The Important Role of Diabody

  • Abstract: The study of diabody began after the birth of monoclonal antibody technology in the 1980s. A bispecific antibody is a non-native antibody that has different specificities for the two arms that bind to the antigen. The construction of diabody is usually carried out using biological methods and cross-linking methods. With the development of antibody engineering and molecular biology techniques, a new class of methods for constructing diabody, genetic engineering, has been developed in recent years. The use of genetic engineering methods can not only construct diabody with multiple functions and multiple uses. As a new secondary guiding system, diabody have potential application value in clinical treatment. Early deficiencies in immunogenicity, structural stability, and antibody quality control have limited the further development of diabody. In recent years, improvements in upstream genetically engineered antibodies and downstream production techniques have overcome the shortcomings of traditional diabody, thereby facilitating the entry of multiple new diabody into clinical development.

    Key words: bispecific antibody, preparation and expression method, mechanism of action

    Development of diabody technology

    • Chemical method

    The chemical cross-linking method is simple and convenient, the construction period is short, the yield is high, the purification is simple, and the bispecific antibody lacking the Fc fragment is suitable for clinical treatment. However, the bispecific antibody constructed by this method can destroy the function of the antibody due to chemical cross-linking, and it is not easy to reach the target site, and the stability in the body is relatively poor. In addition, this method has no continuity and needs to be rebuilt each time.

    • Biological method

    The bispecific antibody constructed by the hybrid-hybridism method has a strict Ig molecular structure and similar various functions and the method can continuously provide a bispecific antibody over a period of time. However, this method also has obvious shortcomings: complex process, long cycle; low yield, complicated purification technology; hybrid-hybridism genome is too large and unstable; cannot be engineered for diabody, so it cannot be prepared for human body various variant antibodies.

    • Genetic engineering method

    The greatest advantage of constructing diabody by genetic engineering is that engineering can be engineered, such as constructing humanized diabody, small molecule diabody, immunoconjugates, and the like. In addition, with the establishment of antibody libraries, it is more and more convenient to construct diabody by this method. It also has the characteristics of short cycle, high yield and easy purification. The genetic engineering method is in a period of vigorous development, and there will be more and better ways to be created.

    Genetically engineered diabody expression system

    • Expressed in prokaryotic cells

    The E. coli transcription system is easy to manipulate, requires less foreign DNA, grows rapidly, and is simple to purify. It is a common system for gene engineering protein expression. However, this system cannot perform glycosylation processing on recombinant antibodies and cannot express active intact antibody molecules, so it is mostly used for the development of small molecule genetic engineering antibodies.

    • Expressed in mammalian cells

    The main advantage of mammalian cell expression is that mammalian cells can correctly and efficiently recognize the synthesis, processing and secretion signals of recombinant proteins, and can correctly assemble, fold and glycosylate antibody polypeptides into active antibody proteins. A commonly used stable cell line is a myeloma cell line or a non-lymphocyte cell line.

    • Express in plant cells

    The technology for producing transgenic plants has been well established to direct foreign proteins to selected organs or subcellular compartments. The production of antibodies by plants offers the possibility of large-scale production of antibodies for therapeutic, diagnostic and affinity binding.

    Mechanism of action of diabody

    • Mediated T cell killing

    An important mechanism of diabody is to mediate T cell killing. In recent years, with the deepening of the understanding of the immune escape mechanism of cancer cells and the rise of cancer immunotherapy, the research on antibody drugs that activate T cells has received much attention. It is generally believed that efficient activation of T cells requires a dual signal, the first signal being from MHC on antigen presenting cells. The antigen complex binds to the T cell receptor TCR-CD3, and the second signal is a non-antigen-specific costimulatory signal produced by the interaction of the T cell with the costimulatory molecule expressed by the antigen presenting cell. Since the expression of MHC on the surface of most cancer cells is down-regulated or even absent, immune killing is escaped. CD3X diabody can bind to T cell surface CD3 molecules and cancer cell surface antigens, respectively, thereby narrowing the distance between cytotoxic T cells (CTLs) and cancer cells, guiding T cells to directly kill cancer cells, and no longer rely on the dual activation signal of the cell. The unique T cell activation pattern of CD3X diabody is considered to be a major advantage in its mechanism of action.

    • Double target blocking

    Another important mechanism of action of diabody is the simultaneous binding of dual targets to block the dual signaling pathway. The mechanism is applied in a wide range of applications, including cancer, autoimmune diseases, inhibition of blood vessel growth and anti-infective treatment. For example, the Trans membrane tyrosine kinase receptor HER family, which plays an important regulatory role in cellular physiology, includes HER1 (erbB1, EGFR), HER2 and many other members, and the surface of many epithelial solid tumor cells is abnormally high. Expression is an important target for tumor targeted therapy. Studies have revealed that homologous or heterodimers between members of the HER family or between different members activate intracellular signals and promote cell proliferation and tumor development.

    Conclusion and outlook

    Diabody have irreplaceable advantages in dual blocking and inducing the mechanism of T cell tumor killing. With the development of upstream genetic engineering technology and downstream production technology, it is now possible to construct diabody that are identical or similar to conventional full-implant molecular structures, and achieve large-scale production, paving the way for the wider application of diabody in the future. Road; more and more diabody are entering clinical research. It is foreseeable that bispecific antibody drugs will become a new growth point for the development of antibody drugs in the future.

    References

    [1] Spasevska I. An outlook on bispecific antibodies: Methods of production and therapeutic benedicts. Biosocial Master Rev, 2014, 1-7.

    [2] Baeuerle PA, Reinhardt C. Bispecific T-cell engineering antibodies for cancer therapy. Cancer Res, 2009, 69(12): 4341-4944