What is Fab fragment?
Fab fragments, also known as antigen-binding fragments, are regions in the structure of antibodies that can bind to antigens. Fab fragment consists of a complete VH and CH1 domains of light and heavy chains. Both light chain and heavy chain have a constant region and a variable region, and there are disulfide bond links between light chain and heavy chain.
Structure and Characteristics of Fab
Natural polyclonal antibodies and monoclonal antibodies are immunoglobulin molecules. IgG molecule consists of two identical 5x104 heavy chains and two identical 2.5x104 light chains. Fab molecule is composed of light chain and heavy chain, about 5x104. The interaction between the two chains and the disulfide bond formed play a stable role in the double-chain structure of Fab molecule. The antigen binding sites of Fab and IgG are composed of six complementarity determination regions (CDB), which are located on light chain and heavy chain respectively, and form the core of antigen binding sites. In the three-dimensional structure of the variable region, two opposite parallel beta-folded peptide chains form the framework region. The outer side is connected by highly variable CDR rings, forming a sandwich structure. The inner side is connected by conservative disulfide bonds, which makes the CDR prominent.
Development of Fab antibody
In 1988, researchers first used genetic engineering technology to design and modify the antibody gene structure at the gene level, expressed the antibody heavy chain variable region and light chain variable region with bacteria, and recombined them in vitro, and successfully obtained functional recombinant molecules. Since then, with the maturation of the technology of manipulating antibodies at the gene level, the rapid development of DNA recombination technology and the deeper understanding of antibodies, the third generation of antibodies——recombinant antibodies, after polyclonal antibodies and monoclonal antibodies, have emerged. Small molecule antibodies in recombinant antibodies have become the main members and research hotspots of the recombinant family because of their many characteristics. Fab antibody is an important discovery of its small molecule antibody.
Like monoclonal antibodies, the development of Fab antibodies has gone through four stages: murine, chimeric, humanized and human monoclonal antibody. Chimeric Fab fragments consist of murine variable region and human constant region. Humanized Fab fragments consist of murine CDR and human frame region.
Acquisition of Fab Antibody
Fab fragments were obtained mainly by chemical, enzymatic and genetic engineering methods. Chemical method is to use chemical reagents to act on the N-terminal of disulfide bond between heavy chains, open the disulfide bond, and obtain two identical antigen binding fragments. Enzymatic digestion of IgG full-length antibodies with papain, pepsin and fig protease is also a common method. Fab fragments of functional antibodies obtained by genetic engineering are mainly expressed in E. coli system, Pichia pastoris system and insect system. Escherichia coli system is the most commonly used one. However, the low expression of antibody Fab fragment in E. coli system is the main factor restricting its large-scale production.
Advantages of Fab Antibody
One of the main advantages of antibody Fab fragment is that it is suitable for expression in E. coli, while IgG full-length antibody is mainly expressed in mammalian cells because of its large molecule and glycosylation modification of CH2 region. E. coli expressing antibody Fab fragment is not only low cost and short cycle, but also conducive to DNA recombination technology to efficiently induce mutation. Through various optimization methods, it can improve the expression, solubility and stability of antibody Fab fragment in a short time, and has higher affinity and stability than the corresponding scFv. However, the low expression of antibody Fab fragment in E. coli is the main factor restricting its large-scale production and application. Through modification and optimization of host cells, expression vectors, expression conditions and purification conditions, it is possible to obtain high expression of genetic engineering antibodies.
Application of Fab Antibody
Compared with complete IgG, Fab fragment lacks Fc fragment, which can selectively bind to antigen but does not precipitate. Because of its low autoimmunity, it can not be recognized by living immune cells, so it can obviously reduce the probability of hypersensitivity reaction and improve the safety of products. Fab antibody fragments have the characteristics of small molecular weight, strong tissue distribution, low immune prototype, and can be operated by genetic engineering. Fab has become an important member of the medical research field. Fab drugs are widely used in prevention, diagnosis, treatment and other fields.
Diversification of therapeutic antibodies is the trend of development in the future. There are several molecular types, such as minimization of effective function antibody molecule, toxin-containing antibody molecule, antibody-drug conjugates, bispecific/trispecific molecule and newly discovered mini-antibody. Among them, the bispecific and trispecific molecules of Fab have just entered the early stage of clinical research. With the diversity of antibody fragments in the future, the efficacy and safety of small molecular antibodies may be better than that of full molecular monoclonal antibodies. There is reason to believe that in the near future there will be safer, more efficient, convenient and inexpensive Fab antibody drugs, which will play a more important role in the field of disease prevention, diagnosis and treatment.
