A Study On Utilization Of Phage Display Technology To Screen Epidermal Growth Factor Receptor Simulated Surface Epitope

Colorectal cancer(CRC)is the 4th most common cancer after lung cancer, gastric cancer and liver cancer. Many studies have established that epidermal growth factor receptor(EGFR) often revealed over expression in CRC. Upon activation,EGFR induces the proliferation, angiogenesis, invasion and metastasis as well as promotion of cell survival of tumor.Indeed it has been widely established that EGFR is an ideal target for anti-tumor drugs.Both Cetuximab and nimotuzumab are molecular antibodfies against EGFR antigen epitope and approved by FDA and SFDA. Their effects are more specific against tumor cells, thus reduces normal tissue damage and significantly improve the patient’s condition. But, there exist some of shortcomings. Firstly they are antibodies with big molecular weight, which reduced their therapeutic effect. Secondly, They are expensive and require long-term use, which gives patients and their families a huge economic burden. If we can imitate the site of the monoclonal antibody drugs combination with target and made peptide vaccine, which can avoided the monoclonal antibody drug shortcomings, improve the therapeutic effect, reduce the financial burden of patients. Comparing with the traditional method of epitope analysis, we think that the phage display technology has unique advantages.Phage random peptide library(PRPL) contains a large number of different sequence structure of the polypeptide, which can be used as a mimetic peptide for any target epitopes. When used peptide library screening the target antigen, it does not need the spatial conformation, which only need to target antigen getting protein polypeptide with antigenicity and immunogenicity reflecting the antigen binding sites of spatial conformation. Smith fused foreign protein gene into the DNA of filamentous phage for the first time, so the foreign protein and the coat protein displayed together on the outside of the phage surface, and this is the phage display technology. At present, the phage display technology has been widely used in the basic research of tumor and tumor treatment for the development of new technology. In this experiment, it analyzed the EGFR epitope in phage twelve peptide library, and nimotuzumab and cetuximab were used as target proteins against EGFR. There is no literature reports to use two kinds of antibody analysis the same antigen epitope at present. After three rounds of phage biopanning experiments, it got monoclonal phage specific antibody of binding to anti tumour drugs. And then, with the aims to obtaining the amino acid sequence of EGFR antigen epitope, it extracted and sequenced the single strand DNA of the phage. Using competitive ELISA method, it used high-frequency monoclonal phage and extracted liquid of EGFR high-expressed of Caco2 cell membrane protein competitive binding to cetuximab and nimotuzumab, for further verifying the affinity activity of the obtained peptide with cetuximab and nimotuzumab. Objective1. Screening the mimic antigen epitopes of EGFR by using the biological washing in phage twelve peptide library.2. The screened short peptides will be implemented bioactivity detection.MethodsBy using nimotuzumab and cetuximab as the target molecule of anti EGFR antigen, we screened the mimic antigen epitopes of EGFR using the biological washing in phage twelve peptide library. After three rounds of biological washing, the amplified selected monoclonal phages were by ELISA method. It selects a higher affinity of positive monoclonal phage with cetuximab or nimotuzumab, and clones and sequences the positive product.By using the competitive ELISA method, high-frequency monoclonal phage and extracted liquid of EGFR high-expressed of Caco2 cell membrane protein competitively binds to cetuximab and nimotuzumab. Results1. Cetuximab, as the target protein, three rounds of "absorption elution amplification" biopanning of it used phage random twelve peptide library. The phage which biopanning on each round were quantified by titer determination. The results showed that the overall recovery rate of phage screened by cetuximab from the first round of(6.67 × 10-6)% increase to the third round(2.80 × 10-3)%, with the enrichment ratio upto 420 times. With the same method of previous, the overall recovery rate of phage screened by nimotuzumab from the first round(8.67 × 10-6)% increase to the third round(1.80 × 10-3)%, with the enrichment ratio upto 208 times.2. In the third round of phage titer measured plate, the randomly selected 30 separate good monoclonal locus coeruleus were amplified respectively, and then, the titer determination was done. The amplified monoclonal phage as testing group and the amplified liquid of phage original library as the negative control group was tested by ELISA. For evaluation, the OD value in testing group more than 2 times that of negative control group was considered positive. Cetuximab has 17 positive results, and nimotuzumab has 21 positive results with the positive rates of 56.67% and 70% respectively. This suggests that cetuximab and nimotuzumab is panning out specific binding phage. Dependence analysis of the randomly selected positive phage clones showed that the OD value has a dose dependece with increasing positive phage inputs.3. Single stranded DNA was extracted from the selected positive phage clones and sequences analysis was done. According to the phage peptide library provides the leader sequence to find the 36 inserting bases of phage, and translate them into amino acids, namely for the 12 peptide sequences of phage display. After sequenced the 17 positive cetuximab phage, three short peptides had a higher frequency, HSFKWLDSPRLR appeared 6 times, HTSSLWHLFRST appeared 4 times and HLFNHNKNLPKR appeared 3 times respectively. After sequenced the 17 positive cetuximab phage, three short peptides had a higher frequency, HSFKWLDSPRLR appeared 8 times, HWKSSYVKWHNV appeared 5 times. The cetuximab and nimotuzumab have a same sequence of HSFKWLDSPRLR.4. The extracted EGFR high-expressed Caco2 cell membrane protein and monoclonal phage of HSFKWLDSPRLR, HTSSLWHLFRST and HLFNHNKNLPKR can competitively binds to cetuximab. These three short peptides appeared different degree of inhibition. And, the extracted liquid of EGFR high-expressed Caco2 cell membrane protein and monoclonal phage of HSFKWLDSPRLR and HWKSSYVKWHNV can competitively binds to nimotuzumab, these two short peptides appeared different degree of inhibition. Moreover, the same sequence of HSFKWLDSPRLR had a higher inhibition rate. ConclusionUsing cetuximab and nimotuzumab can obtain the epidermal growth factor receptor related antigen epitope in phage display random peptide library.The extracted EGFR high-expressed Caco2 cell membrane protein and the screened antigen mimic peptide competitively binds to cetuximab and nimotuzumab.The HSFKWLDSPRLR peptide can be used as EGFR mimotope to develop tumor vaccine in the future.