Process for generating genetically engineered autologous t cells

Abstract

To provide a method for production of autologous genetically engineered T cells for use in cell therapy applications.SOLUTION: Provided is a method for producing genetically engineered autologous T cells expressing at least one protein of interest. The method comprises the steps of: inoculating a closed single use bioreactor bag containing culture media with apheresed donor cells and one or more soluble T cell activators; culturing the cells in the bioreactor bag while continuously rocking at a rate of about 2 RPM; transducing the cells in the bioreactor bag with at least one soluble viral vector comprising a polynucleotide which encodes the protein of interest while continuously rocking at a rate of about 2 RPM; and expanding the cells in the bioreactor bag at a rocking rate of about 2 RPM and increasing a culture volume and rocking rate as needed to maintain a culture until harvest.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the production of autologous genetically modified T cells for use in cell therapy applications. [Background technology]

[0002] Adoptive T cell therapy utilizes genetically modified T cells and has been shown to be an effective and powerful therapeutic treatment in certain hematological indications. Autologous T cells are genetically modified to express one or more cell surface receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), that recognize a protein of interest associated with the surface of a target cell, while retaining and / or enhancing their ability to recognize and kill the target cell. In addition to the ability of genetically modified T cells to recognize and destroy target cells, the success of adoptive T cell therapy benefits from the ability of T cells to persist in the patient and expand greatly in response to target antigens. Genetically modified autologous T cells have shown great success in treating a wide range of cancers.

[0003] In autologous T-cell therapy, the product is live cells from a specific patient that are returned to that patient. Therefore, the manufacturing of autologous cells is more challenging than traditional biopharmaceutical processes, since one lot is manufactured for one patient. Manufacturing can only begin when the patient's sample is received, and care must be taken to ensure that the cells are returned to the patient without significant modifications that would make them a risk rather than an effective treatment. To produce cells in sufficient quantity and quality, manufacturing is more practical and labor intensive, and is carried out multiple times on a small scale. Donor-to-donor variability adds complexity, since the starting material is different for each new lot manufactured.

[0004] The evaluation criteria for autologous formulations are also different from typical recombinant protein formulations. For autologous T cell therapy, product purity, cell phenotype, percentage of modified T cells, potency and specificity are the parameters tested. Integrated closed process platforms using complex automation systems have been developed, but require significant capital investment. Developing an efficient, scalable, reliable and cost-effective process remains one of the major challenges in advancing the use of autologous cells as a therapy, which is largely a highly manual process.

[0005] There is a need for a manufacturing process for producing T cell therapeutics, particularly autologous T cell therapeutics, that reduces the risk of contamination, costs, facility footprint, and inefficiencies and complexities of current manufacturing processes. Autologous T cell therapy is projected to range from $350,000 to $475,000 per patient, not including hospital-related costs. If genetically modified autologous T cells are to achieve their full clinical and commercial potential, the challenges facing the production of clinical grade cells in commercially relevant quantities and quality must be overcome. The invention described herein fulfills this need by providing an efficient and effective manufacturing method for genetically modified T cells for autologous cell therapy. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides a method of producing genetically modified autologous T cells expressing at least one protein of interest, comprising the steps of seeding apheresis donor cells and one or more soluble T cell activators in a closed single-use bioreactor bag containing a culture medium, where the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM, transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding the protein of interest, while continuously rocking at a speed of about 2 RPM, and expanding the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM while increasing the culture volume and rocking speed as necessary to maintain the culture until harvest. In one embodiment, the apheresis donor cells comprise cells derived from peripheral blood. In a related embodiment, the apheresis donor cells comprise nucleated and non-nucleated cells. In one embodiment, the apheresis donor cells include white blood cells and red blood cells. In a related embodiment, the apheresis donor cells also include granulocytes and / or platelets. In one embodiment, the apheresis is leukapheresis. In one embodiment, the apheresis donor cells are washed and resuspended in culture medium. In one embodiment, the at least one T cell activator is an anti-CD3 antibody or a binding fragment thereof. In yet another embodiment, the T cell activator includes an anti-CD3 antibody and an anti-CD28 antibody, or a binding fragment thereof. In one embodiment, the T cell activator includes at least an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, or a binding fragment thereof. In another embodiment, the T cell activator includes at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex. In another embodiment, the concentration of the at least one soluble T cell activator is from at least 0.001 μg / ml to at least 10 μg / ml.In a related embodiment, the concentration of the at least one soluble T cell activator is at least 0.1 μg / ml to at least 5 μg / ml. In yet another embodiment, the at least one soluble T cell activator binds to the at least one donor cell at the time of seeding. In another embodiment, the apheresis donor cells are incubated with the one or more soluble T cell activators before seeding into the bioreactor bag. In a related embodiment, the incubation is for a period of time sufficient to allow binding of the one or more soluble T cell activators to the apheresis donor cells prior to seeding to be fully saturated. In a related embodiment, the apheresis donor cells and the one or more soluble T cell activators are incubated in a transfer bag. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 50 ml. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 10 ml. In a related embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 30 minutes or more. In a related embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 1 hour. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.0E9 to about 1.3E9. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.2E9. In one embodiment, the bioreactor bag is seeded with the apheresis donor cells at a cell density of about 1E6 to about 5E6 nucleated cells / ml. In a related embodiment, the bioreactor bag is seeded with the apheresis donor cells at a cell density of about 2E6. In one embodiment, the bioreactor bag contains at least 300 ml to at least 400 ml of culture medium at the time of seeding. In a related embodiment, the bioreactor bag contains at least 300 ml of culture medium at the time of seeding. In one embodiment, the apheresis donor cells are cultured in the bioreactor bag for about 12 to 24 hours. In one embodiment, the culture medium includes at least one soluble cytokine.In a related embodiment, the soluble cytokine is selected from IL-2, IL-7, IL-15, or IL-21. In a related embodiment, the at least one soluble cytokine is IL-2. In a related embodiment, the IL-2 is at a concentration of about 250 IU / ml to about 350 IU / ml. In a related embodiment, the IL-2 is at a concentration of about 300 IU / ml. In a related embodiment, the soluble cytokine is IL-7 in combination with IL-15 or IL-21. In a related embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 30 ng / ml. In a related embodiment, the concentration of the at least one cytokine is at least 10 ng / ml to at least 20 ng / ml. In one embodiment, the culture medium also includes a WNT pathway activator. In a related embodiment, the WNT pathway activator is TWS117. In a related embodiment, the culture medium includes a mixture of soluble TWS117, IL-7, and IL-21. In one embodiment, the culture medium also includes a soluble glycolytic inhibitor. In a related embodiment, the soluble glycolytic inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the lentiviral vector is added at an MOI of 0.25-10. In a related embodiment, the lentiviral vector is added at an MOI of 1. In one embodiment, the cells are transduced for about 20-24 hours. In one embodiment, after transduction, half of the culture medium is removed from the bioreactor bag and replaced with an equal amount of fresh culture medium. In a related embodiment, the culture is incubated for about 12-24 hours. In one embodiment, during growth, fresh culture medium is added to the bioreactor by fed-batch / perfusion feeding and / or by perfusion. In one embodiment, during growth, the culture is perfused at a rate of one bioreactor bag volume per day. In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased up to 1 liter during growth.In one embodiment, as the cells grow, the volume of culture medium is gradually increased to maintain a cell density of at least 2E6 nucleated cells / ml. In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased to 1 liter during growth to maintain a cell density of at least 4E6 nucleated cells / ml. In one embodiment, as the cells grow, the rocking speed is gradually increased to 6 RPM. In one embodiment, at the start of growth, the volume of culture medium in the closed single-use bioreactor bag is 300 ml, rocked at a speed of 2 rpm at an angle of 2°. In one embodiment, the culture in the closed single-use bioreactor bag is maintained at about 80-100% O2. In one embodiment, the cells are grown for 7-14 days. In one embodiment, the culturing, transduction, and / or growth steps are performed at 34-37°C. In one embodiment, the protein of interest is a cell surface receptor. In a related embodiment, the cell surface receptor is a T cell receptor or a chimeric antigen receptor. In a related embodiment, the cell surface receptor recognizes an antigen target associated with the target cell. In a related embodiment, the target cell is a cancer cell. In one embodiment, the genetically modified autologous T cells are used to treat a condition in a patient in need thereof.

[0007] The present invention provides a pharmaceutical composition comprising genetically modified autologous T cells produced using the above-described method.

[0008] The present invention provides a method of treating a condition in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition as described above.

[0009] The present invention relates to a method for increasing transgene expression in genetically modified autologous T cells expressing a protein of interest, comprising the steps of seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing a culture medium, wherein at least one soluble T cell activator is bound to at least one donor cell at the time of seeding, and the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM, and culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM. and growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture until harvest, wherein expression of the transgene is greater than expression of the transgene in genetically modified autologous T cells derived from an enriched population of T cells from the same apheresis donor cells and expressing the same protein of interest. In one embodiment, the apheresis donor cells comprise cells from peripheral blood. In a related embodiment, the apheresis donor cells comprise nucleated and anucleated cells. In one embodiment, the apheresis donor cells comprise white blood cells and red blood cells. In a related embodiment, the apheresis donor cells also comprise granulocytes and / or platelets. In one embodiment, the apheresis is leukapheresis. In one embodiment, the apheresis donor cells are washed and resuspended in culture medium. In one embodiment, the at least one T cell activator is an anti-CD3 antibody or a binding fragment thereof. In yet another embodiment, the T cell activator comprises an anti-CD3 antibody and an anti-CD28 antibody, or a binding fragment thereof. In one embodiment, the T cell activator comprises at least an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, or a binding fragment thereof.In another embodiment, the T cell activator comprises at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex. In another embodiment, the concentration of the at least one soluble T cell activator is at least 0.001 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration of the at least one soluble T cell activator is at least 0.1 μg / ml to at least 5 μg / ml. In another embodiment, the apheresis donor cells are incubated with one or more soluble T cell activators prior to seeding into the bioreactor bag. In a related embodiment, the incubation is for a period of time sufficient to allow binding of the one or more soluble T cell activators to the apheresis donor cells to be fully saturated prior to seeding. In a related embodiment, the apheresis donor cells and the one or more soluble T cell activators are incubated in a transfer bag. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 50 ml. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 10 ml. In a related embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 30 minutes or more. In a related embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 1 hour. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.0E9 to about 1.3E9. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.2E9. In one embodiment, the apheresis donor cells are seeded in a bioreactor bag at a cell density of about 1E6 to about 5E6 nucleated cells / ml. In a related embodiment, the apheresis donor cells are seeded in a bioreactor bag at a cell density of about 2E6. In one embodiment, the bioreactor bag contains at least 300 ml to at least 400 ml of culture medium at the time of inoculation. In a related embodiment, the bioreactor bag contains at least 300 ml of culture medium at the time of inoculation.In one embodiment, the apheresis donor cells are cultured in the bioreactor bag for about 12 to 24 hours. In one embodiment, the culture medium comprises at least one soluble cytokine. In a related embodiment, the soluble cytokine is selected from IL-2, IL-7, IL-15, or IL-21. In a related embodiment, the at least one soluble cytokine is IL-2. In a related embodiment, the IL-2 is at a concentration of about 250 IU / ml to about 350 IU / ml. In a related embodiment, the IL-2 is at a concentration of about 300 IU / ml. In a related embodiment, the soluble cytokine is IL-7 in combination with IL-15 or IL-21. In a related embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 30 ng / ml. In a related embodiment, the concentration of the at least one cytokine is at least 10 ng / ml to at least 20 ng / ml. In one embodiment, the culture medium also comprises a WNT pathway activator. In a related embodiment, the WNT pathway activator is TWS117. In a related embodiment, the culture medium comprises a mixture of soluble TWS117, IL-7 and IL-21. In one embodiment, the culture medium also comprises a soluble glycolytic inhibitor. In a related embodiment, the soluble glycolytic inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 10. In a related embodiment, the lentiviral vector is added at an MOI of 1. In one embodiment, the cells are transduced for about 20 to 24 hours. In one embodiment, after transduction, half of the culture medium is removed from the bioreactor bag and replaced with an equal amount of fresh culture medium. In a related embodiment, the culture is incubated for about 12 to 24 hours. In one embodiment, during growth, fresh culture medium is added to the bioreactor by fed-batch / perfusion feeding and / or perfusion. In one embodiment, during growth, the culture is perfused at a rate of one bioreactor bag volume per day.In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased to 1 liter during growth. In one embodiment, as the cells grow, the volume of culture medium is gradually increased to maintain a cell density of at least 2E6 nucleated cells / ml. In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased to 1 liter during growth to maintain a cell density of at least 4E6 nucleated cells / ml. In one embodiment, as the cells grow, the rocking speed is gradually increased to 6 RPM. In one embodiment, at the start of growth, the volume of culture medium in the closed single-use bioreactor bag is 300 ml, rocking at a speed of 2 rpm at an angle of 2°. In one embodiment, the culture in the closed single-use bioreactor bag is maintained at about 80-100% O2. In one embodiment, the cells are grown for 7-14 days. In one embodiment, the culturing, transduction, and / or growth steps are performed at 34-37°C. In one embodiment, the protein of interest is a cell surface receptor. In related embodiments, the cell surface receptor is a T cell receptor or a chimeric antigen receptor. In related embodiments, the cell surface receptor recognizes an antigen target associated with the target cell. In related embodiments, the target cell is a cancer cell. In one embodiment, the genetically modified autologous T cells are used to treat a condition in a patient in need thereof.

[0010] The present invention provides a method of treating a patient with genetically modified autologous T cells expressing a protein of interest, comprising the steps of incubating apheresis cells from the patient with one or more T cell activators selected from the group consisting of anti-CD3, anti-CD2 and anti-CD28 antibodies, or binding fragments thereof, to saturate antibody binding, seeding the apheresis cells into a closed single-use bioreactor bag containing culture medium, wherein the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag while continuously rocking at a speed of about 2 RPM, and culturing the cells while continuously rocking at a speed of about 2 RPM. The present invention provides a method comprising the steps of transducing cells in a closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding a protein of interest, growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as necessary to maintain the culture at a desired cell density until harvest, harvesting and formulating the cells for cryopreservation, freezing and storing the cells until needed for administration to a patient, thawing the cells and resuspending them in a medium suitable for infusion, and reintroducing a pharmacologic effective amount of genetically modified autologous T cells expressing the protein of interest into the patient. In one embodiment, the apheresis donor cells comprise cells from peripheral blood. In a related embodiment, the apheresis donor cells comprise nucleated and anucleated cells. In one embodiment, the apheresis donor cells comprise white blood cells and red blood cells. In a related embodiment, the apheresis donor cells also comprise granulocytes and / or platelets. In one embodiment, the apheresis is leukapheresis. In one embodiment, the apheresis donor cells are washed and resuspended in culture medium. In one embodiment, the at least one T cell activator is an anti-CD3 antibody or a binding fragment thereof. In yet another embodiment, the T cell activator comprises an anti-CD3 antibody and an anti-CD28 antibody, or a binding fragment thereof.In one embodiment, the T cell activator comprises at least an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, or binding fragments thereof. In another embodiment, the T cell activator comprises at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex. In another embodiment, the concentration of the at least one soluble T cell activator is at least 0.001 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration of the at least one soluble T cell activator is at least 0.1 μg / ml to at least 5 μg / ml. In yet another embodiment, the at least one soluble T cell activator binds to the at least one donor cell at the time of seeding. In a related embodiment, the incubation is for a period of time sufficient to allow for sufficient saturation of the binding of the one or more soluble T cell activators to the apheresis donor cells prior to seeding. In a related embodiment, the apheresis donor cells and one or more soluble T cell activators are incubated in a transfer bag. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 50 ml. In a related embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 10 ml. In a related embodiment, the apheresis donor cells are incubated with the one or more T cell activators for at least 30 minutes or more. In a related embodiment, the apheresis donor cells are incubated with the one or more T cell activators for at least 1 hour. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.0E9 to about 1.3E9. In one embodiment, the number of nucleated cells in the apheresis donor cells is about 1.2E9. In one embodiment, the apheresis donor cells are seeded in a bioreactor bag at a cell density of about 1E6 to about 5E6 nucleated cells / ml. In a related embodiment, the bioreactor bag is seeded with apheresis donor cells at a cell density of about 2E6. In one embodiment, the bioreactor bag contains at least 300 ml to at least 400 ml of culture medium at the time of seeding.In a related embodiment, the bioreactor bag contains at least 300 ml of culture medium at the time of seeding. In one embodiment, the apheresis donor cells are cultured in the bioreactor bag for about 12 to 24 hours. In one embodiment, the culture medium comprises at least one soluble cytokine. In a related embodiment, the soluble cytokine is selected from IL-2, IL-7, IL-15, or IL-21. In a related embodiment, the at least one soluble cytokine is IL-2. In a related embodiment, the IL-2 is at a concentration of about 250 IU / ml to about 350 IU / ml. In a related embodiment, the IL-2 is at a concentration of about 300 IU / ml. In a related embodiment, the soluble cytokine is IL-7 in combination with IL-15 or IL-21. In a related embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 30 ng / ml. In a related embodiment, the concentration of the at least one cytokine is at least 10 ng / ml to at least 20 ng / ml. In one embodiment, the culture medium also includes a WNT pathway activator. In a related embodiment, the WNT pathway activator is TWS117. In a related embodiment, the culture medium includes a mixture of soluble TWS117, IL-7, and IL-21. In one embodiment, the culture medium also includes a soluble glycolytic inhibitor. In a related embodiment, the soluble glycolytic inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 10. In a related embodiment, the lentiviral vector is added at an MOI of 1. In one embodiment, the cells are transduced for about 20 to 24 hours. In one embodiment, after transduction, half of the culture medium is removed from the bioreactor bag and replaced with an equal amount of fresh culture medium. In a related embodiment, the culture is incubated for about 12 to 24 hours. In one embodiment, during growth, fresh culture medium is added to the bioreactor by fed-batch / perfusion feeding and / or perfusion.In one embodiment, during growth, the culture is perfused at a rate of 1 bioreactor bag volume per day. In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased to 1 liter during growth. In one embodiment, as the cells grow, the volume of culture medium is gradually increased to maintain a cell density of at least 2E6 nucleated cells / ml. In one embodiment, as the cells grow, the volume of culture medium in the bioreactor is gradually increased to 1 liter during growth to maintain a cell density of at least 4E6 nucleated cells / ml. In one embodiment, as the cells grow, the rocking speed is gradually increased to 6 RPM. In one embodiment, at the start of growth, the volume of culture medium in the closed single-use bioreactor bag is 300 ml and is rocked at a rate of 2 rpm at an angle of 2°. In one embodiment, the culture in the closed single-use bioreactor bag is maintained at about 80-100% O2. In one embodiment, the cells are grown for 7-14 days. In one embodiment, the culturing, transduction, and / or expansion steps are performed at 34-37° C. In one embodiment, the protein of interest is a cell surface receptor. In a related embodiment, the cell surface receptor is a T cell receptor or a chimeric antigen receptor. In a related embodiment, the cell surface receptor recognizes an antigen target associated with a target cell. In a related embodiment, the target cell is a cancer cell. In one embodiment, the genetically modified autologous T cells are used to treat a condition in a patient in need thereof. [Brief description of the drawings]

