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Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay
Published on: September 10, 2018
Antibody-dependent cellular cytotoxicity-null effector developed using mammalian and plant GlycoDelete platform
Cho Eun Kang1, Seungeun Lee2, Taeyoung Ahn1
1Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-Ro, Seodaemun-Gu, Seoul, Republic of Korea.
Abstract:
Cancer therapy using immune checkpoint inhibitor antibodies has markedly shifted the paradigm of cancer treatment. However, methods completely eliminating the effector function of these signal-regulating antibodies is urgently required. The heterogeneity of glycan chains in antibodies limits their use as therapeutic agents due to their variability; thus, the development of uniform glycan chains is necessary. Here, we subjected the anti-programmed cell death protein (PD)-1 antibody nivolumab, a representative immune checkpoint inhibitor, to GlycoDelete (GD) engineering to remove the antibody-dependent cellular cytotoxicity (ADCC) of the antibody, leaving only one glycan in the Fc. Glyco-engineered CHO cells were prepared by overexpressing endo-β-N-acetyl-glucosaminidase (Endo T) in CHO cells, in which N-acetyl-glucosaminyl-transferase I was knocked out using Cas9. GD IgG1 nivolumab and GD IgG4 nivolumab were produced using GD CHO cells, and glycan removal was confirmed using mass spectrometry. Target binding and PD-1 inhibition was not altered; however, ADCC decreased. Furthermore, the IgG4 form, determined to be the most suitable form of GD nivolumab, was produced in a plant GD system. The plant GD nivolumab also reduced ADCC without affecting PD-1 inhibitory function. Thus, CHO and plant GD platforms can be used to improve signal-regulating antibodies by reducing their effector function.
Insights
GlycoDelete (GD) engineering uniformly modifies antibodies like nivolumab, reducing immune effector functions while preserving anti-cancer activity. This approach enhances therapeutic antibody development using CHO and plant-based systems.
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.
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