Stability and CTL activity of N-terminal glutamic acid containing peptides

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan -A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutami c acid. It has been reported that the amino group and gamma -carboxylic gro up of glutamic acids, as well as the amino group and gamma -carboxamide gro up of glutamines, condense easily to form pyroglutamic derivatives. To over come this stability problem, several peptides of pharmaceutical interest ha ve been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T ly mphocyte (CTL) activity. Despite the apparent minor modifications introduce d in PyrELA and AcELA, these two derivatives probably have lower affinity t han ELA for the specific class I major histocompatibility complex. Conseque ntly, in order to conserve full activity of ELA, the formation of PyrELA mu st be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceu tical grade peptides. We report here that the hydrochloride salt, shows hig her stability than the acetate salt and may be suitable for use in man. Sim ilar stability data were also obtained for MAGE-3, another N-terminal gluta mic acid containing CTL peptide in clinical development, leading us to sugg est that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.