Targeted Amino Acid Substitution Overcomes Scale-Up Challenges with the Human C5a-Derived Decapeptide Immunostimulant...

EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro⁷ and N-methyl-Leu⁸ (Me-Leu⁸) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro⁷ and Me-Leu⁸ and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro⁷ and/or Me-Leu⁸ with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine⁷ and/or leucine⁸) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro⁷ and/or Me-Leu⁸ with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.