With the development of research on the impact of immunity and inflammation on cancer, the performance of humanized mice has attracted tremendous attention in clinical oncology research. Generally, humanized mice have been divided into two categories, namely, immunocompromised and immunocompetent mouse models. Immunocompromised mouse models include all types of nude mice or severe combined immunodeficiency (SCID) mice. Immunodeficient mice are essential for establishing mouse models with humanized immune systems. Humanized CD34+ mice have the functional human immune system (HIS) and display T-cell dependent inflammatory responses, with no donor cell immune reactivity toward the host, and yield robust multilineage immune systems. In addition to Humanized CD34+ mice, all immunodeficient mice were engrafted with human hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs), and exogenous administrations of human hormones, growth factors, and cytokines were needed in all cases. Genetically humanized mice have human antibody genes that lead to the production of various human immunoglobulins, which are known as humanized mouse antibodies. Herein, we look at humanized mice for antibody research.
In 1983, the first engrafted humanized mice were developed that have peripheral blood lymphocytes (PBL) of humans, and the mouse model was named huPBL model. The huPBL mouse was created by intraperitoneal injection of human peripheral blood lymphocytes into irradiated immunodeficient mouse.
|Humanized mice||Genetic Background||Reference Publication|
|SCID||CB-17||(Mosier et al., 1988)|
|NOD-SCID||NOD||(Shultz et al., 1995)|
|BRG||Balb/c||(Mazurier et al., 1999)|
|NOG||NOD||(Ito et al., 2002)|
|NSG||NOD||(Ishikawa et al., 2005)|
|NRG||NOD||(Pearson et al., 2008)|
|BRGS||Balb/c||(Legrand et al., 2011)|
|SRG||Balb/c X 129||(De La Rochere et al., 2018)|
|B6RGS||C57BL/6||(Shultz et al., 2019)|
|B6RG-CD47||C57BL/6||(Martinov et al., 2021)|
Table 1: Key Humanized Mouse Models and Their Genetic Backgrounds, with Reference Publications
The Major Breakthroughs of Human Antibody Development Using Humanized Mice
1. Disadvantages in clinical application of murine antibody
Monoclonal antibodies of mouse origin are very specific to a single antigen and may not be effective against a pathogen due to mutation(s) in that specific antigen. They are not involved in hemagglutination (no cross-linkage with antigen) due to slight alterations at the antibody binding site.
2. Chimeric antibody technology
The chimeric antibodies were generated as a result of genetic engineering, in which the human-derived constant region of immunoglobulin joined with the variable region of antibodies of mouse hybridoma origin. These chimeric antibodies are the first step towards more humanization. Chimeric antibodies are used to treat patients with worsened multiple myeloma and hematological malignancies. Producing these chimeric antibodies are cheaper than developing a fully humanized mouse and more useful for initial biotherapeutic research. Notably, species-based chimeric antibodies minimize the risk of anti-species reactions in animal models.
3. Phage display technology
A bacteriophage is used to study the DNA-protein, protein-peptide, and protein-protein interactions, and a connection is generated between the encoding genome and protein. In phage display technology, epitope mapping is performed, and ligands separated from these phage libraries are useful for drug design, therapeutic validation, and vaccine development. Phage display-derived data has been used to inform drug development strategies for humanized antibodies in therapeutic clinical applications.
The Current Situation Of Humanized Mice for Antibody Research
1. Challenges of Engraftment-Based Humanization
Following the engraftment with HSC or PBL, mature B-lymphocytes may be impaired, production of immunoglobulins lowered, and MHC class I and class II alleles restrict T-cell generation. Engraftment of mice with mature T-lymphocytes leads to Graft Versus Host Disease (GVHD) and there is human host-specific pathogen involvement. One of the primary limiting factors in the PBMC model is the development of a lethal human anti-mouse graft versus host disease (GvHD), NSG mice engrafted with human PBMC typically develop overt signs of acute GvHD beginning at 3-4 weeks post injection. At present, the development of a humanized mouse model that mitigates GvHD remains the focus of researchers.
2. Advantages of Genetic Humanization
Genetically humanized mouse models are effective in evaluating the immunotherapeutic efficacy, pharmacodynamic experiments, clinical response estimation, and immunological research specifically concerning tumors. Tumor-specific antibodies have a great advantage over chemotherapeutic agents due to their high specificity to the target cells combined with reduced complications from off-target toxicity. The affinity and efficacy of humanized mice antibodies are comparatively higher than antibodies obtained via in-vitro recombinant technology.
3. Achievements in Using Humanized Mouse Models for Antibody Production
Wege et al. found a new way to generate the mouse model that can make human B cell-derived antibodies specifically target novel antigens presented on human cancer cells. In this study, human hematopoietic stem cells and human breast cancer cells were co-injected into immunodeficient NSG mice. The transplanted mice develop the human immune system response, including antibodies in the serum which are specific to the transplanted tumor cells.
One such novel mouse, called RenMab, has receptor-binding domain (RBD)-blocking antibodies with four different epitopes. With the help of humanized mice, the rational antibodies cocktail was produced against COVID-19 that has proven immunogenic and demonstrates hACE2 blocking activity.
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
Cyagen is able to generate large fragment knock-in (LFKI) humanized mouse models that express human antibody genes up to 300 kb, by using our proprietary TurboKnockout® service alongside RMCE and BAC technology.