Method for treating cancer using manufactured t cells

Abstract

The present disclosure relates to a first immunodepletion regimen, a second immunodepletion regimen, and the use of a composition comprising the manufactured T cells in the preparation of a kit for cancer in a subject in need thereof.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on November 15, 2019, with application number 201980089257.X and invention title "Method for Treating Cancer Using Manufactured T Cells".

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

[0003] The field of this invention relates to the use of a first immune depletion regimen, a second immune depletion regimen, and a composition comprising manufactured T cells in the preparation of a kit for use in cancer patients in need. Background Technology

[0004] CD4 in the type I cytokine spectrum + and CD8 + T cells (Th1 and Tc1 cells, respectively) are candidate T cell populations for adoptive T cell therapy. Th1 T cells are promoted by stimulating polarizing cytokines such as IL-12 and IFN-α, which in turn promote TBET transcription factors that partially define the Th1 differentiation state.

[0005] The success of adoptive T-cell therapy depends in part on the persistence of the T-cell population in the host. T-cell persistence is a balance determined by an increased capacity for T-cell proliferation and maintenance of T-cell memory, and a reduced tendency for T-cell apoptotic cell death. Previous studies have demonstrated that T-cells generated in vitro from the pharmacological agent rapamycin (which inhibits the mammalian target mTOR of rapamycin) exhibit these properties: enhanced ability to perform antigen-driven clonal expansion in vivo after adoptive transfer; and improved memory states, such as T-central memory (T-C). CMThe differentiation state is exemplified; and a multi-layered anti-apoptotic phenotype characterized by induced autophagy (including mitophagy) and preferential expression of anti-apoptotic members of the bcl-2 gene family relative to pro-apoptotic members. In summary, these properties of ex vivo rapamycin-resistant cells are associated with enhanced in vivo regulation of transplant responses, including prevention of graft-versus-host disease (GVHD) and graft rejection, as well as the mediation of human-mouse xenogeneic GVHD; notably, these cells have been successfully translated into clinical trials in autologous and allogeneic settings for the treatment of multiple myeloma, demonstrating safety and efficacy against relapsed and refractory multiple myeloma. For these clinical trial studies, the manufacturing process included 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 in vitro cultures; addition of a high-dose oral administration of the mTOR inhibitor rapamycin (1 μM); and addition of IL-2 to the culture during cytokine polarization (IFN-α addition). Cells produced by this method are referred to herein as T-Rapa cells, which are further specifically defined thereafter in this disclosure.

[0006] Cancer development may be associated with alterations in immune function. These alterations include cell-mediated immunity (CMI) suppression, which is associated with an inability to reject tumors, and humoral immunity enhancement, which can amplify tumor promotion and progression. CD4 + T cell subsets, Th1 T cells, and Th2 T cells have different functions and regulate each other. Th1 cells produce interleukin (IL-2) and interferon (IFN-γ) and direct the CMI response, while Th2 cells produce IL-4 and IL-10 and promote local humoral immune responses.

[0007] There is evidence of Th1 / Th2 imbalance in certain cancers, where the proportion of Th2 cells increases significantly when the number of Th1 cells is reduced. Chronic Th1 / Th2 imbalance that favors Th2 cells may lead to cell-mediated immunosuppression, thereby providing a favorable environment for reduced effective immune surveillance and malignant tumor development.

[0008] Idiotype-specific T-cell responses have been found in most patients with early-stage multiple myeloma. These responses include Th1 responses generated with IL-2 and IFN-γ. For example, Th1 immunity is preferentially found in cases of painless disease, and Th2 responses are primarily found in cases of advanced multiple myeloma. Defective Th1 immune responses (mediated by IL-6) and a dysregulated cytokine network are found in patients with multiple myeloma. Myeloma idiotype-specific T helper cells derived from MM patients are consistently non-Th1 phenotypes.

[0009] Despite advances in the treatment of multiple myeloma and recent FDA approvals of new agents and monoclonal antibodies, multiple myeloma remains almost universally fatal. Thus, patients with relapsed / refractory multiple myeloma (RRMM) who are refractory to the first five drugs for multiple myeloma (“penta-refractory”) have only a limited survival of a few months and few treatment options. Multiple myeloma is a disease susceptible to immunotherapy, as evidenced by the long-term observed curative effects of allogeneic stem cell transplantation and many other approaches, including monoclonal antibody therapy, vaccines, and T-cell receptor (TCR)-modified and CAR-modified T-cell therapies. Therefore, this penta-refractory patient population is a suitable candidate for novel T-cell therapies. Additionally, patients with less refractory disease also urgently need novel treatments; that is, even at the second or third relapse, the median progression-free survival is typically less than two years.

[0010] For certain cancers, novel and innovative immunotherapies are needed. Summary of the Invention

[0011] This disclosure relates to a method for treating cancer in a subject.

[0012] In one embodiment, a method includes administering to the subject a therapeutically effective dose of a composition comprising manufactured T cells.

[0013] In another embodiment, the method further includes subjecting the subject to an immune depletion protocol prior to administering a therapeutically effective dose of the composition comprising the manufactured T cells to the subject to reduce at least a portion of the regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of the regulatory T cells and / or late-senescent effector T cells.

[0014] In some embodiments, the immune depletion regimen includes administering a first composition comprising pentostatin to the subject; and administering a second composition comprising cyclophosphamide to the subject.

[0015] In some embodiments, the method includes a first treatment cycle and one or more additional treatment cycles, the first treatment cycle comprising: subjecting the subject to a first immune depletion regimen to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells; each of the one or more additional treatment cycles comprising: subjecting the subject to a second immune depletion regimen to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells; and administering to the subject a therapeutically effective dose of a composition comprising manufactured T cells.

[0016] In any of the foregoing embodiments, the method may further include measuring the subject's creatinine clearance (CrCl) before 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 when CrCl ≥ 60 mL / min / 1.73 m², the dose is 4 mg / m². 2 Administer pentostatin, where when 60 mL / min / 1.73 m² > CrCl ≥ 30 mL / min / 1.73 m², at a dose of 2 mg / m². 2 Pentostatin is administered, but not when CrCl < 30 mL / min / 1.73 m². In some embodiments, the dose of pentostatin may be adjusted based on CrCl such that when 60 mL / min / 1.73 m² > CrCl ≥ 30 mL / min / 1.73 m², the dose of pentostatin is reduced by 50%, and pentostatin is not administered when CrCl < 30 mL / min / 1.73 m².

[0017] In any of the foregoing embodiments, the method may further include measuring the absolute lymphocyte count (ALC) and 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, wherein cyclophosphamide is administered at a dose of 100 mg when ANC is 500-999 per microliter and ALC ≥ 50 per microliter, and wherein cyclophosphamide is not administered when ALC < 50 per microliter or ANC < 500 per microliter. In some embodiments, the dose of cyclophosphamide may be adjusted based on ALC and ANC such that when ANC is 500-999 per microliter and ALC ≥ 50 per microliter, the dose of cyclophosphamide is reduced by 50%, or when ALC < 50 per microliter or ANC < 500 per microliter, cyclophosphamide is not administered. Attached Figure Description

[0018] Figure 1A The CD4+ expression of FoxP3 was depicted on day 0 and under various culture conditions. + The percentage of T cells.

[0019] Figure 1B The CD4+ expression of TBET was depicted on day 0 and under various culture conditions. + The percentage of T cells.

[0020] Figure 2A The cultivation of CD4 under various conditions was described. + and CD8 + T cell production rate after T cells are generated.

[0021] Figure 2B The cultivation of CD4 under various conditions was described. + and CD8 + T cell production rate after T cells are generated.

[0022] Figure 3A The cultivation of CD4 under various conditions was described. + and CD8 + IFN-γ secretion following T cell death.

[0023] Figure 3B The cultivation of CD4 under various conditions was described. + and CD8 + TNF-α secretion following T cell death.

[0024] Figure 4 The image depicts CD4 cells cultured using the T-Rapa method and the method of this disclosure (Rapa-T). + and CD8 + Western blots of p-4EBP1 and actin in T cells (top image). CD4 cells cultured using the T-Rapa method and the method disclosed herein (Rapa-T). + and CD8 + T cells normalize p-4EBP1 levels through actin expression (see figure below).

[0025] Figure 5 The image depicts CD4 cells cultured using the T-Rapa method and the method of this disclosure (Rapa-T). + and CD8 + Western blots of P70S6K and actin in T cells (top image), and CD4 cells cultured using the T-Rapa method and the method disclosed herein. + and CD8 +The normalized P70S6K level in T cells is expressed through actin (see figure below).

[0026] Figure 6 The image depicts CD4 cells cultured using the T-Rapa method and the method of this disclosure (Rapa-T). + and CD8 + Western blots of P-STAT5 and actin from T cells (top image), and CD4 cells cultured using the T-Rapa method and the method disclosed herein. + and CD8 + The normalized P-STAT5 level in T cells is determined by actin expression (see figure below).

[0027] Figure 7A The cultivation of CD4 under various conditions was described. + and CD8 + IFN-γ is secreted on day 6 after T cells are generated.

[0028] Figure 7B The cultivation of CD4 under various conditions was described. + and CD8 + IFN-γ is secreted on day 13 after T cell delivery.

[0029] Figure 8A The cultivation of CD4 under various conditions was described. + and CD8 + TNF-α is secreted on day 6 after T cells are generated.

[0030] Figure 8B The cultivation of CD4 under various conditions was described. + and CD8 + TNF-α is secreted on day 13 after T cells are generated.

[0031] Figure 9A The cultivation of CD4 under various conditions was described. + and CD8 + GM-CSF is secreted on day 6 after T cells are generated.

[0032] Figure 9B The cultivation of CD4 under various conditions was described. + and CD8 + GM-CSF is secreted on day 13 after T cells are generated.

[0033] Figure 10A The cultivation of CD4 under various conditions was described. + and CD8 + IL-2 is secreted on day 6 after T cells are generated.

[0034] Figure 10B The cultivation of CD4 under various conditions was described.+ and CD8 + IL-2 is secreted on day 13 after T cells are generated.

[0035] Figure 11 The images depict CD4 cells cultured under various conditions. + and CD8 + Western blots of P62 and actin from T cells (top image), and CD4 under various conditions. + and CD8 + P62 protein expression normalized by actin expression in T cells (see figure below).

[0036] Figure 12 The images depict CD4 cells cultured under various conditions. + and CD8 + Western blots of p-RAPTOR and actin from T cells (top image), and CD4 cells cultured under various conditions. + and CD8 + The p-RAPTOR level in T cells is normalized by actin expression (see figure below).

[0037] Figure 13 The images depict CD4 cells cultured under various conditions. + and CD8 + BIM and actin proteomic blots of T cells (top image), and CD4 cultured under various conditions. + and CD8 + BIM protein expression normalized by actin expression in T cells (see figure below).

[0038] Figure 14A Flow cytometry analysis was performed to depict the expression of CD45RA on CD4+ cell subsets after T cells were cultured under various conditions.

[0039] Figure 14B Flow cytometry analysis was performed to depict the expression of CD45RA on CD4+ cell subsets after T cells were cultured under various conditions.

[0040] Figure 14C Flow cytometry analysis was performed to depict the expression of CD45RA on CD4+ cell subsets after T cells were cultured under various conditions.

[0041] Figure 14D The study described the effects of CD4 on T cells cultured under various conditions. + Flow cytometry analysis of CD45RA expression in cell subsets.

[0042] Figure 15AFlow cytometry analysis was performed to depict the expression of CD62L, CCR7, and CD127 on CD4+ cell subsets after T cells were cultured under various conditions.

[0043] Figure 15B Flow cytometry analysis was performed to depict the expression of CD62L, CCR7, and CD127 on CD4+ cell subsets after T cells were cultured under various conditions.

[0044] Figure 15C Flow cytometry analysis was performed to depict the expression of CD62L, CCR7, and CD127 on CD4+ cell subsets after T cells were cultured under various conditions.

[0045] Figure 15D Flow cytometry analysis was performed to depict the expression of CD62L, CCR7, and CD127 on CD4+ cell subsets after T cells were cultured under various conditions.

[0046] Figure 16 The fold increase in culture yield of T cells cultured under various conditions was depicted.

[0047] Figure 17A The secretion of IFN-γ on day 6 after culturing T cells under various conditions was described.

[0048] Figure 17B The secretion of IFN-γ on day 13 after culturing T cells under various conditions was described.

[0049] Figure 18A The secretion of TNF-α on day 6 after culturing T cells under various conditions was described.

[0050] Figure 18B The secretion of TNF-α on day 13 after culturing T cells under various conditions was described.

[0051] Figure 19 The CD4 counts on days 6 and 13 after culturing T cells under various conditions were depicted. + Flow cytometry analysis of CD25 expression in T cell subsets.

[0052] Figure 20 The CD4 counts on days 6 and 13 after culturing T cells under various conditions were depicted. + Flow cytometry analysis of CD62L, CCR7 and CD127 expression in T cell subsets.

[0053] Figure 21Flow cytometry expression analyses were performed on the following methods compared to the novel Rapa-T method without bead co-stimulation, the novel Rapa-T method with bead co-stimulation (bead to T cell ratio of 1:3), and the old T-Rapa method (bead to T cell ratio of 3:1): primary and T central memory maps; CD45RA expression; co-expression of CD62L and CCR7; and co-expression of CD62L, CCR7, and CD127.

[0054] Figure 22 Flow cytometry expression analyses were performed on the following methods compared with the new Rapa-T method without bead co-stimulation, the new Rapa-T method with bead co-stimulation (bead to T cell ratio of 1:3), and the old T-Rapa method (bead to T cell ratio of 3:1): expression of IL-2 receptor CD25; and expression of immunosuppressive molecules CTLA4 and TIM3.

[0055] Figure 23 The study depicted cytokine secretion results at the end of manufacturing and after an additional 6 days of culture without the use of inhibitors, comparing the following: secretion of type II cytokine IL-4 and type I cytokine IFN-γ in the new Rapa-T method without bead co-stimulation, the new Rapa-T method with bead co-stimulation (bead to T cell ratio of 1:3), and the old T-Rapa method (bead to T cell ratio of 3:1).

[0056] Figure 24 The results of type I cytokine secretion (IL-2 and IFN-γ) at the end of the manufacturing process and after an additional 6 days of culture under n=11 different culture conditions without the use of inhibitors were depicted to further identify the role of bead-free co-stimulation in the manufacturing of new Rapa-T cell populations.

[0057] Figure 25A Flow cytometry data measurements of CD45RA+ T cells were depicted under various culture conditions.

[0058] Figure 25B Flow cytometry data measurements of CD25+ T cells were depicted under various culture conditions.

[0059] Figure 25C Flow cytometry data measurements of CD28+ T cells were depicted under various culture conditions.

[0060] Figure 25D Flow cytometry data measurements of ICOS+ on T cells were depicted under various culture conditions.

[0061] Figure 25E Flow cytometry data measurements of CD39+ T cells were depicted under various culture conditions.

[0062] Figure 25F Flow cytometry data measurements of CD73+ T cells were depicted under various culture conditions.

[0063] Figure 25G Flow cytometry data measurements of GITR+ on T cells were depicted under various culture conditions.

[0064] Figure 25H Flow cytometry data measurements of LAG3+ T cells were depicted under various culture conditions.

[0065] Figure 25I Flow cytometry data measurements of PD1+ on T cells were depicted under various culture conditions.

[0066] Figure 25J Flow cytometry data measurements of 2B4+ T cells were depicted under various culture conditions.

[0067] Figure 25K Flow cytometry data measurements of LAIR1+ on T cells were depicted under various culture conditions.

[0068] Figure 25L Flow cytometry data measurements of CTLA4+ T cells were depicted under various culture conditions.

[0069] Figure 25M Flow cytometry data measurements of KLRG1+ on T cells were depicted under various culture conditions.

[0070] Figure 25N Flow cytometry data measurements of TIGIT+ on T cells were depicted under various culture conditions.

[0071] Figure 25O Flow cytometry data measurements of TIM3+ in T cells were depicted under various culture conditions.

[0072] Figure 26 Western blot results depicting p-STAT5, p-STAT1, STAT1, p70S6K, p-SGK1, SGK1, Raptor, Rictor, cytochrome C, and actin in cells under various culture conditions were presented.

[0073] Figure 27A The study depicted the measurement of IL-2 secretion from RAPA-T cells and T-RAPA cells 24 hours after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads and exposure to different cytokines.

[0074] Figure 27BThe study depicted the measurement of TNF-α secretion from RAPA-T cells and T-RAPA cells 24 hours after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads and exposure to different cytokines.

[0075] Figure 28 The secretion of IL-2, TNF-α, and IL-13 in RAPA-T cells after co-stimulation with anti-CD3 / anti-CD28 coated beads or soluble anti-CD3 / anti-CD28 microparticles was described.

[0076] Figure 29 The expression of CD4, CD8, CD25, and CTLA4 in RAPA-T cells was depicted by flow cytometry after co-stimulation with anti-CD3 / anti-CD28 coated beads or soluble anti-CD3 / anti-CD28 microparticles.

[0077] Figure 30 The fabricated T-cell therapy protocol is described.

[0078] Figure 31 The pentostatin / cyclophosphamide regimen was described, followed by the manufacture of T-cell infusions.

[0079] Figures 32A-32C The nature of the control group was detailed, namely, subjects who were not randomized to receive Rapa-T cell therapy would receive one of three FDA-approved triple regimens applicable to subjects with second or third relapses of MM: DPd regimen (A); DRd regimen (B); or KRd regimen (C).

[0080] Figure 33 An exemplary workflow for producing the manufactured T cells of this disclosure is described.

[0081] Figure 34A The cytokine secretion of RAPA-T cells after exposure to pancreatic cancer cells was described.

[0082] Figure 34B The cytokine secretion of RAPA-T cells after exposure to lung cancer cells was described.

[0083] Figure 35 The study describes the cytokine secretion of RAPA-T cells under conditions of exposure to pancreatic cancer cells or lung cancer cells, with or without exposure.

[0084] Figure 36 The correlation between checkpoint inhibitor treatment response and the number of tumor mutations in cancer was depicted. Detailed Implementation

[0085] This disclosure provides methods for producing manufactured T cells, manufactured T cells produced by the methods disclosed herein, populations and compositions comprising manufactured T cell populations, and methods for treating a subject with cancer using said manufactured T cells or manufactured T cell populations.

[0086] definition

[0087] The following definitions are provided:

[0088] As used herein, unless the content expressly indicates otherwise, the singular form “a / an” and “described” include plural indicators. The term “or” in the claims and this disclosure is used to mean “and / or” unless it is expressly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

[0089] When used with numerical values, the term “about” is intended to include + / - 10%. As an example rather than a limitation, if the number of amino acids identified is about 200, this would include 180 to 220 (plus or minus 10%).

[0090] The terms “subject,” “patient,” and “individual” are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the present invention can be used in laboratory animals, veterinary applications, and the development of animal models of disease, including but not limited to rodents including mice, rats, and hamsters; and primates.

[0091] The term “sample” is used in its broadest sense herein. Samples including cells, polynucleotides, polypeptides, peptides, antibodies, etc., may include body fluids; soluble fractions of cell preparations, or culture media in which cells are grown; chromosomes, organelles, or membranes isolated or extracted from cells; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; cells; tissues; tissue imprints; fingerprints, skin, or hair; etc.

[0092] "Treatment" is an intervention aimed at preventing the progression of a disease or altering its pathology or symptoms. Therefore, "treatment" refers to both therapeutic treatment and preventative or preventative measures. Patients requiring treatment include those already suffering from the disease and those needing to prevent its development. For example, in the treatment of tumors (e.g., cancer), a therapeutic agent can directly reduce the pathology of tumor cells or make tumor cells more susceptible to other therapeutic agents (e.g., radiation and / or chemotherapy). As used herein, "improvement" refers to symptoms that are close to a normalized value (by way of example and not limitation, a value obtained in a healthy patient or individual), for example, symptoms that differ from a normalized value by less than 50%, preferably symptoms that differ from a normalized value by less than about 25%, more preferably symptoms that differ from a normalized value by less than 10%, and even more preferably symptoms that are not significantly different from a normalized value determined using conventional statistical tests. By way of example and not limitation, improvement or treatment of a patient with an infectious disease organism (e.g., hepatitis B virus) can be determined by the reduction of viral particles in a sample collected from the patient, such as by, for example, a reduction in plaque-forming units (pfu).

[0093] As used in this article, “treatment cycle” can generally refer to any primary treatment cycle, first treatment cycle, second treatment cycle, or one or more additional treatment cycles.

[0094] As used herein, the term "therapeutic effective dose" or "therapeutic effective amount" means the amount of the compound of the present invention that effectively produces the desired therapeutic response. By way of example and not limitation, a dose that effectively delays cancer growth or shrinks cancer or prevents metastasis can be a "therapeutic effective dose." The specific therapeutic effective dose will vary depending on factors such as the specific condition being treated, the patient's physical condition, the type of mammal or animal being treated, the duration of treatment, the nature of any concurrent therapies, and the structure of the specific formulation and compound or its derivatives used.

[0095] As used herein, “immune cells” are intended to include any cell of the immune system that can be measured, 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 monocytes, tumor-infiltrating (TIL) cells, genetically modified immune cells (including hybridomas), drug-modified immune cells, and derivatives, precursors, or progenitor cells of the above cell types.

[0096] "T cells" are a subset of lymphocytes that originate from the thymus and possess heterodimeric receptors associated with proteins of the CD3 complex (e.g., rearranged T cell receptors, heterodimeric proteins on the T cell surface responsible for cell antigen / MHC specificity). T cell responses can be detected by measuring the effects of T cell responses on other cells (e.g., target cell killing, activation of other immune cells such as B cells) or by measuring cytokines produced by T cell responses.

[0097] 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 portion. Where anti-CD3 / anti-CD28 co-stimulation is not provided even through a specific form such as anti-CD3 / anti-CD28 magnetic beads, it should be understood that this also precludes the use of other forms of anti-CD3 / anti-CD28 co-stimulation.

[0098] As used herein, the term "one or more T-Rapa cells" refers to one or more T cells generated by co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a 3:1 ratio (beads to T cells ratio) without any delay between culture initiation and co-stimulation, wherein the cells are grown in X-Vivo or an equivalent medium containing IFN-α (10,000 IU / mL), IL-2 (20 IU / mL), and rapamycin (1 μM) supplemented with 5% AB serum but without the addition of IL-2 signaling inhibitors, wherein the cells are cultured at 37°C for 6 days and then at 1.5 × 10⁻⁶ cells / mL. 6 Start culturing at a concentration of cells / mL. As used with respect to methods, unless otherwise stated, “T-Rapa” refers to the method of producing T-Rapa cells as defined above.

[0099] 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 this disclosure. "Manufactured T cells" may contain CD4+. + CD8 + T cells or both. “Manufactured T cells” does not include T cells collected from patients, i.e., naturally occurring T cells.

[0100] It should be understood that, as used herein, “level of expression,” “expression level,” or equivalent references, when used to refer to results measured by flow cytometry, refer to the frequency of positive cells of a specified type in a population. With respect to “level of expression” or equivalent expression referring to the expression level of a specific cell type, it should be understood that, unless otherwise stated, any decrease or increase mentioned is relative to the corresponding specified cell type.

[0101] As those skilled in the art will recognize, the term “autologous” cell refers to a cell that has the same or similar haplotype as the cell of the subject or “host” to which the cell is administered, such that when these cells are transplanted into a host, no significant immune response against these cells occurs.

[0102] CD4 is a cell surface protein important for T cell receptor recognition of antigenic peptides that bind to MHC class II molecules on the surface of APCs. Upon activation, primordial CD4 T cells differentiate into at least two cell types (Th1 cells and Th2 cells), each characterized by the cytokines they produce. Th1 cells are primarily involved in activating macrophage-mediated cellular immunity and inflammatory responses, while Th2 cells, or helper T cells, are primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for human immunodeficiency virus (HIV). Effector molecules for Th1 cells include, but are not limited to, IFN-γ, GM-CSF, TNF-α, CD40 ligand, Fas ligand, IL-3, TNF-β, and IL-2. Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, IL-13, CD40 ligand, IL-3, GM-CSF, IL-10, TGF-β, and eosinophil chemokine. Activation of Th1 cytokine responses can inhibit Th2 cytokine responses, and conversely, activation of Th2 cytokine responses can inhibit Th1 responses.

