IMMUNOGENICITY OF NEW HETEROBIFUNCTIONAL CROSS-LINKING REAGENTS USED IN THE CONJUGATION OF SYNTHETIC PEPTIDES TO LIPOSOMES

We have investigated the immunogenicity of six thiol-reactive heterobi functional cross-linking reagents that permit the conjugation of cyste ine carrying peptides to the surface of liposome containing monophosph oryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linke rs to limit potential carrier-induced epitopic suppression. For that p urpose the properties of three new polyoxyethylene linkers of differen t lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiop yridine) were compared to known derivatives obtained by reacting the c lassical reagents SMPB and SPDP or N-succinimidyl bromoacetate with ph osphatidylethanolamine. The least immunogenic linkers were the bromoac etate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly e licited the production of anti-peptide IgG antibodies. This immune res ponse was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage between the p eptide and the thiol-reactive moieties of the cross-linkers, i.e. stab le thioether or bio-reducible disulfide bonds.