Study on the Preparation of the Immunotoxin 2E8-NCTD and Its Targeting Killing Effect In Vitro.

Objective: Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated to antibody proteins reported were toxic proteins which presented immunogenicity to patients capable of producing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule of toxin derived from a Chinese medicine Mylabris phalerata Pallas. The chemical name of this agent is 7-Oxabicyclo(2,2,1) heptane-2,3-dicarboxylic anhydride with a molecular weight of 168.15 CAS. 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 in order to provide the experimental fundamentals for the further development of this new targeting agent.

Methods: 2E8 monoclonal antibody 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) to CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometery. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, Norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm6 cells or K562 cells were made.

Results: The purity of the purified 2E8 antibody was more than 99% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm6 cells, while only 0.98% of K562 cells were reacted with this antibody. The new generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm6 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 after 96 h of culture with inhibitory rates of 71.25% and 97.62%, respectively (P < 0.001), while the purified 2E8 antibody (inhibitory rate of 30.29, P > 0.05) didn’t show any significant influences on the growth of Nalm6 cells as compared to that of negative control. No significant inhibitory effects were identified among immunotoxin 2E8-NCTD and 2E8 antibody as compared to that of control group on CD19-K562 cells with inhibitory rates of 15.41% and 4.17%, respectively, P > 0.05, indicating that a significant targeting effect of the 2E8-NCTD against Nalm6 cells was noticed.

Conclusions: The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing efficacy on CD19+ Nalm-6 leukemia cells in vitro, which lays the experimental fundamentals for the further development of this new targeting agent.