T cell response to expression of chimeric antigen receptors and other receptors

Abstract

To provide methods for preparing T cell populations useful for a variety of purposes requiring a T cell population that is active for an extremely long time.SOLUTION: The T cell populations are made rich in nave T cells (TN), memory stem T cells (TSCM) and central memory T cells (TCM). These cell populations can be acquired from peripheral blood mononuclear cells (PBMC) by: 1) depleting unwanted cell populations such as CD14 expressing myeloid cells and CD25 expressing cells; and 2) making them rich in CD62L expressing memory T cells and nave T cells.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates generally to an isolated population of human cells comprising T cells, and in particular to an isolated population of human cells, wherein the T cells comprise central memory T cells, memory stem T cells and naive T cells, and wherein greater than 40% of the T cells are CD45RA+ and greater than 70% of the T cells are CD62L+. [Background technology]

[0002] Adoptive T cell therapy (ACT), which utilizes ex vivo expanded autologous and allogeneic T cells, is an attractive therapeutic approach for the treatment of viral infections, cancer, and autoimmune diseases. Methods that can rapidly generate large numbers of therapeutic T cells are crucial for the efficacy and safety of ACT. Various T cell enrichment methods have been used for ACT, including methods for selection and expansion of defined T cell subsets. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2016 / 044811 [Patent Document 2] WO2104 / 144622 [Patent Document 3] WO2002 / 077029 [Patent Document 4] WO / US2014 / 0288961 [Non-patent literature]

[0004] [Non-Patent Document 1] Berger et al.,2008 Journal of Cellular Immunology 118:4817 [Non-Patent Document 2] Wang et al.,2011 Blood 117:1888 [Non-Patent Document 3] Wang X et al. J Immunotherapy 2012;35:689. [Non-Patent Document 4] Graef et al. Immunity 41:116 [Non-Patent Document 5] Gattioni et al.2014 Immunity 41:7 Summary of the Invention [Problem to be solved by the invention]

[0005] It is desirable to use a T cell population that has relatively high in vivo activity and relatively high proliferative capacity. [Means for solving the problem]

[0006] Described herein are methods for preparing T cell populations useful for expressing chimeric antigen receptors ("CARs") for ACT. The T cell populations are also useful for a variety of other purposes where a highly active and long-lived T cell population is required. The T cell populations prepared by the methods described herein include: naive T cells (T N ), memory stem T cells (T SCM ) and central memory T cells (T CM ) are enriched for T CM / SCM / N Cells or T CM / SCM / N These cell populations can be described as populations of cells that can be obtained from peripheral blood mononuclear cells (PBMCs) by both: 1) depleting unwanted cell populations such as CD14-expressing myeloid cells and CD25-expressing cells; and 2) enriching for CD62L-expressing memory T cells and naive T cells. Thus, the resulting cell populations include naive T cells (T N ) and memory stem T cells (T SCM ), and central memory T cells (TCM ) population is also included. CM / SCM / N The population of cells is similar to previously described T cells in that its preparation does not involve depletion of CD45RA+ T cells. CM Different from a population of cells. CM / SCM / N At the time of preparation, the population of cells was classified as effector memory cells (T EM ) and effector cells (T E ) is relatively free of T CM / SCM / N As a population of cells proliferates, differentiation occurs, e.g., T E Cells are produced.

[0007] Patient-specific autologous T CM / SCM / N Cellular and allogeneic T CM / SCM / N Cells (or allogeneic T CM / SCM / N The cells can be engineered to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the engineered cells can be expanded and used in ACT.

[0008] Described herein are populations of isolated human cells comprising T cells (i.e., cells expressing CD3 or CD3+ cells), including central memory T cells; memory stem T cells, and naive T cells, wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the T cells are CD45RA+ and further, greater than 70% (greater than 75%, 80%, 85% or 90%) of the T cells are CD62L+. In various embodiments: less than 15% (less than 12%, 10%, 8%, or 6%) of the T cells are CD14+ and less than 5% (less than 4%, 3%, or 2%) of the T cells are CD25+; more than 10% (more than 15%, 20%, 25%, 30%, 35%, or 40%) of the T cells harbor a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding a chimeric antigen receptor or a T cell receptor; e.g., a viral vector such as a T cell vector); and at least 40% (45%, 50%, 55%, 60%, 65%, or 70%) of the T cells are CD14+. at least 10% (greater than 15%, 20%, 25%, 30%, 35% or 40%) of the T cells are CD8+ and CD62L+; less than 60% (less than 55%, 50%, 45%, 40%, 35%, 30%, 24%, 20% or 15%) of the T cells are CD45RO+; and at least 80% (greater than 85%, 90%, 95% or 98%) of the cells in the isolated cell population comprising T cells are T cells.

[0009] Also described herein are isolated populations of cells comprising human T cells (i.e., cells expressing CD3 or CD3+ cells), the T cells including central memory T cells and memory stem T cells, wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65%, or 70%) of the T cells are CD45RA+, greater than 40% (greater than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) of the T cells are CD62L+, greater than 70% (greater than 75%, 80%, 85%, or 90%) of the T cells are CD95+, and greater than 10% (greater than 15%, 20%, 25%, 30%, 35%, or 40%) of the T cells comprise a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding a chimeric antigen receptor or a T cell receptor, such as a viral vector). In various embodiments: less than 15% (less than 12%, 10%, 8%, 6%) of the T cells are CD14+ and less than 5% (less than 4%, 3%, or 2%) of the T cells are CD25+; at least 40% (greater than 45%, 50%, 55%, 60%, 65%, or 70%) of the T cells are CD4+ and CD62L+, or CD8+ and CD62L+; at least 1 of the T cells are CD4+ and CD62L+. 0% (greater than 15%, 20%, 25%, 30%, 35% or 40%) of the T cells are CD8+ and CD62L+; less than 60% (less than 55%, 50%, 45%, 40%, 35%, 30%, 24%, 20% or 15%) of the T cells are CD45RO+; and at least 80% (greater than 85%, 90%, 95% or 98%) of the cells in a population of isolated cells that contains T cells are T cells.

