RESUMO
Utilizing decision making biomarkers in drug development requires thorough assay validation. Special considerations need to be taken into account when monitoring biomarkers using immunoassays in the presence of therapeutic antibodies. We have developed robust and sensitive assays to assess target engagement and proof of mechanism to support the clinical progression of a human monoclonal antibody against the neurotoxic amyloid-ß (Aß)42 peptide. Here we present the introduction of novel pre-treatment steps to ensure drug-tolerant immunoassays and describe the validation of the complete experimental procedures to measure total Aß42 concentration (bound and unbound) in cerebrospinal fluid (CSF) and plasma, free Aß42 concentration (unbound) in CSF, and Aß40 concentration in CSF. The difference in composition of the matrices (CSF and plasma) and antigen levels therein, in combination with the hydrophobic properties of Aß protein, adds to the complexity of validation. Monitoring pharmacodynamics of an Aß42 specific monoclonal antibody in a non-human primate toxicology study using these assays, we demonstrated a 1500-fold and a 3000-fold increase in total Aß42 in plasma, a 4-fold and 8-fold increase in total Aß42 in CSF together with a 95% and 96% reduction of free Aß42 in CSF following weekly intravenous injections of 10âmg/kg and 100âmg/kg, respectively. Levels of Aß40 were unchanged. The accuracy of these data is supported by previous pre-clinical studies as well as predictive pharmacokinetic/pharmacodynamics modeling. In contrast, when analyzing the same non-human primate samples excluding the pre-treatment steps, we were not able to distinguish between free and total Aß42. Our data clearly demonstrate the importance of thorough evaluation of antibody interference and appropriate validation to monitor different types of biomarkers in the presence of a therapeutic antibody.