Enhanced Antitumor Effect Against Human Telomerase Reverse Transcriptase (hTERT) By Vaccination With Chemotactic-hTERT

To estabilish a more efficient treatment against most human cancers, we constructed a chemotactic vaccine targeting human telomerase reverse transcriptase(hTERT) and investigated the possibility to improve its antitumor effect and the related immunological mechanism.The cDNA of hTERT was analyzed by peptide-motif scoring system to select epitopes that could potentially bind to HLA classâ… or classâ…¡molecules. Two fragments encoding multiple CTL and Th epitopes were selected and fused as a tumor antigen(named Te). Its N-terminal was ligated with human chemokine CCL21, and the C-terminal was ligated with human IgG Fc. The recombinant construct(CCL21-Te-Fc) was inserted into an expression vector to generate pCCL21-Te-Fc. Both RT-PCR and western-blotting assay confirmed that the recombinant fusion proteins were expressed in the transfected cells. The chemotaxis assay demonstrated that the human CCL21 in all of our constructs was functional and had the ability to attract human and mouse lymphocytes. Furthermore, the chemotatic activity of the fusion protein(CCL21-Te-Fc) was detected in vivo. H&E staining of paraffin-embedded dermis tissues sections suggested that the secreted CCL21-Te-Fc had the same chemotactic activity with that of CCL21 in vivo. In poorly immunogenic mouse melanoma model (B16/Te), DNA (pCCL21-Te-Fc) vaccination significantly inhibited tumor growth but all of the mice were dead by day 52. The immunization with pCCL21-Te-Fc-modified tumor cells (B16/CCL21-Te-Fc) resulted in a higher antitumor effect than DNA vaccination and there were 37.5ï¼…and 25ï¼…of tumor-bearing mice achieved long-term survival in the prophylactic model and in the therapeutic model, respectively.The combined therapy of B16/CCL21-Te-Fc plus anti-4-1BB MAbs further enhanced the immune response, resulting in 75ï¼…of tumor-bearing mice achieved long-term survival in subcutaneous model and few lung nodules in pulmonary metastasis model, suggesting that there was an additive effect from combining B16/CCL21-Te-Fc vaccination and anti-4-1BB. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy of B16/CCL21-Te-Fc plus anti-4-1BB. In order to identify the effector cells involved in the tumor rejection response associated with the combination therapy we depleted NK cells, CD4~+ T cells and CD8~+ T cells, respectively. The CTL activity induced by the treatment of B16/CCL21-Te-Fc plus anti-4-1BB was completely abrogated by the depletion of CD8~+ T cells. Partial abrogation was observed by the depletion of CD4~+ T cells or NK cells. Similar results were obtained in the analysis of local tumor size development. These results indicated that CD8~+ T cells were essential in the antitumor activity induced by B16/CCL21-Te-Fc plus anti-4-1BB MAbs, whereas NK cells and CD4~+ T cells played substantial roles.HLA-A*0201~+ PBMCs transfeced with pCCL21-Te-Fc were used to stimulate autologous lymphocytes in vitro, and the cytotoxicity of the induced CTL was evaluated against a panel of tumor cells with diverse histological origins. Our results showed that the cytotoxicity was dependent on the expression of HLA-A*0201 molecule. And at the same time the CTL only lysed the hTERT-positive cell line but not the hTERT-negative cell line, further demonstrating antigen specificity of the cytotoxic response. Taken together, these data indicated that the selected hTERT might be naturally processed by human antigen-presenting cells and induced hTERT-specific CTL against hTERT-positive human tumor cells.