A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

Abstract

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro . Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea

“…80 and 91) and (ii) the abEC1.1 antibody, which does not interfere with pannexin 1 channels, is selective for Cx26, Cx30 and Cx32 hemichannels, reduces connexin hemichannel currents and abrogates ATP release. 35,36,92 This vast body of experimental results, together with the data presented here, demonstrate that ATP-dependent ATP release through connexin hemichannels sustains Ca 2+ wave propagation not only in the LER, [25][26][27] but also in the GER.…”

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice

“…28-4082-53, GE Healthcare, Pittsburgh, PA, USA) with the ÄKTApurifier 100 system (GE Healthcare) following the Manufacture's instruction. After purification, the buffer was exchanged to phosphate buffer saline (PBS, pH 7.4) and the antibodies were kept in PBS at 4°C [36] .…”

“…The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels [ 35 , 36 ]. Monoclonal antibodies “have made a striking transformation from scientific tools to powerful human therapeutics” [37] , and many are already on the market or under advanced clinical development [38] .…”

“…Next, we formed abEC1.1 by fusing the Cx-binding scFv with the hinge and fragment constant (Fc) domain of human immunoglobulin G1 (IgG1, see Materials and Methods) [35] . We generated also a chimeric version (abEC1.1m) with murine hinge and Fc domain [36] . Both scFv-Fc polypeptides (see Supplementary materials, Fig.…”

“…By performing in silico analysis of Cx hemichannel-antibody interaction, we identified critical amino acids (N54, T55, L56, Q57, P58, P175 and N176) that are conserved in the extracellular domain of Cx26, Cx30 and Cx32 [36] . Of note, even a single a.a. difference in the above a.a. list reduced drastically the inhibitory effects of the antibodies on all other tested Cx hemichannels (Cx30.2/31.3, Cx30.3, Cx31, Cx31.1, Cx37, Cx43 and Cx45) [36] , most of which are expressed in the skin and its appendages [41] , [42] , [43] , [44] , [45] .…”