[0010] Described herein is a method of preparing a population of human cells comprising T cells (i.e., cells expressing CD3 or CD3+ cells) comprising a recombinant nucleic acid molecule, comprising: (a) providing a sample of human cells comprising T cells, the T cells including: central memory T cells; memory stem T cells, and naive T cells, wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the T cells are CD45RA+ and greater than 70% (greater than 75%, 80%, 85% or 90%) of the T cells are CD62L+; (b) activating the population of human cells comprising T cells; and (c) transducing or transducing cells in the population of human cells comprising T cells with the recombinant nucleic acid molecule to provide a population of human cells comprising T cells comprising the recombinant nucleic acid molecule, the method not including the step of depleting cells expressing CD45RA. In various embodiments: the recombinant nucleic acid molecule is a viral vector (e.g., a lentiviral vector, a viral vector encoding a CAR, a viral vector encoding a T cell receptor); the method further comprises culturing the population of human cells comprising T cells comprising the recombinant nucleic acid molecule; the culturing comprises adding exogenous IL-2 and exogenous IL-15; further, the activating comprises exposing the cells to an anti-CD3 antibody and an anti-CD28 antibody; and further, at least 80% (greater than 85%, 90%, 95%, or 98%) of the cells in the population of isolated cells comprising T cells are T cells.

[0011] Described herein is a method for preparing a population of human cells comprising T cells (i.e., cells expressing CD3 or CD3+ cells), including central memory T cells; memory stem T cells, and naive T cells, where greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the cells are CD45RA+ and greater than 70% (greater than 75%, 80%, 85% or 90%) of the cells are CD62L+, the method comprising: (a) providing a population of isolated human cells comprising T cells; (b) isolating cells expressing CD25 and CD3+ cells to prepare a depleted cell population. (c) treating the population of isolated human cells comprising T cells to deplete cells expressing CD14; and (c) treating the depleted cell population to enrich for cells expressing CD62L, thereby preparing a population of human cells comprising T cells, wherein the T cells include central memory T cells; memory stem T cells and naive T cells, and wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the cells are CD45RA+ and further, greater than 70% of the cells are CD62L+, wherein the method does not include depleting cells expressing CD45RA. In various embodiments: the method further comprises activating a population of human cells comprising T cells, and transducing or transducing the activated cells with a recombinant nucleic acid molecule to provide a population of T cells comprising T cells comprising the recombinant nucleic acid molecule; the population of isolated human cells comprising T cells comprises T cells comprising PBMCs; and further, at least 80% (greater than 85%, 90%, 95% or 98%) of the cells in the population of isolated cells comprising T cells are T cells.

[0012] Also described herein are populations of human cells comprising T cells (i.e., cells expressing CD3 or CD3+ cells), including central memory T cells; memory stem T cells, and naive T cells, wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the cells are CD45RA+ and greater than 70% (greater than 75%, 80%, 85% or 90%) of the cells are CD62L+, the populations comprising: providing a population of isolated human cells comprising T cells (e.g., PBMCs from a donor); depleting cells expressing CD25 and depleting cells expressing CD14 to prepare a depleted cell population; and treating the depleted cell population to enrich for cells expressing CD62L, thereby preparing a population of human cells comprising T cells, wherein the T cells include central memory T cells; memory stem T cells and naive T cells, and wherein greater than 40% (greater than 45%, 50%, 55%, 60%, 65% or 70%) of the cells are CD45RA+ and further greater than 70% (greater than 75%, 80%, 85% or 90%) of the cells are CD62L+, wherein the method does not include depleting cells expressing CD45RA. In various embodiments, less than 15% (less than 12%, 10%, 8%, 6%) of the T cells in a population of human cells are CD14+ and less than 5% (less than 4%, 3%, or 2%) of the T cells are CD25+; at least 40% (more than 45%, 50%, 55%, 60%, 65%, or 70%) of the T cells are CD4+ and CD62L+, or CD8+ and CD62L+; at least 10% (more than 15%, 20%, 25%, 30%, 35%, or 40%) of the T cells are CD8+ and CD62L+; and less than 60% (less than 55%, 50%, 45%, 40%, 35%, 30%, 24%, 20%, or 15%) of the T cells are CD45RO+.

[0013] Also described herein are methods of treating cancer, autoimmune or infectious diseases, the methods comprising administering to a patient in need thereof a pharmaceutical composition comprising a population of human cells described herein. Optionally, the cells are autologous to the patient being treated, and optionally, the cells are allogeneic to the patient being treated.

