Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody.

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.