Targeted Amino Acid Substitution Overcomes Scale-Up Challenges with the Human C5a-Derived Decapeptide Immunostimulant EP67

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. Pro(7) and N-methyl-Leu(8) (Me-Leu(8)) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils vs. first-generation EP54. Low coupling efficiency between Pro(7) and Me-Leu(8) 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 Pro(7) and/or Me-Leu(8) with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine(7) and/or leucine(8)) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that, depending on the secreted cytokine and mononuclear phagocyte, EP67 analogs had (i.) similar or lower (29 to 39%) potency and similar, increased (9.5 to 45%), or decreased (5 to 23%) efficacy for IL-6 and TNF-α secretion from mononuclear phagocytes and (ii.) similar potency and similar or decreased (21% to 24%) efficacy for myeloperoxidase secretion from human neutrophils without affecting selective activation of human mononuclear phagocytes. Thus, replacing Pro(7) and/or Me-Leu(8) with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.