Abstract
In October 2018, half of the Nobel Prize in Chemistry was awarded to American scientist George P. Smith and British scientist Gregory P. Winter. The two winners were the phage display technology for peptides and antibodies. Among them, George P. Smith is the creator of phage display technology, which creatively pioneered the platform of this technology, and Gregory P. Winter used this technology to develop the first fully human antibody drug for rheumatoid treatment. Arthritis, psoriasis, etc., is the famous drug king adalimumab. In 2017, its global sales reached 18.5 billion US dollars, with accumulated sales of 115.9 billion US dollars. Phage display technology has a wide range of applications in biomedical research and development, and is the source technology for the development of new biopharmaceuticals.
Introduction
Phage display technology is called the power to control evolution. The most famous thing about biological evolution and evolution theory is Darwin's theory of evolution. "The natural selection of the game, the survival of the fittest", Darwin believes that there is a struggle between the creatures, the adaptors survive, and the discomfort is eliminated, this is the natural choice. It is through biology, variation and natural selection that biology evolves and develops from low to high, from simple to complex. The inheritance and variation of organisms have created the diversity of the population. Some key harsh conditions of the natural environment are used as screening pressures to make the diversity of the population partially lost. At the same time, the population can survive the adaptation to the screening pressure. The interaction between biology and the environment is a complex interaction between multiple trait diversity and multiple conditional screening pressures in complex systems. In contrast, phage display technology is a single trait diversity and selection of replicated organisms. A single screening pressure interacts to purposefully obtain a technique that is responsive to the trait of the screening pressure. Specific to phage antibody display technology, usually by genetic engineering of Escherichia coli filamentous bacteriophage M13 to replicate the genetic diversity of the antibody of the organism, and fusion of the gene-encoded antibody and phage membrane protein on the surface of the phage, select whether The ability to bind to a specific target protein as a screening pressure, a technique for obtaining antibodies and genetic information capable of binding to the specific target protein. The breakthrough contribution of phage antibody display technology lies in establishing a direct link between the in vitro antibody function and its corresponding genetic information, and screening the phage of the functional antibody to obtain the genetic information corresponding to the encoded antibody in the phage.
Diversity of protein and genetic information of antibodies
The antibody protein has a monoclonal antibody in a single Y-shaped monomer or a plurality of Y-shaped monomers, and each Y-shaped monomer consists of four polypeptide chains, comprising two identical heavy chains and two identical light chains. The top of the Y-shaped structure is the variable region, which is the antigen binding site, and the rest is a more conserved constant region. The single antibody variable region is called scFv (single-chain variable fragment). The heavy chain variable region and the light chain variable region are folded into a globular structure by covalent bond and non-covalent bond, and the light and heavy chain variable region is composed of 4 The frame region (FWR) with relatively constant frequency of change and the inclusion of three hypervariable regions (CDRs), the hypervariable region is also called the complementarity determining region, and the six complementary determining regions of the light and heavy chain are close to each other in the three-dimensional structure. Forming a protein cyclic structure is a key region that binds to the antigen.
The light and heavy chain variable regions are encoded by the light chain V gene and the heavy chain V gene, respectively, wherein the light chain V gene has two types, a kappa light chain and a lamda light chain, assembled by a VL fragment and a JL fragment or a VΚ fragment and a JΚ fragment. The heavy chain V gene is assembled from VH fragments, DH and JH fragments. According to the following figures, more than 30 light chain heavy chain V fragments, 23 heavy chain D fragments, and 4-6 light and heavy chain J fragments in the human body are not completely counted, and their arrangement constitutes the diversity of antibody genetic information. Generally, the CDR3 region of the heavy chain of the antibody protein is a key region for antigen binding, and its main body is determined by the DH fragment, but during the assembly of the heavy chain V gene V, D, J, the VH fragment and the JH fragment are also inserted in a small number. The nucleotides of the nucleus enter the CDR3 region, further increasing the diversity of the CDR3. In theory, the diversity of the human antibody genetic information can be as high as 5E13, and the 10 trillion-level antibody library accumulated by humans for a long time. It can be said that it can react with any known or unknown macromolecular small molecule on the earth, which shows the great power of life evolution, and the phage display technology will harness the power of this evolutionary diversity.
