CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells

Abstract

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Finally, compared with conventional antibodies, sdAbs have been implied to have privileged access to recessed sites on membrane proteins, 84 such as ion channels and G protein-coupled receptors ( GPCRs ). While this is an intriguing hypothesis, it has yet to be substantiated by any data.…”

“…Camelid V H Hs generated against the Kv1.3 ion channel targeted extracellular loops, not the channel cavity, 85 and the epitopes of V H Hs against the P2X7 ion channel were not defined. 86 Similarly, camelid V H Hs developed as potential therapeutics against the chemokine receptors CXCR4, 84 CXCR7 87 and ChemR23, 88 as well as V H Hs used as crystallization chaperones for several GPCRs, channels and transporters, 89–95 all appear to bind solvent-exposed extracellular or intracellular loops of these receptors in a manner similar to conventional antibodies and their fragments. By contrast, a synthetic CXCR4-binding “i-body” engineered from an Ig-like NCAM domain was found to penetrate deep into the receptor’s ligand-binding pocket to occupy a truly cryptic, partially transmembrane epitope.…”

Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges

“…Here also, the MAbCross method has been validated experimentally: for an initial antibody published by the group of Dr Martine Smit, a secondary target was found, unrelated to the initial one. 17 In many instances, an antibody with potentially important biologic activity cannot be further developed due to one or more issues related to, for example, toxicity, solubility, production yield, or intellectual property. MAbCross provides a suitable opportunity by finding unrelated antibodies that bind to the same epitope as an initial antibody.…”

“…Moreover, these biparatopic Nbs act as inverse agonists on the constitutively active mutant CXCR4, while the FDA approved CXCR4 antagonist AMD3100 (plerixafor, Mozobil TM ) acts a neutral antagonist. 17 In particular, the expression levels of CXCR4 in tumor cells are elevated, which may be associated with increases in basal activity. Hence, the use of inverse agonistic CXCR4 Nbs could be beneficial.…”

“…Since these biparatopic Nbs bind to distinct and partially overlapping epitopes, these Nbs are suggested to target CXCR4 dimers. 17 Thereafter, Nbs targeting CXCR7, also known to bind CXCL12, were identified. CXCR7, an atypical chemokine receptor now referred to as ACKR3, does not signal via G proteins but recruits b-arrestin, contributing to oncogenic signaling.…”