Establishment Of Recombinant Protein EGFR-single Domain Antibody-iRGD And Evaluation Of Recombinant Protein Together With Anticancer Drugs In The Treatment Of Gastric Cancer

Purpose:With many more antibodies approved for therapy,this class of molecules has become an established treatment modality for cancer.However,improving tumor targeting and penentration of anticancer drugs is very important.A recombinant protein containing anti-EGFR single-domain antibody and tumor penetrating peptide was constructed.And this dissertation focused on:1.developing recombinant protein anti-EGFR-iRGD and evaluating its targeting skills,penetrating ability and antitumor efficacy in 2D cells,3D multicellular spheroids(MCS)and in vivo;2.evaluating its efficacy in improving anticancer drugs'(such as 5-FU,CPT-11,DOX,PTX,bevacizumab,PTX-Liposome)permeability and antiproliferation in multicellular spheroids.Methods:Recombinant protein anti-EGFR-iRGD was constructed by DNA recombinant technology and purified by nickel-nitrilotriacetic acid affinity chromatography.The binding profiles of anti-EGFR-iRGD were analyzed using the SPR-based biosensor.The molecular weight of anti-EGFR-iRGD was then detected by MALDI-TOF.To evaluate the targeting skill,penetrating ability and anticancer efficacy of anti-EGFR-iRGD,the in vitro model,3D MCS of cancer cells and the tumor mass were all used.Moreover,we also tested whether the conjugation of iRGD to the C-terminus of anti-EGFR could enhance the penetration of DOX,nanoparticles into MCS and could improve anticancer efficacy of other cancer drugs(5-FU,CPT-11,DOX,PTX,bevacizumab,PTX-Liposome).Results:Recombinant protein anti-EGFR-iRGD was expressed successfully in E.coli BL21(DE3)and purified by nickel-nitrilotriacetic acid affinity chromatography.The binding profiles of anti-EGFR-iRGD were analyzed using the SPR-based biosensor by flowing it over the surface of human EGFR-extracellular domain.The kD of anti-EGFR-iRGD was 7.09nM.The molecular weight of anti-EGFR-iRGD was detected as 18kDa by MALDI-TOF,which is consistent with the values expected from the recombinant protein sequence.Anti-EGFR-iRGD,which targets EGFR and?v?3,spreads extensively throughout both the multicellular spheroids and the tumor mass.The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines,multicellular spheroids,and mice with no toxicity in important organs.Moreover,anti-EGFR-iRGD could improve doxorubicin(DOX),nanoparticle permeability in multicellular spheroids.Anti-EGFR-iRGD could also improve anticancer efficacy of other cancer drugs(5-FU,CPT-11,DOX,PTX,bevacizumab)in MCS.The synergetic effect of protein anti-EGFR-iRGD of low concentration combined with PTX on human BGC-823 cells is in regard of proliferation,apoptosis,and cell cycle distribution.Our results provide impetus for further studies to provide a cogent basis for potentially using anti-EGFR-iRGD with standard cytotoxic treatment regimens for enhancing therapy of gastric cancer patients.Conclusions:Anti-EGFR-iRGD,which targets EGFR and ?v?3,spreads extensively throughout both the multicellular spheroids and the tumor mass.The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines,multicellular spheroids,and mice.Moreover,anti-EGFR-iRGD could improve anticancer drugs,such as doxorubicin(DOX),bevacizumab,nanoparticle permeability and efficacy in multicellular spheroids.This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD.As a consequence,anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs.