Previous Chapter Review: "Research Status and Progress of Antibody Technology (Part 1)"
With the continuous development of modern biotechnology and considering the biological characteristics of antibody drugs, researchers have continuously optimized and innovated upon traditional antibody technology, aiming to obtain therapeutic antibodies with lower immunogenicity, better human compatibility, and lower costs through simpler and faster methods.
3. Transgenic Mouse Technology
Transgenic mouse technology involves the use of genetic engineering techniques to disrupt endogenous antibody genes in mice and then transfer human antibody genes into the mice. Afterward, the transgenic mice are immunized with the target antigen to express the corresponding antibodies in their bodies. This technology enables the antigen-antibody immune response to occur in the mouse body, ensuring the integrity of antibody class switching, the diversity of antibody clone selection, and the natural mechanism of antibody affinity maturation. Therefore, the antibodies obtained using this technology exhibit excellent affinity, stability, and solubility. Due to the complexity and high technical threshold of this technology, only a few companies have mastered it, including XenoMouse, UltiMab, VelociImmune, and Kymab. XenoMouse, UltiMab, VelociImmune, and Kymab mice were developed by researchers from Cell Genesys (USA), Medarex (USA), Regeneron (USA), and Kymab (UK), respectively. XenoMouse and UltiMab mice, as the first-generation transgenic mice, have similar technologies, both transferring the main genes of human IgH and Igκ while inactivating the mouse's own IgH and Igκ. On the other hand, VelociImmune mice and Kymab mice precisely replace the V region of mouse IgH and Igκ with the corresponding V region of human IgH and Igκ, while preserving all the C regions and other gene expression regulatory elements of the mouse itself.
In 2006, the first drug, Panitumumab, was approved for the treatment of RAS wild-type metastatic colorectal cancer. Also originating from the XenoMouse platform are two human antibodies for autoimmune skin diseases: Secukinumab, an IL-17α antibody, and Brodalumab, an IL-17R antibody, which were approved by the US FDA in 2015 and 2017 for the treatment of psoriasis, respectively. The two antibodies developed by HuMabMouse, Ipilimumab (a CTLA-4 antibody) and Nivolumab (a PD-1 antibody), are used for the treatment of melanoma and were approved in 2011 and 2014, respectively. Among them, Nivolumab was also approved for the treatment of non-small cell lung cancer in 2018. Additionally, Ustekinumab binds to the p40 subunit of cytokines such as IL-12 and IL-23, blocking inflammatory signals to reduce inflammation. This drug was approved for the treatment of severe plaque psoriasis in 2009 and Crohn's disease in 2016. Velocimune mice, the second-generation mouse with humanized constant regions, have contributed to the development of Dupilumab, an IL-4R antibody, and Sarilumab, an IL-6R antibody, for the treatment of eczema and rheumatoid arthritis, both of which were approved in 2017. In 2020, the US FDA approved the first Ebola antibody therapy, Inmazeb, which was developed using transgenic mouse technology. During the COVID-19 pandemic, Regeneron Pharmaceuticals in the US has also utilized this transgenic mouse technology to develop neutralizing antibodies against the novel coronavirus.
Currently, research teams in China are also actively establishing transgenic mouse-related research and development systems. Harbour Mice, cultivated by Harbour Biopharma, have two strains: H2L2 and HCAb. H2L2 mice can produce monoclonal antibodies with two fully human heavy and light chains, while HCAb mice can produce unique heavy chain antibodies with fully human variable region fragments. The Chongqing Institute of Animal Sciences has cultivated CAMouse, which includes two strains: CAMouseHG, a fully human antibody transgenic mouse, and CAMouseH, a fully human single-domain antibody transgenic mouse.
4. Single-cell sequencing technology
Single-cell sequencing technology, also known as single B lymphocyte antibody preparation technology, is a newly developed rapid method for preparing monoclonal antibodies. Based on the characteristics that each B cell contains only one functional heavy chain variable region DNA sequence and one light chain variable region DNA sequence, and each B cell produces only one specific antibody, this technology combines single-cell isolation and identification techniques with PCR technology to form an in vitro expression system for fully human monoclonal antibodies.