[0011] [Figure 1] (A) Growth curves of cells grown in Xuri W25 bioreactors supplemented with IL2 alone or a cocktail of IL7, IL21 and TWS119. (B) Viability of cells grown in Xuri W25 bioreactors supplemented with IL2 alone or a cocktail of IL7, IL21 and TWS119. [Diagram 2]T cell transgene expression from engineered T cells expressed in three systems: Xuri W25 bioreactors ("Xuri"), a combination of a static perfusion bag and a Xuri W25 bioreactor ("Permeable bag") and a G-Rex® plate ("G-Rex"). [Diagram 3] Leukocyte subsets in harvested engineered cells expressed in three systems: Xuri W25 bioreactor ("Xuri"), a combination of a static perfusion bag and Xuri W25 bioreactor ("Permeable bag") and G-Rex plates ("G-Rex"). [Figure 4] Proportions of T cell subsets on days 5 (A) and 10 (B) from modified T cells expressed in three systems: Xuri W25 bioreactor ("Xuri"), a combination of static perfusion bag and Xuri W25 bioreactor ("Permeable bag") and G-Rex plate ("G-Rex"). [Figure 5-1] Shown is the cytotoxic function of modified T cells characterized by their ability to lyse target cells (A), release IFN-gamma in response to target cells (B), and release TNF-alpha in response to target cells (C). [Figure 5-2] Shown is the cytotoxic function of modified T cells characterized by their ability to lyse target cells (A), release IFN-gamma in response to target cells (B), and release TNF-alpha in response to target cells (C). [Figure 6] Resistance to target cell challenge of engineered T cells expressed in three systems: Xuri W25 bioreactor ("Xuri"), a combination of a static perfusion bag and a Xuri W25 bioreactor ("Permeable bag") and a G-Rex plate ("G-Rex") Annexin levels (A), Tim3 (B). [Figure 7] Growth curves (A) and viability (B) of cells grown in medium with 2-DG (medium 1) and without 2-DG (medium 2). [Figure 8] Transgene expression in cells grown in medium with (medium 1) and without (medium 2) 2-DG. [Figure 9] CD25 expression by cells grown in medium with (medium 1) and without (medium 2) 2-DG. [Figure 10] Glucose (A) and lactate (B) concentrations in cultures supplemented with 2-DG (medium 1) and without 2-DG (medium 2). [Figure 11] T differentiation phenotype of cells grown in medium with (medium 1) and without (medium 2) 2-DG. [Figure 12] Leukocyte subsets in harvested T cells from cells grown in medium with (medium 1) and without (medium 2) 2-DG. [Figure 13-1] Potency assay results of engineered TCR T cells. (A) Cytotoxicity. (B) IFN-γ. (C) T cell differentiation. [Figure 13-2] Potency assay results of engineered TCR T cells. (A) Cytotoxicity. (B) IFN-γ. (C) T cell differentiation. [Figure 14] Transgene expression in T cells harvested from enriched and non-enriched processes, gated on CD8+ cells (A) or CD4+ cells (B). [Figure 15] TCR vector copy numbers in CD4+ and CD8+ T cells. [Figure 16] Percentages of leukocytes and leukocyte subsets measured at the end of harvest. [Figure 17] T cell phenotypes upon harvest from enriched and non-enriched cells from three donors. [Figure 18] Growth curves (A) and viability (B) of cells in the bioreactor from enriched and non-enriched donor cells. [Figure 19] Cell growth curves and viability. [Figure 20] Percentages of leukocyte subsets on days 0 and 10. [Figure 21] T cell differentiation during bioprocessing. [Figure 22] Transgene expression in harvested T cells at the cell surface level (A) and DNA level (B) is shown. [Diagram 23] Percentage of target cell killing at different effector to target cell (E:T) ratios. [Figure 24] IFN-gamma release from harvested cells in response to target cells. [Diagram 25] Results of titration of coated anti-CD3 (OKT3) antibodies and pan T cell activation measured by CD69, CD25 and 4-1BB. [Figure 26] Results of pan T cell stimulation by various soluble activators against coated anti-CD3 as measured by CD69, CD25 and 4-1BB. [Figure 27] Transduction efficiency of modified T cells, Pan T cells (A) and apheresis donor cells (B). [Figure 28] Cell proliferation of cells activated by various activators. [Figure 29] Effect of culture conditions on transduction efficiency. [Diagram 30] Cell density and transduction efficiency. [Diagram 31] Multiplicities of infection for TCR transduction. (A) MOI of 1–10; (B) MOI of 0.25–2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Autologous T-cell therapy is a personalized therapy, where genetically modified T cells are derived from the patient's own cells, "autologous cells," which are engineered and then returned to the patient. T cells or T-lymphocytes refer to a type of lymphocyte (the others being B cells and NK cells) that actively participates in the body's immune response. While the use of autologous cells reduces the risk of an immunological response, manufacturing one lot of therapy per patient adds complexity and cost. Also, because autologous T-cell therapy is patient-centric, the therapy is not amenable to large-scale manufacturing and off-the-shelf use, unlike traditional biological therapies.

[0013] Autologous T cell therapy involves treating a patient with the patient's own live T cells that have been engineered to express a protein of interest that recognizes a protein associated with a target cell. Autologous T cell manufacturing processes generally use multiple unit operations, including semi-closed and / or closed steps, to transfer the patient's cells between multiple containers, such as plates, flasks, containers, bags, or other types of containers, typically gas-permeable containers, throughout the manufacturing process. These transfers may involve moving fluids from one container to another by means of syringes or pipettes, centrifuges, etc., which may apply unnecessary stress to the cells through shear forces, exposing the cells to contaminants and loss. In addition, such containers require manual transfers within and between equipment, such as cell separators, biosafety cabinets, and incubators.

[0014] As described herein, a method for producing genetically modified autologous T cells expressing a protein of interest has been developed that generates T cells with high purity of over 98%, shows high transgene expression in CD8 T cells of over 45%, and produces a cell yield of over 10 billion with a viability of over 90% in a fully closed continuous process in 10 days. Starting from apheresis donor cells, T cells are selectively expanded from about 50% at harvest to over 98% after genetic engineering and expansion of activated cells. The resulting T cells are less differentiated and retain a more stem cell or memory-like phenotype, which increases persistence and overall efficacy. The resulting cells are more potent at killing target cells and resistant to target cell challenge.

[0015] Proliferation rates and viability were comparable to cells enriched prior to activation and transduction, but transduction was more efficient and the function and efficacy of cells generated directly from harvested donor cells was greater.

[0016] Genetically modified autologous T cells were generated in closed continuous rocking cultures throughout the activation, transduction and expansion steps using a bioreactor capable of generating rocking motion over the entire period. Rocking was adjusted to accommodate changes in bioreactor volume (increased to 1 liter by the end of expansion) and oxygen levels as cell density increased during the process. This ensured sufficient oxygen and nutrient mass transfer to support high-density cultures, in contrast to methods using static cultures that must be grown at low cell densities (≦2E6 cells / ml) due to limitations of culture medium under static conditions. Both cells rocked in bioreactor bags and the commonly used static G-Rex® gas-permeable culture system had efficient oxygen transport, but the G-Rex® system lacked convection to transfer nutrients to the cells in a timely manner. Bioreactor bags can also utilize perfusion of fresh medium to support very high cell densities (up to 50E6 cells / ml in some cases).

[0017] Compared to cells produced in cultures produced using static gas permeable vessels, or a combination of static (activation and transduction operations) and rocking (growth operations) systems, the methods described herein eliminated over 30% of the hands-on labor and significantly reduced costs. Incubators and other support equipment essential to prepare and maintain static cultures in gas permeable vessels are not required, reducing the manufacturing footprint and risk of cell and equipment contamination from the handling and manipulation these static cultures require. While the growth rates and viability of cells produced by these systems were comparable, the transgene expression, function and efficacy of cells produced by the methods described herein were greater than cells produced in cultures utilizing gas permeable vessels in static cultures, or hybrid static / rocking cultures.

[0018] The donor may be any subject for whom a sample of blood cells is needed to produce genetically modified autologous T cells. The donor may be a patient in need of treatment with a cell population (i.e., an autologous donor) generated by the methods described herein.

[0019] Donor cells may be collected from circulating peripheral blood by any suitable method used in the art, such as extracorporeal methods, venipuncture, or other blood collection methods where blood samples are commonly obtained. In one embodiment, donor cells are collected by apheresis. Apheresis refers to the removal of blood from a donor, separating it into cellular and soluble components, removing certain desired components from the blood, and then returning the remaining blood to the donor. When T cells are the desired cell type, such as for cell therapy applications, leukapheresis is most frequently used, an apheresis process that preferentially removes blood cells (leukocytes) from the donor's peripheral blood. The collected leukapheresis sample may be provided in a Leukopak® container. Multiple blood volumes are processed from the same donor to generate a complete Leukopak®.

[0020] Apheresis separates incoming blood into various blood components using methods such as differential centrifugation. However, due to the close density between blood components, this does not result in a pure population of cells, and cells remain in the sample collected by the apheresis process, and the amount of these remaining cells increases as the amount of blood processed during the apheresis procedure increases. Cells collected by leukapheresis will include nucleated cells such as white blood cells, including monocytes, dendritic cells, lymphocytes (T cells, B cells, and NK cells), and granulocytes, as well as non-nucleated cells such as red blood cells and platelets. Although the percentage of lymphocytes in the collected apheresis sample is more concentrated compared to whole blood, there are also significant numbers of other cells in the sample, such as NK cells, red blood cells, platelets, and granulocytes.

[0021] Apheresis cells are frequently used as a source for harvesting blood cells, such as white blood cells and lymphocytes. However, since these samples contain a mixture of blood cells, for example, a Leukopack® containing apheresis cells may contain tens of billions of red blood cells, which make the harvested cell sample visibly red. Red blood cells are known to affect the further processing of the cells in the sample, for example, by greatly reducing the transfection rate when electroporation is used. "Because of the inherent variability of cell populations in these leukapheresis products, processes for removal of unwanted cells or isolation of specific cell populations have been developed using a variety of techniques, including physical separation by centrifugation, magnetic, fluorescent and acoustic based selection. Cell types can be separated based on size by centrifugation with or without the use of density gradient media systems (e.g., Ficoll), which allows the leukapheresis product to be cleared of undesirable fractions such as granulocytes, platelets and residual red blood cell contaminants. Iyer et al., (2018) Frontiers in Medicine, Vol. 5, 150. In the manufacture of autologous and allogeneic T cells, it is common to further isolate, select and / or enrich for a desired cell population or phenotype from the apheresis sample prior to using the cells in a specific method or application related to cell therapy. The purpose of this further processing is to obtain highly defined cell populations, such as PBMCs, or specific lymphocyte populations for uses such as T cell activation or expansion, e.g., Stroncek et al. Journal of Translational Medicine Please refer to 2014,12:241-249.

[0022] Procedures to obtain the desired cell populations, such as enrichment for specific T cell phenotypes, are among the most expensive unit operations in the autologous T cell manufacturing process, costing an average of about $10,000 per patient in clinical settings, for example, using CliniMAC Plus magnetic separation technology. Even after enrichment, T cell purity only reaches 90-95%, and about 10% of T cells can be lost in the negative fraction (flow-through).

[0023] The standard procedure for separating red blood cells and granulocytes from PBMCs and lymphocytes in collected blood samples, such as apheresis cells, is by the use of density gradient centrifugation. A typical density gradient material used for this purpose is Ficoll®, a hydrophilic polysaccharide, which separates components in blood samples, such as whole blood or blood collected by an apheresis process or other methods. Ficoll® density gradients separate mononuclear cells, dendritic cells and lymphocytes (T cells, B cells and NK cells) found in the buffy coat from red blood cells, platelets and granulocytes found in the pellet. However, different Ficoll® separation media, protocols, operator skills, etc. can affect cell yield, viability and function. In addition, in some processes, cells are manipulated in hyperosmolar Ficoll® solutions and then resuspended in artificial media, which can have significant modifying effects on T cell function. See Mallone et al., Clinical and Experimental Immunology, 163:33-49 (2010). It has also been shown that lymphocytes expressing high binding to autologous red blood cells result in the loss of these T-rosetting cells during Ficoll® separation. See, e.g., Hokland et al. Scand. J. Immunol, 11, 353-356 (1980).

[0024] Furthermore, the further isolated, selected and / or enriched apheresis cells are subjected to further manipulations such as centrifugation, density gradients, etc. as part of the washing and separation process. This can lead to cell damage and loss due to the shear forces they are repeatedly subjected to during these processes. In addition, every time the cells are handled, either manually or as part of an automated process, the risk of cell exposure and contamination increases. In manufacturing facilities where large volumes of samples from different donors are processed for use in autologous cell therapy applications, contamination of the processed samples with foreign donor cells is a serious risk and danger to the donor patient.

[0025] The methods described herein utilize the entire apheresis sample without further isolation, separation and / or enrichment for specific cell populations or phenotypes. The methods begin with billions of cells collected by leukapheresis, including white blood cells, including monocytes, dendritic cells, lymphocytes (T cells, B cells and NK cells) and granulocytes, as well as nucleated cells such as megakaryocytes, and cells without nuclei, such as red blood cells and platelets. Although only about 30-60% of the cells in the apheresis sample are T cells, other supportive cells have a positive effect on the outcome of the method, contributing to the overall health of T cells from day 0 onwards. Genetically modified T cells derived from these apheresis samples, when processed with other cells in the apheresis sample, proliferated faster, had higher transduction efficiency and maintained a more memory-like state compared to the same apheresis cells further enriched for specific T cell populations.

[0026] Further isolation, selection and / or enrichment of any subset of apheresis cells, such as by cell type or phenotype, was found to be unnecessary to achieve genetically modified T cells with high purity and viability and high transgene expression, and did not affect the ability to generate cell densities well over tens of billions in a 10-day process starting from this mixed cell population. The resulting T cells were less differentiated and remained in a more memory-like phenotype increasing persistence and overall efficacy, and were more capable of killing target cells upon challenge. Starting from apheresis cells eliminated the need for additional separation steps, such as magnetic separation or other sedimentation to isolate the cells of interest, and thus allowed the harvesting of genetically modified T cells from apheresis donor cells with minimal sequential manipulation. This significantly reduces the footprint per patient in the manufacturing facility and the time to process the harvested cells. Furthermore, eliminating the need for donor cell selection or enrichment prior to activation results in less manipulation of the donor sample, less exposure to unnecessary chemicals such as Ficoll®, less overall cell loss, less overall cost, less potential damage to the cells, and significantly less risk of contaminating the patient sample. Autologous cell samples are derived from patients with serious diseases. There is always a risk involved when taking cell samples from these patients and returning the resulting genetically modified T cells to the patient. Exposing the cells to excessive manipulation and unnecessary processing, mishandling during intensive hands-on processing, or exposing the patient to further unnecessary risks by returning contaminated cells to the patient is not a viable option.

[0027] The present invention provides a method of producing genetically modified autologous T cells expressing at least one protein of interest, comprising the steps of seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing a culture medium, where the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM, transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding the protein of interest while continuously rocking at a speed of about 2 RPM, and expanding the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM while increasing the culture volume and rocking speed as needed to maintain the culture until harvest.

[0028] In one embodiment, the apheresis donor cells comprise cells from peripheral blood. In a related embodiment, the apheresis donor cells comprise nucleated and non-nucleated cells. In one embodiment, the apheresis donor cells comprise white blood cells and red blood cells. In a related embodiment, the apheresis donor cells also comprise granulocytes and / or platelets. In one embodiment, the apheresis is leukapheresis. In a related embodiment, the apheresis donor cells are provided in Leukopak®.

[0029] The apheresis donor cells are not subjected to further isolation, selection and / or enrichment for any cell population or cell type after apheresis prior to activation of the apheresis donor cells.

[0030] As will be appreciated, harvested cells can be washed to remove the plasma fraction and any apheresis buffer, and the harvested cells can be placed in an appropriate buffer or medium for further processing. For this purpose, automated closed processes are commercially available, such as Sepax C-Pro (GE Healthcare, Pittsburg, PA), Cobe 2991 cell processor (Terumo BCT, Lakewood, CO), CellSaver 5 (Haemonetics, Boston, MA), etc. In one embodiment, apheresis donor cells are washed and resuspended in culture medium prior to seeding. No further washing steps are performed until the expanded genetically modified autologous T cells are harvested for formulation and cryopreservation.

[0031] T cell activation is an antigen-dependent process that results in the proliferation and differentiation of naive T cells into effector cells. Activation is stimulated by primary and co-activation signals. With the appropriate stimulation, T cells proliferate in vitro. T cells require two signals to be fully activated; the primary stimulation is antigen-specific, due to the interaction of the T cell receptor with peptide-HLA molecules on antigen-presenting cells. The second is a non-antigen-specific co-stimulatory signal, due to the interaction between the membrane of the antigen-presenting cell and the T cell.

[0032] As described herein, activation intensity did not seem to correlate with better transduction efficiency. Although bound activators provided the highest signal intensity, they did not result in high transduction rates. Soluble activators, such as mixtures of soluble CD3, CD28 and / or CD2 antibodies, especially combinations of soluble CD3, CD28 and CD2 antibodies, had signal intensities intermediate between bag-bound and bead-bound activators, but they activated T cells with the highest GFP expression and modified TCR.

[0033] One or more soluble T cell activators can be used to generate an activated T cell population. Such T cell activators include antibodies or functional fragments thereof that target T cell stimulators and / or costimulatory molecules. Such T cell activators include, but are not limited to, anti-CD3 antibodies or binding fragments, anti-CD28 antibodies or binding fragments, and anti-CD2 antibodies or binding fragments, or combinations thereof. Also included are anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and anti-human CD2 monospecific tetrameric antibody complexes. Such T cell activators are commercially available from a variety of sources, including Stem Cell Technologies, Vancouver, BC CA, among others.

