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 heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate 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 elicited the production of anti-peptide IgG antibodies. This immune response 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 peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.