 × 10 6 cells / mL.

[0092] Anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies can be used in the step of stimulating the lymphocyte population. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibodies, or functional fragments thereof, can be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)). In some aspects, antibodies can be commercially purchased from vendors known in the art, including, but not limited to, Miltenyi Biotec (e.g., TransAct™ T Cell Reagent Large Scale, #130-109-104), BD Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Additionally, one of skill in the art would understand how to produce anti-CD3 and / or anti-CD28 antibodies by standard methods. In some embodiments, the one or more T cell stimulatory agents used in accordance with the step of stimulating a population of lymphocytes comprises an antibody or functional fragment thereof that targets a T cell stimulatory or costimulatory molecule in the presence of a T cell cytokine. In one aspect, the one or more T cell stimulatory agents comprise an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulatory agent comprises an anti-CD3 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In one particular embodiment, the concentration of the anti-CD3 antibody is about 50 ng / mL.

[0093] The methods described herein may further include transducing a population of activated T cells with a viral vector containing a nucleic acid molecule encoding a protein of interest (e.g., a CAR) using single-cycle transduction to produce a population of transduced T cells. Several recombinant viruses have been used as viral vectors for delivering genetic material to cells. The viral vector that can be used in accordance with the transduction step can be any ecotropic or amphotropic viral vector, including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors. In some embodiments, the method further includes transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce the population of activated T cells is an MSGV1 gamma retroviral vector. According to one aspect of this embodiment, the viral vector is grown in suspension culture in a medium specific for viral vector production, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or supplements for growing viral vectors can be used in the viral vector inoculum according to the methods provided herein. According to some embodiments, a viral vector inoculum is added to the culture medium during the transduction step.

[0094] In some embodiments, one or more T cells can be transduced with a lentivirus. In one embodiment, the lentivirus contains a heterologous gene encoding a protein of interest. In a specific embodiment, the protein of interest can bind to an antigen on the surface of a target cell, for example, on the surface of a tumor cell, and can be a CAR.

[0095] Conditions for transducing a population of activated T cells described herein can include a specific time period at a specific temperature and / or in the presence of a specific level of CO2. In certain embodiments, the temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature for transduction is about 34-38°C. In another embodiment, the temperature for transduction is about 35-37°C. In another embodiment, the temperature for transduction is about 36-38°C. In yet another embodiment, the temperature for transduction is about 36-37°C. In one specific embodiment, the temperature for transduction is about 37°C.

[0096] In certain embodiments, the time for transduction is about 0 to 36 hours after activation. In some embodiments, the time for transduction is about 12 to 16 hours, about 12 to 20 hours, about 12 to 24 hours, about 12 to 28 hours, or about 12 to 32 hours. In other embodiments, the time for transduction is about 20 hours or at least about 20 hours. In one embodiment, the time for transduction is about 16 to 24 hours. In other embodiments, the time for transduction is at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours.

[0097] In certain embodiments, the CO2 level for transduction is about 1.0-10% CO2. In other embodiments, the CO2 level for transduction is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for transduction is about 3-7% CO2. In another embodiment, the CO2 level for transduction is about 4-6% CO2. In another embodiment, the CO2 level for transduction is about 4.5-5.5% CO2. In one specific embodiment, the CO2 level for transduction is about 5% CO2.

[0098] In some embodiments, transducing a population of activated T cells described herein can be performed for a specific time, at a specific temperature, and / or in the presence of a specific level of CO2, in any combination: at a temperature of about 36-38°C, for a period of about 16-24 hours, and in the presence of CO2 at a level of about 4.5-5.5% CO2.

[0099] The methods provided herein can include expanding one or more populations of transduced T cells for a specified period of time to produce a population of engineered T cells. The predetermined expansion time can be any suitable time that allows for the production of (i) a sufficient number of cells in the population of engineered T cells for at least one dose to be administered to a patient, (ii) a population of engineered T cells with a favorable ratio of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This time will depend on the protein of interest expressed by the T cells, the vector used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the predetermined time for expansion can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the expansion time is shorter than expansion methods known in the art. For example, the predetermined time for expansion can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% shorter, or more than 75% shorter. In one embodiment, the time for expansion is about 3 days and the time from enriching the population of lymphocytes to producing engineered T cells is about 6 days.

[0100] Conditions for expanding a population of transduced T cells can include temperature and / or the presence of a certain level of CO2. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature is about 34-38°C. In another embodiment, the temperature is about 35-37°C. In another embodiment, the temperature is about 36-38°C. In yet another embodiment, the temperature is about 36-37°C. In a specific embodiment, the temperature is about 37°C. In certain embodiments, the CO2 level is 1.0-10% CO2. In other embodiments, the CO2 level is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level is about 4.5-5.5% CO2. In another embodiment, the CO2 level is about 5% CO2. In other embodiments, the CO2 level is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, conditions for expanding a population of transduced T cells can include temperature and / or the presence of a certain level of CO2, in any combination. For example, conditions for expanding a population of transduced T cells include a temperature of about 36-38°C and the presence of CO2 at a level of about 4.5-5.5% CO2.

