Anti-tumor Activity Of Monoclonal Antibody And Single-Chain Antibody Targeting The Dimer Interface Of EGFR

Epidermal growth factor receptor?EGFR,HER1 or ErbB1?is a member of the tyrosine kinase type?receptor family.Overexpression or mutation of EGFR could lead to abnormal cell proliferation,angiogenesis,metastasis and anti-apoptosis.And it was highly correlated with the occurrence,development and prognosis of tumor.So,it was one of the hot targets of targeted therapy for malignant tumors.There was low clinical response rate?10-25%?and drug resistance problem on antibody drugs of this target.Which may be related to the high degree of variation in the extracellular domain of EGFR receptor,the heterodimerize between EGFR and other family members receptors,and the competitive inhibition of ligands.Dimerization was a necessary process for the activation of EGFR family receptors,and the interface directly involved in dimerization was a highly conservative region.Therefore,we proposed a targeting strategy for the EGFR dimer interface.The aim was to solve the problem of low response rate and drug resistance caused by receptor mutation and heterodimerization through this targeting strategy antibody.In our previous work,we used?-cyclic peptides from the EGFR dimer interface directly involved in dimerization to prepare polypeptide vaccines and monoclonal antibody EGFR dimer 5G9,which were targeting the EGFR dimer interface.The results showed that both of them could effectively inhibited the growth of EGFR overexpressed tumor cells.In order to further evaluate the feasibility of targeting strategy of EGFR dimer interface,the variable region of EGFR dimer5G9 was used to construct the single chain antibody?EGFR dimer ScFv?.The inhibitory effect of antibodies of different molecular sizes on the growth of tumor cells with different receptor phenotypes in vitro,the ability of homo-and heterodimerization of anti-receptor,the ability of phosphorylation of anti-receptor,and the therapeutic effect on human epithelial carcinoma A431 in vivo were observed.In order to lay the foundation for the research and development of new anti-EGFR therapeutic antibody.Content:1.Construction and efficient preparation of EGFR dimer ScFv for targeting EGFR dimer interface;2.Phenotypic identification of receptor of tumor cell;3.Anti-tumor activity of monoclonal antibody and single chain antibody in vitro;4.Anti-receptor dimerization activity of monoclonal antibody and single chain antibody;5.Anti-receptor phosphorylation activity of monoclonal antibody and single-chain antibody;6.Therapeutic effect of monoclonal antibody on human epidermal carcinoma A431 xenotransplanted tumor in nude mice.Methods:1.Construction and efficient preparation of EGFR dimer ScFvThe VH and VL sequences of the EGFR dimer 5G9 were obtained by means of gene sequencing.The VH and VL connections were ligated with?Gly₄-Ser?₃ linked peptide,and introduced MF-?signal peptide,His-tag and enzyme cutting site.Then,the gene were connected into the expression vector pGAPZ?-A by double-enzyme digestion,and electrically transforming into the expression host Pichia pastoris X-33,and screening high-resistance to obtain the engineering bacteria.The engineering bacteria were fermented at 30?and180 rpm for 72 h.The antibody on the fermentation was crude purified by the ammonium sulfate precipitation method.Then,the pure antibody was purified through the nickel column,and replaced buffer by G25 molecular sieve.The concentration of the protein was determined by the BCA method,and the concentration of the protein were diluted according to the concentration of 1mg/mL,and were stored in a sterile penicillin bottle at 4?for 1-2 months.2.Phenotypic identification of receptor of tumor cellHuman epidermal carcinoma A431,human pancreatic cancer BxPC-3 and mouse fibroblast NIH-3T3 were selected for cell culture.When the cells were cultured to an abundance of 80%,the cells were lysed with NP-40 lysis buffer?containing 10 mmol/L PMSF?for 30 min at Ice bath.The lysates were vortexed and centrifuged,and the supernatants were semi-quantified by the BCA method and diluted at a concentration of 1 mg/mL.The supernatants were added5×SDS-PAGE loading buffer,and the expression of EGFR,HER2 and HER3was detected by 8%SDS-PAGE and Western Blotting analysis.3.Anti-tumor activity of monoclonal antibody and single chain antibody in vitroThe EGFR/HER2 overexpressed A431 cells were selected for culture.When the cells were cultured to an abundance of 80%,the cells were digested.The cells were seeded in 96-well plates and cultured in complete medium for24 h.Then,under the stimulation of 1.12?mol/L of EGF,the different concentrations of antibody were incubated with the cells,and the serum-free culture for 48 h was performed.The proliferation of A431 cells was detected by MTT method and the inhibition rate was calculated to observe the dose-effect relationship of antibody.At the concentration of 2.136?mol/L,the inhibitory effect of antibody on NIH-3T3 and BxPC-3 cells in vitro for 48 h was detected under the same conditions.The solvent group was a negative control,and the Cetuxi group was a positive control.4.Anti-receptor dimerization activity of monoclonal antibody and single chain antibodyThe EGFR/HER2 overexpressed A431 and EGFR/HER2/HER3overexpressed BxPC-3 cells were selected for the experiment of anti-receptor dimerization of monoclonal antibody and single chain antibody.When the cells were cultured to an abundance of 80%,the cells were seeded in 6-well plates and cultured in complete medium for 24 h.Then,the cells were starved by free-serum for 24 h.Under the stimulation of 1.12?mol/L EGF,the cells were incubated with a 2.136?mol/L antibody for a different period of time?2 h