Using an in vitro xenoantibody-mediated complement-dependent cytotoxicity model to evaluate the complement inhibitory activity of the peptidic C3 inhibitor Cp40

Abstract

Simple and reliable methods for evaluating the inhibitory effects of drug candidates on complement activation are essential for preclinical development. Here, using an immortalized porcine aortic endothelial cell line (iPEC) as target, we evaluated the feasibility and effectiveness of an in vitro xenoantibody-mediated complement-dependent cytotoxicity (CDC) model for evaluating the complement inhibitory activity of Cp40, a potent analog of the peptidic C3 inhibitor compstatin. The binding of human xenoantibodies to iPECs led to serum dilution-dependent cell death. Pretreatment of the human serum with Cp40 almost completely inhibited the deposition of C3 fragments and C5b-9 on the cells, resulting in a dose-dependent inhibition of CDC against the iPECs. Using the same method to compare the effects of Cp40 on complement activation in humans, rhesus and cynomolgus monkeys, we found that the inhibitory patterns were similar overall. Thus, the in vitro xenoantibody-mediated CDC assay may have considerable potential for future clinical use.

The renaissance of complement therapeutics

“…Notably, even inhibition of C3 activation does not completely abrogate pathogen-targeted complement mechanisms; C4b opsonization of cells via the classical and lectin pathways can still occur, and a FD inhibitor still allowed substantial bactericidal activity against meningococci in the serum of vaccinated patients 192,193 . The minimum amount of complement activity that is required to confer substantial antimicrobial protection has not been determined; however, case reports in patients with C3 nephritic factor (an autoantibody that stabilizes the alternative pathway C3 convertase and therefore results in increased cleavage of C3) and no history of recurring infections suggest that C3 levels as low as 15% of the normal range are protective 194,195 .…”

New milestones ahead in complement-targeted therapy

“…Even residual complement activity is expected to confer significant antimicrobial protection, given that recurrent infection is not apparent in patients with hypocomplementemia (e.g., due to C3G). It is worth noting that even broad inhibitors such as compstatin do not block complement activity completely, but instead leave some aspects intact, such as tickover, conformational activation of C3 on surfaces, C3 cleavage by coagulation proteases, and/or deposition of the C4b opsonin by initiating pattern recognition complexes [47, 134, 135]. …”

Factor H–IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial PathogenStreptococcus pyogenes

“…Compstatin in turn promoted bacterial survival by blocking complement activation at the level of C3. Of note, although compstatin prevents C3b deposition on the bacteria, at the same time it allows for increased C4b deposition (63), which may serve as an opsonin by binding CR1 (64). The potential relevance of this enhanced C4b deposition is still not clear.…”