COMPARISON OF LINEAR AND BRANCHED PEPTIDE FORMS (MAPS) IN THE INDUCTION OF T-HELPER RESPONSES TO POINT-MUTATED RAS IMMUNOGENS

The utility of multiple antigenic peptides (MAPs) for the induction of antibody and cellular immune responses in animal models has been demo nstrated for a variety of peptide epitopes involved in human disease, However, little is known about immune responses to MAPs constructed wi th antigenic tumor epitopes, nor has peptide specificity in branched f orms been addressed, A potentially important advantage of the MAP syst em over linear peptide immunogens for clinical applications is elimina tion of the need for a protein carrier with its associated toxicity an d immunogenicity. Here, we examined cellular immune responses followin g in vivo administration of MAPs incorporating a 13-mer T helper epito pe from point-mutated ras p21 (ras V12) and compared the potency of th e responses to that of the linear peptide. The Gly --> Val mutation in position 12, which is associated with a range of human carcinomas, re presents a useful system for evaluating the specificity of the immune response, In initial studies with the point-mutated Linear peptide epi tope, optimal in vitro proliferation responses were obtained following sc administration of the peptide in a squalane-containing adjuvant fo rmulation, Comparative immunization studies using point-mutated MAPs b earing two, four, or eight branches were administered either in saline or in adjuvant. These studies showed that adjuvant was required for t he induction of cellular immune responses using both linear and all th ree forms of branched peptides, Moreover, there was no apparent advant age of using any of the MAPs vs linear peptide when equivalent mass am ounts were administered, i.e., the intensity of the immune response wa s no greater using any of the branched structures compared to the line ar form. Specificity of the in vivo responses for both the linear and the MAP immunogens was demonstrated by the higher stimulation indices observed in vitro in the presence of the mutant ras V12 vs the normal ras G12 linear peptide, No apparent cellular immune response to the MA P core structure itself was observed. However, a nonspecific response to the two-branched MAP2G12 structure was observed in some assays, the nature of which is unknown at this time, This work represents the fir st reported investigation of a cellular immune response using MAP immu nogens incorporating a tumor-specific peptide epitope and demonstrates that linear peptides are as efficient as three different MAP structur es in the generation of specific T cell responses.