Introduction and Objectives As a potent ligand for an immunotoxin, many moieties have been tried. However, majority of those moieties were large molecules or proteins which may produce human anti-toxin antibodies or were radioisotopes which may generate the concerns of the radioactive contaminations. Thus a small, non-protein toxin is desired to avoid the above disadvantages. Our hypothesis was that there could be a water soluble toxin in the saline extract of a Chinese medicine Mylabris phalerata Pallas (SEM) and easy to be conjugated with antibody proteins to generate immunotoxin. In this study, the toxin (s) in SEM was used to conjugate with our anti-CD19 monoclonal antibody ZCH-4-2E8 (2E8) protein and targeting efficacy on leukemia cells was tested to elucidate its leukemia-specific targeting efficacy and its mechanisms.

Materials and Methods The physical and biological activities of SEM were tested by heat treatment, dialysis, SDS-PAGE and cell culture analyses. SEM was conjugated with 2E8 antibody using direct incubation at 37°C for 24 hours. Targeting efficacy were evaluated by cell culture. Cell apoptosis was analyzed by using Annexin V-FITC/Propidium Iodide double staining and flow cytometry method.

Results Different concentrations of SEM at 1:200, 1:400, 1:800 and 1:1600 had different cell growth inhibition on both Nalm-6 and K562 cells non-specifically. The concentration at 1:200 generated the most potent cell kill and all the cells were dead after 72 hr culture. Concentrations below 1:800 or lower generated a minimal efficacy of cell kill and the concentration at 1:1600 showed no significant impact as compared to that of control (P > 0.05). Time course investigations showed that the growth inhibition was increased with incubation time. The IC50 for Nalm-6 cells was 1:805 while that for K562 was 1:689. The prudent toxin was heat stable as treatment of 100°C for 5 min did not diminish its cytotoxic activity. The toxin could be dialyzed through the 10kDa pore-size membrane. Electrophoresis analyses with SDS-PAGE showed no protein bands on the gel after staining with Coomassie brilliant blue. The natural conjugation could occur when CD19 antibody supernatant was incubated with the same volume of 1:200 SEM at 37°C for 24 hr. After 144 hr incubation, the inhibition rate of 2E8-SEM (1:2 of 2E8 supernatant with ≤ 1:400 of SEM) on Nalm-6 cells was 55.11% If normal saline was considered as negative control (0%), which was significantly higher than that of non-SEM conjugated 2E8 antibody at 1:2 of supernatant (16.01% of inhibition rate, P < 0.05). The inhibitions were not observed when 2E8-SEM (12.38% of inhibition, P > 0.05) and non-SEM conjugated 2E8 antibody (8.98% of inhibition, P > 0.05) were co-cultured with CD19- K562 cells at the same conditions as compared to that (0%) of normal saline. The cell death was via apoptotic process based on the flow cytometry analysis using Annexin V-FITC/Propidium Iodide double staining.

Conclusions The strong and non-specific cytotoxic water soluble toxin(s) existed in SEM which was able to bind to antibody protein using simple incubation at 37°C showing significant targeting efficacy on Nalm-6 leukemia cells. The further study on this new toxin and the optimal way of conjugation are currently under investigation to improve the binding and the targeting efficacies.

Disclosure: No relevant conflicts of interest to declare.

Author notes

(This work was supported by a grant (Z205166) from Zhejiang Provincial Natural Scientific Fundation).