Affinity maturation is the process to improve antibody affinity for an antigen. In vivo, natural affinity maturation by the immune system takes place by somatic hypermutation and clonal selection. In vitro, in the laboratory affinity maturation, can be obtained by mutation and selection.
Creative Biolabs has gained extensive experience in antibody affinity maturation. 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. We also provide affinity maturation services for single domain antibodies. Two methods, untargeted mutagenesis and oligonucleotide-directed mutagenesis, are employed to construct random or defined 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 -9 to 10 -10. We have successfully obtained a scFv antibody that has an extremely high affinity of 10 -12, whose binding to the antigen is essentially irreversible.
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™.
If the structure of the antibody/antigen complex is available or modeling the structure of the antibody/antigen is possible, 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. Most of the time, we need study the AA sequences of the antibody to find out the conserved sequences (in comparison with the germ-line and antibody subfamily sequences). We may then introduce mutations to the positions in the frame work regions that are not conserved. Supposedly, these regions will be antigen-specific and change in these regions may not increase immunogenicity.
Phage Display Antibody Library Screening
Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. In the first "surface-panning" strategy, decreasing concentrations of antigen is surface immobilized. In the second "solution-sorting" strategy, in which a labeled antigen in solution is used, we have two approaches, selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd.
Antibody Affinity Measurement
We offer Biacore Analysis services for binding kinetic analyses of antibodies. We typically capture the antibody on the chip and run antigen on top of the captured antibody. The antigen will be ran at 6 different concentrations for each antibody and chi-square analysis will be performed on the binding constants we obtain from each antigen concentration. The documentation package will include a real time on-rate (Ka), off rate (Kd), an affinity constant (KD), chi square value and a graph of real-time binding kinetics. We would like to obtain ~50 uL of 1 mg/mL antigen and antibody solutions. We will need ~100 ug of antigen and ~50ug for each antibody. We would need MW information for the antigen as well. It may require special considerations for antigens with repeated or multiple epitopes for affinity determination.>> Learn more about Antibody Affinity Measurement Services
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.
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