Discovery of VH domains that allosterically inhibit ENPP1

Abstract

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.

From mechanism to therapy: the journey of CD24 in cancer

“…Moreover, ENPP1 intersects significantly with the cGAS-STING pathway (117). By degrading cGAMP, a secondary messenger in the STING pathway, ENPP1 dampens the immune response to cancer cells (118,119).…”

Advancements in ovarian cancer immunodiagnostics and therapeutics via phage display technology

“…This journey involves repeated cycles of screening and sequencing against tumor cells or tissues, along with stringent validation of ligand binding affinities. Achieving success in this domain necessitates the synthesis of phage-peptides, which are integral to the development of precision-targeted cancer therapeutics ( 9 ). This review is dedicated to a comprehensive examination and integration of the existing literature on the application of phage display technology in the immunodiagnostics and treatment of ovarian cancer.…”