Humanized Antibodies---a Field with Unlimited Potential

  • Abstract: From the advent of the monoclonal antibody to the present, it has undergone a tortuous development. Humanized antibodies are an important milestone and are accompanied by a series of major technological innovations such as PCR technology, antibody library technology, and genetically modified animals. The form of the humanized antibody has also evolved from the original chimeric antibody, modified antibody, etc. to today's human antibody. The humanization of antibodies has become the development trend of therapeutic antibodies, and various antibody derivatives are also emerging. They overcome the application limitations of the antibodies themselves from different angles, and provide more tools for treating human diseases. Modification of monoclonal antibodies for clinical treatment requires not only a more in-depth study of the mechanism of antibody action, but also a comprehensive and accurate understanding of the regulatory mechanisms of the human immune system. The greatest feature of an antibody drug is that it recognizes the specificity of the antigen. This article mainly introduces the development history and research progress of therapeutic antibodies, especially humanized antibodies, and summarizes the research status of antibody derivatives.

    Keywords: humanized antibody, phage antibody library

    What is a humanized antibody?

    Humanized antibodies are a transitional form from murine monoclonal antibody to fully human antibodies. On the basis of a mouse monoclonal antibody, the corresponding portion of the murine antibody was replaced with a human antibody constant region to form a human mouse chimeric antibody. About 70% of these antibody molecules are human, retaining the characteristics of the parental antibody in terms of antigen specificity and affinity, while the immunogenicity is reduced to about 12%, and the half-life and effector functions in the body are closer to humans. The murine component is further reduced on the basis of the chimeric antibody, and only the CDR regions of the murine antibody are retained, and the rest are replaced with the corresponding portions of the adult antibody, and the human antibody of the modified antibody is 90%, that is, the humanized antibody generally referred to. It is worth noting that CDR grafting often leads to a decrease in affinity and is not applicable to every murine monoclonal antibody.

    Preparation of fully human therapeutic antibodies

    Whole human therapeutic antibodies are the development trend of therapeutic antibodies. At present, the method for producing fully human antibodies has reached a relatively mature stage, mainly including transgenic mouse technology and antibody library technology.

    • Transgenic mouse technology

    In 1989, Bruggemann et al. successfully transferred the human antibody heavy chain gene library into mice, which produced an immune response to human heavy chain proteins. Subsequently, the technology of transgenic mice continued to develop and improve. In 1994, American transgenic mice were introduced as vectors for the production of fully human antibodies.

    In 1997, Abgenix successfully used the technology to prepare the transgenic mouse Xenomouse, a genetically engineered mouse that synthesizes antibodies with human protein sequences in vivo. The transgenic mice established by Medarex have developed antibodies for Phase III clinical trials. When the transgenic mouse technology is used to prepare a fully human antibody, since the antibody is produced in vivo and undergoes normal assembly and maturation processes, it has the advantages of high efficacy and strong target affinity. However, there are also some shortcomings. In addition to the complicated operation, high difficulty, high cost and incomplete human sequence, British scholar Clark believes that the antibody produced by the final step of antibody assembly and glycosylation in mice There may be a pattern of glycosylation. Although there is currently no clinical evidence that glycosylation causes any problems, it directly leads to the fact that these monoclonal antibodies are ultimately not fully human.

    • Phage antibody library technology

    In the 1990s, combinatorial chemistry technology and genetic engineering antibody technology combined to produce antibody library technology. The development of antibody library technology has led to changes in traditional antibody preparation methods, opening up a new world of antibody preparation in vitro, which is currently considered to be the fastest, simple and economical method for preparing human antibodies.

    • The biggest feature of the antibody library

    In addition to high-throughput screening and screening of antibodies  that are human-derived, and the library capacity is greatly increased, it is theoretically possible to screen specific antibodies against any antigen from these large-capacity human antibody libraries, and to solve some The problem of difficult preparation of antibodies against weak immunogens and toxic antigens.

    • Classification of phage antibody libraries
    • Natural antibody library
    • immune antibody library
    • semi-synthetic antibody library
    • synthetic antibody library
    • Antibody library’s powerful screening capabilities

    Specific antibodies against an antigen can be screened directly from the library using a phage antibody library. The choice of the best screening method for different antigens is also different. According to the different characteristics of each screening method, two kinds of alternate screening can be selected to improve the screening positive rate. Throughout recent studies, most of the human antibody libraries constructed have relatively simple source of lymphocyte RNA, usually from healthy volunteers or antibody libraries for certain diseases, so that the diversity of antibody libraries is limited.

    • Other antibody library displays

    In recent years, many other display platform systems have also been used to display antibody libraries, including ribosome display, yeast display, bacterial display, baculovirus display, mammalian cell display, retroviral display, and bacterial periplasmic display. There are also minimal unit display systems such as mRNA displays, DNA display systems, and more.

    • The ribosome display technology frees the display technology from the limitations of the display technology in vivo, and the library capacity is greatly improved, which lays a foundation for high-throughput screening in vitro;
    • The mRNA display technology directly links the mRNA to the expressed protein, so that the screening efficiency is further improved;
    • The DNA display technology DNA template replaces the unstable mRNA to make the display system more stable, and the method is simple and ingenious.

     

    Three techniques have been widely used in antibody screening, affinity maturation, and molecular evolution. Some of the above-mentioned in vitro display technologies are not dependent on any cell system, and are not affected by cloning and cell transformation efficiency, and have a large library capacity, which can make up for the deficiency of traditional in vivo display technology.

    References 

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    [2] Nelson AL, Dhimolea E, Richert JM. Development trends for human monoclonal antibody therapeutic [J]. Drug Discovery, 2010, 9:767-774