Abstract
The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.
Functional characterization of a T-cell receptor BV6+T-cell clone derived from a leprosy lesion