The Role of Somatic Hypermutation in Developing Therapeutic Antibodies

Therapeutic antibody development requires technologies to optimize their affinity, stability, and safety profiles. Maturation of antibodies in B cells during the adaptive immune response is the result of somatic hypermutation (SHM), a process native to B-cells in the adaptive immune system. SHM is driven by activation-induced cytidine deaminase (AID) to introduce mutations into immunoglobulin (Ig) genes, promoting antibody diversity and enabling the selection of antibodies with improved binding characteristics. Recent advances now allow the use of SHM in vitro, coupled with cell surface display techniques, providing an efficient path to affinity maturation for therapeutic antibody candidates.¹

The Role of Somatic Hypermutation in Antibody Evolution

Somatic hypermutation is essential to the natural evolution of antibodies within B cells. In the immune system, SHM introduces point mutations into the variable (V) regions of antibody genes, leading to structural changes in the antibody’s antigen-binding sites. Over successive rounds of mutation and selection, B cells expressing antibodies with enhanced antigen affinity are preferentially expanded. This iterative mutation and selection process enables the development of antibodies with high specificity and binding strength for their target antigens. 

SHM primarily targets "hotspots" within the V regions, particularly in the complementarity-determining regions (CDRs) which are directly involved in antigen recognition. AID initiates SHM by deaminating cytosine bases to uracil in hotspots, often at WRC motifs (W = A/T, R = A/G), triggering error-prone repair pathways that result in a diversity of mutations. This mechanism, when replicated in vitro, allows researchers to engineer antibodies with high affinity for specific therapeutic targets. 

Engineering Antibodies with SHM and Cell Surface Display

Adapting SHM for in vitro affinity maturation of antibodies involves exposing Ig gene sequences to AID-induced mutation, followed by a selection process for those mutations that improve binding affinity. One effective platform for this is mammalian cell surface display, where antibodies or antibody fragments are presented on the surface of mammalian cells. Through fluorescence-activated cell sorting (FACS), cells displaying antibodies with enhanced antigen-binding can be selected, allowing for a rapid enrichment of high-affinity variants. 

The integration of next-generation sequencing (NGS) with SHM-based display systems allows for the analysis of sequence variants that emerge after selection and identifying promising candidates based on computational analysis of their binding potential.  

Related: What is Affinity Maturation?

Humanization and the Role of SHM in Addressing Affinity Loss

While antibodies generated in animal models, such as mice, are effective in initial target binding, they can elicit immune responses when administered to humans, reducing their clinical viability. Humanization, the process of modifying these antibodies to be more "human-like," mitigates immunogenicity risks. This involves replacing non-human framework regions with their human counterparts, while retaining antigen-specific CDRs. However, this process frequently leads to reduced binding affinity and biological activity.  

SHM can reintroduce mutations in humanized antibodies to restore or even improve the lost affinity. By applying SHM to the humanized antibody’s V regions, researchers can screen for variants that recover binding strength without reintroducing non-human sequences. This approach reduces the likelihood of adverse immune reactions, balancing affinity restoration with the need for compatibility in human patients. 

Phage Display and Enzymatic SHM: Expanding Affinity Maturation Tools

Phage display is another powerful platform for the affinity maturation of therapeutic antibodies. In phage display, antibody genes are incorporated into bacteriophage genomes, allowing the expression of antibody fragments on the phage surface. Traditional phage display relies on random mutagenesis techniques, which may generate numerous non-productive variants. However, combining phage display with enzymatic SHM using AID offers a more targeted mutation approach, focusing mutations within the V region's hotspots, where they naturally occur in B cells.²

References:

1. Bowers, P. M., Boyle, W. J., & Damoiseaux, R. (2018). The Use of Somatic Hypermutation for the Affinity Maturation of Therapeutic Antibodies. Methods in Molecular Biology, 479–489. https://doi.org/10.1007/978-1-4939-8648-4_24

2. Jeong, S. L., Zhang, H., Yamaki, S., Yang, C., McKemy, D., Lieber, M., Pham, P., & Goodman, M. (2022). Immunoglobulin somatic hypermutation in a defined biochemical system recapitulates affinity maturation and permits antibody optimization. Nucleic Acids Research, 50(20), 11738–11754. https://doi.org/10.1093/nar/gkac995