[0034] In one embodiment, at least one T cell activator is an anti-CD3 antibody or a binding fragment thereof. In one embodiment, the T cell activator comprises an anti-CD3 antibody and an anti-CD28 antibody, or a binding fragment thereof. In another embodiment, the T cell activator comprises at least an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, or a binding fragment thereof. In another embodiment, the T cell activator comprises at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex.

[0035] In one embodiment, the concentration of the at least one soluble T cell activator is at least 0.001 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration is at least 0.01 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration is at least 0.1 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration is at least 1 μg / ml to at least 10 μg / ml. In a related embodiment, the concentration is at least 5 μg / ml to at least 10 μg / ml. In one embodiment, the concentration of the at least one T cell activator is at least 0.001 μg / ml to at least 1 μg / ml. In a related embodiment, the concentration of the at least one T cell activator is at least 0.01 μg / ml to at least 1 μg / ml. In a related embodiment, the concentration of the at least one T cell activator is at least 0.1 μg / ml to at least 1 μg / ml. In one embodiment, the concentration of the at least one T cell activator is at least 0.001 μg / ml to at least 5 μg / ml. In a related embodiment, the concentration of the at least one T cell activator is at least 0.01 μg / ml to at least 5 μg / ml. In a related embodiment, the concentration of the at least one T cell activator is at least 0.1 μg / ml to at least 5 μg / ml. In a related embodiment, the concentration of the at least one T cell activator is at least 1 μg / ml to at least 5 μg / ml. In one embodiment, the concentration is at least 0.001 μg / ml. In one embodiment, the concentration is at least 0.01 μg / ml. In one embodiment, the concentration is at least 0.1 μg / ml. In one embodiment, the concentration is at least 1 μg / ml. In one embodiment, the concentration is at least 5 μg / ml. In one embodiment, the concentration is at least 10 μg / ml. In one embodiment, the concentration is greater than 10 μg / ml.

[0036] In one embodiment, the culture medium comprises at least one soluble cytokine. Cytokines such as IL-1, IL-2, IL-4, IL-5, IL-7, IL-15 and / or IL-21 can also be used as soluble T cell stimulants. In one embodiment, the soluble cytokine is selected from IL-2, IL-7, IL-15 or IL-21. In one embodiment, the soluble cytokine is IL-7 in combination with IL-15 or IL-21. Such cytokines may be used in concentrations of at least 5 ng / ml to at least 30 ng / ml or more. In one embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 25 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 20 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 15 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 5 ng / ml to at least 10 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 10 ng / ml to at least 20 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 5 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 10 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 15 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 20 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 25 ng / ml. In one embodiment, the concentration of the at least one cytokine is at least 30 ng / ml. In one embodiment, the concentration of the at least one cytokine is greater than 30 ng / ml. Other molecules that affect T cell activation or maturation, such as WNT pathway activators, may also be included. Such activators include the 4,6-disubstituted pyrrolopyrimidine TWS119 (Stemcell Technologies), an inhibitor of the serine / threonine kinase glycogen synthase kinase-3β (Gsk-3β).In one embodiment, the concentration of TWS119 is at least 5 μM to at least 20 μM or more. In one embodiment, the concentration of TWS119 is at least 5 μM to at least 15 μM. In one embodiment, the concentration of TWS119 is at least 5 μM to at least 10 μM. In one embodiment, the concentration of TWS119 is at least 10 μM to at least 20 μM. In one embodiment, the concentration of TWS119 is at least 5 μM. In one embodiment, the concentration of TWS119 is at least 10 μM. In one embodiment, the concentration of TWS119 is at least 15 μM. In one embodiment, the concentration of TWS119 is at least 20 μM. In one embodiment, the concentration of TWS119 is greater than 20 μM. In one embodiment, the culture medium comprises a mixture of soluble TWS117, IL-7 and IL-21.

[0037] In one embodiment, the at least one soluble cytokine is IL-2. In one embodiment, the IL-2 is at a concentration of about 250 IU / ml to about 350 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 250 IU / ml to 300 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 300 IU / ml to 350 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 250 IU / ml, 300 IU / ml or 350 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 250 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 300 IU / ml. In one embodiment, the soluble cytokine is IL-2 at a concentration of about 350 IU / ml.

[0038] During antigen stimulation, T cells transition to glycolytic metabolism to maintain effector function. This occurs in the first few days after adding activator to the cells. During the activation and transduction steps, glycolytic inhibitors can be added along with the activator to inhibit glycolytic metabolism, which can contribute to the generation of T cells that exhibit a less differentiated phenotype, enhanced transduction efficiency, and / or enhanced T cell expression. As described herein, modified T cells derived from 2-deoxy-D-glucose (2-DG)-inhibited T cells produced more Tscm and Tcm compared to T cells derived from media without 2-DG. The inhibitor can be kept in the culture medium until harvest or removed at any time to restore glycolysis and support T cell proliferation. In one embodiment, the culture medium also includes a soluble glycolytic inhibitor. In a related embodiment, the soluble glycolytic inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment, the concentration of 2-DG is about 1 mM to about 3 mM. In related embodiments, the concentration of 2-DG is about 1 mM to about 2 mM. In related embodiments, the concentration of 2-DG is about 2 mM to about 3 mM. In one embodiment, the concentration of 2-DG is about 1 mM or less. In one embodiment, the concentration is about 2 mM. In one embodiment, the concentration of 2-DG is about 3 mM or more.

[0039] The procedure for generating modified T cells includes many unit operations such as cell isolation, selection and / or enrichment, activation, transduction, expansion, harvesting, formulation and cryopreservation of collected donor samples for specific cell types or phenotypes. During each of these steps, multiple vessels or processes may be used to perform each operation. For example, steps such as washing, magnetic antibody labeling, performing selection or enrichment procedures, centrifugation / sedimentation, and enrichment of isolated, selected or enriched cells may be performed using one or more closed system vessels and / or processes to obtain the desired cell type or phenotype. Binding activators for activation and / or binding agents to facilitate transduction are often used, whether they are coated on plates, coated on bags, or bound to beads or other supports. Once stimulation or transduction with these bound activators is complete, the cells must be removed, washed, and transferred to a new vessel. Multiple vessels can be used during expansion to accommodate increased nutrient demands and increased culture volumes. The changeover between different types of vessels involved in these processes is time consuming, expensive, and can expose cells to damage, loss, and contamination.

[0040] As described herein, the present invention provides a closed continuous operation of the entire process from seeding of apheresis donor cells to harvesting of expanded genetically modified T cells. All steps, including activation, transduction and expansion, are performed in a single closed bioreactor system that is constantly rocked at a speed of at least 2 RPM. This not only minimizes manual handling of donor material and reduces the risk of contamination and cell loss, but also allows for automation of feeding and culture operations. Additional equipment such as incubators and separate containers for activation and / or transduction are no longer required for processing of donor cells, greatly reducing the risk of contamination and cell loss, as well as reducing materials / reagents and FTE time, as well as the footprint per patient in the manufacturing facility.

[0041] As described herein, activation, transduction and expansion in a continuous rocking bioreactor utilizing soluble components in a culture medium optimized for T cell expansion resulted in the generation of high densities of modified autologous T cells from washed apheresis donor cells. The use of soluble components such as T cell activators, stimulators and metabolic pathway inhibitors allowed for continuous flow between the various steps in the manufacture of autologous modified T cells, generating modified T cells of the desired phenotype and transgene expression. Rocking during this process also ensured sufficient mass transfer of oxygen and nutrients to support high density cultures, in contrast to static cultures that must grow at low cell densities (≦2E6 cells / ml) due to limitations of culture medium under static conditions. Although both the rocking bioreactor and the commonly used G-Rex® gas permeable culture system have efficient oxygen transport, the G-Rex® system lacks a convective flow that transfers nutrients to the cells in a timely manner. The use of a rocking bioreactor system also allows for perfusion of fresh medium allowing for very high cell densities, in some cases up to 50E6 cells / ml.

[0042] Activation, transduction and expansion of autologous donor apheresis cells, including white and red blood cells, were performed in a closed continuous system in a single continuously rocked bioreactor bag. Bioreactor bags such as the Cellbag™ bioreactor bag (GE Healthcare) are used as part of a rocking bioreactor platform such as the Wave or Xuri cell culture system (GE Healthcare), which in addition to operating as a bioreactor, also have the ability to rock at various speeds and angles that allow efficient mass transfer of oxygen and nutrients without applying excessive shear stress to the T cells. Such bioreactor bags are generally equipped with perfusion filters to filter the incoming culture medium, ports and lines to add culture medium and other components and to remove spent medium and samples, all within a sterile, controlled environment. The bag is also equipped to automatically measure and control culture parameters such as temperature, CO2, O2, pH, metabolic and medium parameters such as glucose and lactate, and interface with the controls of the rocking platform system to control gas and medium flow. The bags allow for automated delivery, whether bolus, fed-batch, fed-batch / perfusion, and / or semi- or continuous perfusion. In addition to reducing the amount of manual handling of the cells, culturing the cells in a closed bioreactor system allows for increased and improved capacity for yield and density of modified T cells.

[0043] The generation of modified autologous T cells for cell therapy applications generally relies on static culture, which utilizes gas-permeable containers, may require extensive manual manipulation, exposes cultures and equipment to contamination risks, and limits nutrient flow to the cells. Static gas-permeable containers, such as bags, flasks, and plates, are widely used in autologous T cell processes, including the transduction step, to generate genetically modified T cells. Examples of such static gas-permeable cell culture bags currently in use include Permalife cell culture bags, Origin Biomedical, Austin Texas, MACS GMP cell differentiation bags, Miltenyi Biotech, Cambridge, MA, Rapid Expansion Flask (G-Rex) bioreactors, other G-Rex® containers, Wilson-Wolf Manufacturing. The containers require an incubator or other environmental control chamber to maintain the desired temperature, and are gas-permeable to allow passive diffusion of O2 from the surrounding incubator environment and CO2 from the culture medium. The containers require manual handling for transfer to and from processing equipment, incubators, or biosafety cabinets, etc., required to maintain the cultures. In addition, the containers require constant connection and reconnection to processing equipment, syringes, pumps, chambers, and other containers and devices to maintain the cultures. Manual handling increases the risk of contamination of the cultures. Furthermore, Permalife cell culture bags are only designed for low density T cell expansion (<2E6 cells / ml) and require extremely large volumes to deliver at the high doses required for T cell therapy. On the other hand, G-rex® can expand T cells at relatively high densities (approximately 10E6 cells / ml), but there are challenges in manipulating the expansion parameters. These two containers also lack the control systems and data recording capabilities to support more advanced and digital manufacturing operations.

[0044] The method of the invention provides for seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing culture medium, where the bioreactor bag is part of a rocking bioreactor platform. The volume of culture medium in the bioreactor bag at the time of seeding can be any predetermined volume. In one embodiment, the volume of culture medium is at least the minimum volume of the bioreactor bag. In one embodiment, the second volume of culture medium is at least 300 ml. In one embodiment, the volume of culture medium is 400 ml or more. In one embodiment, the volume of culture medium is about 300 ml to about 400 ml. In one embodiment, the volume of culture medium is about 325 ml to about 400 ml. In one embodiment, the volume of culture medium is about 350 ml to about 400 ml. In one embodiment, the volume of culture medium is about 375 ml to about 400 ml. In one embodiment, the volume of the culture medium is about 300 ml, about 325 ml, about 350 ml, about 375 ml, or about 400 ml. In one embodiment, the volume of the culture medium is about 300 ml. In one embodiment, the volume of the culture medium is about 325 ml. In one embodiment, the volume of the culture medium is about 350 ml. In one embodiment, the volume of the culture medium is about 375 ml. In one embodiment, the volume of the culture medium is about 400 ml. In one embodiment, the volume of the culture medium is greater than or equal to about 400 ml.

[0045] T cells require cell-cell contact to activate and survive. Insufficient cell concentration during activation can reduce cell-cell contact to a level where activation is insufficient. Apheresis donor cells include nucleated and non-nucleated cells. The number of nucleated cells in an apheresis donor sample can be determined by any known method, for example, by use of an automated system such as the NC-200™ Automated Cell Counter (ChemMetec, Denmark). In one embodiment, the apheresis donor cells include as many nucleated cells as can be fully activated in a bioreactor bag with a rocking speed of 2 RPM. In one embodiment, the apheresis donor cells include about 1.0E9 to about 1.3E9 nucleated cells. In a related embodiment, the apheresis donor cells include about 1.1E9 to about 1.2E9 nucleated cells. In a related embodiment, the apheresis donor cells include about 1.2E9 to about 1.3E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.2E9 to about 1.25E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.0E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.1E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.2E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.25E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.3E9 nucleated cells. In related embodiments, the apheresis donor cells comprise about 1.0E6 nucleated cells, about 1.1E9 nucleated cells, about 1.2E9 nucleated cells, about 1.25E9 nucleated cells, or about 1.3E9 nucleated cells.

[0046] In one embodiment, the invention provides seeding apheresis donor cells into a closed single-use bioreactor bag containing culture medium at a cell density of about 1E6 to about 5E6 nucleated cells / ml. In a related embodiment, the cell density is about 2E6 to about 4E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 to about 4E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 to about 3.75E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 to about 3.5E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 to about 3.35E6 nucleated cells / ml. In one embodiment, the cell density is about 2E6 to about 3E6 nucleated cells / ml. In one embodiment, the cell density is about 2E6 nucleated cells / ml, about 3E6 nucleated cells / ml, about 3.25E6 nucleated cells / ml, about 3.75E6 nucleated cells / ml, or about 4E6 nucleated cells / ml. In one embodiment, the cell density is about 2E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 nucleated cells / ml. In one embodiment, the cell density is about 3.25E6 nucleated cells / ml. In one embodiment, the cell density is about 3.75E6 nucleated cells / ml. In one embodiment, the cell density is about 4E6 nucleated cells / ml. In one embodiment, the cell density is about 3E6 nucleated cells / ml to 4E6 nucleated cells / ml in about 300 ml of culture medium containing soluble cytokines.

[0047] The cells during activation can be cultured at a predetermined temperature. In one embodiment, the cells are cultured at 34-39°C. In one embodiment, the cells are cultured at 34-35°C. In one embodiment, the cells are cultured at 35-37°C. In one embodiment, the cells are cultured at 35-36°C. In one embodiment, the cells are cultured at 36-37°C. In one embodiment, the cells are cultured at 34°C, 35°C, 36°C or 37°C. In one embodiment, the cells are cultured at 34°C. In one embodiment, the cells are cultured at 35°C. In one embodiment, the cells are cultured at 36°C. In one embodiment, the cells are cultured at 37°C.

[0048] The activated cells can be cultured for a predetermined time. In one embodiment, the cells are cultured for at least 12 hours. In one embodiment, the cells are cultured for at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 16 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 14 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 16 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 18 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 18-20 hours. In one embodiment, the cells are cultured for at least 20 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours. In one embodiment, the cells are cultured for at least 13 hours. In one embodiment, the cells are cultured for at least 14 hours. In one embodiment, the cells are cultured for at least 15 hours. In one embodiment, the cells are cultured for at least 16 hours. In one embodiment, the cells are cultured for at least 17 hours. In one embodiment, the cells are cultured for at least 18 hours. In one embodiment, the cells are cultured for at least 19 hours. In one embodiment, the cells are cultured for at least 20 hours. In one embodiment, the cells are cultured for at least 21 hours. In one embodiment, the cells are cultured for at least 22 hours. In one embodiment, the cells are cultured for at least 23 hours. In one embodiment, the cells are cultured for at least 24 hours.

[0049] In one embodiment of the invention, at least one soluble T cell activator binds to at least one donor cell at the time of seeding. In one embodiment, the apheresis donor cells are incubated with one or more soluble T cell activators before seeding into the bioreactor bag. In one embodiment, the incubation is for a period of time sufficient to saturate the binding of the one or more soluble T cell activators to the apheresis donor cells prior to seeding. In one embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 30 minutes or more. In one embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 30 minutes to at least 2 hours or more. In a related embodiment, the apheresis donor cells are incubated with one or more T cell activators for at least 1 hour to at least 2 hours. In one embodiment, the incubation is at least 30 minutes. In one embodiment, the incubation is for about 1 hour. In one embodiment, the incubation is for about 2 hours.

[0050] In one embodiment, the apheresis donor cells and one or more soluble T cell activators are incubated in a transfer bag. A minimal amount of culture medium sufficient to suspend the cells and soluble T cell activators and allow binding of the activators to the nucleated cells, preferably sufficient to retain the total volume of the cells, is used. In one embodiment, the volume of the culture medium in the transfer bag is about 5 ml to about 50 ml. In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml to about 40 ml. In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml to about 30 ml. In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml to about 20 ml. In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml to about 15 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 14 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 13 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 12 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 11 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 10 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 9 ml. In one embodiment of the present invention, the initial volume of the culture medium containing a soluble cytokine and one or more T cell activators is about 5 ml to about 8 ml. In one embodiment of the present invention, the initial volume of culture medium containing soluble cytokines and one or more T cell activators is about 5 ml to about 7 ml.In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml to about 6 ml. In one embodiment of the invention, the initial volume of the culture medium containing the soluble cytokines and one or more T cell activators is about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml, about 20 ml, about 30 ml, about 40 ml, or about 50 ml.

[0051] "Transduction" refers to the process of introducing foreign DNA into cells by viral vectors. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ. As used herein, "vector" refers to any molecule or entity, such as a viral vector, that is useful for transformation of a host cell, such as a T cell, and that contains a nucleic acid sequence that induces and / or controls (in cooperation with the cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto. The vector or vectors are then inserted into a cell for amplification and / or polypeptide expression. Transformation of an expression vector into a selected cell may be achieved via viral transformation. Viral transformed T cells can be obtained by transducing a cell with a viral vector that contains a polynucleotide encoding a protein of interest. Viral vectors include retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors and vaccinia virus vectors.

[0052] The present invention provides for transducing cells in a closed single-use bioreactor bag with at least one viral vector comprising a polynucleotide encoding a protein of interest while continuously rocking at a speed of 2 RPM. The continuous rocking speed of 2 RPM allowed sufficient cell-cell contact required for successful transformation. In one embodiment, the culture is rocked at an angle of 2° at a speed of 2 RPM.

[0053] In one embodiment, the viral vector is a retroviral vector. Retroviral vectors, such as lentiviral vectors, remain in the nucleus as integrated proviruses and are replicated during cell division. In one embodiment, the viral vector is a lentiviral vector.

