G protein-coupled receptors (GPCRs) are critical therapeutic targets, yet their complex structures pose challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy for targeting GPCRs, offering advantages over traditional small molecules and conventional antibodies. However, an incomplete understanding of the structural features governing GPCR-nanobody interactions limits their development. Schlimgen et al. [1] investigated VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3), and determined that ACKR3 binding requires an extended CDR3 loop. This type of extended CDR3 loop is uncommon in most nanobodies but prevalent among those targeting GPCRs. Using combined experimental and computational methods, they mapped the inhibitory ACKR3-VUN701 interface and defined a unique conformational mechanism for GPCR inactivation. The findings from this study provide insights into class A GPCR-nanobody selectivity and offer strategies for the development of these novel therapeutic tools.

Background of GPCR Nanobody Development

Over 800 human G protein-coupled receptors (GPCRs) enable cells throughout the body to respond to a myriad of extracellular signals. The fine-tuned control of these receptors makes them the most abundant class of therapeutic targets. However, only one-eighth of human GPCRs are successfully targeted by current therapeutic strategies. While small molecules and peptides are most widely used for therapeutic targeting of GPCRs, novel molecules are needed to target the remaining receptors. Monoclonal antibodies (mAbs), a successful class of biologics, have been explored as a means to overcome the therapeutic targeting challenges of GPCRs. In the past 4 years, the FDA has approved two GPCR-directed mAb drugs (erenumab - CGRP-R, mogamulizumab - CCR4). Despite this partial success, the extensive binding surface of mAbs is not always suitable for selectively recognizing the relatively small extracellular epitopes displayed by membrane-embedded GPCRs. A recent evaluation of 407 anti-GPCR antibodies revealed that only 61% (248) were target-specific, exacerbating the challenges associated with GPCR biologic drug development.

Variable domain of heavy chain-only immunoglobulins (VHHs), also known as nanobodies, have the potential to bridge the gap between mAbs and small molecules. These small antibodies (12-15 kDa) utilize the smaller antigen-binding determinants of their three complementarity-determining regions (CDRs) to bind GPCR epitopes that are more difficult for mAbs to target. In 2019, the approval of the first nanobody drug, caplacizumab (a von Willebrand factor inhibitor), accelerated the development of this novel class of therapeutic molecules. Nanobodies targeting two GPCRs, the chemokine receptors CXCR4 and CX3CR1, are currently in clinical trials. To fully harness the power of nanobodies as pharmacological tools and potential drugs, understanding how they bind extracellular GPCR epitopes and alter receptor signaling is crucial.

Main Research Content

Using bioluminescence resonance energy transfer (BRET) assays to monitor GPCR activation, Schlimgen et al. [1] characterized the receptor specificity of the nanobody (VUN701) targeting the atypical chemokine receptor 3 (ACKR3), establishing VUN701 as a competitive ACKR3 inhibitor with potential for extracellular therapeutic application. By solving the solution structure of VUN701, Schlimgen et al. [1] discovered an unusual motif enabling its inhibitory function. Although uncommon in most nanobody structures, this unique motif is a common feature among GPCR-targeting nanobodies. Schlimgen et al. [1] mapped the inhibitory ACKR3-VUN701 interface and identified the molecular mechanism underlying the inhibition of ACKR3 and potentially other GPCRs. These findings provide structural insights into how nanobodies inhibit GPCRs and offer strategies for developing potent tools with diverse applications.

Figure 1: BRET-based ACKR3 β-arrestin2 recruitment assays with endogenous ligands CXCL11 and CXCL12 (dashed lines). VUN701 competition assays with increasing concentrations of VUN701 in the presence of indicated chemokine concentrations (solid lines).

Using BRET assays to detect β-arrestin2 recruitment to ACKR3, results showed that VUN701 inhibits CXCL11-dependent signaling with an IC50 = 105 nM. VUN701 also inhibits CXCL12-mediated activation with an IC50 = 157 nM.

Figure 2: Dose-dependent activation of ACKR3 with increasing concentrations of VUN701 (Dose-response curves, Schild plot inset).

Schild analysis of CXCL12-induced β-arrestin2 recruitment to ACKR3 revealed that VUN701 alters the potency of CXCL12 in a concentration-dependent manner without changing the slope or maximal efficacy of CXCL12-mediated β-arrestin2 recruitment.

Figure 3: BRET-based β-arrestin2 recruitment assays for CXCR4 (gray) and CXCR3A (black) with increasing concentrations of CXCL12 and CXCL11 respectively (dashed lines). Chemokine competition assays with increasing concentrations of wild-type VUN701 (solid lines).

VUN701 was unable to inhibit chemokine-GPCR mediated β-arrestin2 recruitment at concentrations up to 10 μM for CXCR4 or CXCR3, the receptors most similar to ACKR3 for CXCL12 and CXCL11, respectively.

Figure 4: BRET-based ACKR3 β-arrestin2 recruitment assay showing activation with CXCL12 (gray). Competition assays with 3.3 nM CXCL12 showing inhibition by wild-type VUN701 (black) and CDR variants ΔCDR1 (blue), ΔCDR2 (green), and ΔCDR3 (red), demonstrating changes in IC50 due to deletion of critical CDR contact residues.

Results indicated that while ΔCDR1 and ΔCDR2 mutants retained the ability to bind and inhibit ACKR3, the ΔCDR3 mutant resulted in a near-complete loss of inhibitory function, identifying the extended CDR3 region of VUN701 as the primary antibody epitope interacting with the GPCR. These findings provide structural insights for the development of therapeutic neutralizing nanobodies, primarily through mechanisms involving blockade of ligand binding to the GPCR receptor, and offer strategies for creating potent tools with diverse applications.

In summary, the inhibition of CXCL11 and CXCL12 function in cell-based assays, along with Schild analysis for CXCL12, indicates that VUN701 is a selective, reversible, competitive antagonist. Direct competition between VUN701 and CXCL11/CXCL12 suggests that these two ligands share overlapping orthosteric epitopes. VUN701 achieves high-affinity GPCR interaction through its extended CDR3 loop, blocking ligand binding to the GPCR and thereby achieving inhibition/blockade of specific signaling pathways. This provides innovative strategies for the development of therapeutic neutralizing monoclonal antibody drugs.

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References

[1] Schlimgen, R.R., Peterson, F.C., Heukers, R. et al. Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies. Nat Commun 15, 4611 (2024).