[Biologic activity of bispecific single chain antibody against human gamma-seminoprotein and CD3 molecule].
OBJECTIVE
To construct a recombinant vector which expresses bispecific single chain antibody (BsscFv) against human gamma-seminoprotein and CD3 molecule and evaluate its biologic activity.


METHODS
The BsscFv gene was constructed by the splicing overlap extensive (SOE) PCR and then a flexible peptide linker was inserted between anti-human gamma-seminoprotein single chain Fv gene and anti-CD3 single chain Fv gene. The fusion gene was subcloned into the pSectag2-B plasmid and was expressed in HeLa cell lines. After being analyzed by SDS-PAGE and Western blot, the expressed product was purified through a Ni(2+)-NTA superflow affinity chromatography column. Flow cytometry (FCM) was used to detect the binding activity of BsscFv to CD3(+) cell line Jurkat cells and prostate carcinoma cells LNCaP. In vitro killing effect on target cells (LNCaP) mediated by BsscFv was determined by chromium(51)-release test. The effect of CTLs mediated by BsscFv on inhibiting tumor growth was observed by utilizing nude mice bearing prostate cancer cells.


RESULTS
DNA sequencing indicated that BsscFv gene consisted of 1,500 bp, encoding 500 amino acids. SDS-PAGE and Western blot analysis showed that the expressed product with relative molecular mass of 61,000 existed in culture supernatant of Hela cells. Flow cytometry analysis demonstrated that the binding rate of BsscFv to LNCaP cells and Jurkat cells was 54.1% and 53.7%, respectively. In vitro, BsscFv mediated cytotoxicity of CTLs to LNCaP cells as confirmed by chromium(51)-release assay. In prostate cancer nude mouse model, BsscFv inhibited tumor's growth as compared with control group.


CONCLUSIONS
The BsscFv against human gamma-seminoprotein and CD3 molecule possesses certain biologic activity, and in vitro and in vivo it can mediate cytotoxicity of CTLs to prostate cancer cells.
Wang
Dong
D
Department of Urology, Fuzhou General Hospital, Fuzhou 350025, China. wangdong1202@medmail.com.cn
Wang
He
H
Wu
Guo-jun
GJ
Wu
Wei-zhen
WZ
Yang
Shun-liang
SL
Lin
Wen-hong
WH
Tan
Jian-ming
JM
D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008297:Male; D051379:Mice; D008819:Mice, Nude; D017430:Prostate-Specific Antigen / Q000276:immunology*; D011471:Prostatic Neoplasms / Q000276:immunology / Q000628:therapy; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D007135:Immunoglobulin Variable Region; D017430:Prostate-Specific Antigen