[0014] A cell population may be depleted for cells that express a particular marker (such as a receptor) by removing (e.g., using selective antibodies and cell sorting techniques) some, most, or nearly all of the cells that express that marker from the population of cells, thereby reducing the relative proportion of cells in the population that express that marker. A cell population may be enriched for cells that express a particular marker (such as a receptor) by collecting (e.g., using selective antibodies and cell sorting techniques) some, most, or nearly all of the cells that express that marker from the population of cells and discarding cells that do not express that marker, thereby reducing the relative proportion of cells in the population that express that marker. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the expression patterns of certain markers for various T cell subsets, and in particular, the expression patterns that distinguish TSCM from TN. [Diagram 2] FIG. 1 shows expression data of specific markers for various T cell subsets. [Diagram 3] FIG. 1 shows the relationship between specific T cell subsets based on CD62L and CD45RO expression. [Figure 4]Figure 1 shows the results of surface phenotyping of cells after CliniMACS™ enrichment for TCM or TCM / SCM / N cells. Cells from the indicated products were stained with fluorochrome-conjugated antibodies specific for the T cell markers CD3 and CD8, the central memory T cell marker CD62L and the naive T cell marker CD45RA as well as the monocyte marker CD14 and the regulatory T cell marker CD24. The percentage of immunoreactive cells (shaded histograms) over isotype control staining (black line) is shown in each histogram. [Diagram 5] Figure 1 shows the results of a study on the proliferation of TCM and TCM / SCM / N cell populations. Viable cell counts at the start of the day of lentiviral transduction are plotted for cell products produced from four of five donors, with TCM-derived products represented by black circles and TCM / SCM / N-derived products represented by white squares. A Log2 scale was used for the Y-axis to better represent the doubling time of cells in culture. [Figure 6]Figure 1 shows the results of surface phenotypic analysis of TCM and TCM / SCM / N cell populations. TCM-enriched or TCM / SCM / N-enriched cells were stimulated with beads, transduced with CD19R(EQ)28Z-T2A-EGFRt_epHIV7 or IL13(EQ)BBZ-T2A-CD19t_epHIV7, and expanded ex vivo. Cells were then stained with fluorochrome-conjugated reagents specific for EGFRt or CD19t transduction markers, or for T cell markers CD3, CD4, and CD8, central memory T cell markers CD27, CD28, and CD62L, and naive T cell marker CD45RA, and analyzed by flow cytometry (grey histograms). The percentage of immunoreactive cells over control staining (open histograms) is shown in each histogram of a representative TCM / TCM / SCM / N pair (A) or as a data point for each cell line in (B), with white symbols for TCM-derived products and grey symbols for TCM / SCM / N-derived products. Red bars depict the mean values ​​for each group, and in addition, p values ​​for paired Student's t-test comparisons of TCM-derived versus TCM / SCM / N-derived cell products are shown for each surface marker. *, P value was not calculated for CD27 due to too few values. Note that due to overlap, only shading is shown for CD3 staining in (B) rather than cell line specific symbols. [Figure 7A] Figure 1 shows the results of a study evaluating the therapeutic effect of CD19R TCM or CD19R TCM / SCM / N pretreatment when administered with Sup-B15 tumor cells. One day after irradiating NSG mice, mice were divided into groups (n=4) and either left untreated or treated intravenously with 2.5x106 mock-transduced PBMCs (no CAR), CD19R TCM or CD19R TCM / SCM / N (approximately 0.5x106 CAR+ cells). On day 2, mice were administered 0.7-1.0x106 M,million ffLuc+Sup-B15 tumor cells intravenously. [Figure 7B]Using Xenogen Living Image, a representative mouse from each group shows relative tumor burden. [Figure 7C] Quantification of average Sup-B15 tumor growth and ffLux flux (photons / sec) in each group of mice over time as monitored by Xenogen imaging is shown. [Figure 8A] Figure 1 shows the results of a study investigating the presence of human T cells in blood 41 days after intravenous pretreatment with CD19R TCM or CD19R TCM / SCM / N. One day after irradiation of NSG mice, mice were divided into groups (n=4) and either left untreated or treated intravenously with 2.5x106 mock-transduced PBMCs (no CAR), CD19R TCM or CD19R TCM / SCM / N (approximately 0.5x106 CAR+ cells). On day 2, mice were administered 0.7-1.0x106 tumor GFP+Sup-B15 tumor cells intravenously. On day 41, the blood of each mouse was assessed for the presence of human CD45+CD3+ cells (i.e. human T cells) by flow cytometry. Representative flow cytometric analyses of each mouse are shown (mouse 1 and mouse 2). Here huCD45-dependent cells were further analyzed for expression of CD3 T cell markers and EGFRt transduction markers (%CAR+ based on upper right quadrant). Gold boxes are used to highlight the GFP+SupB15 population. [Figure 8B]Figure 1 shows the results of a study investigating the presence of human T cells in blood 41 days after intravenous pretreatment with CD19R TCM or CD19R TCM / SCM / N. One day after irradiation of NSG mice, mice were divided into groups (n=4) and either left untreated or treated intravenously with 2.5x106 mock-transduced PBMCs (no CAR), CD19R TCM or CD19R TCM / SCM / N (approximately 0.5x106 CAR+ cells). On day 2, mice were administered 0.7-1.0x106 tumor GFP+Sup-B15 tumor cells intravenously. On day 41, the blood of each mouse was assessed for the presence of human CD45+CD3+ cells (i.e. human T cells) by flow cytometry. Representative flow cytometric analyses of each mouse are shown (mouse 3 and mouse 4). Here huCD45-dependent cells were further analyzed for expression of CD3 T cell markers and EGFRt transduction markers (%CAR+ based on upper right quadrant). Gold boxes are used to highlight the GFP+SupB15 population. [Figure 9] Figure 1 shows the results of a long-term killing assay performed with TCM or TCM / SCM / N cells transduced or mock-transduced with CD19R(EQ)28ZEGFRt_epH1V7 as effector and CD19-negative K562 cells (white bars) or CD19+SupB15 cells (black bars) as targets. The percentage of viable target cells (CD45-negative, FSC-high) remaining after 72 hours compared to mock effector co-cultures, normalized to 100%, is shown. [Figure 10] Figure 1 shows the results of a 5-h degranulation assay performed with TCM or TCM / SCM / N cells transduced or mock-transduced with CD19R(EQ)28ZEGFRt_epH1V7 as effector and CD19-negative K562 cells (white bars) or CD19+SupB15 cells (black bars) as stimulators. The percentage of CD45 / CD8 / EGFRt-dependent cells that were immunoreactive for CD107a is shown. [Figure 11]Figure 1 shows the results of an analysis of IFNγ production by TCM or TCM / SCM / N cells transduced or mock transduced with CD19R(EQ)28ZEGFRt_epH1V7. Co-cultures were carried out for 5 hours with TCM- or TCM / SCM / N-derived cell products transduced or mock transduced with CD19R(EQ)28ZEGFRt_epH1V7 as effector and CD19-negative K562 cells (white bars) or CD19+SupB15 cells (black bars) as stimulators. The percentage of CD45 / CD8 / EGFRt-dependent cells that were immunoreactive for IFNγ is shown. [Figure 12] FIG. 1 shows the results of an efficacy study of IL13 CAR expressed by various T cell populations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The T cell compartment contains subsets of T cells at different stages of differentiation. These subsets include naive T cells (T N Among the stem cell-like subsets are memory stem T cells (T SCM These cells are called central memory cells (T CM ) differentiate into T CM are effector memory cells (T EM ) differentiate into T EM are effector T cells (T E )

[0017] Memory stem T cells (T SCM Memory stem T cells (T) are present at low levels within the T cell compartment but appear to have significant self-renewal and proliferation potential. SCM) are similar to naive T cells (T N ), but their expression of CD95 identifies T N (Figure 1). SCM The T cells were stimulated with CD3 / CD28 beads in the presence of IL-7 and IL-15. N It can be generated from T SCM can also be expanded in the presence of Wnt / β-catenin pathway activation.

[0018] T SCM Central memory T cells (T CM ) are a well-defined subset of memory T cells with high potential for self-renewal and proliferation. CM are effector T cells (T E ) and persists through subsequent adoptive transfer (Non-Patent Document 1; Non-Patent Document 2). CM can be enriched from PBMCs for the production of T cell therapeutics based on their CD45RA-CD45RO+CD62L+ phenotype (Figure 2) (Non-Patent Document 3). CM There is some evidence that T cells adopt adult stem cell-like behavior. CM However, T CM multiply to produce more TCM, and T CM But, T EM / T E Over three generations, T CM Single-cell transmission of has been demonstrated in studies in mice (Non-Patent Document 4; Non-Patent Document 5).

[0019] T CM / SCM / N The cell population is a T cell line that has demonstrated efficacy in CAR-T therapy. CM , and T CM More stem cell-like than T N Not only T SCM Figure 3 shows expression data depicting the relationship of various cell populations to PBMCs.

[0020] The cell populations described herein can be engineered to express, for example, a CAR or a T cell receptor. A CAR is a recombinant biological molecule that contains an extracellular recognition domain, a transmembrane region, and one or more intracellular signaling domains. Thus, the term "antigen" is not limited to molecules that bind to antibodies, but to any molecule that can specifically bind to any receptor. Thus, "antigen" refers to the recognition domain of a CAR. The extracellular recognition domain (also referred to as the extracellular domain, or simply by the recognition element it contains) contains a recognition element that specifically binds to a molecule present on the cell surface of a target cell. The transmembrane region anchors the CAR within the membrane. The intracellular signaling domain contains a signaling domain derived from the zeta chain of the human CD3 complex, and optionally contains one or more co-stimulatory signaling domains. CARs can bind antigens and transduce T cell activation independent of MHC restriction. Thus, CARs are "universal" immune receptors that can treat a population of patients with antigen-positive tumors, regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for the treatment of cancer.