[0101] Each step of the methods provided herein can be performed in a semi-closed or closed system. In certain embodiments, the closed system is a closed-bag culture system, including any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP cell differentiation bag, Origen Biomedical PermaLife cell culture bag). In some embodiments, the cell culture bag used in the closed-bag culture system is coated with a recombinant human fibronectin fragment during the transduction step. The recombinant human fibronectin fragment may contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can be used to increase the gene efficiency of retroviral transduction of immune cells by aiding in colocalization of target cells and viral vectors. In certain embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with the recombinant human fibronectin fragment at a concentration of about 1-60 μg / mL or about 1-40 μg / mL. In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1-20 μg / mL, 20-40 μg / mL, or 40-60 μg / mL. In some embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, or about 20 μg / mL. In other embodiments, the cell culture bag is coated with about 2-5 μg / mL, about 2-10 μg / mL, about 2-20 μg / mL, about 2-25 μg / mL, about 2-30 μg / mL, about 2-35 μg / mL, about 2-40 μg / mL, about 2-50 μg / mL, or about 2-60 μg / mL of a recombinant human fibronectin fragment.In certain embodiments, the cell culture bag is coated with at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of recombinant human fibronectin fragment. In one specific embodiment, the cell culture bag is coated with at least about 10 μg / mL of recombinant human fibronectin fragment. Cell culture bags used in closed bag culture systems can optionally be blocked with human albumin serum (HSA) during the transduction process. In an alternative embodiment, the cell culture bag is not blocked with HSA during the transduction process.

[0102] In other aspects, T cell culture medium, with or without human serum, is used to perform at least one of: (a) contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population; (b) transducing the population of activated T cells; and (c) expanding the population of transduced T cells. In some aspects, each of (a)-(c) is performed using T cell culture medium without added serum. As referred to herein, the term "serum-free medium" or "serum-free culture medium" means that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). Thus, in some embodiments, serum is not added to the culture medium as a separate, distinct component for the purpose of supporting the viability, activation, and growth of the cultured cells. Any suitable culture medium, such as a T cell growth medium, can be used to culture cells in suspension according to the methods described herein. For example, the T cell growth medium can include, but is not limited to, a sterile low-glucose solution containing suitable amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OPTMIZER™ (Life Technologies). In contrast to typical methods for producing engineered T cells, the methods described herein can use culture medium that is not supplemented with serum (e.g., human or bovine). Anti-CD3 / 28 nanomatrix

[0103] The anti-CD3 / 28 nanomatrix of the present disclosure is a nanometer-scale matrix comprising antibodies and / or fragments thereof that bind CD3 and CD28, and is provided by Miltenyi Biotec, Inc. (Auburn, CA) as the TransAct™ T cell reagent (hereinafter "TransAct™"). The TransAct™ reagent is a colloidal reagent consisting of nanoscale iron oxide crystals embedded in a biocompatible polysaccharide matrix with a total diameter of approximately 100 nm. Antibodies against CD3 (clone OKT3) and CD28 (clone 15E8) are covalently attached to the matrix. See, e.g., Casati et al., Cancer Immunol Immunother 2013 Oct;62(10):1563-73; Casati et al., MACS & more, Vol 15, 2013 Feb; and US2014 / 0087462.

[0104] T cells The one or more T cells described herein can be obtained from any source, including, for example, a human donor. The donor may be a subject in need of anti-cancer treatment, e.g., treatment with one or more T cells produced by the methods described herein (i.e., an autologous donor), or may be an individual who provides a lymphocyte sample that will be used to treat a different individual or cancer patient upon production of the population of cells produced by the methods described herein (i.e., an allogeneic donor). The population of lymphocytes can be obtained from the donor by any suitable method used in the art.

[0105] The methods described herein allow for the differentiation of less differentiated and immature cells (e.g., T cells) in a T cell population by contacting one or more cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of the T cell population. SCMThe anti-CD3 / 28 nanomatrix can be used to increase the proportion of naive and immature T cells. As described herein, it has surprisingly been found that potent activation of one or more T cells with an anti-CD3 / 28 nanomatrix, such as T Cell Transact™, increases the proportion of naive and immature T cells in vitro. In particular, after activation, one or more T cells can express one or more genes indicative of undifferentiated or immature T cells. The one or more genes indicative of undifferentiated or immature T cells can be selected from the group CD8, CD45RA, CCR7, CD45RO, CD62L, CD28, CD95, IL-7Rα, CXCR4, TCF7, FOXO1, ID3, BCL6, and any combination thereof. For example, contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) increases the proportion of cells expressing one or more genes indicative of undifferentiated or immature T cells selected from CD62L, CD45RA, CD45RO, and any combination thereof.

[0106] In other embodiments, after contacting one or more T cells with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes), the one or more T cells express CD62L, CD45RA, and / or CD45RO. In one particular embodiment, a greater percentage of the one or more T cells express CD62L, CD45RA, and / or CD45RO after contact with the anti-CD3 / 28 nanomatrix compared to before contact. In one specific embodiment, a greater proportion of one or more T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, relative to a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) express CD62L, CD45RA, and / or CD45RO than a population of T cells contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, relative to a T cell population of 17 volumes or more (e.g., 17.5, 20, 30, or 40 volumes).

[0107] T cell therapy The present disclosure provides methods for increasing the proportion of immature, less differentiated T cells in a population by contacting one or more T cells with a ratio of 1 volume of anti-CD3 / 28 nanomatrix, e.g., T-cell Transact™, to 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cells compared to a population of T cells contacted with a ratio of 1 volume of anti-CD3 / 28 nanomatrix, e.g., T-cell Transact™, to 17 volumes or more (e.g., 17.5, 20, 30, or 40 volumes). In some embodiments, the method further comprises administering one or more T cells prepared using the methods provided herein to a subject in need thereof. One of skill in the art will understand that one or more T cells produced by the methods provided herein can be used in any method of treating a patient, including administering one or more T cells to the patient.

