The therapeutic antibody has become a leading treatment option for cancers and other related diseases. In the past 25 years, antibody therapy has become an important treatment method for various diseases, such as cancer. Notably, from 2018 to 2019, there were about 18 new therapeutic antibody drugs approved for clinical use.
In the late 19th century, researchers first confirmed that animal anti-diphtheria toxin antiserum had an antimicrobial effect. This discovery provided a new idea for antimicrobial infection research. Due to this, in 1901, a German microbiologist called Behring became the first person who won the Nobel Prize in physiology and medicine. In 1975, the successful breakthrough of hybridoma technology made it possible to produce monoclonal antibodies infinitely by using hybridoma cells. The establishment of hybridoma cell technology has aroused great interest in the research and development of therapeutic antibody drugs.
In 1986, the first mouse anti-CD3 antibody (orthoclase OKT3) approved by the FDA was used to prevent acute organ transplantation rejection. However, the mouse antibody had to withdraw from the market in 2011 due to its high toxicity and short half-life.
In 1994, the first chimeric antibody, anti-GPIIb/IIIa antigen-binding fragment (Fab) antibody, was approved for the treatment of cardiovascular diseases that related to inhibition of platelet aggregation. The chimeric antibody was developed by combining the variable region of mouse antibody with the constant region of a human antibody. In 1997, anti-CD20, the first chimeric antibody for the treatment of non-Hodgkin's lymphoma was approved.
In 1997, the first humanized anti-IL-2 receptor antibody was approved, which was also used to prevent organ transplantation rejection. The successful development of humanized antibodies makes it possible for antibody drugs to treat tumor and autoimmunity disease. Herceptin, a humanized anti-HER2 antibody, was approved in 1998 for the treatment of breast cancer with HER2 positive metastasis and adenocarcinoma of the gastroesophageal junction.
In 2002, the first fully human antibody, anti-tumor necrosis factor α (TNF- α) antibody, was successfully approved. This human antibody was constructed by phage display technology and mainly used in the treatment of rheumatoid arthritis. Its clinical application has been extended to ankylosing spondylitis, psoriasis, inflammatory bowel disease (IBD), and ulcerative colitis. In 2006, the first fully human anti-EGFR antibody developed by Xenomouse mouse platform was approved to treat a variety of tumors.
In recent years, immune checkpoint-related molecules have attracted great attention in the research of tumor immunotherapy. In 2011, the first human antibody (yervoy) targeted to CTLA-4 was successfully developed by HuMabMouse platform. In 2014, the fully human anti-pd1 antibody (opdivo) and humanized anti-pd1 antibody (keytruda) for immune checkpoints were approved. At present, these two human antibodies have been used in the treatment of melanoma, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma, and kidney cancer. As the top drugs for the treatment of tumor antibodies, these two anti PD1 antibodies respectively ranked second and third in the global sales of antibody drugs in 2018, and ranked sixth and third in the global sales of all drugs in 2019.
Therapeutic antibodies have quickly become some of the best-selling drugs in the pharmaceutical market. So far, there are at least 570 therapeutic antibody clinical trials being carried out by global biomedical enterprises, of which about 80 antibodies have been approved for clinical application by the FDA, including 30 therapeutic antibodies for tumor treatment. At present, the therapeutic antibody market mainly focuses on the treatment of tumors (～40%), autoimmune diseases (～25%), genetic diseases (～7%), infectious diseases (～6%), cardiovascular diseases (～4%), and blood diseases (～4%). According to the data in 2018, eight of the top 10 best-selling drugs in the world were antibody drugs. The global market value of therapeutic antibody drugs is nearly $115.2 billion, and the sales volume is expected to reach US $300 billion by 2025.
The successful commercial development of human antibodies has greatly improved the clinical tolerance of antibodies and opened the door for a wide range of clinical applications of therapeutic antibodies. At present, the approved therapeutic antibodies are classified according to the degree of antibody humanization, such as a fully humanized antibody, humanized antibody, chimeric antibody, and mouse antibody, which accounts for 51%, 34.7%, 12.5%, and 2.8% respectively.
There is immense potential in the development and application of therapeutic antibodies. Traditionally, antibody drugs are mainly used in the clinical treatment of tumors, autoimmune diseases, and infectious diseases. If we can further elucidate the molecular mechanisms of some specific proteins or molecules involved in the pathogenesis of some special diseases, it will be helpful for broadening the development of more effective and specific therapeutic antibodies.
The trends of therapeutic antibody research can be divided into two types. The first type is the so-called naked antibody, which is directly used to treat diseases. For example, the therapeutic tumor antibody directly attacks tumor cells, induces cell apoptosis, or attacks tumor cell growth microenvironment, or attacks immune checkpoint molecules by mediating ADCC/CDC and other related pathways. In this kind of anti-tumor process, the antibody plays the role of killing tumor cells by recruiting natural killer (NK) cells or other immune cells.
