[Study on p53 tetramerization domain in improving functional affinity and biological activity of antibody].
OBJECTIVE
To fuse the genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and exploit a new way to improve the functional affinity and biological activity of antibody.


METHODS
Genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer BsAb was fused by technique of DNA sub-cloning. The fusion gene confirmed by sequencing was subcloned into the pSectag2-B plasmid. Then the recombinant plasmid was transfected into HeLa cells. The expression products, which were analyzed by both SDS-PAGE and western blotting, were purified with Ni(2+)-NTA superflow affinity chromatography. mBsAb-pSectag2-B plasmid was added into the suspensions of human peripheral blood mononuclear cells (PBMCs) and PC-3 cells respectively. Flow cytometry was used to examine the binding rate of multivalent anti-prostate-cander/anti-CD3 bispecific scFv with PBMCs and PC-3 cells. T cells were isolated from the PBMCs. PC-3 cells were labeled with Na(2)[(51)Cr]O(4) used as target cells. Labeled PC-3 cells, T cells, and different concentrations of mBsAb were mixed. Natural release control well with labeled target cells only and maximum release control well with labeled target cells and 10% SDS were prepared. The supernatants were extracted. gamma calculator was used to calculate the counts per minute (cpm) values to calculate the specific release rate of (51)Cr.


RESULTS
Sequencing showed a fragment from mBsAb-pSectag2-B with the size of 1.7 kb corresponding to the predicted value. SDS-PAGE and Western blotting showed expression of 67 000 D protein in the supernatant of culture fluid of HeLa cells transfected with MBsAb-pSectag2-B plasmid. The binding rates of multivalent anti-prostate-cancer/anti-CD3 bispecific scFv with PBMC and PC-3 cells were 70.4% and 81% respectively, significantly higher than those of anti-prostate-cancer/anti-CD3 bispecific scFv. In the presence of mBsAb the activated T cells lysed PC-3 cells in positive correlation with the antibody concentration and effective cell/target cell ratio and with a lysis rate significantly higher than those of the control groups.


CONCLUSIONS
Multivalent anti-CD3 x anti-prostate-cancer BsAb exhibits much higher functional affinity and biological activity than anti-CD3 x anti-prostate-cancer BsAb, which may break a new path to the improvement of functional affinity and biological activity of antibody.
Wang
Dong
D
Department of Urology, Fuzhou General Hospital of People's Liberation Army, Fuzhou 350025, China.
Wu
Guo-Jun
GJ
Tan
Jian-Ming
JM
Wang
He
H
D018033:Antibodies, Bispecific / Q000276:immunology*; D000915:Antibody Affinity; D017252:CD3 Complex / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000276:immunology; D011471:Prostatic Neoplasms / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000276:immunology*
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D017430:Prostate-Specific Antigen