RSPO2-Based Peptibodies Conjugated with Pyrrolobenzodiazepine Dimer or Camptothecin Analogs Demonstrate Potent Antitumor Activity by Targeting the Three Receptors LGR4/5/6 in Colorectal Cancer and Neuroblastoma.

Leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6) are coexpressed or alternately expressed at high levels in tumor cells of colorectal cancer (CRC) and high-risk neuroblastoma (NB). Simultaneous targeting of all three homologous receptors may improve efficacy or overcome drug resistance due to tumor heterogeneity and cancer cell plasticity. R-spondins (RSPOs) bind to LGR4/5/6 with a high affinity and potentiate Wnt/β-catenin signaling. We previously reported that a mutant RSPO4 furin domain-based peptibody that binds to LGR4/5/6 without potentiating Wnt/β-catenin signaling was able to deliver cytotoxins into cancer cells that express any of the three receptors. Here, we generated a mutant RSPO2 furin domain that retains high-affinity LGR4/5/6 binding without any signaling activity. RSPO2 furin mutant peptibodies conjugated with pyrrolobenzodiazepine dimer (PBD) or camptothecin derivative (CPT2) yielded potent, target-selective cytotoxicity in LGR4/5/6-positive CRC and high-risk NB cell lines in vitro and robust antitumor activity in vivo.