人源抗体基因,小鼠 z-bo 2020-07-22 750

　　At the end of the 19th century, the application of animal-derived serum to treat infections in animal experiments was the first attempt to develop antibody drugs. The successful establishment of hybridoma technology in 1975 greatly promoted the development of basic research on therapeutic antibodies. In 1986, the first mouse-derived antibody therapeutic was approved for clinical use, which accelerated the development of antibodies for biomedical companies as important therapeutics. Therapeutic antibodies have become the main drugs for the treatment of tumors and other human-related diseases. Through the continuous development and improvement of gene editing technology, the establishment of a mouse model technology platform expressing human antibody genes is not only a revolutionary innovation in the development of therapeutic antibody drugs, but also a revolutionary innovation in therapeutic antibody drugs. Development and promotion. It is widely used in clinical practice. In the past 25 years, antibody therapy has become an important clinical treatment for tumors and other human-related diseases. It is particularly noteworthy that about 18 new antibody drugs have been clinically approved in 2018-2019.

　　 1. What historical stage has the development of therapeutic antibodies experienced? What breakthroughs have you made in treating this disease? At the end of the 19th century, researchers first confirmed that animal anti-diphtheria toxin antiserum had antibacterial effects. This discovery opened up new ideas for antibacterial infections. In 1901, the German microbiologist Behring won the Nobel Prize in Physiology or Medicine for the first time. The breakthrough success of hybridoma technology in 1975 made it possible to use hybrid cells to produce unlimited monoclonal antibodies. The establishment of hybridoma cell technology has aroused great interest in the field of therapeutic antibody drug development. In 1986, the first mouse-derived anti-CD3 antibody (OrthocloneOKT3) approved by the FDA was used to prevent acute organ transplant rejection. However, due to the high toxicity and short half-life of mouse-derived antibodies, it is necessary to withdraw from the market in 2011. In 1994, the first chimeric antibody, anti-GPIIb/IIIa antigen-binding fragment (Fab) antibody was approved for the treatment of cardiovascular diseases related to platelet aggregation. Chimeric antibodies are developed by combining the variable regions of mouse antibodies with the constant regions of human antibodies. In 1997, anti-CD20 was the first chimeric antibody for tumor treatment and was approved for the treatment of non-Hodgkin's lymphoma. In 1997, the first humanized anti-IL-2 receptor antibody was approved. It is also used to prevent organ transplant rejection. The successful development of humanized antibodies will enable antibody drugs to treat certain diseases (such as tumors and autoimmune diseases) for a long time. Humanized anti-HER2 antibody (Herceptin) approved in 1998 is used to treat patients with human epidermal growth factor 2 (HER2) positive metastatic breast cancer and gastroesophageal junction adenocarcinoma. In 2002, the first fully human antibody was approved. Anti-tumor necrosis factor alpha (TNF-α) antibody is a fully human antibody constructed by phage display technology. It is mainly used to treat rheumatoid arthritis. At present, clinical applications are being extended to diseases such as ankylosing spondylitis, psoriasis, inflammatory bowel disease and ulcerative colitis. The anti-TNF-α antibody (Humira) developed by AbbVie is not only the best-selling high molecular weight drug in the antibody drug market, but also the best-selling drug among all drugs in the world in 2019. Have. In 2006, the first fully human anti-EGFR antibody successfully developed by the human antibody gene mouse (XenoMouse) platform was approved for the treatment of various tumors. In recent years, molecules related to immune checkpoints have attracted great attention and attention in the field of tumor immunotherapy research and development. In 2011, the human antibody gene mouse HuMabMouse platform successfully developed the first fully human antibody against the immune checkpoint CTLA-4 (Yervoy). In 2014, humanized anti-PD1 antibodies (Opdivo) and humanized anti-PD1 antibodies (Keytruda) for immune checkpoints were successively approved. Currently, the two human antibodies are melanoma, non-small cell lung cancer, head and neck cancer, Hodgkin's lymphoma and kidney cancer. As the ``star'' drugs of anti-tumor drugs, the two anti-PD1 antibodies ranked second and third in global antibody drug sales in 2018, and sixth and third in global sales rankings of all drugs in 2019. Be ranked. Therapeutic antibodies are the best-selling drugs in the pharmaceutical market. So far, biomedical companies around the world have conducted at least 570 clinical trials of therapeutic antibodies, of which the FDA has approved about 80 antibodies for clinical use, 30 of which are therapeutic agents for tumor antibodies. That’s because at present, the therapeutic antibody market is mainly on tumors (about 40%), autoimmune diseases (about 25%), genetic diseases (about 7%), infectious diseases (about 6%), and cardiovascular diseases (about 4%). Used for) and blood diseases (about 4%) and other related diseases. According to 2018 data, 8 of the 10 best-selling drugs in the world are antibody drugs. The global therapeutic antibody drug market is valued at approximately 115.2 billion US dollars. By the end of 2019, sales are expected to reach 150 billion U.S. dollars, and by 2025 it is expected to reach 300 billion U.S. dollars.