[0103] "Cytokines" are proteins produced by cells that influence the behavior of other cells through "cytokine receptors" on the surface of the cell to which the cytokine acts. Cytokines produced by lymphocytes are sometimes called "lymphokines." Cytokines are also characterized as type I (e.g., IL-2 and IFN-γ) and type II (e.g., IL-4 and IL-10).

[0104] The term "modulation" refers to any activity mentioned, such as being increased, enhanced, amplified, activated (acting as an agonist), promoted, reduced, decreased, inhibited, blocked, or antagonized (acting as an agonist). Modulation can increase activity by more than 1, 2, 3, 5, 10, or 100 times the baseline value. Modulation can also reduce its activity below the baseline value.

[0105] "Substrate" refers to any rigid or semi-rigid carrier that binds to nucleic acid molecules or proteins, and includes membranes, filters, chips, glass slides, wafers, fibers, magnetic or non-magnetic beads, gels, capillaries or other tubes, plates, polymers, and microparticles with various surface forms (including pores, grooves, pins, channels, and pores).

[0106] Methods for producing manufactured T cells, manufactured T cells produced by the methods disclosed herein, and compositions comprising a population of manufactured T cells.

[0107] In the method disclosed herein, IFN-α is used to in vitro polarize cultures including T cells toward a Th1-type differentiation phenotype. Th1-type differentiation can be directed toward regulatory T cells (T1-type T cells). REG The polarization of the cell phenotype is weakened, and the Th1 differentiation is promoted by cytokines containing IL-2, which signal via STAT5 to promote partially defined T cells. REG Differentiated FoxP3 transcription factors. In the method disclosed herein, autocrine IL-2 signaling is prevented during Th1 polarization by omitting exogenous use of IL-2 during cell culture and by adding an IL-2 signaling inhibitor to the culture. REG Pollution. In some respects, IL-2 signaling inhibitors are anti-IL-2 receptor monoclonal antibodies.

[0108] In this disclosure, a novel manufacturing method is provided that, compared with other T cell manufacturing methods, incorporates the following interventions: (1) delaying or omitting the addition of anti-CD3 / CD28 beads (or alternatively, any alternative source of anti-CD3 / anti-CD28 co-stimulation, such as nanoparticles or microparticles) for culture to improve T cell yield, wherein the anti-CD3 / CD28 beads or nanoparticles are used for co-stimulation; (2) using a lower ratio of anti-CD3 / CD28 beads to enhance the resistant T cell phenotype, or alternatively, omitting the addition of co-stimulation based on artificial antibody beads, nanoparticles, or microparticles; (3) using an mTOR-inhibiting parenteral formulation (temsirolimus) to improve manufacturing feasibility; and (4) avoiding IL-2 signaling and the resulting Th1-type differentiated T cells by omitting the typical use of exogenous IL-2 during T cell culture and eliminating endogenous autocrine IL-2 signaling. REGCell contamination, for example, can occur through the use of anti-IL-2 receptor monoclonal antibodies (daclizumab and basiliximab, or other agents that inhibit IL-2 receptor signaling) during T cell culture. In parallel culture experiments, T cells generated by this novel combination method (referred to as "manufactured T cells") exhibited a more desirable cell phenotype compared to T cells previously generated in ex vivo cultures.

[0109] Figure 33 An exemplary workflow for producing manufactured T cells according to this disclosure is provided.

[0110] In some embodiments, the method for producing manufactured T cells includes the step of seeding a culture comprising T cells from a subject into a cell population at a certain cell density in a culture medium containing tesimolimus and an IL-2 signaling inhibitor. In some aspects, the culture medium does not yet contain tesimolimus and / or the IL-2 signaling inhibitor, and these components may be added at or approximately simultaneously with seeding. Incubating the culture into the cell population for a first time period without co-stimulation by anti-CD3 / anti-CD28 antibodies, by way of example and not limitation, includes co-stimulation using anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles. After the first time period, the culture into the cell population may be stimulated with anti-CD3 / anti-CD28 antibodies, for example, by adding anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles. When using anti-CD3 / anti-CD28 coated magnetic beads, they may be used at a bead ratio between 1:1 and 1:12. Additionally, IFN-α is added to the culture medium. The culture is then incubated with the cell population for a second time period to produce manufactured T cells. In some aspects, co-stimulation using anti-CD3 / anti-CD28 coated magnetic beads, nanoparticles, or microparticles is not performed. In some embodiments, co-stimulation is not performed.

[0111] In any of the foregoing embodiments, the method for producing manufactured T cells may further include, after harvesting the manufactured T cells: packaging at least a portion of the manufactured T cells in a package; and freezing the package containing the portion of the manufactured T cells. The cryopreservation of the manufactured T cells can be performed using methods known in the art.

[0112] In any of the foregoing embodiments, the method may further include harvesting the culture input cell population from the subject before inoculating T cells from the subject in a culture medium at a certain cell density.

[0113] In any of the foregoing embodiments, the culture medium may be IL-2-free and no IL-2 may be added to the culture medium. In any of the foregoing embodiments, no serum may be added to the culture; for example, the culture is serum-free. In any of the foregoing embodiments, the culture medium may be substantially serum-free.

[0114] In any of the foregoing embodiments, the IFN-α may be added at or approximately simultaneously with the addition of the anti-CD3 / anti-CD28 coated magnetic beads. If no co-stimulation is applied to the culture, the IFN-α may be added, for example, at the start of culture or within 48 hours after the start of culture. By way of example and not limitation, the IFN-α may be added at 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 culture.

[0115] In any of the foregoing embodiments, the cell density may be approximately 1 × 10⁻⁶ per mL. 6 From 50 × 10 T cells per mL 6 Cell density. By way of example and not limitation, the cell density could be approximately 1 × 10⁶ cells per mL. 6 From 5 × 10 cells per mL 6 10 cells per mL, 1×10 6 From 10 × 10 cells per mL 6 10 cells per mL, 1×10 6 15 × 10 cells / mL 6 15 × 10 cells per mL 6 From 10 cells per mL to 22.5 × 10⁻⁶ cells per mL 6 10 × 10 cells per mL 6 From 10 cells per mL to 22.5 × 10⁻⁶ cells per mL 6 10 × 10 cells per mL 6 From 10 cells per mL to 22.5 × 10⁻⁶ cells per mL 6 5 × 10 cells per mL 6 From 10 cells per mL to 22.5 × 10⁻⁶ cells per mL 6 10 cells per mL, 1×10 6 50 × 10 cells / mL 6 10 × 10 cells per mL 6 From 10 cells per mL to 40 × 10⁶ cells per mL 6 10 cells, 20 × 10 per mL6 From 10 cells per mL to 40 × 10⁶ cells per mL 6 10 cells per mL, 1×10 6 1 cell, 2.5 × 10⁶ cells per mL 6 5 × 10 cells per mL 6 7.5 × 10 cells per mL 6 10 × 10 cells per mL 6 12.5 × 10 cells per mL 6 15 × 10 cells per mL 6 17.5 × 10 cells per mL 6 10 cells, 20 × 10 per mL 6 10 cells, 22.5 × 10⁻⁶ per mL 6 1 cell, 25 × 10⁶ cells per mL 6 10 T cells, 30 × 10 per mL 6 1 cell, 35 × 10⁶ cells per mL 6 40 × 10 cells per mL 6 45 × 10 cells per mL 6 1 cell or 50 × 10 per mL 6 Each cell.

[0116] In any of the foregoing embodiments, tesimolimus may be present in the culture medium at a concentration of 0.1-5 μM. In some embodiments, tesimolimus may be present in the culture medium at a concentration of 0.1-1 μM. In any of the foregoing embodiments, tesimolimus may be present in the culture medium at a concentration of 1 μM. By way of example and not limitation, tesimolimus may be present in the culture medium at concentrations of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater. As a further example and not a limitation, tesimolimus may be present in the culture medium at concentrations of about 1-5 μM, 2-5 μM, 3-5 μM, 4-5 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater.

[0117] In any of the foregoing embodiments, tesimolimus may be added to the culture medium once or multiple times during the second time period to maintain the desired concentration. In any of the foregoing embodiments, tesimolimus may be added to the culture medium once. By way of non-limiting example, tesimolimus may be added to the culture medium every 2 days during the second time period. The desired tesimolimus concentration may be between 0.1 and 5 μM. In any of the foregoing embodiments, the desired tesimolimus concentration may be between 0.1 and 1 μM. In any of the foregoing embodiments, the desired tesimolimus concentration may be 1 μM. By way of example and not limitation, the desired concentration of tesiromosin may be at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater. As a further example and not a limitation, the desired concentration of tesiromosin can be about 1-5 μM, 2-5 μM, 3-5 μM, 4-5 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM or greater.

[0118] The IL-2 signaling inhibitor can be any substance that inhibits IL-2 signaling and can be added in an amount sufficient to inhibit IL-2 signaling. In any of the foregoing embodiments, the IL-2 signaling inhibitor can be an anti-IL-2 receptor antibody or a fragment thereof, such as balithimab or dacrolimus. The IL-2 signaling inhibitor can be present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL. By way of non-limiting examples, IL-2 signaling inhibitors may be present at concentrations of about 5 μg / mL to 50 μg / mL, 5 μg / mL to 40 μg / mL, 5 μg / mL to 30 μg / mL, 5 μg / mL to 20 μg / mL, 5 μg / mL to 10 μg / mL, 40 μg / mL to 50 μg / mL, 30 μg / mL to 50 μg / mL, 20 μg / mL to 50 μg / mL, 20 μg / mL to 40 μg / mL, 20 μg / mL to 30 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, or 50 μg / mL.

[0119] In any of the foregoing embodiments, the first time period can be from about 8 hours to about 24 hours. By way of non-limiting example, the first time period can be from about 8 hours to about 20 hours, from 8 hours to about 16 hours, from 8 hours to about 12 hours, from 20 hours to about 24 hours, from 16 hours to about 24 hours, from 12 hours to about 24 hours, from 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0120] In any of the foregoing embodiments, the bead-to-T-cell ratio may be 1:3. In some embodiments, the bead-to-T-cell ratio may be between 1:12 and 1:1, or in the most extreme instance, no beads are added. By way of example and not limitation, ratios of 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, or any range therein, may be used. In some embodiments, co-stimulation of the culture-introduced cell population may be achieved using nanoparticles containing anti-CD3 / anti-CD28, which may be used at concentrations below the recommended concentration. By way of example and not limitation, such nanoparticles may be used at approximately 0.01 to approximately 0.1 times, approximately 0.025 to approximately 0.1 times, approximately 0.05 to approximately 0.1 times, approximately 0.075 to approximately 0.1 times, approximately 0.01 to approximately 0.075 times, approximately 0.01 to approximately 0.05 times, approximately 0.01 to approximately 0.025 times, approximately 0.025 to approximately 0.075 times, approximately 0.025 to approximately 0.05 times, approximately 0.05 to approximately 0.075 times, or approximately 0.01 times, approximately 0.025 times, approximately 0.05 times, approximately 0.075 times, or approximately 0.01 times, approximately 0.025 times, approximately 0.05 times, approximately 0.075 times, or approximately 0.01 times the recommended dosage. By way of example and not limitation, the dosage (per 1×10⁻⁶) may be used at approximately 0.01 times the recommended dosage. 6 Compared to 10 μL per T cell, a reduced dose can be used, as an example rather than a limitation, such as 1.1 μL (a nine-fold reduction) or approximately 0.11 times. T Cell TransAct TM Reagents such as anti-CD3 / anti-CD28 co-stimulation can be used to generate manufactured T cells, alternatively, if anti-CD3 / anti-CD28 co-stimulation is to be used, the source of co-stimulation can be provided by soluble anti-CD3 / anti-CD28 microparticles. By way of example and not limitation, the co-stimulation can be provided by the manufacturer (e.g., The recommended strength for the use of soluble anti-CD3 / anti-CD28 microparticles is 20%. Alternatively, soluble anti-CD3 / anti-CD28 microparticles can be used at 5%, 10%, 15%, 20%, 25%, or 30% of the manufacturer's recommended strength. The specific amount of anti-CD3 / anti-CD28 reagent to be added can be titrated based on the desired functional properties of the final Rapa-T cell product. Specifically, in the presence of the inhibitory molecules described in this disclosure, a sufficient amount of reagent can be added to maintain the viability of T cells in vitro. However, any particular anti-CD3 / anti-CD28 agent should not be added in excess, specifically limited by: inappropriately high levels of T cell activation (increased CD25 expression by flow cytometry relative to T cell cultures using optimal, minimal co-stimulation); 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 (e.g., decreased levels of CD62L and CCR7; increased levels of CD45RO and KLRG).

[0121] In any of the foregoing embodiments, the IFN-α may be added to the culture medium at a concentration of approximately 1,000 IU / mL to 10,000 IU / mL. By way of example and not limitation, concentrations of 2,500 IU / mL to 10,000 IU / mL, 5,000 IU / mL to 10,000 IU / mL, 7,500 IU / mL to 10,000 IU / mL, 1,000 IU / mL to 7,500 IU / mL, 1,000 IU / mL to 5,000 IU / mL, 1,000 IU / mL to 2,500 IU / mL, and 2,500 IU / mL to 7,000 IU / mL may be used. Concentrations of IFN-α were 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. Lower IFN-α concentrations (e.g., 1000 IU / mL) could result in a less pronounced shift to the Th1 phenotype.

[0122] In any of the foregoing embodiments, the second time period can be about 4 to about 8 days, 4 to about 6 days, or 6 to about 8 days. As a non-limiting example, the second time period can be about 4, 5, 6, 7, or 8 days. Without co-stimulation, the incubation period can be about 4 to about 8 days, 4 to about 6 days, or 6 to about 8 days. As a non-limiting example, the second time period can be about 4, 5, 6, 7, or 8 days.

[0123] In any of the foregoing embodiments, the culture medium may further comprise 5% human serum. In some embodiments, the culture medium may further comprise 1%-20% human serum. By way of example and not limitation, the culture medium may comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% human serum. In some embodiments, the culture medium may comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% human serum. In any of the foregoing embodiments, serum may not be added to the culture medium or serum may not be present in the culture medium.

[0124] In any of the foregoing embodiments, the culture medium may further comprise X-Vivo 20 medium. In any of the foregoing embodiments, the culture medium may further comprise TexMACS. Culture medium. Any suitable culture medium can be used to culture T cells.

[0125] In any of the foregoing embodiments, additional culture medium may be added to the culture. By way of example, and not limitation, additional culture medium may be added at any range or time, approximately 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, or between, after the initial seeding of the culture into the cell population. By way of example, and not limitation, the ratio of the amount of culture medium added to the amount of the initial culture medium may be approximately 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 greater, and any range therebetween. By way of example, and not limitation, the amount of culture medium added after the initial seeding of the culture into the cell population may be sufficient to reduce the cell density in the culture to the target cell density. By way of example, and not limitation, the target cell density could be approximately 1 × 10⁻⁶. 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 2×10 7 3×10 7 Or 4×10 7 And any range therein, provided that the initial cell density is greater than the target cell density.

[0126] In any of the foregoing embodiments, the culture input cell population may include about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, or about 5% to about 10% of the total number of cells in the culture input cell population. As a non-limiting example, the culture input cell population may include approximately 5%, 10%, 15%, 20%, 33%, 40%, 50%, 66%, 70%, 75%, 90%, 95%, 98%, or 99% or more of the total number of cells in the culture input cell population.

[0127] In any of the foregoing embodiments, the culture input cell population may further include monocytes. In any of the foregoing embodiments, T cells may be enriched in the culture input cell population. By way of example and not limitation, an automated Ficoll process can be used to subject the culture input cell population to T cell enrichment. Methods for performing the Ficoll process are known in the art and involve the removal of neutrophils and erythrocytes from the sample. Any suitable method can be used to enrich T cells in the cell population.

[0128] In any of the foregoing embodiments, the method may further include harvesting a sample comprising T cells from the subject; and isolating the T cells from the sample to generate the culture-infused cell population. Such samples may contain peripheral blood stem cells (PBSCs) and, by way of example and not limitation, may be obtained by mobilization collection, steady-state apheresis, or simple blood draw. In any of the foregoing embodiments, the sample containing PBSCs and / or the culture-infused cell population may be cryopreserved prior to the preparation of the manufactured T cells. When the subject has a sufficient number of immune cells, steady-state apheresis may be performed, and, by way of example and not limitation, the sufficient number of immune cells may be characterized by a minimum absolute lymphocyte count (ALC). For example, the minimum ALC may be 300 lymphocytes per microliter.

[0129] In any of the foregoing embodiments, the T cells can be isolated by antibody-based purification.

[0130] In any of the foregoing embodiments, the T cells can be enriched by countercurrent centrifugation. This technique is well known in the art.

[0131] In any of the foregoing embodiments, the IFN-α may be added at or approximately simultaneously with the addition of nanoparticles containing anti-CD3 / anti-CD28.

[0132] In any of the foregoing embodiments, anti-CD3 / anti-CD28 antibodies can be removed after culturing by any suitable method. By way of example and not limitation, anti-CD3 / anti-CD28 magnetic beads can be removed by magnetic capture, and soluble anti-CD3 / anti-CD28 microparticles can be removed by adding release buffer and washing the generated T cells.

[0133] In some embodiments, after one week of incubation with anti-CD3 / anti-CD28 magnetic beads, the resulting T cell population exhibited increased IFN-γ secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0134] In some embodiments, after one week of incubation with anti-CD3 / anti-CD28 magnetic beads, the resulting T cell population exhibited increased TNF-α secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0135] In some embodiments, after one week of incubation with anti-CD3 / anti-CD28 magnetic beads, the resulting T cell population exhibited increased GM-CSF secretion relative to T-Rapa cells, with the magnetic beads added at a bead:T cell ratio of 3:1.

[0136] In some embodiments, after one week of incubation with anti-CD3 / anti-CD28 magnetic beads, the resulting T cell population exhibited increased IL-2 secretion relative to T-Rapa cells, the magnetic beads being added at a bead:T cell ratio of 3:1.

[0137] In some embodiments, the manufactured T cell population, relative to the control T cell population and T-Rapa cells, includes an increased percentage of cells that are positive for CD4, CD62L, CCR7, and CD127.

[0138] In some embodiments, the manufactured T cell population exhibits increased 4EBP1 phosphorylation relative to the control T cell population. In some embodiments, the manufactured T cells exhibit increased 4EBP1 phosphorylation relative to control T cells. By way of example, and not limitation, the increase in 4EBP1 phosphorylation relative to the control T cell population or control T cells characterized by the T cells that produced said T cells (i.e., culture-infused T cells) is no more than 50%, no more than 45%, no more than 40%, no more than 35%, or no more than 30%. As a further example, and not a limitation, the increase in 4EBP1 phosphorylation can be between 5%-50%, 5%-45%, 5%-40%, 5%-30%, 5%-20%, 5%-10%, 10%-50%, 10%-45%, 10%-40%, 10%-30%, 10%-20%, 20%-50%, 20%-45%, 20%-40%, 20%-30%, 30%-50%, 30%-45%, 30%-40%, 40%-50%, or any value or range within these ranges. Compared to T-Rapa cells, 4EBP1 phosphorylation is reduced (or weakened). In some embodiments, the increase in 4EBP1 phosphorylation can be measured 32 hours after the start of culture.

[0139] In some embodiments, the manufactured T cell population exhibits decreased P70S6K expression relative to T-Rapa cells and increased P70S6K expression relative to the control T cell population characterized by the cells that produced the manufactured T cells. By way of example and not limitation, the increase may be at least 10%, 20%, 30%, 40%, 50% or more, and the decrease may be 50%, 60%, 70%, 80% or more.

[0140] In some embodiments, the T cell population produced by flow cytometry exhibits reduced IL-2 receptor CD25 expression relative to T-Rapa cells. By way of example and not limitation, the reduction can be at least 50%, 60%, 70%, 80%, 90%, or more.

[0141] In some embodiments, the generated T cells, relative to the culture-infused T cells, can express a unique RNA expression profile, characterized by an increase of 50% or more in the RNA content of dedifferentiation molecules (such as KLF4, KLF10, Nanog, and combinations thereof) and a decrease of 50% or more in the RNA content of differentiation molecules (such as perforin, granzyme B, IFN-□, and combinations thereof).

[0142] In some embodiments, the resulting T cell population, relative to T-Rapa cells, exhibits reduced levels of the following molecules associated with immunosuppression: CTLA4; and TIM3.

[0143] In some embodiments, the manufactured T cell population can be characterized by 10% or less of CD4+ or CD8+ manufactured T cells expressing CTLA4, as measured by flow cytometry. In some embodiments, the manufactured T cell population can be characterized by 5% or less of CD4+ or CD8+ manufactured T cells expressing CTLA4, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of CD4+ or CD8+ T cells in the manufactured T cell population may express CTLA4, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CTLA4 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CTLA4, as measured by flow cytometry. In some embodiments, the reduced frequency of CTLA4 expression in CD4+ or CD8+ T cells can be at least 50% less than the corresponding frequency of CTLA4 expression in CD4+ or CD8+ T-Rapa cells. By way of example, and not limitation, the reduced frequency can be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0144] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing TIM3, as measured by flow cytometry. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing TIM3, as measured by flow cytometry. By way of example and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express TIM3, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing TIM3, as measured by flow cytometry, relative to the corresponding frequency of TIM3 expression in a control T cell population characterized by the T cells that produced the manufactured T cells. In some embodiments, the reduced frequency of TIM3 expression in CD4+ or CD8+ T cells may be at least 50% less than the corresponding frequency of TIM3 expression in CD4+ or CD8+ T cells in the control population. By way of example, and not limitation, the reduced frequency of TIM3 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of TIM3 expression in CD4+ or CD8+ T cells in the control population. In some embodiments, the reduced frequency is observed 6 days after the cells producing the manufactured T cells are seeded into the culture. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of TIM3 expression in CD4+ or CD8+ T cells relative to the corresponding frequency of TIM3 expression in CD4+ or CD8+ T-Rapa cells, as measured by flow cytometry. In some embodiments, the reduced frequency of TIM3 expression in CD4+ or CD8+ T cells may be at least 50% lower than the corresponding frequency of TIM3 expression in CD4+ or CD8+ T-Rapa cells. By way of example, and not limitation, the reduced frequency of TIM3 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower than the corresponding frequency of TIM3 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells producing the manufactured T cells are seeded into the culture.

[0145] In some embodiments, the manufactured T cell population can be characterized by 5% or less of CD4+ or CD8+ manufactured T cells expressing PD1, as measured by flow cytometry. By way of example, and not limitation, 5%, 4%, 3%, 2%, 1%, or less of the manufactured T cell population may express PD1, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a frequency of PD1 expression by CD4+ or CD8+ T cells relative to the corresponding frequency of PD1 expression by CD4+ and CD8+ T-Rapa cells, as measured by flow cytometry. In some embodiments, the reduced frequency of PD1 expression by CD4+ or CD8+ T cells may be at least 50% less than the corresponding frequency of PD1 expression by CD4+ or CD8+ T-Rapa cells. By way of example and not limitation, the reduced frequency of PD1 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of PD1 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency is 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0146] In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ and CD8+ manufactured T cells expressing 2B4, as measured by flow cytometry. By way of example, and not limitation, 5%, 4%, 3%, 2%, 1%, or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express 2B4, as measured by flow cytometry. In some embodiments, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4, as measured by flow cytometry. By way of example, and not limitation, at least 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or more of the CD4+ T cells in the manufactured T cell population may express 2B4, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD8+ T cell 2B4 expression, as measured by flow cytometry, relative to the corresponding frequency of CD8+ T cell 2B4 expression in a control T cell population characterized by the manufactured T cells. By way of example, and not limitation, the reduced frequency of CD8+ T cell 2B4 expression may be at least 50%, 60%, 70%, or 80% less than the corresponding frequency of CD8+ T cell 2B4 expression in the control T cell population. In some embodiments, the reduced frequency occurs 6 days after the cells that will generate the manufactured T cells are seeded into the culture. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cell 2B4 expression, as measured by flow cytometry, relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cell 2B4 expression. By way of example and not limitation, the reduced frequency of 2B4 expression in CD4+ or CD8+ T cells may be at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the corresponding frequency of 2B4 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduction occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0147] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing LAIR1, as measured by flow cytometry. By way of example, and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express LAIR1, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of LAIR1 expression in CD4+ or CD8+ T cells, as measured by flow cytometry, relative to the corresponding frequency of LAIR1 expression in a control T cell population characterized by the T cells that produced the manufactured T cells. By way of example, and not limitation, the reduced frequency of LAIR1 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower than the corresponding frequency of LAIR1 expression in CD4+ or CD8+ T cells in the control T cell population. In some embodiments, the reduced frequency is 6 days after the cells that produce the manufactured T cells are seeded into the culture. In some embodiments, the CD4+ or CD8+ T cells in the manufactured T cell population may exhibit a reduced LAIR1 expression level relative to T-Rapa cells, as measured by flow cytometry. By way of example, and not limitation, the reduction may be at least 30%, 40%, 50%, or more.

