Method for treating cancer using engineered T cells

Abstract

The present disclosure provides methods for treating cancer. The methods for treating cancer can include administering pentostatin and cyclophosphamide, followed by administering manufactured T cells. The present disclosure further provides methods for generating such manufactured T cells, the manufactured T cells, and compositions comprising the manufactured T cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 768,145, filed on November 16, 2018, U.S. Provisional Application No. 62 / 927,034, filed on October 28, 2019, and U.S. Provisional Application No. 62 / 927,079, filed on October 28, 2019, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] CD4 + and CD8 + T cells with a type I cytokine profile (Th1 and Tc1 cells, respectively) are candidate T cell populations for adoptive T cell therapy. Such Th1-type T cells are promoted by polarizing cytokines such as IL-12 and IFN-α that stimulate the STAT1 and STAT4 transcription factors, which in turn promote the TBET transcription factor that partially defines the Th1-type differentiation state.

[0004] The success of adoptive T cell therapy depends in part on the in vivo persistence of the T cell population in the host. T cell persistence is a balance determined by both an increase in the ability of T cells to proliferate and maintain T cell memory and a decrease in the propensity for T cell apoptotic cell death. In previous studies, it has been demonstrated that T cells generated in vitro in the pharmacological agent rapamycin, which inhibits the mammalian target of rapamycin mTOR, exhibit these properties, namely: enhanced ability to undergo antigen-driven clonal expansion in vivo after adoptive transfer; improved memory state, such as T central memory (T CM)Exemplified by a differentiated state; and a multi-faceted anti-apoptotic phenotype characterized by induction of autophagy (including mitophagy) and preferential expression of anti-apoptotic members of the bcl-2 gene family relative to pro-apoptotic members. In summary, these properties of in vitro-generated rapamycin-resistant cells are associated with enhanced in vivo regulation of transplant responses, which in vivo regulation of transplant responses includes prevention of graft-versus-host disease (GVHD) and graft rejection and mediation of human-mouse xenogeneic GVHD; notably, these cells have been successfully translated into clinical trials in autologous and allogeneic settings for the treatment of multiple myeloma and have shown safety and efficacy against relapsed, refractory multiple myeloma. For these clinical trial efforts, the manufacturing process includes the following elements: co-stimulation with anti-CD3, anti-CD28-coated magnetic beads (3 / 28 beads) at a relatively high ratio of 3 beads:1 T cell; addition of both T cells and 3 / 28 beads to in vitro culture; addition of a high dose of orally administered mTOR inhibitor rapamycin (1 μM); and addition of IL-2 to the culture during cytokine polarization (IFN-α addition). The cells generated by this method are referred to herein as T-Rapa cells, which are more specifically defined later in this disclosure.

[0005] Cancer development may be associated with alterations in immune function. Such alterations include cell-mediated immunity (CMI) suppression associated with the inability to reject tumors, and humoral immunity enhancement that can potentiate tumor promotion and progression. CD4 + T cell subsets, Th1 T cells and Th2 T cells have different functions and regulate each other. Th1 cells produce interleukin (IL-2) and interferon (IFN-γ) and direct CMI responses, while Th2 cells produce IL-4 and IL-10 and promote local humoral immune responses.

[0006] There is evidence of a Th1 / Th2 imbalance in certain cancers, in which the proportion of Th2 cells is significantly elevated with a reduction in the number of Th1 cells. A chronic Th1 / Th2 imbalance favoring Th2 potentially leads to cell-mediated immunity suppression, thereby providing a favorable environment for reduced effective immune surveillance and development of malignancy.

[0007] Idiotypic-specific T cell responses have been found in most patients with early multiple myeloma. These responses include Th1 responses with IL-2 and IFN-γ production. For example, Th1-type immunity is preferentially found in cases of painless disease, and Th2-type responses are mainly found in advanced multiple myeloma cases. Defective Th1 immune responses (mediated by IL-6) and dysregulated cytokine networks have been found in multiple myeloma patients. Myeloma idiotypic-specific T helper cells derived from MM patients are consistently of a non-Th1 phenotype.

[0008] Despite the progress made in the treatment of multiple myeloma and the recent FDA approval of new agents and monoclonal antibodies, multiple myeloma is almost universally fatal. As such, patients with relapsed, refractory multiple myeloma (RRMM) who are difficult to treat with the top five drugs for multiple myeloma (“penta-refractory”) have only a limited number of months of survival and few treatment options. As demonstrated by the curative effect of long-term observed allogeneic stem cell transplantation and many other methods, including monoclonal antibody therapy, vaccines, and T cell receptor (TCR)-modified and CAR-modified T cell therapies, multiple myeloma is a disease susceptible to immunotherapy. As such, this penta-refractory patient population is suitable for novel T cell therapies. Additionally, patients with less refractory disease are also in urgent need of novel therapies; that is, even at the second or third recurrence of the disease, the median progression-free survival is usually less than two years.

[0009] For certain cancers, novel and innovative immunotherapies are needed. Summary of the Invention

[0010] The present disclosure relates to a method for treating cancer in a subject.

[0011] In one embodiment, a method includes administering to the subject a therapeutically effective dose of a composition comprising engineered T cells.

[0012] In another embodiment, the method further includes subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or late senescent effector T cells or reduce the function of at least a portion of regulatory T cells and / or late senescent effector T cells prior to administering to the subject a therapeutically effective dose of the composition comprising engineered T cells.

[0013] In some embodiments, the immune depletion regimen includes administering to the subject a first composition comprising pentostatin; and administering to the subject a second composition comprising cyclophosphamide.

[0014] In some embodiments, the method comprises a first treatment cycle and one or more additional treatment cycles, the first treatment cycle comprising: subjecting the subject to a first immunosuppressive regimen to reduce at least a portion of regulatory T cells and / or terminally senescent effector T cells or reduce the function of at least a portion of regulatory T cells and / or terminally senescent effector T cells; each of the one or more additional treatment cycles comprising: subjecting the subject to a second immunosuppressive regimen to reduce at least a portion of regulatory T cells and / or terminally senescent effector T cells or reduce the function of at least a portion of regulatory T cells and / or terminally senescent effector T cells; and administering to the subject a therapeutically effective dose of a composition comprising engineered T cells.

[0015] In any of the foregoing embodiments, the method may further comprise measuring the creatinine clearance rate (CrCl) of the subject prior to administering one or more additional doses of pentostatin, and adjusting the dose of pentostatin administered to the subject based on the CrCl, wherein when CrCl ≥ 60 mL / min / 1.73 m 2 pentostatin is administered, wherein when 60 mL / min / 1.73 m > CrCl ≥ 30 mL / min / 1.73 m, pentostatin is administered at 2 mg / m 2 and wherein when CrCl < 30 mL / min / 1.73 m, pentostatin is not administered. In some embodiments, the dose of pentostatin may be adjusted based on the CrCl such that when 60 mL / min / 1.73 m > CrCl ≥ 30 mL / min / 1.73 m, the dose of pentostatin is reduced by 50%, and wherein when CrCl < 30 mL / min / 1.73 m, pentostatin is not administered.

[0016] In any of the foregoing embodiments, the method may further comprise measuring the absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) prior to administering one or more additional doses of cyclophosphamide, and adjusting the dose of cyclophosphamide administered to the subject based on the ALC and ANC, wherein when ANC > 1000 per microliter, cyclophosphamide is administered at a dose of 200 mg, wherein when ANC is 500 - 999 per microliter and ALC ≥ 50 per microliter, cyclophosphamide is administered at a dose of 100 mg, and wherein when ALC < 50 per microliter or ANC < 500 per microliter, cyclophosphamide is not administered. In some embodiments, the dose of cyclophosphamide may be adjusted based on the ALC and ANC such that when ANC is 500 - 999 per microliter and ALC ≥ 50 per microliter, the dose of cyclophosphamide is reduced by 50%, or when ALC < 50 per microliter or ANC < 500 per microliter, cyclophosphamide is not administered. Brief Description of the Drawings

[0017] Figure 1A Depicts the percentage of CD4 + T cells expressing FoxP3 after day 0 and various culture conditions.

[0018] Figure 1B Depicts the percentage of CD4 + T cells expressing TBET after day 0 and various culture conditions.

[0019] Figure 2A Depicts the T cell yield after culturing CD4 + and CD8 + T cells under various conditions.

[0020] Figure 2B Depicts the T cell yield after culturing CD4 + and CD8 + T cells under various conditions.

[0021] Figure 3A Depicts the IFN-γ secretion after culturing CD4 + and CD8 + T cells under various conditions.

[0022] Figure 3B Depicts the TNF-α secretion after culturing CD4 + and CD8 + T cells under various conditions.

[0023] Figure 4 Depicts the Western blot of p-4EBP1 and actin (upper panel) from CD4 + and CD8 + T cells cultured using the T-Rapa method and the method of the present disclosure (Rapa-T). The p-4EBP1 levels were normalized by actin expression in CD4 + and CD8 + T cells (lower panel).

[0024] Figure 5 Depicts the Western blot of P70S6K and actin (upper panel) from CD4 + and CD8 + T cells cultured using the T-Rapa method and the method of the present disclosure, and the CD4 + and CD8 +P70S6K levels normalized by actin expression in T cells (lower panel).

[0025] Figure 6 Depicts Western blots of P-STAT5 and actin from CD4 + and CD8 + T cells cultured using the T-Rapa method and the method of the present disclosure (Rapa-T) (upper panel), and P-STAT5 levels normalized by actin expression in CD4 + and CD8 + T cells cultured using the T-Rapa method and the method of the present disclosure (lower panel).

[0026] Figure 7A Depicts IFN-γ secretion on day 6 after culturing CD4 + and CD8 + T cells under various conditions.

[0027] Figure 7B Depicts IFN-γ secretion on day 13 after culturing CD4 + and CD8 + T cells under various conditions.

[0028] Figure 8A Depicts TNF-α secretion on day 6 after culturing CD4 + and CD8 + T cells under various conditions.

[0029] Figure 8B Depicts TNF-α secretion on day 13 after culturing CD4 + and CD8 + T cells under various conditions.

[0030] Figure 9A Depicts GM-CSF secretion on day 6 after culturing CD4 + and CD8 + T cells under various conditions.

[0031] Figure 9B Depicts GM-CSF secretion on day 13 after culturing CD4 + and CD8 + T cells under various conditions.

[0032] Figure 10A Depicts IL-2 secretion on day 6 after culturing CD4 + and CD8 + T cells under various conditions.

[0033] Figure 10B Depicts after culturing CD4 under various conditions+ and CD8 + IL-2 secretion on day 13 after CD4

[0034] Figure 11 Depicts Western blots of P62 and actin from CD4 + and CD8 + T cells (upper panel), and P62 protein expression normalized by actin expression in CD4 + and CD8 + T cells (lower panel).

[0035] Figure 12 Depicts Western blots of p-RAPTOR and actin from CD4 + and CD8 + T cells (upper panel), and p-RAPTOR levels normalized by actin expression in CD4 + and CD8 + T cells (lower panel).

[0036] Figure 13 Depicts Western blots of BIM and actin from CD4 + and CD8 + T cells (upper panel), and BIM protein expression normalized by actin expression in CD4 + and CD8 + T cells (lower panel).

[0037] Figure 14A Depicts flow cytometry expression analysis of CD45RA on CD4+ cell subsets after culturing T cells under various conditions.

[0038] Figure 14B Depicts flow cytometry expression analysis of CD45RA on CD4+ cell subsets after culturing T cells under various conditions.

[0039] Figure 14C Depicts flow cytometry expression analysis of CD45RA on CD4+ cell subsets after culturing T cells under various conditions.

[0040] Figure 14D Depicts flow cytometry expression analysis of CD45RA on CD4 + cell subsets after culturing T cells under various conditions.

[0041] Figure 15ADepicts the flow cytometry expression analysis of CD62L, CCR7, and CD127 on CD4+ cell subsets after culturing T cells under various conditions.

[0042] Figure 15B Depicts the flow cytometry expression analysis of CD62L, CCR7, and CD127 on CD4+ cell subsets after culturing T cells under various conditions.

[0043] Figure 15C Depicts the flow cytometry expression analysis of CD62L, CCR7, and CD127 on CD4+ cell subsets after culturing T cells under various conditions.

[0044] Figure 15D Depicts the flow cytometry expression analysis of CD62L, CCR7, and CD127 on CD4+ cell subsets after culturing T cells under various conditions.

[0045] Figure 16 Depicts the fold increase in the culture yield of T cells cultured under various conditions.

[0046] Figure 17A Depicts the IFN-γ secretion on the 6th day after culturing T cells under various conditions.

[0047] Figure 17B Depicts the IFN-γ secretion on the 13th day after culturing T cells under various conditions.

[0048] Figure 18A Depicts the TNF-α secretion on the 6th day after culturing T cells under various conditions.

[0049] Figure 18B Depicts the TNF-α secretion on the 13th day after culturing T cells under various conditions.

[0050] Figure 19 Depicts the flow cytometry expression analysis of CD25 on CD4 + T cell subsets on the 6th and 13th days after culturing T cells under various conditions.

[0051] Figure 20 Depicts the flow cytometry expression analysis of CD62L, CCR7, and CD127 on CD4 + T cell subsets on the 6th and 13th days after culturing T cells under various conditions.

[0052] Figure 21Depicts flow cytometry expression analysis comparing the new Rapa-T method without bead co-stimulation, the new Rapa-T method with bead co-stimulation (bead-to-T cell ratio of 1:3), and the old T-Rapa method (bead-to-T cell ratio of 3:1) for the following: naive and T central memory plots: CD45RA expression; co-expression of CD62L and CCR7; and co-expression of CD62L, CCR7, and CD127.

[0053] Figure 22 Depicts flow cytometry expression analysis comparing the new Rapa-T method without bead co-stimulation, the new Rapa-T method with bead co-stimulation (bead-to-T cell ratio of 1:3), and the old T-Rapa method (bead-to-T cell ratio of 3:1) for the following: expression of the IL-2 receptor CD25; and expression of the immunosuppressive molecules CTLA4 and TIM3.

[0054] Figure 23 Depicts cytokine secretion results at the end of manufacturing and after an additional 6-day culture without inhibitors, comparing the new Rapa-T method without bead co-stimulation, the new Rapa-T method with bead co-stimulation (bead-to-T cell ratio of 1:3), and the old T-Rapa method (bead-to-T cell ratio of 3:1) for the following: secretion of the type II cytokine IL-4; and secretion of the type I cytokine IFN-γ.

[0055] Figure 24 Depicts type I cytokine secretion (IL-2 and IFN-γ) results at the end of manufacturing and after an additional 6-day culture without inhibitors under n = 11 different culture conditions to further identify the role of bead-free co-stimulation in the generation of the new Rapa-T cell population.

[0056] Figure 25A Depicts flow cytometry data measurements for CD45RA+ of T cells under various culture conditions.

[0057] Figure 25B Depicts flow cytometry data measurements for CD25+ of T cells under various culture conditions.

[0058] Figure 25C Depicts flow cytometry data measurements for CD28+ of T cells under various culture conditions.

[0059] Figure 25D Depicts flow cytometry data measurements for ICOS+ of T cells under various culture conditions.

[0060] Figure 25E Depicts flow cytometry data measurements for CD39+ of T cells under various culture conditions.

[0061] Figure 25F Depicts flow cytometry data measurements of CD73+ for T cells under various culture conditions.

[0062] Figure 25G Depicts flow cytometry data measurements of GITR+ for T cells under various culture conditions.

[0063] Figure 25H Depicts flow cytometry data measurements of LAG3+ for T cells under various culture conditions.

[0064] Figure 25I Depicts flow cytometry data measurements of PD1+ for T cells under various culture conditions.

[0065] Figure 25J Depicts flow cytometry data measurements of 2B4+ for T cells under various culture conditions.

[0066] Figure 25K Depicts flow cytometry data measurements of LAIR1+ for T cells under various culture conditions.

[0067] Figure 25L Depicts flow cytometry data measurements of CTLA4+ for T cells under various culture conditions.

[0068] Figure 25M Depicts flow cytometry data measurements of KLRG1+ for T cells under various culture conditions.

[0069] Figure 25N Depicts flow cytometry data measurements of TIGIT+ for T cells under various culture conditions.

[0070] Figure 25O Depicts flow cytometry data measurements of TIM3+ for T cells under various culture conditions.

[0071] Figure 26 Depicts Western blot results of p-STAT5, p-STAT1, STAT1, p70S6K, p-SGK1, SGK1, Raptor, Rictor, cytochrome C, and actin in cells under various culture conditions.

[0072] Figure 27A Depicts IL-2 secretion measurements of RAPA-T cells and T-RAPA cells from supernatants 24 hours after co-stimulation with anti-CD3 / anti-CD28-coated beads and exposure to different cytokines.

[0073] Figure 27BDepicts the measurement of TNF-α secretion of RAPA-T cells and T-RAPA cells from the supernatant after 24 hours of co-stimulation with anti-CD3 / anti-CD28-coated magnetic beads and exposure to different cytokines.

[0074] Figure 28 Depicts the measurement of IL-2, TNF-α, and IL-13 secretion of RAPA-T cells after co-stimulation with anti-CD3 / anti-CD28-coated beads or soluble anti-CD3 / anti-CD28 microparticles.

[0075] Figure 29 Depicts the expression of CD4, CD8, CD25, and CTLA4 of RAPA-T cells measured by flow cytometry after co-stimulation with anti-CD3 / anti-CD28-coated beads or soluble anti-CD3 / anti-CD28 microparticles.

[0076] Figure 30 Depicts the manufactured T cell therapy protocol.

[0077] Figure 31 Depicts the pentostatin / cyclophosphamide protocol followed by manufactured T cell infusion.

[0078] Figures 32A - 32C Details the nature of the control group, i.e., subjects who are not randomly assigned to receive Rapa-T cell therapy will receive one of three FDA-approved triple regimens applicable to subjects with second or third relapse of MM, namely: the DPd regimen (A); the DRd regimen (B); or the KRd regimen (C).

[0079] Figure 33 Depicts an exemplary workflow for generating the manufactured T cells of the present disclosure.

[0080] Figure 34A Depicts the cytokine secretion of RAPA-T cells after exposure to pancreatic cancer cells.

[0081] Figure 34B Depicts the cytokine secretion of RAPA-T cells after exposure to lung cancer cells.

[0082] Figure 35 Depicts the cytokine secretion of RAPA-T cells with or without exposure to pancreatic cancer cells or lung cancer cells.

[0083] Figure 36 Depicts the correlation between checkpoint inhibitor treatment response and the number of tumor mutations in cancer. Detailed Description

[0084] The present disclosure provides methods for generating engineered T cells, engineered T cells generated by the methods disclosed herein, populations and compositions comprising populations of engineered T cells, and methods for treating cancer in a subject using the engineered T cells or populations of engineered T cells.

[0085] Definitions

[0086] The following definitions are provided:

[0087] As used herein, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. The term "or" as used in the claims and the present disclosure is used to mean "and / or" unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive.

[0088] When used in connection with a numerical value, the use of the term "about" is intended to encompass + / - 10%. By way of example and not limitation, if the number of amino acids identified is about 200, this would encompass 180 to 220 (plus or minus 10%).

[0089] The terms "subject", "patient", and "individual" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention can be used in experimental animals, veterinary applications, and the development of animal disease models, including but not limited to rodents such as mice, rats, and hamsters; and primates.

[0090] "Sample" is used herein in its broadest sense. Samples including cells, polynucleotides, polypeptides, peptides, antibodies, etc. can include body fluids; the soluble fraction of a cell preparation, or the medium in which the cells are grown; chromosomes, organelles, or membranes isolated or extracted from cells; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; cells; tissues; tissue imprints; fingerprints, skin, or hair; etc.

[0091] "Treatment" is an intervention that aims to prevent the development of a disorder or alter the pathology or symptoms of a disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Patients in need of treatment include patients who already have the disorder and patients who need to prevent the disorder. For example, in the treatment of a tumor (e.g., cancer), a therapeutic agent can directly reduce the pathology of tumor cells or make the tumor cells more susceptible to treatment with other therapeutic agents (e.g., radiation and / or chemotherapy). As used herein, "improvement" refers to symptoms that approach a normalized value (a value obtained, by way of example and not limitation, in a healthy patient or individual), e.g., symptoms that deviate from the normalized value by less than 50%, preferably by less than about 25% from the normalized value, more preferably by less than 10% from the normalized value, and still more preferably by not significantly different from the normalized value as determined using conventional statistical tests. By way of example and not limitation, improvement or treatment of a patient with an infectious disease organism (e.g., hepatitis B virus) can be determined by a reduction in viral particles in a sample taken from the patient, as measured, for example, by a reduction in plaque forming units (p.f.u.).

[0092] As used herein, "treatment cycle" can generally refer to any primary treatment cycle, first treatment cycle, second treatment cycle, or one or more additional treatment cycles.

[0093] As used herein, the term "therapeutically effective dose" or "therapeutically effective amount" means the amount of a compound of the invention that is effective to produce the desired therapeutic response. By way of example and not limitation, a dose that is effective to delay cancer growth or shrink a cancer or prevent metastasis can be a "therapeutically effective dose". The specific therapeutically effective dose will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapies, if any, and the specific formulation and structure of the compound or its derivatives employed.

[0094] As used herein, "immune cell" is intended to include any cell of the immune system that can be assayed, including but not limited to B lymphocytes (also known as B cells), T lymphocytes (also known as T cells), natural killer (NK) cells, natural killer T (NKT) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor infiltrating (TIL) cells, genetically modified immune cells (including hybridomas), drug-modified immune cells, and derivatives, precursors or progenitors of the above cell types.

[0095] "T cells" are a subset of lymphocytes that originate from the thymus and have a heterodimeric receptor associated with the proteins of the CD3 complex (e.g., rearranged T cell receptor, a heterodimeric protein on the surface of T cells responsible for the antigen / MHC specificity of the cell). T cell responses can be detected by measuring the effect of T cell responses on other cells (e.g., target cell killing, activation of other immune cells such as B cells) or the cytokines produced by T cell responses.

[0096] As used herein, the term "anti-CD3 / anti-CD28" should be understood to refer to anti-CD3 / anti-CD28 antibodies. For example, "anti-CD3 / anti-CD28 magnetic beads" should be understood to refer to magnetic beads having an anti-CD3 / anti-CD28 antibody moiety associated therewith. In the event that it is disclosed that co-stimulation with anti-CD3 / anti-CD28 is not provided even in a specific form such as anti-CD3 / anti-CD28 magnetic beads, it should be understood that this may also exclude the use of co-stimulation with other forms of anti-CD3 / anti-CD28.

[0097] As used herein, the term "one or more T-Rapa cells" refers to one or more T cells generated by co-stimulating with anti-CD3 / anti-CD28-coated magnetic beads at a ratio of 3:1 (bead to T cell ratio) and without a delay between the start of culture and co-stimulation, wherein the cells are grown in X-Vivo or an equivalent medium supplemented with 5% AB serum but without addition of an IL-2 signaling inhibitor and containing IFN-α (10,000 IU / mL), IL-2 (20 IU / mL), and rapamycin (1 μM), wherein the cells are cultured at 37 °C for 6 days and then 6 started at a concentration of 1.5×10

[0098] As used herein, the terms "engineered T cells" and "Rapa-T cells" are used interchangeably and refer to T cells generated by the methods of the present disclosure. "Engineered T cells" can include CD4 + , CD8 + T cells or both. "Engineered T cells" do not include T cells collected from a patient, i.e., naturally occurring T cells.

[0099] It should be understood that, as used herein, the terms "level of expression", "expression level", or equivalent references, when used to refer to results measured by flow cytometry, refer to the frequency of positive cells of a specified type in a population. With respect to the "level of expression" or equivalent expression referring to the expression level of a particular cell type, it should be understood that any decrease or increase mentioned is relative to the corresponding specified cell type, unless otherwise indicated.

[0100] As will be recognized by those skilled in the art, the term "autologous" cells refers to cells that have the same or similar haplotype as the cells of the subject or "host" to which the cells are administered, such that when these cells are transplanted into the host, no significant immune response against these cells occurs.

[0101] "CD4" is a cell surface protein that is important for T cell receptor recognition of antigenic peptides bound to MHC class II molecules on the surface of APCs. Upon activation, naive CD4 T cells differentiate into one of at least two cell types (Th1 cells and Th2 cells), each type being characterized by the cytokines it produces. "Th1 cells" are mainly involved in cell-mediated immunity and inflammatory responses that activate macrophages, while "Th2 cells" or "helper T cells" are mainly involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is a receptor for human immunodeficiency virus (HIV). Effector molecules for Th1 cells include, but are not limited to, IFN-γ, GM-CSF, TNF-α, CD40 ligand, Fas ligand, IL-3, TNF-β, and IL-2. Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, IL-13, CD40 ligand, IL-3, G-CSF, IL-10, TGF-β, and eosinophil chemotactic factor. Activation of the Th1-type cytokine response can inhibit the Th2-type cytokine response, and conversely, activation of the Th2-type cytokine response can inhibit the Th1-type response.

[0102] "Cytokines" are proteins made by cells that affect the behavior of other cells through "cytokine receptors" on the surface of the cells on which the cytokines act. Cytokines made by lymphocytes are sometimes called "lymphokines". Cytokines are also characterized as type I (e.g., IL-2 and IFN-γ) and type II (e.g., IL-4 and IL-10).

[0103] The term "modulate" means that any recited activity is, for example, increased, enhanced, augmented, agonized (acting as an agonist), promoted, decreased, reduced, inhibited, blocked, or antagonized (acting as an agonist). Modulation can increase the activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc. over the baseline value. Modulation can also reduce its activity below the baseline value.

[0104] "Substrate" refers to any rigid or semi-rigid carrier to which a nucleic acid molecule or protein binds and includes membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, capillary or other tubes, plates, polymers, and particles having a variety of surface forms including pores, trenches, pins, channels (chamiels), and pores.

[0105] Methods for generating engineered T cells, engineered T cells generated by the methods disclosed herein, and compositions comprising a population of engineered T cells

[0106] In the methods of the present disclosure, IFN-α is used to ex vivo polarize a culture comprising T cells towards a Th1-type differentiated state phenotype. Th1-type differentiation can be attenuated by polarization towards a regulatory T (T REG ) cell phenotype, the Th1-type differentiation being promoted by a cytokine comprising IL-2 that signals through STAT5 to promote the FoxP3 transcription factor that partially defines the T REG differentiated state. In the methods of the present disclosure, autocrine IL-2 signaling is prevented by omitting the exogenous use of IL-2 during cell culture and by culturing with an IL-2 signaling inhibitor, limiting T REG contamination during Th1-type polarization. In some aspects, the IL-2 signaling inhibitor is an anti-IL-2 receptor monoclonal antibody.