[0054] In one embodiment, the cells can be modified using one or more viral vectors comprising one or more polynucleotide sequences encoding one or more proteins of interest. In one embodiment, the protein of interest is a cell surface receptor. In one embodiment, the cell surface receptor is a chimeric antigen receptor or a T cell receptor. In one embodiment, the cell surface receptor recognizes an antigen target on the surface of a target cell.

[0055] In one embodiment, the lentiviral vector is added at a multiplicity of infection (MOI) of 0.25 to 10. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 5. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 2. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 1. In one embodiment, the lentiviral vector is added at an MOI of 0.25 to 0.5. In one embodiment, the lentiviral vector is added at an MOI of 0.5 to 10. In one embodiment, the lentiviral vector is added at an MOI of 0.5 to 5. In one embodiment, the lentiviral vector is added at an MOI of 0.5 to 2. In one embodiment, the lentiviral vector is added at an MOI of 0.5 to 1. In one embodiment, the lentiviral vector is added at an MOI of 1 to 10. In one embodiment, the lentiviral vector is added at an MOI of 1 to 5. In one embodiment, the lentiviral vector is added at an MOI of 1 to 2. In one embodiment, the lentiviral vector is added at an MOI of 2 to 10. In one embodiment, the lentiviral vector is added at an MOI of 2 to 5. In one embodiment, the lentiviral vector is added at an MOI of 5 to 10. In one embodiment, the lentiviral vector is added at an MOI of 0.25, 0.5, 1, 2, 5 or 10. In one embodiment, the lentiviral vector is added at an MOI of 10. In one embodiment, the lentiviral vector is added at an MOI of 5. In one embodiment, the lentiviral vector is added at an MOI of 2. In one embodiment, the lentiviral vector is added at an MOI of 1. In one embodiment, the lentiviral vector is added at an MOI of 0.5. In one embodiment, the lentiviral vector is added at an MOI of 0.25.

[0056] The cells can be transduced at a predetermined temperature. In one embodiment, the cells are transduced at 34-39°C. In one embodiment, the cells are transduced at 34-35°C. In one embodiment, the cells are transduced at 35-37°C. In one embodiment, the cells are transduced at 35-36°C. In one embodiment, the cells are transduced at 36-37°C. In one embodiment, the cells are transduced at 34°C, 35°C, 36°C or 37°C. In one embodiment, the cells are transduced at 34°C. In one embodiment, the cells are transduced at 35°C. In one embodiment, the cells are transduced at 36°C. In one embodiment, the cells are transduced at 37°C.

[0057] The cells may be transduced for a predetermined period of time. In one embodiment, the cells are transduced for at least 12 hours. In one embodiment, the cells are transduced for at least 24 hours. In one embodiment, the cells are transduced for at least 12 hours to at least 24 hours. In one embodiment, the cells are transduced for at least 12 hours to at least 20 hours. In one embodiment, the cells are transduced for at least 12 hours to at least 18 hours. In one embodiment, the cells are transduced for at least 12 hours to at least 16 hours. In one embodiment, the cells are transduced for at least 12 hours to at least 14 hours. In one embodiment, the cells are transduced for at least 14 hours to at least 24 hours. In one embodiment, the cells are transduced for at least 14 hours to at least 20 hours. In one embodiment, the cells are transduced for at least 14 hours to at least 18 hours. In one embodiment, the cells are transduced for at least 14 hours to at least 16 hours. In one embodiment, the cells are transduced for at least 16 hours to at least 24 hours. In one embodiment, the cells are transduced for at least 16 hours to at least 20 hours. In one embodiment, the cells are transduced for at least 16 hours to at least 18 hours. In one embodiment, the cells are transduced for at least 18 hours to at least 24 hours. In one embodiment, the cells are transduced for at least 18-20 hours. In one embodiment, the cells are transduced for at least 20 hours to at least 24 hours. In one embodiment, the cells are transduced for at least 12 hours. In one embodiment, the cells are transduced for at least 13 hours. In one embodiment, the cells are transduced for at least 14 hours. In one embodiment, the cells are transduced for at least 15 hours. In one embodiment, the cells are transduced for at least 16 hours. In one embodiment, the cells are transduced for at least 17 hours. In one embodiment, the cells are transduced for at least 18 hours. In one embodiment, the cells are transduced for at least 19 hours.In one embodiment, the cells are transduced for at least 20 hours. In one embodiment, the cells are transduced for at least 21 hours. In one embodiment, the cells are transduced for at least 22 hours. In one embodiment, the cells are transduced for at least 23 hours. In one embodiment, the cells are transduced for at least 24 hours.

[0058] As described herein, transduction was performed using a rocking bioreactor with a single-use bioreactor bag, with a rocking speed of 2 RPM, specifically, at an angle of 2° and a rocking speed of 2 RPM. One or more viral vectors containing a polynucleotide encoding a protein of interest may be inoculated directly into the culture in the bioreactor bag while the bag is rocking. The use of gas-impermeable bags, such as those used in rocking bioreactors, is not recommended for use during transduction. "Gas-impermeable containers, such as disposable bioreactors designed to fit rocking motion bioreactors, are not ideal for transduction of cells via viral vectors. Therefore, transduction is currently performed in static cell culture bags or flat containers" see Farid and Jenkins, Bioprocesses for Cell Therapies, Chapter 44, Biopharmaceutical Processing: Development, Design and Implementation of Manufacturing Processes, Eds. Jagschies et al., Elsevier, page 914, 2018.

[0059] However, as described herein, compared to cells transduced in a static gas permeable vessel, cells transduced in a disposable bioreactor bag designed to be part of a rocking motion bioreactor had higher transduction efficiency, were less differentiated, were more memory cell-like, and were more potent at killing target cells than cells transduced in a static gas permeable bag or the static G-Rex® gas permeable system.

[0060] In one embodiment, after transduction, half of the culture medium is removed from the bioreactor bag and replaced with an equal amount of fresh culture medium and rocked at a speed of 2 RPM. In one embodiment, the culture is rocked at an angle of 2° at a speed of 2 RPM.

[0061] The cells can be cultured at a predetermined temperature. In one embodiment, the cells are cultured at 34-39°C. In one embodiment, the cells are cultured at 34-35°C. In one embodiment, the cells are cultured at 35-37°C. In one embodiment, the cells are cultured at 35-36°C. In one embodiment, the cells are cultured at 36-37°C. In one embodiment, the cells are cultured at 34°C, 35°C, 36°C or 37°C. In one embodiment, the cells are cultured at 34°C. In one embodiment, the cells are cultured at 35°C. In one embodiment, the cells are cultured at 36°C. In one embodiment, the cells are cultured at 37°C.

[0062] The cells can be cultured for a predetermined period of time. In one embodiment, the cells are cultured for at least 12 hours. In one embodiment, the cells are cultured for at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 16 hours. In one embodiment, the cells are cultured for at least 12 hours to at least 14 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 14 hours to at least 16 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 20 hours. In one embodiment, the cells are cultured for at least 16 hours to at least 18 hours. In one embodiment, the cells are cultured for at least 18 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 18-20 hours. In one embodiment, the cells are cultured for at least 20 hours to at least 24 hours. In one embodiment, the cells are cultured for at least 12 hours. In one embodiment, the cells are cultured for at least 13 hours. In one embodiment, the cells are cultured for at least 14 hours. In one embodiment, the cells are cultured for at least 15 hours. In one embodiment, the cells are cultured for at least 16 hours. In one embodiment, the cells are cultured for at least 17 hours. In one embodiment, the cells are cultured for at least 18 hours. In one embodiment, the cells are cultured for at least 19 hours. In one embodiment, the cells are cultured for at least 20 hours. In one embodiment, the cells are cultured for at least 21 hours. In one embodiment, the cells are cultured for at least 22 hours. In one embodiment, the cells are cultured for at least 23 hours. In one embodiment, the cells are cultured for at least 24 hours.

[0063] The present invention provides for growing cells in a closed single-use bioreactor bag at a rocking speed of about 2 RPM, while increasing the culture volume and rocking speed as needed to maintain the culture until harvest. T cells require cell-cell contact to survive. Below 2E6 cells / ml, cell-cell contact may not be possible, cell stress may increase, and / or the ability to overcome the agitation of the bioreactor and grow may be reduced. Increasing to 4E6 cells / ml or more allows for an increase in culture volume, which also simplifies the feeding process. A stepwise increase in culture volume allows the culture to grow to 1 liter in a timely manner, with the rocking speed / angle corresponding to the increase in volume and stabilizing the amount of dissolved oxygen in the system to promote cell growth. During activation, T cells consume and release large amounts of glucose and lactate. High levels of lactate appear to be detrimental to T cell activation and growth. As a result, during the early stages of activation and transduction, cell medium exchange is not a parameter that corresponds to cell density, since the cells are not growing very much. During activation / transduction, lactate levels are a critical culture parameter and sufficient turnover of medium is important for T cell activation and proliferation. Semi-continuous medium exchange is used to control metabolite levels in the culture medium.

[0064] T cells can reach higher densities when cultured in a rocking bioreactor platform than in a static gas permeable vessel system. The final yield of the method described herein was 15-35 billion cells in 10-14 days in a 1 L bioreactor. One bioreactor is sufficient to support one patient. Approximately 20 permeable bags (250 ml) or a 4 L G-rex® bioreactor would be required to obtain the same 10 billion modified T cells produced in one liter by the method of the present invention. The cell yield of the method of the present invention is higher than that of other common methods used in practice.

[0065] In one embodiment, as the cells grow, the volume of the culture medium is gradually increased to maintain a cell density of at least 2E6 nucleated cells / ml. In one embodiment, as the cells grow, the volume of the culture medium in the bioreactor is gradually increased to 1 liter during growth to maintain a cell density of at least 4E6 nucleated cells / ml. In one embodiment, the cell density during growth is at least 2E6 cells / ml or more. In one embodiment, the cell density is about 2E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 3.50E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 3.25E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 3.00E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 2.75E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 2.50E6 cells / ml. In one embodiment, the cell density is about 2E6 cells / ml to about 2.25E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 3.50E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 3.25E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 3.0E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 2.75E6 cells / ml. In one embodiment, the cell density is about 2.25E6 cells / ml to about 2.50E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 3.50E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 3.25E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 3.00E6 cells / ml.In one embodiment, the cell density is about 2.50E6 cells / ml to about 2.75E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 2.50E6 cells / ml. In one embodiment, the cell density is about 2.50E6 cells / ml to about 2.75E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 3.50E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 3.25E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 3.00E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 2.75E6 cells / ml. In one embodiment, the cell density is about 2.75E6 cells / ml to about 2.50E6 cells / ml. In one embodiment, the cell density is about 3.0E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 3.0E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 3.0E6 cells / ml to about 3.50E6 cells / ml. In one embodiment, the cell density is about 3.0E6 cells / ml to about 3.25E6 cells / ml. In one embodiment, the cell density is about 3.25E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 3.25E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 3.25E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 3.50E6 cells / ml to about 4.0E6 cells / ml. In one embodiment, the cell density is about 3.50E6 cells / ml to about 3.75E6 cells / ml. In one embodiment, the cell density is about 3.75E6 cells / ml to about 4.0E6 cells / ml.

[0066] In one embodiment, during growth, fresh culture medium is added to the bioreactor by a combination of fed-batch / perfusion feeding and / or by perfusion. In one embodiment, fresh culture medium is added by fed-batch / perfusion until the culture reaches a volume of at least 1 liter, and then switched to perfusion until the culture is harvested. In one embodiment, the culture is perfused throughout the growth period until harvest. In one embodiment, during growth, the perfusion rate is at least one bioreactor bag volume per day. In one embodiment, the perfusion rate is less than one bioreactor bag volume per day. In one embodiment, the perfusion rate is more than one bioreactor bag volume per day.

[0067] In one embodiment, the volume of the culture medium in the bioreactor is gradually increased to 1000 ml during growth to maintain a cell density of about 2E6 to about 4E6 nucleated cells / ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 600 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 700 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 800 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 900 ml to at least 1000 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 600 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 700 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 800 ml to at least 900 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 800 ml.In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 700 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 700 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 600 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 700 ml to at least 800 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 600 ml to at least 700 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 600 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 600 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 600 ml. In one embodiment, the volume of the bioreactor is increased from at least 500 ml to at least 600 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 500 ml.In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 500 ml. In one embodiment, the volume of the bioreactor is increased from at least 400 ml to at least 500 ml. In one embodiment, the volume of the bioreactor is increased from at least 200 ml to at least 400 ml. In one embodiment, the volume of the bioreactor is increased from at least 300 ml to at least 400 ml. In one embodiment, the volume of the bioreactor is at least 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, at least 500 ml, at least 550 ml, at least 600 ml, at least 650 ml, at least 700 ml, at least 750 ml, at least 800 ml, at least 850 ml, at least 900 ml, at least 950 ml, at least 1000 ml, or more than 1000 ml. In one embodiment, the volume of the bioreactor is at least 200 ml. In one embodiment, the volume of the bioreactor is at least 300 ml. In one embodiment, the volume of the bioreactor is at least 400 ml. In one embodiment, the volume of the bioreactor is at least 500 ml. In one embodiment, the volume of the bioreactor is at least 600 ml. In one embodiment, the volume of the bioreactor is at least 700 ml. In one embodiment, the volume of the bioreactor is at least 800 ml. In one embodiment, the volume of the bioreactor is at least 900 ml. In one embodiment, the volume of the bioreactor is at least 1000 ml. In one embodiment, the volume of the bioreactor is greater than 1000 ml.

[0068] In one embodiment, the rocking speed increases with increasing culture volume and / or cell density. In one embodiment, the rocking speed increases from at least 2 RMP to at least 3 RPM. In one embodiment, the rocking speed increases from at least 2 RMP to at least 4 RPM. In one embodiment, the rocking speed increases from at least 2 RMP to at least 5 RPM. In one embodiment, the rocking speed increases from at least 2 RMP to at least 6 RPM. In one embodiment, the rocking speed increases from at least 3 RMP to at least 4 RPM. In one embodiment, the rocking speed increases from at least 3 RMP to at least 5 RPM. In one embodiment, the rocking speed increases from at least 3 RMP to at least 6 RPM. In one embodiment, the rocking speed increases from at least 4 RMP to at least 5 RPM. In one embodiment, the rocking speed increases from at least 4 RMP to at least 6 RPM. In one embodiment, the rocking speed increases from at least 5 RMP to at least 6 RPM. In one embodiment, the rocking speed is at least 2 RPM at a 2° angle. In one embodiment, the rocking speed is at least 3 RPM at a 3° angle. In one embodiment, the rocking speed is at least 4 RPM at a 4° angle. In one embodiment, the rocking speed is at least 5 RPM at a 5° angle. In one embodiment, the rocking speed is at least 6 RPM at a 6° angle.

[0069] In one embodiment, the rocking speed is gradually increased to 6 RPM as the volume of culture medium gradually increases to 1000 ml. In one embodiment, the volume of culture medium in the bioreactor bag at the start of expansion is about 300 ml at a rocking speed of 2 rpm at an angle of 2° and is maintained at a perfusion rate of 1 bioreactor bag volume per day. In one embodiment, when the cell density of the culture reaches 4E6 nucleated cells / ml, the volume of culture medium in the bioreactor bag is increased to 600 ml at an angle of 4°, a rocking speed of 4 rpm and a perfusion rate of 1 bioreactor bag volume per day. In a related embodiment, when the cell density of the culture reaches 4E6 nucleated cells / ml, the volume of culture medium in the bioreactor bag is increased to 1000 ml at an angle of 6°, a rocking speed of 6 rpm and a perfusion rate of 1 bioreactor bag volume per day.

[0070] In one embodiment, the culture in the single-use bioreactor bag is maintained at about 80-100% O2.

[0071] In one embodiment, the cells are grown for 7-14 days. In one embodiment, the cells are grown for 7-13 days. In one embodiment, the cells are grown for 7-12 days. In one embodiment, the cells are grown for 7-11 days. In one embodiment, the cells are grown for 7-10 days. In one embodiment, the cells are grown for 7-9 days. In one embodiment, the cells are grown for 7-8 days. In one embodiment, the cells are grown for 8-14 days. In one embodiment, the cells are grown for 8-13 days. In one embodiment, the cells are grown for 8-12 days. In one embodiment, the cells are grown for 8-11 days. In one embodiment, the cells are grown for 8-10 days. In one embodiment, the cells are grown for 8-9 days. In one embodiment, the cells are grown for 9-14 days. In one embodiment, the cells are grown for 9-13 days. In one embodiment, the cells are grown for 9-12 days. In one embodiment, the cells are grown for 9-11 days. In one embodiment, the cells are grown for 9-10 days. In one embodiment, the cells are grown for 10-14 days. In one embodiment, the cells are grown for 10-13 days. In one embodiment, the cells are grown for 10-12 days. In one embodiment, the cells are grown for 10-11 days. In one embodiment, the cells are grown for 11-14 days. In one embodiment, the cells are grown for 11-13 days. In one embodiment, the cells are grown for 11-12 days. In one embodiment, the cells are grown for 12-14 days. In one embodiment, the cells are grown for 12-13 days. In one embodiment, the cells are grown for 13-14 days. In one embodiment, the cells are grown for 7, 8, 9, 10, 11, 12, 13, 14 days or more. In one embodiment, the cells are grown for at least 7 days. In one embodiment, the cells are grown for at least 8 days. In one embodiment, the cells are grown for at least 9 days. In one embodiment, the cells are grown for at least 10 days. In one embodiment, the cells are grown for at least 11 days. In one embodiment, the cells are grown for at least 12 days. In one embodiment, the cells are grown for at least 13 days. In one embodiment, the cells are grown for at least 14 days.

[0072] In one embodiment, the cells are grown at 34-39°C. In one embodiment, the cells are grown at 34-35°C. In one embodiment, the cells are grown at 35-37°C. In one embodiment, the cells are grown at 35-36°C. In one embodiment, the cells are grown at 36-37°C. In one embodiment, the cells are grown at 34°C, 35°C, 36°C or 37°C. In one embodiment, the cells are grown at 34°C. In one embodiment, the cells are grown at 35°C. In one embodiment, the cells are grown at 36°C. In one embodiment, the cells are grown at 37°C.

[0073] "Modified T cells" and "genetically modified T cells" are used interchangeably and refer to T cells that have been modified by the introduction of additional genetic material, particularly a polynucleotide that encodes a protein of interest, such as a receptor, to be expressed on the cell surface. Examples of cell surface receptors include chimeric antigen receptors (CARs) and T cell receptors (TCRs). The terms "isolated modified T cells" and "isolated genetically modified T cells" refer specifically to T cells that have been modified or engineered, such as genetically modified, or that are not normally found in nature. As used herein, T cells or T lymphocytes refer to a type of lymphocyte that actively participates in the body's immune response.

