28.    Impact of peptide formulations and adjuvant doses in immunizations against an                 endogenous prostate cancer overexpressed antigen: specific CD8+ T cell response             requirements 

Active immunization has been proposed as a promising strategy for treatment of dvanced Prostate Cancer. However, few tumor associated antigens have demonstrated to elicit an efficient immune response and moreover, our knowledge about how to perform such immunizations remains incipient. Previously, we postulated Galectin-1 as a novel immunogen in PCa and immunizations with a Gal- 1 derived peptide induced specific cytotoxicity. This work evaluates different conditions to ameliorate the immunization protocol. First, we decided to comparatively study the use of short peptides (Gal-1 MHC class I and PADRE MHC Class II) vs a long peptide (both peptides joined by a cleavable link). Second, we evaluated the adjuvancy doses. Both peptide formulations combined with
conventionally used doses of adjuvants (900mM PolyU + 25uM CpG) do not induced specific cytotoxicity while 1/32 lower adjuvant doses induced 15% for the long peptide vs 2% for the short ones (p <0.05, t-test) at day 7 post-priming. This prompted us to comparatively characterize at the cellular level these different immunization protocols. Immunization with long peptide and lower doses of adjuvant demonstrated a moderate total lymph node cell burst in contrast to the great expansion seen with conventional doses of adjuvant. Additionally, lower doses of adjuvant decreased Tregs and more importantly, induced a 2.8 fold increase in the absolute numbers of tetramer specific CD8+ T cells. These parameters could explain the higher response obtained. To gain better insights about parameters required for an optimal boost, we demonstrated that low levels of adjuvant are required in re-stimulations but, in this phase, long peptides do not increase the specific cytotoxicity while the short ones do, suggesting that the use of long peptides is only crucial at priming. In summary, our results challenge current ideas about the parameters required to perform a better immunization against an endogenous, tumor-overexpressed antigen.