[0107] In the present disclosure, new manufacturing methods are provided that, compared to other T cell manufacturing methods, incorporate the following interventions: (1) culturing with delayed or no addition of anti-CD3 / CD28 beads (alternatively, any alternative source of anti-CD3 / anti-CD28 co-stimulation such as nanoparticles or microparticles) to increase T cell yield, wherein anti-CD3 / CD28 beads or nanoparticles are used for co-stimulation; (2) using a lower ratio of anti-CD3 / CD28 beads to enhance the resistant T cell phenotype, or alternatively, no co-stimulation with artificial antibody-based beads, nanoparticles, or microparticles; (3) using a parenteral formulation of mTOR inhibition (temsirolimus) to improve manufacturing feasibility; and (4) avoiding IL-2 signaling and the resulting Th1-type differentiation of T by omitting the typical use of exogenous IL-2 during T cell culture and eliminating endogenous autocrine IL-2 signaling REGCell contamination, for example, by using anti-IL-2 receptor monoclonal antibodies (daclizumab and basiliximab or other reagents that inhibit IL-2 receptor signaling) during T cell culture. In parallel culture experiments, T cells generated by this new combination method (referred to as "engineered T cells") exhibit a more desirable cell phenotype compared to T cells previously generated in ex vivo culture.

[0108] Figure 33 An exemplary workflow for generating the engineered T cells of the present disclosure is provided.

[0109] In some embodiments, a method for generating engineered T cells comprises inoculating a culture input cell population comprising T cells from a subject at a certain cell density in a medium comprising temsirolimus and an IL-2 signaling inhibitor. In certain aspects, the medium does not yet contain temsirolimus and / or the IL-2 signaling inhibitor, and these components can be added at or around the time of inoculation. The culture input cell population is incubated for a first period of time without co-stimulation by anti-CD3 / anti-CD28 antibodies, including, by way of non-limiting example, co-stimulation using anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles. After the first period of time, the culture input cell population can be stimulated by anti-CD3 / anti-CD28 antibodies, for example, by adding anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles. In the case of using anti-CD3 / anti-CD28 coated magnetic beads, the anti-CD3 / anti-CD28 coated magnetic beads can be used at a bead ratio between 1:1 and 1:12. Additionally, IFN-α is added to the medium. Then the culture input cell population is incubated for a second period of time to generate engineered T cells. In some aspects, there is no co-stimulation using anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles. In some embodiments, no co-stimulation is performed.

[0110] In any of the foregoing embodiments, the method for generating engineered T cells can further comprise, after harvesting the engineered T cells: packaging at least a portion of the engineered T cells in a package; and freezing the package containing the portion of the engineered T cells. Cryopreservation of the engineered T cells can be performed by methods known in the art.

[0111] In any of the foregoing embodiments, the method can further comprise, before inoculating T cells from the subject at a certain cell density in the medium: harvesting the culture input cell population from the subject.

[0112] In any of the foregoing embodiments, the culture medium may be free of IL-2 and no IL-2 may be added to the culture medium. In any of the foregoing embodiments, no serum may be added to the culture, e.g., the culture is serum-free. In any of the foregoing embodiments, the culture medium may be substantially serum-free.

[0113] In any of the foregoing embodiments, the IFN-α may be added at or about the same time as the anti-CD3 / anti-CD28 coated magnetic beads are added. If the culture is not co-stimulated, the IFN-α may be added, for example, at the start of the culture or within 48 hours after the start of the culture. By way of example and not limitation, the IFN-α may be added 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours after the start of the culture.

[0114] In any of the foregoing embodiments, the cell density may be from about 1×10 6 T cells per mL to 50×10 6 cells per mL. By way of example and not limitation, the cell density may be from about 1×10 6 cells per mL to 5×10 6 cells per mL, from 1×10 6 cells per mL to 10×10 6 cells per mL, from 1×10 6 cells per mL to 15×10 6 cells per mL, from 15×10 6 cells per mL to 22.5×10 6 cells per mL, from 10×10 6 cells per mL to 22.5×10 6 cells per mL, from 10×10 6 cells per mL to 22.5×10 6 cells per mL, from 5×10 6 cells per mL to 22.5×10 6 cells per mL, from 1×10 6 cells per mL to 50×10 6 cells per mL, from 10×10 6 cells per mL to 40×10 6 cells per mL, from 20×106 cells to 40×10 cells per mL 6 cells, 1×10 cells per mL 6 cells, 2.5×10 cells per mL 6 cells, 5×10 cells per mL 6 cells, 7.5×10 cells per mL 6 cells, 10×10 cells per mL 6 cells, 12.5×10 cells per mL 6 cells, 15×10 cells per mL 6 cells, 17.5×10 cells per mL 6 cells, 20×10 cells per mL 6 cells, 22.5×10 cells per mL 6 cells, 25×10 cells per mL 6 T cells, 30×10 cells per mL 6 cells, 35×10 cells per mL 6 cells, 40×10 cells per mL 6 cells, 45×10 cells per mL 6 cells or 50×10 cells per mL 6 cells.

[0115] In any of the foregoing embodiments, the temsirolimus may be present in the culture medium at a concentration of 0.1-5 μM. In some embodiments, temsirolimus may be present in the culture medium at a concentration of 0.1-1 μM. In any of the foregoing embodiments, temsirolimus may be present in the culture medium at a concentration of 1 μM. By way of example and not limitation, temsirolimus may be present in the culture medium at a concentration of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater. As a further example and not limitation, temsirolimus may be present in the culture medium at a concentration of about 1-5 μM, 2-5 μM, 3-5 μM, 4-5 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater.

[0116] In any of the foregoing embodiments, the temsirolimus may be added to the culture medium one or more times during a second time period to maintain a desired concentration. In any of the foregoing embodiments, the temsirolimus may be added to the culture medium once. By way of non-limiting example, the temsirolimus may be added to the culture medium every 2 days during the second time period. The desired temsirolimus concentration may be between 0.1 - 5 μM. In any of the foregoing embodiments, the desired temsirolimus concentration may be between 0.1 - 1 μM. In any of the foregoing embodiments, the desired temsirolimus concentration may be 1 μM. By way of illustration and not limitation, the desired temsirolimus concentration may be at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater. As a further illustration and not limitation, the desired temsirolimus concentration may be about 1 - 5 μM, 2 - 5 μM, 3 - 5 μM, 4 - 5 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater concentration.

[0117] The IL-2 signal transduction inhibitor may be any substance that inhibits IL-2 signal transduction and may be added in an amount sufficient to inhibit IL-2 signal transduction. In any of the foregoing embodiments, the IL-2 signal transduction inhibitor may be an anti-IL-2 receptor antibody or a fragment thereof, such as basiliximab or daclizumab. The IL-2 signal transduction inhibitor may be present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL. By way of non-limiting example, the IL-2 signal transduction inhibitor may be present at a concentration of about 5 μg / mL to 50 μg / mL, 5 μg / mL to 40 μg / mL, 5 μg / mL to 30 μg / mL, 5 μg / mL to 20 μg / mL, 5 μg / mL to 10 μg / mL, 40 μg / mL to 50 μg / mL, 30 μg / mL to 50 μg / mL, 20 μg / mL to 50 μg / mL, 20 μg / mL to 40 μg / mL, 20 μg / mL to 30 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL.

[0118] In any of the foregoing embodiments, the first time period can be from about 8 hours to about 24 hours. By way of non-limiting example, the first time period can be from about 8 hours to about 20 hours, 8 hours to about 16 hours, 8 hours to about 12 hours, 20 hours to about 24 hours, 16 hours to about 24 hours, 12 hours to about 24 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0119] In any of the foregoing embodiments, the bead to T cell ratio can be 1:3. In some embodiments, the bead to T cell ratio can be between 1:12 and 1:1, or in the most extreme examples, no beads are added. By way of illustration and not limitation, ratios of 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, or any range therebetween can be used. In some embodiments, co-stimulation of the cultured input cell population can be achieved using nanoparticles containing anti-CD3 / anti-CD28, and such nanoparticles can be used at a concentration lower than the recommended concentration. By way of illustration and not limitation, such nanoparticles can be used at about 0.01-fold to about 0.1-fold, about 0.025-fold to about 0.1-fold, about 0.05-fold to about 0.1-fold, about 0.075-fold to about 0.1-fold, about 0.01-fold to about 0.075-fold, about 0.01-fold to about 0.05-fold, about 0.01-fold to about 0.025-fold, about 0.025-fold to about 0.075-fold, about 0.025-fold to about 0.05-fold, about 0.05-fold to about 0.075-fold, or about 0.01-fold, about 0.025-fold, about 0.05-fold, about 0.075-fold, or about 0.1-fold of the recommended dose. By way of illustration and not limitation, compared to the recommended dose (10 μL per 1×10 6 T cells), a reduced dose, by way of illustration and not limitation, such as 1.1 μL (a nine-fold reduction) or about 0.11-fold, can be used for reagents such as T Cell TransAct TM and the like. Alternatively, if anti-CD3 / anti-CD28 co-stimulation is to be used for the production of manufactured T cells, a source of co-stimulation can be provided by soluble anti-CD3 / anti-CD28 microparticles. By way of illustration and not limitation, it can be provided by a manufacturer (e.g., Use soluble anti-CD3 / anti-CD28 microparticles at 20% of the strength recommended by Bio-Techne. By additional example, soluble anti-CD3 / anti-CD28 microparticles can be used at 5%, 10%, 15%, 20%, 25%, or 30% of the manufacturer's recommended strength. The specific amount of anti-CD3 / anti-CD28 reagent to be added can be titrated based on the desired functional characteristics of the final Rapa-T cell product. Specifically, in the presence of the inhibitory molecules described in this disclosure, an amount of reagent sufficient to maintain T cell viability in vitro can be added. However, no particular anti-CD3 / anti-CD28 reagent should be added in excess, as defined by: inappropriate high levels of T cell activation (increase in CD25 expression relative to T cell cultures using optimal, minimal costimulation by flow cytometry); inappropriate high levels of T cell checkpoint inhibitor receptor expression by flow cytometry; and inappropriate altered expression of molecules associated with T cell effector memory cells by flow cytometry (such as decreased levels of CD62L and CCR7; such as increased levels of CD45RO and KLRG).

[0120] In any of the foregoing embodiments, the IFN-α can be added to the medium at a concentration of about 1,000 IU / mL to 10,000 IU / mL. By way of example and not limitation, concentrations of 2,500 IU / mL to 10,000 IU / mL, 5,000 IU / mL to 10,000 IU / mL, 7,500 IU / mL to 10,000 IU / mL, 1,000 IU / mL to 7,500 IU / mL, 1,000 IU / mL to 5,000 IU / mL, 1,000 IU / mL to 2,500 IU / mL, 2,500 IU / mL to 7,500 IU / mL, 2,500 IU / mL to 5,000 IU / mL, 5,000 IU / mL to 7,500 IU / mL, 5,000 IU / mL to 10,000 IU / mL, 7,500 IU / mL to 10,000 IU / mL, or 1,000 IU / mL, 2,500 IU / mL, 5,000 IU / mL, 7,500 IU / mL, or 10,000 IU / mL can be used. Lower IFN-α concentrations (such as 1000 IU / mL) can result in an insignificant shift towards a Th1 phenotype.

[0121] In any of the foregoing embodiments, the second time period may be from about 4 days to about 8 days, 4 days to about 6 days, or 6 days to about 8 days. As a non-limiting example, the second time period may be about 4 days, 5 days, 6 days, 7 days, or 8 days. In the absence of co-stimulation, the time period for incubation may be from about 4 days to about 8 days, 4 days to about 6 days, or 6 days to about 8 days. As a non-limiting example, the second time period may be about 4 days, 5 days, 6 days, 7 days, or 8 days.

[0122] In any of the foregoing embodiments, the culture medium may further comprise 5% human serum. In some embodiments, the culture medium may further comprise 1%-20% human serum. By way of example and not limitation, the culture medium may comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% human serum. In some embodiments, the culture medium may comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% human serum. In any of the foregoing embodiments, no serum may be added to the culture medium or there may be no serum in the culture medium.

[0123] In any of the foregoing embodiments, the culture medium may further comprise X-Vivo 20 medium. In any of the foregoing embodiments, the culture medium may further comprise TexMACS medium. Any suitable culture medium may be used for culturing T cells.

[0124] In any embodiment of the foregoing embodiments, other culture medium can be added to culture.As an example and not limitation, can be by culture input cell group initial inoculation in culture after about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or any scope or time therebetween add other culture medium.As an example and not limitation, the amount of the culture medium added relative to the ratio of the amount of initial culture medium can be about 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or larger, and any scope therebetween.As an example and not limitation, the amount of the culture medium added after culture input cell group initial inoculation in culture can be enough to reduce the cell density in culture to the amount of target cell density. By way of example and not limitation, the target cell density may be about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 ,7×10 6 , 8×10 6 ,9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 or 4×10 7 and any range therebetween, provided that the initial cell density is greater than the target cell density.

[0125] In any of the foregoing embodiments, the culture input cell population can include about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, or about 5% to about 10% T cells of the total number of cells in the culture input cell population. As non-limiting examples, the culture input cell population can include about 5%, 10%, 15%, 20%, 33%, 40%, 50%, 66%, 70%, 75%, 90%, 95%, 98% or 99% or more T cells of the total number of cells in the culture input cell population.

[0126] In any of the foregoing embodiments, the cultured input cell population may further comprise monocytes. In any of the foregoing embodiments, the cultured input cell population may be enriched for T cells. By way of example and not limitation, the cultured input cell population may be subjected to T cell enrichment using an automated Ficoll process. Methods of performing the Ficoll process are known in the art and involve removing neutrophils and red blood cells from a sample. Any suitable method may be used to enrich for T cells in a cell population.

[0127] In any of the foregoing embodiments, the method may further comprise harvesting a sample comprising T cells from the subject; and isolating T cells from the sample to produce the cultured input cell population. Such a sample may contain peripheral blood stem cells (PBSC), and by way of example and not limitation, may be obtained by mobilization collection, steady-state apheresis, or simple blood draw. In any of the foregoing embodiments, samples containing PBSC and / or the cultured input cell population may be cryopreserved prior to the preparation of the manufactured T cells. Steady-state apheresis may be performed when the subject has a sufficient number of immune cells, which, by way of example and not limitation, may be characterized by a minimum absolute lymphocyte count (ALC). For example, the minimum ALC may be 300 lymphocytes per microliter.

[0128] In any of the foregoing embodiments, the T cells may be isolated by antibody-based purification.

[0129] In any of the foregoing embodiments, the enrichment of the T cells may be performed by counterflow centrifugal elutriation. This technique is well known in the art.

[0130] In any of the foregoing embodiments, the IFN-α may be added at or about the same time as adding nanoparticles containing anti-CD3 / anti-CD28.

[0131] In any of the foregoing embodiments, the anti-CD3 / anti-CD28 antibody may be removed by any suitable method after culturing. By way of example and not limitation, anti-CD3 / anti-CD28 magnetic beads may be removed by magnetic capture, and soluble anti-CD3 / anti-CD28 microparticles may be removed by adding a release buffer and washing the manufactured T cells.

[0132] In some embodiments, after one week of stimulation incubation with anti-CD3 / anti-CD28 magnetic beads, the manufactured T cell population exhibits an increase in IFN-γ secretion relative to T-Rapa cells, with the beads added at a bead:T cell ratio of 3:1.

[0133] In some embodiments, after one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads, the manufactured T cell population exhibits increased TNF-α secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0134] In some embodiments, after one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads, the manufactured T cell population exhibits increased GM-CSF secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0135] In some embodiments, after one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads, the manufactured T cell population exhibits increased IL-2 secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0136] In some embodiments, relative to the control T cell population and T-Rapa cells, the manufactured T cell population includes an increased percentage of cells that are positive for CD4, CD62L, CCR7, and CD127.

[0137] In some embodiments, relative to the control T cell population, the manufactured T cell population exhibits increased 4EBP1 phosphorylation. In some embodiments, relative to control T cells, the manufactured T cells exhibit increased 4EBP1 phosphorylation. By way of example and not limitation, the increase in 4EBP1 phosphorylation relative to a control T cell population or control T cells characteristic of the T cells (i.e., culture input T cells) from which the T cells are produced is no more than 50%, no more than 45%, no more than 40%, no more than 35%, or no more than 30%. As a further example and not limitation, the increase in 4EBP1 phosphorylation can be between 5%-50%, 5%-45%, 5%-40%, 5%-30%, 5%-20%, 5%-10%, 10%-50%, 10%-45%, 10%-40%, 10%-30%, 10%-20%, 20%-50%, 20%-45%, 20%-40%, 20%-30%, 30%-50%, 30%-45%, 30%-40%, 40%-50%, or any value therebetween or ranges within these ranges. The phosphorylation of 4EBP1 is decreased (or attenuated) compared to T-Rapa cells. In some embodiments, the increase in 4EBP1 phosphorylation can be measured 32 hours after the start of the culture.

[0138] In some embodiments, the manufactured T cell population exhibits reduced P70S6K expression relative to T-Rapa cells and increased P70S6K expression relative to the control T cell population that is characteristic of the cells from which the manufactured T cells are produced. By way of example and not limitation, the increase can be at least 10%, 20%, 30%, 40%, 50% or more and the decrease can be 50%, 60%, 70%, 80% or more.

[0139] In some embodiments, by flow cytometry, the manufactured T cell population exhibits reduced IL-2 receptor CD25 expression relative to T-Rapa cells. By way of example and not limitation, the decrease can be at least 50%, 60%, 70%, 80%, 90% or more.

[0140] In some embodiments, relative to the cultured input T cells, the manufactured T cells can express a unique RNA expression profile characterized by a 50% or greater increase in the RNA content of dedifferentiation molecules (such as KLF4, KLF10, Nanog and combinations thereof) and a 50% or greater decrease in the RNA content of differentiation molecules (such as perforin, granzyme B, IFN-γ and combinations thereof).

[0141] In some embodiments, relative to T-Rapa cells, the manufactured T cell population exhibits reduced levels of the following molecules associated with immunosuppressive effects: CTLA4; and TIM3.

[0142] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing CTLA4, as measured by flow cytometry. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing CTLA4, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express CTLA4, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CTLA4 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CTLA4, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing CTLA4 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CTLA4. By way of example and not limitation, the reduced frequency can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency. In some embodiments, the reduced frequency is 6 days after inoculating the cells that will give rise to the manufactured T cells into the culture.

[0143] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing TIM3, as measured by flow cytometry. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing TIM3, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express TIM3, as measured by flow cytometry. In some embodiments, relative to the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in a control T cell population characteristic of the T cells from which the manufactured T cells are produced, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing TIM3, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing TIM3 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in the control population. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing TIM3 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in the control population. In some embodiments, the reduced frequency is 6 days after the cells from which the manufactured T cells are produced are seeded into the culture. In some embodiments, relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing TIM3, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing TIM3 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing TIM3 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3. In some embodiments, the reduced frequency is 6 days after the cells from which the manufactured T cells are produced are seeded into the culture.

[0144] In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing PD1, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express PD1, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a frequency of CD4+ or CD8+ T cells expressing PD1 relative to the corresponding frequency of CD4+ and CD8+ T-Rapa cells expressing PD1, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing PD1 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing PD1. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing PD1 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing PD1. In some embodiments, the reduced frequency is 6 days after the cells that will give rise to the manufactured T cells are seeded into the culture.

[0145] In some embodiments, the manufactured T cell population can be characterized by 5% or less of CD4+ and CD8+ manufactured T cells expressing 2B4, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express 2B4, as measured by flow cytometry. In some embodiments, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4, as measured by flow cytometry. By way of example and not limitation, at least 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the CD4+ T cells in the manufactured T cell population can express 2B4, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD8+ T cells expressing 2B4, as compared to the corresponding frequency of CD8+ T cells expressing 2B4 in a control T cell population characteristic of the T cells from which the manufactured T cells are produced, as measured by flow cytometry. By way of example and not limitation, the reduced frequency of CD8+ T cells expressing 2B4 can be at least 50%, 60%, 70% or 80% less than the corresponding frequency of CD8+ T cells expressing 2B4 in the control T cell population. In some embodiments, the reduced frequency is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing 2B4, as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing 2B4, as measured by flow cytometry. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing 2B4 can be at least 20%, 30%, 40%, 50%, 60%, 70% or 80% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing 2B4. In some embodiments, the reduction is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture.

[0146] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing LAIR1, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express LAIR1, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing LAIR1 relative to the corresponding frequency of CD4+ or CD8+ T cells expressing LAIR1 in a control T cell population characteristic of the T cells from which the manufactured T cells are produced, as measured by flow cytometry. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing LAIR1 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T cells expressing LAIR1 in the control T cell population. In some embodiments, the reduced frequency is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture. In some embodiments, the CD4+ or CD8+ T cells of the manufactured T cell population can exhibit a reduced LAIR1 expression level relative to T-Rapa cells, as measured by flow cytometry. By way of example and not limitation, the reduction can be at least 30%, 40%, 50% or more.

[0147] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing TIGIT, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express TIGIT, as measured by flow cytometry. In some embodiments, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4, as measured by flow cytometry. By way of example and not limitation, at least 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the CD4+ T cells in the manufactured T cell population can express TIGIT, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing TIGIT, as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIGIT, as measured by flow cytometry. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing TIGIT can be at least 40%, 50%, 60%, 70%, 80% or 90% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIGIT. In some embodiments, the reduced frequency is 6 days after inoculating the cells that will produce the manufactured T cells into the culture.

[0148] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing LAG3, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express LAG3, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing LAG3 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing LAG3 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing LAG3 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3. In some embodiments, the reduced frequency is 6 days after the cells that will give rise to the manufactured T cells are seeded into the culture.

[0149] In some embodiments, the manufactured T cell population can be characterized by the retention level (i.e., substantially the same level) of the positive co-stimulatory molecule CD28 relative to a control T cell population that is characteristic of the T cells that give rise to the manufactured T cells, as measured by flow cytometry. In some embodiments, the frequency of CD28 expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of CD28 expression in CD4+ or CD8+ T cells in the control population. By way of example and not limitation, the frequency of CD28 expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the frequency of CD28 expression in CD4+ or CD8+ T cells in the control population. In some embodiments, the retention is 6 days after the cells that will give rise to the manufactured T cells are seeded into the culture.

[0150] In some embodiments, the manufactured T cell population can be characterized by the retention level (i.e., substantially the same level) of the positive co-stimulatory molecule ICOS relative to a control T cell population characteristic of the T cells from which the manufactured T cells are produced, as measured by flow cytometry. In some embodiments, the frequency of ICOS expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of ICOS expression in CD4+ or CD8+ T cells in the control population. By way of example and not limitation, the frequency of ICOS expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the frequency of ICOS expression in CD4+ or CD8+ T cells in the control population. In some embodiments, the retention is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture.

[0151] In some embodiments, the manufactured T cell population can be characterized by the retention level (i.e., substantially the same level) of CD45RA relative to a control T cell population characteristic of the T cells from which the manufactured T cells are produced, as measured by flow cytometry. In some embodiments, the frequency of CD45RA expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of CD45RA expression in CD4+ or CD8+ T cells in the control population. By way of example and not limitation, the frequency of CD45RA expression in CD4+ or CD8+ T cells in the manufactured T cell population can be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the frequency of CD45RA expression in CD4+ or CD8+ T cells in the control population. In some embodiments, the retention is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture.

[0152] In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing CD25, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express CD25, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD25 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing CD25 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing CD25 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. In some embodiments, the reduced frequency is 6 days after inoculating the cells that will give rise to the manufactured T cells into the culture.

[0153] In some embodiments, the manufactured T cell population exhibits a quiescent and non-senescent phenotype characterized by reduced KLRG1 levels, as measured by flow cytometry. In some embodiments, the reduction in KLRG1 levels is at 6 days after seeding the cells that will give rise to the manufactured T cells into culture. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing KLRG1, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express KLRG1, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing KLRG1 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing KLRG1, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing KLRG1 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing KLRG1. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing KLRG1 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing KLRG1. In some embodiments, the reduced frequency is at 6 days after seeding the cells that will give rise to the manufactured T cells into culture. In some embodiments, the reduced frequency is at 6 days after seeding the cells that will give rise to the manufactured T cells into culture.

[0154] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule CD39 relative to a control T cell population characterized by T cells from which the manufactured T cells are generated. In some embodiments, the manufactured T cell population can be characterized by 20% or less of the CD4+ or CD8+ manufactured T cells expressing CD39, as measured by flow cytometry. By way of example and not limitation, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express CD39, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD39 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing CD39 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing CD39 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39. In some embodiments, the reduced frequency is 6 days after inoculating the cells from which the manufactured T cells are generated into the culture.

[0155] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule CD73 relative to a control T cell population characterized by T cells from which the manufactured T cells are generated. In some embodiments, the manufactured T cell population can be characterized by 20% or less of the CD4+ or CD8+ manufactured T cells expressing CD73, as measured by flow cytometry. By way of example and not limitation, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express CD73, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD73 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD73, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing CD73 can be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD73. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing CD73 can be at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD73. In some embodiments, the reduced frequency is 6 days after inoculating the cells from which the manufactured T cells are generated into the culture.

[0156] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule GITR relative to a control T cell population characteristic of the T cells from which the manufactured T cells are produced. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing GITR, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express GITR, as measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a reduced frequency of CD4+ or CD8+ T cells expressing GITR relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing GITR, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing GITR can be at least 20% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing GITR. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cells expressing GITR can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing GITR. In some embodiments, the reduced frequency is 6 days after inoculating the cells from which the manufactured T cells are produced into the culture.

[0157] In some embodiments, relative to a control T cell culture, the manufactured T cells can exhibit an early differentiation state of cytokine biology, as evidenced by increased secretion of the precursor cytokine IL-2 and increased responsiveness to the homeostatic cytokines IL-7 and IL-15. In this way, the manufacturing methods of the present disclosure describe a process for manufacturing non-help-dependent T cells with increased responsiveness to homeostatic cytokines.