[0108] For example, but not limited to, the methods described herein can enhance the efficacy of T cell therapy, which may be adoptive T cell therapy selected from the group consisting of tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), allogeneic T cell transplantation, engineered allogeneic T cell therapy, non-T cell transplantation, and any combination thereof. Adoptive T cell therapy broadly includes any method of selecting, enriching in vitro, and administering to a patient autologous or allogeneic T cells capable of recognizing and binding tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, enriched in vitro, and administered to a patient. TIL cells can be autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that involves isolating T cells capable of targeting tumor cells from a patient, enriching the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T cell transplants can include transplants of ex vivo expanded naturally occurring T cells or genetically engineered T cells. Non-T cell transplants can include autologous or allogeneic therapy using non-T cells, such as, but not limited to, natural killer (NK) cells.

[0109] In one specific embodiment, the T cell therapy of the present disclosure is an engineered allogeneic T cell therapy. According to this embodiment, the method may include collecting blood cells from a donor. The isolated blood cells (e.g., T cells) are then collected at a concentration of at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6The T cells may be genetically engineered to reduce expression of one or more naturally occurring proteins (e.g., endogenous TCR) contacted with one volume of anti-CD3 / 28 nanomatrix, e.g., T-cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) at a cell density of 1,000,000 cells / ml. The T cells may be engineered to express a chimeric antigen receptor ("engineered CAR T cells") or a T cell receptor ("engineered TCR T cells"). In one particular embodiment, the engineered allogeneic CAR T cells or engineered TCR T cells contacted with one volume of anti-CD3 / 28 nanomatrix, e.g., T-cell Transact™, for a T cell population of 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) are then administered to the subject. In some embodiments, the engineered T cells are directed to a solid or liquid tumor in a subject.

[0110] In one embodiment, one or more T cells can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor can include a binding molecule to a tumor antigen. The binding molecule can be an antibody or its antigen-binding molecule. For example, the antigen-binding molecule can be selected from scFv, Fab, Fab', Fv, F(ab')2, camelid, and dAb, as well as any fragment or combination thereof.

[0111] Chimeric antigen receptors can be engineered to target specific tumor antigens. In some embodiments, the tumor antigen is selected from the group consisting of BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin 18.2 (Claudin 18A2 or Claudin 18 isoform 2), DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Muc17 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast activation protein alpha), Ly6G6D ( Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), CD40, disialogangliosides GD2 and GD3, C-type lectin-like molecule-1 (CLL-1), ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-1, IGF-II, IGF-1 receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, O1, Nkp30, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and AI domain of tenascin-C (TnC)AI) and fibroblast-associated protein (fap), LRP6, melanoma-associated chondroitin sulfate proteoglycan (MCSP), MARTI, MUC1, LMP2, idiotype, NY-ESO-1, Ras mutants, gp100, proteinase 3, bcr-abl, tyrosinase, hTERT, EphA2, ML-TAP, ERG, NA17, PAX3, ALK, androgen receptor, lineage-specific or tissue-specific The antigen may be selected from a heterologous antigen, such as CD3, CD4, CD8, CD24, CD25, CD34, CD79, CD116, CD117, CD135, CD123, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility complex (MHC) molecule, MUC16, PSCA, Trop2, CD171 (L1CAM), CA9, STEAP1, VEGFR2, and any combination thereof.

[0112] The methods provided herein can include T cell therapy, which involves the transplantation of one or more T cells into a patient. The T cells can be administered in a therapeutically effective amount. The T cells, such as engineered CAR T cells, can be administered in a therapeutically effective amount. + T cells or engineered TCRs + A therapeutically effective amount of T cells may be, for example, at least about 10 4 cells, at least about 10 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 , or at least about 10 10 In another embodiment, the T cells, e.g., engineered CAR+ T cells or engineered TCR + A therapeutically effective dose of T cells is approximately 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In one particular embodiment, the T cell is an engineered CAR. + T cells or engineered TCRs + A therapeutically effective dose of T cells is approximately 2 × 10 6cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 cells / kg.

[0113] In some embodiments, the patient is preconditioned prior to administration of T cell therapy. The patient may be preconditioned according to any method known in the art, including, but not limited to, treatment with one or more chemotherapeutic agents and / or radiation therapy. In some embodiments, preconditioning may include any treatment prior to T cell therapy that reduces the number of endogenous lymphocytes, removes cytokine sinks, increases serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhances effector function of T cells administered after conditioning, enhances antigen-presenting cell activation and / or availability, or any combination thereof.

[0114] cancer treatment The disclosed methods can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, remove tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0115] In one embodiment, the present disclosure is directed to a method of treating a tumor in a subject in need of T cell therapy comprising administering to the subject one or more T cells, wherein the one or more T cells are at least about 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6 and contacting the T cell population with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell at a cell density of at least about 0.2 x 10 cells / ml. In another embodiment, the present disclosure is directed to a method of reducing or diminishing tumor size or inhibiting tumor growth in a subject in need of T cell therapy comprising administering to the subject one or more T cells, wherein the one or more T cells are at least about 0.2 x 10 6 cells / ml ~ approx. 5.8x10 6 At a cell density of cells / ml, the T cells are contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for up to 17 volumes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population.

[0116] The cancer that can be treated can include non-vascularized tumor, not yet substantially vascularized tumor, or vascularized tumor.Cancer can also include solid tumor or non-solid tumor.In certain embodiments, cancer can be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenal cortex cancer, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignant tumor such as NHL, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor. tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumors, central nervous system cancers, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma family of tumors, extracranial embryonal cells cystic tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fibrous histiocytoma of bone, malignant and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Posi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract carcinoma involving the NUT gene, mouth cancercancer), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer cancer), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma The cancer may be selected from tumors originating from the following: rhabdomyosarcoma, salivary gland carcinoma, sarcoma, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, nephroblastoma.

