The clinical application of mouse-derived antibodies has revealed some limitations, such as causing human anti-mouse antibody (HAMA) reaction, an allergic reaction that might accelerate clearance of mouse antibodies and cause anaphylaxis. Moreover, it limits antibody-dependent cell-mediated cytotoxicity (ADCC) induced by Fc fragment reaction against murine antibodies, which severely hinders the application of murine derived antibodies in the clinical research.
To reduce the immunogenicity of mouse antibodies, chimeric antibody and human antibody strategies have been developed and quickly become important technologies in the antibody drug research. The establishment of phage display technology allows researchers to successfully screen and obtain the first fully human antibody with high affinity.
Similarly, constructing a transgenic mouse model carrying a human antibody genome has also become an attractive technology platform for the development of human antibodies. In addition, obtaining human antibodies against special diseases with the help of rehabilitated patient B lymphocytes combined with human hybridoma cells, also becomes a novel technical approach to developing therapeutic human antibody research.
The establishment of humanized antibodies first begins with the construction of chimeric antibody technology by combining a mouse antibody variable region with a human antibody constant region. Approximately 30% of the sequences in chimeric antibodies is from mouse and the remaining 70% is from human-derived antibody sequences. This chimeric antibody retains the specificity of antibody binding to antigen. Whereas in complementarity determining region (CDR) shift technology, only the binding epitope sequences in mouse antibodies are retained and the rest are all human antibody components, which comprise 90% of the chimeric antibody sequences. Therefore, the CDR technology, which is less immunogenic than chimeric antibodies, has been considered the gold standard technique for the development of humanized antibodies. This technology not only reduces the incidence of human anti-chimeric antibodies and human anti-CDR antibodies from ~ 40% to ~ 9%, but also lays the foundation for the clinical treatment of those complex diseases that require long-term and repeated treatment (such as tumors and autoimmune diseases). However, the biggest shortcoming of humanized antibody technology is the lack of a universal approach. For example, the humanized CDR shifting process requires a high degree personalization. Moreover, the clinical application of humanized antibodies still have a certain degree of risk for immune rejection or hypersensitivity due to the existence of 10% mouse-derived antibody sequences.
Along with the successful development of humanized antibody technology, phage display technology became used in the early 1990s to develop fully human antibodies. The desired combinatorial expression repertoire of antibodies is constructed by using fusion of phage coat proteins with foreign antibody genes, such as from humans. These human antibody genes are fused with the coat protein of the phage and can be displayed on the surface of the phage together. With the help of specific antigen binding screening methods, phage antibodies that specifically bind to the antigen can be obtained.
The first human antibodies developed using this technology platform are mainly antibody fragments (such as scFv and Fab). In the development of fully human antibodies, an important contribution of phage display technology lies in the fact that it does not rely on in vivo immune responses. Candidate human antibodies with affinity maturation that bind different antigens (e.g., self-antigens, toxins, labile and non-immunogenic antigens, etc.) can be directly obtained through in vitro antibody screening methods.
In the early 90s, researchers succeeded in establishing another fully human antibody development platform with the creation of a mouse model expressing human antibody genes. This technology can introduce human antibody genes into the corresponding location of the mouse antibody genome, effectively replacing it. After immunizing the mouse's immune system with an introduced antigen, fully human antibodies can be synthesized and produced in mice. The successful development of mouse models expressing human antibody genes has greatly driven the advancement of clinical applications for human antibodies.
Compared with phage display technology for developing fully human antibodies, the development timeline of mouse models expressing human antibody is relatively slow in the initial stages of antigen immunization, screening specific antibodies, and preparing hybridoma cells. However, the technology shows its obvious advantages once the initial antibody is obtained; the subsequent antibody optimization process naturally occurring in mice, including high frequency mutation, improves antibody affinity and effectiveness, and also reduces immune rejection. Currently approved clinical applications of human antibodies also demonstrate that antibody drugs developed by human antibody mouse platform perform better in terms of evaluation of relevant indicators of antibody drug resistance (e.g., antibody self-polymerization, specific binding, etc.).
The establishment of a mouse model expressing a human antibody provides a reliable and irreplaceable platform for the development of therapeutic antibody drugs. In this White Paper, our experts review the whole process of antibody drug development and analyze the various strategies used in generating human antibody mouse models.
Outline of Contents
● How are Therapeutic Antibodies Developed?
● Important Considerations in the Humanization of Antibodies
● Human Antibody Discovery Using In-vivo Mouse Models
● Leveraging Humanized Mice for Human Antibody Discovery