The second type of antibody drug method is to further process and modify the antibody to increase its value in the treatment of diseases. Commonly used antibody modification methods and strategies include antibody immunocytokine binding, antibody chemical drug conjugates, antibody radionuclide conjugates, bispecific antibodies, immunoliposomes, and chimeric antigen receptor (CAR) T cell therapy. The purpose of antibody immunocytokine binding is to enhance the specificity of cytokine delivery through the fusion of antibodies and specific cytokine. Antibody-drug conjugates are the combination of antibodies that can specifically recognize tumor targets and small molecule drugs, which can increase the specificity and effectiveness of small molecule drugs and reduce their toxic effects on non-target cells. The combination of antibody and radionuclide also increases the specific therapeutic effect of radiotherapy.
Recently, the development of bispecific antibodies has provided a new strategy that has become an attractive opportunity for antibody therapy. The bispecific antibody strategy is to connect two antigen-binding domains (such as Fabs/scFvs) by protein engineering technology, so that antibodies can recognize two different antigens at the same time. Therefore, by means of gene-editing technology, the single antibody can play a new role in the treatment of diseases, instead of relying on a simple mixture of the original two antibodies. Most of the design strategies of bispecific antibodies are based on the combination of two cytotoxic effector cells in the immune system. At present, two bispecific antibodies have been used in clinical application, one is the anti-CD3 and anti-CD19 antibodies for the treatment of B-cell acute lymphoblastic leukemia (ALL), and the other is the bispecific IgG antibodies for activating hemagglutination factor IX and X for the treatment of type hemophilia. At the same time, nearly 85 bispecific antibodies entered clinical trials, and about 86% of them were bispecific antibodies to evaluate the effect of anti-tumor therapy.
Early research of therapeutic antibodies focuses on how to improve antibody binding, function, and drug characteristics, to find out which antibody is more suitable for clinical application; such approaches include how to improve humanization and affinity maturity of antibody variable region or develop antibody fragments (FAB and scFv) with different therapeutic effects. Subsequently, the research in this area began to transform to how to improve the Fc function of antibody, such as how to improve the antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or inactivate Fc function of the antibody. Antibody Fc engineering has become a very important tool to enhance the specific activity of antibodies and prolong their activity, which can reduce the use of antibody drugs and potential side effects.
In addition, the chimeric antigen receptor (CAR) T-cell therapy is another kind of antibody that is combined with T cells. By targeting T cells to specific cellular targets, we can destroy tumor cells. CAR T-cells are constructed by fusing antibody variable regions (such as scFv) with activation-related molecules of T cells. In 2017, FDA approved the first CAR T-cell therapy drug for clinical treatment of acute lymphoblastic leukemia (ALL) and adult large B-cell lymphoma.
The isolation and screening of human antibodies from single B cells is also a new trend in the field of antibody research and may become a new research approach for the treatment of infectious diseases. The advantage of developing a human antibody through the immortalization process of EBV-transfected single B cells is that only a small number of human peripheral blood cells are needed to quickly isolate and clone potential high-efficiency human antibodies. Faced with the risk of new pathogenic factors, such as the coronavirus infection, the rapid development of antibody library with immunotherapy or diversity has an increasing practical significance. The single B cell sorting technology is the most ideal choice to achieve the purpose of research. At present, antiviral human antibodies have been successfully developed by using the single B cell method. For example, many human antibodies are currently in clinical trials at different stages that may be used against dengue virus, Zika virus, Ebola virus, HIV, and respiratory syncytial virus (RSV).
Contrary to this, there is no FDA-approved human antibody developed by single B cell technology applied in a clinical application so far, and the technology still faces challenges in areas such as antigen labeling technology, sorting antigen configuration, and clone antibody primer design. Combined with the new generation of NGS technology, new diagnostics, pharmacokinetic applications, and clinical treatment progress, the development of human antibody by single B cell technology will also become a very powerful tool to discover therapeutic antibodies with rare characteristics to meet the needs of future biomedical research.
In recent years, the clinical application of multiple antibody combination strategies (a.k.a. antibody cocktail therapy) in the treatment of diseases is also considered to be a promising development direction of antibody therapy for some special diseases. This antibody cocktail therapy is mainly based on the strategy of targeting different epitopes of the same target in tumors or infectious diseases. This therapy can potentially reduce the use of antibodies, increase the synergy of multiple antibodies, and improve the effectiveness and safety of the treatment of diseases. Therefore, the development of antibody cocktail therapy not only has the advantages of specificity, controllable quality, and low side effects of each antibody but also accounts for the advantages of multiple antibody binding sites, strong affinity, and low escape possibility, which has become highly favorable features for the development of human antibody drugs.
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, by using TurboKnockout® RMCE and BAC technology.