　　As far as the market share of antibody drugs is concerned, at present, Genentech (30.8%), Abbvie (20.0%), Johnson & Johnson (13.6%), Bristol-Myers Squibb (6.5%), MerckSharp & Dohme (5.6%), Novartis (5.5). %) and Amgen (4.9%) accounted for about 13%. The successful development of humanized antibodies has significantly improved the clinical resistance of antibodies and opened the door to a wide range of clinical applications of therapeutic antibodies. Currently approved therapeutic antibodies are classified into fully human antibodies, humanized antibodies, chimeric antibodies and mouse antibodies according to the degree of humanization of the antibodies, accounting for 51%, 34.7% and 12.5% and 2.8%, respectively.

　　 2. Why develop human antibodies? What are the research and development technologies and strategies for human antibodies? The clinical application of mouse-derived antibodies not only accelerates the elimination of mouse-derived antibodies, but also causes unpredictable allergic reactions, and may limit the targeting of mice (HAMA). Due to many unfavorable factors (such as reaction), antibody-mediated cytotoxicity (ADCC) caused by the Fc fragment reaction of the source antibody greatly hinders the wide clinical application of mouse antibodies. In order to reduce the immunogenicity of mouse-derived antibodies, research and development strategies and technologies for chimeric and human antibodies have been developed accordingly, and have become an important antibody development technology in the antibody drug market. By screening the first fully human antibody with high affinity, phage display technology was successfully established, which is a combinatorial library of recombinant antigen-binding fragments expressing human antibody genes from phage. Similarly, the use of human antibody genome to construct mouse models makes it the most attractive technology platform for human antibody development. In addition, the technology of combining B lymphocytes and human hybridoma cells to obtain human antibodies against specific diseases in recovered patients is a new technology with potential for human antibody development. The establishment of humanized antibodies began with the construction of chimeric antibody technology. That is, the variable region of the mouse antibody binds to the constant region of the human antibody. About 30% of chimeric antibody sequences are of mouse origin, and the remaining 70% are human antibody sequences. The chimeric antibody retains the specificity of the antibody binding antigen. Compared with chimeric antibody technology, complementarity determining region (CDR) transfer technology only retains the binding epitope sequence of mouse antibody, and the rest are human antibody components. Human antibody sequences account for 90% of antibodies. %. Therefore, the immunogenicity of CDR introduction technology is lower than that of chimeric antibodies, and was once considered the gold standard technology for the development of humanized antibodies. This technology not only reduces the incidence of human anti-chimeric antibodies and human anti-CDR antibodies from about 40% to about 9%, but it can also treat complex diseases that require long-term repeated treatment (such as tumors and autoimmune diseases) in the clinic. . We also provide treatment. basis. However, the biggest disadvantage of humanized antibody technology is the lack of universal methods. For example, the humanized CDR transmission process requires a high degree of personalization. In addition, due to the presence of 10% mouse antibody sequences, the clinical application of humanized antibodies still has the risk of immune rejection or hypersensitivity. Based on the successful development of humanized antibody technology, phage display technology has been used to develop fully human antibodies in the early 1990s. This technology is based on the construction of recombinant peptide and protein platforms and the realization of in vitro display technology. The phage coat protein is fused with the exogenous diversity combination antibody gene to construct the desired antibody combination expression library. These human antibody genes fused with phage coat protein can be displayed on the surface of phage using their specificity, and phage antibodies that specifically bind to antigen can be obtained by antigen binding screening method. The first batch of human antibodies developed using this technology platform are mainly antibody fragments (such as scFv and Fab). An important contribution of phage display technology in the development of complete human antibodies is that it is independent of the immune response in the body. In vitro antibody screening methods can be used to directly obtain binding to various antigens (autoantigens, toxins, unstable antigens, non-immunogenic antigens, etc.). (Etc.) and candidate source antibodies for affinity maturation transformation. In the early 1990s, researchers also successfully established another complete human antibody research and development technology platform, the human antibody gene mouse model. The technology is to introduce or replace the human antibody genome into the mouse antibody genome. After immunizing the immune system of mice with antigens, complete human antibodies can be synthesized and produced in mice. The successful development of the human antibody gene mouse model platform undoubtedly greatly promotes the development of the clinical application of human antibodies. Compared with the "first priority" and "slow" characteristics of phage display technology used to develop fully human antibodies. However, in the early stages of mouse immunization, screening for specific antibodies and preparing hybridoma cells, human antibody genes are very small. The mouse technology platform is relatively slow. However, once the first antibody is obtained, due to frequent genetic mutations, the subsequent antibody optimization process will naturally be completed in mice, which indicates that the technology platform has the ability to improve antibody affinity and efficacy. , Don’t worry about immune rejection. obvious advantage. The currently approved clinical application of human antibodies is also that antibody drugs developed through human antibody gene mouse technology are better in evaluating the relevant indicators of antibody drug preparation (antibody self-polymerization, specific binding, etc.).