According to incomplete statistics, 10 listed antibodies using phage display technology in the antibody development stage are limited by the patent restriction of phage display technology. The use of this technology is only in the hands of a few companies for a period of time, which is also the first use. The phage display technology antibody adalim was listed in 2002 for the next decade and there are few root causes of the market for antibodies using this technology.
Phage display technology advantages and applications
Phage display technology, whether it is the source technology for antibody discovery or the screening tool for antibody characterization in antibody engineering, has the irreplaceable advantages of existing traditional technologies and the application of multi-directional fields.
1. Compared with the traditional murine immune hybridoma technology, the antibody screened by the human natural phage library is fully human and can effectively reduce the risk of immunogenicity. This chronic disease such as rheumatoid which requires long-term regular injection. It is a key factor in the treatment of diseases.
2. The discovery of fully human antibodies relative to transgenic mice requires in vivo immunization of mice, some humans with highly homologous proteins, and more toxic proteins such as the marketed antibody Raxibacumab against anthrax toxin, antigen-labile Proteins, allosteric proteins, and transmembrane proteins are not suitable for in vivo screening, and human phage libraries are in vitro screened for antibody discovery of more target molecules. Moreover, the phage display technology has not undergone in vivo immunization, and is capable of detecting antibodies against a single target multi-epitope (especially a conserved epitope with high homology of human mouse).
3. Relative to immune-based in vivo antibody discovery technology, animal immunization, late hybridoma fusion, monoclonal screening and other cumbersome cell culture processes, phage display for panning, screening process in E. coli, amplification process is very simple, antibody The time period found was short, the risk of unexpected accidents in the experiment was low, and reagent consumables and labor costs were lower.
4. Immunized animals can extract their B cells for the establishment of an immunophage library, combining the advantages of both in vivo immunization and phage in vitro screening. For example, the first listed Nanobody Caplacizumab is an immune alpaca and then screened using phage library technology. This method has a great role in the screening and discovery of tools for scientific research.
5. Random or site-directed mutagenesis of existing antibody gene sequences to construct a mutant library for humanization or affinity maturation, screening for more affinity antibodies or conditionally activating antibodies, such as Genetech's Ranibizumab and Bevacizuma antibodies. Derived from the murine clone monoclonal antibody A4.6.1, the humanization of the Bevacizuma antibody is obtained by transplanting the CDR regions into the human framework region (VLκ1, VHIII) and then site-directed mutagenesis, while the humanization of Ranibizumab is to transplant the CDR regions. The light and heavy chain framework regions of different humans construct high affinity antibodies 2 screened by phage library.
6. Using the existing antibody as a Guided selection, using a natural phage library to screen out a plurality of new sequence antibodies with the same epitope as the antibody acting on the target, and obtaining a new drug consistent with the in vivo efficacy of the guide antibody. Sequence antibodies, such as adalims, were screened by Guided selection using mouse monoclonal antibody MAK195 as a guide, using phage display method.
7. Selecting a relatively fixed combination of antibody light and heavy chain framework regions to construct a diverse array of synthetic phage libraries in the CDR regions. The relatively fixed combination of light and heavy chain framework regions removes potential post-translational modification sites (PTMs), allowing the selected antibodies to be screened. It has better physical and chemical properties and reduces the difficulty of CMC in the later stage. Ylanthia synthetic phage library 4 as MorphoSys.
8. The CAR of chimeric antigen receptor T cell (CAR-T) therapy is usually scFv, which has a similar display environment as the phage displayed scFv library, using phage display technology to find CAR (chimeric antigen receptor) in CART. It is the key to innovative CAR-T therapy.
9. The technology of phage display screening is also widely used in the field of drug research and development.
Phage display technology is the source technology of bio-innovative medicine research and development. It plays a central role in the discovery and optimization of new antibodies and peptides, and can effectively interface with traditional antibody discovery platforms. It can be widely used in antibodies and antibody couples. Combined drugs and CAR-T/TCR-T and other fields.
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