Alternative complement pathway hemolytic assays reveal incomplete complement blockade in patients treated with...

Area of Science:

• Complement System Biology
• Immunology
• Pharmacology

Background:

• Eculizumab is a C5 complement inhibitor used for atypical hemolytic uremic syndrome (aHUS).
• Monitoring complement inhibition is crucial for assessing eculizumab efficacy.
• Previous studies have focused on classical pathway inhibition.

Purpose of the Study:

• To assess the efficacy of eculizumab in blocking the complement terminal pathway (TP) through both classical (CP) and alternative pathways (AP).
• To compare the sensitivity of hemolytic assays (HA) and ELISA in measuring complement inhibition.
• To evaluate C5a and sC5b9 levels in relation to residual complement activity.

Main Methods:

• Kinetic hemolytic assays (HA) using rabbit (APH50) and chicken (AP100) erythrocytes to measure alternative pathway (AP) activation.
• Functional ELISA to assess TP blockade through AP activation.
• Measurement of C5a and sC5b9 levels.
• In vitro experiments with varying eculizumab concentrations.

Main Results:

• Eculizumab efficiently blocked TP via CP activation (<10%) but incompletely via AP activation (rabbit HA >23%, chicken HA >15%).
• Functional ELISA indicated complete TP blockade via AP activation (<10%) in all patients.
• C5a and sC5b9 levels did not correlate with residual AP activity measured by HA.
• ELISA was found to be less sensitive than HA in detecting residual complement activity.

Conclusions:

• Eculizumab demonstrates variable efficacy in inhibiting the complement alternative pathway, depending on the assay used.
• Hemolytic assays provide a more sensitive measure of residual alternative pathway activity compared to ELISA.
• Further research is needed to fully understand the clinical implications of incomplete alternative pathway inhibition by eculizumab.