　　3. What is the relationship between the basic structure of therapeutic antibodies and the treatment of clinical diseases? Among more than 80 commercially available therapeutic antibodies, IgG is the most common therapeutic antibody among the five antibody immunoglobulin (Ig) classes (ΔIgA, IgD, IgG, IgM, IgE). .. IgG structure is Y-type 150 kDa immunoglobulin, composed of two identical heavy and light chains connected by disulfide bonds. The two arms of the Y-shaped structure respectively constitute the two antigen binding domains (Fabs) of the antibody, including the variable regions (Fv) of the antibody heavy chain and light chain. The main chain region of the heavy chain of the antibody Y structure is called the fragment crystallization region (Fc). In this region, IgG antibodies are cell surface Fc receptors, Fcγ receptors (FcγR), complement proteins (C1q) and neonatal FcR (FcRn). IgG antibodies mainly exert their main therapeutic functions by interacting with corresponding "partners", such as antigens, complement, Fcγ receptors and FcRn. Among them, the selective and specific binding between the variable region of the antibody and the antigen is an important functional area for the antibody to exert its therapeutic function. The binding of Fc to its FcγR and complement proteins can lead to Fc-mediated antibody-dependent cytotoxicity (ADCC) and C1q-mediated complement-dependent cytotoxicity (CDC), leading to cell destruction. The combination of antibody Fc and FcRn may have the effect of increasing the circulating half-life of the antibody. Different antibody subtypes may interact with different Fcγ receptors, which may have a significant impact on the functional activity and pharmacokinetics of the antibody. For example, IgG1 is considered to be the most suitable therapeutic antibody subtype, accounting for 80.3% of current clinical antibodies, while other antibody subtypes are IgG4 (12.7%), IgG2 (5.6%) and it accounts for mixed IgG2/4 (1.4%) ). ). IgG3 subtype is the most important in the induction of ADCC and CDC, but there is no approved IgG3 subtype antibody. The short half-life of this subtype antibody in vivo and the long hinge structure of the antibody may increase the complexity of biological processes. The half-life of IgG1, IgG2 and IgG4 antibodies in serum is about 23 days, while the half-life of IgG3 is 2-6 days. In addition, the IgG2 antibody subtype cannot bind to Fcγ receptors, and IgG4 cannot activate the complement response. Among approved therapeutic antibodies, intact antibodies account for about 78%, Fc fusion proteins account for about 15%, and antibody fragments (Fab and scFv) account for about 7%. The development of therapeutic antibody fragments retains their specificity and selectivity, is advantageous in terms of development time and cost, and is also characterized by good invasion of tumor target cells and tissues. However, because this type of antibody lacks the Fc region, it will affect stability, shorten the effective circulation time in the body and affect the therapeutic effect. Compared with polymer drugs, polymer antibody drugs have stronger target specificity, and their toxicity is mainly target toxicity. The method of administration is usually intravenous or subcutaneous injection for 2 weeks or once a month. After the antibody drug is passed, it is absorbed by the lymphatic system, mainly distributed in the blood vessel and intestinal circulation system, and is metabolized by protease hydrolysis and recovered by the antibody FcRn receptor. Mammalian cell expression system is the most common system for the production of human antibodies. The advantage of this expression system is that it can be produced on a large scale, especially for human antibodies that require glycosylation, in order to perform post-translational modifications during the antibody production process. Approximately 63% of therapeutic antibodies approved for clinical use are produced by Chinese Hamster Ovary (CHO) cells. The rest are mouse myeloma cells NSO (18%) and Sp2/0 (11.1%), human embryonic kidney cell line (HEK) 293 (4.2%) and E. coli (4.2%).

