Study On The Preparation Of The Immunotoxin 2E8-NCTD And Its Targeting Killing Effect In Vitro And In Vivo

Acute lymphoblastic leukemia (ALL) is the most common type of hematopoietic malignancies in children, of whom 70% is derived from B-lineage. Combined chemotherapy with multiple agents is currently the main treatment for childhood ALL. Although chemotherapy is effective in most patients with ALL, it may also present some disadvantages such as low selectivity and severe side effects. Therefore it is very necessary to seek the new treatment strategy. For decades of investigation, immunotoxin has been becoming a promising approach in the treatment of cancer, immunotoxin is a group of agents conjugating the monoclonal antibody protein with certain toxins. It can specifically recognize the antigen on the surface of human leukemia cells by which the toxin molecules or drugs may reach cell surface or enter the cytoplasm of the leukemia cells thus to kill. Compared to common chemotherapy, immunotoxins hold a better strategy with high targeting efficacy on tumor cells while they protect the normal tissues from unwanted attack by the chemotherapeutical agents.Our previous studies have shown that CD19 molecule is expressed strongly and homogeneously on B lineage ALL cells from very primitive B precursor cells to mature B lymphocytes, while CD20 is only expressed on mature or late developed immature B cells. These results indicated that CD19 molecule could become the best surface marker for cell targeting of B-lineage malignancies. ZCH-4-2E8 (2E8), a new clone of CD19 mAb generated in this lab, was classified into CD19 category by the6^th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996. Study on 2E8 for leukemia targeted kill may bear a promising future in clinical application.Cantharidin is the most potent component of the cantharis for its cytotoxicity to tumor cells. In more recent studies, Cantharidin has been shown to be active in most cancer cell lines. However, a wide clinical application of this agent is severely restricted due to its renal toxicity. Investigators have developed many cantharidin derivatives, of which norcantharidin is the most common type of these agents. Its anti-cancer activity is similar to Cantharidin, but the renal toxicity is significantly reduced. It has been shown that several pathways including Fas/FasL, Caspase, mitochondria pathway are involved in inducing the apoptotic process of the tumor cells by norcantharidin. Norcantharidin is used to treat maligmancies derived from gastrointestinal tract such as primary liver cancer, esophageal cancer and gastric cancer etc or leucopenia due to its stimulation on the proliferation of bone marrow cells. Most investigators focused on the preparation of different dosage forms of common injection, sustained-released preparation, microemulsion for the treatment of liver cancer and gallbladder carcinoma. Targeting treatment of norcantharidin for hepatoma is only reported by Fuyou Liang, who conjugated the norcantharidin with a monoclonal antibody. Although norcantharidin has great toxicity to hematopoietic malignant cells, it has not been reported to treat the hematopoietic malignancies both at home and abroad.A new anti-CD 19 monoclonal antibody (mAb) named 2E8 is secreted by ZCH-4-2E8 hybridoma. An immunotoxin has been generated through conjugating CD19 mAb protein and Norcantharidin by means of active ester method. To determine whether this new immunotoxin is effective in vitro and in vivo, a serious of experiments on leukemia cells and animal model has been performed. Hopefully, the results of this study could provide a fundamental basis for the clinical targeting therapy of this new antibody. Material and Methods 1. Cell culture: The cell lines of 2E8 and Nalm6 were cultured under the routinecondition of 37°C/5%CO2/100% humidity using RPMI-1640 supplemented with 15% heat inactivated calf serum as the medium.2. Preparation and purification of 2E8 monoclonal antibody: 2E8 hybridoma is injected into the peritoneal cavity of BALB/C mouse primed with sterile paraffin fluid for the preparation of 2E8 ascites. The Bouvet method was used to purify the antibody protein from the ascites. After precipitation of the antibody protein from the ascites by saturated ammonium sulfate, the precipitated protein was dialyzed overnight with sufficient amount of phosphate buffered saline (PBS), followed by gel permeation chromatography for further purification of the antibody. The purity of 2E8 mAb was analyzed by SDS-PAGE method and the concentration of antibody protein was measured using commassie bright blue method.3. Anti-cancer activity of norcanthridin: The norcanthridin solution (1mg/mL) was diluted to different concentrations with sterile normal saline. 1×10⁵ Nalm6 cells were inoculated into each well in a 24-well plate. Cells were cultured and checked at the following four time points (24h, 48h, 72h and 96h). The morphology was observed and the number of cells was enumerated with trypan blue exclusion method at these time points after culture. The results were recorded and growth curves were drawn.4. Preparation of immunotoxin 2E8-NCTD: Preparation of an active ester with NHS (N-Hydroxysuccinimide) and DCC (N, N'-Dicyclohexylcarbodiiraide), followed by linkage with the amino group on the immunoglobulin molecule to produce the immunoconjugate. The binding activity of the immunoconjugate to CD19 antigens on the cell surface were examined by flow cytometery. The optimal linkage ratio of the antibody protein to the active ester was examined.5. Examination the targeting activity of the immunotoxin 2E8-NCTD: First, the expression levels of CD19 antigens on Nalm6 and K562 cells were analyzed by flow cytometry. Secondly, the newly generated immunotoxin was co-cultured with either Nalm6 cells or K562 cells. 4 groups of experiments were set, i.e., PBS group, 2E8 antibody group, norcantharidin group and immunotoxin 2E8-NCTD group. 