Construction Of Engineered Fusion Proteins Consisting Of Anti-EGFR ScFv And Lidamycin And The Study Of Their Antitumor Activity

The epidermal growth factor receptor(EGFR) is a tyrosine kinase receptor of the ErbB family that is overexpressed in many epithelial tumors.EGFR and its ligands serve as a part of intracellular signaling pathways,which play an important role in the formation and the development of tumors.It has been recognized that EGFR is a promising target for cancer therapy.Single chain Fv(scFv) is an engineered antibody fragment.Compared to the complete antibody,scFv shows lower molecular weight,lower immunogenicity,and strong penetration capability,which can penetrate into solid tumor deeply.Due to the lack of Fc,scFv can not kill target tumor cells,thus a "warhead" molecule including drugs,toxins,and cytokines is needed.Lidamycin(LDM),a macromolecular peptide antibiotic produced by Streptomyces globisporus,shows extremely potent cytotoxicity against tumor cells.LDM consists of an active enedinye chomophore(AE) and an apoprotein(LDP),which can be separated and reconstituted without losing its activity.Therefore,LDM is regarded as an ideal "warhead" molecule for antibody targeted drugs.In this study,phage display was used to select strong affinity scFv for EGFR.Fusion protein ER(Fv-LDP) was prepared and energized fusion protein ER(Fv-LDP-AE) was reconstituted.The potent cytotoxicity of fusion protein and its energized protein was observed.1.Construction and screening of phage antibody libraries against EGFR and soluble expression of ER(Fv)Balb/c mice were immunized by human epidermoid carcinoma A431 cells,and total RNA of the splenic cells was extracted.VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker,then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E.The phagemides containing scFv were transformed into electro-competent E.coli TG1 cells.The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified by ELISA.A total of 48 clones from the library was selected randomly and 45 clones were identified positive. After infecting E.coli HB2151 cells with one positive clone,soluble recombinant antibodies about 27 kDa were produced and located in the periplasm and the supernatant. The result of sequencing showed that ER(Fv) gene was 768bp,which encoded 256 amino acid residues.VH and VL including 3 CDRs and 4 FRs respectively were all homologous to mouse Ig.The soluble ER(Fv) showed specific binding activity to purified EGFR and the EGFR located in carcinoma cell membrane.The successful preparation of ER(Fv) will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.2.Construction of fusion protein ER(Fv-LDP) and the study of its activityScFv gene was ligated into the plasmid pET-30a(+)-LDP,then the recombinant plasmid pET-30a(+)-scFv-LDP was transformed into competent E.coli BL21(DE3) star^TM cells.Fusion protein with His-tag was produced in the form of inclusion after IPTG inducing.Finally about 5 mg of purified protein was obtained from 1 L of culture medium.ELISA and immunofluorescence assays showed that ER(Fv-LDP) had strong affinity for A431 and A549 cells that overexpress EGFR.FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase after ER(Fv-LDP) treatment,and the degree of arrest was decided by the dose of fusion protein.The cytotoxicity of ER(Fv-LDP) was checked by MTT.ER(Fv-LDP) showed potent cytotoxicity against tumor cells overexpressing EGFR.Human epidermoid carcinoma A431 xenograft in nude mice was used to investigate inhibitory effects of the fusion protein in vivo. ER(Fv-LDP) showed strong antitumor activity and less side effects.The inhibition rates of ER(Fv-LDP) at dose of 0.5 mg/kg and 5 mg/kg were 54.1%and 67.5%respectively.3.Preparation of energized fusion protein ER(Fv-LDP-AE) and the study of its antitumor activityThe energized fusion protein ER(Fv-LDP-AE) was prepared by the reconstitution of ER(Fv-LDP-AE) and AE.FACS ananlysis of cell cycle showed that cells were also arrested in G2/M phase after ER(Fv-LDP-AE) treatment,and the degree of arrest was decided by protein dose.Apoptosis of tumor cells treated with ER(Fv-LDP-AE) was observed after Hoechst dying.It is observed in MTT that ER(Fv-LDP-AE) had stronger cytotoxicity than LDM.The IC₅₀ value of ER(Fv-LDP-AE) was only 1/7 of that of LDM for A431 cells and 1/10 for A549 cells respectively.In vivo,ER(Fv-LDP-AE) showed stronger antitumor activity than LDM.The inhibition rates of ER(Fv-LDP-AE) at dose of 0.2 mg/kg and 0.3 mg/kg were 75.4%and 85.3%,while that of LDM at 0.05 mg/kg was 64.1%respectively.The successful preparation of energized protein ER(Fv-LDP-AE) will provide a promising EGFR targeted drug for the therapy of cancer.