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Updated: Jun 27, 2026
Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells
Published on: November 9, 2010
[Preparation of the immunotoxin 2E8-norcantharidin and its targeting killing effect in vitro]
Li-xia Li1, Yong-min Tang, Hai-zhong Zhang
1Department of Hematology-Oncology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310003, China.
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.
Insights
A novel immunotoxin, 204-NCTD, shows promising targeted killing effects against CD19+ leukemia cells. This new therapy utilizes a small molecule toxin, norcantharidin, avoiding immunogenicity issues associated with protein toxins.
Area of Science:
- Oncology
- Immunotherapy
- Drug Development
Background:
- Immunotoxins offer a promising therapeutic strategy for hematopoietic malignancies by targeting cancer cells with antibodies linked to toxins.
- Traditional immunotoxins often use protein toxins, which can elicit an immune response in patients, limiting their efficacy.
- Norcantharidin (NCTD), a small molecule toxin, presents a potential alternative due to its lack of immunogenicity.
Purpose of the Study:
- To investigate the targeting efficacy of a novel immunotoxin, 2E8-NCTD, against CD19-positive lymphoid malignant Nalm-6 cells in vitro.
- To evaluate the potential of NCTD as a non-immunogenic toxic moiety for antibody-drug conjugates.
- To provide experimental data for the development of 2E8-NCTD as a new targeted therapeutic agent.
Main Methods:
- Preparation and purification of anti-CD19 monoclonal antibody (mAb) 2E8.
- Conjugation of 2E8 mAb with Norcantharidin (NCTD) using the activated ester method to generate the immunotoxin 2E8-NCTD.
- Assessment of binding activity and CD19 antigen expression via flow cytometry on Nalm-6 and K562 cells.
- In vitro evaluation of the inhibitory effects of 2E8-NCTD, 2E8 mAb, NCTD, and PBS on Nalm-6 and K562 cell growth.
Main Results:
- The purified 2E8 mAb exhibited high purity (>99%) and specific binding to CD19+ Nalm-6 cells (99.34%), with minimal binding to CD19-negative K562 cells (0.98%).
- The generated immunotoxin 2E8-NCTD demonstrated high binding rates (99.90%) to Nalm-6 cells with retained activity.
- 2E8-NCTD significantly inhibited the growth of CD19+ Nalm-6 cells (P < 0.001), while the control groups showed no significant effect, confirming targeted efficacy.
Conclusions:
- The immunotoxin 2E8-NCTD was successfully synthesized using the activated ester method.
- 2E8-NCTD demonstrated excellent in vitro targeting and killing effects on CD19+ Nalm-6 leukemia cells.
- This study provides crucial experimental data supporting the further development of 2E8-NCTD as a novel targeted therapeutic agent for lymphoid malignancies.
