RSPO2-based peptibodies conjugated with pyrrolobenzodiazepine dimer or camptothecin analogs demonstrate potent anti-tumor activity by targeting the three receptors LGR4/5/6

Leucine-rich repeat containing, G protein-coupled receptor 4, 5, and 6 (LGR4/5/6) are three homologous receptors that are co-expressed or alternately expressed at high levels in tumor cells of colorectal cancer (CRC) and high-risk neuroblastoma (NB). Simultaneous targeting of all three receptors may provide increased efficacy or overcome drug resistance due to tumor heterogeneity and cancer cell plasticity. LGR4/5/6 all bind to R-spondins (RSPOs) with high affinity and potentiate Wnt/{beta}-catenin signaling in response. Previously, we showed that a peptibody based on a mutant RSPO4 furin domain that bound to LGR4/5/6 without potentiating Wnt/{beta}-catenin signaling was able to deliver cytotoxins into cancer cells that express any of the three receptors. We have now generated a mutant RSPO2 furin domain that retains high affinity binding to LGR4/5/6 without signaling activity. Peptibodies based on this RSPO2 furin mutant were conjugated with either pyrrolobenzodiazepine dimer (PBD) or camptothecin derivative (CPT2), and the resulting peptibody-drug conjugates (PDCs) showed potent and specific cytotoxic activity in NB and CRC cell lines expressing any of LGR4/5/6 in vitro and robust anti-tumor activity in vivo. The results support the potential of RSPO2-based PDCs for the treatment of CRC, high-risk NB, and other cancers that express any of LGR4/5/6.