Method for treating cancer with manufactured T cells

Abstract

The present disclosure provides a method for treating cancer. The method for treating cancer can include administering pentostatin and cyclophosphamide followed by administration of manufactured T cells. The present disclosure further provides a method for producing such manufactured T cells, the manufactured T cells, and a composition comprising the manufactured T cells.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 768,145, filed on November 16, 2018; U.S. Provisional Patent Application No. 62 / 927,034, filed on October 28, 2019; and U.S. Provisional Patent Application No. 62 / 927,079, filed on October 28, 2019, the entire contents of each of which are incorporated herein by reference.

Background Art

[0002] CD4 + and CD8 + T cells (Th1 and Tc1 cells, respectively) are candidate T cell populations for adoptive T cell therapy efforts. These Th1 - type T cells are promoted by polarizing to cytokines such as IL - 12 and IFN - α that stimulate the STAT1 and STAT4 transcription factors, and then promote the TBET transcription factor that partly defines the Th1 - type differentiated state.

[0003] 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 tendency of T cells towards apoptotic cell death. Previous studies have shown that the ex - vivo production of T cells in the pharmacological agent rapamycin (which inhibits the mammalian target of rapamycin, mTOR) results in these characteristics, namely, an increase in the ability to undergo antigen - driven clonal expansion in vivo after adoptive transfer, T central memory (T CM)It is demonstrated that T cells were generated that exhibit a multifaceted anti-apoptotic phenotype characterized by improvement of memory state as exemplified by differentiation state, induction of autophagy including mitochondrial autophagy, and preferential expression of anti-apoptotic members of the bcl-2 gene family compared to pro-apoptotic members. Collectively, these properties of ex vivo-produced rapamycin-resistant cells are associated with enhancement of in vivo modulation of transplant responses including prevention of graft-versus-host disease (GVHD) and graft rejection, and mediation of human-into-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 been shown to be safe and effective against relapsed and refractory multiple myeloma. For these clinical trials, 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, simultaneous addition of T cells and 3 / 28 beads to ex vivo culture, addition of a high dose of orally administered mTOR inhibitor rapamycin (1 μM), and use of addition of IL-2 to the culture during cytokine polarization (IFN-α addition). The cells produced by this method are referred to herein as T-RAPA cells, which are more specifically defined later in the present disclosure.

[0004] Cancer development can be associated with changes in immune function. Such changes include suppressor cell-mediated immunity (CMI) associated with failure of tumor rejection, and enhancement of humoral immunity that can enhance tumor promotion and progression. CD4 + T cell subsets, Th1 and Th2 T cells, have specific functions and regulate each other. Th1 cells produce interleukin (IL-2) and interferon (IFN-γ) and directly conduct CMI responses, while Th2 cells produce IL-4 and IL-10 and promote local humoral immune responses.

[0005] There is evidence of Th1 / Th2 imbalance in certain cancers, and the proportion of Th2 cells increases significantly at the expense of Th1 cell numbers. A chronic Th1 / Th2 imbalance favoring Th2 can cause suppression of cell-mediated immunity, thereby providing an environment conducive to reduced effective immune surveillance and the development of malignancies.

[0006] Idiotypic-specific T cell responses are seen in most patients with early-stage multiple myeloma. These include Th1 responses by production of IL-2 and IFN-γ. For example, Th1-type immunity is preferentially found in cases of slowly progressive disease, and Th2-type responses are mainly seen in cases of progressive multiple myeloma. A defective Th1 immune response (mediated by IL-6) as well as a dysregulated cytokine network are found in multiple myeloma patients. Myeloma idiotypic-specific T helper cells derived from MM patients are consistently of a non-Th1 phenotype.

[0007] Despite advances in the treatment of multiple myeloma and the recent FDA approval of new drugs and monoclonal antibodies, multiple myeloma remains almost universally fatal. Thus, patients with relapsed / refractory multiple myeloma (RRMM) who are refractory to the top 5 drugs for multiple myeloma ("pentarefractory") have a limited survival of only a few months and few treatment options. Multiple myeloma is a disease amenable to immunotherapy, as demonstrated by the long-observed therapeutic role of allogeneic stem cell transplantation as well as numerous other approaches including monoclonal antibody therapy, vaccines, and T cell receptor (TCR-) and CAR-modified T cell therapies. Thus, this pentarefractory patient population is suitable for novel T cell therapies. In addition, patients with less refractory disease also greatly need novel treatments, i.e., even at the second or third relapse of the disease, the median progression-free survival is typically less than 2 years.

[0008] There is a need for new and innovative immunotherapy approaches for certain cancers. Summary of the Invention

[0009] The present disclosure is directed to a method for treating cancer in a subject.

[0010] In one embodiment, the method comprises administering to the subject, at a therapeutically effective dose, a composition comprising engineered T cells.

[0011] In another embodiment, the method further comprises subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or terminally exhausted effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally exhausted effector T cells, prior to administering to the subject, at a therapeutically effective dose, a composition comprising engineered T cells.

[0012] In some embodiments, the immune depletion regimen comprises administering to the subject a first composition comprising pentostatin and administering to the subject a second composition comprising cyclophosphamide.

[0013] In some embodiments, the method comprises a first treatment cycle and one or more additional treatment cycles, wherein the first treatment cycle comprises subjecting the subject to a first immune depletion regimen to reduce at least a portion of regulatory T cells and / or terminally exhausted effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally exhausted effector T cells, and each of the one or more additional treatment cycles comprises subjecting the subject to a second immune depletion regimen to reduce at least a portion of regulatory T cells and / or terminally exhausted effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally exhausted effector T cells, and administering to the subject, at a therapeutically effective dose, a composition comprising engineered T cells.

[0014] In any of the above embodiments, the method may further include measuring the creatine clearance (CrCl) of the subject prior to administering one or more additional doses of pentostatin to the subject, and adjusting the dose of pentostatin administered to the subject based on the CrCl, wherein pentostatin is administered to a subject having a CrCl of ≥ 60 mL / min / 1.73 m 2 4 mg / m 2 Pentostatin was administered at a rate of 60 mL / min / 1.73 m 2 >CrCl≧30mL / min / 1.73m 2 2 mg / m 2 Pentostatin was administered at 1.73 m 2 In some embodiments, the dose of pentostatin is 60 mL / min / 1.73 m 2 >CrCl≧30mL / min / 1.73m 2 Pentostatin dose may be adjusted based on CrCl, with a 50% reduction in dose if CrCl < 30 mL / min / 1.73 m 2 Pentostatin is not administered in these cases.

[0015] In any of the above embodiments, the method may further include measuring the absolute lymphocyte count (ALC) and the absolute neutrophil count (ANC) before 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 cyclophosphamide is administered at a dose of 200 mg when ANC > 1000 per microliter, cyclophosphamide is administered at a dose of 100 mg when ANC is 500 - 999 per microliter and ALC ≧ 50 per microliter, and cyclophosphamide is not administered when ALC < 50 per microliter or ANC < 500 per microliter. In some embodiments, the dose of cyclophosphamide can be adjusted based on ALC and ANC, such that the dose of cyclophosphamide is reduced by 50% when ANC is 500 - 999 per microliter and ALC ≧ 50 per microliter, or not administered when ALC < 50 per microliter or ANC < 500 per microliter.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] The present disclosure provides a method for producing engineered T cells, engineered T cells produced by the methods disclosed herein, populations and compositions comprising engineered T cell populations, and methods for treating cancer in a subject using the engineered T cells or engineered T cell populations.

[0018] Definitions The following definitions are provided:

[0019] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The use of the term “or” in the claims and the present disclosure is used to mean “and / or” unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0020] The use of the term “about,” when used in conjunction with a numerical value, is intended to include + / −10%. By way of non-limiting example, if some amino acids are specified as about 200, this would include 180-220 (plus or minus 10%).

[0021] The terms "subject", "patient", and "individual" are used interchangeably herein and refer to a mammalian subject to be treated, preferably a human patient. In some cases, the methods of the invention are found to be used in experimental animals for diseases, veterinary applications, and the development of animal models, including but not limited to rodents such as mice, rats, and hamsters, and primates.

[0022] "Sample" is used herein in the broadest sense. Samples can include, but are not limited to, cells, polynucleotides, polypeptides, peptides, antibodies, etc., and can be body fluids, soluble fractions of cell preparations, or media in which cells have been grown, chromosomes isolated or extracted from cells, organelles, or membranes, genomic DNA, RNA, or cDNA, polypeptides, or peptides, cells, tissues, tissue prints, fingerprints, skin, or hair, etc., bound in solution or to a substrate.

[0023] "Treatment" is an intervention performed with the intention of preventing the onset of a disorder or altering a medical condition or symptom. Thus, "treatment" refers to both therapeutic treatment and prevention or prophylactic measures. Those in need of treatment include those already suffering from a disorder and those in whom the disorder should be prevented. For example, in the treatment of a tumor (e.g., cancer), a therapeutic agent can directly reduce the medical condition of tumor cells or make tumor cells more sensitive to treatment with other therapeutic agents, such as radiation and / or chemotherapy. As used herein, "improved" refers to a symptom approaching a normalized value (e.g., a value obtained in a healthy patient or individual), for example, the difference from the normalized value is less than 50%, preferably less than about 25% from the normalized value, more preferably less than 10% from the normalized value, and even more preferably not significantly different from the normalized value as determined using routine statistical tests. By way of non-limiting example, the improvement or treatment of a patient suffering from an infectious organism such as hepatitis B virus can be determined by a decrease in viral particles in a sample taken from the patient, as measured, for example, by a decrease in plaque-forming units (p.f.u.).

[0024] As used herein, "treatment cycle" generally can refer to either a primary treatment cycle, a first treatment cycle, a second treatment cycle, or one or more additional treatment cycles.

[0025] As used herein, the terms "therapeutically effective dose" or "therapeutically effective amount" mean an amount of a compound of the invention effective to produce a desired therapeutic response. By way of example and not limitation, a dose effective to slow cancer growth, or to shrink cancer, or to prevent metastasis may be a "therapeutically effective dose". The specific therapeutically effective dose will vary depending on factors such 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 therapy (if any), as well as the particular formulation used and the structure of the compound or its derivatives.

[0026] As used herein, "immune cell" means 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, as well as derivatives, precursors or progenitors of the above cell types.

[0027] A "T cell" is a subset of lymphocytes that is derived from the thymus and has a heterodimeric receptor associated with the proteins of the CD3 complex (e.g., a rearranged T cell receptor, a heterodimeric protein on the surface of the cell involved in antigen / MHC specificity of the cell). T cell responses can be detected by assays of their effects on other cells (e.g., target cell killing, activation of other immune cells such as B cells) or the cytokines they produce.

[0028] 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 associated anti-CD3 / anti-CD28 antibody moiety. In instances where it is disclosed that anti-CD3 / anti-CD28 co-stimulation is not provided by a particular form such as anti-CD3 / anti-CD28 magnetic beads, it should be understood that co-stimulation by other forms of anti-CD3 / anti-CD28 can also be excluded.

[0029] As used herein, the term "T-Rapa cell(s)" refers to T cell(s) produced by co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a 3:1 ratio (bead:T cell ratio) without delay between the start of culture and co-stimulation. The cells are grown in an equivalent medium containing X-Vivo or IFN-α (10,000 IU / mL), IL-2 (20 IU / mL) and rapamycin (1 μM), supplemented with 5% AB serum but not containing an IL-2 signaling inhibitor. The cells are cultured at 37 °C for 6 days and 6 initiated at a concentration of 1.5×10 cells / mL. When used with respect to the method "T-Rapa", unless otherwise specified, it refers to the method of producing T-Rapa cells as defined above.

[0030] As used herein, the terms "manufactured T cells" and "Rapa-T cells" are used interchangeably to refer to T cells produced by the methods of the present disclosure. "Manufactured T cells" + can include CD4 + CD8 T cells, or both. "Manufactured T cells" do not include T cells obtained from a patient, i.e., naturally occurring T cells.

[0031] As used herein, when used to refer to results measured by flow cytometry, the "level of expression", "expression level", or equivalent reference value is to be understood to refer to the frequency of a particular type of positive cell in a population. Any decrease or increase mentioned, so long as the "expression level" or equivalent refers to the expression level or equivalent of a particular cell type, is to be understood to be in comparison to the corresponding particular cell type, unless otherwise specified.

[0032] As will be recognized by those skilled in the art, the term "autologous" cells means cells of the same or a similar haplotype as the subject or "host" to whom the cells are administered such that no significant immune response to them occurs when they are transplanted into the host.

[0033] "CD4" is a cell surface protein important for the recognition by the T cell receptor of antigen peptides bound to MHC class II molecules on the surface of APCs. When activated, naive CD4 T cells differentiate into at least one of two cell types, Th1 cells and Th2 cells, each type being characterized by the cytokines it produces. "Th1 cells" are mainly involved in activating macrophages with respect to cell-mediated immunity and the inflammatory response, while "Th2 cells" or "helper T cells" are mainly involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is a receptor for the human immunodeficiency virus (HIV). Effector molecules of 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 of 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 eotaxin. Activation of the Th1-type cytokine response can suppress the Th2-type cytokine response, and conversely, activation of the Th2-type cytokine response can suppress the Th1-type response.

[0034] "Cytokine" is a protein produced by cells that affect the behavior of other cells via "cytokine receptors" on the surface of the cells on which the cytokine acts. Cytokines produced by lymphocytes are sometimes referred to as "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).

[0035] The term "modulate" means that any of the recited activities are, for example, increased, enhanced, augmented, agonized (act as an agonist), promoted, decreased, diminished, suppressed, blocked, or antagonized (act as an antagonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc. over a baseline value. Modulation can also decrease the activity below a baseline value.

[0036] "Substrate" refers to any solid or semi-solid support to which a nucleic acid molecule or protein binds and includes membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, capillaries, or wells, trenches, pins, chamiels, and other tubes, plates, polymers, and microparticles having various surface morphologies including pores.

[0037] Methods for producing engineered T cells, engineered T cells produced by the methods disclosed herein, and compositions comprising engineered T cell populations In the methods of the present disclosure, IFN-α is utilized for ex vivo polarization towards a Th1-type differentiated state phenotype of a culture comprising T cells. Th1-type differentiation can be eroded by polarizing towards a regulatory T (T REG ) cell phenotype, which is promoted in part by cytokines including IL-2 that signal via STAT5 to promote the FoxP3 transcription factor that defines the T REG differentiated state. In the methods of the present disclosure, T REGMixing is restricted during Th1 polarization by omitting the exogenous use of IL-2 during cell culture and by preventing autocrine IL-2 signaling through the addition of an IL-2 signaling inhibitor to the culture. In some embodiments, the IL-2 signaling inhibitor is an anti-IL-2 receptor monoclonal antibody.

[0038] The present disclosure provides a new manufacturing method incorporating the following interventions as compared to other methods for manufacturing T cells: (1) delayed addition to culture or no addition of anti-CD3 / CD28 beads (or any alternative source of anti-CD3 / anti-CD28 costimulation such as nanoparticles or microparticles) for improving T cell yield (where anti-CD3 / CD28 beads or nanoparticles are used for costimulation), (2) use of a lower ratio of anti-CD3 / CD28 beads for enhancing the tolerant T cell phenotype or alternatively no addition of bead, nanoparticle or microparticle artificial antibody-based costimulation, (3) use of an oral formulation of mTOR inhibitor (temsirolimus) for enhancing manufacturing feasibility, and (4) by omitting the typical use of exogenous IL-2 during T cell culture and by suppressing endogenous autocrine IL-2 signaling, for example, through the use of an anti-IL-2 receptor monoclonal antibody (daclizumab or basiliximab or other reagent that inhibits the IL-2 receptor signaling receptor) during T cell culture, avoidance of IL-2 signaling and as a result Th1-type differentiation of T REG Avoidance of cell mixing. In parallel culture experiments, T cells generated by this new combination method, referred to as "manufactured T cells", exhibited a more optimal cell phenotype as compared to the aforementioned T cells produced in ex vivo culture.

[0039] Figure 33 provides an exemplary workflow for generating the manufactured T cells of the present disclosure.

[0040] In some embodiments, a method for producing manufactured T cells comprises inoculating a culture input population of cells containing T cells from a subject at a certain cell density in a culture medium containing temsirolimus and an IL-2 signaling inhibitor. In certain embodiments, the culture medium does not already contain temsirolimus and / or an IL-2 signaling inhibitor, and these components can be added simultaneously or almost simultaneously with the inoculation. The culture input population of cells is incubated for a first period without co-stimulation by anti-CD3 / anti-CD28 antibodies (including, but not limited to, co-stimulation by anti-CD3 / anti-CD28-coated magnetic beads, nanoparticles or microparticles). After the first period, the culture input population of cells can be stimulated by anti-CD3 / anti-CD28 antibodies, for example, by adding anti-CD3 / anti-CD28-coated magnetic beads, nanoparticles or microparticles. When anti-CD3 / anti-CD28-coated magnetic beads are used, they can be used at a bead ratio of 1:1 to 1:12. In addition, IFN-α is added to the culture medium. Then, the culture input population of cells is incubated for a second period to obtain the manufactured T cells. In some embodiments, there is no co-stimulation by anti-CD3 / anti-CD28-coated magnetic beads, nanoparticles or microparticles. In some embodiments, co-stimulation is not performed.

[0041] In any of the above embodiments, the method for producing manufactured T cells can further comprise, after collecting the manufactured T cells, packaging at least a part of the manufactured T cells in a package, and freezing the package containing the part of the manufactured T cells. The cryopreservation of the manufactured T cells can be performed by methods known in the art.

[0042] In any of the above embodiments, the method can further comprise collecting a culture input population of the cells from the subject before inoculating the T cells from the subject at a certain cell density in the culture medium.

[0043] In any of the above embodiments, the culture medium cannot contain IL-2, and IL-2 cannot be added to the culture medium. In any of the above embodiments, serum cannot be added to the culture, for example, the culture is serum-free. In any of the above embodiments, the culture medium can be substantially serum-free.

[0044] In any of the above embodiments, the IFN-α can be added simultaneously with, or almost simultaneously with, the addition of anti-CD3 / anti-CD28 coated magnetic beads. If co-stimulation of the culture is not performed, for example, IFN-α can be added at the start of the culture or within 48 hours from the start of the culture. By way of example, but not limited to, IFN-α can 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.

[0045] In any of the above embodiments, the cell density can be from about 1×10 6 T cells per mL to 50×10 6 cells per mL. By way of example, but not limited to, the cell density can 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 6cells, 5×10 per mL 6 cells ~ 22.5×10 per mL 6 cells, 1×10 per mL 6 cells ~ 50×10 per mL 6 cells, 10×10 per mL 6 cells ~ 40×10 per mL 6 cells, 20×10 per mL 6 cells ~ 40×10 per mL 6 cells, 1×10 per mL 6 cells, 2.5×10 per mL 6 cells, 5×10 per mL 6 cells, 7.5×10 per mL 6 cells, 10×10 per mL 6 cells, 12.5×10 per mL 6 cells, 15×10 per mL 6 cells, 17.5×10 per mL 6 cells, 20×10 per mL 6 cells, 22.5×10 per mL 6 cells, 25×10 per mL 6 T cells, 30×10 per mL 6 cells, 35×10 per mL 6 cells, 40×10 per mL 6 cells, 45×10 per mL 6 cells, or 50×10 per mL 6 cells may be.

[0046] In any of the above embodiments, the temsirolimus can be present in the culture medium at a concentration of 0.1 to 5 μM. In some embodiments, temsirolimus can be present in the culture medium at a concentration of 0.1 to 1 μM. In any of the above embodiments, temsirolimus can be present in the culture medium at a concentration of 1 μM. By way of example, but not limitation, temsirolimus can 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 more. As a further example, but not limitation, temsirolimus can be present in the culture medium at a concentration of about 1 to 5 μM, 2 to 5 μM, 3 to 5 μM, 4 to 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 more than that.

[0047] In any of the above embodiments, the temsirolimus can be added to the culture medium one or more times during the second period to maintain a desired concentration. In any of the above embodiments, temsirolimus can be added to the medium once. As a non-limiting example, the temsirolimus can be added to the culture medium every two days during the second period. The desired temsirolimus concentration can be from 0.1 to 5 μM. In any of the above embodiments, the desired concentration of temsirolimus can be from 0.1 to 1 μM. In any of the above embodiments, the desired concentration of temsirolimus can be 1 μM. By way of non-limiting example, the desired temsirolimus concentration can 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 more. As a further example, by way of non-limiting example, the desired temsirolimus concentration can be about 1 to 5 μM, 2 to 5 μM, 3 to 5 μM, 4 to 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 a concentration exceeding that.

[0048] An IL-2 signal transduction inhibitor can be any substance that inhibits IL-2 signal transduction and can be added in an amount sufficient to inhibit IL-2 signal transduction. In any of the above embodiments, the IL-2 signal transduction inhibitor can be an anti-IL-2 receptor antibody or a fragment thereof, for example, basiliximab or daclizumab. The IL-2 signal transduction inhibitor can be present in the culture medium at a concentration of 5 to 50 μg / mL. As non-limiting examples, the IL-2 signal transduction inhibitor can be present at a concentration of about 5 to 50 μg / mL, 5 to 40 μg / mL, 5 to 30 μg / mL, 5 to 20 μg / mL, 5 to 10 μg / mL, 40 to 50 μg / mL, 30 to 50 μg / mL, 20 to 50 μg / mL, 20 to 40 μg / mL, 20 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.

[0049] In any of the above embodiments, the first period can be from about 8 hours to about 24 hours. As non-limiting examples, the first 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.

[0050] In any of the above embodiments, the above bead:T cell ratio can be 1:3. In some embodiments, the bead:T cell ratio can be from 1:12 to 1:1, or in the most extreme case, there can be no addition of beads. By way of non-limiting example, ratios such as 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 between them can be used. In some embodiments, co-stimulation of the cell culture input population can be achieved using anti-CD3 / anti-CD28-containing nanoparticles that can be used at a concentration lower than that recommended. By way of example, but not limitation, such nanoparticles can be 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 at about 0.01-fold, about 0.025-fold, about 0.05-fold, about 0.075-fold, or about 0.1-fold of the recommended dosage. By way of non-limiting example, reagents such as Miltenyi® T Cell TransAct™ can be 1×10 6It can be used at a lower dose compared to the recommended dose of 10 μL per T cell, for example, by way of example, 1.1 μL (a 9-fold decrease) or about 0.11-fold, but is not limited thereto. Alternatively, when using anti-CD3 / anti-CD28 costimulation to produce the manufactured T cells, the costimulation source can be provided by soluble anti-CD3 / anti-CD28 microparticles. By way of example, but not limited to, the soluble anti-CD3 / anti-CD28 microparticles can be used at 20% of the strength recommended by the manufacturer (e.g., Cloudz®; Bio-Techne). As a further example, the 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 added can be dose-set based on the desired functional characteristics of the final Rapa-T cell product. Specifically, a sufficient amount of the reagent can be added to maintain T cell viability in vitro in the presence of the inhibitory molecules described in the present disclosure. However, any particular anti-CD3 / anti-CD28 reagent should not be added in excess as defined by inappropriately high levels of T cell activation (increased CD25 expression by flow cytometry compared to T cell cultures with optimal and minimal costimulation), inappropriately high levels of T cell checkpoint inhibitor receptor expression by flow cytometry, and inappropriately altered expression of molecules associated with T cell effector memory cells by flow cytometry (decreased levels of CD62L and CCR7, e.g., increased levels of CD45RO and KLRG).

