Methods for isolating and proliferating autologous cancer antigen-specific CD8+T cells
Provided is a method for isolating and proliferating autologous cancer antigen-specific CD8 + T cells, and more particularly, a method for selecting an epitope recognized by CD8 + T cells from autologous cancer antigens present in blood of individual cancer patients; and isolating autologous cancer antigen-specific CD8 + T cells by using a peptide of the selected epitope, and a method of massively proliferating CD8 + T cells by using the method. According to the present invention, it is possible to isolate autologous cancer antigen-specific CD8 + T cells by using the peptide of the CD8 T cell epitope of the autologous cancer antigen present in blood of individual cancer patients instead of a heterologous antigen. Therefore, by using T cells recognizing the autologous cancer antigen, it is possible to effectively select and eliminate cancer cells derived from the cancer patient's own cells. Thus, T cells can be applied to treatment and alleviation of cancer diseases without side effects.
1. A method for isolating autologous cancer antigen-specific CD8+T cells, the method comprising
a) culturing peripheral blood mononuclear cells (PBMCs) isolated from blood of a cancer patient in a cell culture medium with three or more peptides, and I1-2, wherein
(i) the cancer patient has a HLA-A*024 allele and an autologous cancer antigen of hTERT, and the three or more peptides comprise SEQ ID NO:1-3;
(ii) the cancer patient has a HLA-A*024 allele and an autologous cancer antigen of WT1, and the three or more peptides are selected from a group consisting of SEQ ID NO: 4-7;
(iii) the cancer patient has a HLA-A*02 allele and an autologous cancer antigen of NY-ESO1, and the three or more peptides are selected from a group consisting of SEQ ID NO: 8-11;
(iv) the cancer patient has a HLA-A*02 allele and an autologous cancer antigen of MAGE-A3, and the three or more peptides are selected from a group consisting of SEQ ID NO:12-15; and
wherein culturing the PBMCs with the three or more peptides induces 4-1BB expression;
b) selecting cultured cells using an anti-4-1BB antibody, thereby isolating autologous cancer antigen-specific CD8+ T cells.
2. The method of claim 1 , wherein autologous cancer antigen-specific CD8 + T cells are less than 0.1% of all T cells present in blood of the cancer patient.
3. The method of claim 1 , wherein the cell culture medium in step a) comprises plasma that is autologous to the cancer patient.
4. The method of claim 1 , wherein the culturing step a) is performed for 12 to 16 days.
5. The method of claim 1 wherein expression of 4-1BB is induced by culturing the cells of step a) for 12-36 hours.
6. The method of claim 1 , wherein the selecting step b) comprises culturing cells in which 4-1BB expression is induced on a culture plate coated with an anti-4-1BB antibody.
7. The method of claim 6 , wherein the selecting step b) is performed for 1 to 20 minutes.
8. The method of claim 1 , wherein the autologous cancer antigen in step a) is hTERT.
9. The method of claim 1 , wherein the autologous cancer antigen in step a) is NY-ESO1.
10. The method of claim 1 , wherein the autologous cancer antigen in step a) is MAGE-A3.
11. The method of claim 1 , wherein the autologous cancer antigen is step a) is WT1.
12. A pharmaceutical composition comprising autologous cancer antigen-specific CD8 + T cells isolated by the method of claim 1 and a pharmaceutically acceptable carrier.
13. A method for treating cancer in a patient in need thereof comprising administering to the patient the pharmaceutical composition of claim 12 , wherein (i) the cancer patient has a HLA-A*024 allele and an autologous cancer antigen of hTERT or WT1, or (ii) the cancer patient has a HLA-A*02 allele and an autologous cancer antigen of NY-ESO1 or MAGE-A3.
14. The method of claim 13 , wherein the cancer is a solid tumor.
15. The method of claim 13 , wherein the cancer is selected from lung cancer, gastric cancer, pancreatic cancer, melanoma, gliobastoma, leukemia, ovarian cancer, brain cancer, spinal cancer, germ cell cancer, breast cancer, and sarcoma.