[0074] The terms "polynucleotide" or "nucleic acid molecule" are used interchangeably throughout and include both single-stranded and double-stranded nucleic acids, and include genomic DNA, RNA, mRNA, cDNA, or synthetic origin or some combination thereof that is not associated with a sequence normally found in nature. The term "isolated polynucleotide" or "isolated nucleic acid molecule" specifically refers to a sequence of synthetic origin or one that is not normally found in nature. An isolated nucleic acid molecule that includes a specific sequence may, in addition to the specific sequence, include sequences that code for up to 10 or up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences. The nucleotides that comprise the nucleic acid molecule may be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and modifications of internucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates and phosphoroamidates.

[0075] The terms "polypeptide" or "protein" are used interchangeably throughout and refer to molecules that contain two or more amino acid residues linked together by peptide bonds. Polypeptides and proteins also include macromolecules that have one or more deletions, insertions, and / or substitutions from the amino acid residues of the native sequence, i.e., polypeptides or proteins that contain molecules produced by naturally occurring non-recombinant cells or by genetically engineered or recombinant cells and have one or more deletions, insertions, and / or substitutions from the amino acid residues of the native protein's amino acid sequence. Polypeptides and proteins also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. Polypeptides and proteins also include modifications, including, but not limited to, glycosylation, lipid conjugation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Polypeptides and proteins of interest may be of scientific or commercial interest, for example, those that are useful as components of cell therapy. Proteins of interest include, inter alia, membrane-bound proteins.

[0076] The present invention provides a method for generating engineered T cells expressing a protein of interest. In one embodiment, the protein of interest is a cell surface receptor. The cell surface receptor is a genetically engineered receptor such as a T cell receptor (TCR) and a chimeric antigen receptor (CAR or CAR-T cell, TRUCK (a chimeric antigen receptor that redirects T cells for universal cytokine-mediated killing), and an armoured CAR (designed to regulate the immunosuppressive environment)), as well as other proteins including antigen binding molecules that interact with their target antigen. These engineered receptors are expressed on the surface of the T cells. In one embodiment, the cell surface receptor is a T cell receptor or a chimeric antigen receptor.

[0077] TCR is a membrane-anchored heterodimeric protein complex that contains an α-chain and a β-chain complexed with an invariant CD3 chain molecule. The variable domains of the TCR α-chain and β-chain each have three complementarity determining regions (CDRs) that recognize peptides derived from proteins presented by / bound within the groove of MHC / HLA molecules, and a constant domain that participates in disulfide bonds to link the chains. CD3 zeta activates T lymphocytes by signaling. TCR has the potential to recognize antigens presented on the surface of many cells, such as cancer cells, inflammatory cells, and cells from other sources.

[0078] CARs are engineered transmembrane proteins that include an extracellular domain, typically an antigen-binding protein or domain that binds to an antigen of interest, a hinge region, a transmembrane domain, and an intracellular (cytoplasmic) domain.

[0079] The extracellular domain may be derived from synthetic or natural sources, such as the proteins described herein. The extracellular domain often includes a hinge portion, sometimes referred to as a "spacer" region. The hinge may be derived from the proteins described herein, particularly the costimulatory proteins described herein, and immunoglobulin (Ig) sequences or other suitable molecules to achieve a desired specific distance from the target cell.

[0080] The transmembrane domain can be fused to the extracellular or intracellular domain of the CAR. The transmembrane domain can be derived from synthetic sources or natural sources, such as the proteins described herein, particularly the costimulatory proteins described herein.

[0081] The intracellular (cytoplasmic) domain can be fused to the transmembrane domain and provide activation of at least one of the normal effector functions of an immune cell. Effector functions include cytolytic activity or helper activity, including secretion of cytokines. The intracellular domain can be derived from a protein described herein, in particular from CD3.

[0082] CARs can be engineered to bind to antigens (such as cell surface antigens) by incorporating an antigen-binding molecule that interacts with the targeted antigen. CARs typically incorporate an antigen-binding domain (such as an scFv) in conjunction with one or more costimulatory ("signaling") domains and one or more activation domains.

[0083] In one embodiment, the antigen-binding molecule is an antibody fragment thereof, more preferably one or more single-chain antibody fragments ("scFv"). scFvs are suitable for use in CARs because they can be engineered to be expressed as part of a single chain with other CAR components. See Krause et al., J. Exp. Med., 188(4):619-626, 1998; Finney et al., Journal of Immunology, 161:2791-2797, 1998.

[0084] CARs incorporate one or more costimulatory (signaling) domains to enhance their efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause, et al. and Finney, et al. (supra); Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).Suitable costimulatory domains include, among other sources, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1, CD154), and the like. la / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrins, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta , IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-l b, ITGAX, CDl-lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The co-stimulatory domain may be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, 41-BB, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or a fragment or combination thereof. The co-stimulatory domain may comprise one or more extracellular portions, transmembrane portions, and intracellular portions.

[0085] CARs also contain one or more activation domains. CD3 zeta is an element of the T cell receptor on natural T cells and has been shown to be a key intracellular activation element in CARs.

[0086] Cell surface molecules can be targeted with traditional CARs. Intracellular molecules can be targeted with TCRs that recognize peptides derived from proteins presented by or bound within the groove of MHC / HLA molecules. Such targets include alpha folate receptor, 5T4, AFP, ADAM17, 17-A, ART-4, alpha vβ6 integrin, BAGE, Bcr-abl, BCMA, B7-H3, B7-H6, CAIX, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8, CDC27m, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70 (CD27L or TNFSF7), CD79a, CD79b, CD123, CD138, CD171, CDK4 / m, cadherin 19 (CD H19), placental-type cadherin (CDH3), CEA, CLL-1, CSPG4, CT, Cyp-B, DAM, DDL3, EBV, EGFR, EGFRvIII, EGP2, EGP40, ELF2M, ErbB2 (HER2) , EPCAM, EphA2, EpCAM, ETV6-AML1, FAP, fetal AchR, FLT3, FRα, G250, GAGE, GD2, GD3, glypican-3 (GPC3), GNT-V, GP-100, HAGE, HBV, HCV, HER-2 / neu, HLA-A, HPV, HSP70, HST-2, hTERT, iCE, IgE, IL-11Rα, IL-13Rα2, kappa, KIAA0205, LAGE, lambda, LDLR / FUT, Lewis-Y, MAGE, MAGE1, MAGEB2, MART-1 / Melan-A, MC1R, MCSP, MUM-1, MUM-2, MUM-3, mesothelin (MSLN), Muc1, Muc16, myosin / m, N A88-A, NCAM, NKG2D ligand, NY-ESO-1, P15, p190 minor bcr-abl, PML / RARa, PRAME, PSA, PSCA, PSMA, RAGE, ROR1, RU1, RU2, SAGE, SART, SSX-1, SSX-2, SSX-3, survivin, TAA, TAG72, TEL / AML1, TEMs, TPI, TRP-1, TRP-2, TRP-2 / INT2, VEGFR2, and WT1.Loeffler et al.,Blood 85(6):2098-2103,15 March 2000;Wolf et al.,Drug Discovery Today10(8):1237-1244,September 2005;Baeuerle&Reinhardt,Cancer Res 69(12):4941-4944,June 15,2009;Baeuerle et al.,Curr.Opin,Mol.Ther.11:22-30,2009;Nagorsen&Baeuele,Exp Cell Res 317:1255-1260,2011;Nagorsen et al.,Pharmacology&Therapeutics 136:334-342,2012;Huehls et al. al.,Immunology and Cell Biology 93:290-296, 2015; and Stieglmaier et al., Expert Opin Biol Ther 15(8):1093-1099, 2015.

[0087] This method can be used to generate T cells expressing CD19 CARs, such as (Kymriah™) and (Yescarta®).

[0088] In one embodiment, the cell surface receptor recognizes an antigen target associated with the target cell, such as a cell surface molecule or an intermolecular molecule. The target cell is any cell that presents the antigen recognized by the gene of interest. In one embodiment, the target cell is a cancer cell. The cancer cell is a cell associated with cancer, for example, a metastatic tumor, a solid tumor, a blood cancer, a brain cancer, a breast cancer, a colon cancer, a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer, a spleen cancer, a stomach cancer, a thyroid cancer.

[0089] High transduction efficiency allows smaller, more effective doses to be delivered to patients. If there is less generation of T cells transduced with the gene of interest, fewer genetically modified T cells will be available for administration to patients, resulting in less effective doses. The methods described herein achieve high transduction rates (high transgene expression >40% in CD8 T cells) at lower lentiviral vector MOI. Fewer lentiviral vectors are required to generate more T cells expressing the protein of interest, making the methods more efficient and cost-effective. The present invention relates to a method for increasing transgene expression in genetically modified autologous T cells expressing a protein of interest, comprising the steps of seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing a culture medium, wherein at least one soluble T cell activator is bound to at least one donor cell at the time of seeding, and the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM, and culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM. and growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture until harvest, wherein expression of the transgene is greater than expression of the transgene in genetically modified autologous T cells derived from an enriched population of T cells from the same apheresis donor cells and expressing the same protein of interest.

[0090] The genetically modified T cells are harvested when the cell density reaches a desired or predetermined state or at a predetermined or desired time point. The genetically modified T cells may be harvested from the bioreactor using methods known in the art, such as separation using gravity or centrifugation. The cells may also be harvested using semi-automated and fully automated cell separation systems, including blood separation systems. Such systems are commercially available and include the Sepax® 2 and Sefia™ S-2000 Cell Processing Systems (GE Healthcare), the Cobe 2991 Cell Processor (Terumo BCT, Lakewood, CO, CellSaver 5 (Haemonetics, Boston, MA), and others. The harvested cells are typically washed and concentrated into a formulation suitable for administration to patients and cryopreservation.

[0091] The methods described herein have yielded over 10 billion with over 90% viability in a 10 day process, and after extending cell growth for several more days, yields of over 20 billion have been seen.

[0092] Samples can be taken during expansion and / or at harvest for quality control testing. Such QC testing can include quantifying transduction efficiency of genetically modified T cells, such as determining cell surface TCR% using flow cytometry and vector copy number using qPCR to measure integration. Viability can also be tested prior to releasing the product for delivery to patients.

[0093] Standard procedures known in the art can be used to cryopreserve the harvested genetically modified T cells for storage and / or preparation for use in human subjects. In one embodiment, the harvested cells were eluted at 2E8 cells / ml in saline supplemented with 1% HSA and further formulated at a 1:1 ratio with Hyclone cryopreservation medium (GE Healthcare) supplemented with 5% human serum albumin.

[0094] A fraction of the formulated modified T cells, as well as any apheresis cells not used in the present methods, can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients.

[0095] When required for treatment, the cryopreserved genetically modified T cells are thawed and diluted in saline buffer or other suitable medium for administration to the donor by infusion in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin. Volume is typically kept to a minimum as much as possible, and the pharma- ceutically acceptable dose is based on the number and viability of the cells, the condition being treated, and the patient. Therapeutic doses are typically millions to billions of cells, depending on the patient's condition and needs, among other factors. An exemplary dose can be ≦100 ml at about 100E6 cells / ml.

[0096] The present invention provides a method of treating a patient with genetically modified autologous T cells expressing a protein of interest, comprising the steps of incubating apheresis cells from the patient with one or more T cell activators selected from the group consisting of anti-CD3, anti-CD2 and anti-CD28 antibodies, or binding fragments thereof, to saturate antibody binding, seeding the apheresis cells into a closed single-use bioreactor bag containing culture medium, wherein the bioreactor bag is part of a rocking bioreactor platform, culturing the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM, culturing the cells without continuous rocking at a speed of about 2 RPM, and culturing the cells without continuous rocking at a speed of about 2 RPM. and transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding a protein of interest; growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture at a desired cell density until harvest; harvesting and formulating the cells for cryopreservation; freezing and storing the cells until needed for administration to a patient; thawing the cells and resuspending them in medium suitable for infusion; and reintroducing a pharmacologic effective amount of the genetically modified autologous T cells expressing the protein of interest into the patient.

[0097] The formulated modified T cells may be administered as a pharmaceutical composition alone or in combination with other components, such as a diluent and / or IL-2 or other cytokines or cell populations.

[0098] Methods are provided for using engineered T cells to treat a condition, symptom, disease, disorder, and the like. The T cells express a gene of interest, such as a cell surface receptor, and recognize an antigen target associated with a target cell associated with the condition, symptom, disease, disorder, and the like. Such conditions, diseases, or disorders include cancer, tumors, solid tumors, hematological diseases, leukemias, lymphomas, viral infections, inflammatory diseases or disorders, and / or autoimmune diseases or disorders. In one embodiment of the invention, the genetically engineered T cells are used to treat a condition in a subject. In one embodiment of the invention, the genetically engineered T cells are used to treat a cancer patient. In some embodiments, the invention provides for the generation of a T cell-mediated immune response in a donor comprising administering to the donor an effective amount of engineered autologous T cells expressing a protein of interest. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered autologous T cells comprise a genetic construct expressing one or more chimeric antigen receptors, T cell receptors, and / or other proteins of interest. In some embodiments, the target cells are tumor cells. In some embodiments, the invention includes methods for treating or preventing a condition, the methods comprising administering to a subject in need thereof an effective amount of genetically modified autologous T cells generated by the methods described herein. In some aspects, the invention includes methods for treating or preventing inflammation and / or autoimmune disorders.

[0099] The terminology used in this application is standard within the art, but definitions of certain terms are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols may be indicated in their SI accepted form. Numerical ranges described herein are inclusive of the numbers defining the range, and include and support each integer within the defined range. Unless otherwise stated, the methods and techniques described herein are generally carried out according to conventional methods well known in the art, and such methods and techniques are described in various general and more specific references cited and discussed throughout this specification. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and academic papers, are expressly incorporated herein by reference.

[0100] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the above and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. What is described in one aspect or embodiment of the invention can be combined with other aspects and / or embodiments of the invention. EXAMPLES

[0101] Example 1 Comparative study of three processes 1: Autologous T cells generated using a closed continuous autologous T cell bioprocessing method from activation to expansion in the Xuri W25 cell culture system. "Xuri W25 bioreactor." 2: G-Rex® Bioprocessing System from Activation to Expansion in 6 and 24-well Plates. "G-Rex". 3: A hybrid bioprocessing system in which activation and transduction are performed in a gas-permeable bag followed by expansion of T cells in the Xuri W25 cell culture system. "Permeable bag".

[0102] Additionally, two different culture media supplemented with IL2 (medium 1) or TWS119+IL7+IL21 (medium 2) were also compared using a closed continuous autologous T cell bioprocessing system and G-Rex® plates.

[0103] The T cell generation was assessed for their purity, phenotype and in vitro characteristics.

[0104] On day 0, a fresh Leukopack® (Hemacare, Northridge, CA) containing 300 ml of fresh leukopheresis product collected from normal peripheral blood was sterilely connected to a CD-600.1 Sepax cell separation kit (GE) and processed under the Culture Wash-Pro program. The Leukopack® contained white blood cells, red blood cells and platelets. The cells were washed in 1 L ClinMACS PBS / EDTA, 5 ml human serum albumin (Miltenyi Biotec, San Diego, CA) and plasma and apheresis buffer were removed using a Sepax CPro cell separation system with a CS-600.1 kit (GE Healthcare) according to the manufacturer's instructions. Based on the initial white blood cell (WBC) count indicated on the donor information sheet attached to the Leukopack®, the cells were eluted with approximately 50 ml of OpTimizer complete medium at a cell density of 150E6 WBC / ml.

[0105] Nucleated cells in the washed leukapheresis samples were counted using an NC-200™ automated cell counter (ChemMetec, Denmark), followed by total viable cell count, viability and immunophenotypic analysis of the washed cells.

[0106] 1) Closed continuous autologous T cell bioprocessing using the Xuri W25 cell culture system. "Xuri W25 Bioreactor" A portion of the washed apheresis donor cells containing 1.2E9 nucleated cells was then transferred to two transfer bags (Charter Medicine, Winston-Salem, NC) using the Sepax C-Pro processing system with the same CD-600.1 kit under the Dilute program. To one bag, approximately 8 ml of Medium 1: (OpTmizer complete medium (ThermoFisher) with 300 IU / mL rhIL2) was added. To the second bag, approximately 8 ml of Medium 2: (OpTmizer complete medium containing 5 μM TWS119 (Cayman Chemical, Ann Arbor, MI), 20 ng / ml IL7 (Stemcell Technologies, Cambridge, MA) and 20 ng / ml IL-21 (Stemcell Technologies)) was added. Each bag was incubated with 7.5 ml of ImmunoCult™ human CD3 / CD28 / CD2 (25 μl / ml cells, Stemcell Technologies) at room temperature for 1 hour to allow saturation of antibody binding. The concentration of Immunocult added was determined in a final activation culture volume of 300 ml as described in the following steps. OpTmizer complete medium: OpTimizer T cell growth medium (ThermoFisher, Waltham, MA), 2.5% Immune Cell SR supplement (ThermoFisher), 2.6% CTS T cell supplement (ThermoFisher), 1% GlutaMax (ThermoFisher), 0.1% Pluronic F68, and 300 IU / ml IL2.

[0107] A 2L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to a Xuri Cell Culture System W25 and 300ml of Medium 1 was added and equilibrated at 37°C, 5% CO2, 0.1 L / min gas flow rate, and 6 rpm rocking speed at an angle of 6°. A second 2L Xuri SP Perf Cellbag bioreactor was also connected to a Xuri Cell Culture System W25 and 300ml Medium 2 was similarly equilibrated.

[0108] The transfer bags containing cells in Medium 1 and Medium 2 were sterilely connected to the Xuri Cellbag feed line and the contents were transferred into the bag by gravity. The cells were then cultured overnight at 37°C, 5% CO2, 0.1 L / min gas flow rate, rocking speed of 2 rpm at a 2° angle to activate the T cells.

[0109] On day 1, nucleated cells in each bag were counted. An amount of lentiviral vector (containing a polynucleotide encoding the TCR) corresponding to an MOI of 1 functional titer was diluted in 10 ml of medium 1 or medium 2 and placed in a transfer bag. The transfer bag was sterile connected to a Xuri Cellbag feed line and the lentivirus was transferred by gravity flow into each of the bioreactors. The cells were incubated for 20-24 h at 37 °C, 5% CO2, a gas flow rate of 0.1 L / min, and a rocking speed of 2 rpm at a 2° angle.

