Process for generating genetically engineered autologous T cells
The present invention relates to production of autologous genetically engineered T cells for use in cell therapy applications.
1. 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 comprise leukocytes and erythrocytes, 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.
2. The method of claim 1 , wherein the apheresed donor cells further comprise cells from peripheral blood.
3. The method of claim 2 , wherein the cell from peripheral blood comprise nucleated and non- nucleated cells.
4. The method of claim 3 , wherein the cells from peripheral blood also comprise granulocytes and/or platelets.
5. The method of claim 1 , wherein the apheresis is leukapheresis.
6. The method of claim 1 , wherein the apheresed donor cells are washed and resuspended in a culture media.
7. The method of claim 1 , wherein the soluble T cell activator comprises an anti CD3 antibody and an anti CD28 antibody, or binding fragments thereof.
8. The method of claim 1 , wherein the soluble T cell activator comprises at least an anti CD3 antibody, an anti CD28 antibody, and an anti CD2 antibody, or binding fragments thereof.
9. The method of claim 1 , wherein the soluble 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.
10. The method of claim 1 , wherein the concentration of at least one soluble T cell activator is at least 0.001 μg/ml to 10 μg/ml.
11. The method of claim 10 , wherein the concentration of at least one soluble T cell activator is at least 0.1 μg/ml to 5 μg/ml.
12. The method of claim 1 , wherein at least one soluble T-cell activator is bound to at least one donor cell at the time of inoculation.
13. The method of claim 1 , wherein the apheresed donor cells are incubated with soluble T cell activators comprising at least an anti CD3 antibody or binding fragments thereof prior to inoculating into the bioreactor bag.
14. The method of claim 13 , wherein the incubation is conducted for a sufficient time to allow for saturation of binding of soluble T cell activators comprising at least an anti CD3 antibody or binding fragments thereof to the apheresed donor cells prior to inoculation.
15. The method of claim 14 , wherein the apheresed donor cells and soluble T cell activators comprising at least an anti CD 3 antibody or binding fragments thereof are incubated in a transfer bag.
16. The method of claim 15 , wherein the volume of culture media in the transfer bag is about 5 ml to about 50 ml.
17. The method of claim 16 , wherein the volume of culture media in the transfer bag is about 5 ml to about 10 ml.
18. The method of claim 15 , wherein the apheresed donor cells are incubated with solube T cell activators comprising at least an anti CD3 antibody or binding fragments thereof for at least 30 minutes or more.
19. The method of claim 18 , wherein the apheresed donor cells are incubated with solube T cell activators comprising at least an anti CD3 antibody or binding fragments thereof, for at least 1 hour.
20. The method of claim 1 , wherein the number of leukocytes within the apheresed donor cells is about 1.0E9 to about 1.3E9.
21. The method of claim 1 , wherein the number of leukocytes within the apheresed donor cells is about 1.2E9.
22. The method according to claim 1 , wherein the bioreactor bag is inoculated with apheresed donor cells at a cell density of about 1E6 to about 5E6 leukocytes.
23. The method according to claim 22 , wherein the bioreactor bag is inoculated with apheresed donor cells at a cell density of about 2E6 leukocytes.
24. The method according to claim 1 , wherein the bioreactor bag contains at least 300 ml to 400 ml of culture media at inoculation.
25. The method according to claim 24 , wherein the bioreactor bag contains 300 ml of culture media at inoculation.
26. The method according to claim 1 , wherein the apheresed donor cells are cultured in the bioreactor bag for about 12-24 hours.
27. The method of claim 1 , wherein the culture media comprises at least one soluble cytokine.
28. The method according to claim 27 , wherein the soluble cytokine selected from IL-2, IL-7, IL-15, or IL-21.
29. The method according to claim 27 , wherein at least one soluble cytokine is IL-2.
30. The method according to claim 29 , wherein the IL-2 is at a concentration of about 250 IU/ml to about 350 IU/ml.
31. The method according to claim 30 , wherein the IL-2 is at a concentration of about 300 IU/ml.
32. The method according to claim 27 , wherein the soluble cytokine is IL-7 in combination with IL-15 or IL-21.
33. The method according to claim 27 , wherein the concentration of at least one soluble cytokine is at least 5 ng/ml to at least 30 ng/ml.
34. The method according to claim 33 , wherein the concentration of at least one soluble cytokine is at least 10 ng/ml to at least 20 ng/ml.
35. The method according to claim 1 , wherein the culture media also comprises a WNT pathway activator.
36. The method according to claim 35 , wherein the WNT pathway activator is TWS 119 .
37. The method according to claim 35 , wherein the culture media comprises a mixture of soluble TWS119, IL-7, and IL-21.
38. The method of claim 1 , wherein the culture media also comprises a soluble glycolysis inhibitor.
39. The method of claim 38 , wherein the soluble glycolysis inhibitor is 2-deoxy-D-glucose (2-DG).
40. The method of claim 1 , wherein the viral vector is a retroviral vector.
41. The method of claim 1 , wherein the viral vector is a lentiviral vector.
42. The method of claim 41 , wherein the lentiviral vector is added at a MOI of 0.25-10.
43. The method of claim 41 , wherein the lentiviral vector is added at a MOI of 1.
44. The method of claim 42 , wherein the cells are transduced for about 18-22 hours.
45. The method of claim 1 , wherein following transduction, half of the culture media is removed from the bioreactor bag and replaced with an equal volume of fresh culture media.
46. The method of claim 45 , wherein the culture is incubated for about 10-22 hours.
47. The method of claim 1 , wherein during expansion, fresh culture media is added to the bioreactor bag by fed batch/perfusion feeding and/or by perfusion.
48. The method of claim 1 , wherein during expansion the culture is perfused at a rate is one bioreactor bag volume per day.
49. The method of claim 1 , as the cells are expanded the volume of the culture media in the bioreactor bag is incrementally increased to 1 liter during expansion.
50. The method of claim 1 , as the cells are expanded the volume of the culture media is incrementally increased to maintain a cell density of at least 2E6 leukocytes/ml.
51. The method of claim 1 , as the cells are expanded the volume of the culture media in the bioreactor bag is incrementally increased to 1 liter during expansion to maintain a cell density of at least 4E6 leukocytes/ml.
52. The method of claim 1 , as the cells are expanded the rocking rate is incrementally increased to 6 RPM.
53. The method of claim 1 , at the start of expansion the volume of culture media in the single use closed bioreactor bag is 300 ml rocking at a rate of 2 rpm at a 2° angle.
54. The method of claim 1 , wherein the culture in the single use closed bioreactor bag is maintained at about 80-100% O 2 .
55. The method of claim 1 , wherein the cells are expanded for 7 to 14 days.
56. The method of claim 1 , wherein the culture, transduction, and/or expansion steps are performed at 34-37° C.
57. The method of claim 1 , wherein the protein of interest is a cell surface receptor.
58. The method of claim 57 , wherein the cell surface receptor a T cell receptor, or chimeric antigen receptor.
59. The method of claim 58 , wherein the cell surface receptor recognizes an antigenic target associated with a target cell.
60. The method of claim 59 , wherein the target cell is a cancer cell.
61. The method of claim 1 , wherein the genetically engineered autologous T cells are used to treat an indication in a patient in need.
62. 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 CD 3 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.
63. 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.
64. The method according to claim 1 , 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.
65. 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.
66. The method according to claim 1 further comprising,
harvesting and formulating the cells for cryopreservation, p 1 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.
67. The method according to claim 65 further comprising,
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.
68. 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.