Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Dr. Joseph Vetro

Second Advisor

Dr. Jonathan Vennerstrom

Third Advisor

Dr. Russell McCulloh

Fourth Advisor

Dr. Geoffrey Thiele

Abstract

EP67 is a human C5a-derived decapeptide agonist of C5a Receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to stimulate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Two N-alkyl amino acids Pro7 and N-methyl-Leu8 (nme-Leu8) within EP67 induce structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils. Cis/trans isomerization at these positions, however, likely limits the activity of EP67 and low coupling efficiency between Pro7 and nme-Leu8 increases scale-up costs for clinical use. Thus, the goals of this study were to (i.) establish a more clinically relevant, high-throughput assay for screening immunostimulant activity in primary human mononuclear phagocytes (monocytes, unpolarized (M0)-monocyte-derived macrophages, monocyte-derived dendritic cells) and neutrophils and (ii.) determine whether replacing Pro7 with cyclohexylalanine (Cha) and/or nme-Leu8 with Leu adversely affects EP67 potency and efficacy in human mononuclear phagocytes and selective activation over human neutrophils. We found that, depending on the secreted cytokine and mononuclear phagocyte, EP67 analogs had (i.) similar or lower EC50 (30% to 40%) and similar, increased (10% to 45%), or decreased (5% to 19%) EMAX for IL-6 and TNF-α secretion from mononuclear phagocytes 24 h after treatment and (ii.) similar EC50 and similar or decreased (21% to 24%) EMAX for myeloperoxidase secretion from human neutrophils without affecting selective activation of human mononuclear phagocytes. Thus, replacing Pro7 and/or nme-Leu8 with transoid-amino acids expected to induce similar structural changes selectively affects potency and efficacy of EP67 without affecting selective activation over human neutrophils and is a suitable strategy for future analogs of EP67.

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