[Expression of human-mouse chimeric antibody ch-BD1 and its affinity to human bladder cancer in vitro and in vivo].
OBJECTIVE
To investigate the expression of human-mouse chimeric antibody ch-BD1 against human bladder cancer and its affinity to human bladder cancer in vitro and in vivo.


METHODS
Three kinds of mutated fragments of dihydrofolate reductase (DHFR) gene were created by techniques of molecular biology to decrease the transcriptional activities and then cloned into pDHL-BD1 so as to construct the vectors pWSD1-BD1, pWSD2-BD1, and pWSD3-BD1 expressing the human-mouse chimeric antibody ch-BD1 against human bladder cancer with decreased expression of DHFR gene. These vectors and pDHL-BD1 were transfected into Chinese hamster ovary cell (CHO)/DHFR- cell respectively. 72 hours later Northern blotting was used to examine their DHFR gene expression. Methotrexate (MTX) of increasing concentrations was added into the culture of transfected CHO/DHFR-cells. The ch-BD1 levels in the supernatants were measured. Purified ch-BD1 was labeled by (99m)TcO(4)(-) and added into the serially diluted solutions of human bladder cancer EJ cells to examine their radioactivities and calculate their affinity constants. EJ cells were injected into the roots of hind limb of 3 Balb/C mice. Four weeks later, (99m)TcO(4)(-)-labeled antibody ch-BD1 were injected into the rats' caudal veins. Radioimmunoimaging was conducted to examine the distribution of the antibody.


RESULTS
The sequence of DHFR gene expression levels from strong to weak in the constructed vectors was as follows: pDHL-BD1 > pWS1-BD1 > pWS3-BD1 > pWS2-BD1. When the concentration of MTX was 10(-6) mol/L the expression level of ch-BD1 was significantly correlated to the expression level of DHFR gene, the lower the baseline expression level of DHFR gene the higher the expression level of ch-BD1. After the serially diluted EJ cells were co-incubated with the (99m)TcO(4)(-)-labeled antibody ch-BD1 the immunoactivity ratio of ch-BD1 was 76%, and that of murine monoclonal antibody was 81%; the affinity constant of ch-BD1 was 3.56 x 10(9) M(-1), and that of murine monoclonal antibody BD1 was 1.22 x 10(9) M(-1). 6 hours after injection of the (99m)TcO(4)(-)-labeled antibody ch-BD1 into the mice body it was mainly distributed in the tumor, 22 hours later, it was specifically distributed in tumor, and 24 hours later it was still concentrated here.


CONCLUSIONS
Decrease of the baseline expression level of DHFR gene effectively increases the amplification of MTX to exogenous gene. The human-mouse chimeric antibody ch-BD1 shows an ideal affinity activity to human bladder cancer in vivo and in vitro and has a certain clinical prospect.
Bai
Yin
Y
Institute of Urology, Peking University, Beijing 100034, China.
Yu
Li-zhang
LZ
Lü
Ying-qian
YQ
Ai
Jun-kui
JK
Zhou
Li-jun
LJ
Zhang
Chun-li
CL
Wang
Yan
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D000915:Antibody Affinity; D016466:CHO Cells; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013667:Technetium; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D011993:Recombinant Fusion Proteins; D013667:Technetium; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate