INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, generalized myasthenia gravis and neuromyelitis optica. We have completed a Phase I study of pozelimab, a fully human anti-C5 IgG4, in healthy volunteers. Pozelimab was well tolerated and resulted in dose-dependent inhibition of hemolytic activity through the classical complement pathway in normal healthy volunteers. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly subcutaneous (SC) regimen following an intravenous (IV) loading dose (ASH2018 abstract). OBJECTIVE: To further characterize the impact of pozelimab on the alternative complement pathway activity, we investigated the effect of pozelimab on alternative pathway-mediated hemolysis using an AH50 assay in the completed first-in-human (FIH) study. In addition, we compared the effect of pozelimab in both alternative and classical pathway hemolysis assays with those of in-house eculizumab and in-house ravulizumab in pooled normal human serum (NHS) samples, ex vivo. METHODS: In total, 56 subjects were randomized to 4 sequential ascending IV single dose cohorts plus 2 sequential ascending SC single dose cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive pozelimab or placebo (6 active: 2 placebo). Serum collected at multiple time-points was utilized to assess the effect of pozelimab on alternative pathway activity. For ex vivo spike experiments, pooled NHS was used to compare the hemolytic function of pozelimab, in-house produced eculizumab and in-house produced ravulizumab. Comparator antibodies were synthesized from published sequence. The alternative pathway (AP) and classical pathway (CP) hemolysis assays were performed based on lysis of rabbit red blood cells (RBCs) and sensitized sheep RBCs, respectively. Both assays measure the amount of hemoglobin released from red blood cells at 412 nm. RESULTS: In the FIH study, baseline AH50 was comparable across treatment groups with a mean of 110 U/mL (standard deviation = 19, n = 56). Pozelimab exposure led to dose-dependent inhibition of AH50. In all 4 IV dosing cohorts, peak suppression of hemolysis was observed at end of infusion (EOI). Maximal suppression of hemolysis was approximately −85% change from baseline. This was achieved with the 30 mg/kg IV group and the repeat dose 15 mg/kg IV + 400 mg SC QW group. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, which was consistent with observed peak concentrations of pozelimab in serum. In an ex vivo spike study, pozelimab, in-house eculizumab and in-house ravulizumab were spiked into 10, 25 or 48% pooled NHS for AP, and 5, 10 or 25% for CP. The results from AP hemolysis assays showed that, for a given concentration of spiked antibody, the maximal suppression of hemolysis for all the antibodies decreased with increased percentage of serum (Figure). The maximal suppression of hemolysis was consistently higher (32-169%) for pozelimab compared with in-house eculizumab, and lower for in-house ravulizumab compared with pozelimab and in-house eculizumab at all serum percentages tested. The results from CP hemolysis assays showed that, although the maximal suppression of hemolysis was similar for all antibodies tested, in-house ravulizumab was required to be at least a log higher in concentration to achieve a similar effect as the other two anti-C5 antibodies. CONCLUSIONS: Ex vivo studies with pooled NHS demonstrate that pozelimab robustly blocks both CP and AP hemolysis. In-house ravulizumab appeared to be less potent compared with in-house eculizumab in both CP and AP hemolysis assays. The Phase I healthy volunteer study of pozelimab demonstrated dose-dependent and significant inhibition of alternative pathway hemolysis, with the maximal suppression of hemolysis approximately −85% change from baseline. Figure Disclosures Devalaraja-Narashimha: Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Huang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Chaudhari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Morton:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Weyne:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. OffLabel Disclosure: The drug is pozelimab and it is a fully human anti-C5 IgG4 being tested a Phase 1 study in healthy volunteers.