[0148] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing TIGIT, as measured by flow cytometry. By way of example, and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express TIGIT, as measured by flow cytometry. In some embodiments, at least 0.1% of the CD4+ T cells in the manufactured T cell population express 2B4, as measured by flow cytometry. By way of example, and not limitation, at least 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the CD4+ T cells in the manufactured T cell population may express TIGIT, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of TIGIT expression in CD4+ or CD8+ T cells, as measured by flow cytometry, relative to the corresponding frequency of TIGIT expression in CD4+ or CD8+ T-Rapa cells. By way of example, and not limitation, the reduced frequency of TIGIT expression in CD4+ or CD8+ T cells may be at least 40%, 50%, 60%, 70%, 80%, or 90% less than the corresponding frequency of TIGIT expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0149] In some embodiments, the manufactured T cell population can be characterized by 10% or less of the CD4+ or CD8+ manufactured T cells expressing LAG3, as measured by flow cytometry. By way of example, and not limitation, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express LAG3, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing LAG3 relative to the corresponding frequency of LAG3 expression in CD4+ or CD8+ T-Rapa cells, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing LAG3 may be at least 50% less than the corresponding frequency of LAG3 expression in CD4+ or CD8+ T-Rapa cells. By way of example and not limitation, the reduced frequency of LAG3 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of LAG3 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0150] In some embodiments, the manufactured T cell population can be characterized by a level of the positive co-stimulatory molecule CD28 relative to (i.e., substantially the same level) of the control T cell population characterized by the T cells that produced the manufactured T cells, as measured by flow cytometry. In some embodiments, the CD28 expression frequency in CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the CD28 expression frequency in CD4+ or CD8+ T cells in the control population, respectively. By way of example and not limitation, the CD28 expression frequency in CD4+ or CD8+ T cells in the manufactured T cell population can be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the CD28 expression frequency in CD4+ or CD8+ T cells in the control population, respectively. In some embodiments, this retention is performed 6 days after the cells that produced the manufactured T cells are seeded into a culture.

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

[0152] In some embodiments, the manufactured T cell population can be characterized by CD45RA levels relative to (i.e., substantially the same level) a control T cell population characterized by the T cells that produced the manufactured T cells, as measured by flow cytometry. In some embodiments, the CD45RA expression frequency in CD4+ or CD8+ T cells in the manufactured T cell population can be within about 20% of the CD45RA expression frequency in CD4+ or CD8+ T cells in the control population, respectively. By way of example and not limitation, the CD45RA expression frequency in CD4+ or CD8+ T cells in the manufactured T cell population can be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the CD45RA expression frequency in CD4+ or CD8+ T cells in the control population, respectively. In some embodiments, this retention is 6 days after the cells that produced the manufactured T cells are seeded into a culture.

[0153] In some embodiments, the manufactured T cell population can be characterized by 5% or less of CD4+ or CD8+ manufactured T cells expressing CD25, as measured by flow cytometry. By way of example, and not limitation, 5%, 4%, 3%, 2%, 1%, or less of the manufactured T cell population may express CD25, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD25 relative to the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25, as measured by flow cytometry. In some embodiments, the reduced frequency of CD4+ or CD8+ T cells expressing CD25 may be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. By way of example and not limitation, the reduced frequency of CD4+ or CD8+ T cell expression of CD25 may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cells expressing CD25. In some embodiments, the reduced frequency is 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0154] In some embodiments, the manufactured T cell population exhibits a quiescent and non-senescent phenotype characterized by reduced KLRG1 levels, as measured by flow cytometry. In some embodiments, the reduction in KLRG1 levels occurs 6 days after the cells that produced the manufactured T cells are seeded into a culture. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing KLRG1, as measured by flow cytometry. By way of example and not limitation, 5%, 4%, 3%, 2%, 1%, or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express KLRG1, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing KLRG1, as measured by flow cytometry, relative to the corresponding frequency of KLRG1 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency of KLRG1 expression in CD4+ or CD8+ T cells may be at least 50% lower than the corresponding frequency of KLRG1 expression in CD4+ or CD8+ T-Rapa cells. By way of example, and not limitation, the reduced frequency of KLRG1 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower than the corresponding frequency of KLRG1 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells that will produce the manufactured T cells are seeded into the culture.

[0155] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule CD39 compared to a control T cell population characterized by the T cells that produced the manufactured T cells. In some embodiments, the manufactured T cell population can be characterized by 20% or less of the CD4+ or CD8+ manufactured T cells expressing CD39, as measured by flow cytometry. By way of example and not limitation, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express CD39, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD39, as measured by flow cytometry, relative to the corresponding frequency of CD39 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency of CD4+ or CD8+ T cell expression of CD39 may be at least 50% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cell expression of CD39. By way of example, and not limitation, the reduced frequency of CD4+ or CD8+ T cell expression of CD39 may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of CD4+ or CD8+ T-Rapa cell expression of CD39. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0156] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule CD73 compared to a control T cell population characterized by the T cells that produced the manufactured T cells. In some embodiments, the manufactured T cell population can be characterized by 20% or less of the CD4+ or CD8+ manufactured T cells expressing CD73, as measured by flow cytometry. By way of example and not limitation, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express CD73, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing CD73, as measured by flow cytometry, relative to the corresponding frequency of CD73 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency of CD73 expression in CD4+ or CD8+ T cells may be at least 50% less than the corresponding frequency of CD73 expression in CD4+ or CD8+ T-Rapa cells. By way of example, and not limitation, the reduced frequency of CD73 expression in CD4+ or CD8+ T cells may be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than the corresponding frequency of CD73 expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0157] In some embodiments, the manufactured T cell population exhibits reduced expression of the immunosuppressive molecule GITR compared to a control T cell population characterized by the T cells that produced the manufactured T cells. In some embodiments, the manufactured T cell population can be characterized by 5% or less of the CD4+ or CD8+ manufactured T cells expressing GITR, as measured by flow cytometry. By way of example, and not limitation, 5%, 4%, 3%, 2%, 1%, or less of the CD4+ or CD8+ T cells in the manufactured T cell population may express GITR, as measured by flow cytometry. In some embodiments, the manufactured T cell population may exhibit a reduced frequency of CD4+ or CD8+ T cells expressing GITR, as measured by flow cytometry, relative to a corresponding frequency of GITR expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency of GITR expression in CD4+ or CD8+ T cells may be at least 20% less than a corresponding frequency of GITR expression in CD4+ or CD8+ T-Rapa cells. By way of example and not limitation, the reduced frequency of GITR expression in CD4+ or CD8+ T cells may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower than the corresponding frequency of GITR expression in CD4+ or CD8+ T-Rapa cells. In some embodiments, the reduced frequency occurs 6 days after the cells that produce the manufactured T cells are seeded into the culture.

[0158] In some embodiments, the manufactured T cells, relative to control T cell cultures, may exhibit an early differentiation state of cytokine biology, as demonstrated by increased secretion of the precursor cytokine IL-2 and increased responsiveness to homeostatic cytokines IL-7 and IL-15. In this way, the manufacturing method of this disclosure describes a process for manufacturing helpless T cells with increased responsiveness to homeostatic cytokines.

[0159] In some embodiments, the generated T cell population exhibits increased IL-2 secretion compared to T-Rapa cultures incubated under the same conditions. In some embodiments, this IL-2 secretion is increased by 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 generated T cell population, after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, secretes at least 500 pg / mL / 1×10⁻⁶ cells / mL. 6 IL-2 per cell per day. For example, under these conditions, the resulting T cell population can secrete approximately 500 pg / mL / 1×10⁻⁶ cells / day.6 Cells / day, 600 pg / mL / 1×10 6 10 cells / day, 700 pg / mL / 1×10 6 Cells / day, 800 pg / mL / 1×10 6 Cells / day, 900 pg / mL / 1×10 6 Cells / day, 1000 pg / mL / 1×10 6 IL-2 per cell / day or more.

[0160] In some embodiments, the generated T cell population may secrete an increased amount of IL-2 when exposed to IL-7 or IL-15. In some embodiments, the generated T cell population, when incubated in the presence of IL-7 or IL-15, is characterized by an increase in IL-2 secretion of at least 1.1-fold, relative to conditions in the absence of IL-7 or IL-15. By way of example and not limitation, the increase may 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, after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3 and exposure to IL-7 at a concentration of 10 ng / mL and IL-15 at a concentration of 10 ng / mL, or both IL-7 and IL-15 (if present), secretes at least 1000 pg / mL / 1×10 6 IL-2 per cell per day. For example, under these conditions, the resulting T cell population can secrete approximately 1000 pg / mL / 1×10⁻⁶ cells / day. 6 Cells / day, 1100 pg / mL / 1×10 6 Cells / day, 1200 pg / mL / 1×10 6 Cells / day, 1300 pg / mL / 1×10 6 Cells / day, 1400 pg / mL / 1×10 6 Cells / day, 1500 pg / mL / 1×10 6 Cells / day, 1600 pg / mL / 1×10 6 1700 cells / day, 1700 pg / mL / 1×10 6 Cells / day, 1800 pg / mL / 1×10 6 Cells / day, 1900 pg / mL / 1×10 6 Cells / day, 2000 pg / mL / 1×10 6One cell / day or more of IL-2. IL-2 secretion is associated with helper-independent T cells. High IL-2 secretion by Rapa-T cells is advantageous by avoiding the need for exogenous IL-2 administration after T-cell adoptive therapy. In some embodiments, IL-7 or IL-15 (if present) is added at 10 ng / mL.

[0161] In some embodiments, the manufactured T cell population exhibits increased in vivo function relative to the control T cell population, the in vivo function being characterized by increased human T cell engraftment in a human-mouse xenograft anti-host disease model.

[0162] In some embodiments, the manufactured T cell population exhibits a reduction in mTORC1 activation, as measured by phosphor-P70S6K. By way of example, and not limitation, the reduction may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% relative to T-Rapa cells 32 hours after the start of culture.

[0163] In some embodiments, the manufactured T cell population exhibits reduced phosphorus-STAT5 relative to T-Rapa cells. By way of example, and not limitation, the reduction may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% relative to T-Rapa cells 32 hours after the start of culture.

[0164] In some embodiments, the manufactured T cell population exhibits reduced phosphoryl-STAT5 levels relative to a control cultured T-Rapa cell population. By way of example, and not limitation, the reduction can be at least 50% relative to a control cultured T-Rapa cell population. In some embodiments, the reduction occurs 48 hours after the manufactured T cells are seeded from an input cell population comprising T cells into a culture. In some embodiments, p-STAT5 levels are measured by Western blotting.

[0165] In some embodiments, the manufactured T cell population exhibits at least a detectable level of STAT1 and phosphor-STAT1. In some embodiments, the levels are measured 48 hours after the manufactured T cells are seeded into a culture from an input cell population including T cells. In some embodiments, the manufactured T cell population exhibits reduced phosphor-STAT5 compared to a control T cell population and at least a certain level of STAT1 and phosphor-STAT1. In some embodiments, the level of STAT1 or p-STAT1 is measured by Western blotting.

[0166] In some embodiments, such as as measured by p70S6K or Raptor expression, the manufactured T cell population exhibits reduced mTORC1 activation relative to a control T cell population from which the manufactured T cell population is derived. By way of example, and not limitation, the reduction can be 50%. In some embodiments, the reduction occurs 48 hours after the manufactured T cells are seeded from an input cell population comprising T cells into a culture. In some embodiments, the reduction is measured by Western blotting.

[0167] In some embodiments, the manufactured T cell population exhibits substantially the same levels of Rictor, SGK1, or phosphorylated SGK1 as the control T cell population. By way of example, and not limitation, the levels of Rictor, SGK1, or phosphorylated SGK1 may be within 50%, 40%, 30%, 20%, 10%, or 5% of the corresponding levels of Rictor, SGK1, or phosphorylated SGK1 in T-Rapa cells. In some embodiments, the levels are measured 48 hours after the manufactured T cells are seeded from an input cell population comprising T cells into a culture. In some embodiments, the levels of Rictor, SGK1, or pSGK1 are at least as measured in the control T cell population. In some embodiments, the levels are measured by Western blotting.

[0168] In some embodiments, the T cell population, after being manufactured and expanded in culture medium for 6 days without inhibitors, exhibits an increased secretion of at least one of IFN-γ, TNF-α, GM-CSF, and IL-2, relative to T-Rapa cells. By way of example and not limitation, the increase may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more.

[0169] In some embodiments, the manufactured T cell population at the end of manufacturing (day 6 of culture), relative to T-Rapa cells, may have an increased number of CD4+ T cells expressing the T cell marker CD45RA. By way of example and not limitation, the increase may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more.

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

[0171] In some embodiments, the manufactured T cells may have reduced expression of one or more checkpoint inhibitor receptors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3. By way of example, and not limitation, the reduced expression may be at least 25% less than the corresponding expression level in T-Rapa cells. By way of further example, and not limitation, the reduced expression may be at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% less than the corresponding expression level in T-Rapa cells. In some embodiments, the expression level of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 in the manufactured T cells may be within about 25% of the corresponding expression level in control T cells characterized by the T cells from which the manufactured T cells were produced. By way of example and not limitation, the expression levels of one or more checkpoint inhibitors selected from CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, and TIM3 may be within approximately 25%, 20%, 15%, 10%, or 5% of the corresponding expression levels in control T cells characterized by T cells that produce T cells. It should be understood that the expression levels of checkpoint inhibitors are compared between the same cell types; for example, CD4+ producing T cells may be compared with CD4+ T-Rapa cells or CD4+ control T cells characterized by T cells that produce T cells.

[0172] In some embodiments, the manufactured T cells have increased CD127 expression relative to control T cells characterized by the cells that produce the manufactured T cells. By way of example and not limitation, this increase can be at least 10%, 20%, 30%, 40%, 50% or more.

[0173] In some embodiments, the manufactured T cell population may have at least 5% CD4+ T cells expressing CD127, as measured by flow cytometry. For example, the manufactured T cell population may have at least 5%, 6%, 7%, 8%, 9%, 10% or more CD4+ T cells expressing CD127, as measured by flow cytometry. In some embodiments, the manufactured T cell population may have an increased frequency of CD4+ T cells expressing CD127 compared to a control T cell population characterized by the cells that produced the manufactured T cell population. By way of example and not limitation, the increase may be at least 50%, 100%, 150%, 200%, 300% or more.

[0174] The manufactured T cells can be characterized by the aforementioned properties to the extent that any of these properties associated with the manufactured T cell population can be associated with a single cell. In any of the foregoing embodiments, the manufactured T cells or the manufactured T cell population may have more than one of the aforementioned properties.

[0175] Methods for treating cancer in subjects

[0176] Patients with relapsed multiple myeloma (MM) have limited survival and a cure remains elusive. Therefore, novel T-cell therapies are suitable candidates for patients with relapsed MM.

[0177] Immunotherapy differs from existing immunotherapies in several important categories. First, the manufactured T-cell products are designed to inhibit mammalian target levels along the rapamycin (mTOR) pathway, thereby converting them into resistance to apoptosis and enrichment for central memory differentiation. Second, the manufactured T-cell products promote CD4+ at high doses. + Th1 and CD8 + Third, the manufactured T-cell products are minimally co-stimulated with monoclonal antibodies or not and express various T-cell receptor (TCR) lineages; thus, the antitumor effect mediated by the manufactured T cells is expected to occur primarily through in vivo clonal expansion to tumor antigens. This mechanism can be advantageous in multiple myeloma, where tumor antigens are unknown or change over time due to high tumor mutation rates. Characterization of the emerging T-cell response in vivo is crucial for improving the understanding of the potential mechanisms of manufactured T-cell therapy and will be evaluated as a second objective of this study. Fourth, the manufactured T-cell therapy will be evaluated against novel immune depletion and immunosuppressive regimens consisting of a combination of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). This PC regimen is relatively unaffected by myeloid cells, thereby allowing repeated treatment cycles with minimal neutropenia; this regimen is advantageous in terms of cost (can be administered in outpatient settings) and safety (reduced infection rate due to preservation of myeloid cells). Multiple myeloma is a disease largely promoted by inflammatory signaling; thus, inflammation suppression mediated by the PC protocol would be an integral part of the protocol's efficacy. Each of these factors was considered in the design of the clinical trial, which focused on T-cell production through multiple infusions following PC conditioning.

[0178] The manufactured T cells express significantly reduced levels of checkpoint inhibitors, thus providing a novel ex vivo method for releasing the immune system from checkpoint inhibition, currently achieved through monoclonal antibody therapy. Following this line of thought, manufactured T-cell therapy is expected to be successful in cancers susceptible to checkpoint inhibitor therapy, including but not limited to melanoma, renal cell carcinoma, bladder cancer, lung cancer, lymphoma, multiple myeloma, and colon cancer. It should also be noted that checkpoint inhibitor monoclonal antibody therapy has relatively high toxicity in patients with multiple myeloma, thus necessitating alternative methods to avoid immune checkpoints, such as manufactured T-cell therapy.

[0179] In addition, the ability of manufactured T cells to undergo extensive clonal expansion with multiple tumor antigens predicts that tumor cells with increased mutation rates and tumors with microsatellite instability will be particularly sensitive to manufactured T cell therapy.

[0180] This disclosure provides a method for treating cancer, the method comprising administering T cells manufactured according to this disclosure at a therapeutically effective dose.

[0181] In some embodiments, the method for treating cancer includes administering a therapeutically effective dose of a composition comprising manufactured T cells to a subject. In some embodiments, the administration of the composition comprising manufactured T cells may be repeated or accumulated at a therapeutically effective dose. In some embodiments, the method further includes harvesting autologous cells from the subject prior to subjecting the subject to the immune depletion protocol. In some embodiments, the method further includes harvesting autologous cells from the subject prior to administering the composition comprising manufactured T cells to the subject.

[0182] In any of the foregoing embodiments, the immune depletion regimen may include administering at least one of pentostatin and cyclophosphamide to the subject. In some embodiments, pentostatin is administered to the subject, and the dose of said pentostatin may be between 0.5-4 mg / m². 2 Between 0.5-3 mg / m² 2 Between 0.5-2 mg / m 2 Between 0.5-1 mg / m² 2 Between 1-4 mg / m 2 Between 2-4 mg / m 2 Between or 3-4 mg / m 2 Between. As a non-limiting example, the dose of said pentostatin is about 0.5 mg / m². 2 1mg / m 2 1.5mg / m 2 2mg / m2 2.5mg / m 2 3mg / m 2 3.5mg / m 2 Or 4mg / m 2 In some embodiments, cyclophosphamide is administered to the subject, and the dose of said cyclophosphamide may be between 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 said 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, said pentostatin and cyclophosphamide are administered to the subject as a single composition. In some embodiments, said single composition is administered intravenously to the subject.

[0183] In any of the foregoing embodiments, the immune depletion regimen may comprise administering to the subject a first composition comprising pentostatin; and administering to the subject a second composition comprising cyclophosphamide. In some embodiments, the first composition is administered at a dose of pentostatin of 1-4 mg / m², wherein the dose is between 0.5-4 mg / m². 2 Between 0.5-3 mg / m² 2 Between 0.5-2 mg / m 2 Between 0.5-1 mg / m² 2 Between 1-4 mg / m 2 Between 1-3 mg / m 2 Between 1-2 mg / m 2 Between or 3-4 mg / m 2 Between. As a non-limiting example, the dose of said pentostatin is about 1 mg / m². 2 1.5mg / m 2 2mg / m 2 2.5mg / m 2 3mg / m 2 3.5mg / m 2 Or 4mg / m 2In some embodiments, the second composition comprises cyclophosphamide, and the second composition is administered in a dose of the cyclophosphamide between 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 the cyclophosphamide is about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg.

[0184] In any of the foregoing embodiments, the effective therapeutic dose may be 1 × 10⁻⁶ per kilogram of the subject's body weight. 5 Up to 5×10 6 cells / kg, 1×10 6 Up to 2.5×10 6 cells / kg, 2.5×10 6 Up to 5×10 6 cells / kg, 1×10 5 Up to 2.5×10 6 cells / kg, 2.5×10 5 Up to 5×10 6 cells / kg, 1×10 5 Up to 2.5×10 5 cells / kg, 2.5×10 5 Up to 5×10 5 cells / kg, 1×10 5 cells / kg, 2×10 5 Cells / kg, 3×10 5 cells / kg, 4×10 5 cells / kg, 5×10 5 cells / kg, 1×10 6 cells / kg, 2×10 6 Cells / kg, 3×10 6 cells / kg, 4×10 6 Cells / kg or 5×10 6 Manufactured T cells per kg. In any of the foregoing embodiments, the composition comprising manufactured T cells is administered to the subject by infusion.

[0185] In any of the foregoing embodiments, by way of example and not limitation, the cancer may be selected from the group consisting of: multiple myeloma, renal cell carcinoma, bladder cancer, lung cancer, liver cancer, lymphoma, gastric cancer, and colon cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is relapsed multiple myeloma. By way of further example and not limitation, the cancer may be sarcoma, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, or colorectal cancer. In some embodiments, the cancer is PD-L1 negative cancer. In some embodiments, the cancer is susceptible to checkpoint inhibitor therapy. In some embodiments, the multiple myeloma is relapsed, refractory multiple myeloma. In some embodiments, the multiple myeloma is quadruple or quintuple refractory multiple myeloma. In some embodiments, the multiple myeloma is condensative multiple myeloma. In some embodiments, the subject has relapsed multiple myeloma. In some aspects, by way of example and not limitation, the subject has relapsed multiple myeloma 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the subject has cryptic multiple myeloma. In some embodiments, the subject has quadruple or pentaple refractory multiple myeloma. In some embodiments, by way of example and not limitation, the subject has one, two, three, four, five or more types of treatment-resistant multiple myeloma.

[0186] In some embodiments, the subjects have previously received treatment and are now experiencing a second or third relapse following a different treatment series selected from the following groups: 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, in which randomization of the control group to standard chemotherapy for patients with second or third relapse is reasonable.

[0187] In a separate embodiment, the subject is highly refractory to multiple standard drugs, and therefore a randomized clinical trial would be impractical. Instead, such highly refractory patients will be treated with Rapa-T therapy in a single-arm phase II clinical trial. Highly refractory status can be quantified using a quadruple or quintuple refractory nomenclature, where such subjects are refractory to four or five of the multiple drugs used to treat multiple myeloma, namely: bortezomib, carfilzomib, lenalidomide, pomalidomide, and daratumumab.

[0188] In the case of an example involving a phase 3 clinical trial for the treatment of MM during a second or third relapse, the primary study objective would involve progression-free survival as the study endpoint, defined as a less than 25% increase in the M protein / free light chain difference in the subject at monthly monitoring.

[0189] In some embodiments, the duration of the first treatment cycle is at least 28 days. In some embodiments, the duration of each of the one or more additional treatment cycles is at least 35 days.

[0190] 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.

[0191] In any of the above embodiments, each of the one or more additional treatment cycles is spaced 0 to 4 weeks apart. In any of the above embodiments, the first treatment cycle is spaced 0 to 4 weeks apart from the first treatment cycle of the one or more additional treatment cycles. In any of the above embodiments, the step of administering a therapeutically effective dose of the composition comprising the manufactured T cells to the subject is performed on days 15, 16, 17, and / or 18 of each of the one or more additional treatment cycles.

