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 wherein the apheresed donor cells comprise leukocytes and erythrocytes, and soluble T cell activators comprising at least an anti CD3 antibody or binding fragments thereof, 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 same closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the same 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 same 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 cells are not removed from the closed single use bioreactor bag and subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype prior to harvest;
• and
• 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 wherein the apheresed donor cells comprise leukocytes and erythrocytes, with T cell activators comprising at least an anti CD3 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 same closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the same 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 same 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;
• wherein the cells are not removed from the closed single use bioreactor bag and subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype, prior to 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.

A method for producing genetically engineered autologous T cells expressing at least one protein of interest, the method comprising:

• inoculating a closed single use bioreactor bag containing culture media with apheresed donor cells wherein the apheresed donor cells are not subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype prior to inoculation, and soluble T cell activators comprising at least an anti CD3 antibody or binding fragments thereof, wherein the bioreactor bag is part of a rocking bioreactor platform,
• culturing the cells in the same closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the same 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 same closed single use bioreactor bag at a rocking rate of about 2 RPM and increasing the culture volume and rocking rate as needed to maintain the culture until harvest;
• wherein the cells are not removed from the closed single use bioreactor bag and subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype prior to harvest.

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, wherein the apheresed donor cells are not subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype prior to inoculation, and soluble T cell activators comprising at least an anti CD3 antibody or binding fragments thereof, 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 same closed single use bioreactor bag continuously rocking at a rate of about 2 RPM,
• transducing the cells in the same 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 same 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 cells are not removed from the closed single use bioreactor bag and subjected to further isolation, selection, and/or enrichment for a desired cell population or phenotype prior to 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.