[0158] In some embodiments, the manufactured T cell population exhibits increased IL-2 secretion compared to a T-Rapa culture incubated under the same conditions. In some embodiments, the increase in IL-2 secretion is at least 1.1-fold. By way of example and not limitation, the increase can be at least 1.1-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold or more. In some embodiments, the manufactured T cell population secretes at least 500 pg / mL / 1×10 6 cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28-coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3. By way of example, under these conditions, the manufactured T cell population can secrete approximately 500 pg / mL / 1×106 cells / day, 600 pg / mL / 1×10 6 cells / day, 700 pg / mL / 1×10 6 cells / day, 800 pg / mL / 1×10 6 cells / day, 900 pg / mL / 1×10 6 cells / day, 1000 pg / mL / 1×10 6 cells / day or more of IL-2.

[0159] In some embodiments, when exposed to IL-7 or IL-15, the engineered T cell population can secrete an increased amount of IL-2. In some embodiments, the engineered T cell population is characterized by at least a 1.1-fold increase in IL-2 secretion when the engineered T cell population is incubated in the presence of IL-7 or IL-15 as compared to incubation in the absence of IL-7 or IL-15. By way of example and not limitation, the increase can be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold or more. In some embodiments, the engineered T cell population secretes at least 1000 pg / mL / 1×10 6 cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3 and exposure to IL-7, IL-15 or both IL-7 and IL-15 (if present) at an IL-7 concentration of 10 ng / mL and an IL-15 concentration of 10 ng / mL. By way of example, under these conditions, the engineered T cell population can secrete approximately 1000 pg / mL / 1×10 6 cells / day, 1100 pg / mL / 1×10 6 cells / day, 1200 pg / mL / 1×10 6 cells / day, 1300 pg / mL / 1×10 6 cells / day, 1400 pg / mL / 1×10 6 cells / day, 1500 pg / mL / 1×10 6 cells / day, 1600 pg / mL / 1×10 6 cells / day, 1700 pg / mL / 1×10 6 cells / day, 1800 pg / mL / 1×10 6 cells / day, 1900 pg / mL / 1×10 6 cells / day, 2000 pg / mL / 1×10 6IL-2 of 1 cell / day or more. IL-2 secretion is associated with non-helper-dependent T cells. The high IL-2 secretion of Rapa-T cells can be advantageous by avoiding the need to administer exogenous IL-2 after T cell adoptive therapy. In some embodiments, IL-7 or IL-15 (if present) is added at 10 ng / mL.

[0160] In some embodiments, the manufactured T cell population exhibits increased in vivo function relative to the control T cell population, the in vivo function being characterized by increased human T cell engraftment in a human-mouse xenogeneic graft-versus-host disease model.

[0161] In some embodiments, the manufactured T cell population exhibits a reduction in mTORC1 activation, as measured by phospho-P70S6K. By way of example and not limitation, the reduction can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% relative to T-Rapa cells 32 hours after the start of culture.

[0162] In some embodiments, the manufactured T cell population exhibits reduced phospho-STAT5 relative to T-Rapa cells. By way of example and not limitation, the reduction can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% relative to T-Rapa cells 32 hours after the start of culture.

[0163] In some embodiments, the manufactured T cell population exhibits reduced phospho-STAT5 relative to a control population of cultured T-Rapa cells. By way of example and not limitation, the reduction can be at least 50% relative to the control population of cultured T-Rapa cells. In some embodiments, the reduction is 48 hours after inoculating the manufactured T cells from an input cell population comprising T cells into the culture. In some embodiments, the p-STAT5 level is measured by Western blot.

[0164] In some embodiments, the manufactured T cell population exhibits at least a detectable level of STAT1 and phospho-STAT1. In some embodiments, the levels are measured 48 hours after inoculating the manufactured T cells from an input cell population comprising T cells into the culture. In some embodiments, the manufactured T cell population exhibits reduced phospho-STAT5 relative to a control T cell population and at least a certain level of STAT1 and phospho-STAT1. In some embodiments, the level of STAT1 or p-STAT1 is measured by Western blot.

[0165] In some embodiments, as measured by p70S6K or Raptor expression, the manufactured T cell population exhibits reduced mTORC1 activation relative to a control T cell population characteristic of the T cells from which the manufactured T cell population is derived. By way of example and not limitation, the reduction can be 50%. In some embodiments, the reduction is 48 hours after inoculating the manufactured T cells from an input cell population comprising T cells into a culture. In some embodiments, the reduction is measured by Western blot.

[0166] In some embodiments, the manufactured T cell population exhibits substantially the same levels of Rictor, SGK1, or phosphorylated SGK1 relative to a control T cell population. By way of example and not limitation, the levels of Rictor, SGK1, or phosphorylated SGK1 can be within 50%, 40%, 30%, 20%, 10%, or 5% of the corresponding levels of Rictor, SGK1, or phosphorylated SGK1 in T-Rapa cells. In some embodiments, the levels are 48 hours after inoculating the manufactured T cells from an input cell population comprising T cells into a culture. In some embodiments, the levels of Rictor, SGK1, or pSGK1 are at least the levels measured in the control T cell population. In some embodiments, the levels are measured by Western blot.

[0167] In some embodiments, relative to T-Rapa cells, the manufactured T cell population exhibits an increase in the secretion of at least one of IFN-γ, TNF-α, GM-CSF, and IL-2 after 6 days of expansion in culture after manufacture in the absence of an inhibitor. By way of example and not limitation, the increase can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more.

[0168] In some embodiments, relative to T-Rapa cells, the manufactured T cell population at the end of manufacture (day 6 of culture) can have an increased number of CD4+ T cells expressing the T cell marker CD45RA. By way of example and not limitation, the increase can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more.

[0169] In some embodiments, the manufactured T cell population can have reduced expression of one or more checkpoint inhibitor receptors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3. By way of example and not limitation, the reduced expression can be at least 25% less than the corresponding expression level in T-Rapa cells. As a further example and not limitation, the reduced expression can be at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% less than the corresponding expression level in the T-Rapa cell population. In some embodiments, the expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 in the manufactured T cell population can be within about 25% of the corresponding expression level in a control T cell population characteristic of the T cells from which the manufactured T cell population is produced. By way of example and not limitation, the expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 can be within about 25%, 20%, 15%, 10%, or 5% of the corresponding expression level in a control T cell population characteristic of the T cells from which the manufactured T cell population is produced. It should be understood that the expression levels of the checkpoint inhibitors are compared between the same cell types, e.g., CD4+ manufactured T cells can be compared to CD4+ T-Rapa cells or CD4+ control T cells characteristic of the T cells from which the manufactured T cell population is produced.

[0170] In some embodiments, the engineered T cells can have reduced expression of one or more checkpoint inhibitor receptors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3. By way of example and not limitation, the reduced expression can be at least 25% less than the corresponding expression level in T-Rapa cells. As a further example and not limitation, the reduced expression can be at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% less than the corresponding expression level in T-Rapa cells. In some embodiments, the expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 in the engineered T cells can be within about 25% of the corresponding expression level in control T cells characteristic of the T cells from which the engineered T cells are generated. By way of example and not limitation, the expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 can be within about 25%, 20%, 15%, 10%, or 5% of the corresponding expression level in control T cells characteristic of the T cells from which the engineered T cells are generated. It should be understood that the expression levels of checkpoint inhibitors are compared between the same cell types. For example, CD4+ engineered T cells can be compared to CD4+ T-Rapa cells or CD4+ control T cells characteristic of the T cells from which the engineered T cells are generated.

[0171] In some embodiments, the engineered T cells have increased CD127 expression relative to control T cells characteristic of the cells from which the engineered T cells are generated. By way of example and not limitation, this increase can be at least 10%, 20%, 30%, 40%, 50%, or more.

[0172] In some embodiments, the engineered T cell population can have at least 5% CD4+ T cells expressing CD127, as measured by flow cytometry. By way of example, the engineered T cell population can have at least 5%, 6%, 7%, 8%, 9%, 10%, or more CD4+ T cells expressing CD127, as measured by flow cytometry. In some embodiments, the engineered T cell population can have an increased frequency of CD4+ T cells expressing CD127 relative to a control T cell population characteristic of the cells from which the engineered T cell population is generated. By way of example and not limitation, the increase can be at least 50%, 100%, 150%, 200%, 300%, or more.

[0173] To the extent that any of the foregoing properties associated with a manufactured T cell population can be associated with an individual cell, the manufactured T cells can be characterized by such properties. In any of the foregoing embodiments, the manufactured T cells or the manufactured T cell population can have more than one of the properties.

[0174] Method for treating cancer in a subject

[0175] Patients with relapsed multiple myeloma (MM) have a limited survival, and curative therapies have remained elusive. As such, patients with relapsed MM are suitable for novel T cell therapies.

[0176] The immunotherapy differs from existing immunotherapy methods in several important categories. First, the manufactured T cell product is engineered to be inhibited at the level of the mammalian target of rapamycin (mTOR) pathway, resulting in conversion to resistance to apoptosis and enrichment for central memory differentiation. Second, the manufactured T cell product is manufactured in high-dose IFN-α that promotes CD4 + Th1 and CD8 + Tc1 differentiation. Third, the manufactured T cell product is costimulated minimally or not at all with monoclonal antibodies and expresses diverse T cell receptor (TCR) repertoires; as such, the anti-tumor effects mediated by the manufactured T cells are expected to occur primarily through in vivo clonal expansion to tumor antigens. This mechanism can be advantageous in multiple myeloma, in which the tumor antigens are unknown or can vary over time due to the high tumor mutation rate. Characterization of the emerging in vivo T cell response is critical to advancing understanding of the underlying mechanisms of the manufactured T cell therapy and will be evaluated as the second objective of this study. And fourth, the manufactured T cell therapy will be evaluated against a novel immune depletion and immunosuppression regimen consisting of the combination of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). This PC regimen relatively spares myeloid cells, thereby allowing repeated treatment cycles with substantially no neutropenia; this regimen has advantages in terms of cost (which can be administered in the outpatient setting) and safety (reduced infection rates due to sparing of myeloid cells). Multiple myeloma is a disease that is largely promoted by inflammatory signaling; as such, the inflammatory inhibition mediated by the PC regimen will be a component that contributes to the efficacy of the regimen. Each of these factors was considered during the design of the clinical trial, which focused on multiple infusions of the manufactured T cells following PC conditioning.

[0177] Engineered T cells express significantly reduced levels of checkpoint inhibitors and thus provide a novel ex vivo method for releasing the immune system from checkpoint inhibition, which is currently achieved by monoclonal antibody therapy. Along these lines, engineered T cell therapy is expected to be successful in cancers susceptible to checkpoint inhibitor therapy, including but not limited to: melanoma, renal cell carcinoma, bladder cancer, lung cancer, lymphoma, multiple myeloma, and colon cancer. It should also be noted that checkpoint inhibitor monoclonal antibody therapy has relatively high toxicity in multiple myeloma patients, thus requiring alternative methods to avoid immune checkpoints, such as engineered T cell therapy.

[0178] In addition, the ability of engineered T cells to undergo extensive clonal expansion against multiple tumor antigens predicts that tumor cells with increased mutation rates and tumors with microsatellite instability will be particularly sensitive to engineered T cell therapy.

[0179] The present disclosure provides methods for treating cancer, the methods comprising administering an engineered T cell of the present disclosure in a therapeutically effective dose.

[0180] In some embodiments, the method for treating cancer includes administering to a subject a therapeutically effective dose of a composition comprising engineered T cells. In some embodiments, the administration of the composition comprising engineered T cells can be repeated or cumulative at a therapeutically effective dose to reach a therapeutically effective dose. In some embodiments, the method further includes harvesting autologous cells from the subject before subjecting the subject to the immune depletion protocol. In some embodiments, the method further includes harvesting autologous cells from the subject before administering the composition comprising engineered T cells to the subject.

[0181] In any of the foregoing embodiments, the immune depletion protocol can comprise administering to the subject at least one of pentostatin and cyclophosphamide. In some embodiments, pentostatin is administered to the subject, and wherein the dose of pentostatin can be between 0.5 - 4 mg / m 2 between, 0.5 - 3 mg / m 2 between, 0.5 - 2 mg / m 2 between, 0.5 - 1 mg / m 2 between, 1 - 4 mg / m 2 between, 2 - 4 mg / m 2 between, or 3 - 4 mg / m 2 between. As a non-limiting example, the dose of pentostatin is about 0.5 mg / m 2 、1 mg / m 2 、1.5 mg / m 2 、2 mg / m2 , 2.5mg / m 2 , 3mg / m 2 , 3.5mg / m 2 or 4 mg / m 2 . In some embodiments, cyclophosphamide is administered to the subject, and the dosage of the cyclophosphamide may be between 50-400 mg, between 50-300 mg, between 50-200 mg, between 50-100 mg, between 100-400 mg, between 200-400 mg, between 300-400 mg, between 200-300 mg, or between 100-200 mg. As a non-limiting example, the dosage of the cyclophosphamide is about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg. In some embodiments, both pentostatin and cyclophosphamide are administered to the subject. In some embodiments, the pentostatin and cyclophosphamide are administered to the subject in the form of a single composition. In some embodiments, the single composition is administered intravenously to the subject.

[0182] In any of the foregoing embodiments, the immunodepletion regimen may comprise administering to the subject a first composition comprising pentostatin; and administering to the subject a second composition comprising cyclophosphamide. In some embodiments, the first composition is administered at a dose of 1-4 mg / m2 of pentostatin, the dose being between 0.5-4 mg / m 2 Between 0.5-3mg / m 2 Between 0.5-2mg / m 2 Between 0.5-1mg / m 2 Between 1-4 mg / m 2 Between 1-3 mg / m 2 Between 1-2 mg / m 2 Between or 3-4 mg / m 2 As a non-limiting example, the dose of pentostatin is about 1 mg / m 2 , 1.5mg / m 2 , 2mg / m 2 , 2.5mg / m 2 , 3mg / m 2 , 3.5mg / m 2 or 4 mg / m 2. In some embodiments, the second composition comprises cyclophosphamide and is administered at a dose of the cyclophosphamide between 50 - 400 mg, between 50 - 300 mg, between 50 - 200 mg, between 50 - 100 mg, between 100 - 400 mg, between 200 - 400 mg, between 300 - 400 mg, between 200 - 300 mg, or between 100 - 200 mg. As a non - limiting example, the dose of the cyclophosphamide is about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg.

[0183] In any of the foregoing embodiments, the therapeutically effective dose can be 1×10 5 to 5×10 6 cells / kg, 1×10 6 to 2.5×10 6 cells / kg, 2.5×10 6 to 5×10 6 cells / kg, 1×10 5 to 2.5×10 6 cells / kg, 2.5×10 5 to 5×10 6 cells / kg, 1×10 5 to 2.5×10 5 cells / kg, 2.5×10 5 to 5×10 5 cells / kg, 1×10 5 cells / kg, 2×10 5 cells / kg, 3×10 5 cells / kg, 4×10 5 cells / kg, 5×10 5 cells / kg, 1×10 6 cells / kg, 2×10 6 cells / kg, 3×10 6 cells / kg, 4×10 6 cells / kg, or 5×10 6 cells / kg of the manufactured T cells. In any of the foregoing embodiments, the composition comprising the manufactured T cells is administered to the subject by infusion.

[0184] In any of the foregoing embodiments, by way of example and not limitation, the cancer can be selected from the group consisting of: multiple myeloma, renal cell carcinoma, bladder cancer, lung cancer, liver cancer, lymphoma, gastric cancer, and colon cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is relapsed multiple myeloma. As a further example and not limitation, the cancer can be sarcoma, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, or colorectal cancer. In some embodiments, the cancer is PDL1-negative cancer. In some embodiments, the cancer is susceptible to checkpoint inhibitor therapy. In some embodiments, the multiple myeloma is relapsed and refractory multiple myeloma. In some embodiments, the multiple myeloma is quadruple or quintuple refractory multiple myeloma. In some embodiments, the multiple myeloma is smoldering multiple myeloma. In some embodiments, the subject has relapsed multiple myeloma. In certain aspects, by way of example and not limitation, the subject has relapsed multiple myeloma 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In some embodiments, the subject has smoldering multiple myeloma. In some embodiments, the subject has quadruple or quintuple refractory multiple myeloma. In some embodiments, by way of example and not limitation, the subject has 1, 2, 3, 4, 5 or more multiple myelomas that are difficult to treat with therapy.

[0185] In some embodiments, the subject has been previously treated and is now in a second or third relapse after receiving a different treatment series selected from the group consisting of: administration of a proteasome inhibitor, administration of an immunomodulatory drug, administration of an alkylating agent, administration of a CD38 monoclonal antibody, and administration of a glucocorticoid. This patient population is considered suitable for evaluation in a Phase 3 randomized clinical trial, where it is reasonable for the control group to receive standard chemotherapy for second or third relapse patients.

[0186] In separate embodiments, the subject is highly refractory to multiple standard drugs and thus a randomized clinical trial is not reasonable. Instead, such highly refractory patients will be treated with Rapa-T therapy in a single-arm Phase II clinical trial. The highly refractory status can be quantified by quadruple or quintuple refractory nomenclature, where such subjects are refractory to 4 or 5 of the top multiple drugs used to treat multiple myeloma, namely: bortezomib, carfilzomib, lenalidomide, pomalidomide, and daratumumab.

[0187] In the case of an embodiment of a Phase 3 clinical trial involving the treatment of MM at the second or third relapse, the primary study objective will relate to progression-free survival as the study endpoint, and the progression-free status is defined as: when monitored monthly, the difference in M protein / free light chain of the subject increases by less than 25%.

[0188] In some embodiments, the duration of the first treatment cycle is at least 28 days. In some embodiments, the duration of each of the one or more additional treatment cycles is at least 35 days.

[0189] In some embodiments, the step of administering pentostatin to the subject is repeated during the first treatment cycle. In some embodiments, the step of administering pentostatin to the subject is performed on the 1st, 4th, 8th, and / or 11th day of the first treatment cycle. In some embodiments, the step of administering cyclophosphamide to the subject is repeated during the first treatment cycle. In some embodiments, the step of administering cyclophosphamide to the subject is performed on the 1st, 2nd, 3rd, 4th, 5th, 8th, 9th, 10th, 11th, and / or 12th day of the first treatment cycle.

[0190] In any of the above embodiments, each of the one or more additional treatment cycles is spaced 0 to 4 weeks apart. In any of the above embodiments, the first treatment cycle is spaced 0 to 4 weeks apart from the first of the one or more additional treatment cycles. In any of the above embodiments, the step of administering a therapeutically effective dose of a composition comprising engineered T cells to the subject is performed on the 15th, 16th, 17th, and / or 18th day of each of the one or more additional treatment cycles.

[0191] In some embodiments, the subject is in the second or third relapse of MM after receiving a regimen consisting of: administration of a proteasome inhibitor, administration of an immunomodulatory drug, administration of an alkylating agent, administration of a CD38 monoclonal antibody, and administration of a glucocorticoid.

[0192] In some embodiments, the subject is in the advanced stage of MM relapse and is quadruple or quintuple refractory, where there is no standard therapy.

[0193] In some embodiments of a Phase 3 clinical trial, the primary study objective will relate to progression-free survival, and progression-free is defined as an increase in the M protein / free light chain difference of less than 25% between treatments.

[0194] In some embodiments, the method includes subjecting the subject to an immunosuppressive depletion regimen to reduce at least a portion of regulatory T cells and / or late senescent effector T cells or reduce the function of at least a portion of regulatory T cells and / or late senescent effector T cells; and after the immunosuppressive depletion regimen, administering to the subject a therapeutically effective dose of a composition comprising engineered T cells.

[0195] In some embodiments, the immunosuppressive depletion regimen includes: administering pentostatin to the subject; and administering cyclophosphamide to the subject; if the creatinine clearance rate of the subject > 30 mL / min / 1.73 m2, administering one or more additional doses of pentostatin to the subject; if the absolute lymphocyte count of the subject is 50 or greater per microliter and the absolute neutrophil count of the subject is 500 or greater per microliter, administering one or more additional doses of cyclophosphamide to the subject.

[0196] In some embodiments, the steps of measuring the CrCl of the subject and adjusting the dose of pentostatin to be administered are performed on day 1, day 4, day 8, and / or day 11 of the immunosuppressive depletion regimen.

[0197] In some embodiments, the steps of measuring the ALC and ANC and adjusting the dose of cyclophosphamide to be administered are performed on day 1, day 2, day 3, day 4, day 5, day 8, day 9, day 10, day 11, and / or day 12 of the immunosuppressive depletion regimen.

[0198] In some embodiments, the step of administering to the subject a therapeutically effective dose of a composition comprising engineered T cells after the immunosuppressive depletion regimen is performed 15 - 18 days after the start of the immunosuppressive depletion regimen.

[0199] In any of the foregoing embodiments, the step of subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or late senescent effector T cells or reduce at least a portion of the function of regulatory T cells and / or late senescent effector T cells and administering to the subject a therapeutically effective dose of a composition comprising engineered T cells after the immune depletion regimen is repeated at least two times. In any of the foregoing embodiments, the step of subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or late senescent effector T cells or reduce at least a portion of the function of regulatory T cells and / or late senescent effector T cells and administering to the subject a therapeutically effective dose of a composition comprising engineered T cells after the immune depletion regimen can be repeated up to 8 times or more. In any of the foregoing embodiments, each step of administering to the subject a therapeutically effective dose of a composition comprising engineered T cells after the immune depletion regimen can be spaced 0 to 9 weeks apart.

[0200] It should also be understood that in any of the foregoing embodiments, when co-stimulation by anti-CD3 / anti-CD28 antibodies is carried out, this co-stimulation can be provided in any form of anti-CD3 / anti-CD28 antibodies. By way of example and not limitation, when co-stimulation by using anti-CD3 / anti-CD28 beads is indicated, anti-CD3 / anti-CD28 nanoparticles or microparticles can be used.

[0201] Examples

[0202] The following examples are provided to better illustrate the methods and resulting engineered T cells of the present disclosure. These examples are not intended to limit or otherwise alter the scope of the methods, cells, and compositions disclosed in the present disclosure.

[0203] Example 1

[0204] Anti-IL-2 receptor blockade and mTOR blockade were used for Th1 enrichment. For adoptive T cell therapy, it is important to engineer T cells that preferentially express a Th1 phenotype, with minimal contamination from cells with a regulatory T (T REG ) cell phenotype. Th1-type cells can be characterized in part by the expression of their cell fate transcription factor TBET, while T REG cells express the FoxP3 transcription factor.

[0205] Methods for promoting TBET while restricting FoxP3 were evaluated. The mTOR inhibitor temsirolimus, which is an FDA-approved intravenous drug for the treatment of refractory renal cell carcinoma, was evaluated. Using an mTOR inhibitor to engineer T cells enriched for a Th1 phenotype might seem paradoxical, since inhibition of mTOR is generally associated with promoting T REGPhenotype is T cell-related. The current experiment differs from previous studies in that temsirolimus is an intravenous formulation and is thus advantageous relative to rapamycin, which is less feasible for cell culture due to its limited solubility in the culture medium.

[0206] The current experiment also differs from past studies in that the combination of temsirolimus and the anti-IL-2 receptor monoclonal antibody daclizumab was evaluated. Both daclizumab and basiliximab are FDA-approved monoclonal antibodies with a common mechanism of action and can thus be used interchangeably in the developed system.

[0207] As indicated, various ratios of anti-CD3, anti-CD28 beads to T cells (1:1 or 1:12) were used; the mTOR inhibitor temsirolimus (1 μM); with or without the anti-IL-2 receptor monoclonal antibody daclizumab (5 μg / ml or 50 μg / ml); and Th1 polarizing cytokine (IFN-α; 10,000 IU / ml) or control regulatory T cell polarization (IL-2 plus TGF-β) were used for in vitro T cell culture. On day 6 of culture, the intracellular expression of the regulatory T cell transcription factor FoxP3 and the Th1 transcription factor T-bet of T cells was evaluated. ( Figures 1A - 1B )

[0208] It was found that adding temsirolimus (at a concentration of 1.0 μM) in the environment of the type I polarizing cytokine IFN-α reduced the resulting T cell expression of FoxP3 relative to the day 0 input T cell population (see Figure 1A ). In addition, it was found that blocking the IL-2 receptor further reduced the expression of FoxP3; using the antibody at 50 μg / ml was more effective than using the antibody at 5 μg / ml, thus indicating a dose-response relationship. If exogenous IL-2 was added in combination with IFN-α, temsirolimus was ineffective in reducing the expression of FoxP3 ( Figure 1A ); in addition, if the culture conditions allowed T REG cell differentiation, temsirolimus and daclizumab were ineffective in restricting the expression of FoxP3, where the culture conditions were: the bead ratio was reduced to 1:12; IFN-α was eliminated from the culture; and exogenous IL-2 plus TGF-β was added ( Figure 1A )

[0209] In addition to restricting FoxP3 expression, the combination of temsirolimus and the anti-IL-2 receptor monoclonal antibody was effective in promoting the Th1 phenotype, as indicated by an increase in T-bet expression ( Figure 1B ). Again, there was a dose-response relationship, where 50 μg / ml daclizumab promoted T-bet expression to a greater extent than 5 μg / ml daclizumab ( Figure 1B)。Although adding IL-2 to IFN-α polarization increased TBET, this was also associated with an increase in FoxP3, thereby indicating a lack of Th1 purity by adding exogenous IL-2. Notably, as expected, T REG Control conditions - IL-2 and TGF-β had reduced TBET expression ( Figure 1B ).

[0210] Thus, the combination of mTOR inhibition and IL-2 receptor blockade represents a new approach for Th1 cell generation.

[0211] Development of combinations of interventions for inhibiting T cells during ex vivo manufacturing: mTOR inhibition; IL-2 receptor blockade; reduction of T cell co-stimulation; and inhibition of T cells before co-stimulation (overnight pre-warming). Given that these results suggest that the combination of mTOR inhibition and IL-2 receptor blockade can promote the generation of Th1-type cells (increase in the TBET to FoxP3 ratio; T REG cell contamination limitation), it is thought that additional interventions may also promote the generation of Th1 cells.

[0212] One benefit of using mTOR inhibition for adoptive T cell therapy is that this intervention can promote the generation of T cells with a more primitive differentiation state, such as the T central memory subset (T CM ) or the T stem cell memory subset (T SCM ). In previous studies using ex vivo rapamycin, it was found that ex vivo rapamycin was effective for generating T CM -phenotype T cells; this result is consistent with the known role of mTOR in the control of T cell memory status. Promoting the T CM and / or T SCM status during manufacturing is important because such T cells with a more primitive differentiation state increase long-term engraftment after adoptive transfer and mediate increased in vivo effects in experimental models. Several other methods have been described for promoting the generation of T cells with a restricted differentiation state, which include: using GSK3 inhibitors to promote WNT signaling; inhibiting ART signaling; and inhibiting PI3 kinase signaling.

