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Data: 29-mar-2012
Autori: Di Puma, Salvatore Davide
Titolo: Progettazione sintesi e valutazione biologica dei lipopeptidi contenenti clusters multimerici dell'epitopo immunodominante pdtrp per l'immunoterapia attiva del cancro
Abstract: During carcinogenesis MUC-1 cell-surface carbohydrates undergo incomplete glycosylation that causes exposure of highly immunodominant peptide PDTRP contained in the tandem repeat sequence of mucin core (GVTSAPDTRPAPGSTAPPAH). The incomplete glycosylation results also in the exposure of the normally cryptic carbohydrate antigens like Tn (GalNAc-O-Ser/Thr). In a previous work, we synthesized a macromolecular construct containing four unit of Tn antigen clustered onto a calix[4]arene scaffold bearing an immunoadjuvant moiety (P3CS). This construct gave in mice higher immune response when compared to monovalent reference compound, and the outlined data clearly showed a so called "cluster effect". The aim of this project was to synthesize multimeric lipopeptide constructs containing clusters of the PDTRP antigen assembled on calixarene scaffolds together with the immunoadjuvant tripalmitoyl-S-glyceryl-Cys-Ser (P3CS). The purpose was to investigate the role of the cluster effect in amplifying the antibody production with respect to a simple concentration increase of antigen units. In addition, the role of the scaffold flexibility was also examined using the relatively rigid tetrameric calix[4]arene and the more flexible calix[8]arene as platforms to realize the constructs. The PDTRP sequence was assembled by solid-phase peptide synthesis using Fmoc-protected amino acids in combination with Barlos s resin. The protected PDTRP-COOH was coupled via glycine spacer to calix[4]arene and calix[8]arene derivatives containing, at the upper rim, 4 and 8 glycine unit respectively, and, at the lower rim, 1 unit of P3CS. These calixarenes constructs were synthesized as previously reported in collaboration with CNR-ICB of Catania (Dr C. Geraci). The final constructs were characterized by 1H-NMR and MALDI-TOF. Five groups of six mice were immunized two times, at 1-week intervals, by intraperitoneal injections of the two synthesized constructs (0.030 micro mol/mouse) or the reference compound, Mono-PDTRP-P3CS, administered at three concentration levels (0.030, 0.120 and 0.240 micro mol/mouse). After 21 days from the first immunization antibodies specific to PDTRP antigen in sera were detected and quantified by end-point dilution ELISA. Mice immunized with the synthesized multimeric constructs showed a substantial and significant production of antibodies. For Octa-PDTRP-Gly-Calix[8]arene-P3CS and Tetra-PDTRP-Gly-Calix[4]-P3CS a significant (p<0.05) increase of the end-point titers of 32- and 8-fold were measured with respect to the mice group immunized using equimolar level of the reference monovalent construct. Even when the concentration of monovalent construct was increased 4- and 8-fold, in order to compare solutions with the same number of PDTRP antigen units, the reactivity elicited by octameric and tetrameric constructs were significantly higher (p < 0.05) with a 8- and 4-fold increase in the end-point titer values. The immunogenic response induced by the Octa-PDTRP-Gly-Calix[8]-P3CS was significantly higher (p<0.05) with respect to the tetrameric construct with an increase of 4-fold in the antibodies production. Novel, well-defined synthetic multivalent lipopeptide vaccine candidates have been designed and synthesized. All the multimeric constructs are able to induce specific PDTRP antibodies production in mice. The most active derivative is the Octa-PDTRP-Gly-Calix[8]-P3CS that ensure an activity double with respect to the tetrameric construct. In addition, the "cluster effect" observed is more evident for the octameric construct that is more flexible with respect to the calix[4]arene construct. The increased conformational flexibility could allow a better arrangement of PDTRP antigens array on the calix[8]arene scaffold, ensuring a good mimicking of the natural motif encountered in vivo in the cancer cells. This reveals perspectives for potential applications in cancer immunotherapy.
InArea 03 - Scienze chimiche

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