Simultaneous targeting of LGR4/5/6 for the treatment of gastrointestinal cancer
Introduction
LGR4/5/6 (leucine-rich repeat containing, G protein-coupled receptor 4, 5, and 6) bind to R-spondins (RSPOs) with high affinity and strongly potentiate Wnt/β-catenin signaling in response. The RSPO-LGR axis has emerged as a major pathway in tumor formation and progression. LGR4 is upregulated in multiple types of solid tumors and LGR5 is enriched in stem cell-like cancer cells that fuel tumor growth and metastasis in colon cancer. We and others showed that antibody-drug conjugates (ADCs) targeting LGR5 stopped tumor growth but could not eradicate tumors, most likely due to plasticity of cancer cells. Remarkably, LGR4/5/6 are frequently co-expressed or alternately expressed at high levels in gastrointestinal cancer with LGR4 being persistently expressed even in LGR5-negative cells. Thus, simultaneous targeting of LGR4/5/6 may overcome tumor heterogeneity and plasticity.
Methods
RSPO’s furin (Fu)-like domain (~110 AA) binds to LGR4/5/6 and two related E3 ubiquitin ligases (RNF43 and ZNRF3) via two separate sub-domains with binding to the E3 ligase being essential to potentiate Wnt signaling. We identified RSPO-Fu mutants with one or two AA changes in the E3 ligase-binding domain that still bind to LGR4/5/6 with high affinity but no longer potentiate Wnt signaling. Such mutant ligands even act as antagonists of wild-type RSPOs due to competitive binding to LGR4/5/6. The mutant ligands were fused to the Fc domain of human IgG1 to create RSPO-Fu peptibodies with increased stability in vivo and higher affinity in receptor binding. The RSPO-Fu peptibodies were then conjugated with cytotoxins and the resulting peptibody drug conjugates (PDCs) were evaluated for anti-tumor potency and efficacy in a panel of cancer cell lines and PDX models in vitro and in vivo.
Results
We generated a series of PDCs using modified RSPO2-Fu domain with the latest generation of camptothecin derivative as payload by a chemoenzymatic method. The PDCs were characterized for binding specificity, purity, integrity, and pharmacokinetics. The PDCs showed excellent potency and efficacy in inhibiting the growth of cancer cell lines expressing any of LGR4/5/6 in vitro and robust anti-tumor effect in vivo in cell line and PDX models. No gross toxicity were observed in vivo at the therapeutically effective dose.
Conclusion
Drug conjugates of modified RSPO-Fu peptibodies are able to target LGR4/5/6 simultaneously and may be developed as novel class of therapeutics for the treatment of gastrointestinal cancers. Further work is need to assess its safety and potential toxicity in vivo.