ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an oncofetal protein prevalently expressed in a variety of hematological and solid malignancies but largely absent in normal adult tissues, making it an attractive ADC target. CS5001/ABL202/LCB71 is an ADC composed of a human monoclonal antibody targeting ROR1, site-specifically conjugated with a proprietary cleavable β-glucuronide linker to a prodrug of PBD dimer. Both linker and prodrug are selectively cleaved by the lysosomal b-glucuronidase, which is overexpressed in many cancerous cells, to allow tumor-selective release of the DNA-crosslinking PBD dimer. In vitro and in vivo pharmacology of CS5001 were evaluated and benchmarked against CS5001-BMK1 (an MMAE-based ROR1 ADC). Binding specificity, affinity and internalization were determined by Octet, ELISA and flow cytometry. Cytotoxicity and predictive biomarkers were evaluated in 20 human cancer cell lines with various levels of ROR1. In vivo efficacy was studied in Jeko-1 mantle cell lymphoma and MDA-MB-231 triple-negative breast cancer xenografts. CS5001 bound to human ROR1, but not ROR2, with a KD value of 1.38 nM. CS5001 has cross reactivity against mouse, rat and cynomolgus ROR1 at similar affinities. Upon binding, CS5001 was rapidly internalized by ROR1-expressing cancer cells at 37°C. CS5001 demonstrated potent cytotoxicity towards ROR1 high expressing cell lines such as Jeko-1 and MDA-MB-231, with an IC50value of 0.161 and 0.900 nM, whereas the IC50 value of CS5001-BMK1 was 10.8 nM and 129 nM, respectively. The growth inhibition activity of CS5001 but not CS5001BMK was significantly correlated with ROR1 density (Pearson correlation= -0.697, P = 6.4 x 10-4). CS5001 potently induced DNA damage in MDA-MB-231 cells, and resulted in increased apoptosis and G2-M cell cycle arrest in a concentration-dependent manner, indicating a mechanism of cytotoxicity consistent with that of the PBD payload. CS5001 exhibited prominent antitumor activity in both Jeko-1 and MDA-MB-231 xenograft models in a dose-dependent manner. In Jeko-1, complete regression (CR) was observed after a single administration of CS5001 at 1mg/kg (approximate 1/5th the maximum tolerated dose (MTD) in mice), whereas no CR was achieved for CS5001-BMK1 at the highest dose (2.5mg/kg, QWx3). When both CS5001 and CS5001-BMK1 were administrated at 1/20th of their respective MTDs, CS5001 (0.25mg/kg, single dose) produced significantly superior efficacy to CS5001-BMK1 (2.5mg/kg, single dose) with tumor growth inhibition (TGI) of 60% and 38%, respectively (p=0.018). In MDA-MB-231, 106% and 65.7% of TGI were observed for CS5001 at 1mg/kg (single dose) and CS5001-BMK1 at 2.5mg/kg (QWx3) respectively. CS5001 exhibited potent and selective cytotoxicity to a variety of ROR1-expressing cell lines and showed remarkable in vivo antitumor activity. ROR1 cytometric density predicts sensitivity to CS5001 in vitro. CS5001 is a promising therapeutic candidate for ROR1-expressing hematological and solid malignancies with precision medicine potential.

Citation Format: Fu Li, Yongwang Li, Lan Zhang, Clarence K. Zhang, Hui-Han A. Hu, Juan Zhang, Ying Pan, Jinwon Jung, Sang Hoon Lee, Hyun-Min Ryu, Yun-Hee Park, Haixiang Yu, Archie N. Tse. CS5001, a novel ROR1-targeting antibody drug conjugate (ADC) armed with tumor-cleavable β-glucuronide linkers and pyrrolobenzodiazepine (PBD) prodrugs for hematological and solid malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA008.