[0051] In any of the above embodiments, the IFN-α can be added to the culture medium up to a concentration of about 1,000 IU / mL to about 10,000 IU / mL. By way of non-limiting example, 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. Low concentrations of IFN-α, such as 1000 IU / mL, may result in a less pronounced shift to the Th1 phenotype.

[0052] In any of the above embodiments, the second period can be about 4 days to about 8 days, 4 days to about 6 days, or 6 days to about 8 days. By way of non-limiting example, the second period can be about 4 days, 5 days, 6 days, 7 days, or 8 days. If co-stimulation is not performed, the incubation period can be about 4 days to about 8 days, 4 days to about 6 days, or 6 days to about 8 days. By way of non-limiting example, the second period can be about 4 days, 5 days, 6 days, 7 days, or 8 days.

[0053] In any of the above embodiments, the above medium can further contain 5% human serum. In some embodiments, the culture medium can further contain 1% - 20% human serum. By way of non-limiting example, the culture medium can contain 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 can contain 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 above embodiments, the serum can be either added to or absent from the culture medium.

[0054] In any of the above embodiments, the above medium can further contain X-Vivo 20 medium. In any of the above embodiments, the above medium can further contain TexMACS (Miltenyi (registered trademark)) medium. Any suitable culture medium can be used for culturing T cells.

[0055] In any of the above embodiments, an additional culture medium can be added to the culture. By way of non-limiting example, the additional culture medium can be added about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, or at any range or time between them after the initial inoculation of the culture input population of cells into the culture. By way of example, and not limitation, the amount of the added culture medium relative to the initial amount of the culture medium can be at a ratio of 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 more, and at any range between them. By way of example, and not limitation, the amount of the culture medium added after the initial inoculation of the culture input population of cells into the culture can be an amount sufficient to reduce the cell density in the culture to the target cell density. By way of non-limiting example, this target cell density can be, when the initial cell density is greater than the target cell density, 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 cells, and can be at any range between them.

[0056] In any of the above embodiments, the cultured input population of the cells can include, with respect to T cells, 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% of the total number of cells in the cultured input population of the cells. As non-limiting examples, the cultured input population of the cells 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 cultured input population of the cells.

[0057] In any of the above embodiments, the cultured input population of the cells can further include monocytes. In any of the above embodiments, the cultured input population of the cells can be enriched for T cells. By way of example, but not limitation, the cultured input population of the cells can be subjected to T cell enrichment using an automated Ficoll procedure. Methods of performing the Ficoll procedure are known in the art and involve removing neutrophils and red blood cells from the sample. Any suitable method for enriching T cells in a cell population can be used.

[0058] In any of the above embodiments, the method can further include collecting a sample containing T cells from the subject and isolating the T cells from the sample to obtain a cultured input population of the cells. Such a sample can contain peripheral blood stem cells (PBSC) and can be obtained, by way of non-limiting example, by mobilized collection, steady-state apheresis, or simple blood draw. In any of the above embodiments, a sample containing PBSC and / or the cultured input population of cells can be cryopreserved prior to the manufactured T cell preparation. Steady-state apheresis can be performed when the subject has a sufficient number of immune cells, which can be characterized, by way of non-limiting example, by a minimum absolute lymphocyte count (ALC). The minimum ALC can be, for example, 300 lymphocytes per microliter.

[0059] In any of the above embodiments, the T cells can be isolated by antibody-based purification.

[0060] In any of the above embodiments, enrichment of the T cells can be performed by counter-flow centrifugal elutriation. Such techniques are well known in the art.

[0061] In any of the above embodiments, the IFN-α can be added at the same time as, or substantially simultaneously with, the addition of anti-CD3 / anti-CD28-containing nanoparticles.

[0062] In any of the above embodiments, the anti-CD3 / anti-CD28 antibody can be removed by any suitable method after culturing. By way of non-limiting example, anti-CD3 / anti-CD28 magnetic beads can be removed by magnetic capture, and cleavable anti-CD3 / anti-CD28 microparticles can be removed by adding a release buffer and washing the manufactured T cells.

[0063] In some embodiments, the manufactured T cell population shows increased IFN-γ secretion compared to T-Rapa cells one week after incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads added at a bead:T cell ratio of 3:1.

[0064] In some embodiments, the manufactured T cell population shows increased TNF-α secretion compared to T-Rapa cells one week after incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads added at a bead:T cell ratio of 3:1.

[0065] In some embodiments, the manufactured T cell population shows increased GM-CSF secretion compared to T-Rapa cells one week after incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads added at a bead:T cell ratio of 3:1.

[0066] In some embodiments, the manufactured T cell population shows increased IL-2 secretion compared to T-Rapa cells one week after incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads added at a bead:T cell ratio of 3:1.

[0067] In some embodiments, the manufactured T cell population includes an increased percentage of cells positive for CD4, CD62L, CCR7, and CD127 compared to the control population of T cells and compared to T-Rapa cells.

[0068] In some embodiments, the manufactured T cell population shows an increase in 4EBP1 phosphorylation compared to the control population of T cells. In some embodiments, the manufactured T cells show increased 4EBP1 phosphorylation compared to control T cells. By way of example, but not limitation, the increase in phosphorylation of 4EBP1, compared to the control population of T cells characteristic of the T cells produced, i.e., the T cells input into the culture, or compared to control T cells, is 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less. As a further example, but not limitation, the increase in 4EBP1 phosphorylation can be 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 between these or a value within these ranges. The phosphorylation of 4EBP1 is decreased (or blunted) compared to T-Rapa cells. In some embodiments, the increase in 4EBP1 phosphorylation can be measured 32 hours after the start of the culture.

[0069] In some embodiments, the manufactured T cell population shows a decrease in P70S6K expression compared to T-Rapa cells and an increase compared to the control population of T cells characteristic of the cell morphology produced by the manufactured T cells. By way of example, but 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.

[0070] In some embodiments, the manufactured T cell population shows a decrease in the expression of the IL-2 receptor CD25 by flow cytometry compared to T-Rapa cells. By way of example, but not limitation, the decrease can be at least 50%, 60%, 70%, 80%, 90% or more.

[0071] In some embodiments, the manufactured T cells can exhibit a unique RNA expression profile that is characterized by an increase of at least 50% in the RNA content of dedifferentiation molecules such as KLF4, KLF10, Nanog, and combinations thereof, and a decrease of at least 50% in the RNA content of differentiation molecules such as perforin, granzyme B, IFN-γ, and combinations thereof, compared to the cultured input T cells.

[0072] In some embodiments, the manufactured T cell population shows a decrease in the levels of the following molecules related to immunosuppressive effects: CTLA4, and TIM3, compared to T-Rapa cells.

[0073] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the CD4+ or CD8+ manufactured T cells expressing CTLA4 are 10% or less. In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the CD4+ or CD8+ manufactured T cells expressing CTLA4 are 5% or less. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD4+ or CD8+ T cells expressing CTLA4, compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CTLA4, when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing CTLA4 can be decreased by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CTLA4. By way of example, and not limitation, the decreased frequency can be decreased by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0074] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing TIM3 are 10% or less. In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing TIM3 are 5% or less. By way of example, but not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population, when measured by flow cytometry, can show a decrease in the frequency of CD4+ or CD8+ T cells expressing TIM3 as compared to the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing TIM3 can be reduced by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in the control population. By way of example, but not limitation, the decreased frequency of CD4+ or CD8+ T cells expressing TIM3 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T cells expressing TIM3 in the control population. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture. In some embodiments, the manufactured T cell population, when measured by flow cytometry, can show a decrease in the frequency of CD4+ or CD8+ T cells expressing TIM3 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing TIM3 can be reduced by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3.As an example, without limitation, the reduced frequency of CD4+ or CD8+ T cells expressing TIM3 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIM3. In some embodiments, the reduction in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0075] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the frequency of CD4+ or CD8+ manufactured T cells expressing PD1 is 5% or less. As an example, without limitation, when measured by flow cytometry, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express PD1. In some embodiments, the manufactured T cell population can exhibit the frequency of CD4+ or CD8+ T cells expressing PD1 as compared to the corresponding frequency of CD4+ and CD8+ T-Rapa cells expressing PD1 when measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing PD1 can be reduced by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing PD1. As an example, without limitation, the reduced frequency of CD4+ or CD8+ T cells expressing PD1 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing PD1. In some embodiments, the reduction in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0076] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the CD4+ or CD8+ manufactured T cells expressing 2B4 are 5% or less. By way of example, but not limited to, when measured by flow cytometry, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express 2B4. In some embodiments, when measured by flow cytometry, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4. By way of example, but not limited to, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD8+ T cells expressing 2B4 as compared to the corresponding frequency of CD8+ T cells expressing 2B4 in a control population of T cells characteristic of the T cells from which the manufactured T cells were produced, when measured by flow cytometry. By way of example, but not limited to, the decreased 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 decrease in frequency is 6 days after the cells giving rise to the manufactured T cells are inoculated into the culture. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD4+ or CD8+ T cells expressing 2B4 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing 2B4, when measured by flow cytometry. By way of example, but not limited to, the decreased 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 decrease is 6 days after the cells giving rise to the manufactured T cells are inoculated into the culture.

[0077] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing LAIR1 are 10% or less. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population, when measured by flow cytometry, can show a decrease in the frequency of CD4+ or CD8+ T cells expressing LAIR1 as compared to the corresponding frequency of CD4+ or CD8+ T cells expressing LAIR1 in a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. By way of example, and not limitation, the decreased 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 population of T cells. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture. In some embodiments, the CD4+ or CD8+ T cells of the manufactured T cell population can show a decrease in the expression level of LAIR1 as compared to T-Rapa cells when measured by flow cytometry. By way of example, and not limitation, the decrease can be at least 30%, 40%, 50%, or more.

[0078] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing TIGIT are 10% or less. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, when measured by flow cytometry, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD4+ or CD8+ T cells expressing TIGIT as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing TIGIT when measured by flow cytometry. By way of example, and not limitation, the decreased 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 decrease in frequency is 6 days after the cells giving rise to the manufactured T cells are inoculated into the culture.

[0079] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing LAG3 are 10% or less. By way of example, and without limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD4+ or CD8+ T cells expressing LAG3 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3 when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing LAG3 can be decreased by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3. By way of example, and without limitation, the decreased frequency of CD4+ or CD8+ T cells expressing LAG3 can be decreased by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing LAG3. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture

[0080] In some embodiments, the manufactured T cell population, when measured by flow cytometry, can be characterized by a preserved level, i.e., a substantially the same level, of the positive costimulatory molecule CD28 as compared to a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the frequency of CD28 expression of CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of CD28 expression of CD4+ or CD8+ T cells in the control population, respectively. By way of example and not limitation, the frequency of CD28 expression of 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 of CD4+ or CD8+ T cells in the control population, respectively. In some embodiments, this preservation occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0081] In some embodiments, the manufactured T cell population, when measured by flow cytometry, can be characterized by a preserved level, i.e., a substantially the same level, of the positive costimulatory molecule ICOS as compared to a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the frequency of ICOS expression of CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of ICOS expression of CD4+ or CD8+ T cells in the control population, respectively. By way of example and not limitation, the frequency of ICOS expression of 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 of CD4+ or CD8+ T cells in the control population, respectively. In some embodiments, this preservation occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0082] In some embodiments, the manufactured T cell population, when measured by flow cytometry, can be characterized by a preserved level, i.e., a substantially the same level of CD45RA, compared to a control T cell population characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the frequency of CD45RA expression of CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the frequency of CD45RA expression of CD4+ or CD8+ T cells in the control population, respectively. By way of example and not limitation, the frequency of CD45RA expression of 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 of CD4+ or CD8+ T cells in the control population, respectively. In some embodiments, this preservation occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0083] In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing CD25 are 5% or less. By way of example, and not limitation, when measured by flow cytometry, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population can express CD25. In some embodiments, the manufactured T cell population can show a decrease in the frequency of CD4+ or CD8+ T cells expressing CD25 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25 when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing CD25 can be reduced by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. By way of example, and not limitation, the decreased frequency of CD4+ or CD8+ T cells expressing CD25 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0084] In some embodiments, the manufactured T cell population exhibits a quiescent and non-senescent phenotype characterized by a reduced level of KLRG1 as measured by flow cytometry. In some embodiments, the reduction in the level of KLRG1 occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture. In some embodiments, the manufactured T cell population can be characterized by having 5% or less of the CD4+ or CD8+ manufactured T cells that express 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 show a decrease in the frequency of CD4+ or CD8+ T cells that express KLRG1 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express KLRG1 as measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells that express KLRG1 can be reduced by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express KLRG1. By way of example and not limitation, the decreased frequency of CD4+ or CD8+ T cells that express KLRG1 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express KLRG1. In some embodiments, the reduction in frequency occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture. In some embodiments, the reduction in frequency occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0085] In some embodiments, the manufactured T cell population exhibits a decrease in the expression of the immunosuppressive molecule CD39 compared to a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the manufactured CD4+ or CD8+ T cells expressing CD39 are 20% or less. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can exhibit a decrease in the frequency of CD4+ or CD8+ T cells expressing CD39 compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39 when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells expressing CD39 can be decreased by at least 50% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39. By way of example, and not limitation, the decreased frequency of CD4+ or CD8+ T cells expressing CD39 can be decreased by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD39. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0086] In some embodiments, the manufactured T cell population exhibits a decrease in the expression of the immunosuppressive molecule CD73 as compared to a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the manufactured T cell population can be characterized in that, when measured by flow cytometry, the percentage of CD4+ or CD8+ manufactured T cells that express CD73 is 20% or less. By way of example, and not limitation, when measured by flow cytometry, 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. In some embodiments, the manufactured T cell population can exhibit a decrease in the frequency of CD4+ or CD8+ T cells that express CD73 as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express CD73 when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells that express CD73 can be reduced by at least 50% as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express CD73. By way of example, and not limitation, the decreased frequency of CD4+ or CD8+ T cells that express CD73 can be reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express CD73. In some embodiments, the decrease in frequency occurs 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0087] In some embodiments, the manufactured T cell population exhibits a decrease in the expression of the immunosuppressive molecule GITR as compared to a control population of T cells characteristic of the T cells from which the manufactured T cells were produced. In some embodiments, the manufactured T cell population can be characterized by having 5% or less of the manufactured CD4+ or CD8+ T cells that express GITR when 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 when measured by flow cytometry. In some embodiments, the manufactured T cell population can exhibit a decrease in the frequency of CD4+ or CD8+ T cells that express GITR as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express GITR when measured by flow cytometry. In some embodiments, the decreased frequency of CD4+ or CD8+ T cells that express GITR can be decreased by at least 20% as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express GITR. By way of example and not limitation, the decreased frequency of CD4+ or CD8+ T cells that express GITR can be decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% as compared to the corresponding frequency of CD4+ or CD8+ T-Rapa cells that express GITR. In some embodiments, the decrease in frequency is 6 days after the cells that give rise to the manufactured T cells are inoculated into the culture.

[0088] In some embodiments, the manufactured T cells can exhibit a cytokine biological early differentiation state as compared to control T cell cultures, as demonstrated by an increase in the secretion of the precursor cytokine IL-2 and an increase in responsiveness to the homeostatic cytokines IL-7 and IL-15. In this way, the manufacturing method of the present disclosure describes a method for manufacturing helper-independent T cells having an increased responsiveness to homeostatic cytokines.

[0089] In some embodiments, the manufactured T cell population exhibits increased IL-2 secretion as compared to T-Rapa cultures incubated under the same conditions. In some embodiments, this 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 / 1×106 cells / day of IL-2 after co-stimulation with CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1 to 1:3. By way of example, the manufactured T cell population can secrete about 500, 600, 700, 800, 900, 1000 or more pg / mL / 1×106 cells / day of IL-2 under these conditions.

[0090] In some embodiments, the manufactured T cell population can secrete increased amounts of IL-2 when exposed to IL-7 or IL-15. In some embodiments, the manufactured T cell population is characterized by at least a 1.1-fold increase in IL-2 secretion when the manufactured T cell population is incubated in the presence of IL-7 or IL-15 as compared to conditions 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 manufactured T cell population is co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3;1 to 1:3, and exposed to IL-7, IL-15, or both IL-7 and IL-15 at a concentration of 10 ng / mL of IL-7 and 10 ng / mL of IL-15, if present, and thereafter secretes at least 1000 pg / mL / 1×10 6 cells / day of IL-2. By way of example, the manufactured T cell population can secrete about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more pg / mL / 1×10 6can secrete IL-2 at the rate of [[number]] cells / day. IL-2 secretion is associated with helper-independent T cells. The high IL-2 secretion of Rapa-T cells can be advantageous by eliminating the need to administer exogenous IL-2 after adoptive T cell therapy. In some embodiments, IL-7 or IL-15, if present, is added at 10 ng / mL.

[0091] In some embodiments, the manufactured T cell population exhibits increased in vivo function compared to the control population of T cells, the in vivo function being characterized by increased human T cell engraftment in a model of xenogeneic graft-versus-host disease from human to mouse.

[0092] In some embodiments, the population of manufactured T cells shows a decrease in mTORC1 activation as measured by phospho-P70S6K. The decrease can be, by way of example and not limitation, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to T-Rapa cells at 32 hours after the start of culture.

[0093] In some embodiments, the manufactured T cell population shows a decrease in phospho-STAT5 compared to T-Rapa cells. The decrease can be, by way of example and not limitation, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to T-Rapa cells at 32 hours after the start of culture.

[0094] In some embodiments, the manufactured T cell population shows a decrease in phospho-STAT5 compared to a control population of cultured T-Rapa cells. The decrease can be, by way of example and not limitation, at least 50% compared to the control population of cultured T-Rapa cells. In some embodiments, the decrease is at 48 hours after the manufactured T cells are inoculated into the culture from the input cell population containing T cells. In some embodiments, the p-STAT5 levels are measured by Western blot.

[0095] 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 the manufactured T cells are inoculated into the culture from the input cell population containing T cells. In some embodiments, the manufactured T cell population exhibits a decrease in phospho-STAT5 as compared to a control population of T cells and as compared to at least a certain level of STAT1 and phospho-STAT1. In some embodiments, the levels of STAT1 or p-STAT1 are those measured by Western blot.

[0096] In some embodiments, the manufactured T cell population exhibits a decrease in mTORC1 activation as measured by p70S6K or Raptor expression as compared to a control population of T cells characteristic of the T cells from which the manufactured T cell population was obtained. The decrease can be, by way of example and not limitation, a 50% decrease. In some embodiments, the decrease is at 48 hours after the manufactured T cells are inoculated into the culture from the input cell population containing T cells. In some embodiments, the decrease is measured by Western blot.

[0097] In some embodiments, the manufactured T cell population exhibits approximately the same levels of Rheb, SGK1, or phosphorylated SGK1 as compared to a control population of T cells. By way of example and not limitation, the levels of Rheb, SGK1 or phosphorylated SGK1 can be within 50%, 40%, 30%, 20%, 10% or 5% of the levels of Rheb, SGK1 or phosphorylated SGK1 in T-Rapa cells, or the corresponding levels. In some embodiments, the levels are at 48 hours after the manufactured T cells are inoculated into the culture from the input cell population containing T cells. In some embodiments, the levels of Rheb, SGK1 or pSGK1 are the levels measured in a control population of T cells. In some embodiments, the levels are measured by Western blot.

[0098] In some embodiments, the manufactured T cell population, after 6 days post-manufacture without inhibitors and after expansion in culture medium, shows an increase in the secretion of at least one of IFN-γ, TNF-α, GM-CSF, and IL-2 as compared to T-Rapa cells. The increase can be, by way of example and not limitation, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more.

[0099] In some embodiments, 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 as compared to T-Rapa cells. The increase can be, by way of example and not limitation, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more.

[0100] 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 reduction in expression can be at least 25% less than the corresponding expression level in T-Rapa cells. As a further example, and not limitation, the reduction in 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 manufactured T cell population has an expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 that is 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 was 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 population of T cells characteristic of the T cells from which the manufactured T cell population was produced. It is to be understood that the expression levels of the checkpoint inhibitors are compared between the same cell types, e.g., CD4+ manufactured T cells are compared to CD4+ T-Rapa cells or CD4+ control T cells characteristic of the T cells from which the manufactured T cell population was produced.

[0101] In some embodiments, the manufactured T cells can have a 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, but not limitation, the reduced expression can be at least 25% less than the corresponding expression level in T-Rapa cells. As a further example, but 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 manufactured T cells can have an expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 that is within about 25% of the corresponding expression level in control T cells characteristic of the T cells from which the manufactured T cells were produced. By way of example, but 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 manufactured T cells were 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 are compared to CD4+ T-Rapa cells or CD4+ control T cells characteristic of the T cells from which the manufactured T cells were produced.

[0102] In some embodiments, the manufactured T cells have an increased expression of CD127 as compared to control T cells characteristic of the T cells from which the manufactured T cells were produced. By way of example, but not limitation, this increase can be at least 10%, 20%, 30%, 40%, 50% or more.

[0103] In some embodiments, the manufactured T cell population can have at least 5% CD4+ T cells that express CD127, as measured by flow cytometry. By way of example, the manufactured T cell population can have at least 5%, 6%, 7%, 8%, 9%, 10% or more CD4+ T cells that express CD127, as measured by flow cytometry. In some embodiments, the manufactured T cell population can have an increased frequency of CD4+ T cells that express CD127 as compared to a control T cell population characteristic of cells from which the manufactured T cell population was produced. By way of example, without limitation, this increase can be at least 50%, 100%, 150%, 200%, 300% or more.

[0104] As long as any of the above properties associated with the manufactured T cell population is associated with a single cell, the manufactured T cell can be characterized by that property. In any of the above embodiments, the manufactured T cell or manufactured T cell population can have two or more of the recited properties.

[0105] Method for treating cancer in a subject Patients with relapsed multiple myeloma (MM) have a limited survival period and curative therapies have been difficult. In this regard, patients with relapsed MM are suitable for novel T cell therapies.

[0106] Immunotherapy differs from existing approaches to immunotherapy in several important categories. First, the manufactured T cell product is manufactured to be inhibited at the level of the mammalian target of rapamycin (mTOR) pathway, which changes the resistance to apoptosis and changes the enrichment for central memory differentiation. Second, the manufactured T cell product is CD4 + Th1 and CD8 +It is produced with high-dose IFN-α that promotes Tc1 differentiation. Third, the produced T cell product is minimally co-stimulated or not co-stimulated with monoclonal antibodies and expresses a diverse T cell receptor (TCR) repertoire. Therefore, the anti-tumor effect mediated by the produced T cells is expected to occur mainly through in vivo clonal expansion against tumor antigens. Such a mechanism may be preferable in multiple myeloma where the tumor antigen is unknown or variable over time due to a high tumor mutation rate. Evaluation of the characteristics of the T cell response that appears in vivo is important for improving the understanding of the potential mechanisms of the produced T cell therapy and will be evaluated as a secondary objective of this study. Fourth, the produced T cell therapy is evaluated with a novel immune depletion and immunosuppression regimen that combines pentostatin and low-dose dose-adjusted cyclophosphamide (PC regimen). This PC regimen relatively spares myeloid cells, thereby enabling repeated treatment cycles without substantial neutropenia. This regimen is advantageous from the perspectives of cost (it can be administered outpatient) and safety (reduction of the infection rate due to myeloid cell preservation). Multiple myeloma is a disease highly promoted by inflammatory signaling. Therefore, the inflammation suppression mediated by the PC regimen is one component contributing to the efficacy of the regimen. Each of these factors was considered during the design of clinical trials focusing on multiple infusions of the produced T cells after PC conditioning.

