Screening, Expression And Preliminary Activity Analysis Of Human Anti TfR Single Chain Antibody

With the intensification of global aging, the incidence of central nervous system disease(Parkinson’s disease, Alzheimer’s disease, brain tumors, etc) increased year by year, that has become one of the major diseases threatening human health. Despite decades of intensive research, the biggest challenge in the treatment of central nervous system disease is still that macromolecular drugs how to through the blood-brain barrier(BBB) effectively. The BBB is mainly composed of the brain capillary endothelial cells and the close connection between cells. Nearly 98 percent of the small molecule drugs and all of the macromolecular drugs are difficult to pass the BBB. It severely limits the effect of drugs in the treatment of central nervous system diseases.In recent years, it has brought the hope for macromolecular drugs in the treatment of central nervous system diseases by the receptor mediated brain targeting drug delivery system. Transferrin receptor(TfR) can mediate the transport of endogenous macromolecules transferrin(Tf) cross the BBB. So it has been a hot target for the study of the transport of macromolecular drugs through the BBB. Studies have shown that the monoclonal antibody OX26 of rat TfR could be able to deliver coupled brain-derived neurotrophic factor(BDNF) into the brain. At present, most of the transporter antibodies acting on human TfR are from murine or humanized chimeric antibody. When applied to people, it is inevitable to produce some adverse reactions. Therefore, it has more advantages and important application value to explore the human-derived transporter antibodies acting on human TfR with better activity and lower immunogenicity.In recent years, we had established surface display human ScFv antibody library based on mammalian cells. Because it used eukaryotic cells, it had the function of post-translational modification which was not possessed by the prokaryotic expression system. So the antibody molecules obtained most similar to the natural human antibody, and with lower immunogenicity. In the present study, we used human TfR as the target protein to screen the mammalian cell display human ScFv antibody library, and further constructed secreted antibody library for functional screening. Finally we expressed the candidate scFv fused with LC and identified its activity for transporting and acrossing the BBB.Firstly, ScFv gene screening was conducted by FITC-labeled TfR by FACS from the mammalian cell display human ScFv antibody library. Plasmids contained the mammalian cell display human ScFv antibody gene library were prepared with which 293 T cells were transfected. 60 hours later, the single chain antibody was fully displayed on the surface of the cell membrane. The FITC labeled human TfR was incubated with the transfected cells at room temperature for 1 hour(the final concentration of TfR was 10 nM). Flow cytometry was employed to sort and select cells with a strong fluorescent light intensity, which represented approximately 0.3% of the total number of cells. The plasmids were extracted from the positive cells and amplified as the antibody plasmid library for the next round of screening. In the second round of screening, the final concentration of TfR was reduced to 1 nM. The remaining procedure was the same as described above, we sorted and selected cells with a strong fluorescent light intensity, which represented approximately 0.2% of the total number of cells. The affinity of antibody was improved by decreasing the concentration of TfR. The screening filtered most negative molecular and the remaining candidate molecules were enrichmented, which was beneficial to the subsequent functional screening. The sequences of the positive clones were identified and analyzed via the IgBlast tool. The results showed that these sequences were functional human Ig variable region genes. The results means that the preliminary screening of the mammalian cell display human Scfv antibody library was successful.Secondly, the candidate ScFv antibody secretedly expressing library was constructied and screened. The degenerate primers used in the construction of the humanized ScFv antibody library were applied to design 28 pairs of primers, which contained BspQI restriction sites. The antibody plasmid library obtained from the second round of screening was utilized as a template for PCR amplification. The PCR product was recovered, and BspQI was used to digest the PCR product and SGLs expression vector in order to promote ScFv fusion with the upstream LC fragment of the vector. The LC fragment could be beneficial to the purification as a purification tag, while it could improve the expression level. It could be used as a substitute for drugs ScFv carried in order to investigate whether scFv can carry macromolecular drugs through the BBB. The plasmid library was transfected into CHO-K1 cells via the electroporation method. The supernatants were detected by ELISA to screen for cell lines with strong expressions. 293 T cells were transfected with pENTER-TfR plasmid. After 60 hours of transfection, 293 T cells with high TfR expression on the cell surface were obtained. Flow cytometry was used to screen out four cell lines with positive expression for the supernatant and showed affinity to TfR. Genomic DNA was extracted from these four cell lines. The recovered PCR product was ligated into T vector. DNA sequence analyses was employed to obtain two different gene sequences. These two sequences were identified and analyzed via the IgBlast tool. The results showed that these two sequences were identical and functional human Ig variable region genes.Thirdly, the TfR-ScFv-LC fusion protein were prepared and the bioactivity-was identified. The positive cell lines expressed 2F8 and 5D9 were inoculated into CD-CHO medium. After cultured for 7 days, the ScFv-LC fusion protein was purified via Capto L affinity chromatography. The concentration of purified protein was determined by BCA Protein Quantification Kit. The concentrations of 2F8 fusion protein and 5D9 fusion protein were 0.6mg/m L and 0.8 mg/m L. These two fusion protein were detected via SDS-PAGE gel electrophoresis. The results showed that the proteins had a molecular weight of 38 kDa, which was consistent with the expected size. The results from Western-blotting indicated that these two fusion protein could form specific binding with goat anti-human IgG labeled by HRP. Measured by ForteBio Octet system, the affinity constants of 2F8 fusion protein and 5D9 fusion protein were 3.18×10-7 mol/L and 7.90×10-7 mol/L. The results showed that the two fusion proteins had good affinity with TfR,and the affinity of 2F8 fusion protein is better than that of 5D9 fusion protein. The affinity of 2F8 fusion protein is better. These two fusion proteins labeled by FITC were incubated with hCMEC / D3 cells. Flow cytometry showed that the two fusion proteins could form specific bindings with the hCMEC/D3 cell, which had a nature expression of TfR receptors. And the affinity of 2F8 fusion protein was better, which was consistent with the result of affinity constant. Laser confocal microscopy results indicated that the two fusion proteins could enter hCMEC/D3 cells via TfR mediated endocytosis. This indicates that the two ScFv molecules have the potential to carry macromolecules through the blood brain barrier.In conclusion, we had successfully constructed an human secreted ScFv-LC antibody library based on the mammalian cell display human ScFv antibody library. And obtained two new ScFv fragments with good affinity to TfR via screening. It showed that this screening mode was effective, and provided methods for the screening of similar molecules. The two fusion proteins could bind with hCMEC/D3 cells and enter the cell via TfR mediated endocytosis. This research would lay the foundation for follow-up study of transporting across blood-brain barrier in vivo.