Introduction: MYCN oncogene is overexpressed in high-risk neuroblastoma and associated with aggressive and refractory disease. Interleukin-21 (IL-21) is known to promote anti-tumor effects by stimulating cytotoxic but not regulatory T cells. Therefore, we developed protein and IL-21-based DNA vaccines with MYCN minigenes encoding for highly antigenic T-cell epitopes of MYCN. To increase proteasomal degradation and MHC class I presentation of MYCN epitopes to cytotoxic T lymphocytes, an upstream ubiquitin sequence was additionally integrated in the DNA vaccine. For evaluation of anti-NB effects in vivo, syngeneic mice will be immunized with protein vaccine in combination with the adjuvant aluminum hydroxide and DNA vaccine using attenuated Salmonella typhimurium SL7207 as vehicle.
Methods and results: For protein vaccine, a DNA sequence containing MYCN minigenes combined with an upstream leader sequence for protein secretion in vitro was synthesized and inserted into a plasmid encoding for human IgG1 constant heavy chain using standard molecular biology techniques. To enable permanent production of the fusion protein, CHO cells will be stably transfected with the generated plasmid. Finally, the fusion protein will be isolated from supernatant for in vivo vaccination.
For DNA vaccine, previously designed MYCN minigenes combined with ubiquitin sequence and IL-21 DNA fragment synthesized from murine spleen RNA were inserted into the respective multiple cloning site of the bicistronic expression vector pIRES allowing simultaneous expression of both inserts.
Correct plasmid assembly of both vaccines was confirmed by gene-specific PCR, restriction and sequence analysis. MYCN epitope expression and IL-21 production was approved in vitro by ELISA and Western blot after transfection of CHO cells with the respective plasmid.
Conclusion: We generated and partly characterized two new MYCN-based DNA and protein vaccines for active immunotherapy against MYCN-expressing neuroblastoma.