　　IV. What are the strategies and methods for establishing human antibody gene mouse models? What are the current developments in the clinical application of this human antibody? The strategy of using the mouse model to develop human antibodies is to use the mouse immune system to rearrange the human antibody genes in the mouse and spontaneously generate somatic hypermutations, resulting in multiple combinations of different immunogens. produce. Then there are certain human antibodies. The establishment of human antibody gene mouse model provides a reliable technology platform for the development of therapeutic antibodies. The gene mouse technology platform has advantages over other human antibody development technologies. The production of human antibodies requires not only the humanization and diversity of antibody combinations, but also the natural optimization of in vivo affinity maturation of antibodies and the screening process of antibody clones. .. Of course, the human antibody Ig gene covers a very wide genomic region, which makes it extremely difficult to construct such a human antibody gene mouse. In 1985, scientists first proposed to introduce human antibody genes into mouse germ cells and establish transgenic mice that produce human antibodies. The proposal of this idea gave rise to new ideas for the production and development of human antibodies. In 1989, scientists first constructed a human antibody heavy chain gene vector containing human IgM antibody heavy chain variable region (including VDJ) and μ chain constant region genes. The plasmid DNA vector of about 25kb was microinjected into the fertilized mouse eggs, and about 4% of the mouse B cells expressed the human antibody μ chain, and the transgenic mice that produced human IgM antibodies were successfully obtained. In 1993, scientists could knock out the heavy (JH) and light (Jk) genes of mouse antibodies, and mate with transgenic mice expressing human IgH and IgL antibodies to produce a variety of combinations. We obtained a human antibody transgenic mouse model .

