Study On An Immunotoxin 2E8-BM Targeted Cell Kill Of B Lineage Leukemia Cells

B lineage acute lymphoblastic leukemia (B-ALL) is the most common type of hematopoietic malignancies in children. The current primary treatment of childhood ALL is combined chemotherapy. Although the present therapies on childhood ALL are effective, severe toxicity and side effects are still the major complications due to the poor treatment selectivity of the chemotherapeutic agents. Over decades of intensive investigations, targeted therapy has been becoming a new promising alternative for cancer treatment in clinical trials. Among various targeted therapy strategies for cancer treatment, immunotoxins possess many advantages. They are a group of artificially-made cytotoxic molecules targeting cancer cells. Each molecule of the immunotoxin consists of a targeting moiety, such as a ligand or a monoclonal antibody (mAb), and an attack moiety, such as protein poisons, radionuclides and small-molecule drugs. Compared to the conventional chemotherapeutic agents, immunotoxins only kill caner cells expressing target molecules while spare those without them. Therefore targeted therapy with immunotoxins can greatly reduce the nonspecific toxicity on normal tissues and side effects in patients.The CD19 is a B lineage-specific surface molecule expressed on the surface of leukemia cells from 99.1% of patients with B lineage ALL while it is absent from other hematopoietic components such as T, NK, granulocytes, monocyte/macrophages, red blood cells, hematopoietic stem/progenitor cells and those from non-hematopoietictissues or organs, which indicates that the CD19 molecule can be as an optimal target for B lineage malignancies. ZCH-4-2E8 (2E8), a new clone of CD19 mAb generated in this lab was assigned into CD19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996. The use of 2E8 mAb to selectively deliver cytotoxic agents to tumors surely has the potential of both improving anti-tumor efficacy and reducing the systemic toxicity of the therapy.The dried body of the Chinese blister beetle (Mylabris phalerata Pallas), known as mylabris, is one of the insect-derived Chinese medicines and has been applied topically to treat malign sores and to relieve blood stasis for more than 2000 years. Mylabris was recently used to treat patients with malignancies particularly liver cancer. But the use of mylabris in traditional Chinese medicine is frequently accompanied with severe side effects, such as a burning sensation of the digestive tract, vomiting, nephritis, cystitis, and so on. Immunotoxins consisting of mAb as directing carriers and cytotoxic ingredient of mylabris as warhead could avoid the systemic side effects caused by the nonspecific cytotoxicity of mylabris. Previous studies showed that the primary active component of Mylabris was cantharidin. But cantharidin is insoluble in cold water, only very slightly soluble in hot water and soluble in organic solvents such as chloroform (1 in 65), acetone (1 in 46) etc, which makes it difficult to be prepared in the generation of an immunotoxin. In addition, due to its unique chemical structure, there are currently no effective chemical methods to link the cantharidin to a mAb protein to generate an immunotoxin. Hence it is very necessary to find out whether any other water-soluble toxic ingredients of mylabris exist besides cantharidin and whether there are any relatively simple methods to conjugate those water-soluble toxins to a mAb protein to generate an immunotoxin.In this study, the new clone of anti-human CD19 antibody, 2E8 was used to conjugate with water-soluble toxins of mylabris (in Chinese BanMao or BM) via direct incubation to generate 2E8-BM immunotoxin. By using this immunotoxin, target killingof CD19+ leukemia cells was carried out and the killing mechanism was investigated. The results of this study have provided a better fundamental for generating second generation of 2E8 immunotoxin such as single chain fragment of variable region immunotoxin (ScFv immunotoxin), human-mouse chimeric antibody immunotoxin or humanized antibody immunotoxin for clinical purpose.Materials and Methods1. Cell cultureThe cell lines of 2E8, Nalm-6 and K562 were cultured under routine conditions with RPMI1640 medium supplemented with 20% heat inactivated calf serum. The viability of the cells was ≥ 95% evaluated by trypan blue exclusion method before they were inoculated into 24-well plates.2. Flow cytometry analysis of the CD19 antigen expression pattern of Nalm-6 and K562 cellsNalm-6 and K562 cells were centrifuged and resuspended in PBS. Add 5×10⁵ cells into each tube. Add 50μl PBS into the negative control tubes and 50μl 2E8 hybridoma tissue culture supernatant into test tubes. Incubate for 20 minutes at 4°C. Wash twice: Add 1 ml PBS; vortex; centrifuge at 250 × g for 5 minutes and remove liquid. Add 2μl GAM-FITC into each tube and incubate for 20 minutes at 4°C in the dark. Wash twice as above and resuspend cells in 200-300 μl of PBS followed by the analysis on a flow cytometer.3. Observation of the effect of water-soluble toxins of mylabris (BM) on the growth of Nalm-6 and K562 cells3.1. Preparation of water-soluble toxins of mylabris (BM stock): Add 1g of dried body of mylabris into a small beaker. Cut it into small pieces and grind it into powder. Add10 ml normal saline (NS) and incubate for 24h at room temperature. Sterilize the solution by a 0.22μm micropore filter and store it at 4°C.3.2. Nalm-6 and K562 cells were inoculated into 24-well plates (10⁵ cells per well), and add more RPMI 1640 medium till the volume of 1800 μl. Add 200μl different diluted BM solution to each well making final BM concentrations ranged from 1:1600 to 1:100 (relative concentration compared to the original BM stock). Add same volume