The continuous development and improvement of gene editing technology has driven the successful establishment of such a platform for mouse models expressing human antibody genes. This has not only led to revolutionary innovations in the research and development of therapeutic antibody drugs, but has also served to promote their wide clinical applications.
How are human antibodies developed using a mouse model? Utilizing the mouse immune system to produce diverse combinations of different immunogens and specific human antibodies through the natural process of recombination in mice and high mutation of somatic cells is a viable strategy.
The establishment of a mouse model expressing the human antibody gene provides a reliable platform for the development of therapeutic antibodies. Compared with other human antibody development and production technologies, in human antibody production, this strategy features the following advantages: 1) antibodies humanization is not required; 2) Multi-antibody portfolio is diverse 3) antibodies undergo affinity maturation in vivo, etc.
In 1985, scientists first proposed introducing human antibody genes into mouse germ cells and producing human antibodies by establishing transgenic mice. The proposal of this idea led to a new method for the production and development of human antibodies. In 1989, scientists constructed human antibody heavy chain gene vectors for the first time, including the heavy chain variable region (including VDJ) and human IgM antibody, and the μ chain constant region. Large plasmid DNA vector of about 25 kb was microinjected into the fertilized eggs of mice, which successfully generated about 4% of mouse B cells expressing human antibody μ chain, and transgenic mice with human μ chain that could produce human IgM antibody. In 1993, scientists knocked out the human heavy chain joining cluster (JH) and light chain joining cluster (JK) genes in mice, then mated with transgenic mice expressing human IgG and IGL antibodies, and successfully obtained a human antibody transgenic mouse model that can produce diverse antibody combinations.
In 1994, the first human antibody mouse platform - HuMabMouse was successfully developed. This mouse model was constructed via knockout of the mouse heavy and light chain (IgH and IgG) genes and introduction of human antibody genes to express the heavy chain and light chain. The whole human IgH genome is about 1.29 mb and the human IgK genome is about 1.39 mb, while the original human antibody heavy chain genome is only about 80 kb. The antibody diversity combination is determined by the V(D)J gene recombination in its germ cells. Therefore, the key technical problems to be solved in the successful development of the technology platform remains in determining how to increase the genomic capacity of imported human antibody and improve the diversity of the gene combination of human antibody.
In 1993, scientists began to use yeast artificial chromosome (YAC) vectors to construct human antibody heavy chain (~220 kb) and light chain (~300 kb) vectors through yeast homologous recombination, and with the help of yeast-embryonic stem (ES) cell fusion method, successfully introduced it into mouse ES cells.
In 1997, YAC with large fragment of heavy chain of human antibody (~ 1 mb) and light chain (~ 700 kb) was introduced into mouse ES cells and mated with mouse antibody gene (variable region and constant region) knockout mice. Scientists successfully constructed a XenoMouse mouse model expressing human antibody genes. This model contained 66 VDJ genes and 32 VJ genes. The XenoMouse and HuMabMouse mouse models have completely ruled out the possible interference of mouse antibody genes on human antibody genes, and also increased the diversity of human antibody gene combinations. However, these two mouse antibody genes have been completely knocked out - that is, the mouse lacks both antibody variable region and constant region genes - which reduces the effectiveness of human antibody production and also affects the class conversion effect of antibodies in mice and the incidence of somatic high frequency mutations..
In 2014, by using bacterial artificial chromosome (BAC) and Cre/loxP recombination technology, scientists successfully constructed the KyMouse mouse model. To achieve this, they inserted the human antibody heavy chain (V-D-J) variable region and light chain (Vk-Jk) variable region into the upstream region of the mouse heavy chain constant region (Cμ) and light chain constant region (Ck), respectively. The KyMouse mouse model was successfully constructed on the basis of not affecting the anti-constant region of the mouse. After antigen stimulation, KyMouse can achieve high frequency somatic cell mutation and produce high affinity human antibody.
In addition, scientists also successfully constructed Veloclmune mouse model. First, a certain number of large fragments of human antibody gene BAC are constructed. Then corresponding BAC vectors are introduced via microinjection into mouse ES cells, so as to achieve the site-specific replacement of human antibody heavy chain and light chain variable region genes with the corresponding mouse antibody variable region genes, while retaining the constant region of the mouse antibody gene.
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