[0107] The manufactured T cells express a significantly reduced level of checkpoint inhibitors, thus providing a novel ex vivo method for liberating the immune system from checkpoint inhibition currently achieved by monoclonal antibody therapy. Along this line, the manufactured T cell therapy is expected to succeed 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 is relatively toxic in multiple myeloma patients, thereby creating a need for alternative approaches to avoid immune checkpoints such as the manufactured T cell therapy.

[0108] In addition, the ability of the manufactured T cells to undergo extensive clonal expansion to a wide variety of tumor antigens predicts that tumor cells with increased mutation rates and tumors with microsatellite instability are particularly sensitive to the manufactured T cell therapy.

[0109] The present disclosure provides a method for treating cancer, including administering the manufactured T cells of the present disclosure in a therapeutically effective dose.

[0110] In some embodiments, the method for treating cancer includes administering to the subject a composition comprising the manufactured T cells in a therapeutically effective dose. In some embodiments, the administration of the composition comprising the manufactured T cells can be repeated either at a therapeutically effective dose or to cumulatively achieve a therapeutically effective dose. In some embodiments, the method further includes collecting autologous cells from the subject prior to administering the immune depletion regimen to the subject. In some embodiments, the method further includes collecting autologous cells from the subject prior to administering the composition comprising the manufactured T cells to the subject.

[0111] In any of the above embodiments, the immune depletion regimen can include administering to the subject at least one of pentostatin and cyclophosphamide. In some embodiments, pentostatin is administered to the subject, and the dose of pentostatin is 0.5 - 4 mg / m 2 、0.5 - 3 mg / m 2 、0.5 - 2 mg / m 2 、0.5 - 1 mg / m 2 、1 - 4 mg / m 2 、2 - 4 mg / m 2 、or 3 - 4 mg / m 2 and can be. 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 / m 2 、2.5 mg / m 2 、3 mg / m 2 、3.5 mg / m 2 、or 4 mg / m 2 is. In some embodiments, cyclophosphamide is administered to the subject, and the dose of cyclophosphamide can be 50 - 400 mg, 50 - 300 mg, 50 - 200 mg, 50 - 100 mg, 100 - 400 mg, 200 - 400 mg, 300 - 400 mg, 200 - 300 mg, or 100 - 200 mg. As a non-limiting example, the dose of 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 a single composition. In some embodiments, the single composition is administered intravenously to the subject.

[0112] In any of the above embodiments, the immune depletion regimen can include 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 to 4 mg / m2 of pentostatin, 0.5 to 4 mg / m 2 , 0.5 to 3 mg / m 2 , 0.5 to 2 mg / m 2 , 0.5 to 1 mg / m 2 , 1 to 4 mg / m 2 , 1 to 3 mg / m 2 , 1 to 2 mg / m 2 , or 3 to 4 mg / m 2 . By way of non-limiting example, the dose of pentostatin is about 1 mg / m 2 , 1.5 mg / m 2 , 2 mg / m 2 , 2.5 mg / m 2 , 3 mg / m 2 , 3.5 mg / m 2 , or 4 mg / m 2 . In some embodiments, the second composition comprises cyclophosphamide and is administered at a dose of 50 to 400 mg, 50 to 300 mg, 50 to 200 mg, 50 to 100 mg, 100 to 400 mg, 200 to 400 mg, 300 to 400 mg, 200 to 300 mg, or 100 to 200 mg of cyclophosphamide. By way of non-limiting example, the dose of cyclophosphamide is about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg.

[0113] In any of the above embodiments, the therapeutically effective dose is 1×10 5 to 5×10 6 , 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 of the produced T cells per kg of the subject's body weight.6 cells / kg, 1×10 5 ~2.5×10 5 cells / kg, 2.5×10 5 ~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. In any of the above embodiments, the composition containing the manufactured T cells is administered to the subject by injection.

[0114] In any of the above embodiments, the cancer can be selected from the group consisting of, but not limited to, multiple myeloma, renal cell cancer, 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, but not limited to, 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 quad or penta 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, the subject has, by way of example and not limitation, multiple myeloma that has relapsed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the subject has smoldering multiple myeloma. In some embodiments, the subject has quad or penta refractory multiple myeloma. In some embodiments, the subject has, by way of example and not limitation, multiple myeloma that is resistant to 1, 2, 3, 4, 5 or more treatments.

[0115] In some embodiments, the subject has been previously treated and is currently at the time of the second or third relapse after receiving a different treatment regimen selected from the group consisting of administration of a proteasome inhibitor, administration of an immunomodulatory agent, 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 random assignment to a control cohort to receive standard chemotherapy for second or third relapse patients is justified.

[0116] In another embodiment, since the subject is highly refractory to multiple standard drugs, a randomized clinical trial is not justified. Rather, such highly refractory patients are treated with Rapa-T therapy in a single-arm phase II clinical trial. The highly refractory state can be quantified by the name of quad- or penta-refractory, whereby such a subject is refractory to 4 or 5 of the top drugs used in the treatment of multiple myeloma, namely bortezomib, carfilzomib, lenalidomide, pomalidomide, and daratumumab.

[0117] In the case of embodiments related to a phase III clinical trial for treating MM at the second or third recurrence, the primary study objective is related to progression-free survival as a study endpoint, and the progression-free state is defined as having an increase in the difference in M protein / free light chain of less than 25% when the subject is monitored monthly.

[0118] In some embodiments, the first treatment cycle has a duration of at least 28 days. In some embodiments, each of the one or more additional treatment cycles has a duration of at least 35 days.

[0119] 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 days 1, 4, 8, and / or 11 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 days 1, 2, 3, 4, 5, 8, 9, 10, 11, and / or 12 of the first treatment cycle.

[0120] In any of the above embodiments, each of the one or more additional treatment cycles is at 0 to 4 week intervals. In any of the above embodiments, the first treatment cycle and the first cycle of the one or more additional treatment cycles are at 0 to 4 week intervals. In any of the above embodiments, the step of administering the composition comprising the manufactured T cells to the subject at a therapeutically effective dose is performed on the 15th, 16th, 17th or 18th day of each of the one or more additional treatment cycles.

[0121] 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, an immunomodulatory agent, an alkylating agent, a CD38 monoclonal antibody, and a glucocorticoid.

[0122] In some embodiments, the subject is quad or penta refractory in the late stage of MM relapse, such that there is no standard therapy.

[0123] In some embodiments related to the Phase 3 clinical trial, the primary study objective is related to progression-free survival, and progression-free is defined as an increase of less than 25% in the difference in M-protein / free light chain between treatments.

[0124] In some embodiments, the method comprises subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or terminally senescent effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally senescent effector T cells, and after the immune depletion regimen, administering to the subject a composition comprising the manufactured T cells at a therapeutically effective dose.

[0125] In some embodiments, the immune depletion regimen comprises administering pentostatin to the subject, administering cyclophosphamide to the subject, and the subject's creatinine clearance is ≧30 mL / min / 1.73m 2If so, administering to the subject one or more additional doses of pentostatin, and if the subject's absolute lymphocyte count is 50 or more per microliter and the subject's absolute neutrophil count is 500 or more per microliter, administering to the subject one or more additional doses of cyclophosphamide, are included.

[0126] In some embodiments, the step of measuring the subject's CrCl and adjusting the dose of pentostatin administered is performed on days 1, 4, 8, and / or 11 of the immunosuppressive regimen.

[0127] In some embodiments, the step of measuring ALC and ANC and adjusting the dose of cyclophosphamide administered is performed on days 1, 2, 3, 4, 5, 8, 9, 10, 11, and / or 12 of the immunosuppressive regimen.

[0128] In some embodiments, the step of administering to the subject a composition comprising the manufactured T cells at a therapeutically effective dose after the immunosuppressive regimen is performed 15 to 18 days after the start of the immunosuppressive regimen.

[0129] In any of the above embodiments, administering an immune depletion regimen to the subject to reduce at least a portion of regulatory T cells and / or terminally senescent effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally senescent effector T cells, and after the immune depletion regimen, administering to the subject a composition comprising the manufactured T cells in a therapeutically effective dose, are repeated at least 2 times. In any of the above embodiments, administering the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or terminally senescent effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or terminally senescent effector T cells, and after the immune depletion regimen, administering to the subject a composition comprising the manufactured T cells in a therapeutically effective dose, can be repeated up to 8 times or more. In any of the above embodiments, each step of administering to the subject a composition comprising the manufactured T cells in a therapeutically effective dose after the immune depletion regimen can be at intervals of 0 to 9 weeks.

[0130] Also, in any of the above embodiments where co-stimulation with anti-CD3 / anti-CD28 antibody is performed, it should also be understood that this co-stimulation can be provided with any form of anti-CD3 / anti-CD28 antibody. By way of example, but not limitation, if it is shown that co-stimulation is performed by using anti-CD3 / anti-CD28 beads, anti-CD3 / anti-CD28 nanoparticles or microparticles can be used.

Examples

[0131] The following examples are provided to better illustrate the methods and resulting manufactured T cells of the present disclosure. These examples are not intended to limit or otherwise modify the scope of the methods, cells, and compositions disclosed herein.

[0132] Example 1 Use of anti-IL-2 receptor blockade and mTOR blockade for Th1 enrichment. In the case of adoptive T cell therapy, regulatory T (T REG)It is important to produce T cells that preferentially express the Th1 phenotype while minimizing contamination from cells with the cell phenotype. Th1 cells can be partially characterized by the expression of the cell fate transcription factor TBET, while T REG cells express the FoxP3 transcription factor.

[0133] A method for promoting TBET while restricting FoxP3 was evaluated. The mTOR inhibitor temsirolimus, an FDA-approved drug administered intravenously for the treatment of refractory renal cell carcinoma, was evaluated. Generally, since inhibition of mTOR has been associated with the promotion of T REG phenotype T cells, it may seem contradictory to use an mTOR inhibitor to produce T cells enriched for the Th1 phenotype. Current experiments are advantageous compared to previous studies because temsirolimus is an intravenous formulation and thus not very suitable for cell culture due to limited solubility in the medium compared to rapamycin.

[0134] Current experiments also differed from past studies because they evaluated the combination of temsirolimus and daclizumab, an anti-IL-2 receptor monoclonal antibody. Both daclizumab and basiliximab are FDA-approved monoclonal antibodies and have a common mechanism of action, so they can be used interchangeably in the developed system.

[0135] As shown, T cells were cultured ex vivo using various ratios of anti-CD3, anti-CD28 beads to T cells (1:1 or 1:12), with or without the mTOR inhibitor temsirolimus (1 μM), the anti-IL-2 receptor monoclonal antibody daclizumab (5 or 50 μg / ml), and either the Th1 polarization cytokine (IFN-α, 10,000 IU / ml) or control regulatory T cell polarization (IL-2 + TGF-β). On day 6 of culture, T cells were evaluated for intracellular expression of the regulatory T cell transcription factor FoxP3 and the Th1 transcription factor TBET. (Figures 1A - 1B).

[0136] In the setting of type I polarized cytokine IFN-α, the addition of temsirolimus (at a concentration of 1.0 μM) was found to decrease T cell expression resulting in FoxP3, as compared to the T cell population seeded on day 0 (see Figure 1A). Furthermore, blockade of the IL-2 receptor further decreased FoxP3 expression, and the use of the antibody at 50 μg / ml was more effective than the use of the antibody at 5 μg / ml, thereby showing a dose-response relationship. When exogenous IL-2 was added in combination with IFN-α, temsirolimus was not effective in decreasing FoxP3 expression (Figure 1A), and furthermore, when the culture conditions were permissive for T REG cell differentiation, i.e., a decrease in the bead ratio to 1:12, removal of IFN-α from the culture; as well as the addition of exogenous IL-2 + TGF-β (Figure 1A), temsirolimus and daclizumab were not effective in restricting FoxP3 expression.

[0137] In addition to restricting FoxP3 expression, the combination of temsirolimus and anti-IL-2 receptor monoclonal antibody was effective in promoting the Th1 phenotype, as indicated by an increase in the expression of TBET (Figure 1B). Again, there was a dose-response relationship, and daclizumab at 50 μg / ml promoted TBET expression to a greater extent than 5 μg / ml (Figure 1B). The addition of IL-2 to IFN-α polarization increased TBET, but this was also associated with an increase in FoxP3, thereby showing a lack of Th1 purity with the addition of exogenous IL-2. Notably, T REG control conditions, IL-2 and TGF-β decreased the expression of TBET, as expected (Figure 1B).

[0138] Therefore, the combination of mTOR inhibition and IL-2 receptor blockade represents a new approach for Th1 cell generation.

[0139] Development of combinations of interventions to inhibit T cells during ex vivo manufacture: mTOR inhibition; IL-2 receptor blockade; reduction of T cell co-stimulation; and inhibition of T cells prior to co-stimulation (overnight pre-incubation). Considering these results demonstrating that the combination of mTOR inhibition and IL-2 receptor blockade can improve the production of Th1-type cells (improvement of the TBET to FoxP3 ratio, T REG cell contamination limitation), additional interventions were considered to potentially improve the production of Th1 cells.

[0140] One advantage of the use of mTOR inhibition in adoptive T cell therapy efforts is that this intervention can promote the production of T cells with more primitive differentiation states, such as the T central memory subset (T CM ) or the T stem cell memory subset (T SCM ). Previous studies using rapamycin ex vivo have found that ex vivo rapamycin is effective in the production of T CM -phenotype T cells; this result is consistent with the known role of mTOR in the control of T cell memory states. It is important to promote the T CM and / or T SCM state during manufacture because T cells with more primitive differentiation states have increased long-term engraftment after adoptive transfer and mediate increased in vivo effects in experimental models. Several other methodologies for promoting the production of T cells with limited differentiation states have been described, including the use of GSK3 inhibitors to promote WNT signaling, inhibition of AKT signaling, and inhibition of PI3 kinase signaling.

[0141] We developed a method of incubating seeded T cells in X-Vivo 20 medium supplemented with 5% human AB serum lacking exogenous cytokines and containing the mTOR inhibitor temsirolimus and IL-2 receptor blockade via addition of monoclonal antibody. This method incorporated an approximately 16-hour "pre-incubation" prior to co-stimulation with anti-CD3, anti-CD28-coated magnetic beads. To our knowledge, this method has not been reported previously.

[0142] As a first step in evaluating these increasingly stringent culture conditions, CD4, as defined by the presence of viable cells at the end of the culture period (a 6-day culture period in the case of Th1 production), + and CD8 + T cell cultures were evaluated for their viability under various conditions. In Figures 2A-2B, as shown, CD4 + and CD8 + T cells were placed in culture under various conditions. The ratio of 3 / 28 beads to T cells was either 3:1, 1:1, or 1:3. T cells were co-stimulated either simultaneously with addition to the culture ("without overnight pre-incubation") or after an overnight 16-hour pre-incubation. 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 either 1.0 or 0.1 μM, or the control mTOR inhibitor rapamycin was added at a concentration of 1.0 μM. Most cultures were also supplemented with IFN-α (10,000 IU / ml), a type I cytokine promoter. As shown, most of the 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 the culture ("day 0 input culture").

[0143] As shown in Figures 2A-2B, changing the culture conditions to include not only mTOR inhibition (use of temsirolimus at either 1.0 or 0.1 μM, control use of rapamycin at 1.0 μM) and IL-2 receptor blockade (daclizumab), but also a decrease in co-stimulation (decrease from a 3:1 bead to T cell ratio to 1:1 and 1:3 ratios) and an overnight pre-incubation resulted in a similar number of viable T cells being obtained compared to control T cell cultures.

[0144] Importance of pre-incubation and high-dose temsirolimus in Th1 / Tc1 production. At the time of cryopreservation of the Th1 / Tc1 cell product (end of culture), the T cells were T CMIn addition to the phenotype, it is important to have a relatively quiescent phenotype. That is, it has previously been found that T cells generated with rapamycin secreted very small amounts of cytokines upon adoptive transfer but resulted in large amounts of cytokines in vivo. Notably, other cells have identified a similar inverse correlation between cell product effector function (minimal) and in vivo effector function (maximal). T cell quiescence at the time of adoptive transfer can improve post-transfer T cell survival and may also be important to reduce the risk of cytokine release syndrome, a cause of morbidity and mortality after other forms of adoptive T cell therapy, particularly gene-modified chimeric antigen receptor (CAR) T cell therapy.

[0145] To evaluate this, we tested the cytokine secretion potential of T cells produced at the end of cell culture (day 6) and then again 1 week after ex vivo expansion after growth in medium containing maximal costimulation (3 / 28 bead-to-T cell ratio, 3:1) and no inhibitors.

[0146] In FIGS. 3A-3B, CD4 + and CD8 +T cells were purified and cultured for 6 days either with or without a 16-hour pre-incubation interval prior to co-stimulation (1:1 ratio of 3 / 28 beads to T cells). As shown, temsirolimus was added at either a concentration of 1.0 or 0.1 μM, and 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 3:1 ratio of 3 / 28 beads and the supernatant was tested for cytokine content by Luminex assay (results are reported as pg per ml of cytokine secreted per million cells per 24 hours). Additionally, the resulting T cells were co-stimulated at a 3:1 ratio of 3 / 28 beads and maintained in culture for 1 week using medium without exogenous cytokines or inhibitors. After this T cell culture, T cells were collected on day 13 of culture, re-stimulated with 3 / 28 beads (ratio 3:1), and the 24-hour supernatant was evaluated for cytokine content as described above. The abbreviation N.A. indicates not applicable (insufficient T cell yield to perform the assay).

[0147] The results are shown in FIGS. 3A - 3B, with IFN-γ secretion shown in FIG. 3A and TNF-α secretion shown in FIG. 3B. In both cases, the T cell cytokine potential on day 6 is shown in the left panel and the T cell cytokine potential on day 13 is shown in the right panel.

[0148] These data indicate that high-dose temsirolimus (1.0 μM) resulted in very low, desirable levels of T cell IFN-γ and TNF-α secretion on day 6, 24 hours after maximal co-stimulation, under both overnight pre-incubation conditions and without pre-incubation. Notably, the use of temsirolimus at a concentration of 0.1 μM only partially decreased the T cell cytokine secretion potential on day 6. Therefore, in our method, temsirolimus needs to be used at a higher concentration, i.e., at least 1 μM. These data also indicate that an overnight pre-incubation intervention alone is not sufficient to yield a resting T cell phenotype. Therefore, to achieve the full desired results, the overnight pre-incubation step needs to be used in combination with high-dose temsirolimus.

[0149] Furthermore, this method results in the desired inverse correlation between initial T cell quiescence and the subsequent increase in effector function that occurs after restimulation. That is, for both the day 13 T cell values of IFN-γ and TNF-α secretion, the condition of the lowest level of cytokine potential on day 6 (combination of pre-incubation + high-dose temsirolimus) resulted in the highest values of cytokine secretion on day 13. Notably, the condition consisting of high-dose temsirolimus without overnight pre-incubation did not result in a sufficient yield to evaluate cytokine secretion potential on day 13 of culture, thus further supporting the value of the high-dose temsirolimus + pre-incubation step for Th1 / Tc1 cell generation.

[0150] The new combination method results in enhanced mTOR inhibition and suppression of STAT5 phosphorylation. The ability of the new combination method (mTOR inhibition, IL-2 receptor blockade, pre-incubation for delayed co-stimulation, and use of lower-intensity co-stimulation) to produce T cells of the desired phenotype depends in part on the increased ability to control the molecular and cellular events previously associated with the rapamycin-resistant T cell phenotype.

[0151] One component of this phenotype is the control of mTOR-dependent signaling events that occur at the 4EBP1 level and that serve to control protein translation. To address this, the T-Rapa method was used to generate Th1 / Tc1 cells, which incorporated the simultaneous addition of T cells to cultures supplemented with high levels of costimulation (3 / 28 bead to T cell ratio, 3:1), high-dose rapamycin (1.0 μM), and cytokine addition (IL-2 + IFN-α). In comparison with controls, we generated T cells using the novel combination method of the present disclosure (16-hour preincubation step, costimulation at reduced levels (1:1 ratio), temsirolimus (1.0 μM) and daclizumab (50 μg / ml), and addition of IFN-α only without IL-2).

[0152] In Figure 4, CD4+ and CD8+ T cells were cultured using our previous methodology ["T-Rapa": simultaneous T cell addition to cultures by addition of high levels of costimulation (3 / 28 bead to T cell ratio, 3:1), high-dose rapamycin (1.0 μM), and cytokine addition (IL-2 + IFN-α)], or the novel combination methodology for the generated T cells ["Rapa-T": 16-hour preincubation step; use of both temsirolimus (1.0 μM) and daclizumab (50 μg / ml) at reduced levels of costimulation (1:1 ratio), and addition of IFN-α only without IL-2]. At 16 and 32 hours of T cell culture, a portion of the T cells was collected, proteins were isolated, and Western blot analysis of the housekeeping gene β-actin and the mTOR pathway molecule phospho-4EBP1 was quantified. The results were compared to proteins obtained from the input T cells on day 0 prior to any T cell activation.

[0153] As shown in Figure 4, the novel combination method for generating the produced T cells ("Rapa-T" in Figure 4) resulted in a decrease in the activation of the mTOR pathway (measured by phosphorylation of 4EBP1) compared to T cells produced using our aforementioned method ("T-Rapa") at both 16 and 32 hours of culture.

[0154] P70S6 kinase is an additional important molecule in the mTOR pathway. In Figures 5 and 6, CD4 + and CD8 + T cells were cultured using our previous methodology ["T-Rapa": high-level costimulation (3 / 28 bead-to-T cell ratio, 3:1), high-dose rapamycin (1.0 μM), and simultaneous T cell addition to culture by addition of cytokines (IL-2 + IFN-α), or a new combinatorial methodology for generating engineered T cells ["Rapa-T": 16-hour preincubation step; costimulation at reduced levels (1:1 ratio), use of both temsirolimus (1.0 μM) and daclizumab (50 μg / ml), and addition of IFN-α only without IL-2]. At 16 and 32 hours of T cell culture, a portion of the T cells was collected, 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 proteins obtained from the input T cells on day 0 prior to any T cell activation.

[0155] In favorable contrast, production using the new combinatorial method for generating engineered T cells significantly blunted the levels of P70S6K (Figure 6, Rapa-T condition). These data provide further evidence that the combinatorial method of Th1 / Tc1 production provides improved control over mTOR activation. Substantial upregulation of P70S6K was found in both 16- and 32-hour T cell cultures using the previous production method for generating rapamycin-resistant T cells (Figure 5, T-Rapa condition).