[0021] CAR can be produced by any means known in the art, but preferably by recombinant DNA technology. Advantageously, the nucleic acid encoding several regions of chimeric receptor can be prepared by standard molecular cloning techniques known in the art (genomic library screening, overlap PCR, primer-assisted ligation, site-directed mutagenesis, etc.), and furthermore, the complete coding sequence can be constructed. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T-lymphocyte cell line, most preferably an autologous T-lymphocyte cell line.

[0022] T CM / SCM / N Various CARs suitable for expression by cells include, for example, those described in Patent Documents 1-4. EXAMPLES

[0023] T CM / SCM / N 1. Preparation of Cells Using various methods, human T CM / SCM / N A population of cells can be generated. For example, T CM / SCM / N The population of cells can be prepared from a mixed T lymphocyte population. The population of T lymphocytes can be allogeneic or autologous to the subject ultimately treated with the cells, and can be obtained from the subject by leukopheresis or blood sampling.

[0024] The following method involves the production of T lymphocytes from T lymphocytes obtained by leukopheresis or other means. CM / SCM / N This is an example of a method that can be used to obtain a population of cells. Peripheral blood is collected by leukopheresis or peripheral blood drawing. Typically, day 1 of a manufacturing cycle is the day that the Ficoll procedure is performed. The leukopheresis product of interest is diluted with EDTA / PBS and the product is centrifuged at 1200 RPM for 10 minutes at room temperature with maximum brake. After centrifugation, the platelet-rich supernatant is removed and the cell pellet is gently vortexed. EDTA / PBS is used to resuspend the vortexed cell pellet in each conical tube. Each tube is then underlaid with Ficoll and centrifuged at 2000 RPM for 20 minutes at room temperature without brake. After centrifugation, the PBMC layer from each tube is transferred to another conical tube. The cells are centrifuged at 1800 RPM for 15 minutes at 4° C. with maximum brake.

[0025] After centrifugation, the cell-free supernatant is discarded and the cell pellet is gently vortexed. The cells are washed twice with EDTA / PBS and three times with PBS each time. The cells are centrifuged at 1200 RPM each time with maximum brake at 4° C. for 10 minutes. After the last PBS wash, the vortexed cell pellet is resuspended in complete X-Vivol5 medium (X-Vivol5™ medium with 10% FBS) and transferred to a transfer bag. The bag with washed PBMCs is left on a rotator overnight at room temperature on the benchtop for immunomagnetic selection the next day.

[0026] Selection procedures are then used to deplete the cell population for cells expressing certain markers and to enrich the cell population for cells expressing certain other markers. These selection steps preferably occur on day 2 of the production cycle. The cell population is substantially depleted for cells expressing CD25 and CD14. Importantly, the cell population is not substantially depleted for cells expressing CD45RA. Briefly, cells are resuspended in labeling buffer (LB; EDTA / PBS with 0.5% HSA) and incubated with anti-CD14 and anti-CD25 Miltenyi antibodies for depletion by CliniMACS®, the composition is mixed gently and then incubated on a rotator for 30 minutes at room temperature on the benchtop.

[0027] The depletion step is performed on a CliniMACS® instrument using the depletion tube set. The harvested cells after the depletion step are transferred to a tube and further centrifuged at 1400 RPM for 15 minutes with maximum brake at 4° C.

[0028] The cell-free supernatant is removed, the cell pellet is gently vortexed and resuspended, and to enrich for cells expressing CD62L, the cell suspension is treated with anti-CD62L-biotin (made at City of Hope Center for Biomedicine and Genetics), mixed gently and incubated on a rotator at room temperature on the benchtop for 30 minutes.

[0029] After the incubation period, LB is added to the tube and the cells are centrifuged at 1400 RPM for 15 minutes with maximum brake at 4°C. The cell-free supernatant is removed and the cell pellet is gently vortexed. LB is added to resuspend the cell pellet in the tube and the resuspended cells are transferred to a new transfer bag. Anti-biotin (Miltenyi Biotec) reagent is added, the mixture is gently mixed and incubated on a rotator at room temperature on the benchtop for 30 minutes.

[0030] The CD62L enrichment step is performed on a CliniMACS® instrument using a tubing set. The product of this enrichment can be frozen for storage and then thawed and activated.

[0031] There is an option to freeze the cells following the selection process to provide an intermediate holding step in manufacturing. The cells are pelleted by centrifugation at 1400 RPM for 15 minutes with maximum brake at 4°C. The cells are resuspended in a Cryostor® and aliquoted into cryovials. The vials will be transferred to a controlled chiller (e.g., Nalgene® Mr. Frosty; Sigma-Aldrich) capable of cooling at approximately 1°C / min, which is immediately transferred to a -80°C freezer. After 3 days in the -80°C freezer, the cells are transferred to a GMP LN2 freezer for storage.

[0032] We found that cryopreserved cells showed good recovery and viability, maintained the appropriate cell surface phenotype when thawed up to 8.5 months after cryopreservation, and could be successfully transduced and expanded in vitro after thawing.

[0033] Alternatively, the freshly enriched Tcm / scm / n cells may be activated, transduced or expanded as described in Example 3 below. EXAMPLES

[0034] T CM and T CM / SCM / N Comparison of yield and recovery of 1) CD62L + Central Memory (T CM ) cells, and 2) central memory (T CM ) population also has stem cell-like memory (T SCM ) population, as well as naive T cells (T N ) with CD62L+T CM / SCM / N A study was performed to evaluate the yield of human peripheral blood mononuclear cells (PBMCs) following CliniMACS / AutoMACS™ enrichment of T cell populations using Guava analysis of viable cell counts and flow cytometric analysis of phenotype.

[0035] Experimental design: T cells were isolated from PBMCs obtained from healthy human donors. CM and T CM / SCM / N We enriched cell lines from T CM / SCM / N Cells were generated by CliniMACS / AutoMACS™ depletion of CD14+ monocytes and CD25+ regulatory T cells followed by AutoMACS™ selection of the CD62L+ memory population. In contrast, T CMThe cells were generated by CliniMACS / AutoMACS™ depletion of CD14+ monocytes, CD25+ regulatory T cells, and CD45RA+ naive and memory stem T cells followed by AutoMACS™ selection of the CD62L+ population. Evaluation of the selection process included viable cell enumeration and flow cytometry analysis, as described in more detail below. Cells were then cryopreserved for future studies.