[0213] A method was developed whereby T cells were plated in X-Vivo 20 medium supplemented with 5% human AB serum and incubated in this medium, which contained no exogenous cytokines and contained the mTOR inhibitor temsirolimus and IL-2 receptor blockade achieved by addition of monoclonal antibodies. This method incorporated an approximately 16-hour "pre-incubation" before co-stimulation with anti-CD3, anti-CD28-coated magnetic beads. It is known that this method has not been reported previously.

[0214] As a first step in evaluating these increasingly stringent culture conditions, the viability of culturing CD4 + and CD8 + T cells was evaluated, as defined by the presence of live cells at the end of the culture interval (manufactured for Th1, 6-day culture interval). In Figures 2A - 2B it, as indicated, CD4 + and CD8 + T cells were placed in culture under various conditions. The 3 / 28 bead to T cell ratio was 3:1, 1:1, or 1:3. T cells were co-stimulated either simultaneously with the addition to the culture (“no overnight pre-warming”) or after a 16-hour overnight pre-warming. In some conditions, the anti-IL-2 receptor monoclonal antibody daclizumab was added at a concentration of 50 μg / ml. For mTOR inhibition, temsirolimus was added at 1.0 or 0.1 μM; alternatively, the control mTOR inhibitor rapamycin was added at a concentration of 1.0 μM. Most cultures were also supplemented with the type I cytokine promoter IFN-α (10,000 IU / ml). As indicated, most culture conditions did not include the addition of exogenous IL-2. T cell yields were calculated after 6 days of culture and compared to the start of culture (“day 0 input culture”).

[0215] As Figures 2A - 2B shown, the culture conditions were modified to include not only mTOR inhibition (using 1.0 μM or 0.1 μM temsirolimus; control using 1.0 μM rapamycin) and IL-2 receptor blockade (daclizumab), but also reduced co-stimulation (from a 3:1 bead to T cell ratio to ratios of 1:1 and 1:3) and overnight pre-warming such that a similar number of live T cells were produced relative to control T cell cultures.

[0216] Importance of pre-warming and high-dose temsirolimus in Th1 / Tc1 manufacture. When cryopreserving Th1 / Tc1 cell products (at the end of culture), it is important that in addition to the T CM cell phenotype, the T cells should also have a relatively quiescent phenotype. That is, it has previously been found that T cells produced by rapamycin secreted very little cytokine upon adoptive transfer, but produced a large amount of cytokine in vivo; notably, others have identified a similar inverse correlation between the effector function of the cell product (minimal) and the effector function in vivo (maximal). T cell quiescence upon adoptive transfer can promote the survival of transferred T cells and may also be important for reducing the risk of cytokine release syndrome, which is the cause of morbidity and death following other forms of adoptive T cell therapy, especially gene-modified chimeric antigen receptor (CAR) T cell therapy.

[0217] To evaluate this, the cytokine secretion potential of the manufactured T cells was tested at the end of cell culture (day 6), and then again one week after ex vivo expansion following propagation in maximum co-stimulation (3 / 28 bead to T cell ratio of 3:1) and in medium without any inhibitors.

[0218] In Figures 3A - 3B , prior to co-stimulation, CD4 + and CD8 + T cells were purified and cultured for 6 days (3 / 28 bead to T cell ratio of 1:1) with or without a 16-hour interval pre-incubation. As indicated, temsirolimus was added at a concentration of 1.0 or 0.1 μM; all cultures were supplemented with IFN-α (10,000 IU / ml) and daclizumab (50 μg / ml). On day 6 of culture, the resulting T cells were co-stimulated for 24 hours using a 3 / 28 bead to resulting T cell ratio of 3:1; cytokine content in the supernatant was tested by Luminex assay (results are described as pg per ml secreted per million cells per 24 hours). Additionally, the resulting T cells were co-stimulated using a 3 / 28 bead to resulting T cell ratio of 3:1 and cultured for one week in medium without any exogenous cytokines or inhibitors; after this T cell culture, on day 13 of culture, the T cells were harvested, re-stimulated using 3 / 28 beads (ratio of 3:1), and cytokine content of the 24-hour supernatant was evaluated as described above. The abbreviation N.A. indicates not applicable (T cell yield was insufficient for measurement).

[0219] Results showed that in Figures 3A - 3B , secretion of IFN-γ was shown in Figure 3A and secretion of TNF-α was shown in Figure 3B . In each case, the left panel shows the T cell cytokine potential on day 6, while the right panel shows the T cell cytokine potential on day 13.

[0220] These data indicate that after 24 hours of maximum co-stimulation under both overnight pre-incubation conditions and no pre-incubation conditions, high-dose temsirolimus (1.0 μM) produces the desired very low levels of day 6 T cell IFN-γ and TNF-α secretion. Notably, use of temsirolimus at a concentration of 0.1 μM only partially reduces the day 6 T cell cytokine secretion potential. Thus, temsirolimus in the method should be used at a higher concentration, i.e., at least 1 μM. These data also indicate that the overnight pre-incubation intervention alone is insufficient to produce a quiescent T cell phenotype. Thus, the overnight pre-incubation step must be used in combination with high-dose temsirolimus to achieve the full desired result.

[0221] In addition, this method induces the desired inverse relationship between initial T cell quiescence and enhanced effector function upon subsequent restimulation. That is, for the day 13 T cell values for both IFN-γ and TNF-α secretion, the condition with the lowest level of day 6 cytokine potential (combination of pre-incubation with high-dose temsirolimus) yields the highest day 13 cytokine secretion values. Notably, the condition consisting of high-dose temsirolimus and no overnight pre-incubation did not yield sufficient productivity at day 13 of culture for assessing cytokine secretion potential; thus, this result further confirms the value of high-dose temsirolimus plus the pre-incubation step for Th1 / Tc1 cell generation.

[0222] The new combination method induces enhanced mTOR inhibition and elimination of STAT5 phosphorylation. The ability of the new combination method (mTOR inhibition; IL-2 receptor blockade; pre-incubation of delayed co-stimulation; and use of lower-intensity co-stimulation) to generate T cells with the desired phenotype depends in part on its enhanced ability to control molecular and cellular events previously associated with the rapamycin-resistant T cell phenotype.

[0223] One component of this phenotype is the control of mTOR-dependent signaling events (such as those occurring at the 4EBP1 level), which contribute to the control of protein translation. To address this, Th1 / Tc1 cells were generated using the T-Rapa method, which simultaneously added T cells along with high-level co-stimulation (3 / 28 bead to T cell ratio of 3:1), high-dose rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture. In a parallel comparison, T cells were generated using the new combination method of the present disclosure (16-hour pre-incubation step; reduced level (ratio of 1:1) of co-stimulation; use of both temsirolimus (1.0 μM) and daclizumab (50 μg / ml); and addition of only IFN-α without IL-2).

[0224] At Figure 4In it, CD4+ and CD8+ T cells were cultured using a previous method ["T-Rapa": simultaneously adding T cells, high-level co-stimulation (3 / 28 bead to T cell ratio of 3:1), high-dose rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture] or a new combination method for the manufactured T cells ["Rapa-T": 16-hour pre-incubation step; low-level co-stimulation (1:1 ratio); using both temsirolimus (1.0 μM) and daclizumab (50 μg / ml); and adding only IFN-α without IL-2]. At the 16th hour and 32nd hour of T cell culture, a portion of the T cells was harvested, proteins were isolated, and Western blot analysis of the housekeeping gene β-actin and the mTOR pathway molecule phosphor-4EBP1 was quantified. The results were compared to the proteins obtained from the input T cells on day 0 before any T cell activation.

[0225] As Figure 4 shown, relative to the T cells manufactured using the previously described method ("T-Rapa"), at both the 16th hour and 32nd hour culture time points, the new combination method for generating the manufactured T cells ( Figure 4 "Rapa-T" in ) reduced mTOR pathway activation (as measured by 4EBP1 phosphorylation).

[0226] P70S6 kinase is another key molecule in the mTOR pathway. In Figure 5 and 6 CD4+ and CD8+ T cells were cultured using a previous method ["T-Rapa": adding T cells, high-level co-stimulation (3 / 28 bead to T cell ratio of 3:1), high-dose rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture simultaneously] or a new combination method for generating the manufactured T cells ["Rapa-T": 16-hour pre-incubation step; low-level co-stimulation (1:1 ratio); using both temsirolimus (1.0 μM) and daclizumab (50 μg / ml); and adding only IFN-α without IL-2]. At the 16th hour and 32nd hour of T cell culture, a portion of the T cells was harvested, proteins were isolated, and Western blot analysis of the housekeeping gene β-actin and the mTOR pathway molecule P70S6K ( Figure 5 ) or phosphorylated STAT5 ( Figure 6 ) was quantified. The results were compared to the proteins obtained from the input T cells on day 0 before any T cell activation.

[0227] In sharp contrast, generation using the new combinatorial method for generating manufacturing T cells greatly reduces the level of P70S6K ( Figure 6 , Rapa-T condition). These data provide further evidence that the combinatorial method of Th1 / Tc1 generation improves the control of mTOR activation. Using the previous method for generating rapamycin-resistant T cells, it was found that P70S6K was significantly upregulated at both the 16th and 32nd hours of T cell culture ( Figure 5 , T-Rapa condition).

[0228] The combinatorial method is associated with an increase in the purity of Th1-type cells (reduced contamination by cells expressing FoxP3). The previous generation method led to a large amount of STAT5 phosphorylation ( Figure 6 , T-Rapa results at the 16th and 32nd hours of culture); in sharp contrast, the combinatorial method eliminated STAT5 phosphorylation ( Figure 6 ; Rapa-T results at the 16th and 32nd hours of culture).

[0229] Thus, the new combinatorial method is also advantageous in increasing the control of mTOR signaling during T cell generation and eliminating signaling events that promote T REG cell contamination (controlling STAT5 phosphorylation).

[0230] The individual components of the Th1 / Tc1 cell generation combinatorial method are further described. Further cultures were established to obtain additional information on the individual contributions of the culture interventions to the resulting Th1 / Tc1 phenotype. For Figures 7-10, as indicated in Figures 7-10, CD4+ and CD8+ T cells were cultured using various 3 / 28 bead ratios, various mTOR inhibition methods, variable addition of anti-IL-2 receptor blockade, variable addition of type I polarizing cytokine IFN-α, and variable use of an initial overnight prewarming step. Supernatants generated by repeated co-stimulation (3:1 bead ratio) were collected on the 6th and 13th days of culture, and Luminex assays were used to test for IFN-γ ( Figures 7A - 7B ), TNF-α ( Figures 8A - 8B ), GM-CSF ( Figures 9A - 9B ), or IL-2 ( Figures 10A - 10B ) content (results are expressed as pg per million cells per ml per 24 hours).

[0231] Figures 7A - 7B IFN-γ secretion results at the end of culture (day 6) and one week after further culture without inhibitor (day 13) are shown. The desired phenotype consists of relatively low cytokine secretion values on day 6 and relatively high cytokine values on day 13. Figures 7A - 7BIt has been demonstrated that culture conditions containing each of the combinatorial elements (low-level costimulation [1:1 ratio]; delayed costimulation after overnight pre-incubation; addition of the polarizing cytokine IFN-α; addition of an mTOR inhibitor [in this experiment, a suboptimal concentration of 0.1 μM was used]; and inclusion of the IL-2 receptor antibody daclizumab) have the desired phenotype, as these conditions result in reduced IFN-γ secretion on day 6 but high IFN-γ secretion on day 13. T cell culture conditions omitting one or more of these elements tend to have higher cytokine values on day 6 and / or lower cytokine values on day 13. Additionally, a previously used method for manufacturing rapamycin-resistant T cells (T-Rapa; Figures 7A - 7B ) expressed a less favorable cytokine secretion pattern (higher values on day 6; lower values on day 13).

[0232] When evaluating the following cytokines, the Th1 / Tc1 manufacturing combinatorial method also produced a favorable cytokine phenotype (decreased values on day 6 combined with increased values on day 13): TNF-α( Figures 8A - 8B );GM-CSF( Figures 9A - 9B );and IL-2( Figures 10A - 10B ).

[0233] Collectively, these results provide further evidence that the new manufacturing method for the engineered T cells has important advantages over the existing T-Rapa method.

[0234] Molecular changes associated with Th1 / Tc1 cell manufacturing using the combinatorial method. Additional experiments were conducted to characterize the molecules that are altered during Th1 / Tc1 cell manufacturing using the combinatorial method. Such information is valuable not only because it can lead to a better understanding of the T cell phenotype, but also because such information can be used as a quality control element during manufacturing. Additionally, such information can be used during the screening of other combinatorial steps that may be used in future manufacturing efforts.

[0235] In previous efforts, it was found that rapamycin-resistant T cells undergo autophagy during T cell manufacturing. It has long been known that autophagy is a direct result of mTOR inhibition: when mTOR is activated, T cells remain in a growth and proliferative state (autophagy signals are turned off); conversely, when mTOR is inhibited, autophagy is promoted, resulting in a reduction in T cell volume, including a reduction in mitochondrial volume (mitophagy). Indeed, autophagy is an essential homeostatic process in T cell biology and is associated with T cell health, as it can reduce energy requirements and can eliminate intracellular organelles and other cellular debris.

[0236] For Figures 11 - 13, human CD4 was cultured with a 16-hour pre-incubation interval before adding 3 / 28 beads at a reduced bead-to-T cell ratio of 1:3 + and CD8 + T cells. As Figures 11 - 13 indicated, various T cell cultures received different methods of mTOR inhibition (1.0 μM rapamycin; 1.0 μM or 0.1 μM temsirolimus), different conditions of exogenous IL-2 addition, and different conditions relative to the addition of the anti-IL-2 receptor monoclonal antibody daclizumab. After the 16-hour pre-incubation interval, cells were harvested from the T cell cultures, proteins were isolated, and p62 ( Figure 11 ), phospho-RAPTOR ( Figure 12 ), or BIM ( Figure 13 ) and the housekeeping gene β-actin were quantified by Western blotting.

[0237] The ability of the combination method to promote autophagy was evaluated, as measured by T cell expression of the autophagy marker p62. As Figure 11 indicated, the combination method included a pre-incubation step, low-level co-stimulation (a bead-to-T cell ratio of 3 / 28 at 1:3), daclizumab blockade of the IL-2 receptor, and upregulation of the autophagy marker p62 by an mTOR inhibitor. If each of these factors was present, autophagy could be achieved by mTOR inhibition using rapamycin (1 μM) or temsirolimus (1.0 or 0.1 μM). Elimination of IL-2 receptor blockade from the protocol substantially attenuated autophagy induction.

[0238] In addition, the combination method also led to inhibition of the mTOR pathway being promoted, as indicated by a decrease in the expression of the phosphorylated form of RAPTOR ( Figure 12 ). Notably, the combination method incorporating high-dose temsirolimus produced a low level of phospho-RAPTOR compared to using low-dose temsirolimus or high-dose rapamycin.

[0239] T cell expression of the pro-apoptotic member BIM of the bcl-2 gene family was also evaluated. bcl-2 family members mainly operate at the mitochondrial level, and as such, mitophagy can affect the balance of bcl-2 family member genes, which helps determine the apoptosis threshold. Mitophagy has been shown to be beneficial in reducing the apoptosis threshold, potentially selectively eliminating mitochondria, while the balance of bcl-2 family molecules is unfavorable. The combination method found to be produced led to a decrease in BIM expression ( Figure 13 ).

[0240] In summary, these experiments indicate that enhanced autophagy, reduced mTOR signaling, and reduced pro-apoptotic molecule expression are associated with the combinatorial approach to Th1 / Tc1 cell manufacturing. These changes may contribute to enhanced in vivo function of the manufactured T cells and can thus be used as a quality control step or for screening future next-generation T cell manufacturing methods.

[0241] The combinatorial approach promotes T cell quiescence and T cell dedifferentiation. Additional experiments were performed to characterize the surface phenotype of Th1 / Tc1 cells manufactured by the combinatorial approach, as defined by a pre-warming step, low-level co-stimulation (3 / 28 bead to T cell ratio of 1:3), daclizumab blockade of the IL-2 receptor, and combination with an mTOR inhibitor. First, the effect of culture variables on the expression of T cells of the CD45RA subtype, a marker of T cell naivety that includes T cells of the stem cell memory subset, was evaluated. Thus, it is desirable to develop a T cell manufacturing method that retains or increases CD45RA expression.

[0242] For Figures 14A - 15D , as Figures 14A - 15D now indicated, CD4+ and CD8+ T cells were cultured using various 3 / 28 bead ratios, various mTOR inhibition methods, variable addition of anti-IL-2 receptor blockade, variable addition of the type I polarizing cytokine IFN-α, and variable use of an initial overnight pre-warming step. On day 6 of culture, the T cells were harvested and the flow cytometric expression of CD45RA ( Figures 14A - 14D ) or CD62L, CCR7, and CD127 ( Figures 15A - 15D ) on the CD4 cell subset of the T cells was evaluated and the results were compared to the expression levels on day 0 of culture ("day 0 input culture").

[0243] As Figures 14A - 14D shown, T cell manufacturing without the key elements of the combinatorial approach (using high co-stimulation at a bead to T cell ratio of 3:1; not using daclizumab; not using an mTOR inhibitor) resulted in a rapid decline in CD45RA expression (see column #2). In stark contrast, the use of all of these components resulted in complete retention of CD45RA expression (see column #4). Notably, T cells propagated using the previously identified manufacturing method did not optimally preserve CD45RA expression (T-Rapa cells; Figure 14B , column #3).

[0244] In addition, it was evaluated whether the combinatorial approach led to other markers of reduced T cell differentiation, including CD62L, CCR7, and CD127. Figure 15A(Column #4) indicates that T cells made using a pre - incubation step, low - level co - stimulation, antibody blockade of the IL - 2 receptor, and an mTOR inhibitor have increased co - expression of these T - cell markers. Notably, T cells propagated using previously identified manufacturing methods do not have optimal increased expression of these three memory markers (T - Rapa cells; Figure 15A , column #3).

[0245] Thus, in terms of manufacturing T cells in a limited differentiation state, the combined method of Th1 / Tc1 cell manufacturing is advantageous, which has clearly and reproducibly shown to mediate increased in - vivo effects.

[0246] The combined method is optimized by reducing the T - cell purity at the start of culture. For T - cell manufacturing, it is important to determine whether the culture input population must be highly purified to a high T - cell content or whether accessory cell populations such as monocytes can be tolerated. From the perspective of financial cost and labor, it is generally desirable to start the culture using a non - highly purified population. However, contaminating cell populations at the start of culture may be detrimental to T - cell expansion or the generation of the desired T - cell phenotype. To evaluate this parameter, cultures are started using the combined method with input populations having T - cell contents of 100%, 66%, 33%, or 10% respectively; the remaining cell populations are mainly monocytes.

[0247] For Figures 16 - 20 , before starting the culture, the input cell population is adjusted such that the purity of T cells is 100%, 66%, 33%, or 10%; the remaining cell populations consist of non - T cell populations contained in peripheral blood mononuclear cells (mainly monocytes). As indicated, T cells are expanded in media variably containing or not containing temsirolimus (at a concentration of 1.0 or 0.1 μM); additionally, the cultures variably include a 16 - hour pre - incubation or no pre - incubation before anti - CD3, anti - CD28 bead co - stimulation (at a ratio of 1:3). Each culture shown is propagated in media containing the anti - IL - 2 receptor monoclonal antibody daclizumab (50 μg / ml) and IFN - α (10,000 IU / ml). At the end of the 6 - day manufacturing interval, the T cells receive high - level co - stimulation (bead - to - T - cell ratio of 3:1). For Figure 16 , after high - level co - stimulation, the T cells are then expanded in inhibitor - free media for one week. At the end of this expansion interval, the amount of T cells is calculated and plotted against the input number on day 0. For Figures 17 - 18, after high - level co - stimulation, the T cells are expanded until day 13 of culture. At days 6 and 13, the T cells are co - stimulated, and the IFN - γ ( Figures 17A - 17B ) or TNF - α ( Figures 18A - 18B) Content (results are shown as pg per million cells per ml per 24 hours). For Figures 19 - 20 , after high-level co-stimulation, T cells were expanded until day 13 of culture. On days 6 and 13, CD25 expression of T cells was evaluated by flow cytometry (results shown are the percentage of CD4+ T cells co-expressing CD25)( Figure 19 ) or the expression of CD62L, CCR7, and CD127 ( Figure 20 ).

[0248] As Figure 16 detailed, cultures starting with reduced T cell purity at the time of culture input resulted in greater T cell expansion capacity, and this relationship occurred in a dose-dependent manner. Notably, T cells propagated using an overnight pre-warming step had greater expansion capacity relative to T cells co-stimulated at the start of culture. These data provide further support for the combination method and indicate that cell populations that may be considered contaminants at the start of culture actually appear to promote the expansion potential of T cells. Thus, the optimized use of the combination method should also include the use of T cells that are not highly enriched at the start of culture; to ensure quality, it is important to control the purity level, and as a non-limiting example, start each culture with an inoculum having a T cell content of 66% or 33%. As Figures 17A - 17B and 18A-18B detailed, T cells manufactured using the combination method and non-highly purified input T cells resulted in a desired cytokine secretion pattern, namely:( Figures 17A - 17B ) reduced IFN-γ secretion at the end of manufacture (day 6), and increased IFN-γ secretion after one week of expansion in inhibitor-free medium (day 13); and( Figures 18A - 18B ) reduced TNF-α secretion at the end of manufacture (day 6), and increased TNF-α secretion after one week of expansion in inhibitor-free medium (day 13).

[0249] In addition, cell surface marker expression in T cells manufactured using the combination method was evaluated using an input population with reduced T cell purity. As Figure 19 shown, such T cells had reduced CD25 expression at the end of manufacture (day 6), consistent with a quiescent phenotype; after one week of culture in inhibitor-free conditions, T cells greatly upregulated CD25. As Figure 20 shown, such T cells also had increased co-expression of the memory markers CD62L, CCR7, and CD127; then after one week of T cell expansion, these markers decreased.

[0250] In summary, these data indicate that it is possible to generate Th1 / Tc1 cells using a combinatorial approach in the case of using input T cells that are highly contaminated with non-T cell populations. Indeed, the purposeful inclusion of such non-T cell populations can be used to increase T cell yields and improve the resulting T cell memory profile.

[0251] Manufactured from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells will be stored in the vapor phase of liquid nitrogen until the cells are thawed and Rapa-T cells are generated. In the case of cells freshly isolated by apheresis or in the future by simple blood collection, the cells will be processed immediately and then can be placed directly into culture or can also be cryopreserved by controlled-rate freezing techniques and stored in the vapor phase of liquid nitrogen for subsequent use.

[0252] T cell culture from cryopreserved cell substrates in freshly isolated cell populations requires some type of T cell enrichment, e.g., by using monoclonal antibodies and column technology (positive or negative selection). Enrichment of the starting cell material used in Rapa-T manufacture does not require such antibody-based methods because T cells are effectively enriched during the culture interval; as such, this method is consistent with recommendations for effective cell therapy at the global level. The initial processing steps for manufacturing Rapa-T cells focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (when applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and to some extent monocytes. These steps are performed in a relatively automated method that primarily uses closed-system technology; this procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves the consistency of the overall manufacturing process, and reduces the risk of contamination of the final product with infectious agents. Processing of the Rapa-T product incorporates the following steps: (1) thawing of the cryopreserved product (when applicable) using a solid-state non-aqueous-based method to reduce infectious agent contamination; (2) automated washing of the cell product using a LOVO-permeable membrane device; (3) integration of RBC lysis using ammonium chloride potassium (ACK) buffer during the LOVO wash step; (4) volume reduction of the cell content using the LOVO method followed by plating of the cells into a closed-system countercurrent centrifugal elutriation (CCE) device (Elutra; Terumo); and (5) pre-programmed operation of the Elutra device for effective removal of granulocytes and monocytes by CCE.

[0253] After lymphocyte enrichment and media purification, the cells are seeded into a dedicated chamber with robust oxygen exchange capabilities (G-Rex vessel; Wilson-Wolf). In addition to having enhanced gas permeability characteristics, the G-Rex vessel is a closed system unit and has the additional advantage of automated closed system media volume reduction (GatheRex liquid handling pump). The lymphocyte-enriched cells are maintained in the G-Rex vessel for 6 days.

[0254] Several specific culture conditions can be utilized to facilitate the generation of CD4+ and CD8+ T cell mixtures with engineered T cell functional attributes in the G-Rex vessel. These specific conditions include: (1) using enriched media (including but not limited to X-Vivo 20; Lonza) further supplemented with 5% human serum; (2) incorporating a 16-hour interval prior to co-stimulation for seeding cells into the G-Rex (seeding cells at a relatively high density of 1.5×10 6 cells per milliliter); (3) during this initial interval, optimizing cell rest by adding the monoclonal antibody basiliximab (which blocks the IL-2 receptor and thereby prevents autologous T cell activation via endogenously produced IL-2) and temsirolimus (a pharmacological inhibitor of mTORC1); (4) after this 16-hour interval, under sub-optimal conditions, the cells are either not co-stimulated or co-stimulated with anti-CD3 / anti-CD28-coated magnetic beads (3 / 28 beads), as defined by a bead-to-T cell ratio of 1:3 (typically, most T cell expansion conditions utilize a 9-fold higher co-stimulation level, i.e., a bead-to-T cell ratio of 3:1); (5) importantly, it is crucial not to wash the T cells after the initial interval; (6) after the interval, in addition to adding 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4+ Th1 and CD8+ Tc1 phenotypes; (7) importantly, it is crucial to avoid adding IL-2, a common additive for T cell culture; and (8) after adding the beads and IFN-α, it is important to leave the cells undisturbed until harvest on day 6 of culture (no cell washing, no further culture additives).

[0255] Cryopreservation of engineered T cells. 1) After 6 days of cell culture in the G-Rex vessel, the volume of the culture can be reduced in a closed system manner using the GatheRex instrument. Subsequently, the cells can be harvested, the 3 / 28 beads can be removed with a handheld magnet, and the cells can be placed in a LOVO device for serial washing to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0256] The washed cells can be reconstituted into cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation is performed in 50 ml cryobags as multiple single-use aliquots. Rapa-T cells are cryopreserved by a GMP-compliant controlled-rate freezing method and, after passing the specified release standard tests, are transported by a certified cryotransporter in the vapor phase of liquid nitrogen.

