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Remodeling domain interfaces to enhance heterodimer formation
1Department of Molecular Oncology, Genentech Inc., South San Francisco, California 94080, USA.
Protein Science : a Publication of the Protein Society
|April 1, 1997
Summary
Protein engineering strategies enhance the production of functional bispecific diabodies. Domain interface modifications improve heterodimer formation, crucial for therapeutic antibody development.
Area of Science:
- Biotechnology
- Protein Engineering
- Immunology
Background:
- Bispecific diabodies offer targeted therapies by engaging multiple antigens.
- Engineering strategies are needed to improve the assembly of functional heterodimers over inactive homodimers.
Purpose of the Study:
- To evaluate domain interface engineering strategies for enhancing functional heterodimer formation in bispecific diabodies.
- To compare disulfide bond stabilization and knob-into-hole mutations for improving diabody assembly.
Main Methods:
- Constructed a disulfide-stabilized diabody using cysteine mutations (VL L46C, VH D101C).
- Designed and modeled eleven "knob-into-hole" diabody variants with sterically complementary mutations.
- Expressed and purified engineered diabodies in Escherichia coli.
- Assessed functional heterodimer fraction, overall yield, and antigen binding affinity.
Main Results:
- Disulfide-stabilized diabody showed improved functional fraction (>96%) but significantly lower yield.
- Knob-into-hole variants demonstrated efficient heterodimer formation (e.g., v5 at 92%) with maintained yields and antigen affinity.
- Interface engineering effectively enhanced functional heterodimer assembly.
Conclusions:
- Domain interface remodeling, via disulfide bonds or knob-into-hole mutations, facilitates functional bispecific diabody production.
- These engineering approaches expand the possibilities for assembling complex protein interactions.