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) selecting a CD8 + T cell epitope of an autologous cancer antigen present in blood of a cancer patient;
b) culturing a peripheral blood mononuclear cell (PBMC) isolated from blood of the cancer patient in a medium together with a peptide of the epitope and IL-2;
c) inducing 4-1BB expression in the cultured cells by adding the peptide same as in step b); and
d) culturing cells in which 4-1BB expression is induced on a culture plate coated with an anti-4-IBB antibody, and then removing unattached cells.
2 . The method of claim 1 , wherein the autologous cancer antigen in step a) is selected from the group consisting of hTERT (GenBank: BAC11010.1), WT1 (GenBank: AA061088.1), NY-ESO1 (GenBank: CAA05908.1), and MAGE-A3 (NCBI Reference Sequence: NP — 005353.1).
3 . The method of claim 1 , wherein the epitope in step b) is a peptide formed by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 15.
4 . The method of claim 1 , wherein the medium in step b) is a medium comprising autoplasma.
5 . The method of claim 1 , wherein the culture in step b) is performed for 12 to 16 days.
6 . The method of claim 1 , wherein the induction of expression in step c) is performed for 12 to 36 hours with culturing.
7 . The method of claim 1 , wherein the culture in step d) is performed for 1 to 20 minutes.
8 . A method for mass culturing autologous cancer antigen-specific CD8 + T cells, the method comprising: suspending autologous cancer antigen-specific CD8 + T cells isolated by the method of claim 1 , and allogenic PBMC irradiated with radiation in a medium comprising IL-2, an anti-CD3 antibody, and autoplasma, and then injecting the suspension into a culture bag; and additionally injecting the medium and culturing.
9 . The method of claim 8 , wherein the PBMC is isolated from a healthy doner.
10 . The method of claim 8 , wherein the culture is performed for 4 to 15 days.
11 . The method of claim 10 , wherein during the culture, the medium is additionally injected on day 4, 7, 9, 11 and 14 of culture.