Binding-Induced Enzyme Activation for Prodrug Therapy of HER2 Variants

PROJECT SUMMARY Strikingly, cancer patients with a variant form of HER2 have a median progression free survival (PFS) of 4.9 months – a stark contrast to generic reports that HER2 targeted therapy gives 90% of patients a PFS of 5+ years. Most resistance is due to HER2 variants – structural mutants that prevent drug binding or drastically decrease the internalization of HER2. Accordingly, most standard and emerging treatments fail for these patients. There is an immediate need for therapeutic approaches that can be specifically designed to treat HER2 variants. Antibody-directed enzyme prodrug therapy (ADEPT), a strategy of conjugating prodrug-activating enzymes to antibodies that target cell surface receptors, nearly reached clinical success. It was limited, in large part, by premature prodrug activation in circulation. We propose to re-engineer the ADEPT strategy, addressing previous shortcomings, to tackle the pressing clinical need resulting from HER2 variants. In our strategy, Complementation Dependent Enzyme Prodrug Therapy (CoDEPT), split-enzyme fragments are fused to two separate antibodies against HER2. The binding of these antibodies to HER2, at distinct epitopes, brings the split- enzyme fragments into proximity, facilitating reconstitution of the active enzyme. The enzyme subsequently activates prodrug at the tumor and should thereby drastically reduce off-site activation and related toxicity. We hypothesize that CoDEPT constructs can reduce tumor growth in a murine breast cancer model. In Aim 1 we will engineer and characterize a library of split-β-lac constructs targeting HER2 variants and select lead CoDEPT constructs. In Aim 2, we will determine kinetic parameters and concentration thresholds that will establish foundational requirements for CoDEPT prodrug delivery and activation. In Aim 3, we will evaluate therapeutic efficacy of select CoDEPT constructs in vitro and in vivo.