Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.