Structural Basis of Nanobody Induced ACKR3 Inhibition

When bound to their small, secreted chemotactic cytokine ligands (chemokines), the G protein-coupled receptor (GPCR) subfamily known as chemokine receptors (CKRs) can promote cell proliferation, survival, and migration towards increasing chemokine concentration gradients. Their strong association with these phenotypic hallmarks of cancer and their upregulation in cancer cells has made these receptors a topic of extensive research in the cancer field. Recent findings show upregulation of the atypical chemokine receptor 3 (ACKR3) in a wide variety of cancers including various breast, lung, and brain cancers, suggesting the involvement of ACKR3 in cancer pathogenesis. Acting as a scavenger receptor for a narrow range of chemokine ligands (CXCL12/CXCL11), binding of ACKR3 to a cognate ligand induces non-canonical β-arrestin dependent signaling, which can lead to receptor-ligand internalization, and cause a unique signaling cascade to directly promote cell proliferation, cell growth, and angiogenesis. Moreover, silencing of ACKR3 in murine models has been shown to significantly decrease the growth and invasiveness of breast, lung, and endothelial cancer. The development of therapeutics capable of selectively inhibiting ACKR3 has now become an area of active interest. Despite this drive, the characteristic promiscuity of the CKR network and a lack of understanding in ACKR3's mechanism of inhibition has left all ACKR3 antagonists to date with varying degrees of partial agonism, low specificity, or low stability. Here we identify and characterize a new antagonist of ACKR3, turning to recombinant variable domain monomers from the unique heavy-chain only camelid antibodies or "nanobodies". These 13 kDa nanobodies have highly conserved structures with the exception of three structural loops which encode the nanobody complimentary-determining regions (CDRs) involved in antigen recognition and protein binding. This intricate binding epitope allows nanobodies to uniquely target complex GPCR epitopes, offering increased receptor specificity and selectivity versus small molecules and traditional antibody therapeutics. Using NMR spectroscopy, we have resolved the solution structure of the nanobody VUN701, an ACKR3 antagonist capable of completely ablating ACKR3 signaling at nanomolar concentrations. The structure of this inhibitor contains an uncharacteristically well-ordered extended beta-hairpin in the CDR3 region. In combination with cell-based assays and molecular modeling, this analysis has allowed us to develop an atomic view of the VUN701-ACKR3 interface. These findings shed light on the mechanism behind ACKR3 inhibition and drive forward the development of highly specific lead compounds for cancer therapy. © FASEB.