Immunogenicity assessment of concizumab in patients with Hemophilia A and b: Assays developed to measure anti-drug antibodies and integrated results from clinical trials Free

Background Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody indicated for patients with hemophilia A/B with and without inhibitors, approved in the US, Europe and other countries for once-daily, subcutaneous prophylaxis in hemophilia A/B with inhibitors. Immunogenicity assessment of biological drugs during clinical trials is a regulatory requirement. It involves validated, multi-tiered assays for anti-drug antibody (ADA) detection and characterization in samples from patients treated with the trial drug. To further understand the efficacy and safety profile of the trial drug, immunogenicity results are subsequently analyzed for correlations with multiple endpoints.

Aim Here, we describe the binding ADA (bADA) and neutralizing antibody (nAb) assays developed to monitor immunogenicity during phase 2 and 3 (ph2/ph3) clinical trials on concizumab. In particular, we present nAb assay optimization and explore an integrated analysis method of bADA, pharmacokinetic (PK) and pharmacodynamic (PD) measures to investigate neutralizing ADAs.

Methods The bADA assay consisted of a bridging format on the Meso Scale Discovery (MSD) platform, with an electrochemiluminescence endpoint. A tiered approach was used to test and further characterize samples: screening, confirmation, and characterization to assess specificity and titer. Different assay modalities were used to assess the in vitro neutralizing potential of ADAs throughout the concizumab ph2/ph3 clinical trials. During ph2, a colorimetric functional assay, based on the enzymatic activity of activated factor X, was used. During ph3, two different modalities of competitive ligand binding assays, based on nAbs preventing binding of concizumab to TFPI, were developed on the MSD platform. For both bADA and nAb assays, samples were pre-treated with acid or purified to overcome potential drug interference. To validate the assay, samples from drug-naïve individuals were used to set the assay cut-points. Control samples (negative: no concizumab reactivity; positive: different levels of Abs spiked into matrix) were used to monitor assay performance. During the ph2/3 clinical trials, ADA sampling was performed at baseline and at regular intervals after the first concizumab dose, together with samples taken for PK/PD (concizumab plasma concentration) endpoints.

Results A robust bADA assay was validated during ph2/ph3 clinical development of concizumab, demonstrating high sensitivity (~1.0 ng/mL ADA) and drug-tolerance, ensuring that sample drug levels did not interfere with ADA detection. Performance parameters of the nAb assay were optimized, with the 3^rd generation of the nAb assay demonstrating improved sensitivity, drug tolerance, and run acceptance rate, compared to previous versions. The bADA and 3^rd generation nAb assay continue to be used in 3 ongoing ph3 trials.

After completion of 2 ph2 trials and including the patients from the 2 ongoing ph3 trials, 71/320 (22.2%) patients exposed to concizumab developed bADAs, with 18/320 (5.6%) exhibiting ADAs with in vitro neutralizing effect. The ADA titer was low in all but 3 patients, with bADAs developing after 12 weeks into concizumab exposure in 2/3 patients. ADA responses were primarily directed against the complementarity-determining region of concizumab and were mostly transient. Overall, ADAs did not appear to have clinical impact on efficacy, safety or PK/PD, except in one patient with inconclusive impact on PK/PD.

Conclusion bADA and nAb assays were successfully developed, validated and implemented to monitor concizumab immunogenicity during clinical development of concizumab, including the three ongoing ph3 trials. The assays meet regulatory requirements and allow a thorough characterization of ADA incidence, magnitude, onset, kinetics, specificity and in vitro neutralizing potential. To date, a favorable immunogenicity profile has been observed during the concizumab ph2/3 clinical trials with no apparent impact of ADAs on efficacy, safety or PK/PD using the validated bADA and nAb assays. Based on the presented integrated analysis of bADAs with PK/PD, the nAb assays may be considered redundant for the interpretation of overall immunogenicity findings.