[Construction and expression of anti-CD3/anti-prostate-cancer bispecific single-chain antibody].
OBJECTIVE
To construct a recombinant vector that expresses anti-CD 3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and study its biological activities and clinical significance.


METHODS
An anti-CD 3/anti-prostate-cancer BsAb was constructed by PCR and molecular biological technique of DNA cloning. The fusion gene, confirmed by sequencing, was subcloned into the pSectag2-B plasmid from the pUC18 vector by digestion with EcoR I and Hind III restriction endonucleases, whose sites exist in both the vectors. Then the recombinant plasmid was transfected into HeLa cells. The expression products in the supernatant were analyzed by both SDS-PAGE and Western blot technique, then were purified with Ni(2+)-NTA superflow affinity chromatography. Its biological activities were examined by flow cytometry (FCM).


RESULTS
A fragment of 1.5 kb was inserted into the pUC18 vector, which was sequenced and verified to be identical with that designed. The expression of anti-CD 3 x anti-prostate-cancer BsAb yielded a soluble protein with an apparent molecular mass of 61 KD. The purification rate of the expressed BsAb was up to 90.537% and the yield of purified BsAb from this procedure was 0.09 mg/ml. The positive binding rates of BsAb to PBMC and to PC-3 cell were 54.1% and 53.7% respectively.


CONCLUSIONS
The anti-CD 3 x anti-prostate-cancer BsAb thus constructed exhibits beneficial biological activities and may play an important role in the treatment of prostate cancer.
Wang
Dong
D
Department of Urology, Fuzhou General Hospital of People's Liberation Army, Fuzhou 350025, China.
Wu
Guo-jun
GJ
Wang
He
H
Yang
Shun-liang
SL
Wu
Wei-zhen
WZ
Xu
Ting-zhao
TZ
Tan
Jian-ming
JM
D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000302:isolation & purification / Q000627:therapeutic use; D000912:Antibodies, Neoplasm / Q000096:biosynthesis*; D017252:CD3 Complex / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000276:immunology* / Q000302:isolation & purification / Q000627:therapeutic use; D011471:Prostatic Neoplasms / Q000276:immunology* / Q000628:therapy; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013601:T-Lymphocytes / Q000276:immunology
D018033:Antibodies, Bispecific; D000912:Antibodies, Neoplasm; D017252:CD3 Complex; D011993:Recombinant Fusion Proteins; D017430:Prostate-Specific Antigen