[0110] On day 2, approximately half the volume of culture medium in each bioreactor bag was replaced using three 50 ml bag washes through the feed and waste lines. Cells were counted daily and viable cell density (VCD) and viability were measured using an NC200™ automated cell counter (ChemMetec), dissolved oxygen and metabolites were also measured. Cell phenotype was determined for all samples tested. Bags were maintained on a rocking bioreactor at 2° angle, 2 rpm, 37° C., 5% CO2, 0.1 L / min gas flow for 24 hours.

[0111] On days 3-7, additional OpTimizer complete medium containing IL-2 (Medium 1) or TWS119 / IL17 / IL21 (Medium 2) was added to both cultures by fed-batch / perfusion feeding to maintain cell density above 2E6 cells / ml to a final volume of 1 L. With increasing volume, the rocking speed and angle was increased to 4 RPM at an angle of 4° to maintain cell density at the desired level.

[0112] Total viable cell density, viability, glucose and lactate measurements were performed daily. Phenotypic analysis was performed on days 3, 5 and 7.

[0113] On days 7–10, both cultures were switched to perfusion feeding at a rate of 1 bag volume / day, where both bioreactor bags were fed with Medium 1. As cell density increased, dissolved O2 levels were maintained at 80% by feedback control.

[0114] Total viable cell density, viability, glucose and lactate were measured daily. Phenotypic analysis was performed on day 10.

[0115] Cells were harvested on day 10. The Xuri SP Per bioreactor bag was sterilely connected to a Selfia5200 cell processing system using the FlexCell program and the CT-800.1 cell processing kit (GE Healthcare). T cells were concentrated to approximately 20 ml at a flow rate of 75 ml / min. One wash cycle was performed with 0.9% saline (Baxter, Deerfield, Ill.) supplemented with 1 vol% human serum albumin (HSA). Washing was performed at 380×g for 5 min.

[0116] Cells were cryopreserved by reconstituting an equal volume of saline + 1% HSA + HyClone™ Cryopreservation Medium (GE Healthcare) supplemented with 5% HSA, freezing to -80°C using a controlled rate freezer Via Freeze (GE Healthcare), and storing in liquid nitrogen.

[0117] 2.G-rex plate "G-rex". Cells were cultured in 24-well or 6-well G-Rex® (WilsonWolf, New Brighton, MN) plates using Medium 1 and Medium 2 according to the manufacturer's protocol. 1E6 cells / well were seeded in G-Rex® plates and activated with ImmunoCult™ human CD3 / CD28 / CD2 (medium with 25ug / ml cells) on day 0. Approximately 24 hours after activation, cells were seeded at 1E6 cells / well (concentration 2E6 cells / mL) and an amount of the same lentiviral vector (containing a polynucleotide encoding a TCR) corresponding to an MOI of 1 functional titer was added to each well. On day 2, Medium 1 and Medium 2 were added to the full volume of the well (6mL total) to dilute the virus. The medium was changed and the cells were fed every 2-3 days.

[0118] Cells were counted daily and viable cell density (VCD) and viability were measured using an NC200™ automated cell counter (ChemMetec), dissolved oxygen and metabolites were also measured. Cell phenotype was determined for all samples tested.

[0119] 3. A hybrid bioprocessing system in which activation and transduction are performed in a gas-permeable bag followed by expansion of T cells in the Xuri W25 cell culture system. "Perfusion bag". Washed apheresis donor cells containing 500E6 nucleated cells were transferred to gas-permeable bags (OriGen, Austin, TX) with 6 ml of ImmunoCult™ human CD3 / CD28 / CD2 and incubated at 37° C., 5% CO2 for 24 hours.

[0120] Nucleated cells were counted. An amount of the same lentiviral vector (containing aTCR-encoding polynucleotide) corresponding to an MOI of 1 functional titer was added directly to the bag using a syringe and incubated overnight at 37° C., 5% CO2.

[0121] The cells were then split into two gas-permeable bags the next day and incubated in the bags at 37° C., 5% CO 2 , up to a total of 1 billion nucleated cells.

[0122] The cells were then seeded into a 2L Xuri SP Perf Cellbag bioreactor bag on a Xuri W25 cell culture system (GE Healthcare) containing 300ml of OpTimizer complete medium with 300IU / ml IL2, rocking at an angle of 6° and a speed of 6 rpm. The bioreactor was scaled up to a volume of 1L 24 hours after seeding. Fresh medium was perfused at 500ml / day for 1 day, followed by 1L / day until harvest.

[0123] Cells were counted daily and viable cell density (VCD) and viability were measured using an NC200™ automated cell counter (ChemMetec), dissolved oxygen and metabolites were also measured. Cell phenotype was determined for all samples tested.

[0124] Cells were harvested, concentrated, and further cryopreserved by formulating at a 1:1 ratio with HyClone™ Cryopreservation Medium (GE Healthcare) supplemented with 5% HSA, freezing to -100°C using a Via Freeze (GE Healthcare), and storing in liquid nitrogen. Viability and recovery at the time of harvest were determined.

[0125] result FIG. 1 shows the growth curves and TCR expression at harvest of T cells generated under a closed continuous autologous T cell bioprocess using the Xuri W25 bioreactor in condition 1. FIG. 1A shows the growth curves of cell growth in the Xuri W25 bioreactor with media supplemented with IL2 only or a cocktail of IL7, IL21 and TWS119. Media with IL2 yielded 16 billion T cells in 10 days, and media with IL7, IL21 and TWS119 yielded 12 billion T cells in 10 days. FIG. 1B shows the viability of cells grown in the Xuri W25 bioreactor with media supplemented with IL2 only or a cocktail of IL7, IL21 and TWS119. Cell viability was over 90% throughout the growth, and no differences in cell viability were observed between the different media conditions.

[0126] Figure 2 shows T cell transgene expression under different conditions. One day after activation, all cells were transduced with the same lentiviral vector at MOI=1. The transgene was more highly expressed in cells transduced in the Xuri W25 bioreactor compared to cells transduced in the permeable bag (hybrid condition 3) or the G-Rex system (condition 2).

[0127] Figure 3 shows leukocyte subsets in T cells recovered from the different test conditions. Analysis of human B cells (CD19), monocytes (CD14), NK cells (CD56 / 16), NKT cells (CD3+Cd56+) and T cells (CD3+Cd56-) was performed by immunophenotyping. T cell purity was greater than 98% in all conditions, and no significant differences were observed in the proportions of non-T cell subsets between the different conditions.

[0128] Figure 4 shows the percentage of T cell subsets on days 5 (A) and 10 (B) under different conditions. Tscm, Tcm, Tem and Tte subsets were analyzed by immunophenotypic analysis based on the expression of CD45RA, CD45RO, CD95 and CCR7. On day 5, T cells generated in the Xuri W25 bioreactor had a higher percentage of Tscm and Tcm than T cells generated in the G-Rex system or permeable bags, and on day 10, the percentage of Tcm was higher than T cells generated in the G-Rex system, showing less phenotypic differentiation.

[0129] FIG. 5 shows the cytotoxic function of modified T cells characterized by their ability to lyse target cells (A), release IFN-gamma in response to target cells (B), and release TNF-alpha in response to target cells (C). Briefly, harvested T cells and peptide-pulsed T2 cells were co-cultured at a 1:1 ratio. T2 cells were luciferase-treated and luminescence was measured using the Steady-Glo® Luciferase Assay System (Promega, Madison, WI). The decrease in luminescence was used to quantitate T2 cell death as an index of modified T cell cytotoxicity. As shown in the figure, modified T cells generated in the Xuri W25 bioreactor under both media conditions (IL2, or IL7, IL21, TWS119) were more capable of killing target cells (A) than modified T cells generated in the static permeable bag and static G-Rex systems, likely due to a stronger release of IFN-gamma (B) and TNF-alpha (C).

[0130] FIG. 6 shows the resistance of modified T cells to target cell challenge, characterized by the levels of annexin (A) and expression of the exhaustion marker Tim3 (B) after co-culture with target cells in vitro at an effector to target (E:T) ratio of 1:1. Externalization of phosphatidylserine in apoptotic cells was detected using recombinant annexin V conjugated to the purple fluorescent Pacific Blue™ dye. Since T cells with an effector phenotype are more susceptible to activation by target cell challenge and undergo apoptosis after activation, lower levels of annexin should indicate T cells with more memory subsets. As shown in the figure, modified T cells generated in the Xuri W25 bioreactor showed lower annexin levels in response to target cell challenge than cells from the G-Rex system or permeable bags, and thus are more memory phenotype-like (A). Furthermore, modified T cells generated in the Xuri W25 bioreactor and permeable bags showed lower expression of the exhaustion marker Tim3 compared to cells from the G-Rex system (B).

[0131] The ability to generate autologous lentiviral engineered T cells was compared between a closed continuous autologous T cell bioprocessing method and the static G-Rex and hybrid gas permeable bag / Xuri systems. The closed continuous method generated T cells with higher transgene expression, higher levels of cytokine secretion, and a more memory-like phenotype when compared to T cells transduced with the static G-Rex system or static gas permeable bag.

[0132] Example 2 Addition of glycolytic inhibitors to the culture medium On day 0, a fresh Leukopack® (Hemacare, Northridge, CA) containing 300 ml of fresh leukopheresis product collected from normal peripheral blood was sterilely connected to a CD-600.1 Sepax cell separation kit (GE Healthcare) and processed under the Culture Wash-Pro program. The Leukopack® contained white blood cells, red blood cells and platelets. The cells were washed in 1 L ClinMACS PBS / EDTA, 5 ml human serum albumin (Miltenyi Biotec) and plasma and apheresis buffer were removed using a Sepax CPro cell separation system with a CS-600.1 kit (GE Healthcare) according to the manufacturer's instructions. The cells were washed and counted as described above in Example 1.

[0133] A portion of the washed apheresis donor cells containing 1.2E9 nucleated cells was transferred to two transfer bags under the Dilute program using a Sepax C-Pro processing system with the same CS-600.1 kit (no new kit was used). To one bag, approximately 8 ml of Medium 1 (OpTmizer complete medium (ThermoFisher) containing 300 IU / ml rhIL2 and 2 mM 2-deoxy-D-glucose (2-DG) (MilliporeSigma, Burlington, MA)) was added. To the second bag, approximately 8 ml of Medium 2 (OpTmizer complete medium (ThermoFisher) containing 300 IU / ml rhIL2) was added. To each, 7.5 ml of Immunocult anti-CD3 / CD28 / CD12 (medium with 25 μl / ml cells, Stemcell Technologies) was added. The amount of activator was determined based on the 300 ml culture volume used in the following incubation / activation steps: The bags were incubated at room temperature for 1 hour to allow saturation of antibody binding.

[0134] A 2L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to a Xuri Cell Culture System W25 and 300ml of Medium 1 was added and equilibrated at 37°C, 5% CO2, 0.1 L / min gas flow rate, and 6 rpm rocking speed at an angle of 6°. A second 2L Xuri SP Perf Cellbag bioreactor was also connected to a Xuri Cell Culture System W25 and 300ml Medium 2 was similarly equilibrated.

[0135] The transfer bags containing cells in Medium 1 and Medium 2 were sterilely connected to the Xuri Cellbag feed line and the contents were transferred into the bag by gravity. The cells were then incubated overnight at 37°C, 5% CO2, 0.1 L / min gas flow rate, rocking speed of 2 rpm at a 2° angle.

[0136] On day 1, nucleated cells in each bag were counted and an amount of lentiviral vector (containing a polynucleotide encoding the TCR) corresponding to an MOI of 1 functional titer was diluted in 10 ml of medium 1 or medium 2 and placed in a transfer bag. The transfer bag was sterile connected to a Xuri Cellbag feed line and the lentivirus was then transferred by gravity flow into each of the bioreactors. The cells were incubated for 20-24 hours at 37 °C, 5% CO2, a gas flow rate of 0.1 L / min, and a rocking speed of 2 rpm at a 2° angle.

[0137] On day 2, approximately half the volume of culture medium in each bioreactor bag was exchanged using three 50 ml bag washes through the feed and waste lines. Cell counts were determined daily, and viable cell density (VCD), viability, dissolved oxygen, and metabolites were measured as described above. Cell phenotypes were determined for all samples tested. Bags were maintained on a rocking bioreactor at 2° angle, 2 rpm, 37°C, 5% CO2, and 0.1 L / min gas flow for 24 hours.

[0138] On days 3-9, additional OpTimizer complete medium containing either IL-2 / 2-DG (Medium 1) and IL2 (Medium 2) was added to 1L by fed-batch / perfusion feeding to maintain cell density above 2E6 cells / ml. With increasing culture volume, the rocking speed and angle was increased to 4 RPM at an angle of 4° to maintain sufficient mass transfer in the bioreactor. When the culture volume reached 1L, Medium 1 containing 2-DG was perfused with OpTimizer complete medium containing only IL2 (Medium 2). In a separate experiment, OpTimizer complete medium containing 2-DG was maintained until harvest.

[0139] Total viable cell density, viability, glucose and lactate measurements were performed daily. Phenotypic analysis was performed on days 3, 5 and 7.

[0140] On days 9-14, both cultures were switched to a perfusion feed of 1 L / day with rocking set at 6 RPM at a 6° angle.

[0141] Total viable cell density, viability, glucose and lactate were measured daily. Phenotypic analysis was performed on days 9 and 14.

[0142] Cells were harvested on day 14. The Xuri SP Per bioreactor bag was sterilely connected to a Selfia5200 cell processing system using the FlexCell program and the CT-800.1 cell processing kit (GE Healthcare). T cells were concentrated to approximately 20 ml at a flow rate of 75 ml / min. One wash cycle was performed with 0.9% saline (Baxter, Deerfield, Ill.) supplemented with 1 vol% human serum albumin (HSA). Washing was performed at 380×g for 5 min.

[0143] Viability and recovery were measured. Cells were concentrated and further formulated in a 1:1 ratio with Hyclone supplemented with 5% human serum albumin, frozen to -80°C using ViaFreeze (GE Healthcare), and cryopreserved by storing in liquid nitrogen.

[0144] result During antigen stimulation, T cells transition to glycolytic metabolism to maintain effector function. However, the role of glucose metabolism in T cell differentiation during in vitro expansion is unclear. In this study, we hypothesized that glucose metabolism, particularly glycolysis, supports T cell differentiation to an effector phenotype during expansion and that pharmacological inhibition of glycolysis with 2-DG can attenuate CD3 / CD28 / CD2 antigen-mediated differentiation and promote a memory phenotype. In this experiment, 2-DG was added to cells along with activators to inhibit glycolysis after T cell activation. 2-DG was then either removed later in the expansion phase to restore glycolysis and support cell proliferation, or maintained throughout the entire process until harvest. Modified T cells derived from 2-DG-inhibited T cell differentiation produced more Tscm and Tcm compared to T cells derived from 2-DG-free medium. This resulted in higher yields of T cells, higher transgene expression, and more memory-like phenotypes of T cells.

[0145] Figure 7 shows the growth curves (A) and viability (B) of cells grown in media supplemented with 2-DG (Medium 1) and without 2-DG (Medium 2). Media containing 2-DG yielded 35 billion T cells in 14 days, whereas media conditions without 2-DG yielded 25 billion T cells in 14 days. Overall viability was greater than 70%, with cells derived from 2-DG media showing slightly higher viability than cells derived from non-2-DG media from days 4 to 10. This decrease in viability from days 4 to 10 was likely due to cell death of non-T cell leukocytes. After the T cell population was enriched to greater than 90% in culture, viability returned to greater than 90% until harvest. Given the inhibition of T cell differentiation into Tems confirmed by 2-DG, the reduced cytotoxic cytokine release associated with the reduction in the Tems population may also contribute to the higher viability in samples containing 2-DG media.

[0146] Figure 8 shows transgene expression in cells grown in medium supplemented with 2-DG (medium 1) and medium without 2-DG (medium 2). Transgene expression by T cells in 2-DG medium was slightly higher than that of T cells in non-2-DG medium (56% vs. 54%) and could be maintained at high levels (50.4%) when cell densities were as high as 35 billion cells / ml. In contrast, transgene expression in T cells in non-2-DG medium decreased to 44.4% at high cell densities (25 billion cells / ml).

[0147] Figure 9 shows CD25 expression by cells grown in medium supplemented with 2-DG (medium 1) and medium without 2-DG (medium 2). Expression of CD25 by T cells in non-2-DG medium showed a stronger response to activation, expressing at higher levels than T cells in 2-DG medium (46% vs. 32%). Expression of CD25 by T cells in both conditions also saturated at ±80%. T cells in 2-DG medium retained CD25 better than cells in non-2-DG medium at days 7 and 9. This may be due to higher transgene expression and T cell yield of T cells generated in 2-DG medium.

[0148] FIG. 10 shows the concentrations of glucose (A) and lactate (B) under different medium conditions. Glucose and lactate were measured using a Cedex Bio Analyzer (Roche, Indianapolis, IN). Inhibition of glycolysis under 2-DG medium conditions (medium 1) was evidenced by higher glucose concentrations (lower glucose consumption) (A) and higher lactate concentrations (B) in the spent medium compared to those from the non-2-DG condition (medium 2). Furthermore, the effect of 2-DG was reversible, as restoration of glucose (A) and lactate (B) levels was seen following removal of 2-DG from the medium from days 7 to 14.

[0149] FIG. 11 shows T cell differentiation phenotype analysis of cells grown in medium supplemented with (Medium 1) and without (Medium 2) 2-DG. 2-DG was added to the medium from day 0 to day 7, and the feed medium was replaced with non-2-DG medium (Medium 1) from day 7 to day 14. T cells in 2-DG medium were less differentiated, as suggested by a higher Tscm subset at day 5 (26% vs. 14%) and a higher total of Tcm and Tscm at day 7 (86% vs. 65%). T cells continued to differentiate after 2-DG was perfused from the culture medium, finishing at a differentiation level comparable to T cells in non-2-DG medium (Medium 1).

[0150] Figure 12 shows leukocyte subsets in T cells recovered under different media conditions. Analysis of human B cells (CD19), monocytes (CD14), NK cells (CD56 / 16), NKT cells (CD3+Cd56+) and T cells (CD3+Cd56-) was performed by immunophenotyping. NKT cells expanded from 5% to ±10%, while CD3+ cell purity in both conditions was greater than 98%, and no significant differences in the proportions of non-T cell subsets were observed between 2-DG and non-2-DG media conditions.

