A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

Abstract

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.

Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen

“…Only Abs detectably hexamerizing in solution could bind C1q or activate complement in solution efficiently, as illustrated by control Abs and variants E430G and E345K (Figures 2C and 2D). Combined with the observation that the described CDC-stimulating single mutations (de Jong et al, 2016) all promoted increased hexamerization in solution when combined into double or triple mutants (Figure 1), this suggests that IgG hexamerization is prerequisite to C1q binding and C1 activation.…”

“…We identified a number of specific single-point mutations in the IgG1 Fc domain that stimulated hexamerization and complement activity of the mutated Ab after its binding to cell surface-expressed Ag (de Jong et al, 2016). Interestingly, several such mutations were found to have additive effects, and a variant combining three mutations (i.e., E345R, E430G, and S440Y [RGY]) was shown to readily assemble into hexamers and induce complement activation in solution in the absence of Ag binding (Diebolder et al, 2014).…”

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy

“…Based on the finding that IgG antibodies can organize into hexamers on the cell surface after antigen binding, thereby building optimal docking sites for C1q, de Jong and colleagues [74,75] translated this natural concept to design antibodies with improved effector functions. Mutations that enhanced hexamer formation and complement activation by IgG1 antibodies were identified ( fig.…”

Molecular Basis of Modern Targeted Therapy for Squamous Cell Carcinoma of the Tongue and Oral Mucosa With Monoclonal Antibodies

“…Моноклональные антитела к VEGF-рецепторам и их лигандам. В настоящее время разрабатываются и проходят клинические испытания высокоспецифичные препараты, направленные на селективное подавление ан-гиогенеза; из них наиболее широкое применение полу-чил бевацизумаб (Авастин, Авегра, Б-Маб) [48] -препа-рат гуманизированных моноклональных антител к VEGF, одобренный FDA для использования в комплексной те-рапии рака [49]. Ингибиторы VEGF предотвращают акти-вацию рецепторов и дальнейшую трансдукцию проанги-огенного сигнала, подавляя пролиферацию и миграцию эндотелиальных клеток, препятствуя формированию со-судистой сети [50].…”