Screening And Preliminary Evaluation Of Antibody-drug Conjugate (ADC) Targeting HER2+

Antibody-drug conjugate(ADC)covalently links cytotoxin to monoclonal antibody,with specificity in antigen recognition.Some technical problems limit the high-quality development of ADCs,such as uneven drug-to-antibody ratio(DAR)and nonspecific coupling sites.To address the aforementioned challenges,this thesis developed a new method for screening ADCs,using streptavidin-biotin system.This system exhibits the advantages of determined DAR value and site-specific coupling.The targeting ability of the prepared ADC toward HER2 receptors was preliminarily examined in molecular and cellular level.To develop this method,first,Avi-Tag was introduced at the chain ends in Herceptin sequence,thus three biotinylated antibodies(Lc-Avi,Hc-Avi and Lc-Avi+Hc-Avi)were successfully prepared.Molecular weight analysis based on SDS-PAGE showed that the above three proteins all contained biotin.Meanwhile,SA-SMCC-DM1 was prepared from commercially available streptavidin(SA)and SMCC-DM1(Succinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-1carboxylate——Mertansine).The optimal reaction ratio and the drug coupling ratio were determined by hydrophobic chromatography and UV spectrum,respectively.Then,three kinds of biotinylated antibodies were mixed with SA-SMCC-DM1 to prepare ADC.Among them,the one prepared by Lc-Avi had the highest content of maytansine,and the best mixing molar ratio was 1:1.Molecular interaction experiments showed that the dissociation equilibrium constant(KD)toward HER2 antigen was roughly the same as that of Herceptin.Cell experiments illustrated that ADC could be specifically endocytosed by HER2 positive cells(SK-BR-3)and produce cytotoxicity effectively.Finally,we designed and prepared monovalent single-streptavidin(m SA).Once mixed with biotinylated antibodies,commercially available SA,consisting of four binding sites,results in a variety of protein cross-linked products.Instead of HEK293,E.coli could express m SA successfully.Molecular interaction experiments showed that m SA and commercially available SA had the same level of biotinylated antibody binding ability.The m SA we expressed would further improve the quality of ADC.