Methods for isolating and proliferating autologous cancer antigen-specific CD8
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 and culturing autologous cancer antigen-specific CD8 + T cells, the method comprising
a) selecting a cancer patient having an autologous cancer antigen of hTERT, WT1, NY-ESO1 or MAGE-A3, and a HLA-A*02 allele or a HLA-A*024 allele, wherein the patient has been determined to have, by flow cytometry analysis after culture of autologous peripheral blood mononuclear cells (PBMCs) with at least one individual peptide selected from SEQ ID NO:1-15 and re-stimulation by the same individual peptide,
(i) at least 11% of T cells which are 4-1BB+CD8+, and
(ii) a ratio of CD8 + T cells to CD8 − T cells of greater than or equal to one;
b) culturing PBMCs isolated from the blood of the cancer patient selected in step a) in a cell culture medium with three or more peptides and IL-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 are 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; or
(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;
c) isolating the cultured cells and re-stimulating the isolated cells by culturing with the same three or more peptides used for the culturing in step b);
d) incubating the re-stimulated cells on a culture plate coated with anti-4-1BB antibody to allow for attachment of activated peptide-specific CD8 T cells, and then removing unattached cells and culturing the remaining cells adhering to the plate;
e) removing whole cells after culture on the anti-4-1BB coated culture plate and suspending the removed cells in a medium comprising irradiated allogeneic PBMCs, IL-2, anti-CD3 antibody, and autoplasma; and
f) expanding the number of T cells by culturing the suspension in step e) with additional injection of medium comprising IL-2 and autoplasma.
2. The method of claim 1 , wherein the medium in step b) is a medium comprising autoplasma.
3. The method of claim 1 , wherein the culture in step b) is performed for 12 to 16 days.
4. The method of claim 1 , wherein the re-stimulating in step c) is performed for 12 to 36 hours with culturing.
5. The method of claim 1 , wherein the incubating in step d) is performed for 1 to 20 minutes.
6. The method of claim 1 , wherein the allogenic PBMCs in step e) are isolated from a healthy donor.
7. The method of claim 1 , wherein the culturing in step f) is performed for 4 to 15 days.
8. The method of claim 1 , wherein the medium during the culturing in step f) is additionally injected on day 4, 7, 9, 11, and 14 of culture.