A Review of Research Progress in Diabody Drugs

  • Antibody drugs have flourished in the past 20 years. At present, there are varieties of antibody drugs marketed globally, and their therapeutic fields have gradually expanded from traditional cancers and autoimmune diseases to anti-infective and metabolic diseases. In 2013, there were 6 antibody drugs in the world's top 10 best-selling drugs, including 3 autoimmune therapeutic drugs and 3 anti-tumor antibodies. The development of monoclonal antibodies has also opened up the exploration of new structures and new functional antibody drugs, in order to further optimize the functional activity of antibody drugs. Antibody-drug coupling, diabody, etc. are all hotspots in the development of current antibody drugs. Monoclonal antibodies are capable of specifically binding to a particular epitope on a target antigen, with the advantage of high affinity and specificity.

     However, traditional antibodies only bind to a single epitope of a single target, so their efficacy is limited. Pharmacological studies have revealed that most complex diseases involve a variety of disease-related signaling pathways, such as tumor necrosis factor TNF, interleukin-6 and other pro-inflammatory cytokines simultaneously mediate immune inflammatory diseases, and the growth of tumor cells is often caused by caused by abnormal upregulation of multiple growth factor receptors. Blockade of a single signaling pathway is usually limited in efficacy and is prone to develop resistance. Therefore, the development of diabody and their analogs capable of simultaneously binding two targets has long been an important field in the development of new structural antibodies. Early deficiencies in immunogenicity, structural stability, and antibody quality control have limited 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 classes of novel diabody into clinical development. The diabody currently under investigation can be divided into dual signal blocking and anti-CD3+ T cell-mediated diabody: structurally, they can be divided into small antibodies consisting of single-chain antibodies or Fab regions; from the production process can be divided into prokaryotic antibody or Fab region composed of small antibodies and whole antibodies; from the production process can be divided into prokaryotic or eukaryotic expression, single cell expression and dual cell line expression combined with in vitro assembly . This article provides a brief review of the new developments in diabody.

    Method for constructing bifunctional antibody--genetic engineering method

    A diabody is a bispecific antibody that is a non-native antibody that binds to the two arms of the antigen with different specificities. A diabody is a bispecific antibody that is a non-native antibody that has different specificities for the two arms that bind to the antigen. The construction of diabody usually uses biological methods and chemical cross-linking methods. With the development of antibody engineering and molecular biology techniques, a new class of methods for constructing diabody--genetic engineering methods has been developed in recent years.

    The use of genetic engineering methods can not only construct diabody with multiple functions and multiple uses, but also make the construction of humanized diabody a reality. Diabody have potential applications as a new secondary targeting system in clinical treatment. In this paper, the development status of diabody construction technology was introduced from three aspects of biological method, chemical method and genetic engineering method, and the prospect was made. A method for preparing a diabody, characterized in that a small molecule drug is chemically coupled with a pathogen or a tumor-specific antigen to prepare a conjugate of a small molecule drug-specific protein, and after immunizing the animal, the animal spleen is taken The cells are fused with the corresponding myeloma cells to prepare a monoclonal antibody, and then the monoclonal antibody against the small molecule drug is screened by ELISA, and the positive hybridoma is also screened for the antigen corresponding to the specific carrier protein. Monoclonal antibodies. The bifunctional monoclonal antibody binds to the small molecule drug and the specific pathogen antigen through a non-covalent bond, and the binding is reversible, and plays a role in directing the small molecule drug to a specific position, so that the lesion is the drug concentration is higher than other parts of the body.

     

    1. Structure and production technology of diabody

    The study of diabody began with hybridoma fusion of monoclonal antibodies in the 1980s to construct tetraploid hybridomas. The two arms bind to the T cell surface CD3 molecule and the cancer cell surface EpCAM receptor, so this technology is also called "trifunctional antibody". Although catumaxomab was approved by the European Union in 2009 for the treatment of malignant ascites caused by EpCAM-positive tumors, the high immunogenicity of murine antibodies greatly limits its clinical application.

     

    1. 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 tumor 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 comes from the binding of the MHC-antigen complex on the antigen-presenting cell to the T cell receptor TCR-CD3, and the second signal is the co-stimulation of T cell and antigen-presenting cell expression. Non-antigen-specific costimulatory signals produced by molecular interactions. Since the expression of MHC on the surface of most cancer cells is down-regulated or even absent, immune killing is escaped. CD3 diabody can bind to T cell surface CD3 molecules and cancer cell surface antigens, respectively, thereby narrowing the distance between cytotoxic T cells and cancer cells, guiding T cells to directly kill cancer cells, and no longer rely on the dual activation of T cells. signal. The unique T cell activation pattern of CD3 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 wider range of applications, including cancer, autoimmune diseases, inhibition of blood vessel growth and anti-infective treatment. For example, the transmembrane tyrosine kinase receptor HER family, which plays an important regulatory role in cellular physiology, includes members such as HER1, HER2, HER3, and HER4, which are highly expressed on the surface of many epithelial-derived solid cancer cells. It is an important target for tumor targeted therapy. The antibodies that have been marketed include Herceptin monoclonal antibody that binds to the HER2 D4 domain, pertuzumab that binds to the HER2 D2 domain, and Erbituximab that binds to HER1/EGFR, and are widely used in breast cancer.

    Clinical treatment of solid cancer such as gastric cancer and colorectal cancer. Studies have revealed that homologous or heterodimers between members of the HER family or between different members activate intracellular signals and promote the development of cell proliferation tumors. Herceptin antibodies block homodimerization of the HER2 receptor. HER2 and HER3 are the strongest dimeric forms of the HER family that activate the initial oncogenic signaling, and will be clinically capable of blocking the use of this dimerized pertuzumab in combination with Herceptin, achieving better than single antibodies. Efficacy reveals the clinical effect of dual target blockade.

     

    Application of diabody drugs

    Diabetic retinopathy (DR) and diabetic macular edema (DME) are the leading causes of blindness in working populations worldwide, and the standard treatment for DME is anti-VEGF. However, DME is a multifactorial disease and cannot rely solely on one pathway of VEGF. Currently, the development of a diabody drug faricimab can treat DME.