Antibody-dependent cellular cytotoxicity-null effector developed using mammalian and plant GlycoDelete platform

Area of Science:

• Immunology
• Biotechnology
• Glycoscience

Background:

• Immune checkpoint inhibitors, such as antibodies targeting programmed cell death protein 1 (PD-1), have revolutionized cancer therapy.
• However, the effector functions of these signal-regulating antibodies, influenced by heterogeneous glycan chains, require precise control for optimal therapeutic use.
• Developing methods for uniform glycan structures is crucial for consistent antibody efficacy and safety.

Purpose of the Study:

• To engineer nivolumab (an anti-PD-1 antibody) using GlycoDelete (GD) technology to eliminate antibody-dependent cellular cytotoxicity (ADCC) while maintaining target binding and inhibitory function.
• To assess the efficacy of GD engineering in both Chinese Hamster Ovary (CHO) and plant-based expression systems.
• To identify the optimal antibody isotype (IgG1 vs. IgG4) for GD-engineered nivolumab.

Main Methods:

• GlycoDelete (GD) engineering of CHO cells by overexpressing endo-β-N-acetyl-glucosaminidase (Endo T) and knocking out N-acetyl-glucosaminyl-transferase I using Cas9.
• Production of GD IgG1 and GD IgG4 nivolumab in engineered CHO cells and subsequent glycan analysis via mass spectrometry.
• Production of GD nivolumab in a plant-based expression system and evaluation of its functional properties.

Main Results:

• Glycan removal was confirmed in GD-engineered nivolumab using mass spectrometry.
• GD engineering successfully reduced ADCC in both IgG1 and IgG4 nivolumab forms without altering PD-1 binding or inhibitory activity.
• The IgG4 isotype of GD nivolumab proved most suitable and maintained reduced ADCC and PD-1 inhibition when produced in a plant system.

Conclusions:

• CHO and plant-based GD platforms are effective for reducing effector functions of signal-regulating antibodies like nivolumab.
• Glyco-engineering offers a viable strategy to create uniform glycan structures, enhancing the therapeutic potential of immune checkpoint inhibitors.
• This approach allows for precise control over antibody effector functions, paving the way for improved cancer immunotherapies.