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Improving antibody affinity by mimicking somatic hypermutation in vitro

Abstract

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 103 and 104 independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.

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Figure 1: Mesothelin binding as judged by ELISA of phage clones selected after panning each of the four libraries.
Figure 2: Amino acid sequence of some of the ELISA-positive phage clones obtained after panning of the different libraries.
Figure 3: Comparison of the BIAcore sensorgrams obtained for the binding of wild-type and mutated scFv immunotoxins to immobilized mesothelin.

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Acknowledgements

The authors are thankful to Byungkook Lee, George Vasmatzis, and James Vincent for valuable criticisms and Jennie Evans for editorial assistance.

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Correspondence to Ira Pastan.

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Chowdhury, P., Pastan, I. Improving antibody affinity by mimicking somatic hypermutation in vitro . Nat Biotechnol 17, 568–572 (1999). https://doi.org/10.1038/9872

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