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The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.

Random mutagenesis is one of the most effective methodologies to generate variant libraries for directed protein evolution. Indeed, this approach requires no structural or mechanistic information and can uncover unexpected beneficial mutations. Here, we describe a new random mutagenesis method based on the use of human error-prone DNA polymerases (pol beta, pol eta and pol iota). This approach allows the random introduction of mutations through a single replication step followed by a selective PCR amplification of the replicated mutated sequences. The libraries generated using this methodology display different mutation rates and complementary mutational spectra. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomization.

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