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

Area of Science:

• Immunology
• Biotechnology
• Antibody Engineering

Background:

• Monoclonal antibody (mAb) therapies often rely on Fc receptor (FcγR) and C1q interactions for efficacy.
• Optimal C1q engagement requires hexameric Fc:Fc interactions on target cells.
• Current methods for enhancing antibody effector functions are limited.

Purpose of the Study:

• To engineer IgG antibodies with enhanced hexamerization capabilities.
• To investigate the role of the IgM tailpiece in promoting IgG hexamerization and complement-dependent cytotoxicity (CDC).
• To develop an 'on-target hexamerization' strategy for improved therapeutic outcomes.

Main Methods:

• Fusion of the C-terminal tailpiece of IgM to human IgG (hIgG).
• Introduction of a C575S mutation in the IgM tailpiece to modulate hexamerization.
• Assessment of C1q recruitment, CDC, off-target activation, and in-vivo efficacy.
• Evaluation of target cell killing in lymph nodes.

Main Results:

• IgM tailpiece fusion induced spontaneous IgG hexamerization, enhancing C1q binding and CDC.
• Spontaneous hexamerization led to off-target complement activation and reduced in-vivo efficacy.
• The C575S mutation facilitated reversible, concentration-dependent hexamerization, enabling 'on-target hexamerization'.
• C575S mutant antibodies showed increased complement activity post-target binding, retaining in-vivo efficacy and enhancing lymph node cytotoxicity.

Conclusions:

• Exploiting the IgM tailpiece is a viable strategy to augment IgG hexamerization and CDC.
• The C575S mutation provides a mechanism for controlled, on-target hexamerization, mitigating off-target effects.
• C575S-tailpiece technology offers a novel format for developing enhanced antibody therapeutics with improved safety and efficacy.