Identification of high affinity HER2 binding antibodies using CHO Fab surface display

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Monoclonal antibody discovery typically uses phage or yeast display, followed by adaptation to mammalian cells for production.
• This host transition can lead to impaired antibody function, necessitating laborious screening and engineering.
• Mammalian cells are crucial for antibody production due to complex post-translational modifications like disulfide bonds and glycosylation.

Purpose of the Study:

• To develop a novel plasmid-based Fab screening platform in Chinese hamster ovary (CHO) cells for antibody selection within the intended production host.
• To circumvent issues associated with transferring antibodies from microbial display systems to mammalian expression systems.
• To enhance the binding affinity of a low-affinity antibody variant targeting the human epidermal growth factor receptor (HER2).

Main Methods:

• A plasmid-based Fab screening platform was established on CHO cells.
• A library of approximately 10^6 unique clones derived from a hu4D5 variant was generated and screened.
• Fluorescence-activated cell sorting (FACS) was employed for two rounds to isolate high-affinity clones.
• Selected clones were analyzed for antigen binding, expression, aggregation, yield, stability, and affinity.

Main Results:

• The screening platform successfully identified antibody variants with up to 400-fold enhanced binding to HER2.
• Four unique clones demonstrated improved antigen binding on CHO cells and as purified human IgG.
• Three of the four improved clones featured free cysteines in the antibody heavy chain's third complementarity-determining region without compromising expression or causing aggregation.
• The enhanced clones exhibited comparable yields and stabilities to the original hu4D5 antibody, with sub-nanomolar affinities.

Conclusions:

• The CHO cell-based Fab screening platform enables antibody selection in the correct host, incorporating essential post-translational modifications.
• This approach is effective for antibody affinity maturation and specificity enhancement, particularly for surface receptor targets.
• While limited by library size for naive screening, the platform is a powerful tool for optimizing existing antibody candidates and engineering other receptors like T-cell receptors and chimeric antigen receptors.