[0257] The release standard tests for Rapa-T cells include standard tests such as the purity of CD3+, CD4+, and CD8+ T cell content (by flow cytometry, the CD3+ T cell content of the final product can be >70%; the CD4+ and CD8+ subsets can be present at levels of 5% respectively). As determined by flow cytometry annexin and 7-AAD assays, >70% of the cells can be viable. In addition, over a minimum 3-day culture interval (ideally, a 14-day culture interval), the cells should be free from bacterial and fungal contamination; furthermore, the cell product should be below the detection limit of bacterial LPS endotoxin.

[0258] In addition to these standard tests, specialized functional tests will also constitute the release criteria for the Rapa-T cell product. Prior to releasing the product and the cell therapy, relative to the cultured input T cells, Rapa-T cells can have the following properties: (1) an enhanced T central memory phenotype, as defined by increased flow cytometry co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitory molecules (such as programmed death 1 (PD1)); (3) a quiescent state, as defined by reduced Th1 / Tc1-type cytokine secretion levels upon maximal co-stimulation; (4) an autophagy signature, as evidenced by reduced mitochondrial mass determined by flow cytometry MitoTracker assay; (5) a resistant phenotype, as evidenced by at least 50% inhibition of downstream targets of mTORC1 and mTORC2; and (6) a multifaceted differential gene expression profile of n = 80 key transcription factors and differentiation molecules.

[0259] Figure 21 It is shown that relative to T-Rapa cells, T cells generated by the new Rapa-T method carry increased expression of naive or T central memory markers, independent of whether the new Rapa-T method uses bead co-stimulation or uses low-level bead co-stimulation (bead-to-T cell ratio of 1:3). In Figure 21Among them, Rapa-T1 cells were generated by culturing in IFN-α, temsirolimus, and basiliximab, as previously described, without bead co-stimulation (the first two columns in each figure) or with co-stimulation using a bead-to-T cell ratio of 1:3 (the third and fourth columns in each figure); the results were compared with cultures using the previous T-Rapa method (using rapamycin and a bead-to-T cell ratio of 3:1; the fifth and sixth columns in each figure). Flow cytometry was performed at the end of the culture, and the results of CD4 + T cell subsets (black columns) and CD8 + T cell subsets (gray columns) were recorded in detail. The results shown are for naive T cell subsets defined by CD45RA+ expression (left figure); for T central memory subsets, as defined by the co-expression of CD62L and CCR7; and for more naive T cell subsets co-expressing CD62L, CCR7, and CD127.

[0260] As Figure 21 detailed in + and CD8 + T cells produced by the method described according to the present disclosure have increased expression levels of naive and T central memory markers by flow cytometry compared to T-Rapa cells, including under conditions where the method does not involve any bead co-stimulation or has a reduced level of co-stimulation compared to the T-Rapa method.

[0261] Figure 22 It is shown that compared to T-Rapa cells, T cells produced by the new Rapa-T method have reduced expression of CD25, CTLA4, and TIM3, regardless of whether the new Rapa-T method uses no bead co-stimulation or uses a low level of bead co-stimulation (a bead-to-T cell ratio of 1:3). In Figure 22 Among them, Rapa-T1 cells were generated by culturing in IFN-α, temsirolimus, and basiliximab, as previously described, without bead co-stimulation (the first two columns in each figure) or with co-stimulation using a bead-to-T cell ratio of 1:3 (the third and fourth columns in each figure); the results were compared with cultures using the previous T-Rapa method (using rapamycin and a bead-to-T cell ratio of 3:1; the fifth and sixth columns in each figure). Flow cytometry was performed at the end of the culture, and the results of CD4 + T cell subsets (black columns) and CD8 + T cell subsets (gray columns) were recorded in detail. The results shown are the results of the expression of the IL-2 receptor CD25 (left figure); for the immunosuppressive and T REG associated molecule CTLA4; and for the immune checkpoint molecule TIM3.

[0262] As Figure 22 detailed herein, CD4 + and CD8 + T cells produced by the methods described according to the present disclosure have reduced levels of the IL-2 receptor CD25 2 (which is associated with T cell activation and T REG cell function) by flow cytometry, as well as reduced levels of the immunosuppressive molecule CTLA4 3 and the immune checkpoint inhibitory molecule TIM3 4 levels, including conditions where the method does not involve any bead co-stimulation or reduced levels of co-stimulation compared to the T-Rapa method.

[0263] Figure 23 Shown are T cells generated by the new Rapa-T method with a pattern similar to Th2 versus Th1 polarization, but with increased quiescence relative to T-Rapa cells, independent of whether the new Rapa-T method uses no bead co-stimulation or low levels of bead co-stimulation (bead to T cell ratio of 1:3). In Figure 23 , Rapa-T1 cells were generated by culturing in IFN-α, temsirolimus, and basiliximab, as previously described, either without bead co-stimulation (the first two bars in each graph) or with co-stimulation using a bead to T cell ratio of 1:3 (the third and fourth bars in each graph); the results were compared to culturing using the previous T-Rapa method (using rapamycin and a bead to T cell ratio of 3:1; the fifth and sixth bars in each graph). Cytokine secretion analysis (measurement of IL-4 and IFN-γ) was performed at the end of the culture, and the results are detailed at the end of T cell production (day 6) and after an additional 6 days of culture without inhibitors (day 12).

[0264] As Figure 23 detailed herein, CD4 + and CD8 +T cells have a substantial Th2 versus Th1 cytokine polarization relative to T-Rapa cells, including under conditions where the method does not involve any bead co-stimulation or reduced levels of co-stimulation compared to the T-Rapa method. Specifically, the secretion level of the Th2-type cytokine IL-4 is low (the values at the end of manufacture [day 6] and after an additional culture period without inhibitor [day 12] are both between 100 pg / ml and 200 pg / ml at day 6 and day 12), and the secretion level of the Th1-type cytokine IFN-γ is high at day 12 of culture (the value is between 1000 pg / ml and 3000 pg / ml). Relative to the old T-Rapa conditions, IFN-γ secretion at the end of manufacture (day 6) is greatly reduced under the new Rapa-T conditions, indicating the favorable property of T cell quiescence in the new Rapa-T manufacturing method, independent of whether the method uses no bead co-stimulation or low levels of bead co-stimulation (bead to T cell ratio of 1:3).

[0265] As Figure 24 detailed in, CD4 + and CD8 + T cells have a Th1 cytokine polarization pattern relative to T-Rapa cells, including under conditions where the method does not involve any bead co-stimulation or reduced levels of co-stimulation compared to the T-Rapa method.

[0266] Figure 24 Demonstrates that relative to T-Rapa cells, the new Rapa-T method generates T cells with a favorable Th1 polarization pattern, independent of whether the new Rapa-T method uses no bead co-stimulation or low levels of bead co-stimulation (bead to T cell ratio of 1:3). In Figure 24 , Rapa-T1 cells were generated by culturing in IFN-α, temsirolimus, and basiliximab, as previously described, without either bead co-stimulation or co-stimulation using a bead to T cell ratio of 1:3; various control cultures were also evaluated, including the previous T-Rapa method (using rapamycin and a bead to T cell ratio of 3:1). Unless otherwise stated, the concentration of all cultures was 9×10 6cells / ml, no bead co-stimulation, no IL-2 addition, with delayed addition of IFN-α, containing 1 μM of temsirolimus and 10 μM of basiliximab, and using X-Vivo 20 medium supplemented with 5% AB serum. The specific culture conditions according to the legend in the figure are as follows: Condition 1, Rapa-T method, as described above; Condition 2, Rapa-T method without serum; Condition 3, Rapa-T method without basiliximab; Condition 4, Rapa-T method without basiliximab and with reduced temsirolimus (0.1 μM); Condition 5, Rapa-T method without temsirolimus or basiliximab; Condition 6, Rapa-T method using input T cells contaminated with high-frequency monocytes (79% of the input cells are monocytes, an increase compared to all other cultures with approximately 10% monocyte contamination); Condition 7, Rapa-T method using monocytes-free contamination (<1%); Condition 8, Rapa-T method with simultaneous addition of IFN-α (no overnight delay); Condition 9, Rapa-T method with 1:3 bead co-stimulation; Condition 10, control T cell condition (no inhibitor; 3:1 beads); and Condition 11, old T-Rapa condition, rapamycin (1 μM), IL-2 added at 20 IU / ml, 3:1 magnetic beads, no overnight pre-incubation. Cytokine secretion analysis (measurement of IL-2 and IFN-γ) was performed at the end of the culture, and the results were detailed at the end of T cell manufacturing (day 6) and after an additional 6 days of culture under inhibitor-free conditions (day 12).

[0267] As Figure 24 detailed in, CD4 + and CD8 + T cells manufactured according to the method described in the present disclosure have a Th1 cytokine polarization pattern relative to T-Rapa cells, including conditions where the method does not involve any bead co-stimulation or reduced levels of co-stimulation compared to the T-Rapa method.

[0268] As Figure 24As depicted, Condition #1, Rapa-T condition without beads, results in a good high level of IL-2 secretion capacity, especially on day 12 after T cell expansion in the absence of inhibitors. A similar pattern was observed in Condition #2, which was carried out in a medium without serum supplementation, indicating the ability to generate Rapa-T cells with or without serum supplementation. The enhanced IL-2 secretion capacity, especially compared to Culture #11 (old T-Rapa condition), indicates that the new Rapa-T manufacturing method produces T cells with a reduced pre-differentiated precursor profile, which can function in vivo in a helper-independent manner. In this regard, Condition #1 is also advantageous compared to Culture #9, which is the new Rapa-T condition with co-stimulation using a bead-to-T cell ratio of 1:3.

[0269] As Figure 24 depicted, Condition #1 (manufactured by Rapa-T condition without beads) is also preferred in terms of IFN-γ secretion at the end of day 6 of manufacturing, as the level is close to the lower limit of detection, indicating that the Rapa-T cell product is in a quiescent state. In contrast, Condition #5 without inhibitors had an IFN-γ secretion of nearly 4000 pg / ml on day 6; similarly, the old T-Rapa condition had a high level of IFN-γ secretion of approximately 6000 pg / ml on day 6. Notably, the new Rapa-T condition with cell co-stimulation using a bead-to-T cell ratio of 1:3 is less quiescent than Condition #1 without beads, as there is an IFN-γ secretion of approximately 200 pg / ml at the end of manufacturing (day 6). Finally, the new Rapa-T manufacturing method (Condition #1 without beads or Condition #9 with beads) shows a favorable increase in IFN-γ secretion capacity on day 12 of culture after a 6-day culture interval in the absence of inhibitors.

[0270] In summary, the new Rapa-T method can result in the generation of T cells with the following phenotypic characteristics: (a) reduced expression of regulatory T cell markers (such as the transcription factor FOXP3) and increased Th1-type transcription factor TBET relative to input normal T cells; (b) increased quiescence, as indicated in part by reduced expression of the IL-2 receptor CD25 and reduced secretion of the inflammatory cytokines IFN-γ and TNF-α at the end of generation, relative to the old T-Rapa method; (c) reduced expression of p-STAT5 and decreased levels of the mTOR pathway molecules p-RAPTOR, p-4EBP1, and p70S6K relative to the old T-Rapa method; (d) increased autophagy markers relative to input normal T cells, including but not limited to altered expression of the molecule p62; (e) increased flow cytometry markers of naive or T central memory populations (including co-expression of CD45RA, CD62L / CCR7, and concomitant expression of CD62L / CCR7 / CD127) relative to input normal T cells; (f) reduced expression of co-inhibitory molecules (including but not limited to CTLA4) relative to the old T-Rapa method; (g) reduced expression of checkpoint inhibitory receptors (including but not limited to TIM3) relative to the old T-Rapa method; and (h) altered RNA expression patterns relative to input normal T cells, including but not limited to increased expression of dedifferentiation markers (including but not limited to Nanog, KLF4, and KLF10) and reduced expression of differentiation markers (including but not limited to perforin, granzyme B, IFN-γ).

[0271] Most phenotypic characteristics of the T cell product manufactured according to the Rapa-T method detailed in the present disclosure can be determined at the end of the culture. However, it is important to note that the T cell product can be cryopreserved, and as such, the phenotypic characteristics of the T cells in the thawed state reflect the actual product that will be adoptively transferred to the subject. The Rapa-T cells in the thawed state can be characterized by: (a) maintaining a quiescent state, as indicated by low-level expression of the IL-2 receptor CD25, which is comparable between the sample at the end of manufacture on day 6 and the thawed sample; (b) the thawed sample will continue to have low-level secretion of the inflammatory cytokines IFN-γ and TNF-α (no increase relative to the sample collected at the end of manufacture); (c) relative to the sample at the end of manufacture on day 6, the thawed sample will maintain an increased flow cytometry marker of the naive or T central memory population (comprising co-expression of CD45RA, CD62L / CCR7, and concomitant expression of CD62L / CCR7 / CD127); (f) the thawed sample will continue to have decreased expression of co-inhibitory molecules (including but not limited to CTLA4) (no increase in the thawed sample relative to the sample collected at the end of manufacture); (g) the thawed sample will continue to have decreased expression of checkpoint inhibitory receptors (including but not limited to TIM3) (no increase in the thawed sample relative to the sample collected at the end of manufacture); and (h) a change in the RNA expression pattern relative to the input normal T cells, including but not limited to an increase in dedifferentiation markers (including but not limited to Nanog, KLF4, and KLF10) and a decrease in differentiation markers (including but not limited to perforin, granzyme B, IFN-γ).

[0272] Manufactured from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells will be stored in the vapor phase of liquid nitrogen until the cells are thawed and Rapa-T cells are manufactured. In the case of cells freshly isolated by apheresis or in the future by simple blood collection, the cells will be processed immediately and then can be placed directly into the culture, or they can also be cryopreserved by controlled-rate freezing technology and stored in the vapor phase of liquid nitrogen for subsequent use.

[0273] T cell culture from cryopreserved cell substrates in freshly isolated cell populations requires some type of T cell enrichment, e.g., by using monoclonal antibodies and column technology (positive or negative selection). Enrichment of the original cell material used in Rapa-T manufacturing does not require such antibody-based methods because T cells are effectively enriched during the culture interval; thus, this method is consistent with recommendations for effective cell therapy at the global level. The initial processing steps for manufacturing Rapa-T cells focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (when applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and to some extent monocytes. These steps are performed in a relatively automated method that mainly uses closed system technology; this procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves the consistency of the entire manufacturing process, and reduces the risk of contamination of the final product by infectious agents. The processing of the Rapa-T product can include the following steps: (1) thawing the cryopreserved product (when applicable) using a solid-state non-aqueous-based method to reduce infectious agent contamination; (2) automatically washing the cell product using a LOVO-permeable membrane device; (3) integrating lysis of RBCs using ammonium chloride potassium (ACK) buffer during the LOVO wash step; (4) reducing the cell content in volume using the LOVO method and subsequently plating the cells into a closed system countercurrent centrifugal elutriation (CCE) device (Elutra; Terumo Corporation); and (5) pre-programmed operation of the Elutra device for effective removal of granulocytes and monocytes by CCE.

[0274] After lymphocyte enrichment and media purification, the cells can be seeded into a dedicated chamber with enhanced oxygen exchange capabilities (G-Rex vessel; Wilson Wolf Corporation). In addition to having enhanced gas permeability characteristics, the G-Rex vessel is a closed system unit and has the additional advantage of automated closed system media volume reduction (GatheRex liquid handling pump). Lymphocyte-enriched cells can be maintained in the G-Rex vessel for 6 days.

[0275] Several specific culture conditions can be utilized to promote the generation of CD4 + and CD8 + T cell mixtures using the functional properties of the manufactured T cells in the G-Rex vessel. These specific conditions include: (1) using enriched media (including but not limited to X-Vivo 20; Lonza) further supplemented with 5% human serum or in some embodiments without added serum; (2) incorporating a 16-hour interval prior to co-stimulation to plate the cells into the G-Rex at a density of 1.5×10 6(3) plating cells at a relatively high density of cells; (3) during this initial rest period, the cells are optimally rested by adding the monoclonal antibody basiliximab (which blocks the IL-2 receptor and thus prevents autologous T cell activation by endogenously produced IL-2) and temsirolimus (a pharmacological inhibitor of mTORC1); (4) after this 16-hour rest period, under suboptimal conditions, the cells are co-stimulated with anti-CD3 / anti-CD28-coated magnetic beads (3 / 28 beads), as defined by a bead-to-T cell ratio of 1:3 (usually, most T cell expansion conditions utilize a 9-fold higher co-stimulation level, i.e., a bead-to-T cell ratio of 3:1; in some cases, it is beneficial to avoid adding any co-stimulatory reagent); (5) importantly, it is crucial not to wash the T cells after the initial rest period; (6) after the rest period, in addition to adding the 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4 + Th1 and CD8 + Tc1 phenotypes; (7) importantly, it is crucial to avoid adding IL-2, which is a common additive for T cell culture; and (8) after adding the beads and IFN-α, it is important to keep the cells undisturbed until they are harvested on day 6 of the culture (no cell washing, no further culture additives).

[0276] Cryopreservation of the manufactured T cells. 1) After 6 days of cell culture in a G-Rex container, the volume of the culture can be reduced in a closed system manner by the GatheRex instrument. Subsequently, the cells can be harvested, the 3 / 28 beads can be removed with a handheld magnet, and the cells can be placed in a LOVO device for continuous washing to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0277] The washed cells can be reconstituted into a cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation is performed in 50 ml cryobags in multiple single-use aliquots. Rapa-T cells are cryopreserved by a controlled-rate freezing method compliant with GMP, and after the Rapa-T cells pass the designated release standard tests, they are transported in the vapor phase of liquid nitrogen by a certified cryotransporter.

[0278] The release standard tests for Rapa-T cells include standard tests such as CD3 + 、CD4 + and CD8 + T cell content purity (by flow cytometry, the CD3+ T cell content of the final product can be >70%; CD4 + and CD8 +Subpopulations may be present at 5% levels, respectively). Cells may be >70% viable as determined by flow cytometry annexin and 7-AAD assays. In addition, there should be no bacterial and fungal contamination within a minimum 7-day culture interval (ideally, a 14-day culture interval); furthermore, the cell product should be below the detection limit for bacterial LPS endotoxins and mycoplasma.

[0279] In addition to these standard tests, specialized functional tests can also constitute release criteria for Rapa-T cell products. Prior to release of products and cell therapy, Rapa-T cells can have the following attributes relative to culture input T cells: (1) enhanced T-central memory phenotype, as defined by increased flow cytometric co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitory molecules such as programmed death 1 (PD1); (3) quiescence, as defined by reduced levels of Th1 / Tc1-type cytokine secretion at maximal co-stimulation; (4) autophagic signature, as demonstrated by reduced mitochondrial mass measured by flow cytometry MitoTracker; (5) resistance phenotype, as demonstrated by at least 50% inhibition of mTORC1 and mTORC2 downstream targets; and (6) multifaceted differential gene expression profiles of n=80 key transcription factors and differentiation molecules.

[0280] Example 2

[0281] Steady-state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were obtained from GE Enrichment was performed on the instrument using an automated Ficoll process, eliminating >95% of the unwanted contaminating neutrophil population. The lymphocyte-enriched cell population was then plated into G-REX culture vessels at an initial cell density in the media and serum supplementation conditions indicated in Table 1 below and incubated in culture for 6 days. Condition 1 represents the control before culture (lymphocytes enriched after automated Ficoll). Cultures were initiated with variable inhibitor conditions by adding temsirolimus, sirolimus, and / or the anti-IL2 receptor monoclonal antibody basiliximab at the doses indicated in Table 1. For conditions 2-5, basiliximab was added at a concentration of 10 μg / mL, and for conditions 6-8, basiliximab was added at a concentration of 20 μg / mL. Some culture conditions were subjected to use 3 / 28 beads were co-stimulated with anti-CD3 and anti-CD28 at a bead:T cell ratio of 0.88:1 (conditions 2-3 and 5-6) or 3:1 (conditions 8-9). Cytokines were added at the indicated times, consisting of either IFN-α alone (10,000 IU / mL) or IFN-α (10,000 IU / mL) in combination with IL-2 (20 IU / mL). Cytokines were added at the start of culture (conditions 8-9) or one day after the start of culture (conditions 2-7). Cultures 3-4 and 6-7 received additional medium two days after the start of culture to dilute them to the indicated final cell density.

[0282] Table 1: Culture conditions

[0283]

[0284] M / mL = millions of cells per milliliter; TEM = temsirolimus; RAPA = 1 μM sirolimus oral solution; XV = X-Vivo TexMACS is a proprietary medium formulation.

[0285] Condition 9 represents the T-Rapa product. It was found that condition 7 provided the best RAPA-T condition among the conditions tested in Table 1. This condition has several key attributes: (1) serum-free medium; (2) very high initial cell density (30 M / mL); (3) very high temsirolimus concentration (4.5 μM); (4) presence of a monoclonal antibody against the IL2 receptor; (5) absence of co-stimulation; (6) cytokine support with IFN-α alone (no IL-2) added one day after the start of culture; and (7) cell dilution on day 2 of culture.

[0286] After 6 days of culture, the resulting T cells were harvested and the specific molecular expression of the resulting T cells within the CD4+ (indicated by black bars) and CD8+ (indicated by grey bars) T cell subsets was evaluated by flow cytometry, as Figures 25A - 25O shown.

[0287] Figure 25A (CD45RA+) demonstrated the importance of the RAPA-T culture conditions for maintaining the expression of initial T cell markers on both CD4+ and CD8+ T cells relative to the input cells of the culture; in sharp contrast, the previous T-RAPA conditions led to a significant reduction in CD45RA+ cells.

[0288] Figure 25B(CD25+) demonstrated the importance of RAPA-T culture conditions for maintaining T cell quiescence in both CD4+ and CD8+ T cells relative to the cultured input cells; in sharp contrast, the previous T-RAPA condition led to a significantly activated T cell state, as indicated by increased CD25 expression.

[0289] Figure 25C (CD28+) and Figure 25D (ICOS+) showed that the RAPA-T and T-RAPA cell products had similar CD4+ and CD8+ T cell expression of these co-stimulatory activation molecules.

[0290] Figures 25E - 25F (CD39+ and CD73+ respectively) showed that the RAPA-T culture conditions led to reduced expression of these ecto-nucleotidase molecules relative to the T-RAPA condition. These molecules exert immunosuppressive effects by metabolizing ATP to adenosine. Therefore, in terms of therapeutic use, the RAPA-T cell product is expected to be advantageous relative to the T-RAPA cell product.

[0291] Figures 25G - 25O The remaining data in indicated that the new RAPA-T method led to a substantial reduction in the expression of molecules associated with immunosenescence (KLRG1), associated with the immunosuppressive regulatory T cell phenotype (GITR), or associated with checkpoint inhibitory functions (LAG3, PD1, 2B4, LAIR1, CTLA4, TIGIT, and TIM3). Each of these molecules in each of the variable culture conditions related to the RAPA-T cell product was substantially reduced relative to the T-RAPA cell product. Condition 7 demonstrated the most severe and consistent reduction of these molecules among the tested conditions.

[0292] Example 3

[0293] T cells were prepared as shown in Example 2 using culture conditions 1-8 corresponding to culture conditions 2-9 of Example 2. On day 2 of the culture interval, the resulting T cells were harvested and evaluated for molecules related to the mTORC1, mTORC2, and STAT pathways in the resulting T cells by Western blot analysis (method according to the manufacturer's instructions; BioTechne Mr.Wes instrumentation).

[0294] For optimal Th1 / Tc1 type production, it is important to limit the activation (phosphorylation) of STAT5, which can drive the regulatory T cell phenotype. As Figure 26As shown, each of the Rapa-T cell culture conditions (conditions 1-6) is relatively devoid of phosphorylated STAT5; in stark contrast, the T-Rapa conditions show a distinct presence of phosphorylated STAT5 (conditions 7-8). The reduction in STAT5 phosphorylation in Rapa-T cells can exceed 75% relative to T-Rapa cells.

[0295] For optimal Th1 / Tc1 type generation, it is important to have active signaling through specific STAT molecules (including STAT1) that drive type I differentiation. As Figure 26 shown, each of the Rapa-T culture conditions shows a detectable level of STAT1 phosphorylation, although the level is reduced in condition 6 (Example 2, condition 7). However, the total STAT1 level is also reduced in condition 6.

[0296] The optimal phenotype of Rapa-T cells can also be characterized by a reduction in molecules associated with the mTORC1 pathway. The Rapa-T condition 6 (corresponding to Example 2, condition 7) is substantially devoid of expression of the molecule p70S6K associated with mTORC1. Providing co-stimulation in other Rapa-T culture conditions (conditions 1, 2, 4, and 5) increased p70S6K expression. Thus, it may be beneficial to avoid co-stimulation during Rapa-T generation.

[0297] The optimal phenotype for Th1 / Tc1 type RAPA-T cell generation may also depend on the preservation of the mTORC2 signaling pathway. In this regard, it is beneficial that the optimal RAPA-T cell condition (condition 6, corresponding to Example 2, condition 7) has the preservation of the expression of the total SGK1 and phosphorylated SGK1 molecules associated with mTORC2. Condition 6 further exemplifies this property of the optimal RAPA-T cell product with a significant reduction in mTORC1 and relative preservation of mTORC2, namely, a significant reduction in the mTORC1-associated subunit molecule Raptor and relative preservation of the mTORC2-associated subunit molecule Rictor.

[0298] Example 4

[0299] Steady-state apheresis was performed to obtain a patient sample containing PBMC. Lymphocytes in the sample were enriched using an automated Ficoll process on an instrument from GE. The lymphocyte-enriched cell population was then plated in G-REX culture vessels under two conditions (one condition corresponding to condition 7 in Table 1 and one condition corresponding to condition 9 (T-RAPA) in Table 1) and incubated in culture for 6 days as in Example 2. After 6 days of culture, the T cells were harvested and at 1×10 and incubated in culture for 6 days as in Example 2. After 6 days of culture, the T cells were harvested and at 1×10 6Cells were re-plated at a concentration of cells / mL to generate 24-hour supernatants. At the time of re-plating, T cells were co-stimulated with anti-CD3 / anti-CD28-coated magnetic beads at bead:T cell ratios of 3:1, 1:1, 1:3, or 1:9. At each of these ratios, 24-hour supernatant generation was performed without addition of cytokines (indicated by a "-" symbol), with addition of rhuIL-2 (100 IU / mL, indicated by "+IL-2"), with addition of rhuIL-7 (10 ng / mL, indicated by "+IL-7"), with addition of rhuIL-15 (10 ng / mL, indicated by "+IL-15"), or with addition of both rhuIL-7 (10 ng / mL) and rhuIL-15 (10 ng / mL) simultaneously (indicated by "+IL-7+IL-15"). Secretion of IL-2 and TNF-α in the cells was measured by known methods according to the manufacturer's instructions (R&D Systems). The results are shown in Figures 27A - 27B in.

