A general method for greatly improving the affinity of antibodies by using combinatorial libraries

Area of Science:

• Protein engineering
• Antibody engineering
• Immunology

Background:

• Antibody affinity maturation is crucial for therapeutic development.
• Optimizing antibody binding sites requires efficient methods to explore combinatorial mutations.
• Previous optimization of the anti-TNF-alpha antibody D2E7 presented a challenge due to its high initial affinity.

Purpose of the Study:

• To apply and validate the Look-through Mutagenesis (LTM) method for antibody affinity maturation.
• To comprehensively map the energetic landscape of an antibody-binding site.
• To generate novel antibody variants with significantly enhanced affinity and function.

Main Methods:

• Look-through Mutagenesis (LTM) was employed to introduce single amino acid mutations across all complementarity determining regions (CDRs).
• Yeast surface display was used to generate and select antibody libraries.
• Combinatorial beneficial mutagenesis and FACS selection were applied to further enhance affinity.

Main Results:

• 38 beneficial substitutions were identified in 21 CDR positions, increasing TNF-alpha binding affinity.
• Engineered antibody variants demonstrated 500- to 870-fold higher affinities compared to the parent antibody.
• Enhanced affinities correlated with a 15- to 30-fold improvement in TNF-alpha neutralization.

Conclusions:

• LTM is a facile and rapid strategy for comprehensive energetic mapping of antibody-binding sites.
• The LTM approach enables significant affinity maturation of antibodies.
• This methodology holds broad applicability for engineering antibodies and other proteins with improved binding properties.