Research Of Screening And Identification Of Human Single Domain Antibodies Against HER2 And FGFR2 Respectively

Objective:HER2 and FGFR2 proteins are over-expressed in many cancer cells, their over-expression are related to cancer development and metastasis, and these two proteins are ideal targets for cancer therapy. Although there are anti-HER2 antibodies on the market for cancer therapy, they are traditional monoclonal antibodies, and due to their large size, their applications are limited. This study isolated human single domain antibodies of small size against HER2, hoping to replace the monoclonal antibody on the market for cancer therapy. This study also isolated human single domain antibodies against FGFR2. Methods:Part one: screening human single domain antibodies against HER2. Human single domain antibody phage library was prepared by the infection of helper phage. HER2-domain II protein fragment was prepared by prokaryotic cell expression. The library was screened against HER2-domain II by phage display. Polyclonal phage ELISA and monoclonal phage ELISA was performed to confirm the positive single domain antibody clones. DNA sequences of the positive single domain antibodies were obtained. The soluble single domain antibodies were expressed in E. Coli and purified. Non-competitive ELISA was performed to detect the affinity of the purified single domain antibodies binding to HER2-domain II.Part two: screening human single domain antibodies against FGFR2. Human single domain antibody phage library was prepared by the infection of helper phage. FGFR2 protein fragment was synthesized. Human single domain antibody phage library was screened against FGFR2 protein fragment by phage display. Polyclonal phage ELISA and monoclonal phage ELISA was performed to confirm the positive single domain antibody clones. DNA sequences of the positive single domain antibodies were obtained. Results:Part one: screening human single domain antibodies against HER2. Human single domain antibody phage library was prepared by infectting helper phage, and the library titer was 3.41×1013/ml. HER2-domain II protein was expressed from prokaryotic cell inclusion body and purified, and SDS-PAGE showed the high protein purity. Non-reducing SDS-PAGE showed that the size of HER2-domain II protein is about 12 kDa. Screening single domain antibody library againt HER2-domain II was performed for 5 rounds by phage display, and the results showed that single domain antibodies capable of binding to HER2-domain II were enriched. Polyclonal phage ELISA showed that single domain antibodies binding to HER2-domain II protein fragment were most enriched at round 3. A total of 196 single domain antibody clones were randomly picked from the screened library, and monoclonal phage ELISA identified 8 positive single domain antibodies. Monoclonal phage ELISA with the irrelevant antigens as controls found that these 8 single domain antibodies had high specificity to HER2-domain II. These 8 single domain antibodies were sequenced, and the results showed that there were only 2 different single domain antibody clones(named as aHER2-1C1 and aHER2-2B4). aHER2-1C1 and aHER2-2B4 were cloned into the expression vector pET22 b, their proteins were expressed and purified from E. Coli, and SDS-PAGE showed high purity. ELISA found that the purified aHER2-1C1 could bind to HER2-domain II,but aHER2-2B4 did not. Non-competitive ELISA showed that the affinity of aHER2-1C1 binding to HER2-domain II was 3.76 uM.Part two: screening human single domain antibodies against FGFR2. FGFR2 protein fragment was synthesized, HPLC showed high protein purity, and MS analysis showed that the size of FGFR2 protein fragment was about 4 kDa. Screening single domain antibody library againt FGFR2 protein fragment was performed for 5 rounds by phage display. The results showed that single domain antibodies capable of binding to FGFR2 protein fragment were enriched. Polyclonal phage ELISA showed that single domain antibodies binding to FGFR2 protein fragment were most enriched at round 5. A total of 168 single domain antibody clones were randomly picked from the screened library, and monoclonal phage ELISA identified 20 positive single domain antibody clones. Monoclonal phage ELISA with the irrelevant antigens as controls found that these 20 single domain antibodies had high specificity to FGFR2 protein fragment. These 20 single domain antibodies were sequenced, and the results showed that there were only 2 different single domain antibody clones(named as aFG-7E5 and aFG-9F8). Conclusions:In this study, human single domain antibodies against HER2 and FGFR2 were isolated respectively by phage display. This study laied a good foundation to use these identified human single domain antibodies for cancer therapy.