Construction Of Bispecific ScFv Targeting The Dimerization Interface Of EGFR-HER2

EGFR?Epidermal Growth Factor Receptor?is a kind of receptor with tyrosine kinase activity.When it is combined with the corresponding ligands such as EGF?Epidermal Growth Factor?,TGF-??Translational Growth Factor-??,the signal will be transmitted to the cells,so that the normal growth and reproduction of cells.Overexpression or mutation of EGFR will lead to abnormal activation of EGFR,which is closely related to the growth,anti apoptosis,migration and invasion of cancer cells,so it has become a hot target of tumor therapy.Cetuximab and Panituzumab,as well as Gefitinib and Imatinib,have been marketed.Due to the high mutation in the target region of these two kinds of drugs,and the competitive binding of monoclonal antibodies to ligands,the drugs currently on the market have low clinical response rate and drug resistance.Therefore,it is urgent to develop new drugs.The main components of EGFR are extracellular region,transmembrane region and intracellular region.The extracellular domain is the ligand binding domain and the intracellular domain is the tyrosine kinase active region.The extracellular domain can be divided into four subdomains,i.e.I,II,III and IV.In the absence of ligand binding,subdomains?and?bind to each other,making the whole molecule inactive.When there is ligand binding,subdomains?and?bind to ligands,and subdomains?and?separate,and subdomains?and?stretch outward to expose the dimer arm that interacts with another molecule.When another EGFR molecule or other EGFR family molecules also expose dimer arms and are close to each other,the dimer arms of the two molecules will interact to form a dimer and transmit the signal downstream.Dimerization is a key process in the activation of EGFR and other EGFR family receptors.Targeting the dimerization interface of EGFR and preventing the dimerization of the two molecules,thus inhibiting the activation of EGFR molecules,can obtain better antitumor effect.On the surface of some tumor cells with abnormal EGFR expression,HER2,another member of EGFR family,is often overexpressed.HER2 is similar to EGFR in structure,but has no ligand binding subdomain,and its extracellular dimerization arm is always exposed.This makes it an important target for EGFR dimerization.In the previous study,our project team constructed a scFv targeting EGFR dimerization interface,and verified that it can effectively inhibit the growth of EGFR overexpressed tumor cells,but the inhibition effect on EGFR and HER2 overexpression tumor cells is not ideal.Based on the previous study of"targeting EGFR dimerization interface",this project proposes to use bispecific antibody to target EGFR and HER2 dimerization interface simultaneously,so as to study the antitumor effect and mechanism of the bispecific antibody,so as to provide new reference for the treatment of tumors with abnormal expression of EGFR.Purpose:1.Construction of recombinant bispecific antibody EGFR-HER2-Dimer-bis-scFv;2.Efficient preparation of bispecific antibody EGFR-HER2-Dimerbis-scFv;3.Specific binding analysis of bispecific antibody EGFR-HER2-Dimer-bis-scFv with tumor cells;4.Analysis of the inhibitory activity of bispecific antibody EGFR-HER2-Dimer-bis-scFv on tumor cells;5.Analysis of anti receptor dimerization and anti receptor phosphorylation of bispecific antibody.Methods:1.Construction of recombinant bispecific antibody EGFR-HER2-Dimer-bis-scFvThe gene sequence of EGFR Dimer 5G9 screened in the laboratory was used as the gene sequence mother of bispecific antibody targeting EGFR dimer interface sequence,and the gene sequence mother of bispecific antibody Pertuzumab targeting HER2 dimerization interface sequence was used.Then His-Tag and restriction site sequence were added to the 3'end of the recombinant sequence,and MF-?signal peptide and restriction site sequence were added to the 5'end of the recombinant sequence.The target gene was amplified by PCR,digested and ligated into pGAPZ?A a expression vector.After identification and purification,the recombinant expression vector was electroporated into Pichia pastoris X-33 strain?pGAPZ?A,X-33?.After high resistance screening and SDS-PAGE detection,the engineering bacteria which could express bispecific antibody targeting EGFR and HER2 dimerization interface were screened.2.Efficient preparation of bispecific antibody EGFR-HER2-Dimer-bis-scFvThe engineering bacteria expressing EGFR-HER2-Dimer-bis-scFv were fermented in a small scale by using microbial fermentation technology.Single factor experiment was used to optimize the fermentation temperature,pH value,liquid volume and carbon source.The optimal fermentation conditions were obtained by measuring OD₆₀₀00 value and SDS-PAGE protein electrophoresis detection.After obtaining its isoelectric point,the bispecific antibody was purified by weak cation exchange chromatography and nickel ion metal affinity chromatography.The sample buffer was replaced by molecular sieve chromatography and preserved.3.Specific binding analysis of bispecific antibody EGFR-HER2-Dimer-bis-scFv with tumor cellsHuman lung cancer NCI-H292 cells overexpressing EGFR and HER2were selected as positive experimental group cells,NIH-3T3 mouse fibroblasts were used as negative control group cells.The cells were starved with 2‰serum medium for 24 h after being cultured in complete medium until the cell abundance was 80%.About 1x10⁶ cells were collected from each bottle and sealed with skim milk powder for 2 h.then,EGFR-HER2-Dimer-bis-scFv with concentration of 1 mg/mL and volume of 0.02136,0.2136,2.136?mol/L prepared in this