[0117] In one embodiment, the method can be used to treat tumors, where the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell present in the bone marrow. This stem cell produces both myeloid progenitor cells and lymphoid progenitor cells, which produce the various types of white blood cells found in the body. White blood cells that arise from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells that arise from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia may affect one or more of these cell types in a patient.

[0118] Thus, in some embodiments, the method can be used to treat lymphoma or leukemia, where the lymphoma or leukemia is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B-cell lymphoma), splenic, diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt's lymphoma, and lymphoblastic lymphoma. In some embodiments, the lymphoma or leukemia is selected from the group consisting of B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma ( For example, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma (IVBCL), and thymic large B-cell lymphoma (IVBCL). lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma Selected from: cutaneous anaplastic large cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T-lymphoblastic leukemia / lymphoma, and Hodgkin's lymphoma.In some embodiments, the cancer is refractory to one or more prior treatments and / or the cancer has recurred after one or more prior treatments.

[0119] In certain embodiments, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In one particular embodiment, the cancer is diffuse large B-cell lymphoma.

[0120] In some embodiments, the cancer is resistant to or has relapsed after one or more of chemotherapy, radiation therapy, immunotherapy (including T-cell therapy and / or treatment with an antibody or antibody-drug conjugate), autologous stem cell transplant, or any combination thereof. In one particular embodiment, the cancer is refractory diffuse large B-cell lymphoma.

[0121] In some embodiments, cancer is treated by administering to a subject one or more T cells, wherein the one or more T cells are at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6 The T cells are contacted with 1 volume of anti-CD3 / 28 nanomatrix, e.g., T Cell Transact™, for 17 volumes or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, or 16 volumes) of T cell population at a cell density of 1000 cells / ml. In some embodiments, the one or more T cells comprise engineered CAR cells or engineered TCR cells. In one embodiment, the engineered CAR cells or engineered T cells treat a tumor in a subject.

[0122] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference.

[0123] The following examples are intended to illustrate various embodiments of the present disclosure. Therefore, the specific embodiments discussed should not be construed as limitations on the scope of the present disclosure. For example, although the following examples are directed to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), those skilled in the art will understand that the methods described herein can be applied to T cells transduced with any CAR. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are included herein. Furthermore, all references cited in this disclosure are incorporated by reference in their entirety as if fully set forth herein.

[0124] Embodiment E1. 1) contacting a volume of anti-CD3 / CD28 nanomatrix with a volume of a starting cell population in a volume ratio, wherein the starting cell population is at least about 0.2x10 6 cells / ml ~ approx. 5.8x10 6 and 2) culturing the cell population in culture medium, wherein the resulting T cell population comprises an increased proportion of stem memory T cells compared to the second T cell population, wherein the second starting cell population at the same cell density is contacted with and cultured with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 1 volume of second starting cell population of 17.5 or more.

[0125] E2. The method of E1, wherein the nanomatrix is ​​Macs® GMP T Cell Transact™ Nanomatrix.

[0126] E3. The method according to any one of embodiments 1 and 2, wherein the starting cell population is obtained from peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion spleen tissue, tumor, mesenchymal tissue, T cell line, or artificial thymic organoid (ATO) cell culture system.

[0127] E4. The method of any one of embodiments 1 to 3, wherein the starting cell population is selected from the group consisting of T lymphocytes, B lymphocytes, helper T cells, tumor-infiltrating lymphocytes, memory T cells, cytotoxic T cells, natural killer T cells, peripheral blood lymphocytes, tumor-infiltrating leukocytes, peripheral blood mononuclear cells, dendritic cells, umbilical cord blood stem cells, pluripotent stem cells, and mesenchymal stem cells.

[0128] E5. The method of any one of embodiments 1 to 4, wherein the starting cell population is peripheral blood mononuclear cells, positively selected CD3, CD4, or CD8 T cells, or negatively selected CD3, CD4, or CD8 T cells.

[0129] E6. The method of any one of embodiments 1 to 5, wherein the starting cell population is peripheral blood mononuclear cells.

[0130] E7. The method of any one of embodiments 1 to 4, wherein the starting cell population is a purified T cell population.

[0131] E8. The starting cell density is at least approximately 0.50x10 6 ~ approx. 6.00x10 6 , about 0.56x10 6 ~approx. 5.72x10 6 , about 0.80x10 6 ~about 4.0x10 6 , approximately 1.00x10 6 ~about 3.0x10 6 , approximately 2.00x10 6 ~about 3.5x10 6 , or approximately 2.50x 6 ~about 3.5x10 6 8. The method according to any one of embodiments 1 to 7, wherein

[0132] E9. The cell density is approximately 2.86 x 10 6 9. The method of any one of embodiments 1 to 8, wherein the concentration is 0.01 to 0.25 μg / mL.

[0133] E10. The starting cell density is approximately 3.00 × 10 6 10. The method of any one of embodiments 1 to 9, wherein the concentration is cells / mL.

[0134] E11. The starting cell density is at least approximately 0.28 x 10 6 ~about 2.86x10 6 , 0.25x10 6 ~approx. 2.00x10 6 , about 0.5x10 6 ~about 2x10 6 , approximately 1.00x10 6 ~approx. 2.00x10 6 , about 0.80x10 6 ~about 1.5x10 6 , or approximately 1.20x10 6 ~about 1.5x10 6 10. The method of any one of embodiments 1 to 9, wherein the concentration is 0.01 to 0.25% by weight of the sera.

