At the base of normal intestinal crypts, stem cells maintain the highly regenerative gut epithelium. These intestinal stem cells are well characterized for their high expression of the G coupled protein receptor LGR5 (also known as GPR49). Together with R-spondins (potent Wnt signaling modulators) and stem cell growth factors, LGR5 forms part of a signaling cascade responsible for the regulation of cellular proliferation. Key mutations in the APC or BRAF pathways of intestinal stem cells lead to lesions and metastatic colorectal cancer if not diagnosed and resected at an early stage. The majority of primary and metastatic tumors arising from these mutations overexpress LGR5. It has been reported that metastatic colorectal cancer (CRC) patients expressing higher levels of LGR5 in tumor biopsies had increased rates of relapse. BNC101 is a first-in-class anti-LGR5 humanized monoclonal antibody. Biacore analysis of BNC101 has demonstrated a high affinity to LGR5 (Kd=16nM) with no cross-reactivity to LGR4 or LGR6 receptors. Immunoprecipitation studies have also shown that BNC101 has no off-target binding. The murine equivalent of BNC101 has demonstrated antitumor activity in multiple CRC patient-derived xenografts. Flow cytometry studies on CRC cell lines have revealed that LGR5 is located intracellularly with a small fraction present on the cell surface. To better understand its mechanism of action, BNC101 was conjugated to Alexa Fluor 647 and incubated with human CRC cell lines. It was shown that BNC101 interacts with membrane-bound LGR5 and is internalized within 5 minutes. Incubation of the cells for 24 hours at clinically relevant concentrations led to accumulation of fluorophore-conjugated BNC101 within the cell. This intracellular accumulation of BNC101 was further demonstrated with receptor recycling kinetic studies whereby only partial receptor recycling occurred, which may be attributed to the large intracellular LGR5 pool. CHO cell lines overexpressing LGR5 with a GFP tag were used to determine co-localization of BNC101 with its LGR5 ligand. BNC101 is currently in a safety and dose escalation phase I clinical trial in patients with recurrent metastatic CRC. We were able to demonstrate BNC101 target engagement with LGR5 for the first time in tumor biopsies following treatment. Tumor biopsies were analyzed by mass spectrometry together with Matrix Assisted Laser Desorption/Ionization; co-localization of BNC101 with LGR5 was observed, providing us with evidence for the first time that BNC101 infiltrates the patient tumor and engages with the overexpressed LGR5 receptor.

Citation Format: Daniel J. Inglis, John Licari, Kristen R. Georgiou, Nicole L. Wittwer, Ross W. Hamilton, Donna M. Beaumont, Michaela A. Scherer, Tina C. Lavranos. Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3910.