Therapeutic antibody-based drugs have advantages, including high specificity against a particular antigen and uniformity. They have attracted much attention in the treatment of various diseases, especially tumors. Mice are most widely used in the development of therapeutic antibodies, due to their low immunological tolerance to human antigens and ease of handling. However, murine antibody-mediated rejection severely affects the safety and efficacy of antibody therapy. Hence, the humanization of murine antibodies is crucial for the development of a new class of therapeutic antibodies.
The development of therapeutic antibodies has been revolutionized with the advent of a humanized mouse model which is capable of producing a fully-human antibody. Mice that have endogenous antibody genes (heavy and light chains) replaced with human antibody genes can be used to develop humanized antibodies in just one step. This humanized mouse model greatly simplifies the antibody development process, reduces development costs and risks, increases the success rate, and significantly improves the safety and efficacy of clinical application of antibody drugs. Biocytogen’s RenMab mouse is the antibody-humanized mouse with the most complete antibody genes replacement in the world. In the RenMab mouse, the parts of the genes that encode for antibody variable domains of H and κ chains are in situ replaced by human sequences using a unique chromosome engineering technique, while the constant domains and critical regulatory elements remain murine. The length and integrity of humanized antibody genes replacement in the RenMab mouse significantly outperform other existing models. The RenMab mouse has normal immune system development comparable to that of the wild-type mouse. Because of the higher diversity of its antibody genes, the RenMab mouse has a higher potential for antibody drug development. With this model, researchers can obtain more antibody drugs with strong specificity, high affinity, and diversity.
Features and Advantages
1. In situ gene replacement ensures that the antibody gene pattern of the RenMab mouse is highly consistent with that of humans
The RenMab mouse was designed and developed based on Biocyogen’s unique chromosome engineering technique. In this model, the parts of the genes that encode for antibody variable domains of H and κ light chains are replaced by human sequences.
H chain: The genes (about 2.6 Mb in length) that encode for murine antibody variable domains were replaced with the human variable domains (about 1 Mb in length, containing all human V/D/J antibody genes, Figure 1A).
κ light chain: Genes that encode for the variable domains of human κ light chain have two copies in opposite directions, namely proximal V cluster and distant V cluster. The coding region of the proximal V cluster is intact, which is frequently selected for antibody gene replacement in other humanized mouse models. Although the distant V cluster lacks the C gene and is considered to be a pseudogene, V and J genes are still potentially functional. We replaced the genes (about 3.2 Mb in length) that encode for murine antibody variable domains with the human variable domains (about 1.6 Mb in length) containing the two opposite-direction gene copies (Figure 1B).
Figure 1. Schematic of humanization in the RenMab mouse
2. Accurate humanization ensures that the RenMab mouse has a completely developed immune system comparable to the wild-type mouse
A comprehensive test of the immune system involving multiple organs and tissues showed that the RenMab mouse is highly comparable to the wild-type mouse in all assays. There is no significant difference in spleen weight between the RenMab mouse and the wild-type mouse. The numbers, differentiation and maturation patterns of immune cells, including B cells, T cells, and NK cells, are also highly comparable between the RenMab mouse and the wild-type mouse (Figure 2). Therefore, immune organs in the RenMab mouse develop normally.
Figure 2. Distribution of immune cells in the spleen and lymph nodes
3. The high diversity of antibody gene expression significantly boosts the abundance of antibody drug development
The RenMab mouse contains full-length sequences of genes that encode for human antibody variable H and κ light chains domains, and can, therefore, express more diverse antibody molecules than other commercially available humanized mouse models. The components of different antibody isotypes in the serum of the RenMab mouse are nearly the same as those of the wild-type mouse (Figure 3). The genes that encode for human antibody variable domains in the RenMab mouse can be widely used, with a diversity of VDJ recombination. Taken together, the RenMab mouse can produce even more therapeutic antibodies, which facilitates the development of antibody drugs.
Figure 3. Isotype determination of antibodies in serum
1) The genes that encode for human antibody variable domains are highly intact
At the DNA level, antibody genes of the RenMab mouse were comprehensively examined by PCR-sequencing techniques. Our data indicate that the RenMab mouse has intact sequences of human antibody genes.
2) Normal development of the immune system
An extensive examination revealed that there is no significant difference in immune organ development between the RenMab mouse and the wild-type mouse. The numbers, differentiation and maturation patterns of immune cells, including B cells, T cells, NK cells, and DC cells, are also highly comparable between the RenMab mouse and the wild-type mouse.
3) The diversity of antibody genes expression
Using Sanger and Next-Generation Sequencing to detect antibody gene expression levels, we found that the utilization of these genes for V/D/J region of the H chain and V/J region of the κ chain by B cells is highly diverse. The utilization of these genes for V/D/J region in the RenMab mouse is similar to that in humans.
4) The diversity of VDJ recombination
Sequencing data showed that the VDJ recombination in B cells is correct and diverse.
5) The diversity of heavy chain CDR3 length
Statistical analysis of the gene sequence for the CDR3 region of the heavy chain in the RenMab mouse showed that this region has a remarkable length polymorphism. The length of the CDR3 region spans between 6-27 amino acid residues, with the highest frequency between 13-18 amino acid residues.
6) The amino acid composition and utilization of the CDR3 heavy chain region of the RenMab mouse is highly similar to that of humans
7) High-frequency somatic hypermutation
DNA sequencing and alignment showed that RenMab mouse antibody genes produce high-frequency somatic hypermutations. The number and location of somatic hypermutations are normal.
8) Normal immune response to antigens
Compared with the non-immunized group, IgG and IgM levels in the serum of the RenMab mouse were significantly upregulated upon immunostimulation by a variety of antigens, indicating that the immune response level of the RenMab mouse is normal.
9) Normal immunoglobulin isotype switching
IgA, IgM, IgG1, IgG2, and IgG3 immunoglobulin isotypes were detected in the RenMab mouse by ELISA, and the components of these isotypes are consistent between the RenMab mouse and the wild-type mouse.
10) Superior antibody specificity and affinity
The fully-human antibody developed from the RenMab mouse has high antigen specificity, and affinity measurements have shown that this antibody can bind antigen at the nmol-pmol level. The RenMab mouse has great potential for developing therapeutic antibodies with high specificity and affinity.