[0151] FIG. 13 shows the results of a potency assay of modified TCR T cells. Luciferase-expressing T2 cells were pulsed with peptides recognized by the modified TCR or DMSO for 4 hours, and after washing the cells, co-culture assays were set up with 20,000 total T cells at a 1:1 (E:T) ratio. (A) After 48 hours, luciferase signals were measured using the Steady Glo® Luciferase Assay System (Promega). Specific cytotoxicity was measured compared to non-peptide T2 cells cultured with TCR T cells. (B) After 24 hours of co-culture, IFN-γ expression was measured using an AlphaLISA detection kit (Perkin Elmer Waltham, MA). At high peptide concentrations (1 μM), there was no significant difference in T2 cell lysis between T cells in 2-DG medium and non-2-DG medium, and at low peptide concentrations (10 nM), slightly higher T2 cell lysis was observed in T cells in non-2-DG medium. In addition, the cumulative release of IFN-γ at 24 hours was higher for T cells in non-2-DG medium across all concentrations of peptides tested. The lower level of IFN-γ in T cells in 2-DG medium at early time points (24 hours) may be due to a delay. Further data (not shown here) suggested that activation in T cells from 2-DG medium had a longer activation time than T cells from non-2-DG medium and could not be captured in the short-term IFN-γ assay. (C) shows the differentiation of T cells under different medium conditions. Culture medium containing 2-DG was used throughout the entire process (10 days). T cells from 2-DG medium were less differentiated as suggested by the higher percentage of Tscm and Tcm subsets recovered compared to T cells from non-2-DG medium (59% vs. 43%). This data suggests that 2-DG is supplemented in the culture medium throughout the entire process up to harvest.

[0152] Example 3 This experiment compared CAR and TCR transduction of cells from a donor apheresis cell sample (non-enriched) with cells from the same apheresis cell sample that had been further enriched for T cells (enriched).

[0153] On day 0, a fresh Leukopack® (Hemacare, Northridge, CA) containing 300 ml of fresh enriched leukoapheresis product drawn from normal peripheral blood was sterilely connected to a CD-600.1 Sepax cell separation kit (GE) and processed under the Culture Wash-Pro program. The Leukopack® contained white blood cells, red blood cells and platelets. The cells were washed to remove plasma and apheresis buffer as described above in Example 1, and nucleated cells were counted using an NC-200™ automated cell counter and equally divided into two transfer bags (Sample 1, enriched T cells) and (Sample 2, non-enriched apheresis donor cells) under the Dilute program using a Sepax C-Pro cell processing system.

[0154] For sample 1 "enriched T cells", T cells were isolated from washed apheresed cells using EasySep™ human pan T negative selection kit (Stemcell Technologies) according to the manufacturer's instructions. Enriched T cells were added to gas-permeable bags at a cell density of 4E6 nucleated cells / ml in 120 ml of Optimizer complete medium containing 300 IU of IL2. Gas-permeable bags were pre-coated with anti-CD3 antibody (Miltenyi Biotec) at 1.23 g / ml in PBS. For activation, soluble anti-CD28 antibody (Miltenyi Biotec) was then added to the permeable bags at a dilution of 1:100 (1 μ / ml).

[0155] For sample 2 "non-enriched apheresis donor cells", the same cell density (4E6 nucleated cells / ml) in 120 ml Optimizer complete medium (containing 300 IU / ml IL-2) was added to a gas-permeable bag pre-coated with anti-CD3 antibody as in sample 1. For activation, soluble anti-CD28 antibody (Miltenyi Biotec) was then added to the permeable bag at a dilution of 1:100 (1 μg / ml).

[0156] Day 1: Nucleated cells in each gas-permeable bag were counted, and an amount of lentiviral vector containing a polynucleotide encoding a TCR corresponding to an MOI of 1 (TCR) functional titer was added to one bag, a lentiviral vector containing a polynucleotide encoding a CAR corresponding to an MOI of 40 (CAR) functional titer was added directly to the second bag, and a lentiviral vector containing a polynucleotide encoding a green fluorescent protein (GFP) was added to the third gas-permeable bag. The bags were incubated overnight at 37 °C, 5% CO2.

[0157] Days 2-6: On day 2, an additional 120 ml of OpTmizer complete medium containing 300 IU / ml IL-2 was added to each gas permeable bag to dilute the lentivirus.

[0158] Cells from each gas permeable bag were split into two bags on day 4 and inoculated into equilibrated Xuri SP Perf bioreactor bags in a Xuri W25 bioreactor (as described in Example 1) on day 6, with a total volume of 800 ml of culture medium. The bioreactor was rocked at a speed of 6 rpm and angle of 6° throughout the entire growth period at 37° C., 5% CO2, and 0.1 L / min gas flow rate.

[0159] Days 7-13 or 14: On day 7, the volume of culture medium in the bioreactor was scaled up to 1 L. The cells were kept in the bioreactor until day 13 or 14, when they were harvested. During growth, OpTimizer complete medium containing 300 IU / ml IL-2 was perfused at a rate of 500 ml / day on day 8, 800 ml / day on day 9, and 1 L / day from day 10 until the time of harvest. Cell counts, viable cell density, cell viability, and metabolites were measured daily.

[0160] Cells were harvested on day 13 or 14. Bioreactor bags were aseptically connected to a Sepax Pro using the Culture Wash program and a CT-600 cell processing kit (GE Healthcare). One wash cycle was performed using saline (Baxter) supplemented with 1 vol% human serum albumin (HSA). Washing was performed at 380×g for 5 min.

[0161] Viability and recovery were measured. Cells were concentrated and further cryopreserved by formulating at a 1:1 ratio with HyClone™ Cryopreservation Medium (GE Healthcare) supplemented with 5% human serum albumin, freezing to -100°C using a Via Freeze (GE Healthcare), and storing in liquid nitrogen.

[0162] result The use of non-enriched apheresis donor cells as a starting material improved transduction efficiency, as measured by the level of surface expression and genomic integration, compared with the use of enriched T cells as a starting material. Furthermore, non-enriched apheresis cells promoted a less differentiated T cell phenotype. Two of the three donors tested were CD4 + and CD8 + Both T cell subsets showed higher percentages of Tscm and Tcm at harvest, and T cell purity was comparable when enriched versus non-enriched T cells were used as starting material.

[0163] Figure 14 shows transgene expression in T cells from enriched and non-enriched cells at harvest. Three different lentiviral vectors delivering CAR, TCR or GFP were transduced into enriched and non-enriched donor cells. Transgene expression was detected by antibody staining for CAR, dextramer staining for TCR, and fluorescent signal for GFP using flow cytometry. CAR, TCR, and GFP were expressed by CD8 T cells derived from the enrichment process. +(A) Dextramer staining is CD8 receptor dependent, and therefore CD4 T cells did not show expression of TCR (B). However, both CAR and GFP expression were higher in CD4 T cells from non-enriched donor cells compared to enriched donor cells. + It was higher in T cells (B).

[0164] Figure 15 shows the CD4 + and CD8 + The vector copy number of TCR in T cells is shown. WPRE and human albumin copies were measured by qPCR, and the vector copy number of TCR was calculated by dividing the ratio of WPRE to human albumin by 2. The vector copy number of TCR was calculated by dividing the CD4 + T cells and CD8 + Transgene integration was also higher in apheresis-derived T cells than in enriched donor cells, as indicated by higher vector copy numbers.

[0165] Figure 16 shows the percentage of leukocytes and leukocyte subsets measured at the end of harvest. All cells were stained for leukocytes (CD45+). Minimal (<1%) B cells (CD19+), NK cells (CD56 / 16+) and monocytes (CD14+) were detected in both unenriched and enriched donor cells. Overall, CD3 + The percentage of cells (>99%) was comparable between non-enriched and enriched donor cells, with the percentage of T cells (CD3+CD56-) being slightly lower in enriched donor cells due to the expansion of NKT cells (CD3+CD56+).

[0166] Figure 17 shows the T cell phenotype upon harvest of enriched and non-enriched donor cells. This experiment was repeated three times with a different donor sample each time. Of the three donor samples tested, T cells harvested from two donors expressed a higher percentage of memory stem cell markers (CD45RA+, CCR7+) and central memory markers (CD45RA-, CCR7+) from non-enriched donor cells compared to cells subjected to T cell enrichment. This difference was due to the CD8 + Significant only in T cells, CD4 + It was not significant in T cells.

[0167] Figure 18 shows the growth curves and viability of cells in the bioreactor from enriched and non-enriched donor cells. Growth (A) and viability (B) were comparable between enriched and non-enriched donor cells.

[0168] Example 4 This example provides a closed, continuous method for generating genetically modified autologous T cells expressing a T cell receptor. On day 0, a fresh Leukopack® (Hemacare, Northridge, CA) containing 300 ml of freshly enriched leukoapheresis product collected from normal peripheral blood was sterilely connected to a CD-600.1 Sepax cell separation kit (GE) and processed under the Culture Wash-Pro program. The Leukopack® contained white blood cells, red blood cells and platelets. The cells were washed in 1 L ClinMACS PBS / EDTA, 5 ml human serum albumin (Miltenyi Biotec, San Diego, CA) and plasma and apheresis buffer were removed using a Sepax CPro with CS-600.1 kit (GE Healthcare) according to the manufacturer's instructions. Based on the initial white blood cell (WBC) count indicated on the donor information sheet attached to the Leukopack®, the cells were eluted with approximately 50 ml of OpTimizer complete medium at a cell density of 150E6 WBC / ml.

[0169] Nucleated cells in the washed leukapheresis samples were counted using an NC-200™ automated cell counter, followed by total viable cell count, viability and immunophenotypic analysis of the washed cells.

[0170] The washed apheresis donor cell sample containing 1.2E9 nucleated cells was then transferred to a transfer bag (Charter Medicine) in approximately 6.5 ml of OpTimizer complete medium containing 300 IU / ml IL-2 using the Sepax C-pro processing system with the same CD-600.1 kit under the Dilute program and incubated with 7.5 ml of ImmunoCult™ human CD3 / CD28 / CD2 (2.5 μg / ml, Stemcell Technologies) at room temperature for 1 hour to saturate antibody binding.

[0171] A 2L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to a Xuri cell culture system W25 and 300ml of OpTimizer complete medium containing 300IU / ml IL-2 was added and equilibrated at 37°C, 5% CO2, 0.1L / min gas flow rate, and a rocking speed of 6 rpm at a 6° angle.

[0172] The transfer bag was sterilely connected to the XuriCellbag feed line and the contents were transferred into the bag by gravity. The cells were then incubated overnight at 37°C, 5% CO2, 0.1 L / min gas flow rate with a rocking speed of 2 rpm at a 2° angle to promote activation.

[0173] On day 1, nucleated cells in the bag were counted. An amount of lentiviral vector (containing a polynucleotide encoding the TCR) corresponding to an MOI of 1 functional titer was diluted in 10 ml of OpTimizer complete medium containing 300 IU / ml of IL2 and placed in a transfer bag. The transfer bag was sterile connected to the Xuri Cellbag feed line and the lentiviral vector was transferred by gravity to the bioreactor. Cells were incubated for 20-24 h at 37 °C, 5% CO2, a gas flow rate of 0.1 L / min, and a rocking speed of 2 rpm at a 2° angle.

[0174] On day 2, approximately half the volume of culture medium in the bioreactor bag was replaced with fresh OpTimizer complete medium containing 300 IU / ml IL2 using three 50 ml washes. Cells were counted daily and viable cell density (VCD) and viability were measured using NC200, dissolved oxygen, metabolites were also measured. Cell phenotype was determined for all samples tested. Bags were maintained on a rocking bioreactor at 2° angle, 2 rpm, 37°C, 5% CO2, 0.1 L / min gas flow for 24 hours.

[0175] Cultures were maintained in 300 ml of OpTimzer complete medium containing 300 IU / ml IL2 by perfusion at a rate of 1 bag volume per day at 37°C, 5% CO2, 0.1 L / min gas flow rate with a rocking speed of 2 rpm at a 2° angle until the cell density reached 4e6 cells / ml on days 3-10.

[0176] At that point, the volume of culture medium was increased to 600 ml and perfused at a rate of one bioreactor bag per day, at a rocking speed of 4 rpm at a 4° angle, at 37°C, 5% CO2, and a gas flow rate of 0.1 L / min, until the cell density again reached 4e6 cells / ml.

[0177] At that point, the volume of culture medium was increased to 1000 ml and perfused at a rate of one bioreactor bag per day, at a rocking speed of 6 rpm at a 6° angle, at 37°C, 5% CO2, and a gas flow rate of 0.1 L / min, until harvest on day 10.

[0178] Total viable cell density, viability, glucose and lactate measurements were performed daily. Phenotypic analysis was performed on days 3, 5 and 7.

[0179] Cells were harvested on day 10. The Xuri SP Per bioreactor bag was sterilely connected to a Selfia5200 cell processing system using the FlexCell program and the CT-800.1 cell processing kit (GE Healthcare). T cells were concentrated to approximately 20 ml at a flow rate of 75 ml / min. One wash cycle was performed with 0.9% saline (Baxter, Deerfield, Ill.) supplemented with 1 vol% human serum albumin (HSA). Washing was performed at 380×g for 5 min.

[0180] The cells were then eluted at 2E8 cells / ml in saline supplemented with 1% HSA and further formulated in a 1:1 ratio with Hyclone™ cryopreservation medium (GE Healthcare) supplemented with 5% human serum albumin. The final cell product was then divided into two cryobags and several cryovials, which were finally frozen in a VIA Freeze™ Quad freezer (GE) at a cooling rate of -1°C / min until the temperature reached -80°C. After freezing, the cells were transferred to liquid nitrogen for long-term storage.

[0181] result Figure 19 shows the cell proliferation curve and survival rate. The decrease in survival rate is believed to be due to the gradual death of non-T cells.

[0182] Figure 20 shows the percentage of leukocyte subsets on days 0 and 10. Cells were stained with an antibody cocktail targeting B cells (CD19), monocytes (CD14), T cells (CD3+, CD56 / 16-), NKT cells (CD3+, CD56 / 16+), NK cells (CD3-, CD56+), CD4 T cells (CD3+, CD4+) and CD8 T cells (CD3+, CD8+) and analyzed by flow cytometry. By the time of harvest (10 days), CD3 cell purity reached 98%, total T cells 98% and NKT cells 10%.

[0183] Figure 21 shows T cell differentiation during bioprocessing. T cell subsets Tscm (CD45RA+, CD45RO-, CCR7+, CD95+), Tcm (CD45RA-, CD45RO+, CCR7+, CD95+), and Tem (CD45RA-, CD45RO+, CCR7-, CD95+) were stained with antibody cocktails and analyzed by flow cytometry at various time points. T cells differentiated similarly to those shown in Examples 1 and 2, with approximately 70% Tem and approximately 30% Tscm+Tcm by the time of harvest.

[0184] FIG. 22 shows transgene expression in harvested T cells at the cell surface level (A) and DNA level (B). Dextramer staining was performed to confirm surface expression of the transgene in transduced and non-transduced T cells. Transgene expression was greater than 50% in transduced T cells (A). DNA was extracted from harvested cells and 2 ng of DNA was used in qPCR reactions targeting WPRE and human albumin. Vector copy number was calculated as the ratio of WPRE copy number to human albumin copy number divided by 2. No detectable copies of the transgene were found in non-transduced T cells and the transgene copy number in transduced T cells was less than 5, within the guidelines for vector copy number implemented by the FDA (B).

[0185] Figure 23 shows the percentage of target cell killing at various effector to target cell (E:T) ratios. Recovered T cells (transduced or non-transduced) were co-cultured with target cells for 24 hours at E:T ratios of 1:1, 1:5, and 1:10. The percentage of target cell killing was calculated from the percentage of target cell death after 24 hours of co-culture. Transduced T cells showed strong killing effect (>70%) against target cells at all E:T ratios tested.

[0186] Figure 24 shows IFN-gamma release from harvested cells. Harvested cells (transduced or non-transduced) were co-cultured with target cells pulsed with various concentrations of peptide for 24 hours. IFN-gamma in the supernatant was measured by Alphalisa assay. Similar levels of IFN-gamma were seen in Examples 1 and 2.

[0187] A closed continuous autologous process rapidly (10 days) generated over 26 billion modified T cells with high transgene expression (>50%) and high CD3 cell purity (98%). The modified T cells exhibited significant killing ability against target cells at a low effector-to-target cell ratio (1:10), likely due to the potent release of IFN-gamma in response to target cells.

[0188] Example 5 Evaluation of transduction efficiency of T cells expressing TCR after stimulation with various reagents On day 0, half of a fresh Leukopack® (Hemacare, Northridge, CA) containing 150 ml leukapheresis product taken from normal peripheral blood was sterilely connected to a CD-600.1 Sepax cell separation kit (GE) and processed under the Culture Wash-Pro program. The Leukopack® contained white blood cells, red blood cells and platelets. The cells were washed in 1 L ClinMACS PBS / EDTA, 5 ml human serum albumin (Miltenyi Biotec, San Diego, CA) and plasma and apheresis buffer were removed using a Sepax C-Pro cell separation system with a CS-600.1 kit (GE Healthcare) according to the manufacturer's instructions.

[0189] Nucleated cells in the harvested samples were counted using an NC-200™ automated cell counter (ChemoMetec), followed by total viable cell count, viability and immunophenotypic analysis of washed cells.

[0190] Starting populations of cells were prepared by isolating pan T cells from washed donor cells using the EasySep™ Human pan T Negative Selection Kit (Stemcell Technologies) according to the manufacturer's instructions, or by using washed donor cells without further selection or enrichment (non-enriched cells). Enriched pan T cells and non-enriched cells were resuspended in OpTmizer complete medium containing 300 IU / ml IL2 (Stemcell Technologies) and added to the transfer bag, unless otherwise stated. In some experiments, IL-7, IL-15, IL-21, and / or TWS1119 (Stemcell Technologies) were used in the activation and proliferation media.

[0191] Gas permeable bags (PL07, Permalife, OriGen, Austin, TX) were prepared by coating with 1 ug / mL anti-CD3 (Clone OKT3 Miltenyi Biotech, Cambridge, MA) in PBS overnight at 4°C and washed with PBS prior to addition of cells. The following conditions were tested:

[0192] 1) In one experiment, isolated pan T cells (2 × 10 6 CD3+ T cells (10000 cells / mL) were seeded in OpTmizer complete medium containing 300 IU / ml IL2 (Stemcell Technologies) and 1 μg / ml anti-CD28 (Miltenyi) in anti-CD3 coated bags. For titration of anti-CD3 or starting cell numbers, anti-CD3 coated plates (Corning) were used. Plates were coated with anti-CD3 at titrated concentrations ranging from 0.0001 to 10 μg / ml. Cells were activated for 20 and 48 hours. These cells were not transduced. CD69, CD25, and 4-1BB levels on CD3+ T cells were measured by flow cytometry at 20 and 48 hours (two replicates per condition).

