Discovery of VH domains that allosterically inhibit ENPP1

Paige E Solomon; Colton J Bracken; Jacqueline A Carozza; Haoqing Wang; Elizabeth P Young; Alon Wellner; Chang C Liu; E Alejandro Sweet-Cordero; Lingyin Li; James A Wells

doi:10.1038/s41589-023-01368-5

Discovery of VH domains that allosterically inhibit ENPP1

Nat Chem Biol. 2024 Jan;20(1):30-41. doi: 10.1038/s41589-023-01368-5. Epub 2023 Jul 3.

Authors

Paige E Solomon¹, Colton J Bracken^{1

2}, Jacqueline A Carozza³, Haoqing Wang^{4

5}, Elizabeth P Young⁶, Alon Wellner⁷, Chang C Liu^{7

8

9}, E Alejandro Sweet-Cordero⁶, Lingyin Li³, James A Wells^{10

11}

Affiliations

¹ Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
² Cartography Biosciences, South San Francisco, CA, USA.
³ Department of Biochemistry, Stanford University Medical School, Stanford, CA, USA.
⁴ Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁵ Macromolecular Structural Knowledge Center, Stanford University, Stanford, CA, USA.
⁶ Division of Pediatric Oncology, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.
⁷ Department of Biomedical Engineering, University of California, Irvine, CA, USA.
⁸ Department of Chemistry, University of California, Irvine, CA, USA.
⁹ Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
¹⁰ Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA. jim.wells@ucsf.edu.
¹¹ Department of Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA. jim.wells@ucsf.edu.

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.

Discovery of VH domains that allosterically inhibit ENPP1

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