On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation

Abstract

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC.

Avidity in antibody effector functions and biotherapeutic drug design

“…In a mutational screening approach, they identified two conserved glutamic acid residues in the Fc region of IgG — E345 and E430 — for which the substitution of any other amino acid enhanced IgG hexamer formation and complement activation for a wide range of cell surface targets. Sopp et al demonstrated that self-assembly dependent on target binding can also be facilitated by extending IgG1 antibodies with the µ-tailpiece mentioned above, but in which the carboxyl-terminal cysteine is mutated to a serine to prevent covalent hexamerization 171 . By systematically depleting individual complement components, Taylor et al showed that antibodies containing hexamer-enhancing mutations require a reduced presence of membrane attack complex-forming complement components to promote CDC compared to the non-mutated parent antibodies 172 .…”

Multivalent ACE2 engineering—A promising pathway for advanced coronavirus nanomedicine development

“…From the IgM perspective, the full-length or tailpiece IgM-Fc could be explored to achieve the avid effect demonstrated in other studies. [21] , [132] In this case, the IgM-Fc fusion may elicit humoral cytolytic reactions and pathogen agglutination (i.e., the formation of aggregated immune complexes) as reported for bacterial pathogens. [133] This agglutination effect results in a superior neutralization activity to IgA-Fc and IgG-Fc.…”

Structure of IgG-Fc hexamer reveals a mutual lock-and-key mode of Fc-Fc interaction

“…Hexameric IgG via IgM tailpiece fusion or Fc-Fc interface mutations have been shown to increase CDC function, crosslinking ability of cell surface receptors or SARS-CoV-2 virus neutralization ability 14,15 . Our hexameric IgG-Fc structure provides detail information of the Fc-Fc interaction and guidance for further engineering for therapeutic application of IgG hexamer.…”