LAUSR

The onco-embryonic receptor tyrosine kinase ROR1is a cell-surface protein expressed on a broad range of solid tumours and haematological malignancies but is largely absent from normal adult tissue. Increased ROR1 expression correlates with poor clinical outcome in a number of cancer indications, consistent with its functional role in tumorigenesis and disease progression. In addition, elevated expression has been associated with increased metastatic potential and drug resistance in breast cancer and non-small cell lung cancer. The oncofetal expression pattern of ROR1 coupled with its role in cancer pathology and therapy resistance makes it a highly attractive target for a protein-drug conjugate approach. Shark-derived Variable New Antigen Receptor domains (VNARs) are the smallest antigen binding domain in the vertebrate kingdom. We have developed a platform to enable VNAR-based drug discovery and used it to identify a series of highly potent protein-drug conjugates that target ROR1. Through screening VNAR phage libraries, high affinity ROR1 specific binders were isolated that bind distinct regions of the extracellular domain and showed species cross reactivity (mouse, rat and human). By exploiting the modular nature of the VNAR domains, multiple different therapeutic formats have been engineered, including multimeric binders and Fc fusion proteins. Subsequent site-specific attachment of highly potent toxins yielded homogeneous VNAR-based drug conjugates which demonstrated potent ROR1-dependant killing of selected cancer cell-lines in vitro with IC50 values in the picomolar range. In a series of in vivo studies, VNAR-hFc fusions conjugated with novel linker payloads were well tolerated in mice and showed significant anti-tumor activity in mouse xenograft models of cancer, including complete and sustained tumour regressions in a patient-derived xenograft (PDX) model of triple negative breast cancer. Lead VNAR-hFc conjugates have been successfully ‘humanized9 guided by de novo structural biology. These engineered VNAR-hFc drug conjugates represent exciting new candidates for the treatment of a variety of both solid tumor and haematological cancer indications. Additionally, bi-paratopic and bi-specific ROR1 binders have been generated which fully leverage the modular design of VNAR-based therapeutics. These provide second generation drug conjugates which can be tailored to specific additional therapeutic applications, further extending the applicability of this approach. Citation Format: Graham Cotton, Jennifer Thom, Paul Trumper, Andrei Kamenski, Stacey Bell, Mark Wappett, Caroline Barelle, Marina Kovaleva, Alicia Campion, Elisa Persiani, Andrew Porter, Estelle McLean, Aidan McCann, Chiara Saladino, Aaron Cranston, Tim Harrison. Exploiting the properties of VNAR domains for the development of novel efficacious protein drug conjugates targeting the oncofetal protein ROR1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 538.