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Protein Engineering by Yeast Surface Display
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A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast
Christian Schröter1, Ralf Günther, Laura Rhiel
1a Institute for Organic Chemistry and Biochemistry ; Technische Universität Darmstadt ; Darmstadt , Germany.
Mabs
|December 20, 2014
Summary
Researchers engineered pH-sensitive antibodies by incorporating histidine, reducing binding at acidic pH while maintaining high affinity at neutral pH. This novel strategy offers a generalizable method for developing advanced protein therapeutics with tunable binding properties.
Area of Science:
- Protein Engineering
- Immunology
- Biochemistry
Background:
- Growing demand for engineered proteins with tunable pH-dependent binding properties.
- Need for robust methods to confer pH-sensitivity to antibodies for therapeutic applications.
Purpose of the Study:
- To develop a novel strategy for engineering pH-sensitive antigen binding into antibody variable domains.
- To demonstrate the efficacy of combinatorial histidine scanning and yeast surface display for this purpose.
Main Methods:
- Utilized combinatorial histidine scanning libraries and yeast surface display for simultaneous screening of high affinity and pH-sensitivity.
- Applied the strategy to engineer pH-dependent binding into adalimumab complementary-determining regions.
- Isolated and combined heavy and light chain mutations to generate full-length antibody variants.
Main Results:
- Successfully generated three full-length antibody variants with sharp, reversible pH-dependent binding profiles.
- Achieved 230- to 780-fold increase in dissociation rate constants at pH 6.0, while retaining sub-nanomolar affinity at pH 7.4.
- Confirmed that histidine substitutions did not impact binding to human neonatal Fc receptor (huFcRn) or thermal stability.
Conclusions:
- The study presents a generalizable strategy for engineering pH-switch functions in antibodies and protein-based therapeutics.
- This approach enables the creation of antibodies with precisely controlled binding characteristics based on pH.
- The developed method excludes conventional negative selection steps, streamlining the engineering process.