[0192] In some embodiments, the subject is experiencing a second or third relapse of multiple myeloma (MM) following a regimen consisting of the group consisting of: administration of a proteasome inhibitor, administration of an immunomodulatory drug, administration of an alkylating agent, administration of a CD38 monoclonal antibody, and administration of glucocorticoids.

[0193] In some embodiments, the subject is in the late stage of relapsed MM and is quadruple or quintuple refractory, where there is no standard therapy available.

[0194] In some embodiments involving phase 3 clinical trials, the primary study objective will involve progression-free survival, which is defined as an increase of less than 25% in the difference between treatments in M ​​protein / free light chain.

[0195] In some embodiments, the method includes subjecting the subject to an immune depletion protocol to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells; and following the immune depletion protocol, administering to the subject a therapeutically effective dose of a composition comprising manufactured T cells.

[0196] In some embodiments, the immune depletion protocol includes: administering pentostatin to the subject; administering cyclophosphamide to the subject; administering one or more additional doses of pentostatin to the subject if the subject's creatinine clearance is >30 mL / min / 1.73 m²; and administering one or more additional doses of cyclophosphamide to the subject if the subject's absolute lymphocyte count is 50 or greater per μL and the subject's absolute neutrophil count is 500 or greater per μL.

[0197] In some embodiments, the steps of measuring the subject's CrCl and adjusting the dose of pentostatin to be administered are performed on days 1, 4, 8, and / or 11 of the immune depletion regimen.

[0198] In some embodiments, the steps of measuring ALC and ANC and adjusting the dose of cyclophosphamide to be administered are performed on days 1, 2, 3, 4, 5, 8, 9, 10, 11 and / or 12 of the immunization depletion regimen.

[0199] In some embodiments, the step of administering a therapeutically effective dose of the composition comprising manufactured T cells to the subject following the immune depletion regimen is performed 15-18 days after the start of the immune depletion regimen.

[0200] In any of the foregoing embodiments, the steps of subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells, and administering a therapeutically effective dose of a composition comprising manufactured T cells to the subject after the immune depletion regimen, are repeated at least twice. In any of the foregoing embodiments, the steps of subjecting the subject to an immune depletion regimen to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells, and administering a therapeutically effective dose of a composition comprising manufactured T cells to the subject after the immune depletion regimen, may be repeated up to eight or more times. In any of the foregoing embodiments, each step of administering a therapeutically effective dose of a composition comprising manufactured T cells to the subject after the immune depletion regimen may be spaced 0 to 9 weeks apart.

[0201] It should also be understood that, in any of the foregoing embodiments, when co-stimulation is performed with anti-CD3 / anti-CD28 antibodies, this co-stimulation can be provided in any form of anti-CD3 / anti-CD28 antibody. By way of example and not limitation, when co-stimulation is indicated to be performed using anti-CD3 / anti-CD28 beads, anti-CD3 / anti-CD28 nanoparticles or microparticles may be used.

[0202] Examples

[0203] The following examples are provided to better illustrate the methods of this disclosure and the resulting manufactured T cells. These examples are not intended to limit or otherwise modify the scope of the methods, cells, and compositions disclosed in this disclosure.

[0204] Example 1

[0205] Anti-IL-2 receptor blockade and mTOR blockade were used for Th1 enrichment. For adoptive T-cell therapy, it is important to generate T cells that preferentially express the Th1 phenotype, particularly those derived from regulatory T cells (T1-T2 receptors). REG Cells with the lowest th1 phenotype have the least contamination. Th1 cells can be partially characterized by the expression of their cell fate transcription factor TBET, while T... REG The cells express the FoxP3 transcription factor.

[0206] Approaches to promote TBET while simultaneously limiting FoxP3 were evaluated. The mTOR inhibitor tesimolimus, an FDA-approved intravenously administered drug for the treatment of refractory renal cell carcinoma, was evaluated. The production of T cells enriched against the Th1 phenotype using an mTOR inhibitor appears contradictory, as inhibition of mTOR is generally associated with promoting T cells. REGPhenotypic T-cell correlation. The current experiment differs from previous studies because tesimolimus is an intravenously formulated formulation, which is advantageous over rapamycin, which is less feasible for cell culture due to its limited solubility in culture media.

[0207] The current experiment also differs from previous studies because it evaluated the combination of tesimolimus and the anti-IL-2 receptor monoclonal antibody dacrolimus. Dacrolimus and balithimab are both FDA-approved monoclonal antibodies with a common mechanism of action and are therefore interchangeable in the systems being developed.

[0208] T cells were cultured in vitro using various ratios of anti-CD3, anti-CD28 beta-T cells (1:1 or 1:12); the mTOR inhibitor tesimolimus (1 μM); with or without the anti-IL-2 receptor monoclonal antibody dacrolimus (5 μg / ml or 50 μg / ml); and Th1 polarizing cytokines (IFN-α; 10,000 IU / ml) or control regulatory T cell polarization (IL-2 plus TGF-β). On day 6 of culture, intracellular expression of the regulatory T cell transcription factor FoxP3 and the Th1 transcription factor TBET was assessed. Figure 1A-1B ).

[0209] It was found that, compared to the T cell population infused on day 0, the addition of tesiromoxetine (1.0 μM) in the presence of the type I polarizing cytokine IFN-α reduced the expression of FoxP3 in the resulting T cells (see [link to study]). Figure 1A Furthermore, it was found that blocking the IL-2 receptor further reduced FoxP3 expression; the 50 μg / ml antibody was more effective than the 5 μg / ml antibody, thus indicating a dose-response relationship. When exogenous IL-2 was added in combination with IFN-α, tesimolimus was ineffective in reducing FoxP3 expression. Figure 1A Furthermore, if the culture conditions allow T REG Cell differentiation was not effectively inhibited by tisirolimus and dacrolimus in limiting FoxP3 expression, and the culture conditions were: a bead ratio reduced to 1:12; elimination of IFN-α from the culture; and addition of exogenous IL-2 and TGF-β. Figure 1A ).

[0210] In addition to limiting FoxP3 expression, the combination of tesimolimus and an anti-IL-2 receptor monoclonal antibody is effective in promoting the Th1 phenotype, as indicated by increased TBET expression. Figure 1B Furthermore, a dose-response relationship was observed, with 50 μg / ml dacrolimus promoting TBET expression to a greater extent than 5 μg / ml dacrolimus. Figure 1BAlthough adding IL-2 to IFN-α polarization increases TBET, this is also associated with an increase in FoxP3, thus indicating a lack of Th1 purity through the addition of exogenous IL-2. It is noteworthy that, as expected, T REG Control conditions—IL-2 and TGF-β showed reduced TBET expression ( Figure 1B ).

[0211] Thus, the combination of mTOR inhibition and IL-2 receptor blockade represents a new approach for Th1 cell production.

[0212] The development of a combination of interventions for suppressing T cells during ex vivo manufacturing: mTOR inhibition; IL-2 receptor blockade; reduced T cell co-stimulation; and T cell inhibition prior to co-stimulation (overnight pre-incubation). These results suggest that the combination of mTOR inhibition and IL-2 receptor blockade can promote the manufacturing of Th1 cells (increased TBET to FoxP3 ratio; T... REG (Limitations of cell contamination) suggest that other interventions may also promote the production of Th1 cells.

[0213] One benefit of using mTOR suppression in adoptive T-cell therapy is that this intervention can promote the production of T cells with a more primitive differentiation state, such as the T central memory subset (T3). CM ) or T stem cell memory subset (T SCM In previous studies using isolated rapamycin, it was found that isolated rapamycin was effective in manufacturing T... CM The phenotype of T cells is effective; this result is consistent with the known role of mTOR in the memory state of control T cells. Promoting T cells during manufacturing CM and / or T SCM T cells in a more primitive differentiation state are important because they increase long-term engraftment after adoptive transfer and mediate increased in vivo effects in experimental models. Several other methods have been described to promote the production of T cells in a restricted differentiation state, including: using GSK3 inhibitors to promote WNT signaling; inhibiting ART signaling; and inhibiting PI3 kinase signaling.

[0214] A method has been developed in which T-cell plates are seeded in X-Vivo 20 medium supplemented with 5% human AB serum, which is free of exogenous cytokines and contains the mTOR inhibitor tesimolimus and IL-2 receptor blockade achieved through the addition of monoclonal antibodies. This method incorporates approximately 16 hours of "pre-incubation" before co-stimulation with anti-CD3, anti-CD28 coated magnetic beads. This method has not been previously reported.

[0215] As a first step in evaluating these increasingly stringent culture conditions, the culture of CD4 under various conditions was evaluated. + and CD8 + The feasibility of T cell production is defined, for example, by the presence of live cells at the end of the culture interval (6 days for Th1 production). Figure 2A-2B In, as indicated, CD4 will be used under various conditions. + and CD8 + T cells were placed in the culture. The ratio of 3 / 28 beads to T cells was 3:1, 1:1, or 1:3. T cells were co-stimulated with the culture at the time of addition (“No overnight pre-incubation”) or after an overnight pre-incubation of 16 hours. Under some conditions, the anti-IL-2 receptor monoclonal antibody dacrolimus was added at a concentration of 50 μg / ml. For mTOR inhibition, tesimolimus was added at 1.0 or 0.1 μM; alternatively, the control mTOR inhibitor rapamycin was added at a concentration of 1.0 μM. Most cultures were also supplemented with the type I cytokine promoter IFN-α (10,000 IU / ml). As indicated, most culture conditions did not include the addition of exogenous IL-2. T cell yield was calculated after 6 days of culture and compared with that at the start of culture (“Day 0 culture input”).

[0216] like Figure 2A-2B The culture conditions shown were modified to include not only mTOR inhibition (using 1.0 μM or 0.1 μM tesimolimus; control using 1.0 μM rapamycin) and IL-2 receptor blockade (dacrolimus), but also reduced co-stimulation (from a 3:1 bead-to-T cell ratio to 1:1 and 1:3 ratios) and overnight pre-incubation, resulting in a similar number of live T cells compared to the control T cell culture.

[0217] The importance of pre-incubation and high-dose tesimolimus in Th1 / Tc1 cell production. When cryopreserving Th1 / Tc1 cell products (at the end of culture), it is important to note that, in addition to T… CM Beyond phenotype, T cells should also exhibit a relatively quiescent phenotype. That is, previously observed rapamycin-generated T cells secrete very few cytokines during adoptive transfer, but produce large amounts of cytokines in vivo; notably, others have identified a similar inverse correlation between the function of cell product effectors (minimum) and the function of effectors in vivo (maximum). T cell quiescence at adoptive transfer can promote post-transfer T cell survival and may also be important in reducing the risk of cytokine release syndrome, a cause of morbidity and death following other forms of adoptive T cell therapy, particularly genetically modified chimeric antigen receptor (CAR) T cell therapy.

[0218] To evaluate this, the cytokine secretion potential of the generated T cells was tested at the end of cell culture (day 6), and then tested again one week after in vitro expansion following maximal co-stimulation (3 / 28 beads to T cell ratio of 3:1) and in a medium without any inhibitors.

[0219] exist Figures 3A-3B In the middle, before co-stimulation, CD4 is... + and CD8 + T cells were purified and cultured for 6 days with or without a 16-hour pre-incubation period (3 / 28 beads to T cells ratio of 1:1). Tesimamol was added at a concentration of 1.0 or 0.1 μM as instructed; all culture homogenates were supplemented with IFN-α (10,000 IU / ml) and dacrolimus (50 μg / ml). On day 6 of culture, the resulting T cells were co-stimulated for 24 hours at a 3:1 ratio of 3 / 28 beads to resulting T cells; cytokine levels in the supernatant were measured by Luminex assay (results described as pg per milliliter per million cells per 24 hours). Additionally, the resulting T cells were co-stimulated with 3 / 28 beads at a ratio of 3:1 and cultured for one week in a medium free of any exogenous cytokines or inhibitors. Following this T cell culture, on day 13, the T cells were harvested, restimulated with 3 / 28 beads (at a 3:1 ratio), and the cytokine content of the 24-hour supernatant was assessed as described above. The abbreviation NA indicates not applicable (T cell yield insufficient for assay).

[0220] The results are shown in Figures 3A-3B In Figure 3A The secretion of IFN-γ was demonstrated and in Figure 3B The diagram illustrates TNF-α secretion. In each case, the left panel shows the T cell cytokine potential on day 6, while the right panel shows the T cell cytokine potential on day 13.

[0221] These data indicate that, 24 hours after maximal co-stimulation under both overnight pre-incubation and no pre-incubation conditions, high-dose tesimolimus (1.0 μM) produces the desired very low levels of day 6 T cell IFN-γ and TNF-α secretion. Notably, the use of 0.1 μM tesimolimus only partially reduces the day 6 T cell cytokine secretion potential. Thus, tesimolimus in this approach should be used at a higher concentration, i.e., at least 1 μM. These data also indicate that overnight pre-incubation alone is insufficient to produce a quiescent T cell phenotype. Therefore, the overnight pre-incubation step must be used in combination with high-dose tesimolimus to achieve the full desired outcome.

[0222] Furthermore, this method elicits an inverse relationship between the expected increase in effector function between initial T cell quiescence and subsequent restimulation. That is, for both IFN-γ and TNF-α secretion, the condition with the lowest level of day 6 cytokine potential (combination of pre-incubation and high-dose tesimolimus) produced the highest day 13 cytokine secretion values ​​for T cell levels on day 13. Notably, the condition consisting of high-dose tesimolimus and no overnight pre-incubation did not produce sufficient yield on day 13 for assessing cytokine secretion potential; thus, this result further confirms the value of high-dose tesimolimus plus pre-incubation for Th1 / Tc1 cell production.

[0223] The novel combination approach resulted in enhanced mTOR inhibition and elimination of STAT5 phosphorylation. The ability of the novel combination approach (mTOR inhibition; IL-2 receptor blockade; pre-incubation with delayed co-stimulation; and co-stimulation with lower intensity) to produce T cells with the desired phenotype depends in part on its enhanced control over molecular and cellular events previously associated with the rapamycin-resistant T cell phenotype.

[0224] A component of this phenotype is the control of mTOR-dependent signaling events, such as those occurring at the 4EBP1 level, which contribute to the control of protein translation. To address this issue, Th1 / Tc1 cells were created using the T-Rapa method, which simultaneously adds T cells, along with high levels of co-stimulation (3 / 28 beads to T cell ratio of 3:1), high-dose rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture. In parallel comparisons, T cells were created using a novel combination method of this disclosure (16-hour pre-incubation step; reduced levels of co-stimulation (1:1 ratio); use of both tesimolimus (1.0 μM) and dacrolimus (50 μg / ml); and addition of IFN-α only without IL-2).

[0225] exist Figure 4In this study, CD4+ and CD8+ T cells were cultured using either the previous method [“T-Rapa”: simultaneous addition of T cells and high levels of co-stimulation (3 / 28 beads to T cell ratio of 3:1), high dose of rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture] or a new combination method targeting the manufactured T cells [“Rapa-T”: 16-hour pre-incubation step; low levels of co-stimulation (1:1 ratio); use of both tesimolimus (1.0 μM) and dacrolimus (50 μg / ml); and addition of IFN-α only without IL-2]. At 16 and 32 hours of T cell culture, a fraction of T cells was harvested, proteins were isolated, and Western blot analysis was performed to quantify the housekeeping gene β-actin and the mTOR pathway molecule phosphorosome-4EBP1. The results were compared to proteins obtained from T cells introduced on day 0 prior to any T cell activation.

[0226] like Figure 4 The demonstrated method presents a novel combination of techniques for producing T cells at both the 16-hour and 32-hour culture time points, compared to T cells produced using the previously described method (“T-Rapa”). Figure 4 The “Rapa-T” in the formula reduces mTOR pathway activation (as measured by 4EBP1 phosphorylation).

[0227] P70S6 kinase is another key molecule in the mTOR pathway. Figure 5 and 6 In this study, CD4+ and CD8+ T cells were cultured using either the previous method [“T-Rapa”: simultaneous addition of T cells and high levels of co-stimulation (3 / 28 beads to T cell ratio of 3:1), high dose of rapamycin (1.0 μM), and cytokines (IL-2 plus IFN-α) to the culture] or a new combination method for producing manufactured T cells [“Rapa-T”: 16-hour pre-incubation step; low-level co-stimulation (1:1 ratio); use of both tesimolimus (1.0 μM) and dacrolimus (50 μg / ml); and addition of IFN-α only without IL-2]. At 16 and 32 hours of T cell culture, a fraction of T cells was harvested, proteins were isolated, and the housekeeping gene β-actin and the mTOR pathway molecule P70S6K were analyzed. Figure 5 ) or phosphorylated STAT5 ( Figure 6 Quantification was performed using Western blot analysis of the proteins. The results were compared with proteins obtained from T cells infused on day 0 prior to any T cell activation.

[0228] In stark contrast, the manufacturing process using novel combined methods to produce T cells resulted in a significant reduction in P70S6K levels. Figure 6 (Rapa-T conditions). These data provide further evidence that the combined approach to Th1 / Tc1 production improves control over mTOR activation. Using previous methods for producing rapamycin-resistant T cells, significant upregulation of P70S6K was found at both 16 and 32 hours of T cell culture. Figure 5 (T-Rapa conditions).

[0229] The combination method was associated with improved purity of Th1 cells (reduced contamination of cells expressing FoxP3). Previous manufacturing methods resulted in excessive STAT5 phosphorylation ( Figure 6 The results of T-Rapa at 16 and 32 hours of culture (in contrast to the results of T-Rapa at 16 and 32 hours of culture) show that the combined approach eliminated STAT5 phosphorylation. Figure 6 (Rapa-T results at 16 and 32 hours of culture).

[0230] Thus, the new combination approach increases control over mTOR signaling during T cell production and eliminates the promotion of T cells. REG It is also beneficial in terms of signal transduction events related to cell contamination (controlling STAT5 phosphorylation).

[0231] The individual components of the Th1 / Tc1 cell generation combination approach are further described. Further cultures were established to obtain additional information regarding the individual contribution of culture interventions to the resulting Th1 / Tc1 phenotype. For Figures 7-10, as indicated, CD4+ and CD8+ T cells were cultured using various 3 / 28 bead ratios, various mTOR inhibition methods, variable additions of anti-IL-2 receptor blockade, variable additions of type I polarizing cytokine IFN-α, and variable use of an initial overnight pre-incubation step. Supernatants produced by repeated co-stimulation (3:1 bead ratio) were collected on days 6 and 13 of culture, and Luminex assays were performed to test IFN-γ (…). Figures 7A-7B ), TNF-α Figures 8A-8B ), GM-CSF ( Figures 9A-9B ) or IL-2 ( Figures 10A-10B The content of ) (the result is expressed as pg per million cells per ml per 24 hours).

[0232] Figures 7A-7B The results of IFN-γ secretion are shown at the end of culture (day 6) and one week after further culture without inhibitors (day 13). The desired phenotype consists of relatively low cytokine secretion values ​​on day 6 and relatively high cytokine values ​​on day 13. Figures 7A-7BCulture conditions containing each of the combined elements (low-level co-stimulation [1:1 ratio]; delayed co-stimulation after overnight pre-incubation; addition of the polarizing cytokine IFN-α; addition of an mTOR inhibitor [in this experiment, a suboptimal concentration of 0.1 μM was used]; and the IL-2 receptor antibody dacrolimus) demonstrated that the desired phenotype was observed, as these conditions resulted in decreased IFN-γ secretion on day 6 but high IFN-γ secretion on day 13. T cell culture conditions omitting one or more of these elements tended to have higher cytokine values ​​on day 6 and / or lower cytokine values ​​on day 13. Additionally, previous methods used to produce rapamycin-resistant T cells (T-Rapa; Figures 7A-7B It expressed a less favorable pattern of cytokine secretion (higher values ​​on day 6; lower values ​​on day 13).

[0233] When assessing the following cytokines, the Th1 / Tc1 manufacturing combination approach also produced favorable cytokine phenotypes (decreased values ​​on day 6 combined with increased values ​​on day 13): TNF-α ( Figures 8A-8B ); GM-CSF ( Figures 9A-9B ); and IL-2 ( Figures 10A-10B ).

[0234] In summary, these results provide further evidence that the new method for manufacturing T cells has significant advantages over existing T-Rapa methods.

[0235] Molecular changes associated with the manufacture of Th1 / Tc1 cells using a combination approach. Additional experiments were performed to characterize the molecules altered during the manufacture of Th1 / Tc1 cells using a combination approach. This information is valuable not only because it can lead to a better understanding of T cell phenotypes, but also because it can be used as a quality control element during manufacturing. Furthermore, this information can be used during the screening of other combination steps that may be used in future manufacturing efforts.

[0236] Previous efforts have revealed that rapamycin-resistant T cells undergo autophagy during T cell production. It has long been known that autophagy is a direct result of mTOR suppression: when mTOR is activated, T cells maintain a state of growth and proliferation (autophagy signaling is turned off); conversely, when mTOR is suppressed, autophagy is promoted, leading to a reduction in T cell volume, including a reduction in mitochondrial volume (mitochondrial autophagy). In fact, autophagy is an essential homeostatic process in T cell biology and is associated with T cell health because it reduces energy demand and removes intracellular organelles and other cellular debris.

[0237] for Figure 11-13Human CD4 cells were cultured with a 16-hour pre-warming interval before adding 3 / 28 beads at a reduced bead-to-T cell ratio of 1:3. + and CD8 + T cells. For example... Figure 11-13 As indicated, various T cell cultures were subjected to different mTOR inhibition methods (1.0 μM rapamycin; 1.0 μM or 0.1 μM tesimolimus), different conditions of exogenous IL-2 addition, and different conditions relative to the addition of the anti-IL-2 receptor monoclonal antibody dacrolimus. After a 16-hour pre-incubation interval, cells were harvested from the T cell cultures, proteins were isolated, and p62 (…) was analyzed by Western blotting. Figure 11 ), Phosphate-RAPTOR ( Figure 12 ) or BIM ( Figure 13 ) and the housekeeping gene β-actin were quantified.

[0238] The ability of combined approaches to promote autophagy was evaluated, as measured by T cell expression of the autophagy marker p62. Figure 11 The indicated combination method comprises a pre-incubation step, low-level co-stimulation (3 / 28 beads to T cells ratio of 1:3), dacrolimus blockade of the IL-2 receptor, and upregulation of the autophagy marker p62 expression by an mTOR inhibitor. If each of these factors is present, autophagy can be induced by mTOR inhibition using rapamycin (1 μM) or tesiromosin (1.0 or 0.1 μM). Removing IL-2 receptor blockade from the protocol substantially attenuates the induction of autophagy.

[0239] Furthermore, the combination approach also led to the inhibition of promoted mTOR pathways, such as the reduction in the expression of phosphorylated forms of RAPTOR as indicated by ( Figure 12 It is noteworthy that the combination approach of using high-dose tesiromix produced low levels of phosphate-RAPTOR compared to using low-dose tesiromix or high-dose rapamycin.

[0240] The production of BIM, a pro-apoptotic member of the bcl-2 gene family, was also evaluated on T cell expression. Bcl-2 family members primarily operate at the mitochondrial level, and thus, mitophagy can influence the homeostasis of bcl-2 family member genes, which helps determine the apoptosis threshold. Mitophagy has been shown to be beneficial in reducing the apoptosis threshold, potentially selectively eliminating mitochondria, while the homeostasis of bcl-2 family molecules is detrimental. The combined production methods were found to lead to reduced BIM expression. Figure 13 ).

[0241] In summary, these experiments indicate that enhanced autophagy, reduced mTOR signaling, and decreased expression of pro-apoptotic molecules are associated with a combined approach to Th1 / Tc1 cell manufacturing. These changes may contribute to enhanced in vivo function of the manufactured T cells and could thus be used as a quality control step or for screening future next-generation T cell manufacturing methods.

[0242] Combination methods promoted T cell quiescence and T cell dedifferentiation. Further experiments were conducted to characterize the surface phenotype of Th1 / Tc1 cells produced via the combination methods, as defined by pre-incubation steps, low-level co-stimulation (1:3 3 / 28 bead-to-T cell ratio), dacrolimus antibody blockade of the IL-2 receptor, and the combination of an mTOR inhibitor. The effects of culture variables on T cell expression of the CD45 isotype RA, a marker of T cell naivety containing stem cell memory subsets, were first assessed. Thus, it is desirable to develop methods for T cell production that preserve or increase CD45RA expression.