　　In 1994, we successfully developed the first human antibody gene mouse HuMabMouse technology platform. The mouse model is based on knocking out the mouse antibody heavy and light chain (IgH and IgK) genes, and is constructed to express human antibody heavy and light chain genes. The entire human antibody heavy chain genome is about 1.29 Mb and the light chain genome is about 1.39 Mb, but the first human antibody heavy chain genome introduced is only about 80 kb. Since the combination of antibody diversity is determined by germline V(D)J genes, the successful development of this technology platform will increase the ability of the human antibody genome and increase the diversity of human antibody gene combinations. These methods are reasonable strategies and the main technical problems to be solved. In 1993, scientists used yeast artificial chromosome (YAC) vectors to construct human antibody heavy chain (about 220 kb) and light chain (about 300 kb) vectors through yeast homologous recombination. I started using embryos. Successfully introduced stem cell (ES) cell fusion method and mouse ES cells. In 1997, large fragments of human antibody heavy chain (approximately 1 Mb) and light chain (approximately 700 kb) YAC were introduced into mouse ES cells and carried out with mouse gene antibodies (variable and constant regions) knockout mice. Reproduction. The XenoMouse mouse model expressing human antibody gene was successfully constructed. This gene mouse contains 66 human antibody heavy chain variable region (VDJ) genes and 32 light chain variable region (VJ) genes. Both XenoMouse and HuMabMouse mouse models completely eliminate the possibility of mouse antibody genes interfering with human antibody genes, and increase the diversity of human antibody gene combinations, but these two mouse antibody genes are knocked out, that is, mice are not only missing The variable region genes of the antibody and the knockout of the constant region genes reduce the effectiveness of human antibody production and reduce the transformation of mouse antibody classes and the effect of somatic cells. Also affect. Frequency of frequent mutations.

　　In 2014, scientists applied bacterial artificial chromosome (BAC) and Cre/loxP recombination technology to introduce the variable regions of human antibody heavy chain (VDJ) and light chain (Vk-Jk) into ES cell lines. Changes in vitro. These regions are inserted upstream of the mouse heavy chain constant region (Cμ) and light chain constant region (Ck), and do not affect the mouse anti-constant region. success. KyMouse mice cause high-frequency mutations in somatic cells after antigen stimulation, thereby producing high-affinity human antibodies. In addition, scientists constructed a large number of large fragments of the human antibody gene BAC, performed a series of microinjection methods, and introduced the corresponding BAC vector into mouse ES cells to produce human antibody heavy and light chains. Realize the gene targeting of the chain variable region. On the basis of retaining the constant region of the mouse antibody gene, we successfully constructed the Veloclmmune mouse model by replacing the corresponding mouse antibody heavy chain and light chain variable region genes. ..

　　 Currently, one is XenoMouse, developed by CellGenesys/Abgenix, 2. It is a biomedical company that uses the human antibody gene mouse technology platform to develop therapeutic antibodies, including HuMAb mice developed by Genpharm/Medarex. Later these two companies were acquired by Amgen in 2005 and Bristol-Myers Squibb in 2009; 3. Acquired KyMouse from Kymab; 4. Acquired VelociMouse from Regeneron; 5. Acquired H2L2 mice from Harbor Biomed; 6. Acquired from Trianni Trianni. Mouse; 7. Ablexis Aliva Mab mice. However, the currently approved human antibodies come from the three-gene mouse technology platform: XenoMouse, HuMAbMouse and VelociMouse. To date, the HuMabMouse mouse platform has developed and approved eight human antibodies, two of which are the anti-CTLA-4 antibody YervoyI and the anti-PD-1 antibody Opdivo in 2011 and 2014. Yes. It was approved and was the first drug to treat patients with melanoma. Anti-CTLA-4 antibody binds to the immune checkpoint inhibitor CTLA-4, inhibits the binding of CTLA-4 to B7 on the surface of APC cells, activates the activity of cytotoxic T lymphocytes, and activates tumor cells. Achieve the killing effect. Similarly, the combination of anti-PD-1 antibody and immune checkpoint inhibitor PD-1 prevents the immunosuppressive effect on tumor-specific T cells and achieves the purpose of tumor treatment. The two anti-IL-12 subunit p40 and IL-23 antibodies approved on the HuMabMouse mouse platform can block the inflammatory response signal, play a role in reducing the inflammatory response, and will be used for clinical treatment of autoimmune diseases. These two antibodies were approved for the treatment of severe psoriasis vulgaris and clonal ileitis in 2009 and 2016, respectively. The Mo XenoMouse mouse platform has developed 7 approved human antibodies. In 2006, the first fully human anti-EGFR antibody was used to treat patients with metastatic colon cancer that express EGFR (without KRAS mutation). Human antibodies can prevent EGFR from binding to its ligands, block EGFR signaling pathways and induce tumor cell apoptosis. In addition, there are two human-derived antibodies for the treatment of autoimmune skin-related diseases, one is human-derived anti-IL-17 antibody, which has the effect of reducing inflammation in patients with psoriasis. The other is a human anti-IL-17 receptor antibody that inhibits IL-17 family cytokines. They were approved by the FDA in 2015 and 2017 for the clinical treatment of psoriasis patients.

