Abstract 4966: Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin® drug candidate

Background: HER2 is an important target for antitumor therapy in several cancers, and while currently available HER2-targeting drugs provide a great treatment improvement they rarely achieve full disease control. We have developed a new HER2-targeting molecule with a unique pro-apoptotic mode of action that may provide additional benefit to patients. The DARPin® molecule MP0274 shows potency in several HER2-expressing PDX models and has been shown to directly induce apoptosis in cells that are addicted to HER2. Here we show through phospho-proteome analyses that MP0274 not only potently inhibits HER2/HER3 downstream signaling, but also shows a very distinct changes in the phospho-proteome pattern compared to approved HER2-targeting drugs. This provides mechanistic support to the unique mode of action of MP0274 that results in direct tumor cell killing without the need of effector-functions like ADCC. Methods: The effect of MP0274 on HER2 signaling was compared to trastuzumab, pertuzumab and a combination of both in the HER2-addicted cancer cell lines. Briefly, cells were incubated with drugs for 18 hours and then lysed. Lysates were analyzed for changes in the total proteome and phospho-proteome by Proteome Sciences’ proprietary SysQuant® Global Phosphoproteomics workflow. Results: Heat-maps of genes of interest indicate that MP0274 has a differential mode of action compared to trastuzumab, pertuzumab or a combination of both. On the total peptide and phospho-peptide level, the samples cluster specifically, based on the cancer cells used as well as drug treatment. Twenty nine unique global phosphorylation sites specific to HER2, including C-terminal tyrosines which are reported to recruit adaptor proteins starting signaling processes after auto-phosphorylation, were identified for MP0274. Several proteins were identified which were differentially expressed and phosphorylated after MP0274 treatment and which are involved in three key downstream signaling pathways activated by HER2/HER3 heterodimers: RAF/MAP kinase cascade, PI3K-induced AKT signaling, and signaling by PLCG1. Conclusions: MP0274 shows a unique and distinct inhibition of the HER2 signaling cascade, different from trastuzumab, pertuzumab and a combination of both. It induces a more profound inhibition of downstream signaling which provides mechanistic support to the finding that MP0274 direct cell killing by induction of apoptosis in HER2-addicted tumor cells. * DARPins are small repeat protein