Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling

Abstract

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

The balance of interleukin (IL)-6, IL-6·soluble IL-6 receptor (sIL-6R), and IL-6·sIL-6R·sgp130 complexes allows simultaneous classic and trans-signaling

“…S1B), which is excellent compared with previous reports using IL-6 and the same cell line (18 -21). Despite the fact that our IL-6 appears to have lower binding affinity to IL-6R as compared with some older reports, analysis of its biological activity demonstrates high specificity and activity of our recombinant IL-6, and recent data from Adams et al (15) match our results very closely. Therefore, we concluded that the K d determined in this study is appropriate.…”

“…In the last 25 years, different K d values for IL-6⅐IL-6R complex formation have been described, ranging from 0.5 to 34 nM (12,15,16). Based on the K d and an equation described by Gaillard et al (12), it is possible to calculate the theoretical proportions of IL-6⅐sIL-6R complexes and free IL-6 for any given concentration.…”

“…Finally, sgp130 is found at concentrations between 100 and 400 ng/ml in the serum of healthy humans (12). Different dissociation constant (K d ) values for IL-6 binding to sIL-6R have been reported, ranging from 0.5 to 34 nM (12,15,16). Depending on the K d , calculations based on intermolecular affinities suggested that some or most systemic IL-6 will be trapped in IL-6⅐sIL-6R or IL-6⅐sIL-6R⅐sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system to increase the serum half-life of IL-6 and/or to restrict systemic IL-6 signaling.…”

Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses

“…Trans-activation was significantly increased after the addition of VHH6, a recently described single-domain antibody that specifically stabilizes IL-6:sIL-6R complexes ( Fig. 3B) (17). However, the VHH6-supported trans-presentation was efficiently inhibited by sgp130Fc.…”

“…Such preformed receptor complexes could hinder sgp130Fc accession. In addition, IL-6 does not bind gp130 in the absence of the (s)IL-6R, and the affinity of gp130 for IL-6:(s)IL-6R complexes is about 100 times stronger than the affinity of IL-6 for (s)IL-6R (13,17,30). Thus, low-affinity binding of IL-6 to IL-6R on the cell surface is rapidly followed by high-affinity binding of the IL-6 complex to membrane-bound gp130.…”