Since the first therapeutic antibody was introduced into clinical practice in 1986, therapeutic antibody has developed rapidly and has become an important part of modern biomedicine. With the development of modern science and technology, therapeutic antibodies have undergone different stages of development, such as mouse-derived antibodies, chimeric antibodies, modified antibodies and surface remodeling antibodies (some humanized antibodies), as well as whole-humanized antibodies. Whole humanized antibody is composed of all human gene-encoded proteins, which has little immunogenicity and side effects and good clinical efficacy. It is the main development direction of antibody engineering at present and in the future.

Mechanisms of antibody therapy


Figure1. Treatment of HIV with therapeutic antibodies

The basic unit of an antibody is a symmetrical structure consisting of four peptide chains, including two identical heavy chains and two identical light chains. The heavy and light chains are composed of a variable region and a constant region respectively. The complementary determinant region (CDR) in the variable region is directly related to the diversity of antibody and antigen binding, while the structure of the constant region is related to the biological activity of the antibody. In a few cases, the binding of antibodies to antigens can directly protect the body, such as neutralizing the toxicity of toxins with antibodies, but in most cases, foreign antigens need to be inactivated or cleared through effectual functions. There are two kinds of effecting functions of antibodies. One is to stimulate inflammatory response by activating complements, which can produce many biological effects, such as cell lysis, immune adhesion and regulation. The other is through the interaction between Fc segment of antibody molecule and Fc receptor on cell surface, mediating regulatory or antibody-dependent cytotoxicity through Fc segment of antibody molecule respectively. In addition, the efficacy and mechanism of therapeutic antibodies depend directly on the antigenic determinants they recognize.

Preparation of therapeutic antibody

  1. Monoclonal antibodies

Homogeneous antibodies produced by cell clones that recognize an antigenic determinant are called monoclonal antibodies and can be regarded as second generation antibodies. Because of its high specificity, affinity, high titer and low cross-reaction in serum, it has played an important role in basic research, clinical diagnosis and treatment, immune prevention and other fields. In the treatment, monoclonal antibodies are mainly used for anti-tumor, anti-organ transplantation rejection, anti-infection, detoxification and so on. In recent years, monoclonal antibodies and nuclides, toxins or drugs have been chemically coupled or recombined into targeted drugs for cancer treatment, which has become the focus of research.

The conventional method of preparing monoclonal antibodies is to immunize mice. Hybridoma can produce unlimited amounts of monoclonal antibodies in experimental animals. For most hybridoma, monoclonal antibodies can be produced in vitro without using animals. There are many kinds of monoclonal antibody production systems in vitro, but hollow fiber system is needed for large-scale production of therapeutic monoclonal antibodies. Its success depends on the inherent characteristics of hybridoma, such as cell growth and monoclonal antibody production capacity. Therefore, it is still difficult to produce a large number of monoclonal antibodies for clinical research, but there are several methods to solve these problems, such as the production of chimeric monoclonal antibodies, humanized monoclonal antibodies and human monoclonal antibodies. Among them, humanized antibodies are an important milestone, accompanied by a series of major technological innovations, such as PCR technology, antibody library technology, genetically modified animals and so on. The form of humanized antibodies has gradually developed from chimeric and modified antibodies to today's human antibodies. Humanization of antibodies has become the development trend of therapeutic antibodies. At the same time, a variety of antibody derivatives are emerging. They have overcome the limitations for the application of antibodies themselves from different angles, and also provide more powerful tools for the treatment of human diseases.

  1. Humanization of mouse antibodies

There are also problems with monoclonal antibody drugs, mainly mouse-derived antibodies used in human body to produce human anti-mouse antibodies (HAMA). Mouse monoclonal antibody is a heterologous protein, which is quickly removed in vivo, and the antibody molecule is large, the amount of antibody reaching the target is insufficient, and the effect of antibody itself is not strong, so the effect of antibody therapy is not ideal. Humanized antibody is a transitional form from the murine monoclonal antibody to the human antibody. Based on the murine monoclonal antibody, the corresponding parts of murine antibody are replaced by constant region of human antibody to form human-mouse chimeric antibody. The light and heavy chain variable region genes of mouse monoclonal antibody are inserted into the expression vector containing the constant region of human antibody by DNA recombination technology, and the human-mouse chimeric antibody was expressed in mammalian cells. The degree of humanization is about 70%. The chimeric antibody retains the mutable region of the heterologous monoclonal antibody, maintains its affinity to the maximum extent and reduces its immunogenicity. However, because the whole variable region of HAMA is heterologous, the heterology of chimeric antibody is obvious, and the effect of solving HAMA is not ideal. On the basis of chimeric antibody, the human component of the modified antibody is reduced further, only the CDR region of the mouse antibody is retained, and the rest is replaced by the corresponding part of the adult antibody. The human component of the modified antibody is up to 90%, which is commonly referred to as humanized antibody.

  1. Human Antibody

Although humanized antibodies solve the most important problem, namely the immunogenicity of mouse antibodies, it also has difficulties that are difficult to overcome. For example, the humanization process is complex and expensive, it requires extensive computer simulation, and it needs to replace different amino acids to restore selectivity and affinity. Therefore, the workload is very large, and it still contains a small amount of mouse-derived ingredients. At present, the complete humanized antibody is the most prevalent, namely human antibody, which is mainly developed through three ways: screening human antibody libraries and preparing whole-body antibodies from genetically engineered mice. Antibody library screening technologies include phage antibody libraries, synthetic antibody libraries and ribosome display technology. Engineering mouse technology includes transgenic mice and transgenic mice to prepare human antibodies.

Application of antibody therapy

With the progress of antibody preparation technology, therapeutic antibodies have gradually turned from the laboratory to the clinical, showing glorious prospects.

  1. Antineoplastic effect

Due to the limitations of polyclonal antibodies themselves, antibodies cannot be used for anti-cancer therapy until the appearance of monoclonal antibodies. The mechanism of monoclonal antibodies killing tumor cells may be antibody dependent cell mediated cell effects and complement dependent cell lysis. Monoclonal antibodies, coupled with drugs, toxins or radioactive substances, have become a new kind of "biological missile" and can be used for targeted therapy, which has attracted more and more attention.

  1. Anti-infective effect

The use of vaccines and immunoglobulins can prevent and treat infectious diseases, but it is ineffective for viral infections, AIDS and some critical situations, such as endotoxin shock, which cannot obtain corresponding vaccines. The main mechanism of passive antibody therapy is to neutralize virus. It has been proved that the effect of antibody neutralizing viruses in vitro and in vivo does not depend on Fc-mediated function and the existence of bivalent antibodies, but may be affected by antibody affinity.

  1. Anti-organ transplant rejection antibody

Immunosuppressive effects are produced by blocking the recognition of alloantigens, the pathway of rejection and a link of molecular mechanism to prevent rejection.

  1. Detoxification

For patients with drug or animal toxin poisoning, immunotherapy uses specific antibodies or fragments against the drug or toxin. Antibody and toxin form a complex that alters the pharmacokinetics of toxin, dissociates toxin and receptor, and flows out gradient from its binding site into the blood circulation.

  1. Construction of idiotypic vaccine

Anti-idiotypic antibodies can mimic antigens (internal imaging) in structure and function, and the independent vaccines based on these can mimic tumor-associated protein antigens and induce anti-tumor immune response.

  1. Application in autoimmune diseases and allergic diseases

Autoimmune diseases are mostly related to the abnormal increase of monoclonal or oligoclonal antibodies. Gene engineering techniques can be used to prepare anti-antibodies against the idiotypes of these abnormal antibodies or to bind to and inhibit their effects with autoantibodies, or to prepare internal image antibodies that mimic antigens for neutralizing autoantibodies in vivo. At present, according to different pathogenesis, treatment methods tend to be diversified.