[0300] CD4+ and CD8+ T cells in an early differentiation state (such as naïve, central memory, or stem cell central memory subsets) can be beneficial for adoptive transfer. Such early-differentiated T cells are functionally characterized in part by their differential responsiveness to the key homeostatic cytokines, namely IL-7 and IL-15. Thus, the ability of a given T cell product to respond to IL-7 and IL-15 is a desirable property.

[0301] Figure 27A The IL-2 secretion profile of the optimal RAPA-T cell product is shown, while the lower figure shows the IL-2 secretion profile of the T-RAPA cell product. Under maximal co-stimulation challenge and without exogenous cytokine support, the RAPA-T cell product secretes approximately 5-fold higher amounts of IL-2 relative to the T-RAPA cell product. Notably, even at very low co-stimulation levels (1:9 bead:T cell ratio), the RAPA-T cell product still secretes substantial amounts of IL-2; in stark contrast, this stimulation condition under T-RAPA conditions yields undetectable levels of IL-2. The ability to secrete IL-2 has been associated with a beneficial non-helper-dependent T cell phenotype, which is a cytokine secretion property observed in T cells in the early stages of differentiation. Finally, addition of IL-7 or IL-15 further enhances the IL-2 secretion capacity under RAPA-T conditions but not under T-RAPA conditions. Thus, RAPA-T cells respond uniquely to the homeostatic cytokines IL-7 and IL-15.

[0302] Figure 27BThe TNF-α secretion profiles of the optimal RAPA-T cell products are shown, while the TNF-α secretion profiles of the previous T-RAPA cell products are shown in the figure below. Under maximal co-stimulation challenge and without the support of exogenous cytokines, the RAPA-T cell products secrete approximately equal amounts of TNF-α relative to the T-RAPA cell products. However, relative to the T-RAPA condition, the addition of IL-7 or IL-15 in co-stimulation results in a higher TNF-α secretion capacity under the RAPA-T condition. Thus, in terms of inducing the secretion of the Th1 / Tc1 effector cytokine TNF-α, RAPA-T cells respond uniquely to the homeostatic cytokines IL-7 and IL-15.

[0303] Example 5

[0304] Human T cells were co-stimulated without the use of any inhibitors ("conventional"; addition of anti-CD23 / anti-CD28 magnetic beads; magnetic bead:T cell ratio of 3:1). Alternatively, T cells were co-stimulated according to the RAPA-T cell condition ("rapamycin-treated"), and either by anti-CD3 / anti-CD28 magnetic beads ("Dynabeads"; magnetic bead:T cell ratio of 1:3) or by soluble co-stimulation microparticles (Biotech company; used at 20% of the manufacturer's recommended dose per 1 × 10 6 cells in 50 μL stock solution). After 6 days of culture, the T cells were harvested, adjusted to 1 × 10 6 cells / mL, and co-stimulated with anti-CD3 / anti-CD28 beads (bead:T cell ratio of 3:1). Then the 24-hour supernatant was collected and tested for the levels of IL-2, TNF-α, and IL-13 by Luminex assay (results are shown as pg per mL per 1 × 10 6 cells per 24 hours). Using the same protocol, the cells were harvested after 6 days of culture and evaluated by flow cytometry for the expression of cell surface markers (including CD4, CD8, CD25, and CTLA4).

[0305] Figure 28 The secretion data of IL-2, TNF-α, and IL-13 are shown. As Figure 28 shown, the results between RAPA-T cells co-stimulated by anti-CD3 / anti-CD28 nanoparticles ("Dynabeads") and soluble anti-CD3 / anti-CD28 microparticles ( reagent; Biotech company) are similar. As previously described, these cells have a Th1-type cytokine profile, as demonstrated by the abundant secretion of IL-2 and TNF-α and the minimal secretion of the Th2-type cytokine IL-13.

[0306] Figure 29 shows the frequencies of cells expressing the cell surface markers CD4, CD8, CD25, and CTLA4 as measured by flow cytometry. As Figure 29 shown, the results were similar among RAPA-T cells co-stimulated by anti-CD3 / anti-CD28 nanoparticles (“Dynabeads”) and soluble anti-CD3 / anti-CD28 microparticles ( reagent; Biotechnology Company). As previously described, these cells are quiescent (as indicated by reduced expression of CD25) and have reduced checkpoint inhibitory receptor expression (as indicated by reduced expression of CTLA4).

[0307] Example 6: Phase III Randomized Clinical Trial of Rapa-T Cell Therapy Figure 30 depicts the randomized Phase 3 protocol. In Figure 30 the upper panel, for patients randomly selected to receive Rapa-T therapy, autologous cells for Rapa-T cell manufacturing will be derived from steady-state apheresis blood components collected after randomization. The immunosuppression protocol will consist of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC protocol). The first PC cycle will be administered alone (without T cell therapy) at the start of the study during the T1.Rapa manufacturing interval and will have a duration of 28 days; cycles two, three, four, and five of PC will be performed prior to each of the four T1.Rapa cell infusions and will have a duration of 35 days. Figure 30 The lower panel in

[0308] Figure 30 details the manufactured T cell therapy that will be administered to all patients randomly assigned to the manufactured T cell cohort. This therapy will involve: (1) collection of immune cells as the manufacturing substrate for the manufactured T cells, which will be obtained from a previously harvested peripheral blood stem cell transplantation procedure or a fresh steady-state apheresis blood components procedure; (2) enrichment of the monocyte population and subsequent incubation of the monocytes under manufactured T cell culture conditions; (3) cryopreservation of a single-use manufactured T cell therapy product that has undergone identity and functionality verification steps; (4) patient treatment with the pentostatin plus cyclophosphamide drug regimen (PC protocol) will first be isolated and then combined with the manufactured T cell therapy to prepare the patient for the manufactured T cell treatment and directly facilitate the anti-tumor effect; and (5) specialized immune monitoring during and after treatment.

[0309] The immune depletion regimen will consist of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). The first PC cycle will be administered alone (without T cell therapy) at the start of the study during the manufactured T cell manufacturing interval and will last for at least 28 days to allow blood cell counts to recover; before each of the four manufactured T cell infusions, PC cycles two, three, four, and five will be conducted, and the duration will be at least 35 days to allow blood cell counts to recover. After completion of cycle 5 of the manufactured T cell therapy, maintenance therapy will no longer be conducted. Depending on the clinical situation, the treatment cycle can be longer than the specified interval, extended to an indefinite interval. By way of example and not limitation, if the patient is in remission, the cycle can be delayed until evidence of disease recurrence. In addition, additional maintenance cycles of the PC regimen plus adoptive manufactured T cell therapy are envisioned to keep the patient in remission, perhaps 1 to 4 treatment cycles per year, or to treat disease recurrence (if it occurs).

[0310] As Figure 30 indicated, for patients with stable disease after 4 cycles of treatment, additional Rapa-T cell manufacturing can be performed, thereby facilitating potential therapy through additional cycles (up to a total of 9 Rapa-T cell therapy cycles).

[0311] Figure 31 The details of the PC chemotherapy regimen are described in detail. Each cycle of PC therapy will consist of a 14-day course before the infusion of Tl.Rapa cells on day 15 of the cycle (Tl.Rapa dose: between 0.1 cells / kg and 5×10 6 cells / kg). For cycle 1, pentostatin (P) will be administered at a dose of 4 mg / m 2 (intravenously) on days 1, 4, 8, and 11; cyclophosphamide (Cy) will be administered at a dose of 200 mg per day from day 1 to day 5 and from day 8 to day 12. For subsequent cycles, the dose of pentostatin will be reduced to 2 mg / m 2 .

[0312] Figures 32A - 32C Details are provided regarding the nature of the control group, i.e., subjects not randomly assigned to receive Rapa-T cell therapy will receive one of three FDA-approved triple regimens applicable to subjects with second or third relapse of MM, namely: the DPd regimen ( Figure 32A ); the DRd regimen ( Figure 32B ); or the KRd regimen ( Figure 32C ).

[0313] For Figures 32A - 32CIn the control group depicted, patients will be recruited at the second or third relapse of multiple myeloma (MM) and subsequently randomized. Patients must be candidates for one of the three FDA-approved regimens used to treat this patient population. Patients randomized to the control group will receive the DPd, DRd, or KRd regimen using the published standard regimens. Specific aspects of these standard regimens have been indicated in the previous section above: Figure 32A The DPd regimen; Figure 32B The DRd regimen; and Figure 32C The KRd regimen.

[0314] The primary objective of the statistical assessment of the efficacy of the manufactured T cells will be to compare the progression-free survival of the recipients of the manufactured T cell therapy with that of the recipients randomly assigned to the standard therapy. In contrast, the secondary objectives will be initially evaluated using descriptive statistics. Eligible patients with second or third relapse of MM will be randomized in a 1:1 manner to receive the standard of care therapy of the FDA-approved triple therapy consisting of DPd, DRd, or KRd, or to receive adoptive T cell therapy with ex vivo manufactured autologous anti-rapamycin Th1 / Tc1 cells (manufactured T cells). N = 65 evaluable patients will be included in each group. The primary study objective is to determine whether the progression-free survival (PFS) of the patients treated in the manufactured T cell group is increased compared to the patients treated in the standard treatment group.

[0315] The progression-free survival (PFS) and overall survival of the patients will be estimated in both groups of patients by the Kaplan-Meier method and presented with pointwise 95% confidence intervals. By inverting the Kaplan-Meier estimates, non-parametric estimates of the median survival and its 95% confidence interval can be obtained. The primary efficacy outcome (PFS) will be tested by a one-sided log-rank test. The final analysis will be conducted when 130 PFS events have occurred in the study or the recruitment target has been reached and all patients have been followed up for at least 12 months. The overall survival (OS) will be measured from the start of treatment; death from any cause will be the event, and the patients will be examined at the date of the last contact.

[0316] The secondary endpoints will be evaluated in a descriptive manner (such as mean, standard deviation, confidence interval, Kaplan-Meier analysis, or other methods characterizing the remission status of multiple myeloma). The objective response rate (ORR) and minimal residual disease rate (MRD) will be estimated as the patient population observed with the corresponding outcomes and presented with 95% confidence intervals.

[0317] Demographic and baseline data will be described descriptively. Categorical data will be presented as frequencies and percentages, while continuous data will be presented using summary statistics (such as mean, median, and standard deviation). Particular attention will be paid to determining previous treatments for multiple myeloma and the degree of refractoriness to individual drugs used.

[0318] Two interim analyses that may be terminated for futility will be conducted when a total of 28 and 55 PFS events (combining the two groups) have occurred. A beta spending method with a quadratic spending function will be used, which is a compromise between the O'Brien-Fleming method and the Pocock boundary. The following table shows the null hypothesis acceptance boundaries for the two interim analyses and one final analysis. If the timing of the interim analysis is modified, the spending function can be used to modify these values.

[0319]

[0320]

[0321] The following table shows the probability of early stopping the trial due to futility as a function of the true effect size:

[0322]

[0323] Collect immune cells for use as substrates for the manufacture of T cells

[0324] For patients randomly assigned to the manufactured T cell therapy group, cells for the manufacture of T cells need to be collected and transported.

[0325] In addition, in the case where the subject has the necessary value of immune cells in the blood as defined by, for example, an absolute lymphocyte count (ALC; a value of at least 300 lymphocytes per microliter), steady-state apheresis will be performed and will consist of a collection of 10 to 15 liters. Apheresis should be performed within 10 days after the start of the study. The apheresis product will be transported immediately (without cryopreservation) to Rapa Therapeutics.

[0326] The first cycle of the PC regimen should start within 10 days after the start of the study.

[0327] Subsequent iterations of the manufactured T cell therapy may become more efficient, and so it may be possible to start the manufacture using a smaller amount of input cells. Such improved methods will involve, at least in part, improved subject preparation and improved manufacturing processes. In such methods, it may be possible to manufacture T cells using starting materials obtained from a single blood draw of 500 mL or less.

[0328] Manufacture of Manufactured T Cells

[0329] In the case of previously collected cell products, such cryopreserved cells will be stored in the vapor phase of liquid nitrogen until the cells are thawed and the manufactured T cells are produced. In the case of cells freshly isolated by apheresis or in the future by simple blood collection, the cells will be processed immediately and then can be placed directly into culture or can also be cryopreserved by controlled-rate freezing technology and stored in the vapor phase of liquid nitrogen for subsequent use.

[0330] T cell culture of cryopreserved cell substrates from freshly isolated cell populations requires a certain type of T cell enrichment. By way of example and not limitation, T cell enrichment can be achieved by using monoclonal antibodies and chromatography column techniques (positive or negative selection). Enrichment of the original cell material used in the manufacture of the manufactured T cells does not require such antibody-based methods, as the T cells are effectively enriched during the culture interval; thus, this method is consistent with the recommendations for effective cell therapy at the global level. The initial processing steps of the manufactured T cells focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (when applicable), the lysis of red blood cells (RBCs), and the centrifugal removal of contaminating granulocytes and to some extent monocytes. These steps are performed in a relatively automated method that primarily uses closed system technology. This procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves the consistency of the entire manufacturing process, and reduces the risk of contamination of the final product by infectious agents. The processing of the manufactured T cell product combines the following steps: (1) thawing the cryopreserved product (when applicable) using a solid-state non-aqueous-based method to reduce infectious agent contamination, as described by Triana E, Ortega S, Azqueta C, et al., “Thawing of cryopreserved hematopoietic progenitor cells from apheresis will be with using a new dry warming device”. Transfusion. 2013;53(1):85-90; (2) automatically washing the cell product using a LOVO permeable membrane device, as described by Mfarrej B, Bouchet G, Couquiaud J, et al., “Pre-clinical assessment of the Lovo device for dimethyl sulfoxide removal and cell concentration in thawed hematopoietic progenitor cell grafts”. Cytotherapy. 2017;19(12):1501-1508; (3) integrating the lysis of RBCs using ammonium chloride potassium (ACK) buffer during the LOVO washing step, as described by Brown WE, Hu JC, Athanasiou KA."Ammonium-Chloride-Potassium Lysing Buffer Treatment of Fully Differentiated Cells Increases Cell Purity and Resulting Neotissue Functional Properties". Described in Tissue engineering Part C, Methods. 2016;22(9):895-903; (4) The cell content was reduced in volume using the LOVO method, and the cells were subsequently plated into a closed system countercurrent centrifugal elutriation (CCE) device (Elutra; Terumo Corporation) as previously described in Stroncek DF, Fellowes V, Pham C, et al., "Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies". J Transl Med. 2014;12:241. (doi):10.1186 / sl2967-12014-10241-γ; and (5) The pre-programmed operation of the Elutra device was used to effectively remove granulocytes and monocytes by CCE.

[0331] After lymphocyte enrichment and media purification, the cells are seeded into a dedicated chamber with robust oxygen exchange capabilities (G-Rex vessel; Wilson Wolf) as described in Bajgain P, Mucharla R, Wilson J, et al., “Optimizing the production of suspension cells using the G-Rex “M” series.” Molecular Therapy Methods & Clinical Development. 2014;1:14015. In addition to having enhanced gas permeability characteristics, the G-Rex vessel is a closed system unit and has the additional advantage of automated closed system media volume reduction (GatheRex liquid handling pump). Lymphocyte-enriched cells are maintained in the G-Rex vessel for 6 days.

[0332] Several specific culture conditions are utilized to facilitate the generation of CD4 + and CD8 + T cell mixtures with engineered T cell functional attributes in the G-Rex vessel. These specific conditions include: (1) using enriched media (including but not limited to X-Vivo 20; Lonza; the media can also be further supplemented with 5% AB serum) further supplemented with 5% human serum, Zhang HD, Song ZL, Li WP. [In vitro cultivation of dendritic cells with serum-free medium], Zhongguo shiyan xue ye xue za zhi. 2006;14(5):985-989; Lonza); (2) incorporating a 16-hour interval between seeding the cells into the G-Rex prior to co-stimulation (at 1.5 × 10 6(3) During this initial hiatus, the cells are optimally rested by adding the monoclonal antibody basiliximab (which blocks the IL-2 receptor and thereby prevents autologous T cell activation by endogenously produced IL-2) and temsirolimus (a pharmacological inhibitor of mTORC1); (4) After this 16-hour hiatus, under suboptimal conditions, the cells are either not co-stimulated or co-stimulated with anti-CD3 / anti-CD28-coated magnetic beads (3 / 28 beads), as defined by a bead-to-T cell ratio of 1:3 (typically, most T cell expansion conditions utilize a 9-fold higher co-stimulation level, i.e., a bead-to-T cell ratio of 3:1); (5) Importantly, it is crucial not to wash the T cells after the initial hiatus; (6) After the hiatus, in addition to adding the 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4 + Th1 and CD8 + Tc1 phenotypes; (7) Importantly, it is crucial to avoid adding IL-2, a common additive for T cell culture; and (8) After adding the beads and IFN-α, it is important to leave the cells undisturbed until harvest on day 6 of culture (no cell washing, no further culture additives).

[0333] Cryopreservation of the manufactured T cell product and verification of identity and function

[0334] After 6 days of cell culture in a G-Rex vessel, the volume of the culture will be reduced in a closed system manner by the GatheRex instrument. Subsequently, the cells will be harvested, the 3 / 28 beads removed with a hand-held magnet, and the cells placed in a LOVO device for serial washing to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0335] The washed cells will be reconstituted into a cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation will be performed in 50 ml cryobags in multiple single-use aliquots. The manufactured T cell dose will range from 0.1 T cells per kg of recipient body weight to 5×10 6Among T cells. Four disposable aliquots will be cryopreserved to allow for the infusion of the manufactured T cells four times consecutively, approximately once a month. The manufactured T cells will be cryopreserved by a controlled-rate freezing method compliant with GMP, as previously described in Hunt CJ. "Cryopreservation of Human Stem Cells for Clinical Application: A Review". Transfusion medicine and hemotherapy: offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie. 2011; 38(2): 107-123, and; and after the manufactured T cells pass the designated release standard tests, the cells will be transported by a certified cryotransporter in the vapor phase of liquid nitrogen.

[0336] The release standard tests for the manufactured T cells include standard tests such as CD3 + , CD4 + and CD8 + T cell content purity (by flow cytometry, the CD3 + T cell content of the final product must be >70%; CD4 + and CD8 + subpopulations must be present at levels of 5% respectively). As determined by flow cytometry Annexin and 7-AAD assays, the cells must be >70% viable. In addition, over a minimum 3-day culture interval (ideally, a 14-day culture interval), the cells must be free from bacterial and fungal contamination; furthermore, the cell product must be below the detection limits for bacterial LPS endotoxin and mycoplasma.

[0337] In addition to these standard tests, specialized functional tests will also constitute the release criteria for the manufactured T cell product. Before releasing the product and the cell therapy, the manufactured T cells can have the following properties relative to the cultured input T cells: (1) an enhanced T central memory phenotype, as defined by increased flow cytometry co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitory molecules (such as programmed death 1 (PD1)); (3) a quiescent state, as defined by reduced Th1 / Tc1-type cytokine secretion levels upon maximal co-stimulation; (4) an autophagy signature, as demonstrated by reduced mitochondrial mass measured by flow cytometry MitoTracker, see Xiao B, Deng X, Zhou W, Tan EK. “Flow Cytometry-Based Assessment of Mitophagy Using MitoTracker”. *Frontiers in cellular neuroscience*. 2016;10:76; (5) a resistant phenotype, as demonstrated by at least 50% inhibition of downstream targets of mTORC1 and mTORC2; and (6) a multifaceted differential gene expression profile of n = 80 key transcription factors and differentiation molecules.

[0338] Subject preparation using the PC regimen

[0339] Patients receiving the manufactured T cell therapy receive the pentostatin plus cyclophosphamide (PC) regimen. Cycle 1 of the PC regimen will be administered during the manufacture of the manufactured T cells and can thus be administered without an accompanying T cell infusion. Cycle 1 is advantageous at two levels: first, it will reduce the number and function of the subject's regulatory T cells and late senescent effector T cells, thereby enhancing future cycles of the manufactured T cell therapy; and second, it will directly mediate anti-tumor effects against multiple myeloma, thereby controlling the disease during the manufacturing interval. After Cycle #1, subsequent cycles of the PC regimen will be followed by adoptive transfer of the manufactured T cells one day after a two-week PC regimen interval (Day 15). These cycles of the PC regimen are additionally advantageous because the cycles will further modulate the subject's biology, including increasing T cell homeostatic cytokines such as IL-7 and IL-15, which will allow for enhanced expansion of the manufactured T cells after adoptive transfer.

[0340] Manufactured T cell infusion follows the PC regimen. Each cycle of the PC therapy will consist of a 14-day course prior to the infusion of the manufactured T cells on Day 15 of the cycle. The dose of the manufactured T cells will be between 1 cell / kg and 5×10 6 cells / kg, including between 1×105 The T-cell dose between 1×10 6 cells / kg and 5×10 2 cells / kg. Pentostatin (P) will be administered at a dose of 4 mg / m

[0341] on days 1, 4, 8, and 11 (intravenously); cyclophosphamide (Cy) will be administered at a dose of 200 mg per day from day 1 to day 5 and from day 8 to day 12. 2 Before administering pentostatin, premedication and prehydration are required. Sixty minutes before administering pentostatin, 1 liter of 0.9% sodium chloride will be used for prehydration. Premedication with antiemetic drugs is required. The antiemetic drug regimen is recommended as follows: (1) Dexamethasone, 12 mg intravenously infused 60 minutes before each pentostatin administration (i.e., on days 1, 4, 8, and 11 of the cycle); (2) In addition, oral dexamethasone can be administered at a dose of 4 mg per day as needed on other days; (3) 8 mg of ondansetron will be intravenously infused 60 minutes before each pentostatin administration; (4) For the remaining treatment, from day 1 to day 14, ondansetron can be administered orally at a dose of 8 mg (tablets) every 12 hours as needed; and (5) For patients with uncontrolled nausea and vomiting, aprepitant can be added to the antiemetic drug regimen as needed. The pentostatin dose will be 4 mg / m

[0342] The pentostatin dose will be adjusted, and the pentostatin dose administered to the patient will be between 1 mg / m 2 - 4 mg / m 2 . The pentostatin dose will be adjusted according to the creatinine clearance rate (CrCl), which can be obtained through a 24-hour urine collection or calculated using the Cockcroft-Gault formula. If the creatinine level increases during pentostatin and cyclophosphamide therapy in the subject, the subsequent dose will be modified as follows: CrCl > 60 mL / min / 1.73 m 2 → administer pentostatin at a full dose of 4 mg / m 2Administer pentostatin; and CrCl < 30 mL → discontinue pentostatin administration. Pentostatin is rarely associated with organ toxicities such as neurotoxicity (seizures, coma) or cardiotoxicity (reduced ejection fraction). As such, particular attention should be paid to assessing any organ toxicities that occur during PC therapy. If pentostatin is associated with any organ toxicity of grade 2 or greater severity, the institutional PI should be contacted to discuss the need for further pentostatin treatment and further protocol treatment.

[0343] Oral cyclophosphamide (Cy) will be used as part of the PC regimen, where the dose of cyclophosphamide will be between 50 - 400 mg. During cycles 1 - 5 of the PC regimen, the dose of Cy will be 200 mg per day on days 1 - 5 and days 8 - 12. Intravenous infusion of 200 mg cyclophosphamide is also permitted due to tolerance issues or financial considerations. Due to cyclophosphamide bladder toxicity, adequate hydration must be maintained during the PC regimen. Patients should drink at least 2 to 4 liters of fluid per day to keep the urine clear in color.

[0344] The cyclophosphamide dose will be adjusted as needed based on complete blood count (CBC) and differential cell values (absolute lymphocyte count [ALC] and absolute neutrophil count [ANC]) obtained on days 1, 4, 8, and 11 of the cycle according to the table below. The established goal of the PC regimen is to achieve immune depletion and immunosuppression while minimizing myeloid cell suppression. To help ensure this goal is met, the cyclophosphamide dose will be adjusted as needed based on ALC and ANC values obtained on the day of pentostatin administration (i.e., days 1, 4, 8, and 11 of the cycle) according to the table below. The symbols in the table are as follows: 1 Pentostatin will not be dose - adjusted based on ALC / ANC values; 2 For ANC values < 500, in addition to reducing the cyclophosphamide dose, the patient will also receive G - CSF treatment until the next ANC measurement; 3 The indicated cyclophosphamide dose will continue daily until the next ALC / ANC measurement (performed on days 1, 4, 8, and 11 of the cycle).

[0345] Variations of the PC regimen are envisioned. First, pentostatin and cyclophosphamide synergize in their immunosuppressive and immunodepleting effects; this synergy may also exist in terms of antitumor effects, although little information is available regarding this possibility. Thus, it is envisioned that the PC regimen can be used as a stand-alone therapy for cancer treatment (including solid tumors); in a previous instance, refractory mesothelioma patients receiving a combination regimen partially composed of the PC regimen had unprecedented antitumor benefits. Second, due to this synergy, it is recommended that co-administration of the two drugs by intravenous infusion be advantageous, preferably by mixing pentostatin and cyclophosphamide into the same intravenous infusion bag for ease of administration and to reduce pharmacy errors. In such an application, it is important to provide a selection of PC mixtures that should cover a variety of clinically relevant pentostatin to cyclophosphamide ratios.

[0346]

[0347] Manufactured T cell infusion

[0348] Premedication is required prior to all manufactured T cell administrations. Prior to the manufactured T cell infusion, diphenhydramine (25 mg to 50 mg intravenous or orally) and acetaminophen (650 mg, orally) are administered 30 - 60 minutes prior.

[0349] The manufactured T cell infusion will occur on day 15 of cycles two through five; however, for logical reasons, the manufactured T cell infusion may be delayed by up to 3 days at most. Additionally, for logical reasons, a delay of up to 4 weeks is allowed, as well as to allow for toxicity recovery. The manufactured T cell dose will be 5×10 6 cells / kg; however, if suboptimal cell yields occur during manufacturing, a dose as low as 0.1×10 6 cells / kg is allowed. The cryopreserved manufactured T cells are thawed and immediately and rapidly (within 30 minutes) administered intravenously by gravity, in accordance with appropriate institutional SOPs for blood product management. This T cell infusion will be performed on an outpatient basis unless unforeseen circumstances require inpatient administration. Steroids are not allowed in the management of DMSO-related toxicities (chills, muscle aches), which may occur immediately after the cell infusion, unless the toxicity is considered life-threatening.