experiment were added as primary antibody and His tag labeled immunofluorescence antibody as secondary antibody.After washing,the binding activity of cells was detected and analyzed by flow cytometry.4.Analysis of the inhibitory activity of bispecific antibody EGFR-HER2-Dimer-bis-scFv on tumor cellsLung cancer cell NCI-H292 was cultured to the appropriate concentration,and then seeded to 96 well plate at the cell concentration of2000 cells/well.After 24 hours of serum starvation culture,EGF with final concentration of 1.1?mol/L was added.Then EGFR-HER2-Dimer-bis-scFv and EGFR-Dimer-scFv with final concentrations of 0.0668,0.134,0.267,0.534,1.068 and 2.136?mol/L were added to culture for 72hours.At the same time,NIH-3T3 cells were cultured with 1.1?mol/L EGF and 2.136?mol/L EGFR-HER2-Dimer-bis-scFv and EGFR-Dimer-scFv for72 hours as negative control.MTT assay was used to detect the proliferation inhibition of the antibody.5.Analysis of anti receptor dimerization and anti receptor phosphorylation of bispecific antibody.NCI-H292 cells overexpressed with EGFR and HER2 were cultured and digested into a 6-well plate.After 24 hours of serum starvation,1.1?mol/L EGF ligand was added,then 2.136?mol/L EGFR-Dimer-scFv and EGFR-HER2-Dimer-bis-scFv were added respectively.After 24 hours,8mmol/L BS³ crosslinking agent was added for 30 min.after lysis,the receptor dimer was detected by SDS-PAGE and Western blot.The cells were cultured at 4?for 6,12 and 24 h respectively.The phosphorylation of EGFR and HER2 was analyzed by Western blot.Results:1.Construction of recombinant bispecific antibody EGFR-HER2-Dimer-bis-scFvBased on the variable region sequences of the monoclonal antibody EGFR-Dimer-5G9 and Pertuzumab,the gene sequence of the bispecific antibody targeting EGFR and HER2 dimerization interface was designed.The gene sequence was 1518 bp in length.After codon optimization and chemical synthesis,it was subcloned into Pichia pastoris expression vector pGAPZ?A.After identification by colony PCR and sequencing,the recombinant expression vector pGAPZ?A-EGFR-HER2-Dimer-bis-scFv was linearized and transformed into Pichia pastoris X-33 cells.SDS-PAGE protein electrophoresis and Western blot results showed that the target protein was correctly expressed,the size was about 55.6 KD,which was consistent with the expected size,could be recognized by anti-His tag antibody,and the specificity was also in line with the expectation.These results indicated that the engineering bacteria with bispecific antibody were successfully constructed.2.Efficient preparation of bispecific antibody EGFR-HER2-Dimer-bis-scFvThe efficient preparation of bispecific antibody is not only related to the fermentation conditions of high density and high expression of engineering bacteria,but also related to the subsequent purification process.The experimental results showed that when the fermentation temperature was 28?,the pH value was 6.0,the liquid volume was 15%,and 2.0%glucose was used as the carbon source,higher biomass and larger expression of target protein could be obtained.Compared with the fermentation conditions before optimization,the protein content of the engineering strain was increased by 1.69 times.Bioinformatics analysis showed that the isoelectric point of the bispecific antibody was 7.96 with his expression tag,which was suitable for purification by cation exchange chromatography and metal ion affinity chromatography.Therefore,the purity of the fermentation supernatant was over 95%after purification by CM weak cation exchange chromatography and Ni²⁺affinity chromatography 6.6 mg/L after optimization.3.Specific binding analysis of bispecific antibody EGFR-HER2-Dimer-bis-scFv with tumor cellsFlow cytometry analysis showed that the bispecific antibody EGFR-HER2-Dimer-bis-scFv could specifically bind to NCI-H292 cells with both EGFR and HER2 overexpression.It did not bind to NIH-3T3,a mouse fibroblast with low expression of EGFR and HER2.It shows that the bispecific antibody has good targeting.4.Analysis of the inhibitory activity of bispecific antibody EGFR-HER2-Dimer-bis-scFv on tumor cellsMTT assay showed that the bispecific antibody EGFR-HER2-Dimer-bis-scFv had a 52.66±1.89%inhibitory effect on the growth of NCI-H292,which overexpressed both EGFR and HER2,in a concentration dependent manner.The inhibition ability of bispecific antibody was significantly higher than that of single chain antibody EGFR-Dimer-sc Fv,and the highest growth inhibition rate of the latter was 45.01±0.55%.5.Analysis of anti receptor dimerization and anti receptor phosphorylation of bispecific antibodyThe results of Western blot showed that the bispecific antibody EGFR-HER2-Dimer-bis-scFv had stronger inhibition of dimer and phosphorylation of EGFR and HER2 receptors than the scFv targeting only EGFR-Dimer-scFv.Conclusions:1.The bispecific antibody EGFR-HER2-Dimer-bis-scFv was successfully and efficiently prepared,and its purity was up to 95%.After the optimization of fermentation conditions,the biomass of engineered bacteria increased by 27.89%,and the expression of the product increased by 1.69 times.It provides a new idea for large-scale production of this type of antibody.2.The bispecific antibody EGFR-HER2-Dimer-bis-scFv can specifically bind to EGFR and HER2 receptors on cell surface.The antibody also inhibited the proliferation of NCI-H292 cells overexpressing EGFR and HER2,which was stronger than the sc Fv only targeting the dimerization interface of EGFR.The reason may be that the bispecific antibody has stronger ability of anti EGFR and HER2 dimerization and anti EGFR and HER2 receptor phosphorylation.