title: Effects of different interchain linkers on biological activity of an anti-prostate cancer single-chain bispecific antibody.
abstract: BACKGROUND
  A single-chain bispecific antibody (scBsAb; an engineered antibody), has promising clinical applications. Nonetheless, the effect of different interchain linkers on its activity is poorly understood.
  
  
  METHODS
  Gene synthesis was used to splice the anti-γ-seminoprotein single-chain antibody (anti-γ-Sm scFv) gene with the anti-CD3 single-chain antibody (anti-CD3 scFv) gene via different interchain peptide linkers. The Phyre2 software was used to predict spatial configuration of different scBsAbs. Eukaryotic expression vectors carrying scBsAbs were constructed by molecular cloning techniques and these plasmids were transfected into HeLa cells with liposomes. scBsAbs were purified by Ni(2+)-NTA agarose and analysed for antigen binding by an enzyme-linked immunosorbent assay (ELISA). Blood pharmacokinetics and inhibition of prostate tumour growth in nude mice were analysed in in vivo experiments.
  
  
  RESULTS
  Bioinformatics analysis and prediction showed that none of the three linkers, Fc, 205C', and HSA, had a significant effect on protein folding of anti-γ-Sm scFv or anti-CD3 scFv. Nevertheless, the spatial structures of the three linkers were noticeably different. Anti-γ-Sm × anti-CD3 scBsAb with an Fc, 205C', or HSA linker was successfully constructed, and these antibodies had similar protein expression levels. ELISA showed that all the three scBsAbs bound to Jurkat cells and the LNCaP membrane antigen, although binding of (205C')scBsAb was weaker than that of the two parental scFvs (P < 0.05). In contrast, binding strength of (HSA)scBsAb and (Fc)scBsAb was close to that of the parental scFvs (P > 0.05). Pharmacokinetic analysis showed that the half-clearance time of the elimination phase (T(1/2β)) for (HSA)scBsAb was the longest: up to 4.4 h. Compared with γ-Sm ScFv, the three scBsAbs all had a much stronger inhibitory effect on the growth of prostate cancer (P < 0.05), but there were no significant differences among the three scBsAbs (P > 0.05).
  
  
  CONCLUSIONS
  HSA is the optimal linker for the anti-γ-Sm × anti-CD3 scBsAb and may improve antigen-binding affinity of antibodies and prolong physiological retention time. Interchain linkers affect the function of scBsAbs; these effects may have important implications for construction of antibodies.
authors.affiliation: Department of Urology, Zhengzhou People's Hospital, No.33 Huanghe Road, Jinshui District, Zhengzhou, 450003, People's Republic of China. chhao1008@sina.com.
authors.forename: Chao-hui
authors.initials: CH
authors.lastname: Hao
authors.affiliation: Department of Urology, Zhengzhou People's Hospital, No.33 Huanghe Road, Jinshui District, Zhengzhou, 450003, People's Republic of China. qianhehan15@126.com.
authors.forename: Qian-he
authors.initials: QH
authors.lastname: Han
authors.affiliation: Department of Urology, Zhengzhou People's Hospital, No.33 Huanghe Road, Jinshui District, Zhengzhou, 450003, People's Republic of China. zhongjie023@yeah.net.
authors.forename: Zhong-jie
authors.initials: ZJ
authors.lastname: Shan
authors.affiliation: Department of Urology, Zhengzhou People's Hospital, No.33 Huanghe Road, Jinshui District, Zhengzhou, 450003, People's Republic of China. jiantingh@163.com.
authors.forename: Jian-ting
authors.initials: JT
authors.lastname: Hu
authors.affiliation: Department of Urology, Zhengzhou People's Hospital, No.33 Huanghe Road, Jinshui District, Zhengzhou, 450003, People's Republic of China. zhangnanhn@yeah.net.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Urology, The First Affiliated Hospital of Zhengzhou University, No.1 East Jianshe Road, Erqi District, Zhengzhou, 450052, People's Republic of China. xuepeizh@yeah.net.
authors.forename: Xue-pei
authors.initials: XP
authors.lastname: Zhang
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D019295:Computational Biology; D005818:Genetic Engineering; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D017430:Prostate-Specific Antigen / Q000276:immunology*; D011471:Prostatic Neoplasms / Q000276:immunology / Q000628:therapy
chemical_list: D018033:Antibodies, Bispecific; D000912:Antibodies, Neoplasm; D007135:Immunoglobulin Variable Region; D017430:Prostate-Specific Antigen