Selection And Analysis Of Human Vh Domain Antibodies Against VEGF And EGFR Respectively

Tumor growth and metastasis in vivo depend on the neovascularization, and vascular endothelial growth factor(VEGF) plays a critical role in these processes. There are several effective antibodies against VEGF on the market such as Bevacizumab and Aflibercept for the treatment of cancers. So, VEGF is a good drug target for cancer therapy.EGFR is highly expressed in some cancer cells. EGFR overexpression could support cancer cell survival, proliferation, invasion and metastasis. There are several antibodies against EGFR on the market for cancer therapy. So, EGFR is also a good drug target for cancer therapy.Although these antibodies on the market are effective for the treatment of cancers, these antibodies are all traditional monoclonal antibodies. Duo to their large molecular size, they have some disadvantages, such as low tissue permeability, inconvenience in the gene engineering, limit to eukaryotic cells for protein expression. The purpose of this thesis is to find better new antibodies, which can replace these antibodies on the market through the step-by-step modification of new antibodies.Human heavy chain domain antibody(hd Ab) has no immunogenicity and low molecular weight. It can penetrate well in tissues and be easy for genetic engineering and therefore is promising in the future in cancer treatment. This thesis is to screen for hd Abs against VEGF and EGFR respectively by phage display technology and preliminary identification of them. This study can lay a solid foundation for the use of hd Abs for the treatment of different cancers.In the first section of this thesis, hdAbs against VEGF were screened and analyzed.(1) The region spanning exon 3 and 4 of VEGF were chosen as an antigen, and this DNA fragment was cloned into expression vector. The protein of this DNA fragment was expressed in E. coli, and the inclusion body containing the protein was successfully purified, and the protein was further processed by denaturing-renaturing.(2) After 5 rounds of selection of hd Abs against the VEGF fragment by phage display technology, the specific hd Ab phages against the VEGF fragment were successfully enriched from the library.(3) Monoclonal phage ELISA was used to select 41 positive hd Abs against the VEGF fragment from total 370 clones. After the verification by unrelated antigens as controls, 17 hd Abs were obtained because of their high specificity.(4) The 17 positive hd Abs were sequenced, and the 17 DNA sequences were compared. Finally, 4 positive hd Ab phage clones were obtained.(5) The DNA fragments of these 4 hd Abs were amplified by PCR and cloned into expression vectors. The hd Ab proteins were expressed in E. Coli. and successfully purified.(6) The binding of purified hd Abs to VEGF were checked by ELISA. Results showed that two hdAbs can not bind to the VEGF, and two hd Abs can bind to the VEGF, but only one hd Ab(aVE207) had a high binding affinity. Western Blotting further verified their binding.(7) Non-competitive ELISA was performed to examine aVE207 KD as 61 nM.In the second section of this thesis, hd Abs against EGFR were screened and analyzed.(1) AA408-437 of EGFR domain III was chosen as an antigen, the peptide of this antigen fragment were synthesized, and liquid chromatography and mass spectrum analysis showed that the synthetic peptide had high purity and correct size.(2) After 5 rounds of selection of hd Abs against the EGFR peptide by phage display technology, the specific hd Ab phages against the EGFR peptide were successfully enriched from the library.(3) Monoclonal phage ELISA was used to select 24 positive hd Abs against EGFR peptide from total 470 clones. After the verification by unrelated antigens as controls, 13 hdAbs were obtained because of their high specificity.(4) The 13 positive hd Abs were sequenced, and the 13 DNA sequences were compared. Finally, 5 positive hd Ab phage clones were obtained.(5) The DNA fragments of these 5 hd Abs were amplified by PCR and cloned into expression vectors. The hd Ab proteins were expressed in E. Coli. and successfully purified.(6) The binding of purified hd Abs to the EGFR peptide were checked by ELISA. Results showed that all hd Abs can bind to the EGFR peptide, but only two hdAbs(aEG3C6,aEG4C6) had a high binding affinity.(7) Non-ompetitive ELISA was performed to examine aEG3C6 and aEG4C6 KD as 62 nM and 102 nM.In conclusion, 4 hd Abs against VEGF were successfully selected by phage display, and they were successfully expressed and purified. Only one(aVE207) out of the 4 hd Abs could bind to VEGF, and KD was 61 nM. In addition, 5 hdAbs against EGFR were also successfully isolated and were also expressed and purified. Two hdAbs(aEG3C6,aEG4C6) out of the 5 hd Abs could bind to EGFR with high affinity, and KD was 62 nM and 102 nM. This study can lay good foundation for further studies on such hd Abs for the treatment of cancers.