[0156] The combination method was associated with an improvement in the purity of Th1 cells (a reduction in contamination by FoxP3-expressing cells). In previous manufacturing methods, substantial STAT5 phosphorylation occurred (Figure 6, T-Rapa was obtained with 16-hour and 32-hour cultures), and in contrast to the control, the combination approach abrogated STAT5 phosphorylation (Figure 6, Rapa-T was obtained with 16-hour and 32-hour cultures).

[0157] Therefore, the new combination method is also advantageous with respect to increased control of mTOR signaling during T cell manufacturing and abrogation of signaling events that promote contamination by T REG cells (control of STAT5 phosphorylation).

[0158] Further description of the individual components of the combination method for Th1 / Tc1 cell generation. Additional cultures were established to obtain additional information regarding the individual contributions of culture interventions to the resulting Th1 / Tc1 phenotype. In Figures 7-10, CD4 + and CD8 + T cells were cultured using various 3 / 28 bead ratios, various methods of mTOR inhibition, variable addition of anti-IL-2 receptor blockade, variable addition of the type I polarization cytokine IFN-α, and variable use of an initial overnight pre-incubation step, as shown in Figures 7-10. Supernatants generated by repeated co-stimulation (3:1 bead ratio) were collected on days 6 and 13 of culture and tested for IFN-γ (Figures 7A-7B), TNF-α (Figures 8A-8B), GM-CSF (Figures 9A-9B), or IL-2 (Figures 10A-10B) content by Luminex assay (results are expressed as pg / ml per million cells per 24 hours).

[0159] Figures 7A-7B show the IFN-γ secretion results at the end of culture (day 6) and one week after further culture in the absence of inhibitor (day 13). The desired phenotype is composed of a relatively low cytokine secretion value at day 6 and a relatively high cytokine value at day 13. In Figures 7A-7B, the culture conditions containing each of the combined elements (low-level co-stimulation [1:1 ratio], delayed co-stimulation after overnight pre-incubation, addition of the polarized cytokine IFN-α, addition of an mTOR inhibitor [in this experiment, use of a sub-optimal concentration of 0.1 μM], and inclusion of the anti-IL-2 receptor antibody daclizumab) demonstrated that this condition had a desirable phenotype in that it decreased IFN-γ secretion at day 6 but was high in IFN-γ secretion at day 13. T cell culture conditions omitting one or more of these elements tended to have high cytokine values at day 6 and / or low values at day 13. Additionally, previous methods for producing rapamycin-resistant T cells (T-Rapa; Figures 7A-7B) expressed a less favorable pattern of cytokine secretion (high values at day 6, low values at day 13).

[0160] The combined method for Th1 / Tc1 production also yielded a favorable cytokine phenotype when the following cytokines were evaluated (combined with a decrease in the value at day 6 and an increase in the value at day 13): TNF-α (Figures 8A-8B), GM-CSF (Figures 9A-9B), and IL-2 (Figures 10A-10B).

[0161] Collectively, these results provide further evidence that the new method of T cell production produced has significant advantages compared to the previous T-Rapa method.

[0162] Molecular changes associated with Th1 / Tc1 cell production using a combinatorial methodology. We conducted additional experiments to characterize the molecules that are altered during Th1 / Tc1 cell production using a combinatorial methodology. Such information is valuable not only because it may lead to an improved understanding of the T cell phenotype, but also because such information can be utilized during manufacturing as a quality control element. In addition, such information can be used during the screening of additional combinatorial steps that can be used in future manufacturing efforts.

[0163] In previous efforts, it was found that rapamycin-resistant T cells underwent autophagy during T cell production. Autophagy has long been known to be a direct result of mTOR inhibition: when mTOR is activated, T cells maintain a growth and proliferative state (the autophagy signal is off), in contrast, when mTOR is inhibited, autophagy is promoted, thereby resulting in a decrease in T cell volume, including a decrease in mitochondrial volume (mitophagy). Indeed, autophagy is an essential homeostatic process in T cell biology and is related to T cell health because it can reduce energy requirements and eliminate intracellular organelles and other cellular debris.

[0164] In Figures 11-13, human CD4 + and CD8 + T cells were cultured using a 16-hour pre-incubation interval, after which 3 / 28 beads were added at a reduced ratio of 1:3 beads to T cells. As shown in Figures 11-13, various T cell cultures were subjected to various methods of mTOR inhibition (1.0 μM rapamycin, 1.0 or 0.1 μM temsirolimus), various conditions of exogenous IL-2 addition, and various conditions related to the addition of the anti-IL-2 receptor monoclonal antibody daclizumab. After a 16-hour pre-incubation interval, cells were collected 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 blot.

[0165] We evaluated the ability of a combinatorial method to promote autophagy as measured by T cell expression of the autophagy marker p62. As shown in Figure 11, a combinatorial method that included a preincubation step, low-level costimulation (1:3 3 / 28 bead-to-T cell ratio), daclizumab blockade of the IL-2 receptor, and an mTOR inhibitor upregulated the autophagy marker p62. Autophagy could be achieved by mTOR inhibition with rapamycin (1 μM) or temsirolimus (1.0 or 0.1 μM) when each of these factors was present. Exclusion of IL-2 receptor blockade from the regimen substantially blunted the induction of autophagy.

[0166] Furthermore, the combinatorial method resulted in improved inhibition of the mTOR pathway as shown by decreased expression of phosphorylated Raptor (Figure 12). Notably, a lower level of phospho-Raptor was obtained with the combinatorial method incorporating high-dose temsirolimus compared to when low-dose temsirolimus or high-dose rapamycin was used.

[0167] We also evaluated the produced T cell expression of BIM, an apoptosis-promoting member of the bcl-2 gene family. Since bcl-2 family members act mainly at the mitochondrial level, mitophagy can affect the balance of bcl-2 family member genes that help determine the apoptotic threshold. Mitophagy has been shown to be beneficial in lowering the apoptotic threshold and potentially selectively eliminates mitochondria with an unfavorable balance of bcl-2 family molecules. We found that the combinatorial production method resulted in decreased expression of BIM (Figure 13).

[0168] In summary, these experiments show that enhanced autophagy, decreased mTOR signaling, and decreased apoptotic promoting molecule expression are associated with a combinatorial method of Th1 / Tc1 cell production. These changes are likely to contribute to an increase in the in vivo function of the produced T cells and can thus be used as a quality control step or to screen for future next-generation methods of T cell production.

[0169] The combinatorial method promotes T cell quiescence and T cell differentiation. We performed additional experiments to characterize the surface phenotype of Th1 / Tc1 cells produced by a combinatorial method defined by a preincubation step, low-level costimulation (1:3 3 / 28 bead-to-T cell ratio), daclizumab blockade of the IL-2 receptor, and incorporation of an mTOR inhibitor. First, we evaluated the effect of culture variables on the T cell expression of CD45 isoform RA, a marker of T cell naivety that includes T cells of the stem cell memory subset. Thus, it is desirable to develop a T cell production method that maintains or increases the expression of CD45RA.

[0170] For FIGS. 14A-15D, CD4 + and CD8 + T cells were cultured using various 3 / 28 bead ratios, various methods of mTOR inhibition, variable addition of anti-IL-2 receptor blockade, variable addition of type I polarized cytokine IFN-α, and variable use of an initial overnight preincubation step as shown in FIGS. 14A-15D. On day 6 of culture, the T cells were collected and evaluated by flow cytometry for the expression of CD45RA (FIGS. 14A-14D) or CD62L, CCR7, and CD127 (FIGS. 15A-15D) in the CD4 cell subset, and the results were compared to the expression levels on day 0 of culture ("day 0 input culture").

[0171] As shown in FIGS. 14A-14D, the production of T cells without the key elements of the combination method (use of high co-stimulation at a bead-to-T cell ratio of 3:1, without the use of daclizumab, without the use of mTOR inhibitors) results in a rapid deterioration of CD45RA expression (see column #2). In contrast, the use of all of these components resulted in the complete preservation of CD45RA expression (see column #4). Notably, T cells expanded using the previously identified production methods we had did not optimally preserve CD45RA expression (T-Rapa cells, FIG. 14B, column #3).

[0172] In addition, it was evaluated whether the combination method resulted in other markers of reduced T cell differentiation, including CD62L, CCR7, and CD127. FIG. 15A (column #4) shows that T cells produced using the pre-incubation step, low levels of co-stimulation, antibody blockade of the IL-2 receptor, and mTOR inhibitors increased the co-expression of these T cell markers. Notably, T cells expanded using the previously identified production methods we had did not optimally increase the expression of these three memory markers (T-Rapa cells, FIG. 15A, column #3).

[0173] Therefore, the combination method for Th1 / Tc1 cell production is advantageous in producing T cells in a restricted differentiation state and clearly and reproducibly shows mediating an increase in in vivo effects.

[0174] The combination method is optimized by reducing the T cell purity at the start of culture. In the case of T cell manufacturing, it is important to determine whether the starting culture population must be highly purified for T cell content or whether accessory cell populations such as monocytes can be tolerated. From the perspective of economic cost and labor, it is generally desirable to start the culture with a less highly purified population. However, cell populations present at the start of culture can have an adverse effect on T cell proliferation or the generation of the desired T cell phenotype. To evaluate this parameter, cultures were initiated using the combination method with starting populations that were either 100%, 66%, 33%, or 10% pure for T cell content (the remaining cell populations were mainly monocytes).

[0175] In FIGS. 16-20, prior to the start of culturing, the input cell population was adjusted such that the purity of T cells was either 100%, 66%, 33%, or 10%, and the remaining cell population was composed of non-T cell populations contained in peripheral blood mononuclear cells (mainly monocytes). As shown, T cells were grown in media variably containing or not containing temsirolimus (at either a concentration of 1.0 or 0.1 μM), and in addition, the cultures variably included a 16-hour pre-incubation or no pre-incubation prior to anti-CD3, anti-CD28 bead co-stimulation (1:3 ratio). Each of the cultures shown was grown 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 production period, the T cells received a high level of co-stimulation (3:1 bead to T cell ratio). In FIG. 16, after the high level of co-stimulation, the T cells were then grown for 1 week in inhibitor-free media. At the end of this growth period, the T cells were counted and graphed compared to the input number on day 0. In FIGS. 17-18, after the high level of co-stimulation, the T cells were grown until day 13 of culture. On both day 6 and day 13, the T cells were co-stimulated and the supernatants were evaluated for IFN-γ (FIGS. 17A-17B) or TNF-α (FIGS. 18A-18B) content over 24 hours (results are shown as pg / ml per 1 million cells per 24 hours). In FIGS. 19-20, after the high level of co-stimulation, the T cells were grown until day 13 of culture. On both day 6 and day 13, the T cells were evaluated by flow cytometry for CD25 expression (results shown are the percentage of CD4+ T cells co-expressing CD25) (FIG. 19), or for the expression of CD62L, CCR7, and CD127 (FIG. 20).

[0176] As detailed in Figure 16, cultures initiated with reduced T cell purity at the time of culture input resulted in a greater volume for T cell proliferation, and this relationship occurred in a dose-dependent manner. Notably, T cells grown using an overnight pre-incubation step had greater proliferation compared to co-stimulated T cells at the start of culture. These data provide further support for the combination method and indicate that the cell population at the start of culture, which might otherwise be considered a contaminant, actually appears to promote the potential for T cell proliferation. Thus, optimized use of the combination method should also include the use of T cells that are not highly enriched at the start of culture, and for quality assurance, it may be important to control the purity level, for example, by starting each culture with an inoculum of 66% or 33% purity with respect to the T cell content. As detailed in Figures 17A - 17B and 18A - 18B, T cells produced using the combination method and non-highly purified input T cells resulted in the desired cytokine secretion pattern, that is, (Figures 17A - 17B) decreased IFN-γ secretion at the end of production (day 6) and increased IFN-γ secretion after 1 week of growth in inhibitor-free medium (day 13), and (Figures 18A - 18B) decreased TNF-α secretion at the end of production (day 6) and increased TNF-α secretion after 1 week of growth in inhibitor-free medium (day 13).

[0177] In addition, the cell surface marker expression in T cells produced by the combination method was evaluated using an input population with reduced T cell purity. As shown in Figure 19, such T cells had decreased CD25 expression at the end of production (day 6), consistent with a resting phenotype. One week after culture without inhibitor, the T cells significantly upregulated CD25. As shown in Figure 20, such T cells also had increased co-expression of the memory markers CD62L, CCR7, and CD127, and subsequently, these markers decreased after 1 week of T cell proliferation.

[0178] In summary, these data demonstrate that the generation of Th1 / Tc1 cells using the combination method is possible using input T cells that are substantially contaminated with non-T cell populations. Indeed, the intentional inclusion of such non-T cell populations can be utilized to improve T cell yields and the resulting T cell memory profile.

[0179] Production from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells are stored in the vapor phase of liquid nitrogen until cell thawing and generation of Rapa-T cells. In the case of cells isolated by apheresis or freshly isolated by simple blood collection in the future, the cells can be processed immediately and then either placed directly into culture or cryopreserved by a controlled-rate freezing technique and stored in the vapor phase of liquid nitrogen for later use.

[0180] T cell culture from cryopreserved cell substrates instead of culturing from newly isolated cell populations requires enrichment of certain types of T cells, for example, by use of monoclonal antibodies and column technology (positive or negative selection). Enrichment of the source cells used in Rapa-T manufacturing does not require such antibody-based methodologies since T cells are efficiently enriched during the culture period. Thus, our method is consistent with recommendations for effective cell therapy at the global level. Initial processing steps for the manufacture of Rapa-T cells focus on removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (if applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and some monocytes. These steps are carried out in a relatively automated manner mainly using closed system technology, which is advantageous for reducing human error, providing detailed manufacturing data for batch records, improving consistency throughout manufacturing execution, and reducing the risk of contamination of the final product with infectious agents. The processing of Rapa-T products incorporates the following steps: (1) thawing of cryopreserved products (if applicable) using a solid, non-aqueous method to reduce contamination with infectious agents, (2) automated washing of the cell product using a LOVO permeable membrane device, (3) incorporation of lysates of RBCs using ammonium chloride-potassium (ACK) buffer during the LOVO washing step, (4) volume reduction of the cell contents using the LOVO method, followed by seeding of the cells into a closed system, counterflow, centrifugal elution (CCE) device (Elutra, Terumo), and (5) pre-programmed operation of the Elutra device for efficient removal of granulocytes and monocytes by CCE.

[0181] After this lymphocyte enrichment and media purification, the cells are seeded into a special chamber (G-Rex vessel, Wilson-Wolf) that has an abundant volume for oxygen exchange. In addition to enhanced gas permeability characteristics, the G-Rex vessel is a closed system unit and has the added 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.

[0182] Using certain specific culture conditions, it is possible to promote the production of a mixture of CD4+ and CD8+ T cells in a G-Rex vessel with the functional attributes of the produced T cells. These specific conditions are as follows: (1) the use of an enriched medium (including but not limited to X-Vivo 20, Lonza) further supplemented with 5% human serum; (2) incorporating a 16-hour resting period for the cells seeded in the G-Rex prior to co-stimulation (the cells are seeded at a relatively high density of 1.5×106 cells per ml); (3) during this initial resting period, the cells are optimally rested by the addition of 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 resting period, 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 1:3 bead-to-T cell ratio under sub-optimal conditions (usually, most T cell proliferation conditions utilize a 9-fold higher level of co-stimulation, a 3:1 bead-to-T cell ratio); (5) importantly, it is essential that the T cells are not washed after the initial resting period; (6) after the resting period, in addition to the addition of 3 / 28 beads, it is essential to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into the CD4+ Th1 and CD8+ Tc1 phenotypes; (7) importantly, it is important to avoid the addition of IL-2, a common additive in T cell cultures, and (8) after the addition of beads and IFN-α, it is important to leave the cells undisturbed until collection on day 6 of the culture (no cell washing, no further culture additives).

[0183] Cryopreservation of the produced T cells. 1) After 6 days of cell culture in a G-Rex vessel, the volume of the culture can be decreased in a closed-system manner by the GatheRex device. Subsequently, the cells are collected, the 3 / 28 beads are removed by a hand-held magnet, the cells are placed in a LOVO device and the cells are continuously washed to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0184] The washed cells can be reconstituted in a cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation is performed in multiple single-use aliquots in 50 ml freezer bags. Rapa-T cells are cryopreserved by a GMP-compliant controlled-rate freezing method and, after passing the designated release criteria tests, are shipped in the vapor phase of liquid nitrogen by a certified cryo-shipper.

[0185] The release criteria tests for Rapa-T cells include standard tests such as the content of CD3+, CD4+, and CD8+ T cell purity (the final product can have a CD3+ T cell content of >70% by flow cytometry, and the CD4+ and CD8+ subsets can be present at 5% levels each). The cells can have a viability of >70% as determined by flow cytometry annexin and 7-AAD assays. Additionally, the cells need to be free of bacterial and fungal contamination with a minimum culture interval of 3 days (ideally 14 days), and further, the cell product needs to be below the detection limit of bacterial LPS endotoxin.

[0186] In addition to these standard tests, the special function tests constitute the release criteria for the Rapa-T cell product. Prior to release of the product and cell therapy, the Rapa-T cells can have the following attributes compared to the cultured input T cells: (1) an enhanced T central memory phenotype 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 defined by a decrease in the level of Th1 / Tc1-type cytokine secretion upon maximal co-stimulation; (4) an autophagy signature demonstrated by a decrease in mitochondrial mass by flow cytometry MitoTracker assay, (5) a resistant phenotype demonstrated by at least 50% inhibition of mTORC1 and mTORC2 downstream targets, and (6) a multifaceted differential gene expression profile of n = 80 major transcription factors and differentiation molecules.

[0187] Figure 21 shows that the new Rapa-T method generates T cells with increased expression of naive or T central memory markers compared to T-Rapa cells, regardless of whether the new Rapa-T method uses no bead co-stimulation or low-level bead co-stimulation (a 1:3 ratio of beads to T cells). In Figure 21, Rapa-T1 cells were generated by culture in IFN-α, temsirolimus, and basiliximab either without bead co-stimulation (the first two columns of each panel) or with 1:3 bead-to-T cell co-stimulation (the third and fourth columns of each panel), as described above, and the results were compared to cultures using the previous T-Rapa method (use of rapamycin and 3:1 beads-to-T cells; the fifth and sixth columns of each panel). Flow cytometry was performed at the end of the culture, and CD4 + T cell subset (black columns) and CD8 +Detailed results were obtained for both T cell subsets (grey columns). The results shown are for the naive T cell subset (left panel) defined by CD45RA+ expression, for the T central memory subset defined by co-expression of CD62L and CCR7, and for a more primitive T cell subset co-expressing CD62L, CCR7, and CD127.

[0188] As detailed in Figure 21, the method, including conditions with no bead co-stimulation or with co-stimulation at reduced levels compared to the T-Rapa method, produced CD4 + and CD8 + T cells that had increased expression levels of naive and T central memory markers by flow cytometry compared to T-Rapa cells.

[0189] Figure 22 shows that the new Rapa-T method generates T cells with decreased expression of CD25, CTLA4, and TIM3 compared to T-Rapa cells, regardless of whether the new Rapa-T method uses no bead co-stimulation or low levels of bead co-stimulation (a 1:3 ratio of beads to T cells). In Figure 22, Rapa-T1 cells were generated by culture in IFN-α, temsirolimus, and basiliximab either without bead co-stimulation (the first two columns of each panel) or with 1:3 bead-to-T cell co-stimulation (the third and fourth columns of each panel) as described above, and the results were compared to cultures using the previous T-Rapa method (use of rapamycin and 3:1 beads to T cells; the fifth and sixth columns of each panel). Flow cytometry was performed at the end of the culture, and detailed results were obtained for both CD4 + T cell subsets (black columns) and CD8 + T cell subsets (grey columns). The results shown are for the expression of the IL-2 receptor CD25 (left panel), for the immunosuppressive molecule and T REG -associated molecule CTLA4, and for the immune checkpoint molecule TIM3.

[0190] As listed in Figure 22, CD4 produced according to the methods described in the present disclosure, including conditions where the method is without bead co-stimulation or with co-stimulation at a reduced level compared to the T-Rapa method + and CD8 + T cells, by flow cytometry, compared to T-Rapa cells, show a decrease in the expression level of IL-2 receptor CD25 related to T cell activation and T REG cell function, and a decrease in the levels of the immunosuppressive molecule CTLA4 2 and the immune checkpoint inhibitor molecule TIM3 3 4

[0191] Figure 23 shows that the new Rapa-T method, whether it does not use bead co-stimulation or uses low-level bead co-stimulation (a 1:3 ratio of beads to T cells), has a similar pattern of Th2 to Th1 polarization compared to T-Rapa cells, but generates T cells with increased quiescence. In Figure 23, Rapa-T1 cells were generated by culture in IFN-α, temsirolimus, and basiliximab either without bead co-stimulation (the first two columns in each panel) or with 1:3 bead to T cell co-stimulation (the third and fourth columns in each panel) as described above, and the results were compared to cultures using the previous T-Rapa method (using rapamycin and a 3:1 bead to T cell ratio; the fifth and sixth columns in each panel). Cytokine secretion analysis (measurement of IL-4 and IFN-γ) was performed at the end of the culture, and the results are listed at the end of T cell production (day 6) and after an additional 6 days of culture without inhibitors (day 12).

[0192] As listed in Figure 23, CD4 produced according to the methods described in the present disclosure, including conditions where the method is without bead co-stimulation or with co-stimulation at a reduced level compared to the T-Rapa method + and CD8 + ​​T cells have a similar Th2 to Th1 cytokine polarization compared to T-Rapa cells. Specifically, on day 12 of culture, the secretion level of the Th2 cytokine IL-4 is low (the values at the end of production [day 6] and after an additional culture period without inhibitor [day 12] are in the range of 100 - 200 pg / ml on both day 6 and day 12), and the secretion level of the Th1 cytokine IFN-γ is high (values of 1000 - 3000 pg / ml). Regardless of whether this method uses bead co-stimulation or uses low levels of bead co-stimulation (a 1:3 bead to T cell ratio), IFN-γ secretion under the new Rapa-T conditions at the end of production (day 6) is significantly reduced compared to the old T-Rapa conditions, thereby demonstrating the favorable characteristics of T cell quiescence in the new Rapa-T production method.

[0193] As listed in Figure 24, CD4 + and CD8 + T cells have a pattern of Th1 cytokine polarization compared to T-Rapa cells.