[0135] E12. The cell density was approximately 1.43 x 10 6 12. The method of any one of embodiments 1 to 8, or 11, wherein the total amount of the saturation factor is 0.015, 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.080, 0.095, 0.100, 0.110, 0.125, 0.130, 0

[0136] E13. The starting cell density was approximately 1.00x10 6 12. The method of any one of embodiments 1 to 8, or 11, wherein the total amount of the saturation factor is 0.015, 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.080, 0.095, 0.100, 0.110, 0.125, 0.130, 0.140, 0.150, 0.160, 0.170, 0.180, 0.190, 0.195, 0.200, 0.210, 0.220, 0.230, 0.240, 0.250, 0.260, 0.270, 0.280, 0.290, 0.300, 0.310, 0.320, 0.330, 0.340, 0.350, 0.360, 0.370, 0.380, 0.390, 0.395 ...

[0137] E14. The method of any one of embodiments 1 to 13, wherein the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the starting cell population is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, or 1:17.

[0138] E15. The method of any of embodiments 1-14, further comprising transducing the starting cell population before, during, or after culturing the starting cell population with the anti-CD3 / CD28 nanomatrix.

[0139] E16. The method of any of embodiments 15, wherein the starting cell population is transduced with a lentiviral vector, a retroviral vector, and / or an adenovirus-associated vector.

[0140] E17. The method of any of embodiments 1-16, further comprising transfecting the starting cell population before, during, or after culturing the starting cell population with the anti-CD3 / CD28 nanomatrix.

[0141] E18. The method of embodiment 17, wherein the transfection step comprises TALEN® mRNA electroporation (EP) and / or CRISPER Cas9 electroporation.

[0142] E19. The method of embodiment 18, wherein the transfecting step comprises disrupting the TCRαβ gene and / or the β2M gene.

[0143] E20. The method of any of embodiments 1-19, wherein the starting cell population is cultured for about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0144] E21. The method of any one of embodiments 1 to 20, wherein the resulting T cell population expresses one or more markers indicative of undifferentiated or immature T cells.

[0145] E22. The method of embodiment 21, wherein the one or more markers indicative of undifferentiated or immature T cells are selected from the group consisting of CD62L, CD45RA, CD45RO, and any combination thereof.

[0146] E23. The method of any of embodiments 1-22, wherein the resulting T cell population comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% more stem memory T cells than the second resulting T cell population.

[0147] E24. The method of any one of embodiments 1 to 23, wherein the starting cell population volume ratio is 1:5 and the second starting cell population volume ratio is 1:20, 1:25, or 1:50.

[0148] E25. The method of any one of embodiments 1 to 24, wherein the starting cell population volume ratio is 1:10 and the second starting cell population volume ratio is 1:20, 1:25, or 1:50.

[0149] E26. The method of any one of embodiments 1 to 24, wherein the starting cell population volume ratio is 1:15 and the second starting cell population volume ratio is 1:20, 1:25, or 1:50.

[0150] E27. The method of any one of embodiments 16 to 26, wherein the lentivirus comprises a heterologous gene encoding a cell surface receptor.

[0151] E28. The method of embodiment 27, wherein the cell surface receptor is capable of binding an antigen on the surface of a target cell.

[0152] E29. The method of embodiment 28, wherein the target cell is a tumor cell.

[0153] E30. The method of 28 or 29, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0154] E31. TCR or CAR expresses 707-AP (707 alanine proline), AFP (alpha(a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), ), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exon 7 / 8), CAMEL (antigen recognized by CTL in melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 mutation), CDK4 / m (cyclin-dependent kinase 4 mutation), CEA (carcinoembryonic antigen), CT (cancer / Testis (antigen)), Cyp-B (Cyclophilin B), DAM (Differentiation antigen melanoma), EGFR (Epidermal growth factor receptor), EGFRvIII (Epidermal growth factor receptor, variant III), EGP-2 (Epithelial glycoprotein 2), EGP-40 (Epithelial glycoprotein 40), Erbb2, 3, 4 (Erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (Elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6 / Acute bone marrow adenoma) Myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100 kD), HAGE (helicase antigen), HER-2 / neu (human epidermal receptor-2 / neutron receptor; neurology);Also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2 mutated), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), Kappa light chain, LAGE (L antigen), LDLR / FUT (low density lipid receptor / GDP-L-fucose: bD-galactosidase 2-aL fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen recognized by T cells-1 / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (myosin mutation), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous mutations 1, 2, 3), NA88-A (NA in patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), oncofetal antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (190KDbcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous epithelial antigen rejecting tumor 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 31. The method of embodiment 30, wherein the antibody is capable of binding an antigen selected from the group consisting of: tyrosinase-related protein 72), TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1), TPI / m (triosephosphate isomerase mutant), TRP-1 (tyrosinase-related protein 1, or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms tumor gene), CD70, FLT3, DLL3, and any combination thereof.

[0155] E33. The method of any one of embodiments 1-32, further comprising administering a therapeutically effective amount of the resulting T cells to a subject in need thereof.

[0156] E34. A method of treating a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more of the resulting T cells described in any of embodiments 1-33.

[0157] E35. A method of reducing or diminishing tumor size or inhibiting tumor growth in a subject in need of T cell therapy, comprising administering to the subject one or more of the resulting T cells described in any of embodiments 1-34.

[0158] E36. The tumor is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, multiple myeloma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia 36. The method of any of embodiments 29-35, wherein the cancer is selected from acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, other B-cell malignancies, and any combination thereof.