[0243] for Figure 14A-15D ,like Figure 14A-15D The instructions now specify the use of various 3 / 28 bead ratios, various mTOR inhibition methods, variable additions of anti-IL-2 receptor blockade, variable additions of the type I polarizing cytokine IFN-α, and variable use of an initial overnight pre-incubation step for culturing CD4+ and CD8+ T cells. On day 6 of culture, T cells are harvested and the CD45RA (CD45RA) content of the CD4 cell subset of T cells is assessed. Figures 14A-14D ) or CD62L, CCR7 and CD127 ( Figures 15A-15D The expression of ) was measured by flow cytometry, and the results were compared with the expression level on day 0 of culture (“day 0 input culture”).

[0244] like Figures 14A-14D As demonstrated, T cell production without the key elements of the combined method (using high co-stimulation at a 3:1 bead-to-T cell ratio; no dacrolimus; no mTOR inhibitor) resulted in a rapid decline in CD45RA expression (see column #2). In stark contrast, the use of all these components resulted in complete preservation of CD45RA expression (see column #4). Notably, T cells proliferated using the previously identified manufacturing method did not exhibit optimally preserved CD45RA expression (T-Rapa cells; Figure 14B , column #3).

[0245] In addition, other markers, including CD62L, CCR7, and CD127, were evaluated to determine whether the combination approach led to a reduction in T cell differentiation. Figure 15A(Column #4) indicates that T cells manufactured using a pre-incubation step, low-level co-stimulation, antibody blockade of the IL-2 receptor, and an mTOR inhibitor increased the co-expression of these T cell markers. Notably, T cells proliferated using previously identified manufacturing methods did not exhibit the optimal increased expression of these three memory markers (T-Rapa cells; Figure 15A , column #3).

[0246] Thus, the combined approach of Th1 / Tc1 cell production is advantageous for the manufacture of T cells in a limited differentiation state, and the method has been clearly and reproducibly demonstrated to mediate increased in vivo effects.

[0247] Optimization of the cohort approach was achieved by reducing the purity of T cells at the start of culture. For T cell production, it is important to determine whether the input population of the culture must be highly purified to a high T cell content, or whether helper cell populations such as monocytes can be tolerated. From a financial cost and labor perspective, it is generally desirable to start the culture with a less purified population. However, contaminating the cell population at the start of culture may be detrimental to T cell expansion or the generation of the desired T cell phenotype. To evaluate this parameter, a cohort approach was used to start the culture using input populations with T cell contents of 100%, 66%, 33%, or 10%; the remaining cell population consisted primarily of monocytes.

[0248] for Figure 16-20 Before culture began, the input cell population was adjusted to achieve a T cell purity of 100%, 66%, 33%, or 10%; the remaining cell population consisted of non-T cells from peripheral blood mononuclear cells (primarily monocytes). As indicated, T cells were expanded in media with or without tesimolimus (1.0 or 0.1 μM); additionally, cultures were optionally pre-incubated for 16 hours prior to co-stimulation with anti-CD3 and anti-CD28 magnetic beads (ratio 1:3) or without pre-incubation. Each culture shown was multiplied in media containing the anti-IL-2 receptor monoclonal antibody dacrolimus (50 μg / ml) and IFN-α (10,000 IU / ml). At the end of the 6-day manufacturing interval, T cells received high-level co-stimulation (magnetic beads to T cells ratio 3:1). Figure 16 Following high-level co-stimulation, T cells were then expanded in inhibitor-free culture medium for one week. At the end of this expansion interval, the number of T cells was calculated and plotted against day 0. For Figures 17-18, T cells were expanded until day 13 of culture following high-level co-stimulation. T cells were co-stimulated on days 6 and 13, and the IFN-γ (IFN-γ) concentration in the supernatant was assessed after 24 hours. Figures 17A-17B ) or TNF-α ( Figures 18A-18B) content (results are shown as pg per million cells per ml per 24 hours). For Figures 19-20 Following high-level co-stimulation, T cells were expanded up to day 13 of culture. CD25 expression in T cells was assessed by flow cytometry on days 6 and 13 (the results shown are the percentage of CD4+ T cells co-expressing CD25). Figure 19 ) or expression of CD62L, CCR7 and CD127 ( Figure 20 ).

[0249] like Figure 16 As detailed in the literature, cultures starting with reduced T cell purity at culture inoculation result in greater T cell expansion capacity, a relationship that occurs in a dose-dependent manner. Notably, T cells proliferated using an overnight pre-incubation step exhibit greater expansion capacity compared to T cells co-stimulated at culture initiation. These data provide further support for combinatorial approaches and indicate that cell populations that might be considered contaminants at culture initiation appear to actually promote T cell expansion potential. Thus, optimized use of combinatorial approaches should also include the use of T cells that are not highly enriched at culture initiation; to ensure quality, it is important to control purity levels, as a non-limiting example, starting each culture with an inoculum content of 66% or 33% T cells. Figures 17A-17B As detailed in 18A-18B, the use of a combination of methods to produce T cells and the introduction of non-purified T cells resulted in the desired cytokine secretion pattern, namely: ( Figures 17A-17B IFN-γ secretion was reduced at the end of manufacturing (day 6) and increased after one week of expansion in inhibitor-free medium (day 13); and ( Figures 18A-18B TNF-α secretion was reduced at the end of manufacturing (day 6) and increased after one week of expansion in inhibitor-free culture medium (day 13).

[0250] In addition, the expression of cell surface markers in T cells produced using a combination approach was evaluated using an input population with reduced T cell purity. Figure 19 As shown, these T cells exhibited reduced CD25 expression at the end of manufacturing (day 6), consistent with the static phenotype; after one week of culture under inhibitor-free conditions, the T cells showed significantly upregulated CD25. Figure 20 As shown, these T cells also exhibit increased co-expression of memory markers CD62L, CCR7, and CD127; these markers then decrease after a week of T cell expansion.

[0251] In summary, these data indicate that it is possible to generate Th1 / Tcl cells using a combination approach when using infused T cells with a high degree of contamination with non-T cell populations. In fact, intentionally including such non-T cell populations can be used to increase T cell yield and improve the memory curve of the resulting T cells.

[0252] Production from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells will be stored in the gas phase of liquid nitrogen until the cells thaw and Rapa-T cells are produced. In the case of cells isolated by apheresis or by simple blood collection in the future, the cells will be processed immediately and can then be directly placed into cultures, or they can be cryopreserved using controlled-rate freezing techniques and stored in the gas phase of liquid nitrogen for later use.

[0253] T cell culture from cryopreserved cell substrates from freshly isolated cell populations requires enrichment of certain types of T cells, for example, using monoclonal antibodies and column technology (positive or negative selection). Enrichment of the original cell material used in Rapa-T cell manufacturing does not require such antibody-based methods, as T cells are efficiently enriched during the culture interval; thus, this approach aligns with global recommendations for effective cell therapy. The initial processing steps for Rapa-T cell manufacturing focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (where applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminated granulocytes and, to some extent, monocytes. These steps are performed in a relatively automated method that primarily utilizes closed-system technology; this procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves consistency throughout the manufacturing process, and reduces the risk of contamination of the final product with infectious agents. The processing of Rapa-T products combines the following steps: (1) thawing the cryopreserved products (where applicable) using a solid-state non-aqueous method to reduce infectious agent contamination; (2) automatically washing the cell products using a LOVO permeable membrane device; (3) integrating RBC lysis with potassium ammonium chloride (ACK) buffer during the LOVO washing step; (4) reducing cell content using the LOVO method, followed by platening the cells into a closed-system countercurrent centrifugation (CCE) device (Elutra; Terumo); and (5) pre-programmed operation of the Elutra device for efficient removal of granulocytes and monocytes via CCE.

[0254] After lymphocyte enrichment and culture medium purification, the cells were seeded into a dedicated chamber with abundant oxygen exchange capacity (G-Rex container; Wilson-Wolf). In addition to enhanced permeability, the G-Rex container is a closed-system unit and offers the additional advantage of automated closed-system media volume reduction (GatheRex liquid handling pump). The lymphocyte-enriched cells were held in the G-Rex container for 6 days.

[0255] Several specific culture conditions can be used to facilitate the production of a mixture of CD4+ and CD8+ T cells in G-Rex containers, utilizing the functional properties of the manufactured T cells. These specific conditions include: (1) the use of a medium enriched with 5% human serum (including, but not limited to, X-Vivo 20; Lonza); and (2) a 16-hour interval between seeding of cells onto G-Rex plates prior to co-stimulation (at a rate of 1.5 × 10⁻⁶ cells per mL). 6 (3) During this initial rest period, the cells were allowed to rest optimally by adding the monoclonal antibody baliximab (which blocks the IL-2 receptor and thereby prevents autonomous T cell activation by endogenously produced IL-2) and tesimolimus (a pharmacological inhibitor of mTORC1); (4) After this 16-hour rest period, under suboptimal conditions, the cells were either not co-stimulated or co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads (3 / 28 beads), as defined by a bead-to-T cell ratio of 1:3 (typically, most T cell expansion conditions utilize a 9-fold higher level of co-stimulation, i.e., a bead-to-T cell ratio). (3:1); (5) Importantly, it is crucial not to wash T cells after the initial interval; (6) After the interval, in addition to adding 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4+Th1 and CD8+Tc1 phenotypes; (7) Importantly, it is crucial to avoid adding IL-2, a common additive for T cell culture; and (8) After adding beads and IFN-α, it is important to keep the cells undisturbed until harvest on day 6 of culture (no cell washing, no further culture additives).

[0256] Cryopreservation of manufactured T cells. 1) After 6 days of cell culture in G-Rex containers, the culture volume can be reduced using a GatheRex instrument in a closed system. Subsequently, cells can be harvested, 3 / 28 beads can be removed using a handheld magnet, and the cells can be placed in a LOVO device for continuous washing to remove >99% of culture additives (tesimolimus, balithimab, IFN-α).

[0257] The washed cells were reconstituted into a cryopreservation medium containing 5% DMSO and 5% pentameric starch. Cryopreservation was performed in multiple single-use aliquots in 50 ml cryopreservation bags. Rapa-T cells were cryopreserved using a GMP-compliant controlled-rate freezing method and transported in the gas phase of liquid nitrogen by a certified cryopreservation transporter after passing specified release criterion tests.

[0258] The standard assay for Rapa-T cell release includes standard tests such as the purity of CD3+, CD4+, and CD8+ T cell content (by flow cytometry, the final product should contain >70% CD3+ T cells; CD4+ and CD8+ subsets should each be present at a level of 5%). As determined by flow cytometry assays for annexin and 7-AAD, >70% of the cells should be viable. Furthermore, the cells should be free from bacterial and fungal contamination for at least a 3-day culture interval (ideally a 14-day interval); additionally, the cell product should be below the detection limit for bacterial LPS endotoxin.

[0259] In addition to these standard tests, specialized functional tests will also constitute the criteria for the release of Rapa-T cell products. Prior to the release of products and cell therapy, Rapa-T cells, relative to culture-infused T cells, may possess the following properties: (1) an enhanced T central memory phenotype, as defined by increased flow cytometry co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitory molecules such as programmed death 1 (PD1); (3) a quiescent state, as defined by reduced secretion levels of Th1 / Tc1 cytokines at maximal co-stimulation; (4) an autophagy signature, as demonstrated by reduced mitochondrial mass as measured by flow cytometry MitoTracker; (5) a resistance phenotype, as demonstrated by at least 50% inhibition of downstream targets of mTORC1 and mTORC2; and (6) a multifaceted differential gene expression profile of n=80 key transcription factors and differentiation molecules.

[0260] Figure 21 The study demonstrated that, compared to T-Rapa cells, the novel Rapa-T method produced T cells with increased expression of naïve or T central memory markers, independent of whether the novel Rapa-T method used bead co-stimulation or low-level bead co-stimulation (bead-to-T cell ratio of 1:3). Figure 21In this study, Rapa-T1 cells were generated by culturing in IFN-α, tesimolimus, and balithiximab, as previously described, without bead co-stimulation (first two columns of each figure) or co-stimulation using a 1:3 bead-to-T cell ratio (third and fourth columns of each figure); the results were compared with cultures using the previous T-Rapa method (using rapamycin and a 3:1 bead-to-T cell ratio; fifth and sixth columns of each figure). Flow cytometry was performed at the end of the culture, and CD4 counts were recorded in detail. + T cell subsets (black bars) and CD8 + The results for T cell subsets (gray bars) are shown. The results are: the initial T cell subsets defined for CD45RA+ expression (left panel); T central memory subsets defined for co-expression of CD62L and CCR7; and more primitive T cell subsets for co-expression of CD62L, CCR7, and CD127.

[0261] like Figure 21 The CD4 manufactured according to the method described in this disclosure, as detailed in the document. + and CD8 + T cells, relative to T-Rapa cells, exhibit increased expression levels of initial and T central memory markers via flow cytometry, including those obtained by the method without involving any bead co-stimulation or reduced levels of co-stimulation compared to the T-Rapa approach.

[0262] Figure 22 The study demonstrated that, compared to T-Rapa cells, the novel Rapa-T method produced T cells with reduced expression of CD25, CTLA4, and TIM3, independent of whether the novel Rapa-T method used bead co-stimulation or low-level bead co-stimulation (a bead-to-T cell ratio of 1:3). Figure 22 In this study, Rapa-T1 cells were generated by culturing in IFN-α, tesimolimus, and balithiximab, as previously described, without bead co-stimulation (first two columns of each figure) or co-stimulation using a 1:3 bead-to-T cell ratio (third and fourth columns of each figure); the results were compared with cultures using the previous T-Rapa method (using rapamycin and a 3:1 bead-to-T cell ratio; fifth and sixth columns of each figure). Flow cytometry was performed at the end of the culture, and CD4 counts were recorded in detail. + T cell subsets (black bars) and CD8 + The results of T cell subsets (gray bars) are shown. The results also show the expression of the IL-2 receptor CD25 (left panel); targeting immunosuppression and T cell subsets. REG Associated molecule CTLA4; and immune checkpoint molecule TIM3.

[0263] like Figure 22 The CD4 manufactured according to the method described in this disclosure, as detailed in the document. + and CD8 + T cells, compared to T-Rapa cells, have a reduced IL-2 receptor CD25 content as determined by flow cytometry. 2 (It is related to T cell activation and T) REG Expression levels of cell function-related molecules and reduced levels of the immunosuppressive molecule CTLA4. 3 and immune checkpoint inhibitory molecule TIM3 4 The level, including the T-Rapa method, is determined under conditions where the method does not involve any bead costimulation or reduces the level of costimulation.

[0264] Figure 23 The novel Rapa-T method produces T cells exhibiting a Th2-to-Th1 polarization pattern, but with increased quiescence relative to T-Rapa cells, independent of the method's absence of bead co-stimulation or the use of low-level bead co-stimulation (bead-to-T cell ratio of 1:3). Figure 23 In this study, Rapa-T1 cells were generated by culturing in IFN-α, tesimolimus, and balithiximab, as previously described, without bead co-stimulation (first two columns of each figure) or co-stimulation using a 1:3 bead-to-T-cell ratio (third and fourth columns of each figure); the results were compared with cultures using the previous T-Rapa method (using rapamycin and a 3:1 bead-to-T-cell ratio; fifth and sixth columns of each figure). Cytokine secretion analysis (IL-4 and IFN-γ measurements) was performed at the end of culture, and the results are detailed at the end of T-cell production (day 6) and after a further 6 days of culture under inhibitor-free conditions (day 12).

[0265] like Figure 23 The CD4 manufactured according to the method described in this disclosure, as detailed in the document. + and CD8 +T cells exhibit a comparable degree of Th2-to-Th1 cytokine polarization compared to T-Rapa cells, including under conditions where no bead co-stimulation or reduced levels of co-stimulation are involved compared to the T-Rapa method. Specifically, the secretion level of the Th2 cytokine IL-4 is low (values ​​between 100 pg / ml and 200 pg / ml on both days 6 and 12 after the end of manufacturing [day 6] and an additional culture period without inhibitors [day 12]), while the secretion level of the Th1 cytokine IFN-γ is high on day 12 of culture (values ​​between 1000 pg / ml and 3000 pg / ml). The significantly reduced IFN-γ secretion at the end of manufacturing (day 6) under the new Rapa-T conditions compared to the old T-Rapa conditions indicates a favorable property of T cell quiescence in the new Rapa-T manufacturing method, regardless of whether the method uses bead co-stimulation or low levels of bead co-stimulation (bead-to-T cell ratio of 1:3).

[0266] like Figure 24 The CD4 manufactured according to the method described in this disclosure, as detailed in the document. + and CD8 + T cells exhibit a Th1 cytokine polarization pattern relative to T-Rapa cells, which is achieved under conditions where no bead co-stimulation or reduced levels of co-stimulation are involved compared to the T-Rapa approach.

[0267] Figure 24 The study demonstrated that, compared to T-Rapa cells, the novel Rapa-T method produced T cells with a favorable Th1 polarization pattern, independent of whether the novel Rapa-T method used bead co-stimulation or low-level bead co-stimulation (a bead-to-T cell ratio of 1:3). Figure 24 In this study, Rapa-T1 cells were generated by culturing in IFN-α, tesimolimus, and balithimab, as previously described, without bead co-stimulation or co-stimulation using a 1:3 bead-to-T cell ratio; various control cultures, including the previous T-Rapa method (using rapamycin and a 3:1 bead-to-T cell ratio), were also evaluated. Unless otherwise stated, all cultures were at a concentration of 9 × 10⁻⁶. 6Cells / ml, without bead co-stimulation, without IL-2 addition, with delayed IFN-α addition, containing 1 μM tesiromixol and 10 μM baribizumab, and using X-Vivo 20 medium supplemented with 5% AB serum. Specific culture conditions according to the illustration are as follows: Condition 1, Rapa-T method, as described above; Condition 2, Rapa-T method without serum; Condition 3, Rapa-T method without baribizumab; Condition 4, Rapa-T method without baribizumab and using reduced tesiromixol (0.1 μM); Condition 5, Rapa-T method without tesiromixol or baribizumab; Condition 6, Rapa-T method using imported T cells contaminated with a high frequency of monocytes (79% of imported cells are monocytes, compared with...). (This was an increase compared to all cultures with approximately 10% mononuclear cell contamination); Condition 7, using the mononuclear cell-free Rapa-T method (<1%); Condition 8, using the Rapa-T method with simultaneous addition of IFN-α (no overnight delay); Condition 9, using the Rapa-T method with 1:3 bead co-stimulation; Condition 10, control T cell conditions (no inhibitor; 3:1 beads); and Condition 11, the old T-Rapa conditions, rapamycin (1 μM), IL-2 added at 20 IU / ml, 3:1 magnetic beads, no overnight pre-incubation. Cytokine secretion analysis (IL-2 and IFN-γ measurements) was performed at the end of culture, and the results were detailed at the end of T cell production (day 6) and after 6 more days of culture under inhibitor-free conditions (day 12).

[0268] like Figure 24 The CD4 manufactured according to the method described in this disclosure, as detailed in the document. + and CD8 + T cells exhibit a Th1 cytokine polarization pattern relative to T-Rapa cells, which is achieved under conditions where no bead co-stimulation or reduced levels of co-stimulation are involved compared to the T-Rapa approach.

[0269] like Figure 24The condition #1, described in the study, is a Rapa-T condition without beads that produces cells with a high level of IL-2 secretion, particularly on day 12 after T cell expansion without inhibitors. A similar pattern was observed in condition #2, which was performed in a medium without serum supplementation, thus indicating the ability to produce Rapa-T cells with or without serum supplementation. The enhanced IL-2 secretion capacity, especially compared to culture #11 (the old T-Rapa condition), indicates that the new Rapa-T manufacturing method produces T cells with a reduced progenitor spectrum that can function in vivo in a helper-free manner. In this respect, condition #1 is also advantageous relative to culture #9, which is a new Rapa-T condition using co-stimulation with a 1:3 bead-to-T cell ratio.

[0270] like Figure 24 As described, Condition #1 (Rapa-T condition manufacturing without beads) was also preferred in terms of IFN-γ secretion at the end of day 6 of manufacturing, as the level was close to the detection limit, thus indicating that the Rapa-T cell products were in a quiescent state. In contrast, Condition #5 without inhibitors had almost 4000 pg / ml of IFN-γ secretion at day 6; similarly, the old T-Rapa condition had a high level of IFN-γ secretion of approximately 6000 pg / ml at day 6. Notably, the new Rapa-T condition co-stimulated with cells using a 1:3 bead-to-T cell ratio showed less quiescence than Condition #1 without beads, as approximately 200 pg / ml of IFN-γ secretion was present at the end of manufacturing (day 6). Finally, the new Rapa-T manufacturing methods (Condition #1 without beads or Condition #9 with beads) showed a favorable improvement in IFN-γ secretion capacity at day 12 of culture after a 6-day culture interval in the absence of inhibitors.

[0271] In summary, the novel Rapa-T approach results in T cell production with the following phenotypic characteristics: (a) decreased expression of regulatory T cell markers (such as transcription factor FOXP3) and increased expression of the Th1 transcription factor TBET relative to infused normal T cells; (b) increased quiescent state relative to the old T-Rapa approach, as indicated in part by decreased expression of the IL-2 receptor CD25 and decreased secretion of inflammatory cytokines IFN-γ and TNF-α at the end of production; (c) decreased expression of p-STAT5 and reduced levels of mTOR pathway molecules p-RAPTOR, p-4EBP1, and p70S6K relative to the old T-Rapa approach; and (d) increased autophagy markers, including but not limited to p62 expression, relative to infused normal T cells. The changes included: (e) an increase in flow cytometry markers of the initial or T central memory population (including co-expression of CD45RA, CD62L / CCR7, and co-expression of CD62L / CCR7 / CD127) relative to the input of normal T cells; (f) a decrease in the expression of co-inhibitory molecules (including but not limited to CTLA4) relative to the old T-Rapa method; (g) a decrease in the expression of checkpoint inhibitory receptors (including but not limited to TIM3) relative to the old T-Rapa method; and (h) changes in RNA expression patterns relative to the input of normal T cells, including but not limited to an increase in dedifferentiation markers (including but not limited to Nanog, KLF4, and KLF10) and a decrease in differentiation markers (including but not limited to perforin, granzyme B, and IFN-γ).

[0272] Most phenotypic characteristics of the T cell products manufactured according to the Rapa-T method detailed in this disclosure can be determined at the end of the culture. However, it is important to note that the T cell products can be cryopreserved, and thus, the phenotypic characteristics of the T cells in the thawed state reflect the actual product to be adopted into the subject. The thawed Rapa-T cells can be characterized by: (a) maintaining a quiescent state, as indicated by low levels of expression of the IL-2 receptor CD25, which is comparable between samples manufactured at the end of day 6 and thawed samples; (b) continuing to have low levels of secretion of the inflammatory cytokines IFN-γ and TNF-α relative to samples manufactured at the end of day 6 (with no increase relative to samples collected at the end of manufacturing); and (c) maintaining the initial or T central memory population (including co-expression of CD45RA, CD62L / CCR7, and concomitant expression of CD62L / CCR7 / CD127) in flow cytometry relative to infused normal T cells. (f) Increased expression of cytokinetic markers; (g) continued reduced expression of co-inhibitory molecules (including but not limited to CTLA4) in thawed samples (no increase in thawed samples compared to samples collected at the end of manufacturing); (h) continued reduced expression of checkpoint inhibitory receptors (including but not limited to TIM3) in thawed samples (no increase in thawed samples compared to samples collected at the end of manufacturing); and (h) altered RNA expression patterns relative to infused normal T cells, including but not limited to increased dedifferentiation markers (including but not limited to Nanog, KLF4, and KLF10) and decreased differentiation markers (including but not limited to perforin, granzyme B, and IFN-γ).

[0273] Production from cryopreserved cell substrates. In the case of previously collected PBSC products, such cryopreserved cells will be stored in the gas phase of liquid nitrogen until the cells thaw and Rapa-T cells are produced. In the case of cells isolated by apheresis or by simple blood collection in the future, the cells will be processed immediately and can then be directly placed into cultures, or they can be cryopreserved using controlled-rate freezing techniques and stored in the gas phase of liquid nitrogen for later use.

