Higashimoto, Yuichiro

Abstract The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8–10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these “Trojan” Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.

Abstract We describe in this study a strategy to produce synthetic vaccines based on a single polypeptide capable of eliciting strong immune responses to a combination CTL and Th epitopes with the purpose of treating malignancies or preventing infectious diseases. This strategy is based on the capacity of Trojan Ags to deliver exogenous Ags into the intracellular compartments, where processing into MHC-binding peptides takes place. Our previous work demonstrated that Trojan Ags containing a CTL epitope localized to intracellular compartments, where MHC class I-binding peptides were generated in a TAP-independent fashion by the action of various exopeptidases and the endopeptidase furin. In this study, we report that Trojan Ags containing several CTL epitopes joined via furin-sensitive linkers generated all of the corresponding MHC class I-binding peptides, which were recognized by CTL. However, Trojan Ags prepared with furin-resistant linkers failed to produce the MHC class I-binding peptides. We also present data indicating that Trojan Ags bearing both CTL and Th epitopes can generate the corresponding MHC class I- and II-binding peptides, which are capable of stimulating T cell responses. Most significantly, in vivo vaccination of mice with a single injection of multiepitope Trojan Ags resulted in strong CTL and Th responses that translated into significant antitumor responses in a model of malignant melanoma. The overall results indicate that Trojan Ags prepared with furin-sensitive linkers are ideal candidates for producing synthetic multiepitope vaccines for the induction of CTL and Th responses that could be used against a variety of diseases, including cancer.