[0036] Summary of enrichment process: Blood products were Ficolled and the resulting PBMCs were subjected to a series of washes in PBS / EDTA. The PBMCs were then resuspended in complete X-Vivol5 medium and, in some cases, transferred to a 300cc transfer bag and then placed on a 3-D rotator overnight. CD14 - / CD25 - / CD45RA - / CD62L + T CM or CD14 - / CD25 - / CD62L + T CM / SCM / N PBMCs were subjected to successive rounds of depletion and selection with CliniMACS / AutoMACS™ to enrich for any of the following populations: CD14+ monocytes, CD25+ regulatory T cells, and T CM The enrichment rounds included magnetic depletion (via CliniMACS™ or AutoMACS™) to remove CD45RA+ naive T cells for ELISA. The remaining cells were then subjected to positive selection (via AutoMACS™) against the CD62L+ population using anti-CD62L-biotin (Dreg 56) reagent. Final cell numbers after both rounds of enrichment were recorded. Samples of the final selected cell population were then analyzed by flow cytometry.

[0037] Overview of Flow Cytometry Assays and Analysis: Flow cytometry analysis of cell populations was performed as follows. Samples were washed in FACS staining solution (FSS) using a tabletop centrifuge, resuspended in FSS, and aliquoted at 100 μL per sample into pre-labeled 12×75 mm FACS tubes (one tube per condition). The required amount of antibody was added to each FACS tube, and the tubes were then incubated for 30 minutes in the dark at 4° C. At the end of the incubation, each tube was washed twice in FSS and resuspended in either 250 μl of FSS or FSS containing DAPI as a viability dye. Samples were then run on a FACS Calibur (Becton Dickenson) or MACSQuant (Miltenyi) instrument for analysis. The percentage of viable immunoreactive cells was calculated via FCS Express software (De Novo Software, Los Angeles, CA).

[0038] <Yield after enrichment>: The recovery of CD3+ T cells from each of the cell products is shown in Table 1 below. Overall, CD14 - / CD25 - / CD45RA - / CD62L + CD3 + T CM The recovery rate from enrichment of CD14 is 2-6%. - / CD25 - / CD62L + CD3 + T CM / SCM / N The recovery rate of the enrichment was 4-30%. CM / SCM / N Cells vs. T CM The cell recovery rate was 1.6-15 times higher.

[0039] [Table 1] Flow cytometry analysis after enrichment: After both rounds of enrichment with CliniMACS™, the phenotype of enriched cells was determined by FACS analysis. The results of cell products obtained from three representative donors are shown in Figure 4. T cells from each donor were CM and T CM / SCM / N The population showed enriched levels of CD3+ and CD62L+ cells and CM are depleted of CD45RA and T CM / SCM / N expressed CD45RA as expected. Depletion of both CD14+ and CD25+ populations was also observed as expected using the selection strategy. Interestingly, the percentage of CD8+ cells was significantly higher than that of the donor-matched T cells. CM When compared with the T CM / SCM / N The mean mean mean age of the 1st group was 18.1 years and the mean mean mean age of the 2nd group was 18.1 years.

[0040] Results: The results of this study demonstrate that CD62L+T CM The cell population of T CM / SCM / N It was demonstrated that CD3+T cell populations could also be efficiently isolated starting from PBMCs, with 2–6% of the starting population being CD3+T cells. CM By eliminating the CD45RA depletion step, CD3+T CM / SCM / N The cells recovered from the starting population are 4-30%. CM / SCM / N The cell recovery ability is T CM This would increase the number of T cells that can be used as a starting population for genetic engineering, as they are 1.6-15 fold more abundant than CD45RA cells. Furthermore, we observed that elimination of the CD45RA depletion step also appears to increase the proportion of CD8 T cells in the enriched product. EXAMPLES

[0041] T CM / SCM / N Lentiviral transduction of cells T CM / SCM / NThe cell population can be genetically engineered to express one or more proteins of interest.For example, cells can be transduced with lentivirus that has the function of expressing one or more proteins of interest.The following is an example of one method that can be used to transduce T cells with lentivirus vector using protamine sulfate / cytokine solution.

[0042] If the cells are frozen following the selection process, they will need to be thawed as the manufacturing process continues. Cryopreserved vials are thawed in a 37°C water bath until most remain liquid. The contents of the vial are placed into a conical tube containing cold X-Vivol5 medium. The cells are centrifuged at 1200 RPM for 10 minutes with maximum brake at 4°C. After centrifugation, the cell-free supernatant is decanted, the pellets are vortexed, and all pellets are mixed into one tube in X-Vivol5 medium to be transduced with lentivirus. Cell counts are determined and the manufacturing process continues with activation of the Dynal beads.

[0043] The cells are centrifuged at 1400 RPM for 10 minutes at 25° C. After centrifugation, the cell-free supernatant is removed and the cell pellet is gently vortexed and resuspended in complete X Vivo15 medium to be transduced with the lentivirus. If thawed cells are used, the centrifugation step is not necessary.

[0044] Tcm / scm / n cells are stimulated with Dynabeads® human T expander CD3 / CD28 (Invitrogen) at a ratio of 1:3 (T cells:beads) and then cultured in the presence of 50 U / mL rhIL-2 (Chiron) and 0.5 ng / ml rhIL-15 (CellGenix).

[0045] After 1-3 days of incubation, T cells are transduced with lentiviral vectors, usually at an MOI of 0.1-1.5, in X-Vivol5 containing 10% FCS with 5 μg / mL protamine sulfate (APP Pharmaceutical), 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15. The day after lentiviral transduction, a volume of freshly prepared media / cytokine master mix containing complete X-Vivol5 medium and cytokines is added (10 μL rhuIL-2 and 0.5 μL rhuIL-15).

[0046] The culture was then cultured at a cell density of 3 x 10 5 From 2 × 10 6 Cultures were maintained at 37°C in 5% CO in X-Vivol5 10% FCS required to maintain viable cells / mL with cytokine supplementation (at final concentrations of 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15) every Monday, Wednesday, and Friday during culture.

[0047] Depletion of CD3 / CD28 Dynabead beads is performed on days 7 to 9 after bead stimulation. Dynal beads are removed using a DynaMag-50 or MPC magnet. After bead removal, a sample of the cell suspension is taken for counting. Based on the counts, the concentration is approximately 0.5 x 10 6 / mL.

[0048] Cultures are expanded until sufficient numbers of cells are achieved, usually for 8-18 days. During this time, the cytokines in the T cell culture medium are completely replaced and added. Starting on day 12, and then every Monday, Wednesday, and Friday, approximately 0.5 x 10 6 Count to maintain concentration of cells / mL.