　　The second-generation human antibody gene Veloclmmune Mouse technology platform has four human antibodies, including anti-IL-4 receptor antibodies, inhibition of IL-4 and IL-13 signaling pathways, and anti-IL-6 receptor antibodies. Receptor antibodies were approved in 2017, which can inhibit the IL-6 signaling pathway, reduce the release of hepatocyte inflammation-related factors, and have a therapeutic effect on rheumatoid arthritis and other autoimmune diseases. New ones that have been obtained and are being treated Among those with coronary pneumonia, this type of antibody may have the effect of reducing the storm of inflammatory factors caused by the virus.

　　 5. What is the future development trend of human antibody research and development? In recent years, the field of therapeutic antibody research and development has developed rapidly and has become the backbone of the drug research and development market. However, it still has great potential for development and application in the field of therapeutic antibodies. Traditionally, antibody drugs are mainly used in the clinical treatment of tumors, autoimmune diseases and infectious diseases. Elucidating the molecular mechanism of specific proteins or molecules related to the pathogenic process of specific diseases in detail will help to develop more widely used, more effective and specific therapeutic antibodies.

　　 The future development trend of therapeutic antibody research and development can be divided into two types. The first category is the so-called naked antibodies, which are directly used to treat diseases. For example, the treatment of tumor antibodies is mediated by ADCC/CDC and other related pathways. Directly attack tumor cells, cause cell apoptosis, or attack the microenvironment where tumor cells grow, or attack immune checkpoint molecules. In this type of anti-tumor process, antibodies play a role in mobilizing natural killer cells and other immune cells to destroy tumor cells. The second type of antibody drugs achieve the purpose of increasing their value in disease treatment by further processing and modifying antibodies. Commonly used antibody modification methods and strategies include antibody-immunocytokine conjugates, antibody-chemical conjugates, antibody-radionuclide conjugates, bispecific antibodies, immunoliposomes and chimeric antigen receptors. included. Antibody immune cytokine binding, such as cell (CAR-T) therapy, aims to enhance the specificity of cytokine delivery by fusing antibodies with specific cytokines. Antibody-drug conjugates bind to small molecule drugs through antibodies that can specifically recognize tumor targets, thereby improving the specificity and efficacy of small molecule drugs, and reducing their toxic effects on non-target cells. Antibodies and radionuclei The combination of vitamins also enhances the specific tumor treatment effect of radiotherapy.

