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.

Fig. 1.

Characterization of CXCR4-specific nanobodies. (A) Dose–response curve of potential specific CXCR4 nanobodies and the CXCR4-specific monoclonal antibody 12G5 in a ¹²⁵I-CXCL12 displacement assay performed with membranes of transiently transfected HEK293T-CXCR4. (B) Displacement assay of ¹²⁵I-238D4 with CXCR4 nanobodies and the CXCR4-specific monoclonal antibody 12G5.

Fig. 2.

The monovalent nanobodies 238D2 and 238D4 are potent antagonists of CXCR4. (A) CXCL12-induced inositol phosphate formation in CXCR4 and Gα_qi5-expressing HEK293T cells preincubated with 238D2 or 238D4 nanobodies. (B) HEK293T cells transfected with CXCR4 together with the pCRE/β-gal reporter gene display CRE activation upon CXCL12 stimulation. The 238D4 nanobody shows competitive antagonism as shown by Schild–Plot analysis (Inset). (C) CXCL12-induced chemotaxis of Jurkat cells is inhibited in the presence of the nanobodies 238D2 and 238D4, as well as with the CXCR4 monoclonal antibody 12G5 in a dose-dependent manner.

Fig. 3.

Epitope mapping of CXCR4-specific nanobodies unmasks species-specific activity. (A) Snake plot representation of human CXCR4 with highlighted amino acids involved in the binding of 238D2 (blue filled circles), 238D4 (red filled circles), 12G5 (green filled circles), CXCL12 (black filled circles), AMD3100 (blue open circles), and HIV-1 (red open circles). Multiple color codes show differential involvement of specific amino acids with various ligands or HIV-1. (B) Sequence alignment of the ECLs of human and mouse CXCR4 (red indicates differences). (C) Membranes of HEK293T transiently transfected with mouse CXCR4 specifically bind to human ¹²⁵I-CXCL12, which can be displaced by AMD3100 and cold CXCL12, but not by the nanobodies 238D2 or 238D4. (D) HEK293T cells transiently transfected with human or mouse CXCR4 were assayed for the binding of specific monoclonal antibodies (12G5 and 247506 in the case of human and mouse CXCR4, respectively) and the nanobodies 238D2 and 238D4. Bound antibodies or nanobodies were detected by flow cytometry using the appropriate conjugated secondary antibodies. MCF, mean channel fluorescence.

Fig. 4.

Bivalent nanobodies show increased affinity and inhibitory potency compared with their monovalent counterparts. (A) Competition binding experiments of ¹²⁵I-CXCL12 on membranes of HEK293T cells transfected with CXCR4 in the presence of monovalent nanobodies 238D2, 238D4, a mixture of 238D2 with 238D4, or the biparatopic nanobodies L3 and L8. (B) CXCL12-induced chemotaxis experiment of Jurkat cells in the presence of monovalent nanobodies 238D2, 238D4, or the biparatopic nanobodies L3 and L8.

Fig. 5.

CXCR4-specific nanobodies behave as neutral antagonists or inverse agonists on the constitutively active mutant of CXCR4. (A) Modulation of the constitutive formation of inositol phosphates in HEK293T cells transfected with CXCR4-N3.35A and Gα_qi5 by CXCL12, AMD3100, 238D2, 238D4, L3, or L8. (B) Dose-dependent inhibition of the constitutive production of inositol phosphates in HEK293T cells transfected with CXCR4-N3.35A and Gα_qi5 by L8. Treatment of the cells with AMD3100 blocks the inverse agonistic activity of L8.

Fig. 6.

CXCR4-specific biparatopic nanobody L8 induces stem cell mobilization in vivo to a similar extent as AMD3100. (A) Two independent monkeys were injected s.c. with 1 mg/kg of the CXCR4-specific antagonist AMD3100 and blood samples were taken over a period of 24 h after administration. (B) Two independent monkeys were injected i.v. with the biparatopic CXCR4-specific nanobody L8 at 10 mg/kg or 25 mg/kg. Blood samples were taken over a period of 24 h after administration. (C) Four independent monkeys were injected i.v. with various amounts of L8 nanobody, namely 0.1 mg/kg, 1 mg/kg, 10 mg/kg, or 25 mg/kg. Blood samples were withdrawn up to 9 h after administration. In all experiments, the presence of CD34⁺ stem cells in the blood was determined by flow cytometry analysis.