Application of targeted proteomics for quantification of protein targets to support PK/PD-modelling and TMDD de-risking of a bispecific agonistic antibody

A bispecific agonistic antibody has been designed to facilitate tumor-selective activation of protein target 1 (PT1) by specifically binding protein target 2 (PT2) expressed on cancer-associated cells (CAC). The bispecific binding mode is designed to increase intra-tumoral PT1 agonism via PT2-mediated receptor hyperclustering while reducing systemic PT1 activation. Pharmacokinetic (PK) studies in non-tumor bearing mice showed strong non-linear pharmacokinetic behavior as it is often observed for antibodies binding to membrane targets. Prediction of human PK for molecules exhibiting this phenomenon referred to as targeted-mediated drug disposition (TMDD) can be very challenging and relies on quantitative knowledge of target expression across species. In the case of PT2 binding, strong PK non-linearity was not originally expected in non-tumor bearing mice where CACs are absent. However, in addition to specific expression on CACs, PT2 has also been described to be expressed in skin, which is a possible explanation for the observed non-linear pharmacokinetic behavior in mice. Therefore, we developed an LC-MS based method to quantify the amount of PT2 in skin samples from different mouse strains, cynomolgus monkey and human. Absolute quantification of PT2 using stable isotope labeled peptides as surrogate standards gave inconclusive results, so we resorted to relative target quantification in skin samples. Although not quantitative, this approach helped to guide the need and choice of animal models for further pharmacokinetic studies and allowed to de-risk the prediction of TMDD in humans. In addition, we aim to develop an absolute quantitative method for the quantification of PT2 in murine and human tumor samples to support quantitative PK/PD modeling approaches. As alternative approach to using peptides as surrogate standards, we will use stable isotope labeled and unlabeled recombinant proteins for the second attempt.