[0193] 2) To compare various activators, isolated pan T cells (2 × 10 61000 cells / mL) were seeded in OpTmizer complete media containing 300IU / ml IL2 (Stemcell Technologies) and 1ug / ml anti-CD28 (Miltenyi) in anti-CD3 coated bags. The following soluble activators were tested against the coated anti-CD3 condition according to the manufacturer's instructions at 25uL of activator per mL of media at 2E6 cells / mL each: Immunocult anti-CD3 / CD28 (Stemcell Technologies, Cambridge, MA), and Immunocult anti-CD3 / CD28 / CD2 (Stemcell Technologies). Dynabeads™ human T-activator CD3 / CD28 (ThermoFisher) was tested in uncoated bags according to the manufacturer's instructions at a 1:1 bead:cell ratio. Cells / activators were incubated for 20 and 48 hours.

[0194] 3) Isolated pan T cells (2 × 10 6Cells / mL) were seeded in OpTmizer complete medium containing 300 IU / ml IL2 (Stemcell Technologies) and 1 μg / ml anti-CD28 (Miltenyi) in the anti-CD3 coated permeable bags described above. The following activators were tested: 1 ug / mL coated anti-CD3 gas permeable bags described above, 1 ug / mL. Soluble anti-CD28 (Miltenyi), soluble Immunocult anti-CD3 / CD28 / CD2 (Stemcell Technologies) (25 uL activator per mL of medium), Dynabeads™ human T activator CD3 / CD28 (ThermoFisher) (1:1 bead:cell ratio), and soluble MACS® GMP T Cell TransAct™ anti-CD3 and anti-CD28 conjugated polymer nanomatrix (Miltenyi Biotech) at a concentration (1:17.5 dilution) were tested in uncoated bags 48 hours after activation, and cells from the coated anti-CD3 bag were transferred to a new bag to remove the stimulus. After activation, cells were transduced as described below. Cells / activators were incubated for 7 days. Modified TCR expression was assessed by flow cytometry using dextramer, which recognizes peptide-MHC class I complexes, gated on CD8+ T cells.

[0195] 4) Washed leukapheresis cells (2 × 10 61000 cells / mL were seeded in OpTmizer complete medium containing 300 IU / mL IL2 (Stemcell Technologies) and 1 μg / mL anti-CD28 (Miltenyi) in anti-CD3 coated permeable bags as described above. The following activators were tested: 1 μg / mL coated anti-CD3 (Miltenyi) and 1 μg / mL soluble anti-CD28 (Miltenyi) and soluble ImmunocultCD3 / CD28 / CD2 (Stemcell Technologies) (25 μL activator per mL of medium). 48 hours after activation, cells were transferred from the coated anti-CD3 bag to a new bag and the stimulus was removed. After activation, cells were transduced as described below. Cells / activators were incubated for 7 days. Transduction efficiency was assessed by flow cytometry using GFP expression and gating on CD8+ or CD4+ T cells.

[0196] 5) Effect of different cytokine cocktails. Fresh cells from leukopheresed material (WLPC) were cultured at 2 × 10 in gas-permeable bags containing Immunocult anti-CD3 / CD28 / CD2 (Stemcell Technologies) in OpTmizer complete medium containing four different cytokine cocktails: 6 Cells were activated at 100 cells / mL with: a) 300 IU / ml IL2, b) 10 ng / mL IL-7 (Stemcell Technologies) and 10 ng / mL IL-15 (Stemcell Technologies), c) 10 μM TWS119 (Stemcell Technologies), 10 ng / mL IL-7 (Stemcell Technologies) and 20 ng / mL IL-21 (Stemcell Technologies), or d) 10 ng / mL IL-7 (Stemcell Technologies) and 20 ng / mL IL-21 (Stemcell Technologies). Modified TCR expression was assessed by flow cytometry using TCR-specific dextramers (Immudex) and gating on CD8+ T cells.

[0197] 6) Effect of starting WLPC density on activation by soluble activators. WLPCs were cultured at four different densities: (a) 1 × 10 in OpTmizer complete medium containing 300 IU / mL IL-2 or 10 μM TWS119 (Stemcell Technologies), 10 ng / mL IL-7 (Stemcell Technologies) and 20 ng / mL IL-21 (Stemcell Technologies). 6 cells / mL, b) 2×10 6 cells / mL, c) 4×10 6 cells / mL, or d) 6 x 10 6 Cells were activated with Immunocult anti-CD3 / CD28 / CD2 (Stemcell Technologies) at 100 cells / mL. Modified TCR expression was assessed by flow cytometry using TCR-specific dextramers (Immudex) and gating on CD8+ T cells.

[0198] If transduced, cells were counted in each bag (from experiments 3, 4, 5, and 6 above) 24 hours after activation. An amount of lentiviral vector (containing polynucleotides encoding TCR and GFP) corresponding to an MOI of a functional titer of 1-2 was added to the bag and incubated overnight at 37°C, 5% CO2.

[0199] Cells were grown in static culture in gas-permeable bags, with cell densities maintained between 1 and 3 million cells / mL by splitting every 2–3 days and transferring to larger gas-permeable bags as needed. Growth was carried out for 6–9 days. Media containing cytokines was changed every 2–3 days.

[0200] Cell counts were performed every 1 to 3 days.

[0201] result Figure 25 shows the results of titration of coated anti-CD3 (OKT3) antibodies and pan T cell activation as measured by CD69, CD25, and 4-1BB. Based on T cell stimulation, 1 μg / ml of anti-CD3 was selected for comparison with soluble activators.

[0202] Figure 26 shows the results of pan T cell stimulation by various soluble activators against coated anti-CD3 as measured by CD69, CD25, and 4-1BB. Dynabeads (anti-CD3 / CD28) showed the strongest stimulation, followed by Immunocult anti-CD3 / CD28 / CD2 at the manufacturer's recommended concentration of 25 μL / mL cells in medium. Interestingly, soluble Immunocult anti-CD3 / CD28 activators were comparable to coated anti-CD3.

[0203] Figure 27 shows that the transduction efficiency of modified T cells was dependent on the stimulatory signal. (A) Starting material: pan T cells. Dextromer staining of modified TCR on CD8+ T cells showed that Immunocult anti-CD3 / CD28 / CD2 gave the highest transduction efficiency. (B) Starting material: apheresis cells: FACS for GFP expression in transduced CD4+ and CD8+ T cells showed that Immunocult anti-CD3 / CD28 / CD2 was superior to coated anti-CD3.

[0204] FIG. 28 shows that cell proliferation was similar between coated apheresis cell activation anti-CD3 / soluble anti-CD28, Immunocult anti-CD3 / CD28 / CD2, TransAct, and Dynabeads over 9 days.

[0205] Figure 29 shows that culture conditions affected the transduction efficiency of modified T cells. The transduction efficiency of apheresis cells grown in culture medium containing IL-2, IL-7 / 15 combination, and IL-7 / 21 combination was similar. A cocktail containing IL-7 / 21 / TWS119 improved the transduction efficiency.

[0206] Figure 30 shows that starting cell density affected transduction efficiency. Although overall proliferation was not affected, a higher starting density of Immunocult anti-CD3 / CD28 / CD2 activated apheresed cells resulted in better transduction efficiency when the culture medium contained IL-2 or a cocktail of IL-7 / 21 / TWS119.

[0207] In this comparison of different T cell activators and cytokines, two different starting cell populations are used: enriched pan T cells and leukapheresis cells. Activation strength did not seem to correlate with good transduction efficiency, since Dynabeads anti-CD3 / CD28 (ThermoFisher) provided the highest signal strength but did not result in high transduction rates. Immunocult anti-CD3 / CD28 / CD2, with a higher signal strength than coated anti-CD3 but lower than Dynabeads, activated T cells with the highest expression of GFP and modified TCR. Activation of leukapheresis cells with Immunocult anti-CD3 / CD28 / CD2 in the presence of different cytokine cocktails affected the transduction efficiency, but transduction of activated and expanded cells in culture medium containing IL-2 or IL-7 / 21 / TWS119 was similar when compared at different cell densities. Cells cultured in IL-2 and IL-7 / 21 / TWS119 were compared and it was found that the IL-7 / 21 / TWS119 conditions promoted increased transduction efficiency. These results have important implications for the use of soluble activators such as Immunocult anti-CD3 / CD28 / CD2 in manufacturing processes that result in a greater number of functionally modified T cells for cell therapy applications.

[0208] Example 6 Optimal MOI for TCR transduction Previously frozen apheresis cells were thawed and activated with ImmunocultCD3 / CD28 / CD2 (25 μl / ml cells, Stemcell Technologies) for 24 h as described above. One day after activation, lentiviral vectors containing polynucleotides encoding TRC were inoculated at 1 × 10 6 Cells were added at a high multiplicity of infection (MOI) of ≥1 (A) or the MOI was reduced to ≤2 (B). Seven days after transduction, cells were assayed for TCR expression. No observable MOI effect was observed at MOIs above 1. Values ​​reported are the average of TCR expression in CD8+ T cells from four different donors. Figures 31A and 31B show that an MOI of 1 was optimal for lentiviral transduction in a large-scale closed bioprocess.

Claims

1. 1. A method for producing genetically modified autologous T cells expressing at least one protein of interest, comprising: seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing culture medium, said bioreactor bag being part of a rocking bioreactor platform; Cultivating the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM; transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding the protein of interest while continuously rocking at a speed of about 2 RPM; Growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture until harvest. The method includes:

2. 10. The method of claim 1, wherein the apheresis donor cells comprise cells derived from peripheral blood.

3. 3. The method of claim 2, wherein the apheresis donor cells comprise nucleated and non-nucleated cells.

4. 10. The method of claim 1, wherein the apheresis donor cells comprise white blood cells and red blood cells.

5. 5. The method of claim 4, wherein the apheresis donor cells also include granulocytes and / or platelets.

6. 2. The method of claim 1, wherein the apheresis is leukapheresis.

7. 10. The method of claim 1, wherein the apheresis donor cells are washed and resuspended in culture medium.

8. The method of claim 1 , wherein the at least one T cell activator is an anti-CD3 antibody or a binding fragment thereof.

9. The method of claim 1 , wherein the T cell activator comprises an anti-CD3 antibody and an anti-CD28 antibody, or a binding fragment thereof.

10. The method of claim 1 , wherein the T cell activator comprises at least an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, or binding fragments thereof.

11. 2. The method of claim 1, wherein the T cell activator comprises at least an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an anti-human CD2 monospecific tetrameric antibody complex.

12. The method of claim 1, wherein the concentration of the at least one soluble T cell activator is from at least 0.001 μg / ml to at least 10 μg / ml.

13. The method of claim 12, wherein the concentration of the at least one soluble T cell activator is from at least 0.1 μg / ml to at least 5 μg / ml.

14. The method of claim 1 , wherein the at least one soluble T cell activator binds to the at least one donor cell at the time of plating.

15. 10. The method of claim 1, wherein the apheresis donor cells are incubated with the one or more soluble T cell activators prior to seeding into the bioreactor bag.

16. 16. The method of claim 15, wherein the incubation is for a time sufficient to allow for saturation of binding of the one or more soluble T cell activators to the apheresis donor cells prior to seeding.

17. 17. The method of claim 16, wherein the apheresis donor cells and the one or more soluble T cell activators are incubated in a transfer bag.

18. 18. The method of claim 17, wherein the volume of culture medium in the transfer bag is from about 5 ml to about 50 ml.

19. 20. The method of claim 18, wherein the volume of culture medium in the transfer bag is from about 5 ml to about 10 ml.

20. 20. The method of claim 17, wherein the apheresis donor cells are incubated with the one or more T cell activators for at least 30 minutes or more.

21. 21. The method of claim 20, wherein the apheresis donor cells are incubated with the one or more T cell activators for at least 1 hour.

22. 2. The method of claim 1, wherein the number of nucleated cells in the apheresis donor cells is about 1.0E9 to about 1.3E9.

23. 2. The method of claim 1, wherein the number of nucleated cells in the apheresis donor cells is about 1.2E9.

24. 10. The method of claim 1, wherein the bioreactor bag is seeded with the apheresis donor cells at a cell density of about 1E6 to about 5E6 nucleated cells / ml.

25. 25. The method of claim 24, wherein the bioreactor bag is seeded with the apheresis donor cells at a cell density of about 2E6.

26. 10. The method of claim 1, wherein the bioreactor bag contains at least 300 ml to at least 400 ml of culture medium at the time of inoculation.

27. 27. The method of claim 26, wherein the bioreactor bag contains at least 300 ml of culture medium at the time of inoculation.

28. 10. The method of claim 1, wherein the apheresis donor cells are cultured in the bioreactor bag for about 12 to 24 hours.

29. The method of claim 1 , wherein the culture medium comprises at least one soluble cytokine.

30. 30. The method of claim 29, wherein the soluble cytokine is selected from IL-2, IL-7, IL-15, or IL-21.

31. 30. The method of claim 29, wherein the at least one soluble cytokine is IL-2.

32. 30. The method of claim 29, wherein the IL-2 is at a concentration of about 250 IU / ml to about 350 IU / ml.

33. 33. The method of claim 32, wherein the IL-2 is at a concentration of about 300 IU / ml.

34. 30. The method of claim 29, wherein the soluble cytokine is IL-7 in combination with IL-15 or IL-21.

35. 30. The method of claim 29, wherein the concentration of the at least one cytokine is at least 5 ng / ml to at least 30 ng / ml.

36. 36. The method of claim 35, wherein the concentration of the at least one cytokine is from at least 10 ng / ml to at least 20 ng / ml.

37. The method of claim 1 , wherein the culture medium also comprises a WNT pathway activator.

38. 38. The method of claim 37, wherein the WNT pathway activator is TWS117.

39. 38. The method of claim 37, wherein the culture medium comprises a mixture of soluble TWS117, IL-7 and IL-21.

40. The method of claim 1 , wherein the culture medium also contains a soluble glycolytic inhibitor.

41. 41. The method of claim 40, wherein the soluble glycolytic inhibitor is 2-deoxy-D-glucose (2-DG).

42. The method of claim 1 , wherein the viral vector is a retroviral vector.

43. The method of claim 1 , wherein the viral vector is a lentiviral vector.

44. 44. The method of claim 43, wherein the lentiviral vector is added at an MOI of 0.25 to 10.

45. 45. The method of claim 44, wherein the lentiviral vector is added at an MOI of 1.

46. 45. The method of claim 44, wherein the cells are transduced for about 20 to 24 hours.

47. 10. The method of claim 1, wherein after transduction, half of the culture medium is removed from the bioreactor bag and replaced with an equal volume of fresh culture medium.

48. 48. The method of claim 47, wherein the culture is incubated for about 12 to 24 hours.

49. 10. The method of claim 1, wherein during growth, fresh culture medium is added to the bioreactor by fed-batch / perfusion feeding and / or by perfusion.

50. 10. The method of claim 1, wherein during growth, the culture is perfused at a rate of one bioreactor bag volume per day.

51. 10. The method of claim 1, wherein as the cells grow, the volume of the culture medium in the bioreactor is gradually increased up to 1 liter during growth.

52. 10. The method of claim 1, wherein as the cells grow, the volume of the culture medium is gradually increased to maintain a cell density of at least 2E6 nucleated cells / ml.

53. 10. The method of claim 1, wherein as the cells grow, the volume of the culture medium in the bioreactor is gradually increased during growth to 1 liter to maintain a cell density of at least 4E6 nucleated cells / ml.

54. 10. The method of claim 1, wherein the rocking speed is gradually increased to 6 RPM as the cells grow.

55. 2. The method of claim 1, wherein at the beginning of the growth, the volume of the culture medium in the closed single-use bioreactor bag is 300 ml and rocked at an angle of 2° at a speed of 2 rpm.

56. The culture in the closed single-use bioreactor bag is maintained at about 80-100% O ２ The method of claim 1 , wherein the

57. The method of claim 1, wherein the cells are grown for 7 to 14 days.

58. The method of claim 1, wherein the culturing, transduction and / or propagation steps are carried out at 34-37°C.

59. The method of claim 1 , wherein the target protein is a cell surface receptor.

60. 60. The method of claim 59, wherein the cell surface receptor is a T cell receptor or a chimeric antigen receptor.

61. 61. The method of claim 60, wherein the cell surface receptor recognizes an antigen target associated with a target cell.

62. 62. The method of claim 61, wherein the target cell is a cancer cell.

63. 10. The method of claim 1, wherein the genetically modified autologous T cells are used to treat a condition in a patient in need thereof.

64. A pharmaceutical composition comprising the genetically modified autologous T cells of claim 1.

65. 65. A method of treating a condition in a patient in need of such treatment, comprising administering to said patient a pharmaceutical composition according to claim 64.

66. 1. A method for increasing transgene expression in genetically modified autologous T cells expressing a protein of interest, comprising: seeding apheresis donor cells and one or more soluble T cell activators into a closed single-use bioreactor bag containing culture medium, wherein the at least one soluble T cell activator is bound to the at least one donor cell at the time of seeding, and the bioreactor bag is part of a rocking bioreactor platform; Cultivating the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM; transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding the protein of interest while continuously rocking at a speed of about 2 RPM; Growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture until harvest. Including, The method wherein expression of the transgene is greater than expression of the transgene in genetically modified autologous T cells derived from an enriched population of T cells from the same apheresis donor cells and expressing the same protein of interest.

67. 1. A method of treating a patient with genetically modified autologous T cells expressing a protein of interest, comprising: incubating apheresis cells from the patient with one or more T cell activators selected from the group consisting of anti-CD3, anti-CD2 and anti-CD28 antibodies, or binding fragments thereof, to saturate antibody binding; seeding the apheresis cells into a closed single-use bioreactor bag containing culture medium, wherein the bioreactor bag is part of a rocking bioreactor platform; Cultivating the cells in the closed single-use bioreactor bag with continuous rocking at a speed of about 2 RPM; transducing the cells in the closed single-use bioreactor bag with at least one soluble viral vector comprising a polynucleotide encoding the protein of interest while continuously rocking at a speed of about 2 RPM; growing the cells in the closed single-use bioreactor bag at a rocking speed of about 2 RPM and increasing the culture volume and rocking speed as needed to maintain the culture at a desired cell density until harvest; harvesting and formulating the cells for cryopreservation; freezing and storing the cells until needed for administration to the patient; and thawing the cells and resuspending them in a medium suitable for injection; and reintroducing into the patient a pharma- tically effective amount of genetically modified autologous T cells expressing said protein of interest. The method includes:

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