What is scFv fragment?



In the early 1980s, with the successful development of recombinant antibodies, the research of antibodies has entered a new stage of development. Since then, people have tried to use genetic engineering technology to produce recombinant antibodies in large quantities. Through genetic engineering, immunoglobulin molecules have been processed and modified at the gene level, and a large number of small molecular antibodies have been developed for research, diagnosis and treatment. These antibodies not only retain the biological activity and characteristics of natural antibodies, but also remove most of the irrelevant structures, making them more suitable for in vivo diagnosis and treatment. ScFv fragment has attracted more and more attention and research due to its advantages of small molecular weight, strong penetration and high affinity.


Single chain antibody fragment (scFv) is composed of heavy chain variable region (VH) and light chain variable region (VL) linked by 15 to 20 amino acids of short peptide (linker). Its relative molecular weight is about 2.5KDa, which is only 1/6 of the complete antibody.


Advantages and disadvantages of scFv fragment
Compared with traditional antibodies, scFv fragment have the following advantages:


(1) Low molecular weight; (2) Good retention of the configuration and location of the binding site of the original antibody and its affinity to antigen activity and accuracy; (3) Good penetration, easy contact and conjugation with target cells through the vascular wall, suitable for the diagnosis and treatment of tumors; (4) Easy molecular modification and direct killing of target cells; (5) Large-scale production by appropriate E coli; It can bypass the routine antibody preparation procedure and obtain human and toxin antibodies which are difficult to achieve by routine methods.
Of course, scFV antibody also has some shortcomings, such as low molecular weight, rapid clearance of immune system antibodies, difficult to achieve the time required for diagnosis or treatment, low affinity, poor stability, etc.


However, there are still many shortcomings in scFV fragment, such as: (1) some types of antibody fragments VH and VL bind by non-covalent bonds, which are likely to form polymers due to the hydrophobicity of the conjugated peptide, so the stability ratio is poor; (2) the apparent affinity of Fab fragment is low; (3) limited to the current synthesis methods, it is not stable, functional, easy to purify and easy to produce. Bispecific antibodies, 4. Although immunotoxins use toxin derivatives to weaken the non-specific binding to a certain extent, they still have dose-dependent specific or non-specific damage to normal cells.


Preparation of scFV
1. Phage display technology
In recent years, bacteriophages have become an important platform for displaying scFV. Phage display technology uses genetic engineering to fuse scFV fragments with encoding genes of phage shell protein, so that scFV can be expressed on the surface of the phage for further identification and screening.
2. Ribosome display technology
Ribosome display technology has many advantages, such as high affinity, fast display speed, simple and easy operation, and is not affected by bacterial transformation efficiency, thus shortening the experimental cycle. Ribosomal display technology is an efficient method for screening scFV in vitro. The main process is to make new scFV and its mRNA link to form complex, enrich RNA with high affinity to antigen, and then obtain amplified products through PCR reverse transcription. However, if the function of DNA-polymerase is not checked in this process, gene mutation is more likely to occur.
3. Yeast surface display technology
Yeast surface display is a powerful method for screening scFV with high specificity and affinity. However, because of the low conversion efficiency of yeast, the experimental cycle becomes longer, which limits the type and yield of screening antibodies. The core of yeast surface display technology is to connect recombinant protein to yeast cell wall. This technology makes full use of the endoplasmic reticulum of yeast cells, so that the recombinant protein can fold accurately in the endoplasmic reticulum.


1. Targeted therapy
Targeted therapy of tumors is the most direct application field of scFv. Because of its low immunogenicity, good targeting, strong penetration and low side effects, it has unique advantages in targeted therapy. For example, antibody-directed edenzyme prodrug therapy (ADEPT) is to connect scFv with drug-activating enzymes in vitro, then inject inactive or inactive prodrug into human body intravenously, and the enzyme linked with antibody activates the activity of prodrug at the tumor cells, avoiding the combination of exfoliated antigens with liver and kidney, resulting in accumulation of antigens, thus leading to cancer group. Drugs in tissues are abundantly enriched.
2. Imaging analysis
ScFV labeled by radioisotopes injects labeled antibodies against tumor-associated antigens or tumor-specific antigens into the body to determine the location of the tumor, and then relies on the internal radiation effect to achieve the role of radioimmunotherapy. Because of its small molecular weight, scFV can quickly and efficiently infiltrate into the intracellular network of tumors, making the image of tumor location clear and clear, and avoiding non-specific damage, it has high specificity and affinity, and is much higher than the clearance rate of conventional antibodies.
3. Study on autoimmune diseases
The mechanism of autoimmune diseases such as rheumatism and AIDS is also related to the synthesis of autoantibodies. Abnormal organisms spontaneously produce high affinity autoantibodies, and at the same time produce pathological changes under the influence of mechanisms such as ADCC or activation of complement system. ScFV has great potential in the treatment of HIV.
4. Gene therapy
The principle of gene therapy is to use gene recombination technology to make scFv antibody express in non-lymphocyte, and to make scFv antibody distribute in nucleus, cytoplasm or some organelles by modifying the antibody molecule appropriately, so as to specifically interfere with or block the replication process of some biological macromolecules such as virus.
ScFv antibody, as the third generation antibody, has shown a broad application space. The establishment of single-chain antibody library technology is known as another revolutionary progress. Its appearance has basically solved the problem of preparation of human antibodies. People can modify the performance of antibodies according to their needs, and various antibodies can be prepared in vitro without in vivo immunization. However, ScFv also has some shortcomings, such as its ability to bind to antigens is not as strong as that of natural antibodies, its half-life is short, its stability is not good, and it can not be used as a long-acting drug for clinical application. With the deepening of ScFv research, there will be more satisfactory solutions to the existing problems. It is believed that in the near future, ScFv will play an important role in medicine, food hygiene and agriculture by virtue of its own advantages.