[Preparation of the immunotoxin 2E8-norcantharidin and its targeting killing effect in vitro].
OBJECTIVE
Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenicity to patients capable of inducing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent.


METHODS
A monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometry. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made.


RESULTS
The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm-6 cells, while only 0.98% of K562 cells reacted with this antibody. The newly generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19+ Nalm-6 cells (P < 0.001), while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups on CD19(-) K562 cells, a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed.


CONCLUSIONS
The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.
Li
Li-xia
LX
Department of Hematology-Oncology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310003, China.
Tang
Yong-min
YM
Zhang
Hai-zhong
HZ
Shen
Hong-qiang
HQ
Qian
Bai-qin
BQ
Luo
Chun-fang
CF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D018941:Antigens, CD19 / Q000276:immunology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006825:Hybridomas; D000922:Immunotoxins / Q000276:immunology*; D020014:K562 Cells; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D019086:Bridged Bicyclo Compounds, Heterocyclic; D000922:Immunotoxins; C069741:norcantharidin