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Kohler is equal to the monoclonal antibody technology of B lymphocyte hybridoma established in 1975, which has made a major breakthrough in the research and application of antibody technology. This technology has been widely used in the research, diagnosis and treatment of diseases. Cloned antibodies, as heterologous proteins, are restricted by the immune system and produce a strong immune response. In addition, hybridoma technology has many shortcomings, such as the production of monoclonal antibodies is too large, and the affinity with the target antigen is not enough. Different antigens need to be vaccinated with monoclonal antibody alone, and cell fusion technology is difficult and inefficient. The most important thing is that when monoclonal antibodies are used in the treatment of human diseases, the monoclonal antibody will be immune to it within two weeks. The antibody prepared by the traditional monoclonal antibody technology has been greatly restricted in application, and its role in disease prevention and control cannot be fully exerted. Therefore, genetic engineering technology and antibody molecular genetics emerged as the times require, and further development has been made. In the mid-1980s, new technologies emerged that combined the genetic structure and function of immunoglobulins with DNA recombination technology. The post-immunoglobulin molecule gene is introduced into the cell for expression. The antibody obtained by the technique removes or reduces the unrelated structure, retains (or increases) the specificity and biological activity of the natural antibody, and reduces or substantially eliminates the immunogenicity of the antibody. The antibody component of the antibody is reduced while retaining the specificity of the original antibody. The existing excellent mouse monoclonal antibody gene is modified, the obtained antibody has high humanization degree, the production process is simple, the price is easy to obtain, and the rare product is easy to obtain. Antibodies have broad clinical applications.
In 1984, Morrison et al. created a mouse/human chimeric antibody technology, which has led to the further development of genetically engineered antibody technology. In the third generation of antibodies, humanized antibodies, small molecule antibodies, antibody fusion proteins and some Some special types of antibodies have overcome the shortcomings of the previous two generations of antibody technology to a certain extent. In addition, the construction of phage antibody libraries, ribosome display libraries, and the like allows specific antibodies to be obtained without antigen immunization.
1. Humanized genetically engineered antibodies Early attempts were made to produce human monoclonal antibodies using human hybridoma cells, but due to the instability of human hybridoma cells, the low affinity of human monoclonal antibodies, and ethical controversies. This technique is rarely used.
1.1. A method for reducing the immunogenicity of a murine monoclonal antibody by a chimeric antibody is to link the variable region of the murine immunoglobulin to the constant region of the corresponding human immunoglobulin, thus producing a mouse/human chimerism. The antibody and the humanized region are between 60% and 70%. The recombinant region is ligated with the variable region gene of the murine monoclonal antibody and the human constant region gene is inserted into the appropriate expression plasmid, and the corresponding plasmid is transfected. Post-cell expression. The chimeric antibody produced has the function of binding antigen, and at the same time reduces the heterogeneity of murine monoclonal antibody. Such chimeric antibody is not much different in affinity from the corresponding murine monoclonal antibody, and human The immune response to it will be reduced. However, since it still retains the heterogeneity of the murine immunoglobulin variable region, clinical experiments have shown that the chimeric antibody also produces human anti-chimeric antibody (HACA) when applied. The immune response of the variable region.
1.2. Humanized antibodies In order to reduce the immunogenicity of chimeric antibodies and make chimeric antibodies more humanized, CDR grafting technology has been developed. This technology replaces other regions of immunoglobulin with human immunoglobulins, but only retains mice. The CDR portion of the immunoglobulin variable region, which minimizes the immunogenicity of the murine antibody. The antibodies produced are called humanized antibodies or modified antibodies. Such manipulations often require complex DNA manipulations, and some patients An immune response to a heterologous CDR is also generated. To reduce this reaction, only the most specific Amino Acid residue portion of the CDR can be grafted to the backbone portion of the human antibody. Although the affinity of the antibody is primarily dependent on the CDR, the antibody The other parts will also have an effect on affinity. In order to maintain the original affinity after immunoglobulin remodeling, it is also necessary to improve the immunoglobulin framework.
2. Small molecule antibodies have developed techniques for small molecule antibodies in order to break through the limitations imposed by the oversize of monoclonal antibodies. The goal of these techniques is to obtain antigen-binding fragments (Fabs) of antibodies and variable regions (Fv) of antibodies. The fragment can be obtained by cleavage of the antibody, or by amplifying the relevant gene of the immunoglobulin and cloning and expressing it in the bacteria. The small molecule antibody has small molecular weight, strong penetrability, low immunogenicity and short half-life. There are several types of research and more practical prospects.
2.1, single-chain antibody (scFv)
This technique links the heavy chain variable region to the light chain variable region with a linker peptide and expresses it into a single-stranded polypeptide using a prokaryotic expression system and folds into a novel antibody consisting of a heavy chain, light chain variable region. The antibody is only 1/6 the size of intact IgG, and the antigen binding site does not change, thus retaining the complete binding specificity. Based on this advantage, ScFv has better tissue penetration and can enter the general antibody. The site of arrival can be better applied in clinical application and disease treatment. In addition, single-chain antibodies possess polypeptide linkers, which can be designed as special functional sites, such as metal complexes, linked toxins or drugs, etc. For imaging and clinical treatment.
2.2. Multivalent antibodies The multiple antigen binding sites of the antibody have different specificities and can bind different antigen molecules, which changes the deficiency that traditional antibodies can only bind to a single antigen molecule. At present, researchers pay more attention to bispecific. A monoclonal antibody (BsAb) that uses genetic engineering and chemical coupling techniques to rearrange the coding sequences of two antibodies that recognize different antigens to produce a novel recombinant antibody that specifically binds to both antigens simultaneously. Bispecific monoclonal antibody. In this antibody, the two arms recognize two different antigens, respectively, thus constructing a bispecific that binds to both the tumor cell surface antigen and the killer T cell surface antigen. Monoclonal antibody, which brings two different cells close to each other, thereby contributing to the lethal effect of killer T cells on tumor cells.
2.3, Fab fragment In the antibody, the Fab segment mainly plays the role of binding antigen, and the genetic engineering method is used to link the Fd gene to the 5' end of the light chain gene through the interchain disulfide bond, and connect the bacterial signal sequence. The expressed protein is cleaved by the signal peptidase and undergoes stereo folding to become a heterodimer, which functions as a normal Fab. The antibody is only 1/3 of IgG and has no Fc segment, and is immunized. It has low originality, good penetrating power, and is often used as a carrier and development for guiding drugs.
3. Phage antibody technology
In the early 1990s, based on the PCR technology, the expression of antibody Fab fragments in E. coli and the rapid development of phage display technology, an antibody library technology based on molecular biological methods appeared. Among them, phage antibody library technology performance More superior.
The basic procedure of the phage antibody (PhAb) technology is to amplify a full set of antibody variable region genes or antibody fragments (Fab, Fv or scFv) genes by PCR and ligated into the phage capsid protein gene and into the vector, which can The fusion protein is formed with the coat protein of the filamentous phage, and the antibody Fab fragment or single-chain antibody is expressed on the surface of the phage, and then the specific antibody and the gene encoding the same are obtained from a plurality of antibodies by affinity screening of the antigen. Through cell hybridization, mice are no longer immunized to obtain antibodies, and specific human antibody genes can be obtained in a few weeks to produce humanized antibodies. Phages cloned and assembled using B lymphocyte complete antibody variable region genes The population is called a phage antibody library. Depending on the size of the antibody fragment, the antibody library can be divided into Fab library, single-chain antibody (scFv) library, single-domain antibody library, diabody library, dsFv library and minibody library, etc. scFv and single domain The antibody molecule is small, has strong penetrability, short half-life in vivo, and low immunogenicity. Both diabody and minibody are bivalent antibodies, and their ability to bind antigen is much higher than that of single-chain antibody. Immuno-imaging and therapeutic antibody formats.
The technology is simple to operate, does not require cell hybridization or complex PCR technology, has a short cycle and low cost; can simultaneously screen several antibodies using different antigens, and can be selected in millions to hundreds of millions of molecules; suitable for antibodies, hormones The preparation of proteins such as enzymes, drugs, and random peptides is even more interesting. This technology can obtain antibodies that cannot be produced by immune animals due to the influence of immune tolerance mechanisms. Phage antibody technology has greatly promoted the application of genetically engineered antibodies. Development, their emergence has brought new hope for the prevention, diagnosis and treatment of human and animal diseases.
4. Ribosome display technology and mRNA display technology The key step of ribosome display technology is to construct a ScFv antibody gene library by molecular biology technology, and then transcribe into mRNA; translate and express in E. coli and other in vitro expression systems, and finally cure. The antigen is subjected to affinity screening to obtain a high affinity ScFv from the translated mRNA-ScFv complex. The improved RD technique, because there is no stop codon on the mRNA, the protein and the mRNA encoding it are simultaneously bound during translation. On the ribosome, a protein-mRNA-ribosomal complex is formed, so that the phenotype and genotype of the protein are coupled in the form of this complex, and after screening, the screening protein can be enriched 100 to 1000 times. No cells are used, so the efficiency is significantly improved compared to other technologies, greatly shortening the experimental cycle, convenient and fast.
In 1997, Roberts et al. designed a method similar to RD, namely mRNA display technology (RNA display, also known as RNA-peptide fusion), which uses a puromycin molecule to share an mRNA molecule with its encoded polypeptide. The valence is first linked to the 3' end of the single-stranded DNA ligator and then to the mRNA encoded by the library. Thus, when the mRNA is translated in vitro, the ribosome reaches the binding site of mRNA and DNA and stabilizes. The puromycin enters the ribosome aminoacylation site, is coupled to the encoded polypeptide by the action of aminoacyltransferase, and is then screened with the immobilized antigen molecule for the RNA-polypeptide complex.
5. Transgenic animal technology In recent years, transgenic animal technology has developed rapidly and its application range has become wider and wider. For example, the IgH and Igκ sites of mouse embryonic stem cells are inactivated after deletion of JH and Cκ regions by homologous recombination technology. The purpose of the corresponding gene silencing of the host animal can be achieved. The deletion of the JH-/- and Cκ-/- genes of the hybrids of the two regions is inactivated, and then the human antibody gene is cloned into the yeast artificial chromosome. And integrated into the embryonic cells of the mouse, such cells are microinjected into the mother to obtain hybrid progeny containing human antibody IgH and Igκ site sequences. After immunization, the human antibody affinity produced by these hybrid mice It has high specificity, good use effect and safety, and excellent therapeutic effect on chronic diseases such as autoimmune diseases and cancer.
6. Transgenic plant technology Compared with mammalian cell lines or transgenic animals, plant antibodies (Plantibody) have the advantages of low cost, high efficiency, safety, etc. In 1995, Ma et al. prepared an antibody produced in tobacco plants. The secreted dimer IgA/G (SIgA/G) recognizes the cell surface protein of Streptococcus mutans. It has been proven that the antibody can be used to treat gum disease and prevent Streptococcus mutans from colonizing the oral cavity. In 2002, Bouquin et al. successfully prepared a humanized IgG1 antibody against RhD antigen in transgenic mustard plant cells, which has been shown to have broad diagnostic and therapeutic applications.
Antibody preparation technology has entered a new era in the development of genetic engineering antibody technology, which can be widely used in many fields of life science, for viral diseases, organ transplantation, tumors, autoimmune diseases, toxic diseases, metamorphosis It plays an important role in reactive diseases, etc. These techniques make the preparation of antibodies simple, convenient, stable and inexpensive, and provide a good basis for large-scale applications. In addition, the obtained antibodies can be widely used in protein purification engineering. With the continuous development of molecular biology and immunology technology, genetically engineered antibody technology will be increasingly used in disease prevention, diagnosis and treatment, which provides strong support for improving the quality of human life.
Research progress of genetically engineered recombinant antibody technology--《Chinese Journal of Microbiology and Immunology》2016年04期
In the long process of antibody research, the Chinese microbial strain query network has developed three generations of different levels of antibody preparation technology. Among them, the polyclonal antibody prepared by antigen-improving higher vertebrates is called the first generation antibody; it is produced by hybridoma technology. A monoclonal antibody directed against a particular antigenic determinant, referred to as a second-generation antibody; a recombinant DNA technique or a gene mutation method is used to engineer the coding sequence of an antibody gene to produce an original sequence in nature. The antibody protein molecule is called a genetically engineered antibody, that is, a third-generation antibody. This genetic engineering is called antibody engineering.