An immunoinhibition approach to overcome the impact of pre-existing antibodies on cut point establishment for immunogenicity assessment of moxetumomab pasudotox.

Immunogenicity can impact PK, PD, efficacy and safety of biopharmaceuticals, and is often evaluated as a secondary objective in clinical studies. Methods to detect anti-drug antibodies (ADA) and neutralizing ADA (NAb) are semi-quantitative and utilize cut points to determine positive or negative samples. Assay cut points are established by the statistical analysis of treatment-naïve subject specimens that are assumed ADA and NAb-negative. Pre-existing antibodies to various biopharmaceuticals have been observed in treatment-naïve subjects and may artificially elevate the cut point, resulting in compromised assay sensitivities, inaccuracy in immunogenicity reporting and ultimately misleading assessment of the impact of immunogenicity on clinical outcomes. Although several approaches such as removal of pre-existing antibody samples or increasing the sample dilution could be used for cut point establishment to mitigate impact of pre-existing antibodies, they each have limitations, especially when a high prevalence of pre-existing antibodies is observed. Here we describe an innovative approach used to establish cut points for ADA and NAb assays of moxetumomab pasudotox (moxetumomab), a recombinant anti-CD22 immunotoxin, to which a high prevalence of pre-existing antibodies was observed. In order to overcome the challenges associated with this high prevalence and prevent establishment of an artificially elevated cut point, we developed an immunoinhibition approach that allowed generation of pseudo ADA and NAb-negative populations for cut point determination. Immunoinhibition was performed by adding excess moxetumomab (for ADA) or a non-CD22 binding PE38-containing immunotoxin, CAT-5001 (for NAb), to treatment-naive samples prior to evaluating samples for cut point establishment. This approach successfully eliminated pre-existing antibody activity in treatment-naive samples, enabling establishment of more accurate ADA and NAb assay cut points. A comparative analysis of the clinical immunogenicity results using cut points derived with immunoinhibition and without immunoinhibition (conventional method) demonstrated that the immunoinhibition approach markedly improved detection sensitivity and accuracy of immunogenicity characterization in patient samples. This innovative approach provides an alternative, practical solution for immunogenicity assay cut point establishment when biopharmaceuticals have a high prevalence of pre-existing antibodies.