[0049] Final harvest and cryopreservation is performed as follows: Once the expanded cells reach the required viable cell count required for clinical administration or research purposes, the cell product is cryopreserved. The cells are counted and a sample in culture is collected for mycoplasma testing. The remaining cells are centrifuged at 1800 RPM for 20 minutes at 4°C with maximum brake. The cell-free supernatant is removed and all cell pellets are gently vortexed, resuspended and combined in Isolyte buffer (Braun). The tubes are centrifuged at 1800 RPM for 20 minutes at 4°C with maximum brake. The cells are resuspended in Isolyte for a second wash.

[0050] After the second Isolyte wash, the cell-free supernatant is removed, and the cell pellet is gently vortexed and resuspended in an appropriate amount of cryopreservation medium, CryoStor®5 (BioLife Solutions), to bring the cells to the appropriate concentration. The cells are cryopreserved by placing them in a controlled cooling device (e.g., Nalgene® Mr. Frosty; Sigma-Aldrich) or a controlled rate freezer system (Custom Biogenics) capable of cooling at approximately 1° C. / min. Upon completion of the freezing procedure, the cassette(s) / bag and cryovial are immediately transferred into an LN2 freezer for storage. EXAMPLES

[0051] Bead-stimulated, lentivirally transduced, and expanded T CM Cells and T CM / SCM / N Cell Comparison CD62L+T CM population or CD62L+T CM / SCM / NStudies were performed to evaluate the ex vivo proliferation and cell surface phenotype of human peripheral blood mononuclear cells (PBMCs) that had been enriched using CliniMACS / AutoMACS™ for either of these populations (including naïve, central memory and stem cell-like memory T cells) and then bead-stimulated using methods proposed for clinical use and lentivirally transduced with either CD19R(EQ)28Z-T2A-EGFRt_epHIV7, a lentivirus expressing a CAR targeting CD19R, or IL13(EQ)BBZ-T2A-CD19t_epHIV7, a lentiviral vector targeting IL-13Ralpla (see US Pat. Nos. 5,993,333 and 5,993,333 for details of the vectors and the expressed CARs). Proliferation was evaluated by monitoring viable cell counts, and cell surface phenotype was further evaluated by flow cytometry analysis.

[0052] Experimental Design: Chimeric antigen receptor (CAR) T cell products generated from PBMCs of three separate human donors were generated using methodology expected to be suitable for clinical use. Briefly, T cells enriched from PBMCs (essentially as described in the Examples) were CM Cells and T CM / SCM / N Cells were activated by CD3 / CD28 bead stimulation and further transduced with either CD19R(EQ)28Z-EGFRt_epHIV7 or IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentivirus. CD3 / CD28 beads were removed after 7-9 days and then the complete X-Vivol5 medium required for cell growth was added to expand T cell cultures and further maintained for up to 29 days (cell density of 0.3x10 6 From 2 × 10 6The cell population was maintained at 100% viable cells / mL. Cytokines were replenished every Monday, Wednesday and Friday of culture. Viable cell counts were monitored during this expansion process. The final cell product was then assessed for surface markers traditionally used for cell product release, such as CD3, and cleaved EGFR (EGFRt) or cleaved CD19 (CD19t) as transduction markers, as well as T cell markers CD4, CD8, CD27, CD28, CD45RA and CD62L.

[0053] Overview of stimulation, transduction and proliferation: Concentrated T CM Cells or T CM / SCM / N Cells were activated with CD3 / CD28 beads and further transduced with lentivirus using GMP grade CD19R(EQ)28Z-T2A-EGFRt_epHIV7 or GMP or research grade IL13(EQ)BBZ-T2A-CD19t_epHIV7 at 0.3 to 3.0 multiplicity of infection (MOI). Cells were placed in culture in complete X-Vivol5 medium with 50U / mL rhIL-2 and 0.5ng / mL rhIL-15. Cultures were then cultured at a cell density of 0.3x10 6 From 2x10 6 Complete X-Vivol5 medium was added as required to maintain viable cells / mL, and cytokine supplementation (rhIL-2 and rhIL-15) was added every Monday, Wednesday, and Friday of culture. After 7-9 days of bead stimulation, CD3 / CD28 Dynabeads were removed from the culture using a DynaMag-50 magnet. Viable cell numbers were monitored during this expansion process, and viable cell counts were recorded during the last day of culture and cryopreservation. The various products are listed in Table 2.

[0054] [Table 2] Flow cytometry: Samples were washed in FACS staining solution (FSS) using a tabletop centrifuge, resuspended in FSS, and aliquoted at 100 μL per sample into pre-labeled 12x75mm FACS tubes (one tube per condition). The required amount of antibody was added to each FACS tube, and the tubes were then incubated for 30 minutes in the dark at 4°C. In some cases, samples were washed and then stained with fluorochrome-conjugated streptavidin as a secondary reagent. At the end of the incubation, each tube was washed twice in FSS and resuspended in 250 μl FSS or in 140 μL FSS and 70 μL of diluent DAPI (made by mixing 6.9 μL of stock solution with 25 mL of FSS). Samples were then run on a FACS Calibur (Becton Dickenson) or MACSQuant (Miltenyi) instrument for analysis. The percentage of immunoreactive cells was calculated via FCS Express software (De Novo Software, Los Angeles, Calif.).

[0055] Viable cell count analysis results: Following bead stimulation and lentiviral transduction with CD19R(EQ)28Z-EGFRt_epHIV7, the cell products were grown under conditions that support T cell proliferation. Growth curves are depicted in FIG. 5, and the final cell numbers and fold expansion based on total viable cell numbers for each product are shown in Table 3. Overall, T CM / SCM / N The fold expansion of the derived cell product is determined by its T CM Similar to its derived counterpart.

[0056] [Table 3] Flow cytometric analysis of the final product: Flow cytometric analysis of each cell product after ex vivo expansion revealed that all of the final cryopreserved cell products would pass our traditional product specifications of ≥80% CD3 for identity and ≥10% EGFRt or CD19t for potency (Figure 6A). Indeed, T cells from each donor were significantly higher in the 30% EGFR+ / - donor population than in the 30% EGFR+ / - donor population. CM / SCM / N The derived products were immunoreactive with both CD3 and EGFRt / CD19t, similar to their corresponding T CM It was very similar to the product derived from

[0057] Flow cytometric analysis of other T cell markers (Figure 6A) revealed that although all cell products contained both CD4 and CD8 T cell subsets, T CM / SCM / N The derived CAR T cell line was CM A relatively high percentage of CD8 cells was found compared to the T-derived counterpart. CM The cell products derived from T CM / SCM / N The derived cell products also expressed the memory-associated markers CD27, CD28, and CD62L. However, expression of CD27 was generally conserved in T CM / SCM / N Furthermore, CD45RA expression levels were higher in T CM / SCM / N The concentrations were significantly higher in the products derived from .

