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Targeted mutagenesis could streamline antibody engineering

January 20, 2021

Monoclonal antibodies represent an important class of therapies for an ever-increasing number of diseases, infections, and health conditions. That’s why scientists around the world are working to improve the processes required to engineer new antibody candidates for therapeutic use. The BioXp® system for DNA synthesis is a crucial tool for these efforts because it allows for affordable, rapid generation of full-length antibody constructs.

In a recent publication in the journal mAbs, scientists from GigaGen report a fascinating comparison of two different mutagenesis methods to determine how well they perform for in vitro affinity maturation experiments. Their work has broad implications for the antibody engineering community, which currently lacks standardized protocols for this kind of work.

The team, including lead author Jan Fredrik Simons, senior author David Johnson, and collaborators, describes the comparison of two techniques with four human antibodies for immuno-oncology-related proteins. Those include “random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions,” according to the paper.

As expected, the random mutagenesis approach led to a much larger number of variant sequences to test than the targeted approach. What the scientists wanted to know was whether the larger pool of candidates translated to more successful candidates. “A larger library of variants requires more effort to screen, but theoretically increases the probability that the library contains an affinity-matured variant,” they write.

As part of this project, the team used the BioXp® system to create synthetic oligos, generate full-length constructs using the Gibson Assembly® process, and introduce them into vectors for expression.

Their findings could help all researchers streamline their antibody engineering and screening workflows. “Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved [single-chain variable fragment] affinity with similar efficiency,” the scientists report. In part, that’s because “most random V(D)J mutations lead to no change in affinity,” they add.

“One method that might capture synergistic or compensatory mutations involves consecutive rounds of mutagenesis interspersed by rounds of selection,” the team concludes, suggesting that they might proceed with exhaustive targeted mutagenesis “followed by one or two rounds” of random mutagenesis.

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