[0350] It is conceivable that even smaller amounts of manufactured T cell infusions, less than 0.1×10 6 cells / kg, may also be clinically relevant (by way of example and not limitation, one log lower, at 1×10 5cells / kg). First, manufacturing will be optimized to generate manufactured T cells that mediate further enhanced in vivo effects, thereby reducing the required T cell dose; this will be advantageous, in part because T cell collection can occur by simple blood draw, and in part because of improved manufacturing feasibility. Second, as further improvements are made to the PC regimen, the manufactured T cells for adoptive transfer will have further improved in vivo selective advantage relative to host cells, thereby effectively reducing the required dose of manufactured T cells.

[0351] Specialized immune monitoring during and after therapy

[0352] Peripheral blood mononuclear cells and bone marrow cells will be sent to the Rapa therapeutic agent so that immune monitoring tests can be performed; the purpose of these tests is to explore the mechanism of action of the manufactured T cell therapy and to develop biomarkers that will predict the efficacy of the manufactured T cells. In one effort, the ability of the manufactured T cell receptor to produce various Th1-type and Th2-type cytokines in response to various stimuli will be evaluated, the various stimuli including autologous multiple myeloma tumor cells or known or suspected tumor antigens, such as molecules in the cancer-testis antigen (CTA) family. The CTA gene family is numerous and has been shown to be associated with multiple myeloma; since the sequences of the CTA genes are known and the relevance of specific CTA genes has been characterized in multiple myeloma, it can be demonstrated that the manufactured T cell therapy can specifically induce T cell-mediated immunity against a range of CTA antigens. Measurement of such cytokine responses can be performed using RNA expression analysis, secretion analysis by ELISA or Luminex multiplex assay, flow cytometry or ELISPOT assay. Antigen-specific immunity can also be quantified using antibody production assays or cytolysis assays.

[0353] It will be evaluated whether T cells obtained after manufacturing a T cell therapy have enhanced reactivity against autologous multiple myeloma cells relative to T cells obtained before the manufactured T cell therapy. One obstacle to this endeavor is that the propagation of patient-specific multiple myeloma cell lines is generally unsuccessful. To overcome this obstacle, the following will be used to propagate myeloma cells: specialized containers, such as those described in Zhang W, Gu Y, Sun Q, et al., "Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche". PLoS One. 2015;10(5), and a culture medium supplemented with a combination of factors known to enhance multiple myeloma proliferation and survival, including IL-6, CD40 ligand, and acquisition of resistance to carfilzomib. Patient-specific multiple myeloma cells can be used alone as stimulants in the assessment of the anti-tumor reactivity of immune T cells; alternatively, such tumor cells can be brought into an apoptotic state and the contents loaded into professional antigen-presenting cells, which can be manufactured from patient-specific monocytes collected from the elutriation process during the manufacture of the manufactured T cells.

[0354] In addition, it is envisioned that T cell receptor (TCR) repertoire analysis will be useful as a biomarker for manufactured T cell therapies. Preferably, such repertoire analysis will be performed by RNA sequencing rather than the more commonly used DNA sequencing. Unlike most targeted T cell therapies, manufactured T cell therapies are polyclonal approaches because the manufacturing process does not preferentially transfer T cell reactivity to any particular tumor antigen. As such, any beneficial anti-tumor effects following manufactured T cell therapies are expected to result from in vivo clonal expansion to multiple tumor antigens; given this biology, successful manufactured T cell therapies will result in different TCR repertoires when comparing the pre-treatment and post-treatment repertoires of a patient. In other cancer therapy settings, such as monoclonal antibody therapies that relieve checkpoint inhibition, successful therapies are associated with the emergence of new TCR clone specificities, referred to as an offset of the TCR repertoire that can be determined by quantifying the Morisito index, Robert L, Harview C, Emerson R, et al., “Distinct immunological mechanisms of CTLA-4 and PD-1 blockade revealed by analyzing TCR usage in blood lymphocytes”. OncoImmunology. 2014;3:e29244. In a similar manner, with successful manufactured T cell therapies, there will be an offset of the TCR repertoire; the persistence of the TCR offset beyond the manufactured T cell therapy interval will be consistent with long-term immunity of T cells to malignancy. With manufacturing advancements, improved forms of manufactured T cells will be produced; in such cases, the TCR offset will be more extensive, will occur with fewer treatment cycles, and will be more persistent in the post-treatment interval.

[0355] Protocol Inclusion Criteria for Multiple Myeloma Therapy

[0356] Male or female patients aged > 18 years may be eligible for manufactured T cell therapy. There is no formal upper age limit. However, patients over 65 years of age with a history of cardiovascular conditions or symptoms (even if not fully meeting the exclusion criteria detailed below) should be evaluated by a cardiologist at a multi-center site. Such subjects will then be considered on a case-by-case basis. The overall patient performance status should be at least fair good health, as quantified by an ECOG performance status < 2.

[0357] The patient must be definitively diagnosed with multiple myeloma by histological or cytological studies. In addition, the disease must be symptomatic, and the patient must be in the second or third relapse of the disease after receiving a medicament from the following classes: proteasome inhibitors, immunomodulatory drugs, alkylating agents, CD38 monoclonal antibodies, and glucocorticoids.

[0358] Patients in the second or third relapse of the disease are in a relatively advanced stage of the disease. However, by demonstrating the safety and efficacy of the manufactured T cell therapy, it is conceivable that earlier multiple myeloma patients in the overall framework of the therapy will benefit from the manufactured T cell therapy. By way of example and not limitation, the manufactured T cell therapy can be used as an alternative to high-dose chemotherapy in combination with autologous hematopoietic cell transplantation and can also be used in a large number of patients who are not suitable for transplantation. In addition, it is conceivable to perform the manufactured T cell therapy during the earliest stage of multiple myeloma progression before clinical symptoms, i.e., during early detection in the smoldering disease stage.

[0359] On the other hand, it is conceivable that the Rapa-T cell therapy described herein will be applicable to the treatment of patients in more advanced stages of multiple myeloma and patients with highly refractory diseases. Specifically, the Rapa-T cell therapy can be used to treat penta-refractory MM, which is defined as a patient with relapsed MM who is refractory to five of the previous multiple drugs used to treat MM, namely: lenalidomide, pomalidomide, bortezomib, carfilzomib, and daratumumab.

[0360] Since there is no standard therapy option for penta-refractory MM patients, the clinical protocol for evaluating the Rapa-T cell therapy in this case will be a single-arm phase II study, similar to the studies performed previously in Chen C, Siegel D, Gutierrez M, et al., “Safety and efficacy of selinexor in relapsed or refractory multiple myeloma and Waldenstrom macroglobulinemia”. Blood. 2018;131(8):855 - 863 for evaluating novel anticancer drugs. For this phase II study, the Rapa-T cell therapy will be performed as Figure 30 、 31 and as described in 32A - 32C. The statistical objective of the study will be to determine whether the Rapa-T cell therapy can induce a significant at least partial remission rate of refractory MM, as defined by a rate of at least 30%.

[0361] There must be a source of autologous T cells that has the potential to be sufficient to produce the manufactured T cells. Specifically, the patient must have a sufficient number of previously cryopreserved PBSC units available for manufacturing (defined by a CD34 + total content of > 2 million cells / kg) or a sufficient number of circulating T cells that can be collected by steady-state apheresis (defined by an ALC of greater than 300 cells per microliter).

[0362] The patient must be at least two weeks from myeloma therapy, major surgery, radiotherapy, and participation in other investigational trials and have recovered from the clinically significant toxicities of these previous treatments (CTCAE toxicity graded 2 or lower). The cardiac ejection fraction (EF) determined by MUGA or 2D echocardiogram must be within institutional normal limits and the EF level must be at least 40%. Renal function as measured by serum creatinine must be less than or equal to 2.5 mg / dl. Liver function must be adequate as measured by AST and ALT less than or equal to 3 times the upper limit of normal and total bilirubin less than or equal to 1.5 (unless due to Gilbert's disease). Pulmonary function must be adequate as defined by a corrected DLCO greater than or equal to 50% of that expected in a pulmonary function test. There must be no history of abnormal bleeding tendency. Voluntary written consent must be obtained before any study-related procedures that are not part of standard medical care, and it should be understood that the patient may withdraw consent at any time without affecting future medical care.

[0363] Randomized Phase III Trial: Standard of Care Therapy

[0364] To demonstrate the benefits of the manufactured T cell therapy, a randomized study will be conducted to formally compare the outcomes of the manufactured T cell therapy with the standard of care therapy, which consists of the DPd, DRd, or KRd regimens; the regimens will be administered as per their FDA-approved status for MM patients in second or third relapse, as specified in the literature.

[0365] Example 7

[0366] Steady-state apheresis is performed to obtain a patient sample containing PBMCs. The lymphocytes in the sample are passed through a Enrichment is carried out using an automated Ficoll process on the instrument. The lymphocyte-enriched cell population is then plated in G-REX culture vessels and cultured for 6 days in TexMACS medium (serum-free supplement; IL-2-free supplement) and containing IFN-α, temsirolimus, and basiliximab. At the end of manufacturing, the resulting Th1 / Tc1 cells are exposed to pancreatic cancer cells (MIA-Paca2 cell line) or to lung cancer cells (H23 cell line) that have undergone apoptosis by exposure to etoposide. Pulsed with this tumor lysate and then cultured for 7 days in IL-2 (200 IU / mL), a second exposure to the tumor lysate is carried out at the 7th day of said culture. After an additional 7-day culture interval in medium containing IL-2, a third exposure to the tumor lysate is carried out, and the cytokine content of the resulting 24-hour supernatant is tested by Luminex assay (results are shown as pg / mL / 1×10 6 cells / 24 hours). The results of the cytokine assay are shown in Figure 34A -B. Condition A indicates pulsing with a suboptimal tumor lysate formulation; condition B represents the optimal formulation of the tumor lysate. "<" indicates a value below the detection limit.

[0367] As Figure 34A -B shows, RAPA-T cells can be further characterized by their ability to respond to tumor cells containing solid tumors such as pancreatic cancer cells and lung cancer cells. As Figure 34A -B further shows, RAPA-T cells can maintain a characteristic Th1 cytokine phenotype, as demonstrated by high-level secretion of IFN-γ and GM-CSF and reduced secretion of the Th2 cytokines IL-4 and IL-10.

[0368] In another experiment, at the end of manufacturing, the resulting Th1 / Tc1 cells are exposed to pancreatic cancer cells (MIA-Paca2 cell line) or to lung cancer cells (H23 cell line) that have undergone apoptosis by exposure to etoposide. Pulsed with this tumor lysate and then cultured for 7 days in IL-7 (20 ng / mL) and IL-15 (10 ng / mL), a second exposure to the tumor lysate is carried out at the 7th day of said culture. After an additional 7-day culture interval in medium containing IL-7 and IL-15, a third exposure to the tumor lysate is carried out, and the cytokine content of the resulting 24-hour supernatant is tested by Luminex assay (results are shown as pg / mL / 1×10 6cells / 24 hours). Control cultures (“RAPA-201, no tumor”) consisted of engineered resistant Th1 / Tc1 cells propagated in medium containing IL-7 and IL-15 but not pulsed with tumor lysate. The results of the cytokine assays are shown in Figure 35 as shown.

[0369] As Figure 35 shown, RAPA-T cells can be further characterized by their ability to respond to tumor cells including solid tumors such as pancreatic cancer cells and lung cancer cells. This in vitro sensitization to solid tumor cell lines can be readily demonstrated by culturing and expanding in medium supplemented with IL-7 and IL-15, two homeostatic cytokines that have been shown to selectively drive effector functions of RAPA-T cells. RAPA-T cell cytokine secretion against tumor cells can maintain a characteristic Th1 cytokine phenotype as demonstrated by high levels of secretion of IFN-γ, GM-CSF, and TNF-α.

[0370] As Figure 36 shown, many cancers such as renal cell carcinoma, liver cancer, lung cancer, bladder cancer, and gastric cancer have been shown to respond to checkpoint inhibitor therapy such as monoclonal therapies against checkpoint inhibitor molecules such as PD-1 and CTLA4 that can induce solid tumor remission. In further experiments, engineered T cell therapy will be performed on patients (n = 7) with renal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, and low-mutated PDL1-negative or low-mutation rate cancers according to Simon's two-stage design. If any cohort has at least one responsive patient, the cohort will be expanded to a cohort of 20 patients.

[0371] Without being bound by theory, it is expected that Rapa-T cells can provide therapeutic benefits in cancer because the cells have reduced or no checkpoint inhibitor receptors. It is suspected that some cancers may be unresponsive to certain therapies due to checkpoint inhibitor receptors other than PD1 and CTLA4. Thus, it can be expected, without being bound by theory, that Rapa-T cells may be effective in treating other cancers due to the lack of additional checkpoint inhibitor receptors.

[0372] Example 8

[0373] Rapa-T cells were manufactured by culturing for 6 days, and the post-Ficoll cell population was cultured in a medium containing temsirolimus (2 μM) and the anti-IL-2 receptor monoclonal antibody basiliximab (30 μg / mL). After 24 hours, IFN-α (20,000 IU / mL) was added to the culture, and the culture was not supplemented with IL-2. No form of anti-CD3 / anti-CD28 co-stimulation was used in the culture.

[0374] In contrast, for the "control": the post-Ficoll cell population was cultured in a medium without temsirolimus and without basiliximab. On the day of the start of the culture, anti-CD3 / anti-CD28 coated beads were used to co-stimulate the cells at a bead to T cell ratio of 3:1. IL-2 (20 IU / mL) and IFN-α (20,000 IU / mL) were added to the medium on the day of the start of the culture. The mean fluorescence intensity (MFI) of BTLA, CTLA4, PD1, and TIM3 was measured by flow cytometry for the CD4+ and CD8+ T cell subsets of the culture input population, Rapa-T cells, and control cells. The data are shown in Table 2 below. A decrease in checkpoint inhibitor receptor expression was observed between Rapa-T cells and control cells, and for each checkpoint, the MFI was approximately the same for Rapa-T cells and the culture input cells.

[0375] Table 2: Mean Fluorescence Intensity

[0376]

[0377] Manufacturing Example:

[0378] 1. A method for generating manufactured T cells, the method comprising:

[0379] Inoculating a culture input cell population comprising T cells from a subject at a certain cell density in a medium comprising temsirolimus and an IL-2 signaling inhibitor;

[0380] Adding IFN-α to the medium;

[0381] Incubating the T cells and the medium for a certain period of time to generate manufactured T cells;

[0382] Harvesting the manufactured T cells.

[0383] 2. The method according to Example 1, further comprising:

[0384] Adding additional medium to the T cells and the medium.

[0385] 3. The method according to embodiment 2, wherein the additional medium is added about 48 hours after inoculating the culture input cell population into the medium.

[0386] 4. The method according to any one of embodiments 2 to 3, wherein the amount of the additional medium added to the culture is sufficient to reduce the cell density of the cells in the culture to a target cell density, wherein the cell density of the culture input cell population at the time of inoculation is greater than 9×10 6 cells / mL, and wherein the target cell density is about 9×10 6 cells / mL.

[0387] 5. The method according to embodiment 1, wherein no anti-CD3 / anti-CD28 co-stimulation is performed.

[0388] 6. The method according to embodiment 2, wherein when the culture input cell population is inoculated into the medium, the ratio of the amount of the additional medium added to the culture to the amount of the medium is between 1:1 and 3:1.

[0389] 7. The method according to any one of embodiments 1 to 6, further comprising, after harvesting the manufactured T cells:

[0390] packaging at least a portion of the manufactured T cells in a package; and

[0391] freezing the package containing the portion of the manufactured T cells.

[0392] 8. The method according to any one of embodiments 1 to 7, wherein the medium does not contain IL-2 and no IL-2 is added to the medium.

[0393] 9. The method according to any one of embodiments 1 to 8, wherein the IFN-α is added to the medium at about the same time as inoculating the culture input cell population or within 24 hours of inoculating the culture input cell population.

[0394] 10. The method according to any one of embodiments 1 to 9, wherein the cell density is at least 1.5×10 6 cells per mL.

[0395] 11. The method according to any one of embodiments 1 to 9, wherein the cell density is about 7.5×10 6 cells per mL.

[0396] 12. The method according to any one of embodiments 1 to 9, wherein the cell density is about 30×10 6 cells per mL.

[0397] 13. The method according to any one of embodiments 1 to 12, wherein the temsirolimus is present in the culture medium at a concentration of about 4.5 μM.

[0398] 14. The method according to any one of embodiments 1 to 12, wherein the temsirolimus is added to the culture medium one or more times during the time period to maintain a desired concentration.

[0399] 15. The method according to embodiment 14, wherein the temsirolimus is added to the culture medium every 2 days during the time period.

[0400] 16. The method according to any one of embodiments 14 to 15, wherein the desired concentration is about 4.5 μM.

[0401] 17. The method according to any one of embodiments 1 to 16, wherein the IL-2 signal transduction inhibitor is an anti-IL-2 receptor antibody or a fragment thereof.

[0402] 18. The method according to embodiment 17, wherein the IL-2 signal transduction inhibitor is basiliximab or daclizumab.

[0403] 19. The method according to any one of embodiments 1 to 18, wherein the IL-2 signal transduction inhibitor is present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL.

[0404] 20. The method according to any one of embodiments 1 to 19, wherein the IFN-α is added to the culture medium to a concentration of 1,000 IU / mL to 10,000 IU / mL.

[0405] 21. The method according to any one of embodiments 1 to 20, wherein the time period is about 4 days to about 8 days.

[0406] 22. The method according to any one of embodiments 1 to 20, wherein the time period is 6 days.

[0407] 23. The method according to any one of embodiments 1 to 22, wherein the culture medium is substantially serum-free.

[0408] 24. The method according to any one of embodiments 1 to 23, wherein serum is not added to the culture medium.

[0409] 25. The method according to any one of embodiments 1 to 22, wherein the culture medium further comprises 5% human serum.

[0410] 26. The method according to any one of embodiments 1 to 25, wherein the culture medium comprises TexMACS medium.

[0411] 27. The method according to any one of embodiments 1 to 26, wherein the cultured input cell population comprises T cells that account for no more than 66% of the total number of cells in the cultured input cell population.

[0412] 28. The method according to any one of embodiments 1 to 26, wherein the cultured input cell population comprises T cells that account for about 50% to about 95% of the total number of cells in the cultured input cell population.

[0413] 29. The method according to any one of embodiments 1 to 28, wherein the cultured input cell population further comprises monocytes.

[0414] 30. The method according to any one of embodiments 1 to 29, further comprising:

[0415] harvesting a sample comprising T cells from the subject; and

[0416] isolating T cells from the sample to produce the cultured input cell population.

[0417] 31. The method according to embodiment 30, wherein the cultured input cell population comprises T cells that account for about 99% or more of the total number of cells in the cultured input cell population.

[0418] 32. The method according to any one of embodiments 30 to 31, wherein the T cells are isolated by antibody-based purification.

[0419] 33. The method according to any one of embodiments 1 to 29, further comprising:

[0420] harvesting a sample comprising T cells from the subject; and

[0421] enriching T cells in the sample to produce the cultured input cell population.

[0422] 34. The method according to embodiment 33, wherein the enrichment is performed by countercurrent centrifugal elutriation or the Ficoll process.

[0423] 35. The method according to any one of embodiments 33 to 34, wherein the cultured input cell population comprises T cells that account for about 70% of the total number of cells in the cultured input cell population.

[0424] 36. The method according to any one of embodiments 1 to 29, further comprising, before inoculating the culture input cell population comprising T cells from the subject at a certain cell density in a culture medium:

[0425] Harvesting the culture input cell population from the subject.

[0426] 37. A manufactured T cell produced by the method according to any one of embodiments 1 to 36.

[0427] 38. A method for producing a manufactured T cell, the method comprising:

[0428] Inoculating a culture input cell population comprising T cells from a subject at a certain cell density in a culture medium comprising temsirolimus and an IL-2 signaling inhibitor;

[0429] Incubating the culture input cell population with the culture medium for a first period of time without co-stimulating the culture input cell population with anti-CD3 / anti-CD28;

[0430] After incubation for the first period of time, adding anti-CD3 /

[0431] Anti-CD28-coated magnetic beads at a bead:T cell ratio between 1:1 and 1:12 to the T cells and the culture medium to stimulate the T cells;

[0432] Adding IFN-α to the culture medium;

[0433] Incubating the culture input cell population in the culture medium containing the anti-CD3 / anti-CD28-coated magnetic beads and IFN-α for a second period of time to produce a manufactured T cell;

[0434] Isolating the anti-CD3 / anti-CD28-coated magnetic beads from the manufactured T cells; and

[0435] Harvesting the manufactured T cells.

[0436] 39. The method according to embodiment 38, further comprising, after harvesting the manufactured T cells:

[0437] Packaging at least a portion of the manufactured T cells in a package; and

[0438] Freezing the package containing the portion of the manufactured T cells.

[0439] 40. The method according to any one of embodiments 38 to 39, wherein the culture medium does not contain IL-2 and no IL-2 is added to the culture medium.

[0440] 41. The method according to any one of embodiments 38 to 40, wherein the IFN-α is added at or about the same time as the anti-CD3 / anti-CD28 coated magnetic beads are added.

[0441] 42. The method according to any one of embodiments 38 to 41, wherein the cell density is at least 1.5×10 6 cells per mL.

[0442] 43. The method according to any one of embodiments 38 to 41, wherein the cell density is about 7.5×10 6 cells per mL.

[0443] 44. The method according to any one of embodiments 38 to 41, wherein the cell density is about 30×10 6 cells per mL.

[0444] 45. The method according to any one of embodiments 38 to 44, wherein the temsirolimus is present in the culture medium at a concentration of 1 μM.

[0445] 46. The method according to any one of embodiments 38 to 44, wherein the temsirolimus is added to the culture medium one or more times during the second time period to maintain a desired concentration.

[0446] 47. The method according to embodiment 46, wherein the temsirolimus is added to the culture medium every 2 days during the second time period.

[0447] 48. The method according to any one of embodiments 46 to 47, wherein the desired concentration is 1 μM.

[0448] 49. The method according to any one of embodiments 38 to 48, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof.

[0449] 50. The method according to embodiment 49, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab.

[0450] 51. The method according to any one of embodiments 38 to 50, wherein the IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL.

[0451] 52. The method according to any one of embodiments 38 to 51, wherein the first time period is about 8 hours to about 24 hours.

[0452] 53. The method according to any one of embodiments 38 to 51, wherein the first time period is 16 hours.

[0453] 54. The method according to any one of embodiments 38 to 53, wherein the bead:T cell ratio is 1:3.

[0454] 55. The method according to any one of embodiments 38 to 54, wherein the IFN-α is added to the culture medium at a concentration of 1,000 IU / mL to 10,000 IU / mL.

[0455] 56. The method according to any one of embodiments 38 to 55, wherein the second time period is from about 4 days to about 8 days.

[0456] 57. The method according to any one of embodiments 38 to 55, wherein the second time period is 6 days.

[0457] 58. The method according to any one of embodiments 38 to 57, wherein the culture medium further comprises 5% human serum.

[0458] 59. The method according to any one of embodiments 38 to 58, wherein the culture medium comprises TexMACS medium.

[0459] 60. The method according to any one of embodiments 38 to 59, wherein the culture input cell population comprises T cells accounting for no more than 66% of the total number of cells in the culture input cell population.

[0460] 61. The method according to any one of embodiments 38 to 60, wherein the culture input cell population comprises T cells accounting for about 50% to about 95% of the total number of cells in the culture input cell population.

[0461] 62. The method according to any one of embodiments 38 to 61, wherein the culture input cell population further comprises monocytes.

[0462] 63. The method according to any one of embodiments 38 to 62, further comprising:

[0463] harvesting a sample comprising T cells from the subject; and

[0464] isolating T cells from the sample to produce the culture input cell population.

[0465] 64. The method according to embodiment 63, wherein the culture input cell population comprises T cells accounting for about 99% or more of the total number of cells in the culture input cell population.

[0466] 65. The method according to any one of embodiments 63 to 64, wherein the T cells are isolated by antibody-based purification.

[0467] 66. The method according to any one of embodiments 38 to 62, further comprising:

[0468] harvesting a sample comprising T cells from the subject; and

[0469] enriching T cells in the sample to produce the culture input cell population.

[0470] 67. The method according to embodiment 66, wherein the enrichment is performed by counterflow centrifugal elutriation or the Ficoll process.

[0471] 68. The method according to any one of embodiments 66 to 67, wherein the culture input cell population comprises T cells accounting for about 70% of the total number of cells in the culture input cell population.

[0472] 69. The method according to any one of embodiments 38 to 62, further comprising, before inoculating the culture input cell population comprising T cells from the subject at a certain cell density in a culture medium:

[0473] harvesting the culture input cell population from the subject.

[0474] 70. A manufactured T cell produced by the method according to any one of embodiments 38 to 69.

[0475] 71. A method for producing a manufactured T cell, the method comprising:

[0476] inoculating a culture input cell population comprising T cells from a subject at a certain cell density in a culture medium comprising temsirolimus and an IL-2 signaling inhibitor;

[0477] incubating the culture input cell population with the culture medium for a first period of time without co-stimulating the culture input cell population with anti-CD3 / anti-CD28;

[0478] after incubation for the first period of time, adding nanoparticles containing anti-CD3 / anti-CD28 to the T cells and the culture medium at a dose of about 0.01-fold to about 0.1-fold of the recommended dose to stimulate the T cells;

[0479] adding IFN-α to the culture medium;

[0480] incubating the culture input cell population in the culture medium containing the nanoparticles containing anti-CD3 / anti-CD28 and IFN-α for a second period of time to produce a manufactured T cell;

[0481] harvesting the manufactured T cell.

[0482] 72. The method according to embodiment 71, further comprising, after harvesting the manufactured T cells:

[0483] Packaging at least a portion of the manufactured T cells in a package; and

[0484] Freezing the package containing the portion of the manufactured T cells.

[0485] 73. The method according to any one of embodiments 71 to 72, wherein the culture medium does not contain IL-2 and no IL-2 is added to the culture medium.

[0486] 74. The method according to any one of embodiments 71 to 73, wherein the IFN-α is added at or about the same time as adding the nanoparticles containing anti-CD3 / anti-CD28.