[0159] E37. 1) Contacting a volume of anti-CD3 / CD28 nanomatrix with a volume of an initial PBMC population at a volume ratio, wherein the initial PBMC population is at least about 0.5x10 6 cells / ml ~ approx. 2.0x10 6 and 2) culturing the PBMC population in culture medium for 14 to 18 days, wherein the resulting PBMC population contains an increased proportion of stem memory T cells compared to the second PBMC population, wherein the second starting PBMC population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20 volumes of the second starting PBMC population, and cultured for the same number of days.

[0160] E38. 1) contacting a volume of anti-CD3 / CD28 nanomatrix with a volume of a starting purified T cell population in a volume ratio, wherein the starting T cell population is at least about 0.80x10 6 cells / ml ~ approx. 1.60x10 6 and 2) culturing the PBMC population in culture medium for 11 to 18 days, wherein the resulting purified T cell population contains an increased proportion of stem memory T cells compared to the second purified T cell population, wherein the second starting purified T cell population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20, 25, or 50 volumes of the second starting T cell population, and cultured for the same number of days.

[0161] E39. 1) contacting a volume of anti-CD3 / CD28 nanomatrix with a volume of an initial T cell population at a volume ratio, wherein the initial T cell population is at least about 0.80x10 6 cells / ml ~ approx. 1.60x10 6 and 2) culturing the T cell population in culture medium for 11 to 18 days, wherein the resulting T cell population contains an increased number of T cells compared to the second T cell population, wherein the second starting T cell population at the same cell density is contacted with anti-CD3 / CD28 nanomatrix at a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20, 25, or 50 volumes of the second starting T cell population, and cultured for the same number of days. [Example]

[0162] Example 1 Preparation of ex vivo activated allogeneic T cells from PBMC populations Donor blood was collected and separated into its component parts by apheresis. PBMCs were then concentrated on a Ficoll®-hypaque step gradient and cryopreserved. On day 0, Ficoll®-isolated and frozen human PBMCs were thawed and washed once with culture medium containing 10% human serum. The cells were then cultured in medium with 5% human serum and incubated overnight at 37°C and 5% CO2. On day 1, the resulting PBMCs were approximately 3 x 10 6 Cell density below 1.5 x 10 cells / mL and divided into test fractions 6 Activated in cells / mL as follows: [Table 1]

[0163] For the control, test groups 1-2, cells were cultured in the presence of mTransAct, which is provided in the mTransAct CD3 / CD28 Kit (Cat. No. 130-020-008, Miltenyi Biotec, Auburn, CA), in which the anti-CD3 and anti-CD28 antibodies are mouse monoclonal antibodies against CD3 and CD28, respectively.

[0164] For test groups 3 to 5, cells were cultured in the presence of Transact™ (Miltenyi Biotec, Auburn, CA), in which the nanomatrix is ​​conjugated with humanized anti-CD3 and anti-CD28 antibodies (hereinafter "h TransAct™") at the indicated volume ratios.

[0165] For all test groups, cells were activated with Transact™ at the cell culture volume ratios shown in Table 1. The culture medium in which the cells were activated consisted of X-vivo15 (Lonza) and 100 IU / ml of 5% human serum (Gemini) plus IL-2 (Miltenyi Biotec).

[0166] On day 4, the activation culture medium was diluted with fresh culture medium or completely removed by centrifugation, as shown in Table 1. For test groups 1 and 5, cells were diluted with at least one volume of fresh culture medium to one volume of activation culture medium. For the remaining test groups, centrifugation was performed at 540 g for 5 minutes at room temperature. The cells were then washed once with culture medium.

[0167] Additionally, on day 4, activated cells can be genetically modified by lentiviral vector transduction for CAR introduction using methods known in the art. Briefly, for test groups 1 and 5, a lentiviral vector (LVV #1831P, Lentigen Technology, Gaithersburg, MD) was added to the culture medium at a dilution of the activation culture medium with fresh cell culture medium, and cells were transduced according to the manufacturer's instructions. For the remaining test groups, cells were transduced according to the manufacturer's instructions. Briefly, cells were harvested, washed once, and resuspended in culture medium supplemented with lentiviral vector (Lentigen Technology, Gaithersburg, MD) at 1-10% v / v of the lentiviral vector. For both methods, the cell density for transduction was 1 x 10 6 cells / mL.

[0168] Cells were grown in T-flasks from day 4 to day 6. On day 6, cells were further gene-edited by TALEN® mRNA electroporation (EP) to disrupt the TCRαβ gene and knock out its expression. TALEN® mRNA electroporation was performed using the AgilePulse® Electroporation System (available from BTX®, a division of Harvard Biosciences, Holliston, MA) according to the manufacturer's instructions.

[0169] Allogeneic T cells, including genetically modified allogeneic CAR-T cells, were expanded in G-Rex® 10 (Wilson Wolf, St. Paul, MN) between days 8 and 18 according to the manufacturer's instructions.

[0170] Cell growth and yield Actual cell yields were measured on days 1, 4, 6, 8, and 18 using an automated cell counter NucleoCounter NC-200 (Chemometec, Allerod, Denmark). Results from days 1 to 18 are shown in Figure 1. Results from days 1, 4, 6, and 8 before cell expansion in G-Rex® are shown in Figure 2, and results from days 8 to 18 of expansion in G-Rex® are shown in Figure 3.

[0171] Total cell growth fold was determined and results from days 1 through 18 are shown in Figure 4. Figure 5 shows the growth fold at each process step for each test group, with test groups 3 through 5 having higher growth folds than test groups 6 and 7 from days 8 through 18. Figure 6 shows the growth fold results at each process step for each test group from days 0 through 6.

