Molecular basis for the antagonistic activity of an anti-CXCR4 antibody

Abstract

Antagonistic antibodies targeting the G-protein C-X-C chemokine receptor 4 (CXCR4) hold promising therapeutic potential in various diseases. We report for the first time the detailed mechanism of action at a molecular level of a potent anti-CXCR4 antagonistic antibody (MEDI3185). We characterized the MEDI3185 paratope using alanine scanning on all 6 complementary-determining regions (CDRs). We also mapped its epitope using CXCR4 mutagenesis to assess the relative importance of the CXCR4 N-terminal peptide, extracellular loops (ECL) and ligand-binding pocket. We show that the interaction between MEDI3185 and CXCR4 is mediated mostly by CDR3H in MEDI3185 and ECL2 in CXCR4. The MEDI3185 epitope comprises the entire ECL2 sequence, lacks any so-called ‘hot-spot’ and is remarkably resistant to mutations. The structure of MEDI3185 variable domains was modeled, and suggested a β-strand/β-strand interaction between MEDI3185 CDR3H and CXCR4 ECL2, resulting in direct steric hindrance with CXCR4 ligand SDF-1. These findings may have important implications for designing antibody therapies against CXCR4.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3

“…This indicates that the most significant interactions between CXCR4 and antibodies and nanobodies might be electrostatic in nature. However, several studies report nonsignificant effects from single-point mutations in ECL2, while the impact of large substitutions or multiple simultaneous mutations show marked impairment on their binding (Xu et al, 2007;Peng et al, 2016a). These observations indicate that the epitope of these antibodies may be dispersed throughout ECL2 (Fig.…”

“…This explains why often small molecules are not able to displace antibodies or nanobodies. To illustrate, AMD3100 does not affect the binding of MEDI3185 to CXCR4 (Peng et al, 2016a).…”

“…In this context, it is worth noting that for the anti-CXCR4 antibody MEDI3185 the paratope and binding epitope have been described in detail. The interaction of the antibody is likely to be into a b-strand/b-strand fashion between complementarity-determining region 3 (CDR3) of the heavy chain and the ECL2 of CXCR4, which explains the inhibitory activity of the antibody through steric hindrance with CXCL12 (Peng et al, 2016a). This binding mode appears to be different from those of small molecules and peptides and might be beneficial for inhibition of chemokine receptors.…”

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4

“…Additional CXCR4 inhibitors that have been well described in the literature include the small molecule MSX-122 (20) and peptide BL-8040 (21). Nanobodies (6) and various other peptides, small molecules, and antibodies (22,23) have also been described (24).…”

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update

“…These can be broadly divided into those obtained by XFEL, such as 5-HT 2B [75] and GCG [76], and X-ray crystallography, such as TSHR [77], GIP [78] and EP4 [79]. Other studies have conducted homology modeling in combination with intensive epitope mapping to identify putative important contact residues of functional antibodies, e.g., CXCR4 [80], or by obtaining a crystal structure of the Fab fragment and modeled in combination with a GPCR homology model, e.g., FPR1 [81], but the greatest accuracy is obtained from high-resolution crystallography approaches. The knowledge gained from such studies can inform decisionmaking in the drug discovery process.…”