[0487] 75. The method according to any one of embodiments 71 to 74, wherein the cell density is at least 1.5×10 6 cells per mL.

[0488] 76. The method according to any one of embodiments 71 to 74, wherein the cell density is about 7.5×10 6 cells per mL.

[0489] 77. The method according to any one of embodiments 71 to 74, wherein the cell density is about 30×10 6 cells per mL.

[0490] 78. The method according to any one of embodiments 71 to 77, wherein the temsirolimus is present in the culture medium at a concentration of 1 μM.

[0491] 79. The method according to any one of embodiments 71 to 78, wherein the temsirolimus is added to the culture medium one or more times during the second time period to maintain a desired concentration.

[0492] 80. The method according to embodiment 79, wherein the temsirolimus is added to the culture medium every 2 days during the second time period.

[0493] 81. The method according to any one of embodiments 71 to 80, wherein the desired concentration is 1 μM.

[0494] 82. The method according to any one of embodiments 71 to 81, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof.

[0495] 83. The method according to embodiment 82, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab.

[0496] 84. The method according to any one of embodiments 71 to 83, wherein the IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL.

[0497] 85. The method according to any one of embodiments 71 to 84, wherein the first time period is from about 8 hours to about 24 hours.

[0498] 86. The method according to any one of embodiments 71 to 84, wherein the first time period is 16 hours.

[0499] 87. The method according to any one of embodiments 71 to 86, wherein the IFN-α is added to the culture medium to a concentration of 1,000 IU / mL to 10,000 IU / mL.

[0500] 88. The method according to any one of embodiments 71 to 87, wherein the second time period is from about 4 days to about 8 days.

[0501] 89. The method according to any one of embodiments 71 to 87, wherein the second time period is 6 days.

[0502] 90. The method according to any one of embodiments 71 to 89, wherein the culture medium further comprises 5% human serum.

[0503] 91. The method according to any one of embodiments 71 to 90, wherein the culture medium comprises TexMACS medium.

[0504] 92. The method according to any one of embodiments 71 to 91, wherein the culture input cell population comprises T cells accounting for no more than 66% of the total number of cells in the culture input cell population.

[0505] 93. The method according to any one of embodiments 71 to 91, wherein the culture input cell population comprises T cells accounting for about 50% to about 95% of the total number of cells in the culture input cell population.

[0506] 94. The method according to any one of embodiments 71 to 93, wherein the culture input cell population further comprises monocytes.

[0507] 95. The method according to any one of embodiments 71 to 94, further comprising:

[0508] harvesting a sample comprising T cells from the subject; and

[0509] T cells are isolated from the sample to generate the culture input cell population.

[0510] 96. The method according to embodiment 95, wherein the culture input cell population comprises about 99% or more T cells of the total number of cells in the culture input cell population.

[0511] 97. The method according to any one of embodiments 95 to 96, wherein the T cells are isolated by antibody-based purification.

[0512] 98. The method according to any one of embodiments 71 to 94, further comprising:

[0513] harvesting a sample comprising T cells from the subject; and

[0514] enriching T cells in the sample to generate the culture input cell population.

[0515] 99. The method according to embodiment 98, wherein the enrichment is carried out by countercurrent centrifugal elutriation or Ficoll process.

[0516] 100. The method according to any one of embodiments 98 to 99, wherein the culture input cell population comprises about 70% T cells of the total number of cells in the culture input cell population.

[0517] 101. The method according to any one of embodiments 71 to 94, further comprising, before inoculating T cells from the subject at a certain cell density in a culture medium:

[0518] harvesting the culture input cell population from the subject.

[0519] 102. A manufactured T cell produced by the method according to any one of embodiments 71 to 101.

[0520] 103. A manufactured T cell population that exhibits a reduced level of phosphorylated STAT5 form relative to a control manufactured T cell population produced in the presence of exogenous IL-2, wherein the observed reduction is at least 50% less and preferably 90% or more less.

[0521] 104. A manufactured T cell population that exhibits a transition in differentiation from an effector memory state to a T central memory state, as indicated by an increase in the frequency of T cells co-expressing CD62L and CCR7 of at least 25% relative to the culture input T cells.

[0522] 105. A manufactured T cell population that exhibits a resting state, such as CD4 + and CD8+ Indicated by the frequency of T cells co-expressing the IL-2 receptor CD25 at a rate of less than 5% and more preferably less than 1%.

[0523] 106. A manufactured T cell population that exhibits a quiescent state, as indicated by T cells that secrete low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, such as after a stimulation process using high-level co-stimulation (bead-to-T cell ratio of 3:1), where the levels contained in the culture supernatant are <100 pg / ml per 24 hours per 1 × 10 6 cells, as defined.

[0524] 107. A manufactured T cell that transitions from a quiescent state to a high-level inflammatory cytokine-secreting state, as defined by an increase in IFN-γ and TNF-α secretion of at least 5-fold and more preferably 20-fold relative to the secretion level on day 6 after a 6-day expansion cycle in the absence of inhibitors.

[0525] 108. A manufactured T cell population that expresses low levels of the immunosuppressive molecule CTLA4, such as by flow cytometry of at least less than 10% CTLA4 + and more preferably less than 5% CTLA4 + of CD4 + and CD8 + T cell expression, as defined.

[0526] 109. A manufactured T cell population that expresses low levels of the immunosuppressive molecule TIM3, such as by flow cytometry of at least less than 10% TIM3 + and more preferably less than 2% TIM3 + of CD4 + and CD8 + T cell expression, as defined.

[0527] 110. The method according to any one of embodiments 1 to 29, further comprising, before inoculating the culture input cell population comprising T cells from the subject in the medium at a certain cell density:

[0528] Harvesting the culture input cell population from the subject.

[0529] 111. The method according to any one of embodiments 1 to 29, further comprising, before inoculating the culture input cell population comprising T cells from the subject in the medium at a certain cell density:

[0530] Isolating T cells from a sample comprising T cells from the subject to produce the culture input cell population.

[0531] 112. The method according to any one of embodiments 1 to 29, further comprising, before inoculating the culture input cell population comprising T cells from the subject into the culture medium at a certain cell density:

[0532] Enriching a sample comprising T cells from the subject to produce the culture input cell population.

[0533] 113. The method according to any one of embodiments 38 to 62, further comprising, before inoculating the culture input cell population comprising T cells from the subject into the culture medium at a certain cell density:

[0534] Isolating T cells from a sample comprising T cells from the subject to produce the culture input cell population.

[0535] 114. The method according to any one of embodiments 38 to 62, further comprising, before inoculating the culture input cell population comprising T cells from the subject into the culture medium at a certain cell density:

[0536] Enriching a sample comprising T cells from the subject to produce the culture input cell population.

[0537] 115. The method according to any one of embodiments 71 to 94, further comprising, before inoculating the culture input cell population comprising T cells from the subject into the culture medium at a certain cell density:

[0538] Isolating T cells from a sample comprising T cells from the subject to produce the culture input cell population.

[0539] 116. The method according to any one of embodiments 71 to 94, further comprising, before inoculating the culture input cell population comprising T cells from the subject into the culture medium at a certain cell density:

[0540] Enriching a sample comprising T cells from the subject to produce the culture input cell population.

[0541] 117. The method according to any one of embodiments 1 to 36, 38 to 69 and 71 to 100, wherein the IFN-α is added 24 hours after inoculating the culture input cell population into the culture medium.

[0542] 118. A manufactured T cell produced by the method according to any one of embodiments 110 to 117.

[0543] 119. A manufactured T cell population, wherein 10% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express CTLA4, as measured by flow cytometry.

[0544] 120. A manufactured T cell population, characterized in that, relative to the CD4 + or CD8 + frequency of CTLA4 expression by T-Rapa cells, the frequency of CTLA4 expression by CD4 + or CD8 + T cells is reduced, as measured by flow cytometry.

[0545] 121. The manufactured T cell population according to embodiment 120, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0546] 122. A manufactured T cell population, wherein 10% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express TIM3, as measured by flow cytometry.

[0547] 123. A manufactured T cell population, characterized in that, relative to the CD4 in a control T cell population characteristic of the T cells from which the manufactured T cell population is produced + or CD8 + frequency of T cells, the frequency of CD4 + or CD8 + T cells expressing TIM3 is reduced, as measured by flow cytometry.

[0548] 124. The manufactured T cell population according to embodiment 123, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0549] 125. A manufactured T cell population, characterized in that, relative to the CD4 + or CD8 + frequency of TIM3 expression by T-Rapa cells, the frequency of CD4 + or CD8 + T cells expressing TIM3 is reduced, as measured by flow cytometry.

[0550] 126. The manufactured T cell population according to embodiment 125, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0551] 127. A manufactured T cell population, wherein 5% or less of the CD4 in the manufactured T cell population +or CD8 + The T cells express PD1, as measured by flow cytometry.

[0552] 128. A manufactured T cell population, characterized in that, relative to CD4 + or CD8 + The frequency of PD1 expression by CD4 + or CD8 + T cells is reduced, as measured by flow cytometry.

[0553] 129. The manufactured T cell population according to embodiment 128, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0554] 130. A manufactured T cell population, wherein 5% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express 2B4, as measured by flow cytometry.

[0555] 131. The manufactured T cell population, wherein 5% or less of the CD8 + T cells in the manufactured T cell population express 2B4, as measured by flow cytometry.

[0556] 132. A manufactured T cell population, characterized in that, relative to the corresponding frequency of CD8 + T cells in a control T cell population characteristic of the T cells used to generate the manufactured T cell population, the frequency of CD8 + T cells expressing 2B4 is reduced, as measured by flow cytometry.

[0557] 133. The manufactured T cell population according to embodiment 132, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0558] 134. A manufactured T cell population, characterized in that, relative to the corresponding frequency of CD4 + or CD8 + T-Rapa cells expressing 2B4, the frequency of CD4 + or CD8 + T cells expressing 2B4 is reduced, as measured by flow cytometry.

[0559] 135. The manufactured T cell population according to embodiment 134, wherein the reduced frequency is at least 20% less than the corresponding frequency.

[0560] 136. A manufactured T cell population, wherein 10% or less of the CD4 +or CD8 + T cells express LAIR1, as measured by flow cytometry.

[0561] 137. A manufactured T cell population, characterized in that, relative to the corresponding frequency of CD4 + or CD8 + T cells in a control T cell population characteristic of the T cells from which the manufactured T cell population is produced, the frequency of CD4 + or CD8 + T cells expressing LAIR1 is reduced, as measured by flow cytometry.

[0562] 138. The manufactured T cell population according to embodiment 137, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0563] 139. A manufactured T cell population, characterized in that, relative to the corresponding frequency of CD4 + or CD8 + T-Rapa cells expressing LAIR1, the frequency of CD4 + or CD8 + T cells expressing LAIR1 is reduced, as measured by flow cytometry.

[0564] 140. The manufactured T cell population according to embodiment 139, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0565] 141. A manufactured T cell population, wherein 10% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express TIGIT, as measured by flow cytometry.

[0566] 142. A manufactured T cell population, characterized in that, relative to the corresponding frequency of CD4 + or CD8 + T-Rapa cells expressing TIGIT, the frequency of CD4 + or CD8 + T cells expressing TIGIT is reduced, as measured by flow cytometry.

[0567] 143. The manufactured T cell population according to embodiment 142, wherein the reduced frequency is at least 40% less than the corresponding frequency.

[0568] 144. A manufactured T cell population, wherein 10% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express LAG3, as measured by flow cytometry.

[0569] 145. A manufactured T cell population, characterized in that, relative to the corresponding frequency of LAG3 expression by CD4 + or CD8 + T-Rapa cells, the frequency of LAG3 expression by CD4 + or CD8 + T cells is reduced, as measured by flow cytometry.

[0570] 146. The manufactured T cell population according to embodiment 144, wherein the reduced frequency is at least 50% less than the corresponding frequency.

[0571] 147. A manufactured T cell population, wherein 1% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express CD25, as measured by flow cytometry.

[0572] 148. A manufactured T cell population, wherein 5% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express KLRG1, as measured by flow cytometry.

[0573] 149. A manufactured T cell population, wherein 20% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express CD39, as measured by flow cytometry.

[0574] 150. A manufactured T cell population, wherein 20% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express CD73, as measured by flow cytometry.

[0575] 151. A manufactured T cell population, wherein 4% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express GITR, as measured by flow cytometry.

[0576] 152. The manufactured T cell population according to any one of embodiments 108 to 109 and 119 to 150, wherein the frequency of CD4 + or CD8 + T cells expressing CD28 or ICOS in the manufactured T cell population is substantially the same as that of CD4 + or CD8 +The corresponding frequencies of T cells expressing CD28 or ICOS are the same.

[0577] 153. A manufactured T cell population according to any one of Examples 108 to 109 and 119 to 150, wherein the frequency of CD4 + or CD8 + T cells expressing CD28 or ICOS is within 10% of the corresponding frequency of CD4 + or CD8 + T cells expressing CD28 or ICOS in a control T cell population characteristic of the T cells used to generate the manufactured T cell population.

[0578] 154. A manufactured T cell population that secretes at least 500 pg / mL / 1×10 6 cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3.

[0579] 155. A manufactured T cell population that secretes at least 1000 pg / mL / 1×10 6 cells / day of IL-2 after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, and with IL-7, IL-15, or a combination of IL-7 and IL-15 (if present) at a concentration of 10 ng / mL each of IL-7 and IL-15.

[0580] 156. A manufactured T cell population that secretes an increased amount of IL-2 relative to a control T cell population or T-Rapa cells after co-stimulation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15 (if present) at a concentration of 10 ng / mL each of IL-7 and IL-15 (if present).

[0581] 156. A manufactured T cell population that expresses: at least 75% less phosphorylated STAT5 relative to a cultured T-Rapa cell population, detectable levels of STAT1 or phosphorylated STAT1, at least 50% reduction in p70S6K and Raptor, and levels of Rictor, SGK1, and phosphorylated SGK1 that do not differ by more than 50% from a cultured T-Rapa cell population.

[0582] 157. A manufactured T cell population that exhibits reduced mTORC1 activation, wherein the mTORC1 activation is characterized by at least one of the following:

[0583] (a) A reduced phosphorylated P70S6K level relative to a control T cell population, or

[0584] (b) A reduced Raptor level relative to a control T cell population; and

[0585] The manufactured T cell population exhibits mTORC2 molecule retention, which is characterized by substantially the same levels of Rictor, SGK1, or phosphorylated SGK1.

[0586] 157. A manufactured T cell population that expresses: phosphorylated STAT5 that is at least 75% less relative to a population of cultured T-Rapa cells, detectable levels of STAT1 or phosphorylated STAT1, a reduction of at least 50% in p70S6K and Raptor, and levels of Rictor, SGK1, and phosphorylated SGK1 that do not differ by more than 50% from a population of cultured T-Rapa cells.

[0587] 158. A manufactured T cell population that exhibits reduced mTORC1 activation, which is characterized by at least one of the following:

[0588] (a) A reduced phosphorylated P70S6K level relative to T-Rapa cells, or

[0589] (b) A reduced Raptor level relative to T-Rapa cells; and

[0590] The manufactured T cell population exhibits mTORC2 molecule retention, which is characterized by substantially the same levels of Rictor, SGK1, or phosphorylated SGK1 relative to T-Rapa cells.

[0591] 159. The manufactured T cell population according to embodiment 158, which is further characterized by reduced STAT5 phosphorylation and detectable levels of STAT1 or phosphorylated STAT1 relative to a control T cell culture.

[0592] 160. A manufactured T cell population that is characterized by reduced STAT5 phosphorylation and detectable levels of STAT1 or phosphorylated STAT1 relative to a control T cell culture.

[0593] 161. A manufactured T cell population that has one or more of the following properties:

[0594] After a one-week stimulation incubation using anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, IFN-γ secretion is at least 50% higher relative to T-Rapa cells;

[0595] After one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, TNF-α secretion is increased by at least 50% relative to T-Rapa cells;

[0596] After one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, GM-CSF secretion is increased by at least 50% relative to T-Rapa cells;

[0597] After one week of stimulation and incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, IL-2 secretion is increased by at least 50% relative to T-Rapa cells;

[0598] The percentage of cells positive for CD4, CD62L, CCR7, and CD127 is increased by at least 50% relative to a control T cell population characteristic of the T cells used to generate the manufactured T cell population;

[0599] The phosphorylation of 4EBP1 is increased by no more than 50% relative to a control T cell population characteristic of the T cells used to generate the T cell population;

[0600] The expression of p70S6K or Raptor is decreased by at least 50% relative to a population of T-Rapa cells cultured under the same conditions;

[0601] The expression of p-STAT5 is decreased by at least 50% relative to a population of T-Rapa cells cultured under the same conditions;

[0602] Detectable levels of STAT1 and p-STAT1 expression;

[0603] The expression of p70S6K is increased by at least 10% relative to a control T cell population characteristic of the cells used to generate the manufactured T cell population;

[0604] The expression of CD25 is decreased by at least 50% relative to a population of T-Rapa cells;

[0605] 10% or fewer CD4 + or CD8 + T cells express CTLA4, as measured by flow cytometry;

[0606] 10% or fewer CD4 + or CD8 + T cells express TIM3, as measured by flow cytometry;

[0607] 5% or fewer CD4 + or CD8 + T cells express PD1, as measured by flow cytometry;

[0608] 5% or less of CD4 + or CD8 + T cells express 2B4, as measured by flow cytometry;

[0609] 10% or less of CD4 + or CD8 + T cells express LAIR1, as measured by flow cytometry;

[0610] 10% or less of CD4 + or CD8 + T cells express TIGIT, as measured by flow cytometry;

[0611] 10% or less of CD4 + or CD8 + T cells express LAG3, as measured by flow cytometry;

[0612] 5% or less of CD4 + or CD8 + T cells express CD25, as measured by flow cytometry;

[0613] 5% or less of CD4 + or CD8 + T cells express KLRG1, as measured by flow cytometry;

[0614] 20% or less of CD4 + or CD8 + T cells express CD39, as measured by flow cytometry;

[0615] 20% or less of CD4 + or CD8 + T cells express CD73, as measured by flow cytometry;

[0616] 5% or less of CD4 + or CD8 + T cells express GITR, as measured by flow cytometry;

[0617] The expression level of CD28 is within about 20% of a control T cell population characteristic of the T cells that gave rise to the manufactured T cell population;

[0618] The expression level of ICOS is within about 20% of a control T cell population characteristic of the T cells that gave rise to the manufactured T cell population;

[0619] The expression level of CD45RA is within about 20% of a control T cell population characteristic of the T cells that gave rise to the manufactured T cell population;

[0620] CD4 positive for CD45RA + T cells increase by at least 50%, as measured by flow cytometry;

[0621] IL-2 secretion increases by at least 1.1-fold relative to T-Rapa cultures incubated under the same conditions;

[0622] After co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, secrete at least 500 pg / mL / 1×10 6 cells / day of IL-2;

[0623] When incubated in the presence of IL-7, IL-15 or a combination of IL-7 and IL-15, IL-2 secretion relatively increases by at least 1.1-fold, wherein the IL-7 and IL-15 (when present) are added at 10 ng / mL respectively;

[0624] After incubation in the presence of IL-7, IL-15 or a combination of IL-7 and IL-15, secrete at least 1000 pg / mL / 1×10 6 cells / day of IL-2, wherein the IL-7 and IL-15 (when present) are added at 10 ng / mL respectively;

[0625] Relative to the corresponding expression levels of the T-Rapa cell population, the expression of one or more checkpoint inhibitors selected from the following is reduced by at least 25%: CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3 and combinations thereof;

[0626] The expression levels of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3 and combinations thereof are within 25% of the corresponding expression levels in a control T cell population characterized by the cells that gave rise to the manufactured T cell population;

[0627] At least 5% of CD4 + T cells express CD127;

[0628] Relative to a control T cell population characterized by the cells that gave rise to the manufactured T cell population, the frequency of CD4 + T cells expressing CD127 increases by at least 50%;

[0629] Relative to the cultured input T cells, the frequency of T cells co-expressing CD62L and CCR7 increases by at least 25%;

[0630] CD4 + and CD8 + the frequency at which CD4 and CD8 T cells co-express the IL-2 receptor CD25 at a rate of less than 5%, and more preferably at a rate of less than 1%;

[0631] secrete low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, as defined by levels in the culture supernatant of less than 100 pg / ml per 24 hours per 1 × 10 6 cells following stimulation using high levels of co-stimulation (bead to T cell ratio of 3:1);

[0632] in the absence of inhibitors, after a 6-day expansion cycle, the secretion of IFN-γ and TNF-α is increased by at least 5-fold and more preferably by 20-fold relative to the secretion level on day 6; and

[0633] combinations thereof.

[0634] 162. A manufactured T cell having one or more of the following properties:

[0635] after one week of incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, the IFN-γ secretion is increased by at least 50% relative to T-Rapa cells;

[0636] after one week of incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, the TNF-α secretion is increased by at least 50% relative to T-Rapa cells;

[0637] after one week of incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, the GM-CSF secretion is increased by at least 50% relative to T-Rapa cells;

[0638] after one week of incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, the IL-2 secretion is increased by at least 50% relative to T-Rapa cells;

[0639] the phosphorylation of 4EBP1 is increased by no more than 50% relative to a control T cell population characteristic of the T cells from which the T cell population is generated;

[0640] the expression of p70S6K or Raptor is reduced by at least 50% relative to a population of T-Rapa cells cultured under the same conditions;

[0641] the expression of p-STAT5 is reduced by at least 50% relative to a population of T-Rapa cells cultured under the same conditions;

[0642] Detectable levels of STAT1 and p-STAT1 expression;

[0643] The expression of p70S6K is increased by at least 10% relative to a control T cell population characteristic of the cells used to generate the manufactured T cell population;

[0644] The expression of CD25 is decreased by at least 50% relative to the T-Rapa cell population;

[0645] The expression level of CD28 is within about 20% of a control T cell population characteristic of the T cells used to generate the manufactured T cell population;

[0646] The expression level of ICOS is within about 20% of a control T cell population characteristic of the T cells used to generate the manufactured T cell population;

[0647] The expression level of CD45RA is within about 20% of a control T cell population characteristic of the T cells used to generate the manufactured T cell population;

[0648] CD4 + T cells that are positive for CD45RA are increased by at least 50%, as measured by flow cytometry;

[0649] IL-2 secretion is increased by at least 1.1-fold relative to a T-Rapa culture incubated under the same conditions;

[0650] After co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, secrete at least 500 pg / mL / 1×10 6 cells / day of IL-2;

[0651] When incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, IL-2 secretion is relatively increased by at least 1.1-fold, where the IL-7 and IL-15 (when present) are added at 10 ng / mL each;

[0652] After incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, secrete at least 1000 pg / mL / 1×10 6 cells / day of IL-2, where the IL-7 and IL-15 (when present) are added at 10 ng / mL each;

[0653] The expression of one or more checkpoint inhibitors selected from the following is decreased by at least 25% relative to the corresponding expression levels in the T-Rapa cell population: CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3, and combinations thereof;

[0654] The expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3, and combinations thereof is within 25% of the corresponding expression level in a control T cell population characterized by the cells that gave rise to the manufactured T cell population;

[0655] Express CD127;

[0656] Secrete low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, such that after stimulation with high levels of co-stimulation (bead-to-T cell ratio of 3:1), the levels contained in the culture supernatant are defined as <100 pg / ml per 24 hours per 1×10 6 cells;

[0657] In the absence of inhibitors, after a 6-day expansion cycle, the secretion of IFN-γ and TNF-α increases by at least 5-fold and more preferably by 20-fold relative to the secretion levels on day 6; and

[0658] Combinations thereof.

Claims

1. Use of a composition comprising engineered T cells in the preparation of a medicament for treating cancer in a subject in need thereof, wherein: A therapeutically effective dose of the composition comprising engineered T cells is administered to the subject, wherein the engineered T cells are generated by: (a) inoculating a culture input cell population comprising T cells from the subject in a medium comprising temsirolimus and an IL-2 signaling inhibitor, wherein the temsirolimus is present in the medium at a concentration of at least 1 μM, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody, and wherein the medium does not contain IL-2 and no IL-2 is added to the medium; (b) adding IFN-α to the medium; and (c) incubating the T cells and the medium for a period of time to generate the engineered T cells, and wherein the cancer is selected from multiple myeloma, renal cell carcinoma, bladder cancer, lung cancer, liver cancer, lymphoma, gastric cancer, colon cancer, sarcoma, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, and colorectal cancer.

2. The use according to claim 1, wherein the multiple myeloma is smoldering multiple myeloma or relapsed, refractory multiple myeloma.

3. The use according to claim 2, wherein the refractory multiple myeloma is quadruple or quintuple refractory multiple myeloma.

4. The use according to claim 1, wherein the medicament is administered to the subject by infusion.

5. The use according to claim 1, wherein the cancer is selected from lung cancer and pancreatic cancer.

6. The use according to claim 1, wherein the cancer is PDL1-negative cancer.

7. The use according to any one of claims 1 to 2, wherein the generation of the engineered T cells is without anti-CD3 / anti-CD28 co-stimulation.

8. The use according to any one of claims 1 to 2, wherein the IFN-α is added to the medium at the same time as inoculating the culture input cell population or within 24 hours of inoculating the culture input cell population.

9. Use according to any one of claims 1 to 2, wherein the cell density of the cell population of the culture input during inoculation is at least 1.5 × 10 6 cells per mL.

10. The use according to any one of claims 1 to 2, wherein the temsirolimus is present in the medium at a concentration of 4.5 μM.

11. The use according to any one of claims 1 to 2, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab.

12. The use according to any one of claims 1 to 2, wherein the IL-2 signaling inhibitor is present in the medium at a concentration of 5 μg / mL to 50 μg / mL.

13. The use according to any one of claims 1 to 2, wherein the IFN-α is added to the medium to a concentration of 1000 IU / mL to 10000 IU / mL.

14. The use according to any one of claims 1 to 2, wherein the period of time is 4 days to 8 days.

15. The use according to any one of claims 1 to 2, wherein the period of time is 6 days.

16. Use according to any one of claims 1 to 2, wherein the culture medium is serum-free.

17. Use according to any one of claims 1 to 2, wherein the culture medium further comprises 5% human serum.

18. Use according to any one of claims 1 to 2, wherein the cultured input cell population comprises T cells accounting for no more than 66% of the total number of cells in the cultured input cell population.

19. Use according to any one of claims 1 to 2, wherein the cultured input cell population comprises T cells accounting for 50% to 95% of the total number of cells in the cultured input cell population.

Images