0.5×10⁵ cells were inoculated into each well and cultured under routine conditions. The cell morphology was examined and the number of the cells was enumerated with trypanblue exclusion method at the time of 24h, 48h, 72h and 96h after culture. Third, the effects using different concentrations of immunotoxin 2E8-NCTD on Nalm6 cells were determined by the method as described above.6. Establishment of the Acute leukemia animal model: Nude mice were used to establish the leukemia animal model. 2 mg of cyclophosphamide was intraperitoneally injected into each mouse. 24 hours later, 5×10⁶ Nalm6 cells was inoculated via the tail vein and the mice were monitored daily. When the mice were paralyzed or dying, they were sacrificed and the organs including the liver, spleen, lung, heart, kidney, brain, bone marrow, pancreas, testes etc were removed and fixed with formalin. The morphology of tissues was checked by routine pathological method.7. The effect of 2E8 antibody and immunotoxin 2E8-NCTD on leukemia animal model7.1 Early disease was defined as the nude mice were inoculated with Nalm6 cells at the time of 24h before treatment with 2E8mAb and 2E8-NCTD. The mice were injected with antibody and 2E8-NCTD via their tail veins. A total of 120 μg of the 2E8 Mab or the 2E8-NCTD in a short course of successive 3 days with 1 dose per day was administered intravenously. The living conditions of the mice were monitored daily.7.2 Advanced disease was defined as the nude mice were inoculated with Nalm6 cells at the time of two weeks before treatment with 2E8 Mab or 2E9-NCTD. The mice were injected with antibody and 2E8-NCTD via their tail veins, and their living conditions were monitored daily.Results1. Purification and identification of 2E8 antibody: From the curves of recording paper, two peaks could be identified after chromatography separation. The latter big peak was that of the 2E8 antibody protein while the first peak was proven to be the noisy proteins. After the purified 2E8 antibody was analyzed by electrophoresis in the presence of DTT and stained, two clear bands were observed in the gel with the molecular masses of 79kDa (heavy chain) and 23.7 (light chain), respectively with a purity of more than 99%.2. Anti-cancer activity of norcanthridin: Cytotoxicity experiments were performed on Nalm6 cells with different concentrations of norcantharidin. As a result, the 10 μg/mL and 5 μg/ml groups demonstrated greater inhibitory effect than control group (p<0.005). No significant differences were observed among 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL and control groups (p>0.05).3. Exploration of the optimal linkage ratio of the antibody protein to the active ester: The immunotoxin generated with 46 μg of antibody protein and 20 μL (0.01mmol/mL) of active ester was proven to be the optimal linkage ratio, which kept the best recognition of CD19 antigen with the greatest cytotoxicity potency on Nalm6 cells.4. Determination of the CD19 expression levels on Nalm6 and K562 by flow cytometry: 2E8 antibody in the supernatant reacted with 99.34% of Nalm6 cells, while only 0.98% of K562 cells were reacted with this antibody.5. Comparision of the effects among 2E8-NCTD, 2E8 antibody and norcantharidin on Nalm6 cells: Purified 2E8 antibody didn't show any influences on the growth of Nalm6 cells as compared to control group (p=0.082), while both of the 2E8-NCTD and norcantharidin were shown to have a significant inhibitory effects on the growth of Nalm6 cells. Also 2E8-NCTD was shown to have greater inhibitory effect on the cell growth than 2E8 antibody (p<0.05), but no significant differences were found between the effects of the 2E8-NCTD and the norcantharidin (p>0.05).6. Comparision of the effect among 2E8-NCTD, 2E8 antibody and Norcantharidin on K562 cells: Among these groups, only the norcantharidin group (4.6 μg/mL) was shown to present significant inhibition of the K562 cell growth as compared to the control while no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups, which indicated that the immunotoxin did not show any killing effect on K562 cells due to the absence of the CD19 antigen on their surface.7. The effect of different concentrations of 2E8-NCTD on Nalm6 cells: Greater killing effect on Nalm6 cells were observed in the doses of 15 μg/mL, 10 μg/mL and 5 μ g/mL of the immunotoxin as compared to the control group (p<0.005). Similar resultsas control group were obtained with less than 5 μg/mL of the immunotoxin (p>0.05), indicating that the killing effect of immunotoxin was in a dose and time dependent manner.8. Establishment of acute leukemia animal model: Nude mice were used to establish the animal model of human leukemia. After the tumor (Nalm6) cells were inoculated intravenously, the mean survival of the mice were 19.4±0.55 days (n = 6) without intervention. The mice became paralyzed rapidly two weeks after inoculation followed by weight loss with bent spines, hogback, cachexia until death. The histopathological examination showed that the tumor cells infiltrated liver, spleen, kidney, lung, meninges, interior cerebrum were observed, particularly the liver and kidney were the most affected organs.9. The effect of 2E8 antibody and immunotoxin 2E8-NCTD on leukemia animal model 9.1 In the advanced disease, mice receiving PBS or 2E8 antibody at the time of two weeks after inoculation had a median survival time of 19 days, the median death time of both PBS and 2E8 antibody groups was 30 days with no statistically significance between these two groups.9.2. In the early disease model, mice treated with 2E8 antibody and 2E8-NCTD resulted in median survival time of 29 days and 27 days respectively, while those receiving PBS also had a median survival time of 29 days, indicating that no significant effect of treatment among groups was noted. Reasons for these results are not clear which requires further investigation. Conclusions1. 2E8 antibody and FITC labeled antibody is successfully prepared;2. The immunotoxin 2E8-NCTD is successfully synthesized using active ester method with an excellent targeting killing effect in vitro;3. Acute leukemia animal model had been successfully established in the nude mice however, treatment was not successful with unknown reason necessitating further investigation.