[0058] Conclusion: In summary, these data show that CM -Enriched population is also T CM / SCM / N We show that the enriched population is also amenable to CD3 / CD28 bead stimulation, lentiviral transduction, and in vitro expansion. CM and T CM / SCM / N The fold expansion was similar between the derived counterparts, so more T CM / SCM / N It is important to note that the ability to start with low cell numbers may result in shorter expansion times to reach sufficient cell numbers for clinical use.

[0059] Flow cytometry analysis revealed that T CM The cell population of TCM / SCM / N Flow cytometry analysis revealed that T cell populations were transduced with similar efficiency and expressed the EGFRt or CD19t transgenes. CM The cell population of T CM / SCM / N We also confirmed that this cell population expresses the T cell markers CD3, CD4, and CD8, as well as the T cell memory markers CD27, CD28, and CD62L. CM / SCM / N The higher percentage of CD45RA+ cells observed in the population may be due to the selection process being CM This was as expected since the cells were depleted of CD45RA. EXAMPLES

[0060] Intravenously delivered T transduced with CD19R(EQ)28Z-T2A-EGFRt_epHIV7 CM cell populations and T CM / SCM / N In vivo efficacy in cell populations Unselected PBMCs, CD62L+T, transduced and expanded with a lentiviral vector expressing a CD19-targeted CAR (CD19R(EQ)28Z-T2A-EGFRt_epHIV7 lentiviral vector) using methods suitable for clinical use. CM cells and CD62L+T CM / SCM / N A study was conducted to evaluate the in vivo efficacy of the cells. The in vivo antitumor efficacy of these cells administered intravenously (iv) was examined in immunodeficient NSG mice using a CD19-expressing SUP-B15 human acute lymphocytic leukemia cell line engineered to express green fluorescent protein (GFP) and firefly luciferase (ffLuc) reporter genes (PMID:22407828).

[0061] Experimental design: T cells were lentivirally transduced with CD19R(EQ)28Z-T2A-EGFRt_epHIV7 and grown for 21 days. CM Origin or T CM / SCM / NT cell lines derived from the CAR T cells were used in this study (see Examples 2 and 4 above). Freshly thawed CAR T cells administered intravenously were then evaluated for their ability to control the in vivo proliferation of intravenously implanted Sup-B15 cells. Tumor burden was measured by Xenogen imaging, as was T cell persistence measured by flow cytometry analysis of peripheral blood.

[0062] Summary of in vivo xenograft study: Female NSG mice (10-12 weeks old) were irradiated with 300 rads on day 0. On day 1, mice were divided into groups (n=4) and either left untreated or irradiated with 2.5x10 6 Mock-transduced PBMCs from HD270 (no CAR), HD270 CD19R T PBMC , HD270 CD19R T CM Or HD270 CD19R T CM / SCM / N The mice were treated intravenously (i.e., via the tail vein) with either 0.5x10 6 CD19R / EGFRt+T in CAR+ cells PBMC , CD19R / EGFRt+T CM or 0.625x10 6 CD19R / EGFRt+T CM / SCM / N The average of HD270 CD19R T CM and T CM / SCM / N The phenotypic analysis of the derived cell lines defines the degree of differentiation. On day 2, mice were challenged intravenously with two tumor cells per one CAR+ T cell GFP:ffLuc+Sup-B15 tumor cells. The growth of this human acute lymphocytic leukemia cell line was then monitored over time by Xenogen imaging and quantification of firefly luciferase (ffLuc) flux (photons / second). On day 41, retro-orbital bleeding was performed and blood was further evaluated for the presence of human CD3-expressing T cells by flow cytometry analysis using a MACSQuant instrument and FCS Express software (De Novo Software, Los Angeles, CA).

[0063] <Evaluation of antitumor efficacy in vivo>: In Fig. 7A and Fig. 7B, which depict the results of this study, CD19R T CM / SCM / N The cells showed stronger antitumor activity against ffLuc+Sup-Bl5 tumors, whereas CD19R T CM It can be seen that intravenous administration of cells only showed a transient therapeutic effect.

[0064] Analysis of T cells in peripheral blood: To assess the persistence of T cells in the peripheral blood of these mice, flow cytometry analysis was performed on post-orbital bleeds taken 40 days after intravenous T cell administration. As shown in Figure 8, CAR+ T cells were able to express T CM / SCM / N The presence of CAR+ human T cells correlated with the absence of GFP-expressing SupB15 tumor cells, which were only detectable at this time point in the mice that received the cells.

[0065] These studies demonstrated the in vivo antitumor efficacy of CD19R T CM CD19R T cell lines derived from CM / SCM / N It can also be observed in cell lines derived from CD19R T CM / SCM / N Antitumor responses in cell-treated mice were observed in CD19R T CM The antitumor response of treated mice was demonstrated to be greater than that of CD19R T cells. CM / SCM / N This partially correlated with the ability to detect CAR+ T cells in the blood of treated mice. EXAMPLES

[0066] T transduced with CD19R(EQ)28Z-T2A-EGFRt_epHIV7 CM Cells and T CM / SCM / N Cellular effector activity Using methods anticipated to be suitable for clinical use, we transduced and expanded CD62L+ central memory (T CM ) cells or CD62L+T CM / SCM / NStudies were performed to assess the CD19-specific cytolytic activity, degranulation and cytokine production of cells. Cytolytic activity was assessed using a flow cytometry-based long-term killing assay and a 5-h degranulation assay. Cytokine production was assessed by intracellular staining of T cells after 5 h of co-culture with stimulator cells.

[0067] <Experimental Design>:T CM Cells and T CM / SCM / N Cells were lentivirally transduced with CD19R(EQ)28Z-T2A-EGFRt_epHIV7 and expanded for up to 21 days (see Examples 2 and 4). The CD19-specific effector function of these cell products was assessed by long-term killing assays to assess target cell destruction after 72 hours of co-incubation with effector T cells, and by a 5-hour degranulation assay to assess cell surface CD107a recruitment as a marker of T cell cytotoxic activity. Cytokine production by the freshly thawed final cell products after 5 hours of in vitro stimulation was then assessed by flow cytometric analysis of intracellular staining for IFN-γ.

