Generation and characterization of antagonistic anti-human CD39 nanobodies

Front Immunol. 2024 Mar 25:15:1328306. doi: 10.3389/fimmu.2024.1328306. eCollection 2024.

Abstract

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

Keywords: CD39; ENTPD1; VHH; nanobody; single-domain antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Monophosphate
  • Adenosine Triphosphate / metabolism
  • Humans
  • Single-Domain Antibodies* / pharmacology

Substances

  • Single-Domain Antibodies
  • Adenosine Triphosphate
  • Adenosine Monophosphate
  • Adenosine Diphosphate

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID: 335447717 - SFB 1328 to AG (A01), TM (A13), ET (A14), FK-N (Z02), FH (Z02) and BR (Z02), a grant from “Hermann und Lilly Schilling-Stiftung für Medizinische Forschung” to TM, a grant from the Mildred Scheel Cancer Career Center HaTriCS4 to SM and a grant from the T. von Zastrow Foundation to BR and TM.