Studies On The Antitumor Efficacy Of An EGFR-Targeting Recombinant Fusion Protein And Its Antibody-drug Conjugate Against Esophageal Cancer And The Mechanism Of Action

Objective:Esophageal cancer is one of the most common malignant tumors in the world,and the incidence of esophageal squamous cell carcinoma(ESCC)is relatively high in China.Therefore,there is an urgent need to explore new effective therapeutic drugs and promising therapeutic strategies.Epidermal growth factor receptor(EGFR)is one of the ideal targets for cancer therapy.Taking EGFR as the therapeutic target,the preparation of EGFR targeted recombinant fusion protein and its antibody-drug conjugate for the treatment of esophageal cancer may bring good clinical benefits.In previous studies,we constructed a novel recombinant fusion protein(Fv-LDP-D3)and its antibody-drug conjugate(Fv-LDP-D3-AE),in which Fv is an anti-EGFR single-chain variable fragment,D3 is the domain Ⅲ of human serum albumin(HSA),LDP is the apoprotein of Lidamycin(LDM),and AE is the active enediyne chromophore of LDM.The results of previous studies showed that it had a significant antitumor effect on K-Ras mutant pancreatic cancer.However,the anticancer efficacy of Fv-LDP-D3 and Fv-LDP-D3-AE against esophageal cancer and the mechanism of action have not yet investigated.In this study,we evaluated the antitumor activity of Fv-LDP-D3 and Fv-LDP-D3-AE against esophageal cancer in vitro and in vivo,and explored the underlying molecular mechanisms.Research methods:The recombinant fusion protein Fv-LDP-D3 was prepared by genetic engineering and fermentation technology.The active enediyne chromophore AE and the apoprotein LDP of lidamycin were separated in vitro,and then AE was assembled into Fv-LDP-D3 through molecular assembly,so as to prepare the antibody-drug conjugate(Fv-LDP-D3-AE).The binding activity of Fv-LDP-D3 to esophageal cancer cells was analyzed by cell ELISA and cellular immunofluorescence.The macropinocytosis mediated uptake of the recombinant fusion protein Fv-LDP-D3 by esophageal cancer cells was detected by fluorescence microscope.Small animal in vivo imager was used to observe the capability of the recombinant fusion protein to accumulate in tumor sites.The in vitro activity and mechanism of action of the recombinant fusion protein and its antibody-drug conjugate were determined by CCK-8 assay,flow cytometry,western blot and other experiments.Finally,the in vivo anti-tumor effect of the drug was evaluated with esophageal squamous cell carcinoma KYSE150 and KYSE520 xenografts in athymic mice.Research results:ELISA and immunofluorescence analysis indicated that recombinant fusion protein could bind to esophageal cancer cells in vitro.Fluorescence microscopy showed that the recombinant fusion protein Fv-LDP-D3 entered into human esophageal cancer cells through a macropinocytosis-mediated pathway.The recombinant fusion protein Fv-LDP-D3 suppressed the proliferation of esophageal cancer cells by inhibiting the signaling of p-EGFR and IMPDH2.In addition,Fv-LDP-D3 induced apoptosis.In vivo imaging showed that Fv-LDP-D3 exhibited tumor site-specific biodistribution in the KYSE520 nude mouse xenograft model,with targeted enrichment at the tumor site for 624 h.Fv-LDP-D3-AE,a relevant antibody drug conjugate prepared after assembling the active enediyne chromophore AE,displayed highly potent antiproliferative effect against all tested esophageal cancer cell lines.Fv-LDP-D3-AE inhibited migration and invasion of esophageal cancer cell,induced autophagy,damaged mitochondrial structure,induced apoptosis,incurred DNA damage,and caused G2/M arrest.The results of in vivo experiment showed that the tumor inhibition rate of Fv-LDPD3-AE(0.2 mg/kg)was 64.3%in KYSE150 xenograft model.Moreover,in the KYSE520 xenograft model,the tumor inhibition rates of Fv-LDP-D3 at 20 mg/kg and 40 mg/kg were 72.9%and 81.2%;and those of Fv-LDP-D3-AE at 0.25 mg/kg and 0.5 mg/kg were 69.4%and 88.5%,respectively.The tumor inhibition rate of the combined administration group was 81.0%.Histo-pathological observation showed that the recombinant fusion protein and its antibody drug conjugate did not cause obvious damage to major organs of the treated mice,indicating that the recombinant fusion protein and its antibody drug conjugate exerted strong anti-tumor effect in vivo at a tolerated dosage level.Conclusions:The present study demonstrated that the recombinant fusion protein FvLDP-D3 which consists of the Fv fragment of an anti-EGFR antibody,the apoprotein of lidamycin(LDP),and the third domain of human serum albumin(D3)and its pertinent drug conjugate(Fv-LDP-D3-AE)which is prepared by integrating the active enediyne chromophore(AE)into the fusion protein molecule both exhibited strong anti-tumor activity against esophageal cancer cells in vitro and effectively inhibited the growth of cancer xenografts in vivo.The current findings revealed that the recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugates Fv-LDP-D3-AE may be potential candidates for the treatment of esophageal squamous cell carcinoma.