[0274] T cell culture from cryopreserved cell substrates from freshly isolated cell populations requires enrichment of certain types of T cells, for example, using monoclonal antibodies and column technology (positive or negative selection). Enrichment of the original cell material used in Rapa-T cell manufacturing does not require such antibody-based methods, as T cells are efficiently enriched during the culture interval; thus, this approach aligns with global recommendations for effective cell therapy. The initial processing steps for Rapa-T cell manufacturing focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (where applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminated granulocytes and, to some extent, monocytes. These steps are performed in a relatively automated method that primarily utilizes closed-system technology; this procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves consistency throughout the manufacturing process, and reduces the risk of contamination of the final product with infectious agents. The processing of Rapa-T products may include the following steps: (1) thawing the frozen products (where applicable) using a solid-state non-aqueous method to reduce infectious agent contamination; (2) automatically washing the cell products using a LOVO permeable membrane device; (3) integrating the lysed RBCs with potassium ammonium chloride (ACK) buffer during the LOVO washing step; (4) reducing the cell content using the LOVO method, followed by platening the cells into a closed-system countercurrent centrifugation (CCE) device (Erutra; Terumo); and (5) pre-programming the Erutra device to efficiently remove granulocytes and monocytes via CCE.

[0275] After lymphocyte enrichment and culture medium purification, cells can be seeded into a dedicated chamber with abundant oxygen exchange capacity (G-Rex container; Wilson Wolf). In addition to enhanced permeability, the G-Rex container is a closed system unit and offers the additional advantage of automated closed-system media volume reduction (GatheRex liquid handling pump). Lymphocyte-enriched cells can be held in the G-Rex container for 6 days.

[0276] Several specific culture conditions can be used to promote CD4+ expression in G-Rex containers by utilizing the functional properties of manufactured T cells. + and CD8 + Preparation of T-cell mixtures. These specific conditions include: (1) enriched culture media (including, but not limited to, X-Vivo 20; Lonza) further supplemented with 5% human serum or, in some embodiments, without added serum; (2) 16-hour intervals between seeding to G-Rex cell plates prior to co-stimulation (at a rate of 1.5 × 10⁶ cells per mL). 6(3) During this initial rest, cells are optimally rested by adding the monoclonal antibody baliximab (which blocks the IL-2 receptor and thereby prevents autonomous T cell activation by endogenously produced IL-2) and tesimolimus (a pharmacological inhibitor of mTORC1); (4) After this 16-hour rest, under suboptimal conditions, 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 (generally, most T cell expansion conditions utilize a 9-fold higher level of co-stimulation, i.e., a bead-to-T cell ratio of 3:1; in some cases, it is beneficial to avoid adding any co-stimulatory agents); (5) Importantly, it is crucial not to wash T cells after the initial rest; (6) After the rest, in addition to adding 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4. + Th1 and CD8 + Tc1 phenotype; (7) Importantly, it is crucial to avoid adding IL-2, which is a common additive for T cell culture; and (8) After adding bead and IFN-α, it is important to keep the cells undisturbed until harvest on day 6 of culture (without cell washing and no further culture additives).

[0277] Cryopreservation of manufactured T cells. 1) After 6 days of cell culture in G-Rex containers, the culture volume can be reduced using a GatheRex instrument in a closed system. Subsequently, cells can be harvested, 3 / 28 beads can be removed using a handheld magnet, and the cells can be placed in a LOVO device for continuous washing to remove >99% of culture additives (tesimolimus, balithimab, IFN-α).

[0278] The washed cells were reconstituted into a cryopreservation medium containing 5% DMSO and 5% starch pentapolymer. Cryopreservation was performed in multiple single-use aliquots in 50 ml cryopreservation bags. Rapa-T cells were cryopreserved using a GMP-compliant controlled-rate freezing method and transported in the gas phase of liquid nitrogen by a certified cryopreservation transporter after passing specified release criterion tests.

[0279] The standard assay for Rapa-T cell release includes standard tests such as CD3. + CD4 + and CD8 + T cell purity (by flow cytometry, the final product's CD3+ T cell content can be >70%; CD4+ T cells can be >70%). + and CD8 +Subpopulations may be present at a level of 5% (each subpopulation may exist at a level of 5%). As determined by flow cytometry assays for annexin and 7-AAD, cells may be >70% viable. Furthermore, the cells should be free from bacterial and fungal contamination for a minimum of 7-day culture intervals (ideally, 14-day culture intervals); additionally, cell products should be below the detection limits for bacterial LPS endotoxins and mycoplasma.

[0280] In addition to these standard tests, specific functional tests can also constitute the criteria for the release of Rapa-T cell products. Prior to the release of products and cell therapy, Rapa-T cells, relative to culture-infused T cells, can possess the following properties: (1) an enhanced T central memory phenotype, as defined by increased flow cytometry co-expression of CD62 ligand and CCR7; (2) low-level expression of checkpoint inhibitory molecules such as programmed death 1 (PD1); (3) a quiescent state, as defined by reduced secretion levels of Th1 / Tc1 cytokines at maximal co-stimulation; (4) an autophagy signature, as demonstrated by reduced mitochondrial mass as measured by flow cytometry MitoTracker; (5) a resistance phenotype, as demonstrated by at least 50% inhibition of downstream targets of mTORC1 and mTORC2; and (6) a multifaceted differential gene expression profile of n=80 key transcription factors and differentiation molecules.

[0281] Example 2

[0282] Steady-state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were analyzed using GE's [technology / method / technology]. Enrichment was performed using an automated Ficoulin process on the instrument, eliminating >95% of unwanted contamination from neutrophils. The lymphocyte-enriched cell population was then plated into G-REX culture vessels at the initial cell density under the medium and serum supplementation conditions indicated in Table 1 below, and incubated in culture for 6 days. Condition 1 represents the control before culture (lymphocytes enriched after automated Ficoulin). Cultures were initiated under variable inhibitory conditions by adding tesimolimus, sirolimus, and / or the anti-IL2 receptor monoclonal antibody bailiximab at the doses indicated in Table 1. For conditions 2–5, bailiximab was added at a concentration of 10 μg / mL, and for conditions 6–8, bailiximab was added at a concentration of 20 μg / mL. Some culture conditions were accepted. 3 / 28 beads were co-stimulated against CD3 and CD28 at a bead:T cell ratio of 0.88:1 (conditions 2-3 and 5-6) or 3:1 (conditions 8-9). Cytokines, consisting of IFN-α alone (10,000 IU / mL) or a combination of IFN-α (10,000 IU / mL) and IL-2 (20 IU / mL), were added at the indicated time. Cytokines were added at the start of culture (conditions 8-9) or one day after the start of culture (conditions 2-7). Cultures 3-4 and 6-7 received additional culture medium two days after the start of culture to dilute it to the indicated final cell density.

[0283] Table 1: Culture Conditions

[0284]

[0285] M / mL = millions of cells per milliliter; TEM = tesirobolis; RAPA = 1 μM tesirobolis oral solution; TexMACS is a proprietary culture medium formulation.

[0286] Condition 9 represents the T-Rapa product. Condition 7 was found to provide the optimal RAPA-T condition among the conditions tested in Table 1. This condition has several key attributes: (1) serum-free medium; (2) very high initial cell density (30 M / mL); (3) very high tesiromix concentration (4.5 μM); (4) presence of a monoclonal antibody against the IL2 receptor; (5) absence of co-stimulation; (6) cytokine support with only IFN-α (without IL-2) added one day after the start of culture; and (7) cell dilution on day 2 of culture.

[0287] After 6 days of culture, the obtained T cells were harvested, and the expression of specific molecules in the CD4+ (indicated by black bars) and CD8+ (indicated by gray bars) T cell subsets was assessed by flow cytometry, such as... Figure 25A-25O As shown.

[0288] Figure 25A (CD45RA+) demonstrated the importance of RAPA-T culture conditions for maintaining the expression of initial T cell markers on both CD4+ and CD8+ T cells relative to culture input cells; in stark contrast, previous T-RAPA conditions resulted in a significant reduction in CD45RA+ cells.

[0289] Figure 25B(CD25+) demonstrated the importance of RAPA-T culture conditions for maintaining T cell quiescence in both CD4+ and CD8+ T cells relative to culture-introduced cells; in stark contrast, previous T-RAPA conditions resulted in a significantly activated T cell state, as indicated by increased CD25 expression.

[0290] Figure 25C (CD28+) and Figure 25D (ICOS+) indicates that RAPA-T and T-RAPA cell products have similar CD4+ and CD8+ T cell expression to these activating co-stimulatory molecules.

[0291] Figure 25E-25F The results (CD39+ and CD73+, respectively) show that RAPA-T culture conditions, relative to T-RAPA conditions, result in reduced expression of these ecto-nucleotidase molecules. These molecules exert immunosuppressive effects by metabolizing ATP into adenosine. Therefore, for therapeutic use, RAPA-T cell products are expected to be advantageous compared to T-RAPA cell products.

[0292] Figure 25G-25O The remaining data indicate that the novel RAPA-T method results in a significant reduction in the expression of molecules associated with immunosenescence (KLRG1), the immunosuppressive regulatory T cell phenotype (GITR), or checkpoint inhibitory functions (LAG3, PD1, 2B4, LAIR1, CTLA4, TIGIT, and TIM3). Each of these molecules was significantly reduced in every variable culture condition associated with RAPA-T cell products relative to T-RAPA cell products. Condition 7 demonstrated the most severe and consistent reduction in these molecules in the tested condition.

[0293] Example 3

[0294] T cells were prepared as shown in Example 2 using culture conditions 1-8, corresponding to culture conditions 2-9 in Example 2. On day 2 of the culture interval, the resulting T cells were harvested, and the molecules associated with the mTORC1, mTORC2, and STAT pathways in the resulting T cells were evaluated by Western blot analysis (according to the manufacturer's instructions; BioTechne Mr.Wes Instrumentation).

[0295] For optimal Th1 / Tc1 phenotype production, it is important to limit the activation (phosphorylation) of STAT5, which drives the regulatory T cell phenotype. Figure 26As shown, each of the Rapa-T cell culture conditions (conditions 1-6) is relatively free of phosphorylated STAT5; in stark contrast, the T-Rapa conditions (conditions 7-8) show a significant presence of phosphorylated STAT5. The reduction in STAT5 phosphorylation in Rapa-T cells can exceed 75% compared to T-Rapa cells.

[0296] For optimal Th1 / Tc1 type fabrication, it is important to conduct active signaling through specific STAT molecules (including STAT1) that drive type I differentiation. For example... Figure 26 As shown, each of the Rapa-T culture conditions exhibits detectable levels of STAT1 phosphorylation, although the levels described in condition 6 are reduced (Example 2, condition 7). However, the total STAT1 level is also reduced in condition 6.

[0297] The optimal phenotype of Rapa-T cells can also be characterized by a reduction in molecules associated with the mTORC1 pathway. Rapa-T condition 6 (corresponding to Example 2, condition 7) shows virtually no expression of p70S6K, a molecule associated with mTORC1. Providing co-stimulation in other Rapa-T culture conditions (conditions 1, 2, 4, and 5) increases p70S6K expression. Therefore, avoiding co-stimulation during Rapa-T cell production may be beneficial.

[0298] The optimal phenotype produced by Th1 / Tc1 RAPA-T cells can also depend on the preservation of the mTORC2 signaling pathway. In this respect, it is advantageous that the optimal RAPA-T cell conditions (condition 6, corresponding to Example 2, condition 7) preserve the expression of total SGK1 and phosphorylated SGK1, molecules associated with mTORC2. Condition 6 further illustrates this property of optimal RAPA-T cell products with a significant reduction in mTORC1 and a relative preservation of mTORC2, namely, a significant reduction in the mTORC1-associated subunit molecule Raptor and a relative preservation of the mTORC2-associated subunit molecule Rictor.

[0299] Example 4

[0300] Steady-state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were analyzed using GE's [technology / method / technology]. Enrichment was performed using an automated Ficoulcan process on the instrument. The lymphocyte-enriched cell population was then plated in G-REX culture vessels under two conditions (one corresponding to condition 7 in Table 1 and the other to condition 9 (T-RAPA) in Table 1) and incubated in culture for 6 days as in Example 2. After 6 days of culture, T cells were harvested and cultured at 1 × 10⁻⁶ cells / day. 6Re-plate at a concentration of 1 cell / mL to produce a 24-hour supernatant. During re-platening, T cells were co-stimulated with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio of 3:1, 1:1, 1:3, or 1:9. At each of these ratios, 24-hour supernatant was generated with no cytokines added (indicated by the "-" symbol), added rhuIL-2 (100 IU / mL, indicated by "+IL-2"), added rhuIL-7 (10 ng / mL, indicated by "+IL-7"), added rhuIL-15 (10 ng / mL, indicated by "+IL-15"), or both added simultaneously (indicated by "+IL-7+IL-15"). IL-2 and TNF-α secretion in cells was measured using known methods according to the manufacturer's instructions (Luminx). Results are shown in... Figures 27A-27B middle.

[0301] Early-differentiated CD4+ and CD8+ T cells (such as naive, central memory, or stem cell central memory subsets) may be beneficial for adoptive transfer. These early-differentiated T cells are functionally characterized in part by their differential responses to key homeostatic cytokines IL-7 and IL-15. Thus, the ability of a given T cell product to respond to IL-7 and IL-15 is a desirable property.

[0302] Figure 27A The figure below shows the IL-2 secretion profile of the optimal RAPA-T cell product, while the figure below shows the IL-2 secretion profile of the T-RAPA cell product. Under maximum co-stimulatory challenge and without the support of exogenous cytokines, the RAPA-T cell product secretes approximately 5 times more IL-2 than the T-RAPA cell product. Notably, even at very low co-stimulatory levels (a 1:9 pearl:T cell ratio), the RAPA-T cell product still secretes substantial amounts of IL-2; in stark contrast, this stimulation condition under T-RAPA conditions produces undetectable levels of IL-2. IL-2 secretion capacity has been associated with a beneficial helper-independent T cell phenotype, a cytokine secretion characteristic observed in early-differentiation T cells. Finally, the addition of IL-7 or IL-15 further enhanced IL-2 secretion capacity under RAPA-T conditions but not under T-RAPA conditions. Thus, RAPA-T cells respond uniquely to the homeostatic cytokines IL-7 and IL-15.

[0303] Figure 27BThe figure below shows the TNF-α secretion profile of the optimal RAPA-T cell product, while the figure below shows the TNF-α secretion profile of the previous T-RAPA cell product. Under maximal co-stimulatory challenge and without the support of exogenous cytokines, RAPA-T cells secrete approximately the same amount of TNF-α as T-RAPA cells. However, the addition of IL-7 or IL-15 to the co-stimulatory response results in a higher TNF-α secretion capacity under RAPA-T conditions compared to T-RAPA conditions. Thus, RAPA-T cells exhibit a unique response to the homeostatic cytokines IL-7 and IL-15 in inducing the secretion of the Th1 / Tc1 effector cytokine TNF-α.

[0304] Example 5

[0305] Human T cells were co-stimulated without the use of any inhibitors (“standard”; with the addition of anti-CD23 / anti-CD28 magnetic beads; bead:T cell ratio of 3:1). Alternatively, T cells were co-stimulated according to RAPA-T cell conditions (“rapamycin-treated”) and via anti-CD3 / anti-CD28 magnetic beads (“Dynabeads”; bead:T cell ratio of 1:3) or via Soluble co-stimulatory microparticles (Biotechnology Company; use at 20% of the manufacturer's recommended dose) Each 1×10 6 50 μL per cell (Storage solution). After 6 days of culture, T cells were harvested and adjusted to 1×10⁻⁶. 6 Cells / mL were co-stimulated with anti-CD3 / anti-CD28 beads (bead:T cell ratio of 3:1). Supernatant was then collected after 24 hours, and IL-2, TNF-α, and IL-13 levels were measured by Luminex assay (results are shown as 1 × 10⁻⁶ cells / mL per 24 hours). 6 (pg per cell per mL). Using the same protocol, cells were harvested after 6 days of culture and flow cytometry was performed to assess the expression of cell surface markers (including CD4, CD8, CD25, and CTLA4).

[0306] Figure 28 The secretion data for IL-2, TNF-α, and IL-13 are shown. Figure 28 The image shows a mixture of anti-CD3 / anti-CD28 nanoparticles (“Dynabeads”) and soluble anti-CD3 / anti-CD28 microparticles (…). The results were similar between co-stimulated RAPA-T cells (reagent; biotechnology company). As previously described, these cells possess a Th1 cytokine profile, as demonstrated by the abundant secretion of IL-2 and TNF-α and the minimal secretion of the Th2 cytokine IL-13.

[0307] Figure 29 The frequency of cells expressing cell surface markers CD4, CD8, CD25, and CTLA4, as measured by flow cytometry, is shown. Figure 29 The image shows a mixture of anti-CD3 / anti-CD28 nanoparticles (“Dynabeads”) and soluble anti-CD3 / anti-CD28 microparticles (…). The results were similar between co-stimulated RAPA-T cells (reagent; biotechnology company). As previously described, these cells were quiescent (as indicated by reduced CD25 expression) and had reduced expression of checkpoint inhibitory receptors (as indicated by reduced CTLA4 expression).

[0308] Example 6: Phase III Randomized Clinical Trial of Rapa-T Cell Therapy Figure 30 The stochastic third-phase protocol scheme is described. Figure 30 In the diagram above, for patients randomly selected to receive Rapa-T therapy, the autologous cells used for Rapa-T cell production will be derived from steady-state apheresis blood components collected after randomization. The immune depletion regimen will consist of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). The first PC cycle will be administered alone (without T-cell therapy) at the start of the study during the Tl.Rapa production interval and will last for 28 days; PC cycles two, three, four, and five will be performed before each of the four Tl.Rapa cell infusions, and will last for 35 days. Figure 30 The diagram below depicts the extended T1.Rapa cell therapy used to stabilize disease. For T1.Rapa cell recipients whose disease is stable after cycle 4, another batch of T1.Rapa cells will be created to allow up to four additional T1.Rapa cell therapy cycles (cycles 6 through 9).

[0309] Figure 30 The manufactured T-cell therapy is detailed and will be administered to all patients randomly assigned to a manufactured T-cell cohort. This therapy will involve: (1) collecting immune cells as substrates for the manufacture of the manufactured T-cells, said immune cells being obtained from a previously harvested peripheral blood stem cell transplantation procedure or a fresh steady-state apheresis procedure; (2) enriching the monocyte population and subsequently incubating the monocytes under manufactured T-cell culture conditions; (3) cryopreserving a single-use manufactured T-cell therapy product, said product undergoing identity and functional verification steps; (4) patients receiving treatment on a pentostatin plus cyclophosphamide regimen (PC regimen) will be isolated first and then combined with the manufactured T-cell therapy to prepare patients for manufactured T-cell therapy and directly promote antitumor effects; and (5) dedicated immune surveillance during and after treatment.

[0310] The immune depletion regimen will consist of pentostatin and low-dose, dose-adjusted cyclophosphamide (PC regimen). The first PC cycle will be administered alone (without T-cell therapy) at the start of the study during the manufactured T-cell production interval and will last for a minimum of 28 days to allow for recovery of blood cell counts. PC cycles two, three, four, and five will be administered before each of the four manufactured T-cell infusions, and will last for a minimum of 35 days to allow for recovery of blood cell counts. Maintenance therapy will cease after cycle five of manufactured T-cell therapy. Depending on clinical circumstances, treatment cycles may be longer than the specified intervals, extending indefinitely. As an example, and not a limitation, cycles may be delayed if the patient is in remission until evidence of disease relapse is received. Furthermore, additional maintenance cycles of the PC regimen plus adoptive manufactured T-cell therapy are envisioned to maintain the patient in remission, perhaps with 1 to 4 treatment cycles per year, or to treat disease relapse (if it occurs).

[0311] like Figure 30 As indicated, for patients whose disease has stabilized after four cycles of treatment, additional Rapa-T cell production can be performed, thereby promoting the potential therapy through additional cycles (up to a total of nine Rapa-T cell therapy cycles).

[0312] Figure 31 The details of the PC chemotherapy regimen are described in detail. Each cycle of PC therapy will begin with an infusion of Tl.Rapa cells on day 15 of the cycle (Tl.Rapa dose: between 0.1 cells / kg and 5 × 10⁻⁶ cells / kg). 6 The 14-day course consists of cells / kg. For cycle 1, pentostatin (P) will be administered at 4 mg / m² on days 1, 4, 8, and 11. 2 The dosage of pentostatin will be administered intravenously; cyclophosphamide (Cy) will be administered at a dose of 200 mg daily from day 1 to day 5 and from day 8 to day 12. For subsequent cycles, the dose of pentostatin will be reduced to 2 mg / m². 2 .

[0313] Figures 32A-32C The nature of the control group was detailed, namely, subjects who were not randomized to receive Rapa-T cell therapy would receive one of three FDA-approved triple regimens applicable to subjects with second or third relapses of MM, namely: the DPd regimen ( Figure 32A ); DRd scheme ( Figure 32B ); or KRd scheme ( Figure 32C ).

[0314] for Figures 32A-32CThe control group described herein will be recruited and subsequently randomized at the second or third relapse of multiple myeloma (MM). Patients must be candidates receiving one of three FDA-approved regimens for this patient population. Patients randomized to the control group will receive the DPd, DRd, or KRd regimen using published standard regimens. Specific aspects of these standard regimens have been indicated in the section above: [ Figure 32A DPd scheme; Figure 32B ]DRd scheme; and [ Figure 32C KRd scheme.

[0315] The primary objective of the statistical evaluation of the efficacy of manufactured T cells will be to compare progression-free survival (PFS) in recipients of manufactured T cell therapy relative to those randomly assigned to standard-of-care therapy. Secondary objectives will be assessed using descriptive statistics. Eligible patients with second or third relapsed MM will be randomized 1:1 to receive standard-of-care therapy with FDA-approved triple therapy consisting of DPd, DRd, or KRd, or to receive adoptive T cell therapy using ex vivo manufactured autologous anti-rapamycin Th1 / Tc1 cells (manufactured T cells). N = 65 evaluable patients will be included in each cohort. The primary study objective is to determine whether the manufactured T cell cohort has an increased PFS compared to patients receiving standard-of-care therapy.

[0316] Progression-free survival (PFS) and overall survival will be estimated in both groups using the Kaplan-Meier method and presented as pointwise 95% confidence intervals. Nonparametric estimates of median survival and its 95% confidence intervals can be derived by inverting the Kaplan-Meier estimates. The primary power outcome (PFS) will be tested using a one-sided log-rank test. A final analysis will be performed when 130 PFS events occur in the study, or when the recruitment target is reached and all patients have been followed for at least 12 months. Overall survival (OS) will be measured from the start of treatment; death from any cause will be considered an event and will be examined on the date of last contact.

[0317] Secondary endpoints will be assessed in a descriptive manner, such as mean, standard deviation, confidence interval, Kaplan-Meil analysis, or other methods characterizing response status in multiple myeloma. Objective response rate (ORR) and minimum residual disease (MRD) will be estimated for the observed patient population with corresponding outcomes and presented with 95% confidence intervals.

[0318] Demographic and baseline data will be descriptively summarized. Categorical data will be presented as frequencies and percentages, while continuous data will be presented using summary statistics such as mean, median, and standard deviation. Particular attention will be paid to identifying prior treatments for multiple myeloma and the degree of refractory to any single drug used.

[0319] Two interim analyses, potentially terminating due to invalidity, will be performed when a total of 28 and 55 PFS events occur (combining the two groups). A β error rate deduction method with a quadratic error rate deduction function will be used, a compromise between the O'Brien-Fleming method and the Pocock bounds. The null hypothesis acceptance bounds for the two interim analyses and one final analysis are shown below. These values ​​can be modified using the error rate deduction function if the timing of the interim analyses is changed.

[0320]

[0321] The table below shows the probability of early termination of the trial due to invalidity as a function of the true effect size:

[0322]

[0323] Collect immune cells as substrates for the production of T cells.

