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GD2 Peptide Mimotope DNA Vaccines for Anti-Neuroblastoma Immunotherapy.

The tumor-associated antigen disialoganglioside-GD2 is expressed on neuroblastoma and melanoma and is an established target for passive immunotherapy. The aim of this study was to develop an active immunization strategy leading to the induction of a humoral anti-GD2 immune response. However, carbohydrates and glycolipids are T cell-independent antigens (TI) and usually evoke a poor immune response in tumor-bearing hosts.

Here, we describe the identification, characterization and in vivo efficacy of cyclic peptides mimicking the structure of glycolipid GD2, i.e. GD2 mimotopes, in order to overcome T-cell independency. First, GD2 peptide mimotopes were identified by biopanning experiments of a phage-display library displaying circular decapeptides against the human/mouse chimeric anti-GD2 antibody (Ab) ch14.18. Thirteen independent phage clones were isolated which bind to ch14.18 with high specificity. Competitive binding of phages expressing GD2 peptide mimotopes to ch14.18 antibody revealed two superior peptide candidates, mimotope A (MA) and and mimotope D (MD), which were subjected to further evaluation. Second, two plasmid DNA minigene vaccines were generated by overlapping PCR encoding for MA and MD, respectively. The plasmids were based on pSecTag2-A also including a kappa leader sequence, a T-cell helper epitope from HIV-1 gp 120 (T1) and a myc-tag. Minigene expression was demonstrated following transfection of COS-7 cells in western-blots and GD2 mimikry was determined in solid phase ELISA experiments. Third, the efficacy of these mimotope DNA vaccines to induce a tumor protective anti-GD2 immune response was tested in the syngeneic NXS2 model of neuroblastoma expressing ganglioside GD2. The DNA vaccination was accomplished with attenuated Salmonella typhimurium (SL 7207) used as an oral vaccine carrier. Only mice receiving the mimotope DNA vaccines revealed a decrease in primary tumor growth by 50% and a dramatic reduction of spontaneous liver metastases with a mean liver weight of 1g in both groups (MA and MD) in contrast to negative controls (3g). Interestingly, mice immunized with KLH conjugated peptide mimotopes A and D revealed an increased rate of s.c. tumor growth and spontaneous liver metastasis with average liver weights of 5 (MA) and 7 (MD), respecively, suggesting the induction of tolerance using this peptide vaccine approach. Finally the highest anti-GD2 humoral immune response was observed in sera of mice from both GD2 mimotope DNA vaccine groups, consistent with the anti-tumor reponse observed in vivo. Based on these data, we belive that GD2 mimotope DNA vaccines may provide a useful strategy for active immunization against neuroblastoma.