Therapeutic Targeting The Notch3 Receptor With Antibody Drug Conjugates

Activation and mutation of the NOTCH signaling pathway is oncogenic in many tissue types and the target of multiple anti-cancer therapies currently in clinical development. Initial therapeutic strategies designed to target the NOTCH pathway have focused on inhibition of aberrant signaling, but can have undesirable side-effects or insufficient anti-tumor activity. Antibody drug conjugates (ADCs) are an emerging therapeutic modality that equips antibodies with potent cytotoxic payloads that can be directly delivered to tumor cells. We have developed and compared anti-NOTCH3 ADCs using two different classes of therapeutic anti-NOTCH3 antibodies that target the juxtamembrane Negative Regulatory Region (NRR). The first class is unable to stabilize the NOTCH3-NRR in an auto-inhibitory conformation in the presence of ligand and does not block ADAM protease cleavage of the receptor. The second class contains an antibody that exhibits potent signaling inhibition by stabilizing the NRR in an inactive state. Despite antagonizing NOTCH3 signaling, the inhibitory anti-NOTCH3 antibody was unable to regress preclinical tumor xenografts with active NOTCH3 signaling. To enhance their potency, both classes of anti-NOTCH3 antibodies were conjugated to an auristatin-based microtubule inhibitor through a cleavable linker. Unexpectedly, the inhibitory anti-NOTCH3 antibody demonstrated more rapid trafficking to the lysosome than the non-inhibitory antibody suggesting that the two antibodies have distinct internalization routes with important implications for NOTCH3-ADC pharmacology. Pharmacodynamic biomarker analysis demonstrated anti-NOTCH3 ADCs disrupted the mitotic spindle apparatus, induced cell cycle arrest and triggered apoptosis. Anti-NOTCH3 ADCs exhibited robust anti-tumor activity and induce prolonged tumor regressions in preclinical models of breast, lung and ovarian cancer regardless of their ability to block signaling. Furthermore, anti-NOTCH3 ADC treatment was able to regress OVCAR3 ovarian tumor xenografts that were refractory to a platinum-based agent or relapsed anti-VEGF therapy. Our studies demonstrate that anti-NOTCH3 ADCs had enhanced efficacy compared to other NOTCH signaling inhibitors and also allowed targeting of tumors that over-expressed NOTCH3 but were not necessarily addicted to its signaling. The safety and efficacy of the non-inhibitory anti-NOTCH3 ADC, PF-06650808, is currently being examined in a Ph1 clinical trial (Protocol B7501001). Citation Format: Kenneth G. Geles, Yijie Gao, Latha Sridharan, Andreas Giannakou, Ting-Ting Yamin, Jonathan Golas, Judy Lucas, Manoj Charati, Xiantang Li, Magali Guffroy, Tim Nichols, Kai Wang, Max Follettie, Andreas Maderna, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Therapeutic targeting the NOTCH3 receptor with antibody drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1697. doi:10.1158/1538-7445.AM2015-1697