[0194] Figure 24 shows that the new Rapa-T method generates T cells with a favorable pattern of Th1 polarization compared to T-Rapa cells, regardless of whether the new Rapa-T method uses no bead co-stimulation or uses low levels of bead co-stimulation (a 1:3 ratio of beads to T cells). In Figure 24, Rapa-T1 cells were generated by culture in IFN-α, temsirolimus, and basiliximab either without bead co-stimulation or with 1:3 bead to T cell co-stimulation, and various control cultures, including the previous T-Rapa method (using rapamycin and a 3:1 bead to T cell ratio), were also evaluated. All cultures were at 9×10 6at a concentration of cells / ml, without bead co-stimulation, without IL-2 addition, with a delay in IFN-α addition, containing temsirolimus at a concentration of 1 μM and basiliximab at a concentration of 10 μM, and using X-Vivo 20 medium supplemented with 5% AB serum unless otherwise specified. The specific culture conditions according to the legend in the figure are as follows: Condition 1, the above-described Rapa-T method; Condition 2, serum-free Rapa-T method; 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 a high frequency of monocytes (79% of the input cells are monocytes, increased from all other cultures with approximately 10% monocyte contamination); Condition 7, Rapa-T method without monocyte contamination (<1%); Condition 8, Rapa-T method using simultaneous addition of IFN-α (without overnight delay); Condition 9, Rapa-T method containing 1:3 bead co-stimulation; Condition 10, control T cell condition (without inhibitor, 3:1 beads); and Condition 11, old T-Rapa condition, rapamycin (1 μM), IL-2 at 20 IU / ml, 3:1 beads, without overnight pre-incubation. Cytokine secretion analysis (measurement of IL-2 and IFN-γ) was performed at the end of the culture, and the results are listed at the end of T cell production (day 6) and after an additional 6 days of culture without inhibitor (day 12).

[0195] As listed in Figure 24, CD4 produced according to the methods described in the present disclosure, including conditions where the method is without bead co-stimulation or with co-stimulation at a reduced level compared to the T-Rapa method + and CD8 + T cells have a pattern of Th1 cytokine polarization compared to T-Rapa cells.

[0196] As shown in Fig. 24, Condition #1, a Rapa-T condition manufactured without beads, had a favorable high level of IL-2 secretion ability, particularly on the 12th day after T cell proliferation in the absence of an inhibitor. A similar pattern was observed in Condition #2 performed in serum-free medium, thereby indicating the ability to produce Rapa-T cells with or without serum supplementation. In particular, compared with Culture #11 (old T-Rapa condition), the increased IL-2 secretion ability indicates that the new Rapa-T manufacturing method produces T cells with a reduced differentiated precursor profile that can function in an in vivo helper-independent manner. Condition #1 was also advantageous in this regard compared with Culture #9, a new Rapa-T condition manufactured at a bead-to-T cell co-stimulation ratio of 1:3.

[0197] As shown in Fig. 24, Condition #1, a Rapa-T condition manufactured without beads, had a level close to the lower limit of detection, thereby indicating that the Rapa-T cell product was in a quiescent state and was also favorable with regard to IFN-γ secretion on the 6th day after the end of manufacturing. In comparison, Condition #5 without an inhibitor had IFN-γ secretion of approximately 4000 pg / ml on the 6th day. Similarly, the old T-Rapa condition had a high level of IFN-γ secretion of approximately 6000 pg / ml on the 6th day. It should be noted that at the end of manufacturing, there was IFN-γ secretion of approximately 200 pg / ml on the 6th day, so the new Rapa-T condition using 1:3 bead-to-T cell co-stimulation was less quiescent than Condition #1 without beads. Finally, the new Rapa-T manufacturing method (either Condition #1 without beads or Condition #9 with beads) had a good increase in IFN-γ secretion ability on the 12th day of culture after a 6-day culture interval in the absence of an inhibitor.

[0198] In summary, the new Rapa-T method can result in the production of T cells having the following phenotypic characteristics: (a) a decrease in the expression of regulatory T cell markers such as the transcription factor FOXP3 and an increase in the Th1-type transcription factor TBET, compared to input normal T cells; (b) an increase in the quiescent state, as indicated in part by a decrease in the expression of the IL-2 receptor CD25 and a decrease in the secretion of the inflammatory cytokines IFN-γ and TNF-α at the end of production, compared to the old T-Rapa method; (c) a decrease in the expression of p-STAT5 and a decrease in the levels of the mTOR pathway molecules p-rapamycin, p-4EBP1, and p70S6K, compared to the old T-Rapa method; (d) an increase in markers of autophagy, including but not limited to a change in the expression of the molecule p62, compared to input normal T cells; (e) an increase in flow cytometry markers of the naive or T central memory population, including co-expression of CD45RA, CD62L / CCR7, and co-expression of CD62L / CCR7 / CD127, compared to input normal T cells; (f) a decrease in the expression of co-inhibitory molecules, including but not limited to CTLA4, compared to the old T-Rapa method; (g) a decrease in the expression of checkpoint inhibitory receptors, including but not limited to TIM3, compared to the old T-Rapa method; and (h) 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), compared to input normal T cells, including but not limited to a changed RNA expression pattern.

[0199] Most of the phenotypic characterization of the T cell product produced according to the Rapa-T method detailed in this disclosure can be confirmed at the end of culture. However, the T cell product can be cryopreserved, and thus it is important to note that the phenotypic characterization of the T cells in the thawed state reflects the actual product that will be adoptively transferred to the subject. The Rapa-T cells in the thawed state can be characterized by the following: (a) Maintenance of a quiescent state, as indicated by low-level expression of IL-2 receptor CD25 that is equivalent between the end of day 6 of the manufacturing sample and the thawed sample; (b) The thawed sample continues to secrete low levels of the inflammatory cytokines IFN-γ and TNF-α (not increased compared to the sample collected at the end of manufacturing) compared to the end of day 6 of the manufacturing sample; (c) The thawed sample maintains an increase in flow cytometry markers of naive or T central memory populations, including co-expression of CD45RA, CD62L / CCR7, and co-expression of CD62L / CCR7 / CD127, compared to the input normal T cells; (f) The thawed sample continues to have decreased expression of co-inhibitory molecules, including CTLA4 (no increase in the thawed sample compared to the sample collected at the end of manufacturing), but not limited to these; (g) The thawed sample continues to have decreased expression of checkpoint inhibitory receptors, including TIM3 (no increase in the thawed sample compared to the sample collected at the end of manufacturing), but not limited to these; and (h) Compared to the input normal T cells, there are increased dedifferentiation markers (including, but not limited to, Nanog, KLF4, and KLF10), as well as decreased differentiation markers (including, but not limited to, perforin, granzyme B, and IFN-γ), including, but not limited to, such changed RNA expression patterns.

[0200] Manufacture from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells are stored in the vapor phase of liquid nitrogen until cell thawing and manufacture of Rapa-T cells. In the case of cells isolated by apheresis or freshly isolated by simple blood collection in the future, the cells are immediately processed and can then be placed directly into culture or cryopreserved by controlled-rate freezing techniques and stored in the vapor phase of liquid nitrogen for later use.

[0201] T cell culture from cryopreserved cell substrates instead of culture from newly isolated cell populations requires enrichment of certain types of T cells, for example, by use of monoclonal antibodies and column technology (positive or negative selection). Enrichment of the source cells used in Rapa-T manufacture does not require such antibody-based methodologies since T cells are efficiently enriched during the culture period; thus, our method is consistent with recommendations for effective cell therapy at the global level. The initial processing steps for manufacture of Rapa-T cells focus on removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (if applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and some monocytes. These steps are carried out in a relatively automated manner, mainly using closed-system technology, which is advantageous in reducing human error, providing detailed manufacturing data for batch records, improving consistency throughout manufacturing execution, and reducing the risk of infectious agent contamination of the final product. Processing of the Rapa-T product can include the following steps: (1) thawing (if applicable) of the cryopreserved product using a solid, non-aqueous method to reduce contamination by infectious agents, (2) automated washing of the cell product using a LOVO permeable membrane device, (3) incorporation of the RBC lysate using ammonium chloride-potassium (ACK) buffer during the LOVO wash step, (4) volume reduction of the cell contents using the LOVO method, followed by seeding of the cells into a closed-system, counterflow, centrifugal elution (CCE) device (Elutra, Terumo), and (5) pre-programmed operation of the Elutra device for efficient removal of granulocytes and monocytes by CCE.

[0202] After this lymphocyte enrichment and media purification, the cells can be seeded into a special chamber (G-Rex vessel, Wilson-Wolf) having an abundant volume for oxygen exchange. In addition to enhanced gas permeability characteristics, the G-Rex vessel is a closed-system unit and has the added advantage of automated closed-system media volume reduction (GatheRex liquid handling pump). The lymphocyte-enriched cells can be maintained in the G-Rex vessel for 6 days.

[0203] Using certain specific culture conditions, the production of a mixture of CD4+ and CD8+ T cells in a G-Rex vessel having the functional attributes of the manufactured T cells can be promoted. These specific conditions are as follows: (1) the use of an enriched medium (including but not limited to X-Vivo 20, Lonza) supplemented with 5% human serum or, in some embodiments, without the addition of serum, (2) incorporating a 16-hour resting period for the cells seeded in the G-Rex prior to co-stimulation (the cells are seeded at a relatively high density of 1.5×10 6 T cells per ml), (3) during this initial resting period, the cells are optimally rested by the addition of 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 resting period, 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 under sub-optimal conditions (usually, most T cell proliferation conditions utilize a 9-fold higher level of co-stimulation, a 3:1 bead-to-T cell ratio, and in some cases, it is beneficial to avoid the addition of any co-stimulation reagent), (5) importantly, it is essential that the T cells are not washed after the initial resting period, (6) after the resting period, in addition to the addition of 3 / 28 beads, it is essential to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into the CD4+ Th1 and CD8+ Tc1 phenotypes, (7) importantly, it is important to avoid the addition of IL-2, a common additive to T cell cultures, and (8) after the addition of beads and IFN-α, it is important to leave the cells undisturbed (without cell washing, without further culture additives) until collection on day 6 of the culture.

[0204] Cryopreservation of manufactured T cells. 1) After 6 days of cell culture in a G-Rex vessel, the volume of the culture can be reduced in a closed-system manner using a GatheRex instrument. Subsequently, the cells are collected, the 3 / 28 beads are removed using a hand-held magnet, and the cells are placed in a LOVO device for continuous washing to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0205] The washed cells can be reconstituted in a cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation is performed in multiple single-use aliquots in a 50 ml freezer bag. Rapa-T cells are cryopreserved by a GMP-compliant controlled-rate freezing method and shipped in the vapor phase of liquid nitrogen by a certified cryo-shipper after the Rapa-T cells pass the designated release criteria tests.

[0206] The release criteria tests for Rapa-T cells include standard tests such as the content of CD3 + , CD4 + , and CD8 + T cell purity content (the final product can have a >70% CD3 + T cell content by flow cytometry, and the CD4 + and CD8 + subsets can each be present at the 5% level). The cells can have a >70% viability as determined by flow cytometry annexin and 7-AAD assays. Additionally, the cells need to be free of bacterial and fungal contamination with a minimum culture interval of 7 days (ideally 14 days), and furthermore, the cell product needs to be below the detection limits of bacterial LPS endotoxin and mycoplasma.

[0207] In addition to these standard tests, the special function tests can constitute the release criteria of the Rapa-T cell product. Prior to release of the product and cell therapy, the Rapa-T cells can have the following attributes compared to the cultured input T cells: (1) an enhanced T central memory phenotype 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 resting state defined by a decrease in the level of Th1 / Tc1-type cytokine secretion upon maximal co-stimulation; (4) an autophagy signature demonstrated by a decrease in mitochondrial mass by flow cytometry MitoTracker assay, (5) a resistant phenotype demonstrated by at least 50% inhibition of mTORC1 and mTORC2 downstream targets, and (6) a multifaceted differential gene expression profile of n = 80 major transcription factors and differentiation molecules.

[0208] Example 2 Steady state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were enriched by excluding >95% of the unwanted contaminating neutrophil population using an automated Ficoll procedure on a GE Sepax® device. The lymphocyte-enriched cell population was then seeded into G-REX culture vessels at an initial cell density under the culture medium and serum supplementation conditions shown in Table 1 below and incubated in culture for 6 days. Condition 1 represents the pre-culture control (enriched lymphocytes after Sepax® automated Ficoll). Cultures were also initiated under variable inhibitor conditions by adding temsirolimus, sirolimus, and / or the anti-IL2 receptor monoclonal antibody basiliximab at the doses shown in Table 1. Basiliximab was added at 10 μg / mL for Conditions 2-5 and 20 μg / mL for Conditions 6-8. In some culture conditions, anti-CD3, anti-CD28 co-stimulation was received using Dynal® 3 / 28 beads at a bead:T cell ratio of 0.88:1 (Conditions 2-3 and 5-6) or 3:1 (Conditions 8-9). A cytokine consisting of either IFN-α alone (10,000 IU / mL) or a combination of IFN-α (10,000 IU / mL) and IL-2 (20 IU / mL) was added at the indicated times. Cytokine addition was either at the start of culture (Conditions 8-9) or 1 day after the start of culture (Conditions 2-7). Cultures 3-4 and 6-7 received additional medium 2 days after the start of culture and were diluted to the indicated final cell density.

Table 1

[0209] Condition 9 represents the T-Rapa product. Condition 7 was found to provide the optimal RAPA-T condition among those tested in Table 1. This condition had several important 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 anti-IL2 receptor monoclonal antibody, (5) absence of co-stimulation, (6) cytokine support with IFN-α alone (no IL-2) added 1 day after the start of culture, and (7) cell dilution on day 2 of culture.

[0210] As shown in FIGS. 25A to 25O, 6 days after culturing, the obtained T cells were collected and evaluated for the expression of specific molecules in CD4+ (indicated by the black column) and CD8+ (indicated by the gray column) T cell subsets by flow cytometry.

[0211] FIG. 25A (CD45RA+) demonstrates the importance of the RAPA-T culture conditions with respect to maintaining naive T cell marker expression in both CD4+ and CD8+ T cells as compared to the cultured input cells. As a positive control, the previous T-RAPA conditions resulted in a significant decrease in CD45RA+ cells.

[0212] FIG. 25B (CD25+) demonstrates the importance of the RAPA-T culture conditions with respect to maintaining T cell quiescence in both CD4+ and CD8+ T cells as compared to the cultured input cells. As a positive control, the previous T-RAPA conditions resulted in a significantly activated T cell state as indicated by the increase in CD25 expression.

[0213] FIG. 25C (CD28+) and FIG. 25D (ICOS+) show that the RAPA-T and T-RAPA cell products showed similar CD4+ and CD8+ T cell expression of these co-stimulatory molecules of activation.

[0214] FIGS. 25E to 25F (CD39+ and CD73+ respectively) show that the RAPA-T culture conditions resulted in a decrease in the expression of these ectonucleotidase molecules as compared to the T-RAPA conditions. These molecules exert an immunosuppressive effect by metabolizing ATP to adenosine. Therefore, the RAPA-T cell products are expected to be advantageous compared to the T-RAPA cell products for therapeutic use.

[0215] The remaining data in FIGS. 25G to 25O show that the new RAPA-T method significantly reduces the expression of molecules associated with immune senescence (KLRG1), immunosuppressive regulatory T cell phenotype (GITR), or checkpoint inhibitory functions (LAG3, PD1, 2B4, LAIR1, CTLA4, TIGIT, and TIM3). Each of the variable culture conditions associated with the RAPA-T cell product was significantly reduced in each of these molecules compared to the T-RAPA cell product. Condition 7 showed the deepest and most consistent reduction among these molecules of the tested conditions.

[0216] Example 3 Using culture conditions 1 to 8 corresponding to culture conditions 2 to 9 of Example 2, T cells were prepared as in Example 2. On the second day of the culture period, the obtained T cells were collected and evaluated by Western blot analysis (method according to the manufacturer's instructions; BioTechne Mr.Wes instrumentation) for molecules related to the mTORC1, mTORC2, and STAT pathways.

[0217] 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 shown in FIG. 26, each of the Rapa-T cell culture conditions (conditions 1 to 6) was relatively lacking in phosphorylated STAT5, and in contrast, the T-Rapa conditions showed a significant presence of phosphorylated STAT5 (conditions 7 to 8). The reduction in STAT5 phosphorylation of Rapa-T cells compared to T-Rapa cells can be over 75%.

[0218] For optimal Th1 / Tc1 type production, it is important to have active signaling through specific STAT molecules that promote type I differentiation, including STAT1. As shown in FIG. 26, each of the Rapa-T culture conditions showed detectable levels of STAT1 phosphorylation, although the levels were somewhat reduced in condition 6 (Example 2, condition 7). However, in condition 6, the level of total STAT1 also decreased.

[0219] The optimal phenotype of Rapa-T cells can also be characterized by a decrease in molecules associated with the mTORC1 pathway. Rapa-T condition 6 (corresponding to condition 7 in Example 2) was essentially lacking in the expression of the mTORC1-related molecule p70S6K. The provision of 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 the Rapa-T manufacturing process.

[0220] The optimal phenotype for Th1 / Tc1-type RAPA-T cell production can 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 condition 7 in Example 2) has the preservation of the expression of the mTORC2-related molecules total SGK1 and phosphorylated SGK1. This feature of the optimal RAPA-T cell product regarding the marked decrease in mTORC1 due to the relative preservation of mTORC2 is further exemplified by condition 6, namely, the marked decrease in the mTORC1-related subunit molecule raptor, and the relative preservation of the mTORC2-related subunit molecule rictor.

[0221] Example 4 Steady-state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were enriched by using an automated Ficoll procedure on a GE Sepax® instrument. Next, the lymphocyte-enriched cell population was seeded in G-REX culture vessels under two conditions, one corresponding to condition 7 in Table 1 and the other corresponding to condition 9 in Table 1 (T-RAPA), and incubated for 6 days in culture as in Example 2. After 6 days of culture, T cells were collected and re-seeded at a concentration of 1×106 cells / mL for 24-hour supernatant production. At the time of re-seeding, the 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. In each of these ratios, 24-hour supernatant production was performed with no cytokine addition (indicated by the “−” symbol), addition of rhuIL-2 (100 IU / mL, indicated by “+IL-2”), addition of rhuIL-7 (10 ng / mL, indicated by “+IL-7”), addition of rhuIL-15 (10 ng / mL, indicated by “+IL-15”), or addition of both rhuIL-7 (10 ng / mL) and rhuIL-15 (10 ng / mL) (indicated by “+IL-7+IL-15”). IL-2 and TNF-α secretion were measured intracellularly by known methods (Luminex) according to the manufacturer's instructions. The results are shown in FIGS. 27A–27B.

[0222] CD4+ and CD8+ T cells in an early state of differentiation, such as naive, central memory, or stem central memory subsets, may be beneficial for adoptive transfer. Such early-differentiated T cells are partially characterized by their differential responses to the major 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 feature.

[0223] Figure 27A shows the IL-2 secretion profile of the optimal RAPA-T cell product, while the lower panel shows the IL-2 secretion profile of the T-RAPA cell product. With the maximum co-stimulation challenge and without exogenous cytokine support, the RAPA-T cell product secreted approximately 5-fold higher amounts of IL-2 compared to the T-RAPA cell product. Notably, even at very low levels of co-stimulation (1:9 beads:T cell ratio), the RAPA-T cell product secreted substantial IL-2, whereas in the control, this stimulation condition in the T-RAPA state resulted in undetectable levels of IL-2. The IL-2 secretion capacity is associated with a beneficial helper-independent T cell phenotype, which is the cytokine secretion characteristic observed in T cells in the early stages of differentiation. Finally, the addition of either IL-7 or IL-15, but not under the T-RAPA condition, further enhanced the IL-2 secretion capacity in the RAPA-T condition. Thus, RAPA-T cells respond uniquely to the constitutive cytokines IL-7 and IL-15.

[0224] Figure 27B shows the TNF-α secretion profile of the optimal RAPA-T cell product, while the lower panel shows the TNF-α secretion profile of the previous T-RAPA cell product. With the maximum co-stimulation challenge and without exogenous cytokine support, the RAPA-T cell product secreted approximately equal amounts of TNF-α compared to the T-RAPA cell product. However, the addition of IL-7 or IL-15 to the co-stimulation resulted in a higher ability to secrete TNF-α in the RAPA-T condition compared to the T-RAPA condition. Thus, RAPA-T cells respond uniquely to the constitutive cytokines IL-7 and IL-15 in terms of inducing the secretion of the Th1 / Tc1 effector cytokine TNF-α.

[0225] Example 5 Human T cells were costimulated without any inhibitor (``conventional'', addition of anti-CD23 / anti-CD28 beads; 3:1 bead:T cell ratio). Alternatively, the T cells were costimulated according to the RAPA-T cell condition with costimulation provided by either anti-CD3 / anti-CD28 beads (``Dynabeads'', 1:3 bead:T cell ratio) or Cloudz® soluble costimulatory microparticles (Biot-Techne; use of Cloudz® at 20% of the manufacturer's recommended dose, 50 μL of Cloudz® stock per 1×106 cells) (``rapamycin treatment''). After 6 days of culture, the T cells were collected, adjusted to 1×10 6 cells / mL, and costimulated using anti-CD3 / anti-CD28 beads (3:1 bead:T cell ratio). The supernatant was then collected after 24 hours and tested for the content of IL-2, TNF-α, and IL-13 by Luminex assay (results are shown as pg / mL per 1×106 cells per 24 hours). Using the same protocol, cells after 6 days of culture were collected and evaluated by flow cytometry for the expression of cell surface markers including CD4, CD8, CD25, and CTLA4.

[0226] Figure 28 shows the IL-2, TNF-α, and IL-13 secretion data. As shown in Figure 28, the results between RAPA-T cells costimulated with anti-CD3 / anti-CD28 nanoparticles (``Dynabeads'') and RAPA-T cells costimulated with soluble anti-CD3 / anti-CD28 microparticles (Cloudz® reagent, Biot-Techne) are similar. These cells have a Th1 cytokine profile, as evidenced by substantial secretion of IL-2 and TNF-α with minimal secretion of the Th2-type cytokine IL-13, as described above.

[0227] Figure 29 shows the frequencies of cells expressing the cell surface markers CD4, CD8, CD25, and CTLA4, measured by flow cytometry. As shown in Figure 29, the results between RAPA-T cells co-stimulated with anti-CD3 / anti-CD28 nanoparticles (“Dynabeads”) and RAPA-T cells co-stimulated with soluble anti-CD3 / anti-CD28 microparticles (Cloudz® reagent, Bio-Techne) are similar. These cells are quiescent (as indicated by the decreased expression of CD25) and have decreased expression of checkpoint inhibitory receptors (as indicated by the decreased expression of CTLA4), as described above.

[0228] Example 6: Phase III randomized clinical trial of Rapa-T cell therapy Figure 30 shows the randomized phase 3 protocol schema. In the upper panel of Figure 30, for patients randomly assigned to Rapa-T therapy, autologous cells for Rapa-T cell manufacturing are derived from steady-state apheresis collected after randomization. The immunodepletion regimen consists of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). The first PC cycle is administered alone (without T cell therapy) at the time of trial enrollment during the T1.Rapa manufacturing interval and has a 28-day duration, and PC cycles 2, 3, 4, and 5 are administered prior to each of the four T1.Rapa cell infusions and have a 35-day duration. The lower panel of Figure 30 shows extended T1.Rapa cell therapy for stable disease. For T1.Rapa cell recipients with stable disease after cycle 4, an additional lot of T1.Rapa cells is manufactured, allowing up to four additional cycles of T1.Rapa cell therapy (cycles 6-9).

