Antibody affinity maturation

We have gained extensive experience in affinity maturation of small antibodies derived from phage display library screening. We usually take scFv as the antibody format in affinity maturation. Also, a monovalent display phagemid system is used to reduce the avidity effects during antigen-binding screening. Two methods, untargeted mutagenesis and oligonucleotide-directed mutagenesis, are employed to construct random or constrained sub-libraries to introduce a large number of mutants of the original antibody. Antibody binders of higher affinity are then selected by increasing the screening stringency. By constructing a series of sub-libraries of a scFv/Fab antibody, our proprietary protocol allows increase of the affinity of the scFv antibodies from 10 [-8] to 10 [-9]. We have successfully obtained a scFV antibody that has an extremely high affinity of 10 [-12], whose binding to the antigen is essentially irreversible.

Untargeted mutagenesis

We use an error-prone PCR integrated DNA-shuffling approach to mutate mainly CDR regions during sub-library construction. If the potential of introducing immunogenic mutations to framework positions is not a concern, we usually use this approach to create mutations at completely random positions across the entire VH and VL fragments.In these cases, the genetic diversity of the sub-library is further increased via passage through our proprietary bacterial mutator strain, CD-affi.

Oligonucleotide-directed mutagenesis

If the structure of the antibody/antigen is available, certain positions can be randomized at a defined diversity [such as full randomization with all 20 amino acids or biased randomization with selected amino acids at fixed percentages] to improve the affinity. We are able to create any sub-libraries to incorporate the defined mutations using trimer codon technology.

In order to fish out the antibody mutants that have increased affinity, two library screening strategies are available. In the first “surface-panning” method, decreasing concentrations of antigen is surface immobilized. In the second “solution-sorting” method, a sub-library is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Affinity-improved variants are finally validated one by one using ELISA or Biacore methods.

Peptide affinity maturation

Alanine scanning mutagenesis is our favorite method in affinity maturation of peptide binders. In this method, each single AA of a selected binding peptide will be replaced with an alanine, and then the binding of the modified peptides to the target protein will be assayed using BIACore technology. The non-essential AAs will be specifically identified. After that, we will create a directed/constrained peptide sub-library that contains random sequences on the non-essential AA positions. Here, again, we frequently randomize the non-essential residues using “NNK” or “trimer codon” strategy. Mutants with increased binding affinity are identified by enhancing the screening stringency, followed by phage ELISA.