Antibodies produced by single-cell sequencing technology have advantages such as full human origin, high antigen specificity, high affinity, and rich genetic diversity, and are widely used in various fields such as tumor treatment, treatment of pathogenic microorganism infections, detection and treatment of autoimmune diseases, and research on the human immune system. Single-cell sequencing technology can achieve high-throughput screening and sequencing, and is increasingly being applied in the field of antibody drug screening. Currently, there are no approved drugs on the market, but several drugs developed using this technology are in clinical trials, such as sevirumab for the treatment of cytomegalovirus infection and pritumumab for cancer treatment.
Among them, aducanumab, a research drug developed by the American biotechnology company Biogen, has attracted much attention. This monoclonal antibody targets amyloid beta oligomers and is used for the treatment of Alzheimer's disease. In July 2020, the company submitted a biologics license application for aducanumab to the FDA, which was accepted in August 2020 and granted priority review. The drug is currently under review by European Union and Japanese regulatory agencies.
During the COVID-19 pandemic, single-cell sequencing technology has also become the mainstream technology for developing neutralizing antibodies against the novel coronavirus. The Eli Lilly company in the United States has collaborated with teams from AbCellera Biologics in Canada, the Korea National Institute of Health Research, and various Chinese teams from the Institute of Microbiology of the Chinese Academy of Sciences, Tsinghua University, and Peking University to conduct related research. Among them, the combined neutralizing antibody therapy consisting of LY-CoV555 (developed by the Eli Lilly team) and JS016 (developed by the Institute of Microbiology of the Chinese Academy of Sciences) was approved for emergency use authorization by the US FDA on February 9, 2021.
Analysis of Advantages and Disadvantages of Different Antibody Technologies
Hybridoma monoclonal antibody technology, antibody library display technology, transgenic mouse technology, and single-cell sequencing technology are four commonly used antibody technologies, each with its own advantages and disadvantages.
Hybridoma monoclonal antibody technology is mature, convenient, and cost-effective, but it is limited to the screening of mouse-derived antibodies. Mouse-derived antibodies can be recognized by the human immune system, causing severe adverse reactions. Therefore, antibodies screened using hybridoma technology often require further humanization modification. Compared to hybridoma monoclonal antibody technology, antibody library display technology eliminates the need for cell fusion, expands screening capacity, and can directly obtain humanized antibody genes. With the rapid development of antibody library display technology, various expression systems, including in vivo, in vitro, and eukaryotic systems, have been derived, providing more convenient platforms for screening and identifying specific antibodies. However, current antibody library display technology still cannot be widely used for the display of complete IgG antibodies, requiring fragmentation and reconstruction of the complete antibody molecule, which may lead to the selection of non-specific binding or low-affinity antibodies. Moreover, antibody libraries are difficult to store and transport, which is a major obstacle to the sharing of related research materials. Among the antibody drugs approved by the US FDA, only about 10% are screened using this technology.
Although the first fully human antibody originated from phage display technology, transgenic mouse technology is still the most widely used mainstream technology in the field of fully human antibody drugs. It has the advantages of high efficiency, speed, and good immunogenicity to human proteins. Antibodies produced using this technology generally have a higher success rate, and fully human monoclonal antibodies have been developed for the treatment of various diseases such as psoriasis, melanoma, and hypercholesterolemia. Single-cell sequencing technology has the advantages of being fast, high-throughput, and not limited by transformation efficiency. It can directly screen fully human, highly specific, and highly affine antibodies from B cells. With the application of some advanced instruments in single-cell sequencing platforms, this technology has become the mainstream technology for screening virus-like antigenic antibodies.
References
[1] Wu Ruijun, Sang Xiaodong, Li Zhifei, Ao Yi, Fan Ling. Current Status and Prospects of Antibody Technology Research and Development [J]. Chinese Journal of Pharmacology and Toxicology, 2021, 35(05): 374-381.
[2] Feng Jiannan, Qiao Chunxia. Progress in the Development of Human Therapeutic Antibody Technology [J]. Journal of Nanjing Medical University (Natural Science Edition), 2020, 40(11): 1571-1574.
About the Author
Xiaomichong, a researcher in pharmaceutical quality, has been dedicated to pharmaceutical quality research and verification of drug analysis methods for a long time. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection analysis and verification of analytical methods.
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