[0229] Figure 30 details the manufactured T cell therapy administered to all patients randomly assigned to the manufactured T cell cohort. This therapy includes the following: (1) collection of immune cells used as a substrate for the manufacture of manufactured T cells, obtained from either a previously harvested peripheral blood stem cell transplantation procedure or a fresh steady-state apheresis procedure; (2) enrichment of the mononuclear cell population and subsequent incubation of the mononuclear cells under manufactured T cell culture conditions; (3) cryopreservation of a single-use manufactured T cell therapeutic agent, through identity and functionality verification steps; (4) treatment of patients with the pentostatin + cyclophosphamide drug regimen (PC regimen), initially alone and then in combination with both the manufactured T cell therapy, to prepare the patients for the manufactured T cell therapy and directly promote the anti-tumor effect; and (5) specialized immune monitoring during and after treatment.

[0230] The immune depletion regimen consists of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). The first PC cycle is administered alone (without T cell therapy) at the time of study enrollment during the interval of manufactured T cell production, with a minimum duration of 28 days to allow for blood cell recovery. PC cycles 2, 3, 4, and 5 are administered prior to each of the four manufactured T cell infusions, with a minimum duration of 35 days to allow for blood cell recovery. After completion of cycle 5 of the manufactured T cell therapy, maintenance therapy is not performed. Treatment cycles can be extended beyond the indicated intervals, up to an indefinite interval, depending on the clinical situation. By way of non-limiting example, if a patient is in remission, the cycle can be delayed until evidence of disease recurrence appears. Further, additional maintenance cycles of the PC regimen and adoptive T cell therapy are presumably envisioned to maintain the patient in a state of remission, perhaps by administering 1 to 4 therapy cycles per year, or to treat the disease if recurrence occurs.

[0231] As shown in FIG. 30, for patients with diseases that are stable after 4 - cycle therapy, additional production of Rapa - T cells can be performed, thus facilitating potential therapy in additional cycles of up to a total of 9 Rapa - T cell therapy cycles.

[0232] FIG. 31 lists the details of the PC chemotherapy regimen. Each cycle of PC therapy consists of a 14 - day course immediately before the T1.Rapa cell infusion on day 15 of the cycle (T1.Rapa dose: 0.1 - 5×10 6 cells / kg). For cycle 1, pentostatin (P) is administered at a dose of 4 mg / m 2 per day on days 1, 4, 8, and 11 (intravenously), and cyclophosphamide (Cy) is administered at a dose of 200 mg per day on days 1 - 5 and 8 - 12. In subsequent cycles, the pentostatin dose is reduced to 2 mg / m 2 .

[0233] FIGS. 32A - 32C detail the nature of the control group, namely, subjects not randomly assigned to Rapa - T cell therapy who receive one of three FDA - approved triplet regimens suitable for subjects with MM at the second or third relapse, namely, the DPd regimen (FIG. 32A), the DRd regimen (FIG. 32B), or the KRd regimen (FIG. 32C).

[0234] For the control cohorts shown in FIGS. 32A - 32C, patients are enrolled and then randomly assigned at the second or third relapse of multiple myeloma (MM). Patients need to be candidates to receive one of three FDA - approved regimens for treating this patient population. Patients randomly assigned to the control cohort receive either the DPd, DRd, or KRd regimen using the published standard regimens. The specific embodiments of these standard regimens are shown below: [FIG. 32A] DPd regimen, [FIG. 32B] DRd regimen, and [FIG. 32C] KRd regimen.

[0235] Statistical evaluation of the manufactured T - cell efficacy The primary objective is to compare the progression-free survival in recipients of manufactured T cell therapy to recipients randomized to standard therapy. Secondary objectives are evaluated in a preliminary manner using descriptive statistics when making the comparison. Eligible MM patients with second or third relapse are randomized 1:1 to receive either standard therapy with an FDA-approved triplet regimen consisting of either DPd, DRd, or KRd, or adoptive T cell therapy with ex vivo manufactured autologous rapamycin-resistant Th1 / Tc1 cells (manufactured T cells). The primary study objective occurring in each cohort of N = 65 evaluable patients is to determine whether patients treated in the manufactured T cell cohort have increased progression-free survival (PFS) compared to patients treated in the standard therapy cohort.

[0236] Progression-free survival (PFS) and overall patient survival are estimated using the Kaplan-Meier method in both groups and are presented with 95% confidence intervals at each time point. The non-parametric estimates of the median survival time and its 95% confidence interval are obtained by taking the reciprocals of the Kaplan-Meier estimates. The primary efficacy outcome (PFS) is tested by a one-sided log-rank test. The final analysis is performed when 130 PFS events have occurred in the study, or when the recruitment goal is achieved and all patients have been followed for at least 12 months. Overall survival (OS) is measured from the start of treatment, and death from any cause is also an event, with patients censored on the date of their last contact.

[0237] Secondary endpoints are evaluated by descriptive means such as mean, standard deviation, confidence intervals, Kaplan-Meier analysis, or other methods that characterize the multiple myeloma remission status. The objective response rate (ORR) and minimal residual disease rate (MRD) are estimated as the observed proportion of patients with the corresponding outcome and are presented with 95% confidence intervals.

[0238] Describe the demographic and baseline data descriptively. Categorical data are presented as frequencies and percentages, and continuous data are presented using summary statistics such as mean, median, and standard deviation. Of particular note is the determination of previous treatments for multiple myeloma and the degree of refractoriness to the individual drugs utilized.

[0239] Two interim analyses that may be terminated for futility are conducted when 28 and 55 PFS events occur overall (combining the two groups). A beta error-spending approach with a quadratic error-spending function, which is a compromise between the O’Brien-Fleming and Pocock boundaries, is used. The following table shows the acceptance boundaries for the null hypothesis for the two interim analyses and one final analysis. These values are changed using the error-spending function if the timing of the interim analyses is changed.

Table 2

[0240] The following table shows the probability of early termination of the trial for futility as a function of the true effect size.

Table 3

[0241] Collection of immune cells used as a substrate for manufactured T cell production The collection and shipment of cells for manufactured T cell production are required for patients randomly assigned to the manufactured T cell therapy cohort.

[0242] Furthermore, if the subject has the required value for immune cells in the bloodstream defined by the absolute lymphocyte count (ALC, a value of at least 300 lymphocytes per microliter), steady-state apheresis is performed, which consists of a collection of 10 to 15 liters. Apheresis needs to be performed within 10 days from the trial registration. The apheresis product is shipped immediately to Rapa Therapeutics (without cryopreservation).

[0243] The first cycle of the PC regimen needs to start within 10 days after the trial registration.

[0244] Subsequent iterations of the manufactured T cell therapy can be more efficient and thus use a smaller number of input cells to initiate manufacturing. Such improved methods include, at least in part, improved host conditioning and improved manufacturing processes. With such methods, it becomes possible to manufacture T cells using starting materials obtained from a simple blood draw of 500 mL or less.

[0245] Manufacture of the manufactured T cells In the case of previously collected cell products, such cryopreserved cells are stored in the gas phase of liquid nitrogen until cell thawing and manufacture of the manufactured T cells. In the case of cells isolated by apheresis or freshly by simple blood draw in the future, the cells are processed immediately and then can be placed directly into culture or cryopreserved by a controlled-rate freezing technique and stored in the gas phase of liquid nitrogen for later use.

[0246] T cell culture from cryopreserved cell substrates from newly isolated cell populations requires a certain type of T cell enrichment. By way of non-limiting example, enrichment of T cells can be achieved by using monoclonal antibodies and column technology (positive or negative selection). Enrichment of the source cells used in the production of manufactured T cells does not require such antibody-based methodologies as the T cells are efficiently enriched during the culture period. Thus, our method is consistent with the recommendations for effective cell therapy at the global level. The initial processing steps for the production of manufactured T cells focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (if applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and some monocytes. These steps are carried out in a relatively automated manner mainly using closed system technology. This procedure is advantageous as it reduces human error, provides detailed production data for batch records, improves the consistency of the entire manufacturing process, and reduces the risk of infectious agent contamination of the final product. The processing of manufactured T cell products incorporates the following steps: (1) thawing of cryopreserved products (if applicable) using a solid, non-aqueous method as in 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 to reduce infectious agent contamination, (2) 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.Automated washing of cell products using a LOVO permeable membrane device as in 2017;19(12):1501-1508, (3) integration of lysates of RBCs using ammonium-chloride-potassium (ACK) buffer as described in Brown WE, Hu JC, Athanasiou KA. Ammonium-Chloride-Potassium Lysing Buffer Treatment of Fully Differentiated Cells Increases Cell Purity and Resulting Neotissue Functional Properties. Tissue engineering Part C, Methods. 2016;22(9):895-903 during the LOVO washing step, (4) volume reduction of cell contents using the LOVO method 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 / s12967-12014-10241-y, subsequent seeding of the closed system of cells into a counter-current, centrifugal elutriation (CCE) device (Elutra, Terumo), and (5) pre-programmed operation of the Elutra device for efficient removal of granulocytes and monocytes by CCE.

[0247] After this lymphocyte enrichment and media purification, the cells were seeded into special chambers (G-Rex vessels; Wilson-Wolf) with an abundant volume for oxygen exchange, 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 enhanced gas permeability characteristics, the G-Rex vessels are a closed-system unit and have the added advantage of automated closed-system media volume reduction (GatheRex liquid handling pump). The lymphocyte-enriched cells are maintained in the G-Rex vessels for 6 days.

[0248] Using several specific culture conditions, the production of a mixture of CD4 + and CD8 + T cells with functional attributes of the manufactured T cells in G-Rex vessels is promoted. These specific conditions are as follows: (1) use of enriched media (X-Vivo 20, Lonza, including but not limited to, the media may also be 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 shi yan xue ye xue za zhi. 2006;14(5):985-989; Lonza), (2) incorporating a 16-hour resting period for the cells seeded in the G-Rex prior to co-stimulation (the cells are at 1.5 × 10 6seeded at a relatively high density of individual cells), (3) during this initial resting period, the cells are optimally rested by the addition of 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 resting period, 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 1:3 bead-to-T cell ratio under sub-optimal conditions (usually, most T cell proliferation conditions utilize a 9-fold higher level of co-stimulation, a 3:1 bead-to-T cell ratio), (5) importantly, it is essential that the T cells are not washed after the initial resting period, (6) after the resting period, in addition to the addition of 3 / 28 beads, it is essential 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 important to avoid the addition of IL-2, a common additive to T cell cultures, and (8) after the addition of beads and IFN-α, it is important to leave the cells undisturbed (no cell washing, no further culture additives) until collection on day 6 of the culture.

[0249] Cryopreservation and verification of identity and function of the manufactured T cell product After 6 days of cell culture in a G-Rex vessel, the volume of the culture is decreased in a closed-system manner by a GatheRex instrument. Subsequently, the cells are collected, the 3 / 28 beads are removed by a handheld magnet, the cells are placed in a LOVO device and the cells are continuously washed to remove >99% of the culture additives (temsirolimus, basiliximab, IFN-α).

[0250] The washed cells are reconstituted in a cryopreservation medium containing 5% DMSO and 5% pentastarch. Cryopreservation is performed in multiple single-use aliquots in 50 ml freezer bags. The manufactured T cell dose is 0.1 - 5 × 10 per kg of recipient body weight 6Individual T cells. To enable four consecutive approximately monthly infusions of the manufactured T cells, four single-use aliquots are cryopreserved. The manufactured T cells are cryopreserved by a GMP-compliant controlled-rate freezing method 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 the cells are shipped in the vapor phase of liquid nitrogen by a certified cryoshipper after the manufactured T cells pass the designated release criteria tests.

[0251] The release criteria tests for the manufactured T cells include standard tests such as the content of CD3 + , CD4 + , and CD8 + T cell purity (the final product must have a CD3+ T cell content of >70% by flow cytometry, and the CD4 + and CD8 + subsets must each be present at the 5% level). The cells must have a viability of >70% as determined by flow cytometry annexin and 7-AAD assays. Additionally, the cells must be free of bacterial and fungal contamination with a minimum culture interval of 3 days (ideally 14 days), and furthermore, the cell product must be below the detection limits for bacterial LPS endotoxin and mycoplasma.

[0252] In addition to these standard tests, the special function tests constitute the release criteria for the manufactured T cell product. Before release of the product and cell therapy, the manufactured T cells can have the following attributes compared to the cultured input T cells: (1) an enhanced T central memory phenotype defined by increased flow cytometry co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitor molecules such as programmed death-1 (PD1); (3) a quiescent state defined by a decrease in the level of Th1 / Tc1-type cytokine secretion upon maximal co-stimulation; (4) an autophagy signature demonstrated by a decrease in mitochondrial mass by flow cytometry MitoTracker assay, 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 demonstrated by at least 50% inhibition of mTORC1 and mTORC2 downstream targets, and (6) a multifaceted differential gene expression profile of n = 80 major transcription factors and differentiation molecules.

[0253] Preparation of the host using the PC regimen Patients undergoing manufactured T cell therapy receive a pentostatin and cyclophosphamide (PC) regimen. Cycle 1 of the PC regimen is administered during the interval of manufactured T cell production and is thus administered without an accompanying T cell infusion. Cycle 1 is advantageous at two levels: First, it reduces the number and function of host regulatory T cells and terminally senescent effector T cells, thereby enhancing future cycles of the manufactured T cell therapy. Second, it directly mediates the anti-tumor effect against multiple myeloma, thereby controlling the disease during the production interval. After Cycle #1, subsequent cycles of the PC regimen are followed by adoptive transfer of manufactured T cells on the day after the 2-week PC regimen interval (day 15). These cycles of the PC regimen further modulate host biology, including increased T cell homeostasis cytokines such as IL-7 and IL-15, which is further advantageous as it allows for improved proliferation of the manufactured T cells after adoptive transfer.

[0254] Following the PC regimen, an infusion of manufactured T cells follows. Each cycle of the PC therapy consists of a 14-day course immediately prior to the infusion of manufactured T cells on day 15 of the cycle. The manufactured T cell dose is 1×10 5 ~5×10 6 cells / kg, containing a T cell dose of 1 - 5×10 6 cells / kg. Pentostatin (P) is administered at a dose of 4 mg / m2 on days 1, 4, 8, and 11 (by intravenous injection), and cyclophosphamide (Cy) is administered at a dose of 200 mg / day on days 1 - 5 and 8 - 12.

[0255] Premedication and prehydration are required before pentostatin administration. Prehydration with 1 liter of 0.9% sodium chloride is performed 60 minutes before pentostatin. Premedication with an antiemetic is required. Recommended antiemetic regimens are as follows: (1) dexamethasone, 12 mg by IV injection 60 minutes before each pentostatin dose (i.e., on days 1, 4, 8, and 11 of the cycle); (2) additionally, oral dexamethasone may be administered at a dose of 4 mg per day on other days as needed; (3) ondansetron may be administered at a dose of 8 mg by IV injection 60 minutes before each pentostatin dose; (4) in the remaining treatment, ondansetron may be administered at an oral dose of 8 mg (tablets) every 12 hours from day 1 to day 14 as needed; and (5) in patients with intractable nausea and vomiting, aprepitant may be added to the antiemetic regimen as needed. The dose of pentostatin is 4 mg / m 2 and each dose of pentostatin is administered intravenously over 30 to 60 minutes.

[0256] The dose of pentostatin is changed, and the dose of pentostatin administered to the patient is 1 to 4 mg / m 2 The dose of pentostatin is changed based on creatinine clearance (CrCl), which is obtained by 24-hour urine or calculated by the Cockcroft-Gault formula. If the subject experiences an increase in creatinine level during pentostatin and cyclophosphamide therapy, subsequent doses are changed as follows: CrCl ≥ 60 mL / min / 1.73m 2 In this case, pentostatin 4 mg / m 2 (total dose) is administered, and when CrCl < 60 mL but ≥ 30 mL / min / 1.73m 2 In this case, the dose of pentostatin is reduced by 50% to 2 mg / m 2Administer, and if CrCl < 30 mL, hold pentostatin. Pentostatin is rarely associated with organ toxicities such as neurotoxicity (seizures, lethargy) or cardiotoxicity (decreased ejection fraction). Therefore, special attention is required for the evaluation of organ toxicities that occur during PC treatment. If pentostatin is associated with grade 2 or higher organ toxicity, contact the facility PI to discuss whether additional pentostatin therapy and additional protocol therapy are needed.

[0257] Use oral cyclophosphamide (Cy) as a component of the PC regimen, and the dose of cyclophosphamide will be 50 - 400 mg. The dose of Cy is 200 mg per day on days 1 - 5 and 8 - 12 during cycles 1 - 5 of the PC regimen. Intravenous injection of 200 mg of cyclophosphamide is acceptable due to tolerance issues or economic considerations. Due to cyclophosphamide bladder toxicity, sufficient hydration must be maintained during the PC regimen. To maintain clear urine color, patients should drink at least 2 - 4 liters of fluid per day.

[0258] The dose of cyclophosphamide is adjusted according to the following table based on the 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 as needed. The stated goal of the PC regimen is to achieve immune depletion and immunosuppression while minimizing myeloid cell suppression. To ensure this goal, the dose of cyclophosphamide is adjusted according to the following table based on the ALC and ANC values obtained on the pentostatin administration days (i.e., days 1, 4, 8, and 11 of the cycle) as needed. The notations in the table are as follows: 1 Pentostatin is not dose - adjusted based on ALC / ANC values. 2 If ANC value < 500, in addition to reducing the cyclophosphamide dose, the patient receives G - CSF therapy until the next ANC measurement. 3The cyclophosphamide dosage shown is continued daily until the next ALC / ANC measurement (performed on days 1, 4, 8, and 11 of the cycle).

[0259] Modifications of the PC regimen are envisioned. First, pentostatin and cyclophosphamide are synergistic in terms of their immunosuppressive and immunodepleting effects. Such a synergistic effect is likely to exist also in terms of the antitumor effect, but there is little information regarding this possibility. Accordingly, it is envisioned that the PC regimen can be used as a monotherapy for cancer treatment, including solid tumors. In one previous example, patients with refractory mesothelioma who received a combination regimen partially constituted by the PC regimen had an unprecedented antitumor effect. Second, due to this synergistic effect, it is proposed that it is advantageous to administer the two drugs simultaneously by intravenous infusion, preferably by mixing pentostatin and cyclophosphamide in the same intravenous infusion bag to facilitate administration and reduce pharmacy errors. In such an application, it would be important to provide options of PC mixtures encompassing various clinically relevant pentostatin-to-cyclophosphamide ratios.

Table 4

[0260] Infusion of manufactured T cells Premedication is required prior to all manufactured T cell administrations. Diphenhydramine (25 - 50 mg IV or PO) and acetaminophen (650 mg, PO) are administered 30 - 60 minutes before the manufactured T cell infusion.

[0261] The manufactured T cell infusion is performed on day 15 of cycles 2 - 5, but due to logistic reasons, a delay of up to 3 days in the manufactured T cell infusion may occur. Furthermore, a delay of up to 4 weeks between cycles is allowed to enable logistics and recovery from toxicity. The manufactured T cell dosage is 5×10 6 cells / kg, but if a suboptimal cell yield occurs during manufacturing, 0.1×106 A low dose of <0.1×10> cells / kg is acceptable. The cryopreserved manufactured T cells are thawed and immediately and rapidly administered intravenously by gravity (within 30 minutes) according to the SOP of the appropriate facility for blood product administration. This T cell infusion is performed in an outpatient setting unless there are unforeseen circumstances requiring hospitalization of the patient. Steroids are not permitted in the management of DMSO-related toxicity (chills, myalgia) that may occur immediately after cell infusion unless the toxicity is considered life-threatening.

[0262] 0.1×10 6 Small amounts of manufactured T cell infusions of less than <0.1×10> cells / kg are also assumed to be clinically relevant (but not limited to, for example, 1 log lower, at 1×10 5 cells / kg). First, the manufacturing is optimized to yield manufactured T cells that mediate a further enhanced in vivo effect, thereby reducing the required T cell dose. This would be advantageous in part because T cell collection may be performed by simple blood draw and in part because manufacturing feasibility is improved. Second, as described above, with further improvement of the PC regimen, adoptively transferred manufactured T cells have further improved in vivo selective advantages compared to host cells, thereby effectively reducing the required dose of manufactured T cells.

[0263] Special immune monitoring during and after treatment Peripheral blood mononuclear cells and bone marrow cells are sent to Rapa Therapeutics so that immunomonitoring 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 predict the efficacy of the manufactured T cells. In one effort, we evaluate the manufactured T cell recipients for their ability to produce various Th1- and Th2-type cytokines in response to various stimuli, including autologous multiple myeloma tumor cells or known or suspected tumor antigens such as molecules within the cancer-testis antigen (CTA) family. The CTA family of genes is numerous and has been shown to be associated with multiple myeloma. Since the sequences of the CTA genes are known and the association of specific CTA genes has been characterized in multiple myeloma, the manufactured T cell therapy can be demonstrated to specifically induce T cell-mediated immunity against a diverse range of CTA antigens. Measurement of such cytokine responses can be performed using RNA expression analysis, secretion analysis by ELISA or Luminex multiplex assays, flow cytometry, or ELISPOT assays. Antigen-specific immunity can also be quantified by the use of antibody production assays or cell lysis assays.

[0264] We evaluate whether T cells obtained after manufactured T cell therapy have an increased reactivity against autologous multiple myeloma cells compared to T cells obtained before manufactured T cell therapy. One obstacle to this endeavor is that the proliferation of patient-specific multiple myeloma cell lines usually does not succeed. To overcome this obstacle, we use a special container as 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 medium supplemented with a combination of factors known to enhance the growth and survival of multiple myeloma, including acquisition of resistance to IL-6, CD40 ligand, and carfilzomib, to grow myeloma cells. Patient-specific multiple myeloma cells can be used alone as stimulants in the evaluation of the anti-tumor reactivity of immune T cells, or such tumor cells can be made apoptotic by loading professional antigen-presenting cells that can be made from patient-specific monocytes collected from the elution procedure during the manufacture of the produced T cells.

[0265] In addition, T cell receptor (TCR) immune repertoire analysis is believed to be useful as a biomarker for manufactured T cell therapy. Preferably, such repertoire analysis will be performed by RNA sequencing rather than the more commonly used DNA sequencing. Unlike the majority of targeted T cell therapies, manufactured T cell therapy is a polyclonal method because the manufacturing process does not preferentially shift T cell reactivity to any particular tumor antigen. Therefore, any beneficial antitumor effect after manufactured T cell therapy is expected to be derived from in vivo clonal expansion to the diversity of tumor antigens. Given this biological nature, the success of manufactured T cell therapy will result in a different TCR repertoire when comparing a patient's pre-treatment repertoire with their post-treatment repertoire. In other cancer therapy settings, such as monoclonal antibody therapy to resolve checkpoint blockade, successful treatment is associated with the emergence of new TCR clonal specificities, known as TCR repertoire skewing, which can be confirmed by quantification of 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. Similarly, successful manufactured T cell therapy results in TCR repertoire skewing, and persistence of TCR skewing beyond the interval of manufactured T cell therapy is consistent with long-term T cell immunity against malignancies. Advances in manufacturing have generated improved forms of manufactured T cells, in which case TCR skewing will be more extensive, occur with a reduced number of treatment cycles, and be more durable in the post-treatment interval.

[0266] Protocol Selection Criteria for the Treatment of Multiple Myeloma Male or female patients aged 18 years or older may be candidates for the manufactured T cell therapy. There is no formal upper age limit. However, patients 65 years of age or older with a history of cardiovascular pathology or symptoms (even if they do not clearly meet the exclusion criteria detailed below) require evaluation by a cardiologist at multiple institutional sites. Such subjects are considered on a case-by-case basis. The performance status of the overall patient population must be at least of moderate health, as quantified by an ECOG performance status ≤2.