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

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

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

[0327] Subsequent iterations of manufactured T-cell therapies could become more efficient, allowing for the initiation of manufacturing with a smaller amount of input cells. Such improved methods would involve at least in part improved host preparation and improved manufacturing processes. In such methods, it would be possible to manufacture T-cells using starting material obtained from a single blood draw of 500 mL or less.

[0328] Manufacturing of T cells

[0329] In the case of previously collected cell products, such cryopreserved cells will be stored in the gas phase of liquid nitrogen until the cells are thawed and the production of T cells is completed. In the case of freshly isolated cells obtained by apheresis or by simple blood collection in the future, the cells will be processed immediately and can then be placed directly into cultures, or they can be cryopreserved using controlled-rate freezing technology and stored in the gas phase of liquid nitrogen for later use.

[0330] T cell culture from cryopreserved cell substrates derived from freshly isolated cell populations requires some form of T cell enrichment. By way of example, and not limitation, T cell enrichment can be achieved using monoclonal antibodies and column chromatography techniques (positive or negative selection). Enrichment of the original cell material used in the manufacture of manufactured T cells does not require such antibody-based methods, as T cells are efficiently enriched during the culture interval; thus, this method aligns with global recommendations for effective cell therapy. The initial processing steps for manufactured T cells focus on the removal of dimethyl sulfoxide (DMSO) used in the cryopreservation step (where applicable), lysis of red blood cells (RBCs), and centrifugation to remove contaminating granulocytes and, to some extent, monocytes. These steps are performed in a relatively automated method that primarily utilizes closed-system technology. This procedure is advantageous because it reduces human error, provides detailed manufacturing data for batch records, improves consistency throughout the manufacturing process, and reduces the risk of contamination of the final product with infectious agents. The processing of the manufactured T-cell products combines the following steps: (1) Thawing the cryopreserved products (where applicable) using a solid-state, non-aqueous method to reduce infectious agent contamination, such as Triana E, Ortega S, Azqueta C et al., “Thawing of cryopreserved hematopoietic progenitor cells from apheresis will be with using a new drywarming device”. Transfusion. 2013; 53(l):85-90; (2) Automated washing of cell products using a LOVO permeable membrane device, such as 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”. (3) During the LOVO washing step, RBC lysis was integrated using potassium ammonium chloride (ACK) buffer, 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; (4) Cell content was reduced by volume using the LOVO method, followed by plated cells in a closed-system counter-current centrifugation (CCE) apparatus (Erutra; Terumo), as previously described in Stroncek DF, Fellowes V, Pham C et al., "Counter-flowelutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies." Journal of Translational Medicine (J The description in TranslMed.)》2014;12:241.(doi):10.1186 / sl2967-12014-10241-γ; and (5) the pre-programmed operation of the Erutra device for the efficient removal of granulocytes and monocytes via CCE.

[0331] After lymphocyte enrichment and culture medium purification, cells were seeded into a dedicated chamber with abundant oxygen exchange capacity (G-Rex container; Wilson & Wolfe), as described in Bajgain P, Muchala 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 permeability, the G-Rex container is a closed-system unit and offers the additional advantage of automated closed-system media volume reduction (GatheRex liquid handling pump). Lymphocyte-enriched cells were held in the G-Rex container for 6 days.

[0332] Several specific culture conditions were used to promote CD4+ expression in G-Rex containers using the functional properties of manufactured T cells. + and CD8 + Preparation of T-cell mixtures. These specific conditions included: (1) the use of enriched medium further supplemented with 5% human serum (including but not limited to X-Vivo 20; Lonza; the medium may also be further supplemented with 5% AB serum), Zhang HD, Song ZL, Li WP. [In vitro cultivation of dendritic cells with serum-free medium], Chinese Journal of Experimental Hematology. 2006; 14(5):985-989; Lonza); (2) a 16-hour interval between seeding to G-Rex plates prior to co-stimulation (at 1.5 × 10⁻⁶ cells per mL). 6(3) During this initial rest, cells are optimally rested by adding the monoclonal antibody baliximab (which blocks the IL-2 receptor and thereby prevents autonomous T cell activation by endogenously produced IL-2) and tesimolimus (a pharmacological inhibitor of mTORC1); (4) After this 16-hour rest, under suboptimal conditions, 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 (typically, most T cell expansion conditions utilize a 9-fold higher level of co-stimulation, i.e., a bead-to-T cell ratio of 3:1); (5) Importantly, it is crucial not to wash T cells after the initial rest; (6) After the rest, in addition to adding 3 / 28 beads, it is crucial to add the polarizing cytokine IFN-α at a high dose (10,000 IU / ml) to promote differentiation into CD4+. + Th1 and CD8 + Tc1 phenotype; (7) Importantly, it is crucial to avoid adding IL-2, which is a common additive for T cell culture; and (8) After adding bead and IFN-α, it is important to keep the cells undisturbed until harvest on day 6 of culture (without cell washing and no further culture additives).

[0333] Cryopreservation of manufactured T-cell products and verification of their identity and function

[0334] After 6 days of cell culture in G-Rex containers, the culture volume will be reduced using a GatheRex instrument in a closed system. Cells will then be harvested, 3 / 28 beads will be removed using a handheld magnet, and the cells will be placed in a LOVO device for continuous washing to remove >99% of the culture additives (tesimolimus, balithimab, IFN-α).

[0335] The washed cells will be reconstituted into a cryopreservation medium containing 5% DMSO and 5% starch pentamericate. Cryopreservation will be performed in multiple single-use aliquots in 50 ml cryopreservation bags. The dose of T cells produced will be between 0.1 T cells per kilogram of recipient body weight and 5 × 10⁻⁶ cells per 500 ml cryopreservation bags. 6Between individual T cells. Four disposable aliquots will be cryopreserved to allow for four consecutive infusions of the manufactured T cells, approximately once a month. The manufactured T cells will be cryopreserved using 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: Official Organ of the German Society for Transfusion Medicine and Immunohistology, 2011; 38(2):107-123, and; and after the manufactured T cells pass the specified release criteria tests, the cells will be transported in the gas phase of liquid nitrogen by a certified cryopreservation transporter.

[0336] The standard test for the release of manufactured T cells includes standard tests such as CD3. + CD4 + and CD8 + T cell purity (by flow cytometry, final product CD3 content) + T cell count must be >70%; CD4 + and CD8 + Subpopulations must be present at a level of 5% (each subpopulation must be present). As determined by flow cytometry assays for annexin and 7-AAD, >70% of the cells must be viable. Furthermore, the cells must be free from bacterial and fungal contamination for a minimum of 3-day culture interval (ideally 14-day); additionally, cell products must be below the detection limits for bacterial LPS endotoxin and mycoplasma.

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

[0338] Main preparation for using the PC solution

[0339] Patients receiving manufactured T-cell therapy receive the pentostatin plus cyclophosphamide (PC) regimen. Cycle 1 of the PC regimen is administered during the manufacture of manufactured T cells and therefore can be administered without accompanying T-cell infusion. Cycle 1 is advantageous on two levels: first, it reduces the number and function of host regulatory T cells and late-senescent effector T cells, thereby enhancing future cycles of manufactured T-cell therapy; and second, it directly mediates antitumor effects against multiple myeloma, thereby controlling the disease during the manufacture interval. Following cycle #1, subsequent cycles of the PC regimen will be followed by adoptive transfer of manufactured T cells one day after the two-week PC regimen interval (day 15). These cycles of the PC regimen are additionally advantageous because they further modulate host biology, including increasing T-cell homeostatic cytokines such as IL-7 and IL-15, which will allow for the promotion of manufactured T-cell expansion after adoptive transfer.

[0340] The PC regimen involves the infusion of manufactured T cells. Each cycle of PC therapy will consist of a 14-day process preceding the infusion of manufactured T cells on day 15 of the cycle. The dose of manufactured T cells will be between 1 cell / kg and 5 × 10⁻⁶ cells / kg. 6 Between 1 × 10⁶ cells / kg, including those between 1 × 10⁶ cells / kg.5 cells / kg and 5×10 6 The T-cell dose was between [number] cells / kg. Pentostatin (P) was administered at a dose of 4 mg / m² on days 1, 4, 8, and 11. 2 The dosage (intravenous); cyclophosphamide (Cy) will be administered at a dose of 200 mg daily from day 1 to day 5 and from day 8 to day 12.

[0341] Preoperative medication and prehydration are required before administering pentostatin. Prehydration will be performed using 1 liter of 0.9% sodium chloride 60 minutes before pentostatin administration. Preoperative medication with antiemetics is required. The recommended antiemetic regimen is as follows: (1) Dexamethasone 12 mg intravenously 60 minutes before each pentostatin administration (i.e., day 1, day 4, day 8, and day 11 of the cycle); (2) In addition, oral dexamethasone may be administered as needed on other days at a dose of 4 mg daily; (3) Ondansetron 8 mg intravenously 60 minutes before each pentostatin administration; (4) For the remainder of treatment, ondansetron may be administered as needed every 12 hours at a dose of 8 mg (tablets); and (5) For patients whose nausea and vomiting are not controlled, aprepitant may be added to the antiemetic regimen as needed. The pentostatin dose will be 4 mg / m². 2 Each pentostatin dose will be administered intravenously over 30-60 minutes.

[0342] The dosage of pentostatin will be adjusted, and the dose of pentostatin administered to patients will be between 1 mg / m². 2 -4 mg / m 2 The dosage of pentostatin will be adjusted based on creatinine clearance (CrCl), which can be obtained from 24-hour urine or calculated using the Cockcroft-Gault formula. If a subject's creatinine level increases during pentostatin and cyclophosphamide therapy, the subsequent dose will be modified as follows: CrCl > 60 mL / min / 1.73 m² → 4 mg / m² 2 Pentostatin (full dose); CrCl < 60 mL / min / 1.73 m² but > 30 mL / min / 1.73 m² → reduce the dose by 50% to 2 mg / m². 2Administer pentostatin; and discontinue pentostatin administration if CrCl < 30 mL. Pentostatin is rarely associated with organ toxicity, such as neurological toxicity (seizures, coma) or cardiotoxicity (reduced ejection fraction). Therefore, special attention should be paid to evaluating any organ toxicity that occurs during PC therapy. If pentostatin is associated with any grade 2 or higher organ toxicity, the institutional PI should be contacted to discuss whether further pentostatin treatment and further treatment regimens are needed.

[0343] Oral cyclophosphamide (Cy) will be used as part of the PC regimen, with the dose of cyclophosphamide ranging from 50 to 400 mg. During PC regimen cycles one through five, the dose of Cy will be 200 mg daily on days 1-5 and days 8-12. Intravenous infusion of 200 mg cyclophosphamide is also permitted for tolerability issues or financial considerations. Due to cyclophosphamide cystotoxicity, adequate hydration must be maintained during the PC regimen. Patients should drink at least 2 to 4 liters of fluid daily to maintain clear urine color.

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

[0345] Variations of the PC regimen are envisioned. First, pentostatin and cyclophosphamide have a synergistic effect in their immune depletion and immunosuppressive effects; this synergy may also exist in their antitumor effects, although little information is available about this possibility. Thus, the PC regimen is envisioned as a standalone therapy for cancer treatment (including solid tumors); in a previous instance, patients with refractory mesothelioma receiving a combination regimen consisting partly of the PC regimen experienced unprecedented antitumor benefits. Second, due to this synergistic effect, it is advantageous to administer both drugs simultaneously via intravenous infusion, preferably by mixing pentostatin and cyclophosphamide in the same intravenous infusion bag, to facilitate administration and reduce pharmacy errors. In such applications, it is important to provide options for the PC mixture, which should cover a variety of clinically relevant pentostatin to cyclophosphamide ratios.

[0346]

[0347]

[0348] Manufacturing T-cell infusion

[0349] Preoperative medication is required before administering any manufactured T cells. Diphenhydramine (25 mg to 50 mg intravenously or orally) and acetaminophen (650 mg orally) are administered 30–60 minutes prior to the infusion of the manufactured T cells.

[0350] The manufactured T-cell infusion will occur on day 15 of cycles two through five; however, for logical reasons, the manufactured T-cell infusion may be delayed by up to 3 days. Furthermore, for logical reasons, a delay of up to 4 weeks is permitted, as well as to allow for toxicity recovery. The manufactured T-cell dose will be 5 × 10⁻⁶. 6 Cells / kg; however, if a suboptimal cell yield occurs during manufacturing, a yield as low as 0.1 × 10⁻⁶ cells / kg is permissible. 6 The dosage is 1 cell / kg. The cryopreserved manufactured T cells are thawed and administered intravenously immediately and rapidly (within 30 minutes) by gravity, according to the appropriate institutional SOP for the administration of blood products. This T-cell infusion will be performed in an outpatient setting unless there are unforeseen circumstances requiring hospitalization. Steroids are not permitted for the management of DMSO-related toxicities (chills, muscle aches) that may occur immediately after cell infusion unless life-threatening toxicity is deemed possible.

[0351] It can be imagined that below 0.1×10 6 A smaller amount of manufactured T-cell infusion per kg of cells may also be clinically relevant (by way of example and not limitation, a lower logarithm, in the range of 1×10⁻⁶ cells / kg).5 (cells / kg). First, manufacturing will be optimized to produce manufactured T cells that mediate further enhanced in vivo effects, thereby reducing the required T cell dose; this will be advantageous, partly because T cell collection can be achieved through a simple blood draw and partly because of the improved manufacturing feasibility. Second, as detailed above, with further improvements to the PC protocol, adoptive transfer of manufactured T cells will have a further improved in vivo selectivity advantage relative to host cells, thereby effectively reducing the required manufactured T cell dose.

[0352] Specialized immune monitoring during and after treatment

[0353] Peripheral blood mononuclear cells and bone marrow cells will be fed into the Rapa therapeutic agent to enable immune surveillance assays; these assays aim to explore the mechanism of action of the manufactured T-cell therapy and to develop biomarkers that will predict the efficacy of the manufactured T cells. In one effort, the ability of the manufactured T-cell receptor to produce a variety of Th1 and Th2 cytokines in response to a variety of stimuli, including autologous multiple myeloma tumor cells or known or suspected tumor antigens, such as molecules in the cancer-testis antigen (CTA) family. The CTA gene family is numerous and has been shown to be associated with multiple myeloma; since the sequences of CTA genes are known and the association of specific CTA genes has been characterized in multiple myeloma, it can be demonstrated that the manufactured T-cell therapy can specifically induce T-cell-mediated immunity against a range of CTA antigens. Such cytokine responses can be measured using RNA expression analysis, secretion analysis via ELISA or Luminex multianalyte methods, flow cytometry, or ELISPOT assays. Antigen-specific immunity can also be quantified using antibody production assays or cell lysis assays.

[0354] The evaluation will assess whether T cells obtained after manufacturing T-cell therapy exhibit enhanced responsiveness to autologous multiple myeloma cells compared to T cells obtained before manufacturing T-cell therapy. One obstacle to this effort is that the propagation of patient-specific multiple myeloma cell lines is often unsuccessful. To overcome this obstacle, the following will be used to propagate myeloma cells: specialized containers, such as those 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 culture media supplemented with a combination of factors known to enhance multiple myeloma proliferation and survival, including IL-6, CD40 ligand, and carfilzomib resistance. Patient-specific multiple myeloma cells can be used alone as a stimulant in assessing the antitumor responsiveness of immune T cells; alternatively, such tumor cells can be induced into an apoptotic state and their contents loaded into specialized antigen-presenting cells, which can be manufactured from patient-specific mononuclear cells collected during the manufacturing of manufactured T cells.

[0355] Furthermore, it is envisioned that T-cell receptor (TCR) immune repertoire analysis could be used as a biomarker for manufactured T-cell therapies. Preferably, such repertoire analysis would be performed via RNA sequencing rather than the more commonly used DNA sequencing. Unlike most targeted T-cell therapies, manufactured T-cell therapies are polyclonal because the manufacturing process does not preferentially transfer T-cell reactivity to any particular tumor antigen. Thus, any beneficial antitumor effect expected after manufactured T-cell therapy stems from clonal expansion to multiple tumor antigens in vivo; given this biology, successful 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 that deactivates checkpoint inhibition, successful therapies are associated with the emergence of novel TCR clone specificities, referred to as TCR repertoire shifts that can be determined by quantifying the Morisito index (Robert L, Harview C, Emerson R, et al., “Distinct immunological mechanisms of CTLA-4 and PD-1 blockade revealed by analyzing TCR usage in blood lymphocytes”, *OncoImmunology*, 2014; 3:e29244). Similarly, with successful manufacturing of T-cell therapies, there will be TCR repertoire shifts; the persistence of these shifts beyond the manufacturing T-cell therapy interval will be consistent with long-term T-cell immunity against malignancies. With advancements in manufacturing, improved forms of manufactured T cells will emerge; in this case, TCR shifts will be more widespread, occur with fewer treatment cycles, and be more persistent across treatment intervals.

[0356] Inclusion criteria for multiple myeloma treatment regimens

[0357] Male or female patients older than 18 years may be eligible for manufactured T-cell therapy. There is no formal upper age limit. However, patients 65 years of age or older with a history of cardiovascular symptoms or symptomatic care (even if they do not fully meet the exclusion criteria detailed below) should be evaluated by a cardiologist in a multicenter setting. Such subjects will then be considered on a case-by-case basis. The overall patient performance status should be at least moderately good health, quantified as ECOG performance status <2.

[0358] The patient must be definitively diagnosed with multiple myeloma through histological or cytological studies. Furthermore, the disease must be symptomatic, and the patient must be experiencing a second or third relapse after receiving medication from the following classes: proteasome inhibitors, immunomodulatory drugs, alkylating agents, CD38 monoclonal antibodies, and glucocorticoids.

[0359] Patients experiencing a second or third relapse are at a relatively late stage of the disease. However, by demonstrating the safety and efficacy of manufactured T-cell therapy, it is conceivable that patients with multiple myeloma at an earlier stage within the overall therapeutic framework will benefit from it. By way of example, and not limitation, manufactured T-cell therapy could be used as an alternative to high-dose chemotherapy combined with autologous hematopoietic cell transplantation, and could also be used for a large number of patients ineligible for transplantation. Furthermore, it is conceivable that manufactured T-cell therapy could be administered during early detection at the earliest stage of multiple myeloma progression, before clinical symptoms appear, i.e., during the accumulating disease phase.

[0360] On the other hand, it is conceivable that the Rapa-T cell therapy described herein would be applicable to the treatment of patients with more advanced stages of multiple myeloma and those with highly refractory disease. Specifically, Rapa-T cell therapy can be used to treat pentadal refractory MM, defined as patients with relapsed MM who are refractory to five of the first few drugs used to treat MM, namely: lenalidomide, pomalidomide, bortezomib, carfilzomib, and daratumumab.

[0361] Since there are no standard treatment options for patients with pentadal refractory multiple myeloma (MM), the clinical protocol for evaluating Rapa-T cell therapy in this case will be a single-arm phase II study, similar to the previous study 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, to evaluate a novel anticancer drug. For this phase II study, Rapa-T cell therapy will be administered, such as… Figure 30 , 31 As described in 32A-32C. The statistical objective of the study will be to determine whether Rapa-T cell therapy can induce a significant, at least partial, remission rate in refractory MM, as defined by a rate consistent with at least 30%.

[0362] There must be a potential source of autologous T cells sufficient to manufacture the T cells. Specifically, the patient must have a sufficient number of previously cryopreserved PBSC units (composed of >2 million cells / kg CD34) available for manufacturing. + Total content (defined as ALC) or a sufficient number of circulating T cells (defined by greater than 300 cells per microliter) that can be collected by steady-state apheresis.

[0363] Patients must be at least two weeks post-multiple myeloma therapy, major surgery, radiation therapy, or participation in other investigative trials, and must have recovered from clinically significant toxicities of these prior treatments (CTCAE toxicity resolution of 2 or lower). Cardiac ejection fraction (EF) as determined by MUGA or 2D echocardiography must be within institutional limits, with an EF level of at least 40%. Renal function, as measured by serum creatinine, must be less than or equal to 2.5 mg / dL. Liver function must be adequate, as measured by AST and ALT less than or equal to 3 times the upper limit of normal and total bilirubin less than or equal to 1.5 mg / dL (unless due to Gilbert's disease). Lung function must be adequate, as defined by a corrected DLCO greater than or equal to 50% of the expected value in a pulmonary function test. There must be no history of abnormal bleeding tendencies. Voluntary written consent must be obtained before any investigation-related procedures not included in standard medical services, and patients should understand that they can withdraw their consent at any time without affecting future medical services.

[0364] Randomized Phase III Trials: Standards of Care

[0365] To demonstrate the benefits of the manufactured T-cell therapy, randomized studies will be conducted to formally compare the results of the manufactured T-cell therapy with standard of care consisting of a DPd, DRd, or KRd regimen; the standard of care will be administered according to the literature, based on its FDA-approved status for patients with second or third relapses of MM.

[0366] Example 7

[0367] Steady-state apheresis was performed to obtain patient samples containing PBMCs. Lymphocytes in the samples were analyzed using GE's [technology / method / technology]. Enrichment was performed using an automated Ficoecher process on the instrument. The lymphocyte-enriched cell population was then plated in G-REX culture vessels and cultured for 6 days in TexMACS medium (without serum supplementation; without IL-2 supplementation) containing IFN-α, tesiromoximide, and balithimab. At the end of the fabrication, the resulting Th1 / Tc1 cells were exposed to pancreatic cancer cells (MIA-Paca2 cell line) or to lung cancer cells (H23 cell line) that had undergone apoptosis through exposure to etoposide. The cells were pulsed with this tumor lysate and then cultured in IL-2 (200 IU / mL) for 7 days, with a second exposure to the tumor lysate at day 7. After a further 7-day culture interval in IL-2-containing medium, a third exposure to the tumor lysate was performed, and the cytokine content of the resulting 24-hour supernatant was measured using a Luminex assay (results expressed as pg / mL / 1×10⁻⁶). 6 (Per cell / 24 hours shown). Results of cytokine assays are in Figure 34A -B is shown. Condition A indicates a pulse with the second-best tumor lysate formulation; Condition B indicates the optimal formulation of tumor lysate. "<" indicates a value below the detection limit.

[0368] like Figure 34A As shown in -B, RAPA-T cells can be further characterized by their ability to respond to tumor cells containing solid tumors, such as pancreatic cancer cells and lung cancer cells. Figure 34A -B further demonstrates that RAPA-T cells can maintain a characteristic Th1 cytokine phenotype, as evidenced by high levels of IFN-γ and GM-CSF secretion and reduced secretion of Th2 cytokines IL-4 and IL-10.

[0369] In another experiment, at the end of the fabrication process, 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 through exposure to etoposide. The cells were pulsed with this tumor lysate and then cultured for 7 days in IL-7 (20 ng / mL) and IL-15 (10 ng / mL), with a second exposure to the tumor lysate at day 7. A third exposure to the tumor lysate was performed after a further 7-day culture interval in medium containing IL-7 and IL-15, and the cytokine levels in the resulting 24-hour supernatant were measured using a Luminex assay (results expressed in pg / mL / 1×10⁻⁶). 6(Cells / 24 hours shown). The control culture (“RAPA-201, tumor-free”) consisted of manufactured resistant Th1 / Tc1 cells that were multiplied in a medium containing IL-7 and IL-15 but not subjected to pulses of tumor lysis buffer. Results of cytokine assays were presented in... Figure 35 As shown in the image.

[0370] like Figure 35 As shown, RAPA-T cells can be further characterized by their responsiveness to tumor cells containing solid tumors, such as pancreatic cancer cells and lung cancer cells. This in vitro sensitization of solid tumor cell lines can be readily demonstrated by culturing and expanding the cells in media supplemented with IL-7 and IL-15, two homeostatic cytokines that have been shown to selectively drive effector functions of RAPA-T cells. As demonstrated by high levels of secretion of IFN-γ, GM-CSF, and TNF-α, RAPA-T cell cytokine secretion from tumor cells can maintain a characteristic Th1 cytokine phenotype.

[0371] like Figure 36 As shown, many cancers (such as renal cell carcinoma, liver cancer, lung cancer, bladder cancer, and stomach cancer) have been shown to respond to checkpoint inhibitor therapies, such as monoclonal therapies targeting checkpoint inhibitor molecules (such as PD-1 and CTLA4, which can induce remission in solid tumors). In further experiments, according to Simon's phase 2 design, patients (n=7) with renal cell carcinoma, lung cancer, liver cancer, stomach cancer, bladder cancer, and low-mutation PD-L1-negative or low-mutation-rate cancers will receive manufactured T-cell therapy. If any cohort has at least one responsive patient, the cohort will be expanded to a cohort of 20 patients.

