Optimizing Complement Inhibitor Monitoring in PNH and Beyond

Introduction: There are now 6 FDA approved therapies for paroxysmal nocturnal hemoglobinuria (PNH). All are effective in controlling complement-mediated intravascular hemolysis (IVH) but associated with potential for breakthrough hemolysis (BTH). Differentiating pharmacokinetic (PK) from pharmacodynamic BTH and defining the exact contribution of extravascular hemolysis (EVH) to persistent anemia remains an ongoing diagnostic challenge. Outside the CH50 for eculizumab, there are no clinically available tests for monitoring of complement blockade. CH50 has variable accuracy for ravulizumab monitoring and cannot assess AP inhibitors. To address this challenge, we developed the bioluminescent modified Ham (bio mHam), a highly sensitive assay for functional, pathway-specific changes in complement activity that can monitor therapeutic complement blockade of the classical (CP) and alternative (AP) pathways.

Methods: Serum was collected from PNH patients on eculizumab or ravulizumab at various time points in the dosing cycle. For the purpose of demonstrating functional complement inhibition, we generated a cell-based complement “biosensor'' by selective removal of major membrane-bound complement regulators in an autonomously bioluminescent HEK293 cell line: a PIGA knockout (PIGAKO, deficient in CD55 and CD59) and double knockout (DKO, knockout of PIGA and CD46). The assay is performed in gelatin veronal buffer with calcium and magnesium (GVB⁺⁺) for assessment of all complement pathways or GVB-MgEGTA to isolate AP activity. Serum is added to cells and luminescence is monitored serially. Luminescence of cells incubated with the patient serum after 1 hour is compared to the sample’s heat inactivated control.

Results: In vitro addition of C5 inhibitors (eculizumab, ravulizumab, crovalimab) to healthy control serum demonstrated a dose-dependent increase of complement blockade in GVB⁺⁺ buffer. In isolated AP buffer, we observed dose-dependent inhibition with increasing concentrations of eculizumab, danicopan (factor D inhibitor), iptacopan (factor B inhibitor), and pegcetacoplan (C3 inhibitor). We examined functional inhibition of complement on the DKO cell line in PNH patients receiving ravulizumab at various points in the dosing cycle: pre-infusion (n=11), mid-cycle (n=6), and post-infusion (n=14). The mean relative luminescence (RL) at all points was >90% (range 73-100%), indicating inhibition of complement activity. There was no statistically significant difference between the time points, but there was a trend towards increased inhibition post-infusion (pre-infusion RL 90.5% vs post-infusion RL 97.6%, p=0.07). Lastly, we collected serial samples from a PNH patient in the setting of BTH and steady state pegcetacoplan and iptacopan therapy. The bio mHam (all pathways buffer on PIGAKO), performed in the setting of BTH due to 2 missed doses of pegcetacoplan and fever, showed complement-dependent cytolysis which correlated inversely with lactate dehydrogenase (RL 30% at baseline, 32% at 1 week, 44% at 2 weeks, and 54% at 6 weeks). Complement activity in the week 6 sample was completely blocked (>90% RL) by spiking the serum with eculizumab or sutimlimab (CP inhibitor). The bio mHam performed in AP-only buffer showed complete blockade by 2 weeks (RL 73% at baseline, 78% at 1 week, and 100% at weeks 2 and 6 weeks), consistent with the subcutaneous administration route and prior PK steady state data. Interestingly, in the baseline and 1-week serum, either danicopan or sutimlimab completely inhibited AP activity. Complement blockade with sutimlimab in the AP assay may indicate a CP stimulus driving AP amplification during his acute hemolytic event. Further, this suggests that pegcetacoplan is primarily an AP inhibitor at PNH dosing. He was later transitioned to iptacopan, and serum demonstrated inhibition of the AP (90% RL), whereas there was still complement activity in all pathway buffer (64% RL; 98% with addition of eculizumab).

Conclusion: The bio mHam can monitor PNH patients on any FDA approved complement inhibitor. Using this assay, EVH can be more precisely defined as full blockade of complement activity with persistent evidence of hemolysis. Furthermore, the assay has applications for not only PNH but also other diseases in which complement inhibitors are indicated, such as atypical hemolytic uremic syndrome.