[0267] Patients must have a confirmed diagnosis of multiple myeloma by histological or cytological studies. Furthermore, the disease must be symptomatic, and the patient must be in the second or third relapse of the disease after receiving drugs such as proteasome inhibitors, immunomodulatory drugs, alkylating agents, CD38 monoclonal antibodies, and glucocorticoids.

[0268] Patients in the second or third relapse of the disease are in a relatively advanced stage. However, based on the demonstration of the safety and efficacy of the manufactured T cell therapy, we envision that patients with early-stage multiple myeloma in the treatment algorithm will benefit from the manufactured T cell therapy. By way of non-limiting example, the manufactured T cell therapy can be used as an alternative to high-dose chemotherapy in combination with autologous hematopoietic cell transplantation or can be used in a significant number of patients for whom transplantation is ineligible. Furthermore, the manufactured T cell therapy can be envisioned during the earliest point of multiple myeloma progression prior to clinical symptoms, i.e., during early detection at the smoldering disease stage.

[0269] On the other hand, the Rapa-T cell therapy described herein is envisioned to be applicable to the treatment of patients with more advanced-stage multiple myeloma and patients with highly refractory disease. Specifically, the Rapa-T cell therapy can be utilized for the treatment of penta-refractory MM, which is defined as a patient with resistance to five of the top drugs used to treat relapsed MM, namely lenalidomide, pomalidomide, bortezomib, carfilzomib, and daratumumab.

[0270] Patients with penta-refractory MM do not have standard treatment options for therapy. Therefore, the clinical protocol for evaluating Rapa-T cell therapy in this setting is a single-arm phase II trial similar to those previously conducted 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 new anticancer agents. For this phase II trial, Rapa-T cell therapy is administered as described in Figures 30, 31, and 32A - 32C. The statistical objective of this study is to determine whether Rapa-T cell therapy can induce a significant proportion of at least partial remission of penta-refractory MM, as defined by a proportion consistent with at least 30%.

[0271] There is a need to have a potential source of autologous T cells that is potentially sufficient to produce the manufactured T cells. Specifically, the patient needs to have either a sufficient number of previously cryopreserved PBSC units (>2 million cells / kg total CD34 + content, as defined) available for manufacture, or a sufficient number of circulating T cells (defined by an ALC of >300 cells per microliter) that can be collected by steady-state apheresis.

[0272] The patient must have been at least two weeks since myeloma treatment, major surgery, radiotherapy, or participation in other trials, and must have recovered from clinically significant toxicities of these previous treatments (resolution of CTCAE toxicity to a value of 2 or less). The ejection fraction (EF) of the heart by MUGA or 2D echocardiogram must be within the normal range of the facility, and the EF level must be at least 40%. Renal function measured by serum creatinine must be 2.5 mg / dl or less. Liver function, when measured by AST and ALT, must be no more than three times the upper limit of normal, and total bilirubin must be 1.5 or less (except when due to Gilbert's disease). Lung function must be appropriate as defined by a corrected DLCO of 50% or more predicted by pulmonary function testing. There must be no history of abnormal bleeding tendency. Prior to performing any study-related procedures that are not part of standard medical care, the patient must give voluntary written consent after understanding that they can cancel their consent at any time without compromising future medical care.

[0273] Randomized Phase III Trial: Standard Therapy To demonstrate the benefits of the manufactured T-cell therapy, a randomized trial is conducted and the results of the manufactured T-cell therapy are formally compared to a standard therapy regimen consisting of either the DPd, DRd, or KRd regimen, which is administered as per that defined in the literature according to the FDA-approved status of MM patients at the second or third relapse.

[0274] Example 7 Steady state apheresis was performed to obtain a patient sample containing PBMC lymphocytes. The lymphocytes in the sample were enriched by using an automated Ficoll procedure on a Sepax® device by GE. Subsequently, the lymphocyte-enriched cell population was seeded into G-REX culture vessels and cultured for 6 days in TexMACS medium (serum-free, IL-2-free), containing IFN-α, temsirolimus, and basiliximab. At the end of production, the resulting Th1 / Tc1 cells were exposed to either pancreatic cancer cells (MIA-Paca2 cell line) or lung cancer cells (H23 cell line) that had undergone apoptosis by exposure to etoposide. After pulsing with this tumor lysate, the cells were cultured for 7 days in IL-2 (200 IU / mL), at which point a secondary exposure to the tumor lysate was performed. After an additional 7-day culture interval in IL-2-containing medium, a third exposure to the tumor lysate was performed, and the resulting 24-hour supernatant was tested for cytokine content by Luminex assay (results are shown as pg / mL per 1×10 6 cells per 24 hours). The results of the cytokine assay are shown in FIGS. 34A - 34B. Condition A shows pulsing with a sub-optimal formulation of the tumor lysate; condition B represents the optimal formulation of the tumor lysate. "<" indicates that the value was below the detection limit.

[0275] As shown in FIGS. 34A - 34B, RAPA-T cells can be further characterized by their ability to respond to tumor cells, including solid tumors such as pancreatic and lung cancer cells. As further shown in FIGS. 34A - 34B, RAPA-T cells can maintain a characteristic Th1 cytokine phenotype, as demonstrated by high-level secretion of IFN-γ and GM-CSF, and decreased secretion of the Th2 cytokines IL-4 and IL-10.

[0276] In another experiment, at the end of production, the obtained Th1 / Tc1 cells were exposed to either pancreatic cancer cells (MIA-Paca2 cell line) or lung cancer cells (H23 cell line) that had undergone apoptosis by exposure to etoposide. Following pulsing with this tumor lysate, the cells were cultured for 7 days in IL-7 (20 ng / mL) and IL-15 (10 ng / mL), at which point they were exposed to the tumor lysate a second time. After an additional 7-day culture interval in IL-7- and IL-15-containing medium, the cells were exposed to the tertiary tumor lysate, and the resulting 24-hour supernatant was tested for cytokine content by Luminex assay (results are shown as pg / mL per 1 × 10 6 cells per 24 hours). Control cultures ("RAPA-201, no tumor") consisted of produced resistant Th1 / Tc1 cells that had grown in IL-7- and IL-15-containing medium but had not been pulsed with tumor lysate. The results of the cytokine assay are shown in Figure 35.

[0277] As shown in Figure 35, RAPA-T cells can be further characterized by their ability to respond to tumor cells, including solid tumors such as pancreatic and lung cancer cells. This in vitro sensitization to solid tumor cell lines can be readily demonstrated by culturing expansion in medium supplemented with IL-7 and IL-15, two constitutive cytokines that have been shown to selectively drive the effector functions of RAPA-T cells. Secretion of RAPA-T cell cytokines to tumor cells can maintain a characteristic Th1 cytokine phenotype, as evidenced by high-level secretion of IFN-γ, GM-CSF, and TNF-α.

[0278] As shown in FIG. 36, many cancers such as renal cell carcinoma, liver cancer, lung cancer, bladder cancer, and gastric cancer have been shown to respond to checkpoint inhibitor therapies such as monoclonal therapies against checkpoint inhibitor molecules such as PD-1 and CTLA4 that can induce remission in solid tumors. In further experiments, under Simon's two-stage design, patients with renal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, and low-variant PDL1-negative or low-variant rate cancers (n = 7) are administered the manufactured T cell therapy. If any cohort has at least one responsive patient, the cohort is expanded to a 20-patient cohort.

[0279] Without being bound by theory, it is expected that Rapa-T cells can provide a therapeutic benefit in cancer because the cells have reduced or no checkpoint inhibitor receptors. It is suspected that a particular cancer may be non-responsive to a particular treatment due to checkpoint inhibitor receptors other than PD1 and CTLA4. Thus, without being bound by theory, it is expected that Rapa-T cells may be effective in the treatment of other cancers due to the absence of additional checkpoint inhibitor receptors.

[0280] Example 8 Rapa-T cells were produced by culturing for 6 days using the cell population after Ficoll cultured in a medium containing temsirolimus (2 μM) and the anti-IL-2 receptor monoclonal antibody basiliximab (30 μg / mL). After 24 hours, the culture was supplemented with IFN-α (20,000 IU / mL), and there was no IL-2 supplementation of the culture. There was no form of anti-CD3 / anti-CD28 co-stimulation used in the culture.

[0281] In contrast, for the "control": The cell population after Ficoll was cultured in a medium containing no temsirolimus and no basiliximab. The cells were co-stimulated with anti-CD3 / anti-CD28 coated beads at a bead-to-T cell ratio of 3:1 on the day of culture initiation. The medium was supplemented with IL-2 (20 IU / mL) and IFN-α (20,000 IU / mL) on the day of culture initiation. The mean fluorescence intensity (MFI) for BTLA, CTLA4, PD1, and TIM3 was measured by flow cytometry for CD4+ and CD8+ T cell subsets in 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 seen between Rapa-T cells and control cells, and the MFI for each checkpoint was approximately the same for Rapa-T cells and culture input cells.

