Antibody humanization is a technique used to modify non-human (often murine, or mouse-derived) antibodies to make them more similar to human antibodies. The goal of this process is to reduce the risk of immunogenicity—where a patient’s immune system recognizes the antibody as foreign, triggering an unwanted immune response. In clinical applications, minimizing immunogenicity is key to ensuring the safety and efficacy of antibody-based treatments.
The primary challenge with non-human antibodies, particularly murine (mouse) antibodies, is the potential for patients to develop a human anti-mouse antibody (HAMA) response. When a murine antibody is administered, the immune system can recognize it as foreign, causing complications from immune complex formations and resulting in the antibody treatment being ineffective and can even lead to adverse reactions.
The Development of Chimeric Antibodies
One of the first solutions developed to address immunogenicity was the creation of chimeric antibodies. Chimeric antibodies combine the variable regions of murine antibodies (responsible for antigen recognition) with human constant regions (responsible for effector functions). This hybrid antibody maintains its specificity for the target antigen but is less likely to elicit a strong immune response due to the presence of human components.
Using genetic engineering, researchers cloned the genes encoding the variable regions of murine antibodies and fused them with the genes encoding the constant regions of human antibodies. This allowed the antibody to retain its binding affinity and specificity for the target antigen, while incorporating human elements to reduce immunogenicity. Chimeric antibodies represented a significant step forward, yet they still contained around 30% murine sequences, which could still provoke a HAMA response in some cases.
As antibody therapeutics became more widely used, the need for further reductions in murine content led to the development of CDR grafting. CDR (complementarity determining region) grafting involves transferring only the critical antigen-binding regions of a murine antibody to a fully human antibody framework. This strategy reduces the murine content to less than 10%, further lowering the risk of immunogenicity.
In the CDR grafting process, the CDRs—regions of the antibody that directly interact with the target antigen—are carefully selected from the murine antibody. These regions are then "grafted" onto a human antibody framework that has high structural similarity (homology). The human framework helps maintain the overall structure and stability of the antibody while supporting the murine-derived CDRs in recognizing and binding to the antigen.
However, CDR grafting is not without its challenges. While the CDRs are responsible for binding the antigen, the framework regions also play a role in maintaining the proper conformation of the antibody. Therefore, during the humanization process, careful consideration must be given to ensuring that the grafted CDRs maintain their binding affinity and structural integrity when placed in the human antibody framework.
Beyond CDR grafting, several other humanization strategies have been explored to further refine the process and improve therapeutic outcomes. One approach is the use of fully human antibodies generated through transgenic mice or phage display libraries. In transgenic mice, the murine genes for antibody production are replaced with human genes, allowing the mice to produce fully human antibodies. This eliminates the need for humanization altogether and provides a streamlined pathway to developing antibodies with minimal immunogenicity.
Our High-throughput Fully Human Antibody Discovery Platform integrates Cyagen’s HUGO-Ab™ mice with Biointron’s AbDrop™ microdroplet-based single B cell screening. This powerful combination accelerates the discovery and development of fully human antibodies, reducing the time from target identification to therapeutic candidate to just three months.
Another method involves using advanced bioinformatics tools to predict and mitigate potential immunogenic regions within an antibody. By identifying amino acid sequences that are more likely to trigger an immune response, researchers can design antibodies with fewer immunogenic epitopes, making them more suitable for clinical use.
Yaghoub Safdari, Safar Farajnia, Mohammad Asgharzadeh & Masoumeh Khalili (2013) Antibody humanization methods – a review and update, Biotechnology and Genetic Engineering Reviews, 29:2, 175-186, https://doi.org/10.1080/02648725.2013.801235
Harding, F. A., Stickler, M. M., Razo, J., & DuBridge, R. B. (2010). The immunogenicity of humanized and fully human antibodies: Residual immunogenicity resides in the CDR regions. MAbs, 2(3), 256-265. https://doi.org/10.4161/mabs.2.3.11641