　　 Recently, the development of bispecific antibodies has brought new strategies and opportunities for antibody therapy, which are extremely attractive. Bispecific antibodies use protein engineering technology to connect two antigen binding domains (Fab/scFv, etc.) to each other, so that one antibody can recognize two different antigens at the same time. Therefore, with the help of gene editing technology, an antibody can play a new function in disease treatment, not just a mixture of two original antibodies. Most bispecific antibody design strategies are based on the combination of two cytotoxic effector cells against pathogens in the immune system. Currently, two bispecific antibodies are in clinical application. One is a targeting antibody against CD3 and CD19 for the treatment of B-cell acute lymphoblastic leukemia (ALL), and the other is a dual-specific IgG antibody for treatment of activated coagulation factors IX and X. At the same time, nearly 85 Two kinds of bispecific antibodies are in clinical trials, and about 86% are bispecific antibodies used to evaluate the efficacy of anti-tumor therapy. The preliminary research and development of therapeutic antibodies is to improve the degree of humanization of antibody variable regions and their affinity maturity, or to develop various therapeutic effects, such as antibody binding that is more suitable for clinical use. Focus on methods to improve function and drug properties, including antibody fragments (Fab and scFv). Then in this field, research on methods to improve the function of antibody Fc, such as ADCC, ADCP (antibody-dependent cells), has been conducted. Phagocytosis), CDC or antibody inactivates Fc. Antibody Fc engineering has become a very important tool to increase the specific activity of antibodies and extend their shelf life. This achieves the goal of reducing antibody drug use and potential side effects. In addition, Chimeric Antigen Receptor (CAR) T cell therapy is another technical application that combines antibodies and T cells. By targeting T cells to specific targets, the purpose of destroying tumor cells can be achieved. CAR-T cells are constructed by fusing antibody variable regions (such as scFv) with molecules related to T cell activation. In 2017, the FDA approved the first CAR-T cell therapy for the clinical treatment of acute lymphoblastic leukemia (ALL) and adult large B-cell lymphoma. Isolation and screening of human antibodies from a single B cell is also a new trend of research and development in this field, which may be a new research field in the treatment of infectious diseases. The advantage of developing human antibody technology through EBV transfection of single B cell immortalization process is the rapid isolation and cloning of a small number of human peripheral blood cells required for potentially effective human antibodies.. Faced with the risk of new pathogens, such as the recent emergence of new coronaviruses The rapid development of infection, immunotherapy or various combinatorial antibody libraries is more practical. Single B cell sorting technology is the best choice to achieve this R&D goal. However, the current application of single B cells has successfully developed antiviral human antibodies. For example, anti-dengue virus, anti-Dika virus, anti-Ebola virus, anti-HIV virus and anti-respiratory syncytial virus (RSV) human antibodies. Many of these human antibodies are currently undergoing clinical trials in various stages (Phase I/II/III). However, so far, FDA-approved human antibodies developed by a single B-cell technology have not yet entered clinical applications, and the technology still faces challenges that need to overcome related problems. I'm. Antigen labeling technology, antigen component selection, cloning antibody primer design, etc. Combine next-generation NGS sequencing technology, new diagnostic methods, pharmacokinetic applications, and clinical therapies. The use of single B cell technology to develop human antibodies is rare for discovery and discovery The characteristic therapeutic antibody is extremely powerful. It will also be an excellent tool to meet the design requirements and development goals of next-generation therapeutic antibodies. In recent years, the clinical application of a combination of multiple antibodies (antibody mixture therapy) in the treatment of diseases is also considered to be the development direction of antibody therapy for specific diseases. This so-called antibody cocktail therapy was originally based on a strategy of targeting different epitopes of the same target in a tumor or infection. Although this type of therapy may be beneficial in reducing the amount of antibody used, it can enhance the synergistic effect of multiple antibodies, thereby achieving the purpose of improving the efficacy and safety of the disease. .. Therefore, the development of antibody cocktail therapy not only makes full use of the specificity, controllable quality and low side effects of each antibody, but also benefits from multiple antibody binding sites, strong affinity and low avoidance potential. It has also been taken into account and is of human origin. A supplement beneficial to the development of antibody drugs. In addition, other non-traditional human antibody gene mouse technology platforms have successfully established people's attention. For example, it is beneficial for multispecific human antibodies. The R&D technology platform includes HCAb (HarbourBioMed) containing only heavy chains or human antibody gene mouse models (OmniFlic) containing only light chains, and the establishment of other similar genetic animal models, such as human antibody gene rat models (OmniRat? ), Human antibody gene chicken model (OmniChicken?), human antibody gene cattle model (TcBovine?) and other corresponding technology platforms.