[0172] cell phenotype Cell phenotype is analyzed on days 1 and 18. Cells are labeled with fluorescent antibodies against relevant targets according to the manufacturer's instructions.

[0173] Fluorescently labeled cells were then analyzed by an LSRFortessa™ cytometer (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions, and data were analyzed using FlowJo software version 10 (FlowJo, Inc., Ashland, OR).

[0174] PBMC cell subsets were determined for each of the test groups and relevant controls on day 1 using commercially available antibodies against CD5 (BD Horizon), CD14 (Biolegend), CD56 (BD Biosciences), and CD19 (Biolegend).

[0175] CD8 T cell subsets were determined for each of the test groups and relevant controls on day 1 using commercially available antibodies against CD8 (BD Biosciences), CD45RA (Biolegend), and CD62L (BD Biosciences).

[0176] CD4:CD8 cell ratios were determined for each of the test groups and relevant controls on day 1 using commercially available antibodies against CD4 (BD Biosciences) and CD8 (BD Biosciences).

[0177] CD45RA + , CD62L + Total CD5 + Cell subsets were determined for each of the test groups and relevant controls on day 18 using commercially available antibodies against CD5 (BD Horizon), CD45RA (Biolegend), and CD62L (BD Biosciences).

[0178] CD45RO + , CD62L + Total CD5 +Cell subsets were determined for each of the test groups and relevant controls on day 18 using commercially available antibodies against CD5 (BD Horizon), CD45RO (Biolegend), and CD62L (BD Biosciences).

[0179] For each test group and relevant controls, the percentage of CAR+% and TCRαβ-% cells will be determined on day 18 using a fluorochrome-conjugated CAR anti-idiotypic antibody and a commercially available antibody against TCRαβ (Biolegend).

[0180] Cell viability was monitored from day 0 to day 18 for each of the test groups and relevant controls using a NucleoCounter® NC-200™ (Chemometec, Allerod, Denmark) according to the manufacturer's instructions.

[0181] Cell diameter was monitored from day 0 to day 18 for each of the test groups and relevant controls using a NucleoCounter® NC-200™ (Chemometec, Allerød, Denmark) according to the manufacturer's instructions.

[0182] Example 2 Preparation of ex vivo activated allogeneic T cells from purified T cell populations Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from anonymous blood donors using Ficoll®-Paque PLUS (GE Healthcare Life Sciences) and SepMate™-50 tubes (STEMCELL Technologies) according to the protocol provided by STEMCELL Technologies. Pan T cells were then isolated from freshly prepared PBMCs using a pan T cell isolation kit (Miltenyi Biotec) according to the protocol provided by Miltenyi Biotec. Pan T cells were stored in liquid nitrogen until ready for use.

[0183] Primary human pan-T cells (10–30 × 10 6 ) were thawed on day 0 and cultured at approximately 2 × 10 6 The T cells were resuspended at 1000 cells / mL and incubated in a humidified incubator at 37°C, 5% CO2 for 30 minutes. After the 30-minute incubation, the T cells were counted and the cell density was adjusted using T cell transduction medium. The T cells were then mixed with either beads from a T cell activation / expansion kit (Miltenyi Biotec) at a 1:1 bead-to-T cell ratio or beads from a T cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at volumetric dilutions of 1:10, 1:15, 1:20, 1:25, or 1:50. Recombinant human IL-2 (Miltenyi Biotec) was added to a final concentration of 100 U / mL. The T cells were then returned to the 37°C incubator.

[0184] Two days later (day 2), T cells were counted and 5 × 10 5The cells were resuspended in T cell transduction medium at 1000 cells / mL and fresh IL-2 was added. The T cells were then transduced with a lentiviral vector encoding the FMC63-41BB-CD3ζ anti-CD19 CAR and returned to a 37°C incubator. Untransduced (UT) controls were generated in parallel. On day 5, transduction efficiency was confirmed by flow cytometry, and FMC63-41BB-CD3ζ anti-CD19 CAR T cells and UT control T cells were transferred to G-Rex® 6-well plates (Wilson Wolf). T cell culture medium—X-Vivo™ 15 (Lonza) + 5% human serum AB (clot-off) (Gemini BioProducts) and 100 IU / mL recombinant human IL-2 (Miltenyi Biotec)—was added up to 35 mL.

[0185] Immunophenotyping was performed by flow cytometry using an LSRFortessa™ X-20 Cell Analyzer (BD Biosciences), and data were analyzed using FlowJo® v10 (FlowJo) software. The following antibodies were used: Alexa Fluor (AF) 700 anti-human CD25 antibody (BioLegend #302622), BUV395 anti-human CD62L antibody (BD Bioscience #565219), BV605 anti-human CD8a antibody (BioLegend #301040), BV786 anti-human CD4 antibody (BD Bioscience #563914), PE anti-human CD137 (4-1BB) antibody (BioLegend #309804), and peridinin chlorophyll (PerCP) / Cy5.5 anti-human CD45RO antibody (BioLegend #304222).

Claims

1. A method for increasing the proportion of stem memory T cells in a cell population, comprising the following steps a) and b): a) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting cell population in a volume ratio; i) the starting cell population is 1.00 x 10 6 cells / ml to a cell density of 2.00 x 10 cells / ml; and ii) the volume ratio is 1 volume of anti-CD3 / CD28 nanomatrix to 5-15 volumes of the starting cell population; and b) culturing the cell population contacted with the anti-CD3 / CD28 nanomatrix in step a) in a culture medium containing human serum and / or IL-2; Including, the starting cell population is peripheral blood mononuclear cells or a purified T cell population; the cultured cell population of step b) comprises an increased proportion of stem memory T cells compared to the second cell population; the stem memory T cells express one or more markers selected from the group consisting of CD62L, CD45RA, CD45RO, or any combination thereof; The second cell population is obtained from a second starting cell population having the same cell density as the starting cell population by the same method as steps a) and b), except that in step a), the second starting cell population is used instead of the starting cell population, and the volume ratio in step a) is a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to the volume of the second starting cell population of 17.5 or more.