Table 5

[0282] Manufacturing embodiments: 1. A method for producing manufactured T cells, comprising: seeding a culture input population of cells containing T cells from a subject at a certain cell density into a culture medium containing temsirolimus and an IL-2 signaling inhibitor; adding IFN-α to the culture medium; incubating the T cells and the culture medium for a certain period of time to obtain manufactured T cells; collecting the manufactured T cells. 2. The method according to embodiment 1, further comprising adding additional culture medium to the T cells and the culture medium. 3. The method according to embodiment 2, wherein the additional culture medium is added at about 48 hours after seeding the culture input population of cells in the culture medium. 4. The amount of additional culture medium added to the culture is sufficient to reduce the cell density of the cells in the culture to the target cell density, and the cell density of the culture input population of cells at the time of seeding exceeds 9x10 6 cells / mL, and the target cell density is about 9x10 6The method according to any one of Embodiments 2 to 3, which is cells / mL. The method according to Embodiment 1, wherein anti-CD3 / anti-CD28 costimulation is not performed. The method according to Embodiment 2, wherein the amount of additional culture medium added to the culture is in a ratio of 1:1 to 3:1 with respect to the amount of culture medium when the inoculated population of the cell culture is inoculated into the culture medium. 7. After collecting the produced T cells, packaging at least a part of the produced T cells in a package, freezing the package containing the part of the produced T cells, the method according to any one of Embodiments 1 to 6 further comprising. 8. The method according to any one of Embodiments 1 to 7, wherein the culture medium does not contain IL-2 and IL-2 is not added to the culture medium. 9. The method according to any one of Embodiments 1 to 8, wherein the IFN-α is added to the culture medium almost simultaneously with inoculating the inoculated population of the cells or within 24 hours after inoculating the inoculated population of the cells. 10. The cell density is at least 1.5×10 6 cells per mL, the method according to any one of Embodiments 1 to 9. 11. The cell density is about 7.5×10 6 cells per mL, the method according to any one of Embodiments 1 to 9. 12. The cell density is about 30×10 6 cells per mL, the method according to any one of Embodiments 1 to 9. 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. 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 period to maintain the desired concentration. 15. The method according to Embodiment 14, wherein the temsirolimus is added to the culture medium every two days during the period. 16. The method according to any one of embodiments 14 to 15, wherein the desired concentration is about 4.5 μM. 17. The method according to any one of embodiments 1 to 16, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof. 18. The method according to embodiment 17, wherein the IL-2 signaling inhibitor is basiliximab or daclizumab. 19. The method according to any one of embodiments 1 to 18, wherein the IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 to 50 μg / mL. 20. The method according to any one of embodiments 1 to 19, wherein the IFN-α is added to the culture medium at a concentration of 1,000 to 10,000 IU / mL. 21. The method according to any one of embodiments 1 to 20, wherein the period is about 4 days to about 8 days. 22. The method according to any one of embodiments 1 to 20, wherein the period is 6 days. 23. The method according to any one of embodiments 1 to 22, wherein the culture medium is substantially serum-free. 24. The method according to any one of embodiments 1 to 23, wherein no serum is added to the culture medium. 25. The method according to any one of embodiments 1 to 22, wherein the culture medium further contains 5% human serum. 26. The method according to any one of embodiments 1 to 25, wherein the culture medium contains TexMACS medium. 27. The method according to any one of embodiments 1 to 26, wherein the input cell population for culturing contains T cells accounting for 66% or less of the total number of cells in the input cell population for culturing. 28. The method according to any one of embodiments 1 to 26, wherein the input cell population for culturing contains T cells accounting for about 50% to about 95% of the total number of cells in the input cell population for culturing. 29. The method according to any one of embodiments 1 to 28, wherein the input cell population for culturing further contains monocytes. 30. Collecting a sample containing T cells from the subject; The method according to any one of Embodiments 1 to 29, further comprising isolating T cells from the sample to obtain a cell culture input population. 31. The method according to Embodiment 30, wherein the cell culture input population contains about 99% or more T cells out of the total number of cells in the cell culture input population. 32. The method according to any one of Embodiments 30 to 31, wherein the T cells are isolated by antibody-based purification. 33. Collecting a sample containing T cells from the subject, The method according to any one of Embodiments 1 to 29, further comprising enriching the sample for T cells to obtain a cell culture input population. 34. The method according to Embodiment 33, wherein the enrichment is performed by counterflow centrifugal elution or Ficoll procedure. 35. The method according to any one of Embodiments 33 to 34, wherein the cell culture input population contains about 70% T cells out of the total number of cells in the cell culture input population. 36. Before inoculating the cell culture input population containing T cells from the subject into a culture medium at a certain cell density, The method according to any one of Embodiments 1 to 29, further comprising collecting the cell culture input population from the subject. 37. Engineered T cells produced by the method according to any one of Embodiments 1 to 36. 38. A method for producing engineered T cells, comprising: Inoculating a cell culture input population containing T cells from a subject at a certain cell density into a culture medium containing temsirolimus and an IL-2 signaling inhibitor; Incubating the cell culture input population and the culture medium for a first period without co-stimulation of the cell culture input population with anti-CD3 / anti-CD28; After the incubation in the first period, adding anti-CD3 / anti-CD28-coated magnetic beads to the T cells and the culture medium at a bead:T cell ratio of 1:1 to 1:12 to stimulate the T cells; Adding IFN-α to the culture medium; Incubating the input population of cells in the culture medium containing the anti-CD3 / anti-CD28 coated magnetic beads and IFN-α for a second period to obtain the produced T cells, Separating the anti-CD3 / anti-CD28 coated magnetic beads from the produced T cells, Collecting the produced T cells, a method comprising. 39. After collecting the produced T cells, Packaging at least a portion of the produced T cells into a package, Freezing the package containing the portion of the produced T cells, further comprising the method according to embodiment 38. 40. The method according to any one of embodiments 38 to 39, wherein the culture medium does not contain IL-2 and IL-2 is not added to the culture medium. 41. The method according to any one of embodiments 38 to 40, wherein the IFN-α is added at the same time as, or substantially simultaneously with, the addition of the anti-CD3 / anti-CD28 coated magnetic beads. 42. The cell density is at least 1.5×10 per mL 6 cells, the method according to any one of embodiments 38 to 41. 43. The cell density is about 7.5×10 per mL 6 cells, the method according to any one of embodiments 38 to 41. 44. The cell density is about 30×10 per mL 6 cells, the method according to any one of embodiments 38 to 41. 45. The temsirolimus is present in the culture medium at a concentration of 1 μM, the method according to any one of embodiments 38 to 44. 46. The temsirolimus is added to the culture medium one or more times during the second period to maintain the desired concentration, the method according to any one of embodiments 38 to 44. 47. The temsirolimus is added to the culture medium every two days during the second period, the method according to embodiment 46. 48. The desired concentration is 1 μM, the method according to any one of embodiments 46 to 47. 49. The method according to any one of embodiments 38 to 48, wherein the IL-2 signal transduction inhibitor is an anti-IL-2 receptor antibody or a fragment thereof. 50. The method according to embodiment 49, wherein the IL-2 signal transduction inhibitor is basiliximab or daclizumab. 51. The method according to any one of embodiments 38 to 50, wherein the IL-2 signal transduction inhibitor is present in the culture medium at a concentration of 5 to 50 μg / mL. 52. The method according to any one of embodiments 38 to 51, wherein the first period is about 8 hours to about 24 hours. 53. The method according to any one of embodiments 38 to 51, wherein the first period is 16 hours. 54. The method according to any one of embodiments 38 to 53, wherein the bead:T cell ratio is 1:3. 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 to 10,000 IU / mL. 56. The method according to any one of embodiments 38 to 55, wherein the second period is about 4 days to about 8 days. 57. The method according to any one of embodiments 38 to 55, wherein the second period is 6 days. 58. The method according to any one of embodiments 38 to 57, wherein the culture medium further contains 5% human serum. 59. The method according to any one of embodiments 38 to 58, wherein the culture medium contains TexMACS medium. 60. The method according to any one of embodiments 38 to 59, wherein the cultured input population of cells contains T cells that are 66% or less of the total number of cells in the cultured input population of cells. 61. The method according to any one of embodiments 38 to 60, wherein the cultured input population of cells contains T cells that are about 50% to about 95% of the total number of cells in the cultured input population of cells. 62. The method according to any one of embodiments 38 to 61, wherein the cultured input population of cells further contains monocytes. 63. Collecting a sample containing T cells from the subject, The method according to any one of Embodiments 38 to 62, further comprising isolating T cells from the sample to obtain a cultured input population of the cells. 64. The method according to Embodiment 63, wherein the cultured input population of the cells comprises about 99% or more T cells out of the total number of cells in the cultured input population of the cells. 65. The method according to any one of Embodiments 63 to 64, wherein the T cells are isolated by antibody-based purification. 66. Collecting a sample containing T cells from the subject, The method according to any one of Embodiments 38 to 62, further comprising enriching the sample for T cells to obtain a cultured input population of the cells. 67. The method according to Embodiment 66, wherein the enrichment is performed by counterflow centrifugal elution or Ficoll procedure. 68. The method according to any one of Embodiments 66 to 67, wherein the cultured input population of the cells comprises about 70% T cells out of the total number of cells in the cultured input population of the cells. 69. Before inoculating the cultured input population of the cells containing T cells from the subject into a culture medium at a certain cell density, The method according to any one of Embodiments 38 to 62, further comprising collecting the cultured input population of the cells from the subject. 70. Engineered T cells produced by the method according to any one of Embodiments 38 to 69. 71. A method for producing engineered T cells, comprising: Inoculating a cultured input population of cells containing T cells from a subject into a culture medium containing temsirolimus and an IL-2 signaling inhibitor at a certain cell density; Incubating the cultured input population of the cells and the culture medium for a first period without co-stimulation of the cultured input population of the cells with anti-CD3 / anti-CD28; After the incubation in the first period, adding anti-CD3 / anti-CD28-containing nanoparticles to the T cells and the culture medium at a recommended dose of about 0.01-fold to about 0.1-fold to stimulate the T cells; Adding IFN-α to the culture medium; Incubating the input population of cells in a culture medium containing anti-CD3 / anti-CD28-containing nanoparticles and IFN-α for a second period to obtain the produced T cells, collecting the produced T cells, a method comprising. 72. After collecting the produced T cells, packaging at least a portion of the produced T cells in a package, freezing the package containing the portion of the produced T cells, further comprising the method according to embodiment 71. 73. The method according to any one of embodiments 71 to 72, wherein the culture medium does not contain IL-2 and IL-2 is not added to the culture medium. 74. The method according to any one of embodiments 71 to 73, wherein the IFN-α is added simultaneously with or substantially simultaneously with the addition of the anti-CD3 / anti-CD28-containing nanoparticles. 75. The cell density is at least 1.5×10 per mL 6 cells, the method according to any one of embodiments 71 to 74. 76. The cell density is about 7.5×10 per mL 6 cells, the method according to any one of embodiments 71 to 74. 77. The cell density is about 30×10 per mL 6 cells, the method according to any one of embodiments 71 to 74. 78. The temsirolimus is present in the culture medium at a concentration of 1 μM, the method according to any one of embodiments 71 to 77. 79. The temsirolimus is added to the culture medium one or more times during the second period to maintain the desired concentration, the method according to any one of embodiments 71 to 78. 80. The temsirolimus is added to the culture medium every two days during the second period, the method according to embodiment 79. 81. The desired concentration is 1 μM, the method according to any one of embodiments 71 to 80. 82. The method according to any one of embodiments 71 to 81, wherein the IL-2 signal transduction inhibitor is an anti-IL-2 receptor antibody or a fragment thereof. 83. The method according to embodiment 82, wherein the IL-2 signal transduction inhibitor is basiliximab or daclizumab. 84. The method according to any one of embodiments 71 to 83, wherein the IL-2 signal transduction inhibitor is present in the culture medium at a concentration of 5 to 50 μg / mL. 85. The method according to any one of embodiments 71 to 84, wherein the first period is from about 8 hours to about 24 hours. 86. The method according to any one of embodiments 71 to 84, wherein the first period is 16 hours. 87. The method according to any one of embodiments 71 to 86, wherein the IFN-α is added to the culture medium at a concentration of 1,000 IU / mL to 10,000 IU / mL. 88. The method according to any one of embodiments 71 to 87, wherein the second period is from about 4 days to about 8 days. 89. The method according to any one of embodiments 71 to 87, wherein the second period is 6 days. 90. The method according to any one of embodiments 71 to 89, wherein the culture medium further contains 5% human serum. 91. The method according to any one of embodiments 71 to 90, wherein the culture medium contains TexMACS medium. 92. The method according to any one of embodiments 71 to 91, wherein the cultured input population of the cells contains T cells accounting for 66% or less of the total number of cells in the cultured input population of the cells. 93. The method according to any one of embodiments 71 to 91, wherein the cultured input population of the cells contains T cells accounting for about 50% to about 95% of the total number of cells in the cultured input population of the cells. 94. The method according to any one of embodiments 71 to 93, wherein the cultured input population of the cells further contains monocytes. 95. Further comprising collecting a sample containing T cells from the subject, isolating T cells from the sample to obtain the cultured input population of the cells. The method according to any one of embodiments 71 to 94. 96. The method according to embodiment 95, wherein the cultured input population of cells comprises at least about 99% T cells of the total number of cells in the cultured input population of cells. 97. The method according to any one of embodiments 95 to 96, wherein the T cells are isolated by antibody-based purification. 98. Collecting a sample containing T cells from the subject, Enriching the sample for T cells to obtain the cultured input population of cells, the method according to any one of embodiments 71 to 94, further comprising. 99. The method according to embodiment 98, wherein the enrichment is performed by counterflow centrifugal elution or Ficoll procedure. 100. The method according to any one of embodiments 98 to 99, wherein the cultured input population of cells comprises about 70% T cells of the total number of cells in the cultured input population of cells. 101. Before inoculating T cells from the subject into the culture medium at a certain cell density, Collecting the cultured input population of cells from the subject, the method according to any one of embodiments 71 to 94, further comprising. 102. Engineered T cells produced by the method according to any one of embodiments 71 to 101. 103. An engineered T cell population that exhibits a reduced level of phosphorylated STAT5 compared to a control population of engineered T cells produced in the presence of exogenous IL-2, wherein the observed reduction is at least 50% less, more preferably at least 90% less, an engineered T cell population. 104. An engineered T cell population that shows a shift in differentiation towards the T central memory state away from the effector memory state, as indicated by an increase of at least 25% in the frequency of T cells co-expressing CD62L and CCR7 compared to cultured input T cells. 105. CD4 co-expressing the IL-2 receptor CD25 at a rate of less than 5%, more preferably less than 1% + and CD8 + An engineered T cell population present in a quiescent state, as indicated by the frequency of T cells. 106. A manufactured T cell population that exists in a resting state, as indicated by T cells that secrete low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, and that contains, per 24 hours, 1×10 6 per cell defined by <100 pg / ml in the culture supernatant after a stimulation procedure using a high level of co-stimulation (a bead-to-T cell ratio of 3:1). 107. A manufactured T cell that transitions from a resting state to a state of high-level inflammatory cytokine secretion, defined by an increase in IFN-γ and TNF-α secretion that is at least 5-fold, more preferably 20-fold, compared to the secretion level on day 6 after a 6-day expansion period in the absence of an inhibitor. 108. A manufactured T cell population that expresses low levels of the immunosuppressive molecule CTLA4, defined by flow cytometry of less than 10%, more optimally less than 5%, of CTLA4 + on CD4 + and CD8 + T cell expression. + 109. A manufactured T cell population that expresses low levels of the checkpoint inhibitor molecule TIM3, defined by flow cytometry of less than 10%, more optimally less than 2%, of TIM3 + on CD4 + and CD8 + T cell expression. + 110. Before inoculating a culture input population of cells containing T cells from the subject into a culture medium at a certain cell density, collecting the culture input population of cells from the subject, the method according to any one of Embodiments 1 to 29, further comprising this step. 111. Before inoculating a culture input population of cells containing T cells from the subject into a culture medium at a certain cell density, isolating T cells from a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 1 to 29, further comprising this step. ​​Before inoculating the culture input population of the cells containing T cells from the subject into the culture medium at a certain cell density, Enriching a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 1 to 29, further comprising. Before inoculating the culture input population of the cells containing T cells from the subject into the culture medium at a certain cell density, Isolating T cells from a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 38 to 62, further comprising. Before inoculating the culture input population of the cells containing T cells from the subject into the culture medium at a certain cell density, Enriching a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 38 to 62, further comprising. Before inoculating the culture input population of the cells containing T cells from the subject into the culture medium at a certain cell density, Isolating T cells from a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 71 to 94, further comprising. Before inoculating the culture input population of the cells containing T cells from the subject into the culture medium at a certain cell density, Enriching a sample containing T cells from the subject to obtain a culture input population of cells, the method according to any one of Embodiments 71 to 94, further comprising. The method according to any one of Embodiments 1 to 36, 38 to 69, and 71 to 100, wherein IFN-α is added 24 hours after the culture input population of cells is inoculated into the culture medium. Manufactured T cells produced by the method according to any one of Embodiments 110 to 117. A manufactured T cell population, wherein when measured by flow cytometry, 10% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express CTLA4, the manufactured T cell population. A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing CTLA4 when measured by flow cytometry. + or CD8 + A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing CTLA4 as compared to the corresponding frequency of CTLA4-expressing CD4 or CD8 T-Rapa cells. + or CD8 + A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing CTLA4 as compared to the corresponding frequency of CTLA4-expressing CD4 or CD8 T-Rapa cells. 121. The T cell population produced according to embodiment 120, wherein the reduced frequency is at least 50% less than the corresponding frequency. 122. A T cell population produced, wherein when measured by flow cytometry, 10% or less of the CD4 or CD8 T cells in the produced T cell population express TIM3. + or CD8 + A T cell population produced, wherein when measured by flow cytometry, 10% or less of the CD4 or CD8 T cells in the produced T cell population express TIM3. 123. A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T cells in a control T cell population characteristic of the T cells produced in the produced T cell population when measured by flow cytometry. + or CD8 + A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T cells in a control T cell population characteristic of the T cells produced in the produced T cell population when measured by flow cytometry. + or CD8+T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T cells in a control T cell population characteristic of the T cells produced in the produced T cell population when measured by flow cytometry. 124. The T cell population produced according to embodiment 123, wherein the reduced frequency is at least 50% less than the corresponding frequency. 125. A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T-Rapa cells expressing TIM3 when measured by flow cytometry. + or CD8 + A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T-Rapa cells expressing TIM3 when measured by flow cytometry. + or CD8 + A T cell population produced, characterized by a reduced frequency of CD4 or CD8 T cells expressing TIM3 as compared to the corresponding frequency of CD4 or CD8 T-Rapa cells expressing TIM3 when measured by flow cytometry. 126. The T cell population produced according to embodiment 125, wherein the reduced frequency is at least 50% less than the corresponding frequency. 127. A T cell population produced, wherein when measured by flow cytometry, 5% or less of the CD4 or CD8 T cells in the produced T cell population express PD1. + or CD8 + A T cell population produced, wherein when measured by flow cytometry, 5% or less of the CD4 or CD8 T cells in the produced T cell population express PD1. 128. When measured by flow cytometry, CD4 expressing PD1 + or CD8 + T-Rapa cells, compared to the corresponding frequency of CD4 expressing PD1 + or CD8 + A manufactured T cell population characterized by a reduced frequency of T cells. 129. The manufactured T cell population according to embodiment 128, wherein the reduced frequency is at least 50% less than the corresponding frequency. 130. A manufactured T cell population, which, when measured by flow cytometry, has 5% or less of CD4 + or CD8 + T cells in the manufactured T cell population expressing 2B4. 131. A manufactured T cell population, which, when measured by flow cytometry, has 5% or less of CD8+ T cells in the manufactured T cell population expressing 2B4. 132. When measured by flow cytometry, CD8 in a control T cell population characteristic of the T cells produced by the manufactured T cell population + T cells, compared to the corresponding frequency of CD8 T cells expressing 2B4 + A manufactured T cell population characterized by a reduced frequency of T cells. 133. The manufactured T cell population according to embodiment 132, wherein the reduced frequency is at least 50% less than the corresponding frequency. 134. When measured by flow cytometry, CD4 expressing 2B4 + or CD8 + T-Rapa cells, compared to the corresponding frequency of CD4 expressing 2B4 + or CD8 + A manufactured T cell population characterized by a reduced frequency of T cells. 135. The manufactured T cell population according to embodiment 134, wherein the reduced frequency is at least 20% less than the corresponding frequency. 136. A manufactured T cell population, wherein when measured by flow cytometry, 10% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express LAIR1. 137. A manufactured T cell population, wherein when measured by flow cytometry, the CD4 + or CD8 + in the manufactured T cell population has a reduced frequency of CD4 + or CD8+ T cells expressing LAIR1 compared to the corresponding frequency of CD4 138. The manufactured T cell population according to embodiment 137, wherein the reduced frequency is at least 50% less than the corresponding frequency. 139. A manufactured T cell population, wherein when measured by flow cytometry, the CD4 + or CD8 + in the manufactured T cell population has a reduced frequency of CD4 + or CD8 + T cells expressing LAIR1 compared to the corresponding frequency of CD4 140. The manufactured T cell population according to embodiment 139, wherein the reduced frequency is at least 50% less than the corresponding frequency. 141. A manufactured T cell population, wherein when measured by flow cytometry, 10% or less of the CD4 + or CD8 + T cells in the manufactured T cell population express TIGIT. 142. A manufactured T cell population, wherein when measured by flow cytometry, the CD4 + or CD8 + in the manufactured T cell population has a reduced frequency of CD4 + or CD8 + T cells expressing TIGIT compared to the corresponding frequency of CD4 143. The manufactured T cell population according to embodiment 142, wherein the reduced frequency is at least 40% less than the corresponding frequency. 144. A manufactured T cell population, wherein when measured by flow cytometry, 10% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express LAG3, the manufactured T cell population. 145. When measured by flow cytometry, CD4 expressing LAG3 + or CD8 + Compared to the corresponding frequency of T-Rapa cells, a manufactured T cell population characterized by a reduced frequency of CD4 + or CD8 + T cells. 146. The manufactured T cell population according to embodiment 144, wherein the reduced frequency is at least 50% less than the corresponding frequency. 147. A manufactured T cell population, wherein when measured by flow cytometry, 1% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express CD25, the manufactured T cell population. 148. A manufactured T cell population, wherein when measured by flow cytometry, 5% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express KLRG1, the manufactured T cell population. 149. A manufactured T cell population, wherein when measured by flow cytometry, 20% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express CD39, the manufactured T cell population. 150. A manufactured T cell population, wherein when measured by flow cytometry, 20% or less of the CD4 in the manufactured T cell population + or CD8 + T cells express CD73, the manufactured T cell population. 151. A manufactured T cell population, wherein when measured by flow cytometry, 4% or less of the CD4 in the manufactured T cell population +or CD8 + A manufactured T cell population in which T cells express GITR. 152. CD4 T cells expressing CD28 or ICOS in the manufactured T cell population + or CD8 + The frequency of T cells is that of CD4 T cells expressing CD28 or ICOS in a control T cell population characteristic of the T cells from which the manufactured T cell population was produced + or CD8 + The manufactured T cell population according to any one of embodiments 108 - 109 and 119 - 150, wherein the frequency is substantially the same as the corresponding frequency of T cells. 153. CD4 T cells expressing CD28 or ICOS + or CD8 + The frequency of T cells is that of CD4 T cells expressing CD28 or ICOS in a control T cell population characteristic of the T cells from which the manufactured T cell population was produced + or CD8 + The manufactured T cell population according to any one of embodiments 108 - 109 and 119 - 150, wherein the frequency is within 10% of the corresponding frequency of T cells. 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 of 3:1 - 1:3. 155. Anti - CD3 / anti - CD28 - coated magnetic beads at a bead:T cell ratio of 3:1 - 1:3, and IL - 7, IL - 15, or a combination of IL - 7 and IL - 15, and when present, co - stimulated at a concentration of 10 ng / mL for each of IL - 7 and IL - 15, and then secretes IL - 2 at least 1000 pg / mL / 1×10 6 cells / day of IL - 2. A manufactured T cell population. 156. A manufactured T cell population that secretes an increased amount of IL - 2 compared 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, and when present, at a concentration of 10 ng / mL for each of IL - 7 and IL - 15. A manufactured T cell population that expresses at least 75% less phosphorylated STAT5, detectable levels of STAT1 or phosphorylated STAT1, at least 50% decreased p70S6K and Raptor, and levels of Rictor, SGK1, and phosphorylated SGK1 that differ from the cultured T-Rapa cell population by 50% or less, compared to the cultured T-Rapa cell population. 157. A manufactured T cell population, (a) a decreased level of phosphorylated P70S6K compared to a control population of T cells, or (b) a decreased level of Raptor compared to a control population of T cells, characterized by at least one of the above, showing decreased mTORC1 activation, and A manufactured T cell population showing conservation of mTORC2 molecules, characterized by substantially the same levels of Rictor, SGK1, or phosphorylated SGK1. 157. A manufactured T cell population that expresses at least 75% less phosphorylated STAT5, detectable levels of STAT1 or phosphorylated STAT1, at least 50% decreased p70S6K and Raptor, and levels of Rictor, SGK1, and phosphorylated SGK1 that differ from the cultured T-Rapa cell population by 50% or less, compared to the cultured T-Rapa cell population. 158. A manufactured T cell population, (a) a decreased level of phosphorylated P70S6K compared to T-Rapa cells, or (b) a decreased level of Raptor compared to T-Rapa cells, characterized by at least one of the above, showing decreased mTORC1 activation, and A manufactured T cell population showing conservation of mTORC2 molecules, characterized by substantially the same levels of Rictor, SGK1, or phosphorylated SGK1 as T-Rapa cells. 159. The manufactured T cell population according to embodiment 158, further characterized by decreased STAT5 phosphorylation and detectable levels of STAT1 or phosphorylated STAT1 compared to a control T cell culture. A manufactured T cell population characterized by decreased STAT5 phosphorylation compared to a control T cell culture and detectable levels of STAT1 or phosphorylated STAT1. 161. A manufactured T cell population having one or more of the following characteristics, the characteristics being At least a 50% increase in the secretion of IFN-γ compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in the secretion of TNF-α compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in the secretion of GM-CSF compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in the secretion of IL-2 compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1; At least a 50% increase in the percentage of cells positive for CD4, CD62L, CCR7, and CD127 compared to a control population of T cells characteristic of the T cells from which the manufactured T cell population was produced, An increase in 4EBP1 phosphorylation of 50% or less compared to a control population of T cells characteristic of the T cells from which the T cell population was produced, At least a 50% decrease in the expression of p70S6K or Raptor compared to a T-Rapa cell population cultured under the same conditions, At least a 50% decrease in the expression of p-STAT5 compared to a T-Rapa cell population cultured under the same conditions, Detectable levels of STAT1 and p-STAT1 expression, At least a 10% increase in the expression of p70S6K compared to a control population of T cells characteristic of the cells from which the manufactured T cell population was produced, At least a 50% decrease in the expression of CD25 compared to a T-Rapa cell population. When measured by flow cytometry, CD4 + or CD8 + T cells are 10% or less, When measured by flow cytometry, CD4 expressing TIM3 + or CD8 + T cells are 10% or less, When measured by flow cytometry, CD4 expressing PD1 + or CD8 + T cells are 5% or less, When measured by flow cytometry, CD4 expressing 2B4 + or CD8 + T cells are 5% or less, When measured by flow cytometry, CD4 expressing LAIR1 + or CD8 + T cells are 10% or less, When measured by flow cytometry, CD4 expressing TIGIT + or CD8 + T cells are 10% or less, When measured by flow cytometry, CD4 expressing LAG3 + or CD8 + T cells are 10% or less, When measured by flow cytometry, CD4 expressing CD25 + or CD8 + T cells are 5% or less, When measured by flow cytometry, CD4 expressing KLRG1 + or CD8 + T cells are 5% or less, When measured by flow cytometry, CD4 expressing CD39 + or CD8 + T cells are 20% or less, When measured by flow cytometry, CD4 expressing CD73 + or CD8 + T cells are 20% or less, When measured by flow cytometry, CD4 T cells expressing GITR + or CD8 + T cells are 5% or less, The expression level of CD28 is within about 20% of the control population of T cells characteristic of the T cells produced by the produced T cell population, The expression level of ICOS is within about 20% of the control population of T cells characteristic of the T cells produced by the produced T cell population, The expression level of CD45RA is within about 20% of the control population of T cells characteristic of the T cells produced by the produced T cell population, When measured by flow cytometry, at least a 50% increase in CD4 T cells positive for CD45RA + At least a 50% increase in CD4 T cells, Compared to T-Rapa cultures incubated under the same conditions, IL-2 secretion is increased by at least 1.1-fold, After co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1 to 1:3, IL-2 secretion is at least 500 pg / mL / 1×10 6 Cells / day, When incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, where IL-7 and IL-15 are each added at 10 ng / mL, IL-2 secretion is increased by at least 1.1-fold, When incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, where IL-7 and IL-15 are each added at 10 ng / mL, after incubation, IL-2 secretion is at least 1000 pg / mL / 1×10 6 Cells / day, At least a 25% decrease in the expression of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3, and combinations thereof, compared to the corresponding expression levels of the T-Rapa cell population, 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, which is within 25% of the corresponding expression level in a control population of T cells characteristic of the cells produced by the manufactured T cell population. CD4 expressing CD127 + At least 5% T cells CD4 expressing CD127, compared to a control population of T cells characteristic of the cells produced by the manufactured T cell population + At least a 50% increase in the frequency of T cells At least a 25% increase in the frequency of T cells co-expressing CD62L and CCR7 compared to the cultured input T cells CD4 co-expressing IL-2 receptor CD25 at less than 5%, more preferably less than 1% + And CD8 + The frequency of T cells 1×10 per 24 hours contained in the culture supernatant after a stimulation procedure using high-level co-stimulation (3:1 bead-to-T cell ratio) 6 Secretion of low-level inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, defined as <100 pg / ml per cell An increase in IFN-γ and TNF-α secretion after a 6-day expansion period in the absence of inhibitor, which is at least 5-fold, more preferably 20-fold, compared to the secretion level on day 6, and The manufactured T cell population, which is these combinations. 162. A manufactured T cell having one or more of the following characteristics, the characteristics being At least a 50% increase in IFN-γ secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a 3:1 bead:T cell ratio; At least a 50% increase in TNF-α secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a 3:1 bead:T cell ratio; At least a 50% increase in the secretion of GM-CSF after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, compared to T-Rapa cells; At least a 50% increase in the secretion of IL-2 after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, compared to T-Rapa cells; Less than a 50% increase in 4EBP1 phosphorylation, compared to a control population of T cells characteristic of the T cells produced by the T cell population; At least a 50% decrease in the expression of p70S6K or Raptor, compared to a T-Rapa cell population cultured under the same conditions; At least a 50% decrease in the expression of p-STAT5, compared to a T-Rapa cell population cultured under the same conditions; Detectable levels of STAT1 and p-STAT1 expression; At least a 10% increase in the expression of p70S6K, compared to a control population of T cells characteristic of the T cells produced by the T cell population produced; At least a 50% decrease in the expression of CD25, compared to a T-Rapa cell population; CD28 expression levels within about 20% of a control population of T cells characteristic of the T cells produced by the T cell population produced; ICOS expression levels within about 20% of a control population of T cells characteristic of the T cells produced by the T cell population produced; CD45RA expression levels within about 20% of a control population of T cells characteristic of the T cells produced by the T cell population produced; CD4 positive for CD45RA when measured by flow cytometry + At least a 50% increase in T cells; At least a 1.1-fold increase in IL-2 secretion, compared to a T-Rapa culture incubated under the same conditions; After costimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1 to 1:3, IL-2 secretion is at least 500 pg / mL / 1×10 6 cells / day, When present, IL-2 secretion is increased by at least 1.1-fold when incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL. When present, after incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL, the secretion of IL-2 is at least 1000 pg / mL / 1×10 6 cells / day. A decrease of at least 25% in the expression of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3, and combinations thereof, as compared to the corresponding expression levels of the T-Rapa cell population. Expression levels of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3, and combinations thereof, which are within 25% of the corresponding expression levels in a control population of T cells characteristic of the cells produced by the manufactured T cell population. Expressing CD127. Low-level secretion of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, defined as <100 pg / ml per cell, contained in the culture supernatant after a stimulation procedure using a high level of co-stimulation (3:1 bead-to-T cell ratio). 6 An increase in IFN-γ and TNF-α secretion after a 6-day expansion period in the absence of inhibitor, which is at least 5-fold, more preferably 20-fold, compared to the secretion levels on day 6, and Combinations thereof, the manufactured T cells. Combinations thereof, the manufactured T cells.

Claims

1. A composition for treating cancer in a subject in need of cancer treatment, comprising T cells produced in a therapeutically effective dose, wherein the produced T cells are derived from T cells from the subject, which have been cultured ex vivo in a culture medium comprising temsirolimus, an anti-IL-2 receptor antibody, and IFN-α, wherein the cancer is (a) smoldering multiple myeloma, (b) relapsed / refractory multiple myeloma, (c) quad or penta-refractory multiple myeloma, or (d) 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 selected from the group consisting of.

2. The therapeutically effective amount is 1×10 ５ to 5×10 ６ engineered T cells per kg of body weight of the subject, the composition according to claim 1.

3. The composition according to any one of claims 1 to 2, wherein the cancer is selected from lung cancer and pancreatic cancer.

4. The produced T cells are a population of produced T cells having one or more of the following characteristics, wherein the characteristics are at least a 50% increase in the secretion of IFN-γ compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, at least a 50% increase in the secretion of TNF-α compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, at least a 50% increase in the secretion of GM-CSF compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, at least a 50% increase in the secretion of IL-2 compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, at least a 50% decrease in the expression of p70S6K or raptor compared to the T-Rapa cell population, at least a 50% decrease in the expression of p-STAT5 compared to the T-Rapa cell population, detectable levels of STAT1 and p-STAT1 expression, at least a 50% decrease in the expression of CD25 compared to the T-Rapa cell population, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 10% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 10% or less. When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 5% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 5% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 10% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 10% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 10% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 5% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 5% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 20% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 20% or less, When measured by flow cytometry, CD4 ＋ or CD8 ＋ T cells are 5% or less, When measured by flow cytometry, an increase of at least 50% in CD4 ＋ T cells that are positive for CD45RA, at least a 1.1-fold increase in IL-2 secretion compared to the T-Rapa cell population 3:1 to 1:3 bead:T cell ratio, after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads, IL-2 secretion is at least 500 pg / mL / 1×10 ６ cells / day, If present, at least a 1.1-fold increase in IL-2 secretion when incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL, If present, after incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL, the secretion of IL-2 is at least 1000 pg / mL / 1×10 ６ cells / day, CD4 co-expressing less than 5%, more preferably less than 1% of the IL-2 receptor CD25 ＋ and CD8 ＋ frequency of T cells Secretion of low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacture, defined as < 100 pg / ml per cell, in the culture supernatant contained per 24 hours after a stimulation procedure using high-level co-stimulation (3:1 bead-to-T cell ratio). ６ ​ An increase in IFN-γ and TNF-α secretion after a 6-day expansion period in the absence of an inhibitor, at least 5-fold, more preferably 20-fold, compared to the secretion level on day 6, and, A composition according to any of claims 1 to 3, which is these combinations.

5. A manufactured T cell or population thereof having one or more of the following characteristics, wherein the manufactured T cell is a T cell derived from a subject in need of treatment for cancer, cultured ex vivo in a culture medium containing temsirolimus, an anti-IL-2 receptor antibody, and IFN-α, and the characteristics are At least a 50% increase in IFN-γ secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in TNF-α secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in GM-CSF secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% increase in IL-2 secretion compared to T-Rapa cells after 1 week of incubation using stimulation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, At least a 50% decrease in the expression of p70S6K or raptor compared to the T-Rapa cell population, At least a 50% decrease in the expression of p-STAT5 compared to the T-Rapa cell population, Detectable levels of STAT1 and p-STAT1 expression, At least a 50% decrease in the expression of CD25 compared to the T-Rapa cell population, When measured by flow cytometry, an increase of at least 50% in CD4 T cells that are positive for CD45RA ＋ and At least a 1.1-fold increase in IL-2 secretion compared to the T-Rapa cell population, 3:1 to 1:3 bead:T cell ratio, after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads, IL-2 secretion is at least 500 pg / mL / 1×10 ６ cells / day, If present, at least a 1.1-fold increase in IL-2 secretion when incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL, If present, after incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, with IL-7 and IL-15 each added at 10 ng / mL, the secretion of IL-2 is at least 1000 pg / mL / 1×10 ６ cells / day, Secretion of low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of production, defined as < 100 pg / ml per cell, in the culture supernatant contained per 24 hours after a stimulation procedure using high-level co-stimulation (3:1 bead-to-T cell ratio). ６ ​ An increase in IFN-γ and TNF-α secretion after a 6-day expansion period in the absence of an inhibitor that is at least 5-fold, more preferably 20-fold, compared to the secretion level on the 6th day, and, The produced T cells or a population thereof, which are these combinations.

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