Construction and prospect of phage antibody library

Phage antibody library

The construction principle of phage antibody library
Phage antibody library is a successful example of phage display on genetically engineered antibody applications. The use of PCR to obtain antibody gene fragments is the key to phage antibody library. At present, common antibody genes are derived from hybridoma cells, in vitro immune cells, sensitized or non-sensitized B lymphocytes, etc., and total RNA amplification is performed from the above cells to obtain the variable regions of light chain and heavy chain of a full set of antibodies. After the gene of variable region is cloned into the phage genome, the antibody gene encoding peptide and the phage coat protein are fused, and it can be obtained as a single-chain variable region fragment (scFv) or as an antigen-binding fragment (Fab). Phage antibody repertoires on the surface of phage. Specific antibodies can be screened from the phage antibody library by adsorption elution enrichment. In addition to phage, there is a class of library expression vector phagemid. The phagemid is used as an expression vector, and a helper phage such as VCSM13 is used to provide a structural protein required for packaging, and a mixed expression type phage can be produced. Compared with traditional filamentous phage antibody libraries, phagemids are suitable for the expression of a large number of soluble recombinant proteins, and thus are ideal for the expression of genetically engineered antibodies.
Classification of phage antibody libraries
According to the source of the gene, it can be divided into two different types of antibody libraries, namely, an immunological antibody library and a non-immune antibody library, which in turn includes a natural library, a semi-synthetic library, and a fully synthetic library. The immune antibody library refers to an antibody library constructed by lymphocytes after immunization (including vaccination, microbial infection, autoimmune disease, tumor, etc.). Since lymphocytes used to construct these antibody libraries have undergone antigen selection and affinity maturation processes in vivo, highly specific high affinity antibodies can be screened. Many research groups use immunological libraries to obtain small molecule antibody fragments. This method is superior to conventional hybridoma technology, especially for scanning large numbers of clones, which means that the selected antibody fragments have high affinity. Kits prepared by immunological libraries are now in commercial use.

Application of phage antibody library
Since the advent of phage antibody library, its wide application prospects and large development potential have attracted the attention of researchers. The phage antibody library not only enhances the ability to prepare larger numbers of antibodies, but also modifies the antibody to meet the requirements of antibody quality such as affinity and immunogenicity.

1. Preparation of small molecule antibodies

A variety of different forms of antibodies have been produced, such as single-chain variable fragment antibodies (ScFv), Fab antibodies, bispecific antibodies (BsAb), and the like, depending on the function and application requirements of the antibody. Compared with traditional intact antibodies, ScFv and Fab antibodies are easily expressed in prokaryotic cells due to their smaller molecular structure, and can be expressed in various expression systems, without Fc segment, which can reduce the widespread distribution of Fc receptors, the adverse effects of the body, etc. Therefore, these two types of antibodies are two of the more common types of phage antibody libraries that have been constructed. Currently, ScFv and Fab antibodies which have been prepared by the antibody library method for clinical diagnosis and treatment include virus neutralizing antibodies, anti-tumor antibodies, autoantibodies, anti-hormone antibodies and the like.

2. Preparation of humanized antibodies

The application of murine antibodies to humans is highly likely to produce human anti-mouse antibodies, which greatly limits the application and development of murine monoclonal antibodies, and therefore studies the humanization of murine monoclonal antibodies. At first, phage library of human antibodies was constructed by immunizing animals, and specific antibodies were screened from the prepared immunological library. With the deepening of research, the VH and VL gene fragments of human B lymphocytes could be cloned by RT-PCR. And after amplification, and random combination into the human expression vector, the establishment of a large antibody library is ready. This method is a good solution to the inefficiency of human hybridomas. It is simple and easy to perform, and has a large screening capacity, which provides a new way for the preparation of fully humanized antibodies. For example, Yuan et al. used a Mullerian-type inhibitory receptor as a target antigen, and a non-immune antibody library was used to prepare a fully humanized antibody for diagnosis and treatment of ovarian cancer.

3. Improved antibody performance

One of the outstanding advantages of phage antibody library is its ability to improve antibodies. It is mainly reflected in the following points: it can aim at a specific performance, and the performance is improved by screening the mutation library without knowing the primary structure or having the unknown sequence of peptide performance. The antibody library can mimic the antibody affinity maturation process in vivo by introducing mutations or screening for high affinity clones to increase antibody affinity. According to this idea, Hoet et al. greatly improved the affinity of the prepared chimeric/fully synthetic CDR antibody compared to the pre-engineered CDR antibody. Phage antibody library can also be used to increase the amount of antibody expression. Ho et al. used this technique in experiments to increase the expression efficiency of anti-CD22 antibodies and increase the affinity by more than 10 times.

Prospect of phage antibody library development
With its unique advantages, phage antibody library technology has been widely used in many fields of medicine and life sciences, providing scholars with a new research direction. By simulating the natural antibody library, it eliminates the cumbersome and time-consuming immune process, which not only greatly improves the preparation ability of the monoclonal antibody required for scientific research and clinical, but also provides a powerful screening ability for any antigen and even unknown antigen. However, at present, the technology also has the preference of codons, limited library capacity, and limitation of amino acid modification by host bacteria. Therefore, it is necessary to construct a library with large storage capacity and good diversity, and to develop long-term non-destructive preservation of antibody library and establish new ones. The screening method will be the direction of future research. It is believed that the ever-improving phage antibody library will have more excellent performance and wider application.