A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway.

Abstract

Wnt signaling is important in embryonic development and tumorigenesis. These biological effects can be exerted by the activation of the β-catenin-dependent canonical pathway or the β-catenin-independent non-canonical pathway. DKK1 is a potent inhibitor of Wnt signaling by competing with Wnt binding to LRP5/6 co-receptors. DKK1 is tumorigenic in multiple cancer types and immunosuppressive in NK cells. Emerging evidence indicates that DKK1 is involved in T cell differentiation and induces cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms by which DKK1 affects cancer and immune cells have received considerable attention. Using Twist's precision DNA writing technologies, we created phage display libraries with a diversity greater than 1 × 10¹⁰ individual members, and machine learning models were utilized for optimal discovery. We found that anti-DKK1 antibodies blocked the binding of DKK1 to LRP co-receptors. Binding of antibodies to different cysteine-rich domains (CRDs) of hDKK1 leads to different activation effects. In vitro functional assays showed that the interaction of Wnt with LRP5/6 co-receptors was restored in the presence of anti-DKK1 antibodies binding to DKK1 C-terminal CRD2, resulting in the upregulation of Wnt canonical TCF/LEF signaling and reactivation of osteoblast differentiation. Moreover, anti-DKK1 antibodies binding to DKK1 N-terminal CRD1 induced Wnt non-canonical JNK phosphorylation, immune cell activation, and tumor cell cytotoxicity. In vivo studies indicated that these anti-DKK1 antibody leads targeting DKK1 CDR1 are potent in inhibition of tumor growth and may have promising efficacy as cancer immunotherapy due to activation of the Wnt non-canonical pathway.