of NS to the control wells. Experiments were carried out in triplicates.3.3. Incubate the cells at 37°C in a humidified atmosphere of 5% CO₂ for 24h, 48h, 72h and 96h respectively before live cell number was counted by trypan blue exclusion method. Cell morphology was observed under the inverted phase contrast microscope and photographs were taken when necessary.4. Thermostability test of BMPut BM solution in a water bath at 100°C for 5 min to see if it still retains its cytotoxicity to Nalm-6 and K562 cells. Cell culture and live cell number counting were performed as above.5. Molecule size determination test of BM cytotoxic component(s)Dialyze the BM solution with NS by a dialysis tubing system (MWCO 10kDa) to see whether the active form of toxin(s) is larger or smaller than 10kDa in its molecular weight. Cell culture and live cell number counting were performed as above.6. Observation of the effect of the 2E8-BM immunotoxin on the growth of Nalm-6 and K562 cells6.1. Preparation of 2E8-BM immunotoxin: Directly add same volume of BM (1:200) solution to the 2E8 culture supernatant and mix it. Incubate for 24h at 4°C, 25°C and 37°C, respectively. Free BM in the mixture was removed by dialysis with NS for 24h.Concentrate the 2E8-BM solution to 1/10 of its original volume by a Amicon Ultra centrifugal filter devices (100k NMWL). Sterilize the solution with a 0.22μm micropore filter and store it at 4°C.6.2. Flow cytometry analysis of the binding affinity of 2E8 antibody in the form of 2E8-BM immunotoxin: The method was described as above.6.3. Experimental design: The experiment was designed as following 4 groups: (1) NS group: add same volume of NS as 2E8-BM; (2) 2E8 group: add same dose of 2E8 antibody as 2E8-BM; (3) BM group: add same dose of BM as 2E8-BM; (4) 2E8-BM group: add 2E8-BM generated at different temperature.6.4. Cell culture and live cell number counting: The method was performed as above.6.5. Flow cytometry analysis: Two-color flow cytometry was performed to determine the apoptosis as soon as possible (usually within 1 hour) after staining the cells with Annexin V-FITC/PI following the instructions of the Annexin V-FITC Kit.7. Statistical analysisData were expressed in mean±SD and analyzed with SPSS13.0 statistical kit. One-Way ANOVA (Dunnet or LSD) was used to compare two groups. P values ≤ 0.05 were defined as statistical significance. Probit Analysis was used to determine IC50.Results1. CD19 antigen expression pattern on Nalm6 and K562 cellsFlow cytometry analysis showed that the positive cell percentages of 2E8 on the Nalm-6 and K562 cells were 99.56% and 1.32%, respectively with the mean fluorescence intensity (MFI) of 2E8 on both the cell lines of 261.29 and 43.80, respectively.2. Cytotoxic effect of BM on Nalm-6 and K562 cells and its other related biologicalfeaturesAn unknown water-soluble form of toxin did exist in the NS extracted solution of mylabris. BM was named for this unknown toxin due to its Chinese medicine name of BanMao. BM was able to kill both Nalm-6 and K562 cells in a non-selective manner, indicating that its cytotoxicity was nonspecific. The cytotoxicity of BM on both cell lines was in a marked dose-response and time-response manner. After incubation with various concentrations of BM for 72h, the IC50s for Nalm-6 and K562 cells were 1:805 and 1:689, respectively. BM showed good thermostability. Bathing in 100°C water for 5 min didn't affect its cytotoxic effect, indicating that BM was very likely not protein in nature. The cytotoxicity of BM was completely lost after dialysis with sufficient amount of NS, indicating that the molecular weight(s) of the putative form of the active toxin(s) in the BM should be less than 10 kDa.3. The binding affinity of 2E8 antibody in the form of 2E8-BM immunotoxin2E8-BM generated at different temperature conditions all well remained its CD19 antigen binding affinity.4. Influence of 2E8-BM immunotoxin on the growth of CD19+ Nalm-6 and CD19-K562 cells2E8-BM could inhibit the growth of Nalm-6 significantly as compared to the controls with same dose of 2E8 mAb or same volume of NS (P<0.05) while this growth inhibition on CD19- K562 cells was not observed (P>0.05), indicating the significant targeting effect by 2E8-BM exerting on the CD19+ rather than CD19- cells. Unfortunately, the 2E8-BM agents generated in this study were not able to inhibit the growth of Nalm-6 completely as the same dose of unconjugated BM did. Their inhibition rates at 96h were only around 50%, significantly lower than unconjugated BM form with 100% cell kill, indicating that the further improvement of the efficiencyof conjugation between 2E8 and BM is warranted.5. Exploration of the mechanism of 2E8-BM target killing effect on CD19+ leukemia cellsThe two-color (Annexin V-FITC/PI) flow cytometry was used to determine the apoptotic cells after treatment with 2E8-BM conjugates. The results showed that the number of apoptotic cells in 2E8-BM group was significantly increased as compared to the negative control. Therefore, the primary mechanism of 2E8-BM cell kill exerted on Nalm-6 cells is via apoptosis induction.Conclusions1. An unknown water-soluble form of toxin named BM does exist in the normal saline extractions of mylabris. The potent cytotoxicity of BM is nonspecific with a good thermostability and dialyzability.2. BM can be conjugated to 2E8 mAb protein through a simple direct incubation method, but its conjugation efficiency remains to be further improved.3. 2E8-BM immunotoxin was successfully prepared. It is able to significantly inhibit the growth of CD19+ Nalm-6 cells leaving the CD19- K562 cells unaffected, indicating the significant targeted effect by 2E8-BM on CD19+ cells.4. The primary mechanism of 2E8-BM cell kill exerted on Nalm-6 cells is via apoptosis induction.