Cryopreservation of apoptotic cancer cells for use in immunotherapy against cancer
Described herein is a reliable method for preparing a potent vaccine useful for immunotherapy comprising the step of cryopreserving a population of cells undergoing immunogenic cell death, and using such cells to activate dendritic cells for use in immunotherapy. In a specific embodiment, the method comprises cryopreserving cancer cells undergoing cell death, which can be used to prepare a pharmaceutical composition for immunotherapy against cancer.
1. A method for preparing a pharmaceutical composition for use in immunotherapy comprising:
a) inducing immunogenic cell death in a population of cancer cells;
b) cryopreserving the cancer cells undergoing immunogenic cell death in a cryopreservant, wherein the cells undergoing immunogenic cell death are cryopreserved within six hours after induction of immunogenic cell death;
c) thawing the cryopreserved cancer cells, wherein the thawed cells are put back into culture; and
d) pulsing immature dendritic cells with the thawed cancer cells, wherein the thawed cancer cells retain one or more of the hallmarks of immunogenic cell death.
2. The method of claim 1 , wherein the cryopreserved cells undergoing immunogenic cell death are put back into culture for at least 1 hour and not more than 6 hours.
3. The method of claim 1 , wherein the cells undergoing immunogenic cell death are cryopreserved within 30 minutes to 4 hours after induction of immunogenic cell death.
4. The method of claim 1 , wherein the cells undergoing immunogenic cell death are cryopreserved within 1.5 hours to 2.5 hours after induction of immunogenic cell death.
5. The method of claim 1 , wherein the cancer cells are from a cancer cell line.
6. The method of claim 1 , wherein the cancer cells originate from multiple cancer cell lines.
7. The method of claim 1 , wherein the cancer cells have been expanded from cell lines expressing cancer antigens.
8. The method of claim 1 , wherein the immunogenic cell death is induced by high hydrostatic pressure, anthracyclines, anti-EGFR antibodies, Big Potassium channel antagonists, bortezomib, cardiac glycosides, cyclophosphamide, GADD43/PP1 inhibitors and mitomycin, irradiation by UV light or gamma rays, oxaliplatin, photodynamic therapy with hypericin, poly(I:C), or thapsigargin and cisplatin.
9. The method of claim 1 , wherein the cell death is induced by high hydrostatic pressure.
10. The method of claim 1 , wherein the cryopreservant contains dimethyl sulphoxide at a concentration of at least 5%.
11. The method of claim 1 , wherein the cryopreservant contains glycerol at a concentration of at least 5%.
12. The method of claim 1 , wherein the cells undergoing immunogenic cell death are cryopreserved at a temperature below −75° C.
13. The method of claim 1 , wherein the cryopreservation is performed by slow freezing.
14. The method of claim 1 , wherein the thawing is performed for between 5 seconds and 10 minutes at a temperature between 32° C. and 40° C.
15. The method of claim 1 , wherein the immunotherapy is for the treatment of a solid tumor cancer.
16. The method of claim 15 , wherein the solid tumor cancer is prostate cancer, ovarian cancer, lung cancer, renal cancer, colon cancer, breast cancer or glioblastoma.
17. The method of claim 1 , wherein the one or more of the hallmarks of immunogenic cell death are selected from the group consisting of calreticulin, HSP70, and HSP90.
18. The method of claim 1 , wherein the immature dendritic cells were differentiated from monocytes obtained by leukapheresis.