[0372] Unbound by theory, Rapa-T cells are expected to provide therapeutic benefits in cancer because these cells have reduced or no checkpoint inhibitor receptors. It is suspected that some cancers may be unresponsive to certain treatments due to checkpoint inhibitor receptors other than PD1 and CTLA4. Therefore, it can be anticipated that, unbound by theory, Rapa-T cells could be effective in treating other cancers due to the lack of additional checkpoint inhibitor receptors.

[0373] Example 8

[0374] Rapa-T cells were created through a 6-day culture, and the resulting Felix Collins cell population was cultured in a medium containing tesimolimus (2 μM) and the anti-IL-2 receptor monoclonal antibody balithimab (30 μg / mL). After 24 hours, the culture was supplemented with IFN-α (20,000 IU / mL), but no IL-2 was added. No form of anti-CD3 / anti-CD28 co-stimulation was used in the culture.

[0375] In contrast, for the "control": the post-Felix Cole cell population was cultured in medium without tesimolimus and baliximab. On the day of culture initiation, cells were co-stimulated with anti-CD3 / anti-CD28 coated beads at a bead-to-T-cell ratio of 3:1. IL-2 (20 IU / mL) and IFN-α (20,000 IU / mL) were supplemented to the medium on the day of culture initiation. The mean fluorescence intensity (MFI) of BTLA, CTLA4, PD1, and TIM3 was measured by flow cytometry for the CD4+ and CD8+ T-cell subsets of the culture-injected population, Rapa-T cells, and control cells. The data are shown in Table 2 below. A reduction in checkpoint inhibitor receptor expression was observed between Rapa-T cells and control cells; for each checkpoint, the MFI was approximately the same for Rapa-T cells and culture-injected cells.

[0376] Table 2: Average Fluorescence Intensity

[0377]

[0378] Manufacturing Example:

[0379] 1. A method for producing manufactured T cells, the method comprising:

[0380] In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density.

[0381] Add IFN-α to the culture medium;

[0382] The T cells and culture medium are incubated for a certain period of time to produce the manufactured T cells;

[0383] Harvest the T cells produced.

[0384] 2. The method according to Example 1 further includes:

[0385] Additional culture medium was added to the T cells and the culture medium.

[0386] 3. The method according to Example 2, wherein the additional culture medium is added approximately 48 hours after the culture is inoculated into the cell population in the culture medium.

[0387] 4. The method according to any one of Examples 2 to 3, wherein the amount of additional culture medium added to the culture is sufficient to reduce the cell density of the cells in the culture to a target cell density, wherein the cell density of the culture input cell population at the time of inoculation is greater than 9 × 10⁻⁶. 6 100 cells / mL, and wherein the target cell density is approximately 9 × 10⁻⁶. 6 Cells / mL.

[0388] 5. The method according to Example 1, wherein anti-CD3 / anti-CD28 co-stimulation is not performed.

[0389] 6. The method according to Example 2, wherein when the culture input cell population is inoculated into the culture medium, the ratio of the amount of additional culture medium added to the culture to the amount of the culture medium is between 1:1 and 3:1.

[0390] 7. The method according to any one of Examples 1 to 6, further comprising, after harvesting the manufactured T cells:

[0391] At least a portion of the manufactured T cells are packaged in a package; and

[0392] The package containing the portion of the manufactured T cells is frozen.

[0393] 8. The method according to any one of Examples 1 to 7, wherein the culture medium does not contain IL-2 and no IL-2 is added to the culture medium.

[0394] 9. The method according to any one of Examples 1 to 8, wherein the IFN-α is added to the culture medium at approximately the same time as the inoculation of the cell population or within 24 hours of the inoculation of the cell population.

[0395] 10. The method according to any one of Examples 1 to 9, wherein the cell density is at least 1.5 × 10⁻⁶ cells per mL. 6 Each cell.

[0396] 11. The method according to any one of Examples 1 to 9, wherein the cell density is about 7.5 × 10⁻⁶ per mL. 6 Each cell.

[0397] 12. The method according to any one of Examples 1 to 9, wherein the cell density is about 30 × 10⁻⁶ per mL. 6 Each cell.

[0398] 13. The method according to any one of Examples 1 to 12, wherein the tesimolimus is present in the culture medium at a concentration of about 4.5 μM.

[0399] 14. The method according to any one of Examples 1 to 12, wherein the tesimolimus is added to the culture medium once or multiple times during the time period to maintain a desired concentration.

[0400] 15. The method according to Example 14, wherein the tesimolimus is added to the culture medium every 2 days during the said time period.

[0401] 16. The method according to any one of Examples 14 to 15, wherein the desired concentration is about 4.5 μM.

[0402] 17. The method according to any one of Examples 1 to 16, wherein the IL-2 signaling inhibitor is an anti-IL-2 receptor antibody or a fragment thereof.

[0403] 18. The method according to Example 17, wherein the IL-2 signaling inhibitor is balithimab or dacrolimus.

[0404] 19. The method according to any one of Examples 1 to 18, wherein the IL-2 signaling inhibitor is present in the culture medium at a concentration of 5 μg / mL to 50 μg / mL.

[0405] 20. The method according to any one of Examples 1 to 19, wherein the IFN-α is added to the culture medium to a concentration of 1,000 IU / mL to 10,000 IU / mL.

[0406] 21. The method according to any one of Examples 1 to 20, wherein the time period is about 4 days to about 8 days.

[0407] 22. The method according to any one of Examples 1 to 20, wherein the time period is 6 days.

[0408] 23. The method according to any one of Examples 1 to 22, wherein the culture medium is substantially free of serum.

[0409] 24. The method according to any one of Examples 1 to 23, wherein serum is not added to the culture medium.

[0410] 25. The method according to any one of Examples 1 to 22, wherein the culture medium further comprises 5% human serum.

[0411] 26. The method according to any one of Examples 1 to 25, wherein the culture medium comprises TexMACS culture medium.

[0412] 27. The method according to any one of Examples 1 to 26, wherein the culture input cell population comprises T cells comprising no more than 66% of the total number of cells in the culture input cell population.

[0413] 28. The method according to any one of Examples 1 to 26, wherein the culture input cell population comprises T cells comprising about 50% to about 95% of the total number of cells in the culture input cell population.

[0414] 29. The method according to any one of Examples 1 to 28, wherein the culture input cell population further comprises mononuclear cells.

[0415] 30. The method according to any one of Examples 1 to 29, further comprising:

[0416] Samples including T cells were harvested from the subject; and

[0417] T cells were isolated from the sample to generate the culture input cell population.

[0418] 31. The method according to Example 30, wherein the culture input cell population comprises T cells comprising approximately 99% or more of the total number of cells in the culture input cell population.

[0419] 32. The method according to any one of Examples 30 to 31, wherein the T cells are isolated by antibody-based purification.

[0420] 33. The method according to any one of Examples 1 to 29, further comprising:

[0421] Samples including T cells were harvested from the subject; and

[0422] T cells were enriched in the sample to generate the culture input cell population.

[0423] 34. The method according to Example 33, wherein the enrichment is performed by countercurrent centrifugation or a Fico process.

[0424] 35. The method according to any one of Examples 33 to 34, wherein the culture input cell population comprises T cells comprising approximately 70% of the total number of cells in the culture input cell population.

[0425] 36. The method according to any one of Examples 1 to 29, further comprising, prior to seeding the culture comprising T cells from the subject into the cell population at a certain cell density in a culture medium:

[0426] The culture was harvested from the subject and fed into a cell population.

[0427] 37. A manufactured T cell produced by the method according to any one of Examples 1 to 36.

[0428] 38. A method for producing manufactured T cells, the method comprising:

[0429] In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density.

[0430] The culture was introduced into the cell population and incubated with the culture medium for a first time period without co-stimulation of the culture with anti-CD3 / anti-CD28.

[0431] After incubation for the first time period, anti-CD3 / anti-CD28 coated magnetic beads were added to the T cells and culture medium at a bead:T cell ratio between 1:1 and 1:12 to stimulate the T cells.

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

[0433] The culture was incubated in the culture medium containing the anti-CD3 / anti-CD28 coated magnetic beads and IFN-α for a second time period to produce manufactured T cells;

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

[0435] Harvest the T cells produced.

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

[0437] At least a portion of the manufactured T cells are packaged in a package; and

[0438] The package containing the portion of the manufactured T cells is frozen.

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

[0440] 41. The method according to any one of Examples 38 to 40, wherein the IFN-α is added at or approximately simultaneously with the addition of the anti-CD3 / anti-CD28 coated magnetic beads.

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

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

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

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

[0445] 46. ​​The method according to any one of Examples 38 to 44, wherein the tesimolimus is added to the culture medium once or multiple times during the second time period to maintain a desired concentration.

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

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

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

[0449] 50. The method according to Example 49, wherein the IL-2 signaling inhibitor is balithimab or dacrolimus.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0463] Samples including T cells were harvested from the subject; and

[0464] T cells were isolated from the sample to generate the culture input cell population.

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

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

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

[0468] Samples including T cells were harvested from the subject; and

[0469] T cells were enriched in the sample to generate the culture input cell population.

[0470] 67. The method according to Example 66, wherein the enrichment is performed by countercurrent centrifugation or a Fico process.

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

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

[0473] The culture was harvested from the subject and fed into a cell population.

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

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

[0476] In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density.

[0477] The culture was introduced into the cell population and incubated with the culture medium for a first time period without co-stimulation of the culture with anti-CD3 / anti-CD28.

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

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

[0480] The culture was incubated in the culture medium containing nanoparticles containing anti-CD3 / anti-CD28 and IFN-α for a second time period to produce manufactured T cells;

[0481] Harvest the T cells produced.

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

[0483] At least a portion of the manufactured T cells are packaged in a package; and

[0484] The package containing the portion of the manufactured T cells is frozen.

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

[0486] 74. The method according to any one of Examples 71 to 73, wherein the IFN-α is added at or approximately simultaneously with the addition of nanoparticles containing anti-CD3 / anti-CD28.

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

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

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

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

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

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

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

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

[0495] 83. The method according to Example 82, wherein the IL-2 signaling inhibitor is balithimab or dacrolimus.

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

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

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

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

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

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

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

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

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

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

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

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

[0508] Samples including T cells were harvested from the subject; and

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

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

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

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

[0513] Samples including T cells were harvested from the subject; and

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

[0515] 99. The method according to Example 98, wherein the enrichment is performed by countercurrent centrifugation or a Fico process.

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

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

[0518] The culture was harvested from the subject and fed into a cell population.

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

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

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

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

[0523] 106. A manufactured population of T cells exhibiting a quiescent state, as indicated by T cells secreting low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacturing, such that, after stimulation with high levels of co-stimulation (a bead-to-T cell ratio of 3:1), the culture supernatant contains levels of 1 × 10⁻¹¹ cells per 24 hours. 6 Limited to cells <100 pg / ml.

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

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

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

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

[0528] The culture was harvested from the subject and fed into a cell population.

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

[0530] T cells, including those from the subject, are isolated to generate the culture input cell population.

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

[0532] The sample, including T cells, from the subject was enriched to produce the culture input cell population.

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

[0534] T cells, including those from the subject, are isolated to generate the culture input cell population.

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

[0536] The sample, including T cells, from the subject was enriched to produce the culture input cell population.

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

[0538] T cells, including those from the subject, are isolated to generate the culture input cell population.

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

[0540] The sample, including T cells, from the subject was enriched to produce the culture input cell population.

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

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

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

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

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

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

[0547] 123. A manufactured T cell population, characterized by a higher CD4 content compared to a control T cell population with characteristics of T cells used to generate said manufactured T cell population. + or CD8 + The corresponding frequency of T cells, CD4 + or CD8 + The frequency of TIM3 expression on T cells is reduced, as measured by flow cytometry.

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

[0549] 125. A manufactured population of T cells, characterized by being relative to CD4 + or CD8 + T-Rapa cells express the corresponding frequency of TIM3 and CD4. + or CD8 + The frequency of TIM3 expression on T cells is reduced, as measured by flow cytometry.

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

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

[0552] 128. A manufactured population of T cells, characterized by being relative to CD4 + or CD8 + T-Rapa cells express the corresponding frequencies of PD1 and CD4. + or CD8 + The frequency of PD1 expression on T cells is reduced, as measured by flow cytometry.

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

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

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

[0556] 132. A manufactured T cell population, characterized in that, relative to a control T cell population characterized by T cells that produce said manufactured T cell population, CD8+ is present in... + The corresponding frequency of T cells, CD8 + The frequency of 2B4 expression on T cells is reduced, as measured by flow cytometry.

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

[0558] 134. A manufactured population of T cells, characterized by being relative to CD4 + or CD8 + T-Rapa cells express the corresponding frequency of 2B4, CD4 + or CD8 + The frequency of 2B4 expression on T cells is reduced, as measured by flow cytometry.

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

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

[0561] 137. A manufactured T cell population, characterized in that, relative to a control T cell population characterized by T cells that produce said manufactured T cell population, CD4+ is present in... + or CD8 + The corresponding frequency of T cells, CD4 + or CD8 + The frequency of LAIR1 expression on T cells is reduced, as measured by flow cytometry.

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

[0563] 139. A manufactured population of T cells, characterized by being relative to CD4 + or CD8 + T-Rapa cells express the corresponding frequency of LAIR1 and CD4. + or CD8 + The frequency of LAIR1 expression on T cells is reduced, as measured by flow cytometry.

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

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

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

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

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

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

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

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

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

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

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

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

[0576] 152. A population of T cells manufactured according to any one of Examples 108 to 109 and 119 to 150, wherein the manufactured T cell population contains CD4+. + or CD8 + The frequency of T cell expression of CD28 or ICOS is substantially the same as that of the control T cell population characterized by the T cells that produced the manufactured T cell population, with CD4+ expression being significantly higher. + or CD8 +T cells express CD28 or ICOS at the same frequency.

[0577] 153. A population of T cells manufactured according to any one of Examples 108 to 109 and 119 to 150, wherein CD4 + or CD8 + The frequency of T cell expression of CD28 or ICOS in a control T cell population characterized by T cells that produced the manufactured T cell population was lower than that of CD4. + or CD8 + T cells express CD28 or ICOS at frequencies within 10% of their corresponding frequencies.

[0578] 154. A manufactured T cell population, said manufactured T cell population, after co-stimulation with antiCD3 / antiCD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, secretes at least 500 pg / mL / 1×10⁻⁶ cells / mL. 6 IL-2 per cell per day.

[0579] 155. A manufactured T cell population, said manufactured T cell population, after co-stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a bead:T cell ratio between 3:1 and 1:3, and with IL-7, IL-15, or a combination of IL-7 and IL-15 (if present) at a concentration of 10 ng / mL each, secretes at least 1000 pg / mL / 1×10⁻¹¹ T cells. 6 IL-2 per cell per day.

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

[0581] 156. A manufactured T cell population expressing: at least 75% less phosphorylated STAT5, detectable levels of STAT1 or phosphorylated STAT1, at least 50% less p70S6K and Raptor compared to a cultured T-Rapa cell population, and Rictor, SGK1 and phosphorylated SGK1 levels that differ from those of a cultured T-Rapa cell population by no more than 50%.

[0582] 157. A manufactured population of T cells, said manufactured population of T cells exhibiting reduced mTORCl activation, said mTORCl activation being characterized by at least one of the following:

[0583] (a) Reduced phosphorylated P70S6K levels relative to the control T cell population, or

[0584] (b) Reduced Raptor levels relative to the control T cell population; and

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

[0586] 157. A manufactured T cell population expressing: at least 75% less phosphorylated STAT5, detectable levels of STAT1 or phosphorylated STAT1, at least 50% less p70S6K and Raptor compared to a cultured T-Rapa cell population, and Rictor, SGK1 and phosphorylated SGK1 levels that differ from those of a cultured T-Rapa cell population by no more than 50%.

[0587] 158. A manufactured population of T cells, said manufactured population of T cells exhibiting reduced mTORCl activation, said mTORCl activation being characterized by at least one of the following:

[0588] (a) Reduced phosphorylated P70S6K levels relative to T-Rapa cells, or

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

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

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

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

[0593] 161. A manufactured population of T cells having one or more of the following properties:

[0594] After one week of incubation with anti-CD3 / anti-CD28 magnetic beads at a bead:T cell ratio of 3:1, IFN-γ secretion relative to T-Rapa cells increased by at least 50%.

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

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

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

[0598] The percentage of cells positive for CD4, CD62L, CCR7, and CD127 is increased by at least 50% compared to a control T cell population characterized by producing the manufactured T cell population.

[0599] Compared to a control T cell population characterized by T cells producing a T cell population, 4EBP1 phosphorylation increased by no more than 50%.

[0600] Compared to T-Rapa cell populations cultured under the same conditions, the expression of p70S6K or Raptor was reduced by at least 50%;

[0601] Compared to T-Rapa cell populations cultured under the same conditions, p-STAT5 expression was reduced by at least 50%;

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

[0603] Compared to a control T cell population characterized by cells that produce the manufactured T cell population, p70S6K expression is increased by at least 10%.

[0604] Compared to the T-Rapa cell population, CD25 expression was reduced by at least 50%;

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

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

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

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

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

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

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

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

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

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

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

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

[0617] The expression level of CD28 is within approximately 20% of that of a control T cell population characterized by T cells that produce the manufactured T cell population.

[0618] The expression level of ICOS was within approximately 20% of that of a control T cell population characterized by T cells that produced the manufactured T cell population.

[0619] The expression level of CD45RA was within approximately 20% of that of a control T cell population characterized by T cells that produced the manufactured T cell population.

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

[0621] Compared to T-Rapa cultures incubated under the same conditions, IL-2 secretion increased by at least 1.1 times;

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

[0623] When incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, IL-2 secretion increased by at least 1.1-fold, wherein IL-7 and IL-15 (in the presence) were added at 10 ng / mL, respectively.

[0624] After incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, secretion should be at least 1000 pg / mL / 1×10 6 IL-2 per cell / day, wherein IL-7 and IL-15 (in the presence) are added at 10 ng / mL;

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

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

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

[0628] Compared to a control T cell population characterized by cells that produce the manufactured T cell population, CD4 + The frequency of CD127 expression on T cells increases by at least 50%;

[0629] Compared to culture-infused T cells, the frequency of co-expression of CD62L and CCR7 in T cells increased by at least 25%;

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

[0631] At the end of the manufacturing process, low levels of the inflammatory cytokines IFN-γ and TNF-α are secreted, such as after stimulation with high levels of co-stimulation (bead-to-T cell ratio of 3:1), with levels in the culture supernatant of 1 × 10⁻⁶ cells per 24 hours. 6 The limit is defined as <100 pg / ml per cell;

[0632] In the absence of inhibitors, after a 6-day amplification cycle, IFN-γ and TNF-α secretion increased at least 5-fold and more preferably 20-fold compared to the secretion levels on day 6; and

[0633] Its combination.

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

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

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

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

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

[0639] Compared to a control T cell population characterized by T cells producing a T cell population, 4EBP1 phosphorylation increased by no more than 50%.

[0640] Compared to T-Rapa cell populations cultured under the same conditions, the expression of p70S6K or Raptor was reduced by at least 50%;

[0641] Compared to T-Rapa cell populations cultured under the same conditions, p-STAT5 expression was reduced by at least 50%;

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

[0643] Compared to a control T cell population characterized by cells that produce the manufactured T cell population, p70S6K expression is increased by at least 10%.

[0644] Compared to the T-Rapa cell population, CD25 expression was reduced by at least 50%;

[0645] The expression level of CD28 is within approximately 20% of that of a control T cell population characterized by T cells that produce the manufactured T cell population.

[0646] The expression level of ICOS was within approximately 20% of that of a control T cell population characterized by T cells that produced the manufactured T cell population.

[0647] The expression level of CD45RA was within approximately 20% of that of a control T cell population characterized by T cells that produced the manufactured T cell population.

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

[0649] Compared to T-Rapa cultures incubated under the same conditions, IL-2 secretion increased by at least 1.1 times;

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

[0651] When incubated in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, IL-2 secretion increased by at least 1.1-fold, wherein IL-7 and IL-15 (in the presence) were added at 10 ng / mL, respectively.

[0652] After incubation in the presence of IL-7, IL-15, or a combination of IL-7 and IL-15, secretion should be at least 1000 pg / mL / 1×10 6 IL-2 per cell / day, wherein IL-7 and IL-15 (in the presence) are added at 10 ng / mL;

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

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

[0655] Expressing CD 127;

[0656] At the end of the manufacturing process, low levels of the inflammatory cytokines IFN-γ and TNF-α are secreted, such as after stimulation with high levels of co-stimulation (bead-to-T cell ratio of 3:1), with levels in the culture supernatant of 1 × 10⁻⁶ cells per 24 hours. 6 The limit is defined as <100 pg / ml per cell;

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

[0658] Its combination.

Claims

1. Use of a first immune depletion regimen, a second immune depletion regimen, and a composition comprising manufactured T cells in the preparation of a kit for use in cancer patients in need, wherein: The subjects underwent a first treatment cycle and one or more additional treatment cycles. The first treatment cycle includes: The subject was subjected to a first immune exhaustion protocol to reduce at least a portion of regulatory T cells and / or late senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late senescent effector T cells; Each of the one or more additional treatment cycles includes: The subject was subjected to a second immune exhaustion protocol to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells, or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells; and The subject is given a therapeutically effective dose of a composition comprising manufactured T cells.

2. Use of an immune depletion protocol and a composition comprising manufactured T cells in the preparation of a kit for treating cancer in subjects of need, wherein: Subjecting the subject to an immune exhaustion protocol to reduce at least a portion of regulatory T cells and / or late-senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and / or late-senescent effector T cells; and Following the immune depletion protocol, the subject is administered a therapeutically effective dose of a composition comprising manufactured T cells.

3. A method for producing manufactured T cells, the method comprising: In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density. Add IFN-α to the culture medium; The T cells and culture medium are incubated for a certain period of time to produce the manufactured T cells; Harvest the T cells produced.

4. A manufactured T cell produced by the method according to claim 3.

5. A method for producing manufactured T cells, the method comprising: In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density. The culture was introduced into the cell population and incubated with the culture medium for a first time period without co-stimulation of the culture with anti-CD3 / anti-CD28. After incubation for the first time period, anti-CD3 / anti-CD28 coated magnetic beads were added to the T cells and culture medium at a bead:T cell ratio between 1:1 and 1:12 to stimulate the T cells. Add IFN-α to the culture medium; The culture was incubated in the culture medium containing the anti-CD3 / anti-CD28 coated magnetic beads and IFN-α for a second time period to produce manufactured T cells; The anti-CD3 / anti-CD28 coated magnetic beads were isolated from the manufactured T cells; and Harvest the T cells produced.

6. A manufactured T cell produced by the method according to claim 5.

7. A method for producing manufactured T cells, the method comprising: In a culture medium containing tesimolimus and an IL-2 signaling inhibitor, a culture population including T cells from the subject was seeded at a certain cell density. The culture was introduced into the cell population and incubated with the culture medium for a first time period without co-stimulation of the culture with anti-CD3 / anti-CD28. After incubation for the first time period, nanoparticles containing anti-CD3 / anti-CD28 were added to the T cells and culture medium at a dose of 0.01 to 0.1 times the recommended dose to stimulate the T cells; Add IFN-α to the culture medium; The culture was incubated in the culture medium containing nanoparticles containing anti-CD3 / anti-CD28 and IFN-α for a second time period to produce manufactured T cells; Harvest the T cells produced.

8. A manufactured T cell produced by the method according to claim 7.

9. A manufactured T cell population that exhibits a reduced level of STAT5 phosphorylation relative to a control manufactured T cell population produced in the presence of exogenous IL-2, wherein the observed reduction is at least 50% less.

10. A manufactured population of T cells exhibiting a shift from an effector memory state to a T central memory state, as indicated by an increase of at least 25% in the frequency of T cells co-expressing CD62L and CCR7 relative to culture-infused T cells.

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