　　6. How is the development of new human antibodies against coronavirus?

In order to cope with the challenges posed by the current global novel coronavirus pandemic, scientists around the world have also applied technology platforms such as single B cell isolation and screening and human antibody gene mice to create new ones. We are accelerating humans against coronavirus. Development of neutralizing antibodies. Successfully treated the new coronary heart disease. For example, the anti-coronavirus neutralizing antibody (LY-CoV555) developed by EliLilly is the first anti-coronavirus antibody drug in global clinical trials. The domestic anti-coronavirus antibody (JS016) developed by Junshi Biology has also been announced to enter clinical trials. The new neutralizing antibodies against the novel coronavirus developed by Eli Lilly and Junshi Bio are single B cells isolated from the peripheral blood of patients with novel coronavirus pneumonia. In addition, a research team led by the Institute of Microbiology of the Chinese Academy of Sciences recently proved that dozens of fully human antibody genes isolated and identified from patients with new coronary pneumonia were finally screened, two of which were ideally effective and neutralized two. An ideal. According to reports, specific antibodies have been obtained. New coronavirus activity. In a study on a new model of rhesus monkey coronavirus infection, two human antibodies effectively blocked the new coronavirus infection, significantly reduced the load of the new coronavirus in the airway of rhesus monkeys, and protected The lungs are protected from virus infection. It has been confirmed that it can be done. Human antibodies are also in clinical trials. Considering the potential escape of new coronavirus mutations and the clinical use of antibody drugs, many domestic and foreign research teams have positioned antibody cocktail therapy first in the development of new coronavirus neutralizing antibody drugs. I'm. egeneron has applied the VelocImmune human antibody gene mouse platform, and has developed two anti-coronavirus antibodies with high neutralizing activity and non-competitiveness, combined with the technology of isolating single B cells from the peripheral blood of recovered patients. Preclinical studies have shown that the combination of antibody cocktail therapy and two human antibodies can effectively neutralize the currently known mutants of the new coronavirus. The two anti-coronavirus antibodies bind non-competitively to the spike protein S receptor binding domain (RBD), reducing the chance of the mutant virus escaping the antibody treatment. Antibody cocktail therapy has recently entered phase III clinical trials.

　　 HarborBioMed is also one of the first companies to develop antibodies against new coronavirus infections. Use the H2L2 complete human antibody gene mouse technology platform to screen human antibodies that can effectively resist the new coronavirus infection. Because the antibody targets the conserved epitope shared by the new coronavirus and SARS virus, the antibody is expected to play a preventive and therapeutic effect on coronaviruses of the same subgenus. The ability of the antibody to neutralize the virus does not depend on inhibiting the binding of the new coronavirus RBD to the ACE2 receptor, so it forms a "cocktail therapy" that binds to the new coronavirus receptor. It is expected to combine with other antibodies to "produce a synergistic anti-coronavirus infection effect.

At present, there are more than 100 kinds of antibody drug therapies, of which the traditional form of antibody accounts for about 81%, and the other forms are various forms of antibody drugs (single domain antibodies, fusion antibodies, monoclonal antibodies, etc.) and related drugs, as well as from the initial infection Anti-inflammatory drugs to systemic organ failure. Target-related antibody drugs covering coronary pneumonia (including, for example, anti-cytokine storm antibody IL-6R, GM-CSF and C5, etc.). In the development of corresponding neutralizing antibodies, the new coronavirus spike S protein is the most concerned target: the current technical strategy for developing anti-COVID-19 therapeutic antibodies is based on single B cell isolation and screening technology and human-derived application technologies, such as antibodies Gene mouse model and phage display. Platform: At present, the research and development of most related antibody drugs is still in the preclinical stage, and almost 20% of antibody drugs have participated in clinical trials. Due to its high efficiency and specificity, it is still difficult to avoid some side effects in actual clinical applications, for example, the emergence of transient anti-drug antibodies (ADA), leading to severe paralysis. As well as the obstacles to drug removal, with the continuous development and improvement of new antibody development technologies and strategies, it will accelerate the development of human antibodies and expand the scope of antibody drugs to treat various human-related diseases. I think it will benefit more disease patients.