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Updated: Sep 25, 2025
Protein Engineering by Yeast Surface Display
Published on: November 29, 2024
Guidelines, Strategies, and Principles for the Directed Evolution of Cross-Reactive Antibodies Using Yeast Surface
Sara Linciano1, Ee Lin Wong1, Ylenia Mazzocato1
1Department of Molecular Sciences and Nanosystems, Ca' Foscari University of Venice, Mestre, Italy.
Abstract:
The ability of cross-reactive antibodies to bind multiple related or unrelated targets derived from different species provides not only superior therapeutic efficacy but also a better assessment of treatment toxicity, thereby facilitating the transition from preclinical models to human clinical studies. This chapter provides some guidelines for the directed evolution of cross-reactive antibodies using yeast surface display technology. Cross-reactive antibodies are initially isolated from a naïve library by combining highly avid magnetic bead separations followed by multiple cycles of flow cytometry sorting. Once initial cross-reactive clones are identified, sequential rounds of mutagenesis and two-pressure selection strategies are applied to engineer cross-reactive antibodies with improved affinity and yet retained or superior cross-reactivity.
Insights
This study details using yeast surface display to evolve cross-reactive antibodies. These engineered antibodies show enhanced therapeutic potential and improved preclinical to clinical translation for drug development.
Area of Science:
- Biotechnology and Immunology
- Protein Engineering
- Antibody Therapeutics
Background:
- Cross-reactive antibodies bind multiple targets, enhancing therapeutic efficacy and toxicity assessment.
- Facilitating transition from preclinical models to human clinical studies is crucial for antibody therapeutics.
- Yeast surface display is a powerful platform for antibody evolution.
Purpose of the Study:
- To provide guidelines for the directed evolution of cross-reactive antibodies.
- To utilize yeast surface display technology for engineering antibodies with improved properties.
- To enhance the transition of antibody therapies from preclinical to clinical stages.
Main Methods:
- Isolation of cross-reactive antibodies from a naive library using magnetic bead separations and flow cytometry.
- Application of sequential mutagenesis and two-pressure selection strategies.
- Leveraging yeast surface display for antibody engineering and screening.
Main Results:
- Identification of initial cross-reactive antibody clones.
- Engineering of antibodies with improved affinity while maintaining or enhancing cross-reactivity.
- Demonstration of a viable method for directed evolution of cross-reactive antibodies.
Conclusions:
- Directed evolution using yeast surface display is effective for generating cross-reactive antibodies.
- Engineered cross-reactive antibodies offer potential for superior therapeutic efficacy and safety profiles.
- This methodology aids in advancing antibody-based therapeutics towards clinical applications.
