Process for generating genetically engineered autologous t cells

A method for increasing the transgene expression in genetically engineered autologous T cells expressing a protein of interest, the method comprising

• inoculating a closed single use bioreactor bag containing culture media with apheresed donor cells and one or more soluble T cell activators, wherein at least one soluble T-cell activator is bound to at least one donor cell at the time of inoculation and the bioreactor bag is part of a rocking bioreactor platform,
• culturing the cells in the closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the closed single use bioreactor bag with at least one soluble viral vector comprising a polynucleotide which encodes the protein of interest continuously rocking at a rate of about 2 RPM, and
• expanding the cells in the closed single use bioreactor bag at a rocking rate of about 2 RPM and increasing the culture volume and rocking the rate as needed to maintain the culture until harvest, wherein the transgene expression is greater than the transgene expression of genetically engineered autologous T cells derived from an enriched population of T cells from the same apheresed donor cells and expressing the same protein of interest.

A method of treating a patient with genetically engineered autologous T cells expressing a protein of interest comprising,

• incubating apheresed cells from the patient with one or more T cell activators selected from the group consisting of an anti CD3 antibody, an anti CD2 antibody, and an anti CD28 antibody or binding fragments thereof, to allow for saturation of antibody binding,
• inoculating a closed single use bioreactor bag containing culture media with the apheresed cells, wherein the bioreactor bag is part of a rocking bioreactor platform,
• culturing the cells in the closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the closed single use bioreactor bag with at least one soluble viral vector comprising a polynucleotide which encodes the protein of interest continuously rocking at a rate of about 2 RPM, and
• expanding the cells in the closed single use bioreactor bag at a rocking rate of about 2 RPM, increasing the culture volume and rocking the rate as needed to maintain the culture at a desired cell density until harvest.
• harvesting and formulating the cells for cryopreservation,
• freezing the cells and storing until needed for administering to the patient,
• thawing and resuspending the cells in a suitable media for infusion, and
• reintroducing a pharmaceutically effective amount of the genetically engineered autologous T cells expressing the protein of interest into the patient.