[0068] Long-term killing assay summary: Samples of each T cell product were thawed the day before the assay and left overnight in the presence of 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15 before the 72-hour killing assay. Briefly, each T cell line and target cell line were counted and seeded in a 1:1 ratio of CAR+T cells to target cells in 96-well tissue culture-treated round-bottom plates, specifically 25,000:25,000 in 200 μL of X-Vivol5 medium with 10% FBS per well. After 72 hours of culture, cells were harvested by trypsinization, washed with cold FACS buffer, and then stained with anti-human CD45 to detect T cells using DAPI as a viability dye. Samples were run and analyzed on a MACSQuant (Miltenyi) instrument, and the number of viable immunoreactive cells was calculated via FlowJo software (FlowJo, LLC, Ashland, OR). Final results were then graphed as the percentage of CD45-negative, forward scatter-high tumor cells remaining in culture compared to mock-transduced effector cell co-cultures, normalized to 100%.

[0069] Summary of Degranulation Assay and Intracellular Cytokine Analysis: A 5-hour degranulation assay was performed after freshly thawed samples of each T cell product. T cells were counted and resuspended in medium containing Golgi Stop, a protein transport inhibitor that blocks resorption of CD107a from the cell surface. T cells were then seeded in a 1:1 ratio with tumor cells (with Golgi Stop) in 96-well plates. To each well, an antibody against CD107a was added, and the plate was then incubated at 37°C for 5 hours. Additional antibodies were then added to each condition to stain for CD45, CD4, CD8, and EGFRt transgenes. Cells were then fixed and permeabilized for staining with antibodies specific for IFN-γ or isotype control antibodies. Samples were run and analyzed on a MACSQuant (Miltenyi) instrument, and the percentage of immunoreactive cells was calculated via FlowJo software (FlowJo, LLC, Ashland, OR).

[0070] Determination of cytolytic activity by long-term killing assay: CD19R(EQ)28Z-T2A-EGFRt_epHIV7-transduced or mock-transduced T CM Cells and T CM / SCM / N The cells were plated together with CD19-negative K562 cells or CD19+ SupB15 cells for 72 hours, respectively. The results are shown in Figure 9. CM Both cells and CD19R T CM / SCM / N Both cells show a similar reduction in detectable SupB15 target in the culture medium compared to their respective mock-transduced controls, and indeed this reduction in SupB15 target is significantly greater than that observed with the K562 target, further supporting the CD19 specificity of this cytolytic activity.

[0071] Determination of effector activity by degranulation assay: CD19R(EQ)28Z-T2A-EGFRt_epHIV7-transduced or mock-transduced T CM Cells and T CM / SCM / N The cells were seeded with either CD19-negative K562 cells or CD19+ SupB15 cells, respectively. The results are shown below (Figure 10). CM and CD8+CD19R T CM / SCM / N It was demonstrated that the cells exhibited similar CD19-specific degranulation (i.e., CD107a expression) following stimulation with SupB15 cells. The antigen specificity of this activity was further demonstrated by examining mock-transduced cells that were not stimulated to degranulate by CD19-expressing SupB15 cells (Figure 10).

[0072] Determination of effector activity by intracellular cytokine assay: CD19R(EQ)28Z-T2A-EGFRt_epHIV7-transduced or mock-transduced T CM Cells and T CM / SCM / N The cells were plated together with CD19-negative K562 cells or CD19+SupB15 cells, respectively. The results are shown in FIG. 11. CM and CD8+CD19R T CM / SCM / Nhas been demonstrated to exhibit similar CD19-specific intracellular IFN-γ properties. The antigen specificity of this activity was further demonstrated by examining pseudotransduced cells that were not stimulated to express IFN-γ by CD19-expressing SupB15 cells.

[0073] These data show that T cells transduced with CD19R(EQ)C28Z-T2A-EGFRt_epHIV7 CM cells as well as T CM / SCM / N cells were confirmed to exhibit cytolytic activity, degranulation, and cytokine (i.e., IFN-γ) production after co-culture with CD19-expressing target cells.

Example

[0074] T cells transduced with <IL13(EQ)BBZ-T2A-CD19t_epHlV7 CM cells and T CM / SCM / N cells effector activity> CD62L+ T cells transduced with a lentiviral vector expressing a CAR targeting IL13Rα2 CM cells and CD62L+ T CM / SCM / N cells were evaluated for their in vivo efficacy. The CAR contains a human IL-13 variant and is expressed by a lentiviral vector (IL13(EQ)BBZ-T2A-CD19t_epHIV7). A cell population expressing this vector was prepared as previously described in Example 4 and evaluated in a mouse model of glioblastoma in immunodeficient NSG mice using the IL13Rα2+ primary low passage GBM tumoroid line PBT030-2 engineered to express the firefly luciferase (ffLuc) reporter gene.

[0075] Briefly, on day 0, male NSG mice (10 - 12 weeks old) were stereotactically injected with 1×10 5 ffLuc+ PBT030-2 cells in both the left and right hemispheres and allowed to engraft for 6 days. Next, the groups of mice were left untreated or received 1×10 6 CAR+ IL13(EQ)BBζ / CD19t+ T CM / SCM / N, CAR+IL13(EQ)BBζ / CD19t+T CM , mock-transduced T CM / SCM / N or mock-transduced T CM PBT030-2 tumor growth was monitored over time by Xenogen imaging and quantification of ffLuc flux (photons / sec).

[0076] As shown in FIG. 12, T cells transduced with a CAR directed against IL-IL13Rα2 CM / SCM / N have demonstrated that similarly transduced T CM was better than.

Claims

1. 1. A method for preparing a population of human cells comprising central memory T cells; memory stem T cells, and naive T cells, the method comprising: (a) providing a sample of human cells comprising T cells; (b) treating a sample of human cells comprising said T cells to deplete cells expressing CD25 and cells expressing CD14 and to prepare a population of said depleted cells; and (c) treating the population of depleted cells to enrich for cells expressing CD62L to prepare a population of human cells comprising central memory T cells; memory stem T cells, and naive T cells; wherein cells expressing CD45RA are not depleted.

2. 2. The method of claim 1, further comprising activating the population of human cells comprising T cells and introducing or transfecting a nucleic acid molecule into the activated cells to provide a population of human cells comprising T cells harboring the nucleic acid molecule.

3. The method of claim 1 , wherein the sample of human cells comprising T cells comprises PBMCs.

4. The method of claim 2 , wherein the nucleic acid molecule is a vector.

5. 3. The method of claim 2, further comprising culturing said population of human cells comprising T cells carrying said nucleic acid molecule.

6. The method of claim 5, wherein the culturing step comprises the addition of exogenous IL-2 and exogenous IL-15.

7. 3. The method of claim 2, wherein activating the population of human cells comprising T cells comprises exposing the cells to an anti-CD3 antibody and an anti-CD28 antibody.

8. The method of claim 4 , wherein the vector is a viral vector.

9. The method of claim 8 , wherein the viral vector is a lentiviral vector.

10. The method of any one of claims 2 and 4 to 9, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a chimeric antigen receptor or a T cell receptor.

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