2. The method of claim 1 , wherein the starting cell population is peripheral blood mononuclear cells.

3. The starting cell population is CD4 + and / or CD8 + The method of claim 1 or 2, wherein the purified T cell population comprises T cells.

4. The cell density of the starting cell population is 1.50 x 10 6 The method according to any one of claims 1 to 3, wherein the concentration is 0.01 to 0.15 cells / mL.

5. The cell density of the starting cell population is 1.20 x 10 6 ~1.5 x 10 6 The method according to any one of claims 1 to 3, wherein the concentration is 0.01 to 0.15 cells / ml.

6. The cell density of the starting cell population was 1.43 x 10 6 The method according to claims 1 to 3, wherein the concentration is cells / mL.

7. 7. The method of any of claims 1 to 6, wherein the volume ratio of the volume of anti-CD3 / 28 nanomatrix to the volume of the starting cell population is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, or 1:

15.

8. 8. The method of any of claims 1 to 7, wherein the starting cell population is cultured for 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

9. 9. The method of any of claims 1 to 8, wherein the cell population comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% more stem memory T cells than the second cell population.

10. A method described in any of claims 1 to 9, wherein the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the starting cell population is 1:5, and the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the second starting cell population is 1:20, 1:25, or 1:

50.

11. A method described in any of claims 1 to 9, wherein the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the starting cell population is 1:10, and the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the second starting cell population is 1:20, 1:25, or 1:

50.

12. A method described in any of claims 1 to 9, wherein the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the starting cell population is 1:15, and the volume ratio of the volume of the anti-CD3 / 28 nanomatrix to the volume of the second starting cell population is 1:20, 1:25, or 1:

50.

13. 1. A method for increasing the proportion of stem memory T cells in a PBMC population, said method comprising the steps of: a) inducing a proliferation of stem memory T cells in a PBMC population; a) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting PBMC population in a volume ratio; i) the starting PBMC population is 1.00 x 10 6 cells / ml~2.00×10 6 is the cell density in cells / ml, and ii) the volume ratio is 1 volume of anti-CD3 / CD28 nanomatrix to 10-15 volumes of the starting PBMC population; and b) culturing the PBMC population contacted with the anti-CD3 / CD28 nanomatrix in step a) in a culture medium containing human serum and / or IL-2 for 14 to 18 days; Including, the cultured PBMC population of step b) comprises an increased proportion of stem memory T cells compared to the second PBMC population; the stem memory T cells express one or more markers selected from the group consisting of CD62L, CD45RA, CD45RO, or any combination thereof; The second PBMC population is obtained from a second starting PBMC population having the same cell density as the starting PBMC population by the same method as steps a) and b), except that in step a), the second starting PBMC population is used instead of the starting PBMC population, and the volume ratio in step a) is a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20 volumes of the second starting PBMC population.

14. 1. A method for increasing the proportion of stem memory T cells in a purified T cell population, the method comprising the steps of: a) inducing a cell population containing stem memory T cells; a) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting purified T cell population in a volume ratio; i) the starting purified T cell population is 1.00 x 10 6 cells / ml~1.60×10 6 is the cell density in cells / ml, and ii) the volume ratio is 1 volume of anti-CD3 / CD28 nanomatrix to 10-15 volumes of the starting purified T cell population; and b) culturing the starting purified T cell population contacted with the anti-CD3 / CD28 nanomatrix in step a) in a culture medium containing human serum and / or IL-2 for 11 to 18 days; Including, the cultured purified T cell population of step b) comprises an increased proportion of stem memory T cells compared to the second purified T cell population; the stem memory T cells express one or more markers selected from the group consisting of CD62L, CD45RA, CD45RO, or any combination thereof; The second purified T cell population is obtained from a second starting purified T cell population having the same cell density as the starting purified T cell population by the same method as steps a) and b) above, except that in step a), the second starting purified T cell population is used instead of the starting purified T cell population, and the volume ratio in step a) is a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20, 25, or 50 volumes of the second starting purified T cell population.

15. 1. A method for increasing the total number of T cells in a T cell population, said method comprising the steps of: a) and b): a) contacting a volume of an anti-CD3 / CD28 nanomatrix with a volume of a starting T cell population in a volume ratio; i) the starting T cell population is 1.00 x 10 6 cells / ml~1.60×10 6 is the cell density in cells / ml, and ii) the volume ratio is 1 volume of anti-CD3 / CD28 nanomatrix to 10-15 volumes of the starting T cell population; and b) culturing the T cell population contacted with the anti-CD3 / CD28 nanomatrix in step a) in a culture medium containing human serum and / or IL-2 for 11 to 18 days; Including, the cultured T cell population of step b) comprises an increased number of T cells compared to the second T cell population; The second T cell population is obtained from a second initial T cell population having the same cell density as the initial T cell population by the same method as steps a) and b), except that in step a), the second initial T cell population is used instead of the initial T cell population, and the volume ratio in step a) is a ratio of 1 volume of anti-CD3 / CD28 nanomatrix to 20, 25, or 50 volumes of the second initial T cell population.

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