and12 h?.Then,BS³ cross-linking agent was used to in situ couple the dimer.And the cells were lysed with NP-40 lysis buffer?containing a protease-phosphatase inhibitor?for 30 min at Ice bath.The lysates were vortexed and centrifuged,and the supernatants were semi-quantified by the BCA method and diluted at a concentration of 1 mg/mL.The supernatants were added 5×SDS-PAGE loading buffer,and the expression of receptor homo-and heterodimers,the expression of receptor homo-and heterodimers of phosphorylation were detected by 8%SDS-PAGE and Western Blotting analysis.5.Anti-receptor phosphorylation activity of monoclonal antibody and single-chain antibodyThe EGFR/HER2 overexpressed A431 and EGFR/HER2/HER3overexpressed BxPC-3 cells were selected for the experiment of anti-receptor phosphorylation of monoclonal antibody and single chain antibody.When the cells were cultured to an abundance of 80%,the cells were seeded in 6-well plates and cultured in complete medium for 24 h.Then,the cells were starved by free-serum for 24 h.Under the stimulation of 1.12?mol/L EGF,the cells were incubated with a 2.136?mol/L antibody for a different period of time?2 h and 12 h?.And the cells were lysed with NP-40 lysis buffer?containing a protease-phosphatase inhibitor?for 30 min at Ice bath.The lysates were vortexed and centrifuged,and the supernatants were semi-quantified by the BCA method and diluted at a concentration of 1 mg/mL.The supernatants were added 5×SDS-PAGE loading buffer,and the expression of total protein and phosphorylated protein of EGFR,HER2 and HER3 were detected by 8%SDS-PAGE and Western Blotting analysis.6.Therapeutic effect of monoclonal antibody on human epidermal carcinoma A431 xenograft tumor in nude miceThe xenograft tumor model of nude mice was established with A431 cells.The negative control group?solvent?,the experimental group?EGFR dimer 5G9?and the positive control group?Cetuximab?were set up in this experiment,with9 rats in each group.When the tumor volume reached 100 mm³,the nude mice was intraperitoneally injected at a dose of 25 mg/kg,once every 3 days,for a total of 6 times.The volume of the tumor and the weight of the mouse were recorded once every 3 days,and was drawn the growth curve.On the 9 day after the end of the administration,the mice were sacrificed and the tumor tissues were photographed and weighed.Results:1.Construction,expression and preparation of single chain antibody EGFR dimer ScFvThe results showed that the EGFR dimer ScFv was efficiently expressed by Pichia pastoris X-33.SDS-PAGE and Western Blotting analysis showed that the purified antibody had a specific target band of His tag at the attachment of29.0 kDa and purity of antibody>99.5%.2.Phenotypic identification of receptor of tumor cellWestern Blotting analysis showed that NIH-3T3 was an EGFR/HER2/HER3 normal expression or low expression cell,A431 was an EGFR/HER2 overexpression and HER3 low expression cell,BxPC-3 was EGFR/HER2/HER3 overexpressing cells.3.Anti-tumor activity of monoclonal antibody and single chain antibody in vitroThe results of the MTT assay show that the EGFR dimer 5G9 and the EGFR dimer ScFv could significantly inhibit the growth of A431 cells,and the inhibition rate was dependent on the dose.At the concentration of 2.136?mol/L,the inhibition rates of EGFR dimer 5G9 and EGFR dimer ScFv were 47.09±0.94%and 48.92±0.31%,respectively.At the concentration of 2.136?mol/L,the inhibition rate of EGFR dimer 5G9 and EGFR dimer ScFv on BxPC-3 cells was50.17±0.75%and 49.06±0.49%,respectively.The inhibition rate of NIH-3T3cells was 7.19±0.50%and 6.81±0.48%,respectively.4.Anti-receptor dimerization activity of monoclonal antibody and single chain antibodyThe results showed that active dimer bands of two cells were not significantly reduced after 2 h incubation with the EGFR dimer 5G9 or the EGFR dimer ScFv.And active dimer bands of two cells were significantly reduced after 12 h incubation with the cells.5.Anti-receptor phosphorylation activity of monoclonal antibody and single chain antibodyThe results showed that total phosphorylated bands of two cells were not significantly reduced after 2 h incubation with the EGFR dimer 5G9 or the EGFR dimer ScFv,and total phosphorylated bands of two cells were significantly reduced after 12 h incubation with the cells.6.Therapeutic effect of monoclonal antibody on human epidermal carcinoma A431 xenograft tumor in nude miceAt the end of the final experiment,the average tumor volume of the control group was 3082.5±276.1 mm³,and the average tumor volume of the EGFR dimer 5G9 group was 1726.8±187.6 mm³.The inhibition rate of the EGFR dimer 5G9 was 43.98%?P<0.01?.From the beginning of the tumor to the end of the experiment,there was no significant decrease in body weight and no death occurred.At the end of the experiment,the mean tumor wet-weight of the control group was 2.33±0.08 g,and the average tumor wet-weight of the EGFR dimer5G9 group was 1.31±0.13 g.Compared with the control group,the mean tumor wet-weight of the EGFR dimer 5G9 group was significantly lower than that in the control group?P<0.001?.Conclusions:1.A single chain antibody based on monoclonal antibody EGFR dimer5G9 was successfully constructed,and a highly efficient preparation method was established by using Pichia pastoris secretory expression system.2.Both the monoclonal antibody and the single chain antibody targeting the EGFR dimer interface could effectively inhibit the growth of the EGFR overexpressing tumor cell,the receptor homo-and hetero-dimerization and the receptor phosphorylation,and the time accumulation effect was present.3.The monoclonal antibody EGFR dimer 5G9 could significantly inhibit the growth of human epidermal carcinoma A431 xenograft tumor in nude mice.