Characterisation of VHH Antibodies Allosteric Mechanism On IL-6

Signal transduction in response to IL-6 requires binding of the cytokine to its receptors at three distinct receptor-binding sites, referred to as site I (contacting the gp80), site 2 (contacting gp130 between domain 2 and domain 3), and site 3 (contacting the domain 1 of gp130). This leads to the activation of the JAK/STAT signalling cascade. VHH 15 and VHH 56 are nanobodies that bind at the same locality remote from the site II or site III interfaces on IL-6, resulting in the regulation of IL-6 activity via an allosteric mechanism.

VHH 15 is an antagonist to IL-6, thus has therapeutic benefits as IL-6 is implicated in various inflammatory and autoimmune diseases. The goal of this study was to characterise the allosteric regulation of VHH 15 on IL-6 function using a structure - function approach. This work explores the effects of R102 and Y103 in the CDR3 of VHH 15 towards IL-6 interaction with the gp130 receptor, which controls the signalassembly of transducing complex. These residues were chosen as they protrude into a crevice on IL-6. Assays revealed that VHH 15R102A had weaker binding affinity to IL-6, and VHH 15Y103A had a binding affinity that is comparable to VHH 15. Both variants cause weaker IL-6 binding to site II of gp130 comparative to wild type, therefore the variants are more potent inhibitors of IL-6 signalling. Crystal structures of the IL-6:VHH 15R102A reveal the absence of the long R102 side chain not only resulted in the loss of interactions between R102 and Helix B and E of IL-6, but generated a new Y103 side chain configuration. This arrangement of Y103 is identical to N102 in the CDR3 of VHH 56. Moreover, removal of Y103 causes R102 in VHH 15Y103A to become flexible.

These new arrangements cause alterations to the CE loop orientation in IL-6 bound to variants that resemble the loop in IL-6:VHH 56. This explains the weak site II binding and the low IL-6 activity observed for IL-6:variant complexes, an effect homologous with VHH 56 binding. These results highlight the influence of R102 and Y103 in VHH 15:IL- 6 binding of gp130. The presented structural and binding data indicates the presence of VHH 15 allosteric inhibition of IL-6 function mediated by changes in IL-6 structure due to association of anti-IL-6 VHH.