Affinity Maturation in Antibody

Although the potential of therapeutic antibodies in biomedical research has been demonstrated, in clinical applications, the therapeutic effect is often limited by the lack of affinity between the antibody and the target. However, in many cases, screening for antibodies with high affinity progresses slowly. In this situation, increasing the antigen-binding affinity of existing antibodies has become the best choice. With advanced antibody engineering platform established by Creative Biolabs, we provide our clients with antibody affinity maturation services, which is currently a common strategy for increasing the antigen-binding capacity of antibodies.

Antibody Affinity Modification

Protein engineering of therapeutic antibodies can confer desired biological and physicochemical properties such as low immunogenicity, high affinity and specificity, optimal effector function, and good solubility and stability. The two most common modifications currently used for candidate antibodies are antibody humanization and affinity maturation. Complementation determining region (CDR) grafting is a widely used humanization strategy. However, direct transplantation of murine CDRs into human framework receptor sequences typically results in loss of affinity, and thus reverse mutations that support framework region residues (cursor region residues) of the CDR loop structure are often required.

Fig.1 Antibody affinity modification.

Antibody Affinity Maturation

For in vitro affinity maturation, three diversification approaches are typically used:

• Random mutagenesis by error-prone PCR. The degree of mutagenesis is controlled by controlling the number of gene doubling events that occur in the PCR reaction. Our error-prone PCR method is used to optimize de novo protein evolution to improve folding stability, solubility, and ligand binding affinity.
• Randomization of targeted residues using degenerate oligonucleotides. In the targeted randomization approach, CDRs are randomized logical targets in most cases due to CDR-H3 and CDR-L3 tend to dominate antibody interactions. One of the main effects associated with targeted randomization is the selection of locations that are not essential for antigen binding can enhance affinity when amino acids are optimally substituted. Alanine scanning can help determine the residue to be randomized, especially when the CDRs are very long. Sometimes, the alanine mutation itself increases the affinity of the antibody.
• Chain shuffling. Chain shuffling, also known as strand replacement technology, is a relatively simple technique for in vitro affinity maturation of antibodies. Based on the principle of random pairing of antibody variable regions, we keep one strand unchanged while replacing the other strand, and then screen for antibody molecules with high affinity after substitution.

The first step in antibody affinity engineering is to identify key residues for antigen-antibody interactions. Creative Biolabs assessed the effects of mutations by performing alanine scanning mutagenesis in the CDR3 regions of the heavy and light chains (CDR-H3 and CDR-L3) and then analyzing the binding activity of the purified Fab protein by indirect ELISA. After constructing a heavy or light chain CDR3 variant library using the above method, we can perform affinity modification of the candidate antibody.

A complete affinity matured service process can increase antibody binding capacity by a factor of 10 to 100. Our services can also help clients screen for potential immunogenicity and provide antibodies that avoid T cell epitopes.

If you have any questions about our protein engineering service, you can contact us by email or send us an inquiry to find a complete solution.

For Research Use Only.