Construction Of The Immunotoxin MAIL4R-CTX And Detection Of Its Anti-pancreatic Cancer Activity

1. ObjectiveTo explore an effective new approach that against pancreatic cancer with a novel immunotoxin ,we purify a effective Cobra venom cytotoxin(CTX) from cobra (Naja Naja atra)venom. Then CTX was selected as a "warheads" and anti-IL4-R monoclonal antibody (MAIL4R) as a carrier . With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX was connected together and the immunotoxin MAIL4R-CTX was obtained. The selective killing effects on pancreatic carcinoma cells which has high expression of IL-4R was observed and compared so as to provide experimental data for new treatment method of pancreatic cancer.2. Materials and methods1. Isolation and Purification of CTX: In this study, the crude venom of cobra venom(Naja atra) was first isolated on SP-Sephadex C-25 cation-exchange column, The effect of tenth～thirteenth protein peaks to the rat isolated heart perfuse preparations and the rat isolated phrenic nerve-diaphragm preparations then be observed; protein peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each; SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains be used to assess the purity of peak XIII and its mol. wt of them were calculated.2. Immunotoxin MAIL4R-CTX preparation: In room temperature, the cytotoxin of Naja naja atra reacted with SPDP to form CTX-PDP. Then we made the anti-interleukin4-receptor monoclonal antibody and SPDP have the same reaction to form anti-IL-4-receptor monoclonal antibody-PDP, and it was reduced to monoclonal antibody-SH by DTT. In the end, the monoclonal antibody-SH was thoroughly mixed with CTX-PDP in room temperature for at least 24 hours to produce a new type immunotoxin MAIL4R-CTX. 3. Identified the composition of immunotoxin MAIL4R-CTX by the means of SDS-PAGE polyacrylamide gel electrophoresis and double immunodiffusion and dotimmunobindingassay.4. Immunohistochemical technique was used to detect the expression of IL-4R in normal and pancreatic cancer specimens and immunocytochemical technique was used to detect the expression of IL-4R in human pancreatic cancer cell lines (PANC-1 and BxPC-3) and human NSCLC cells.5. Binding ability of the conjugate to BxPC-3 and PANC-1 cells with highly expressed IL-4R and H1299 cells without IL-4R expression was measured by DAB method.8. Using MTT method, Cytotoxic activity of AIL-4R ,CTX and CTX-AIL-4R were tested in PANC-1,BXPC-3 and H1299 cell lines in vitro respectively.3. Results1. Isolation and Purification of CTX: The crude venom of cobra venom(Naja atra) was isolated on SP-Sephadex C-25 cation-exchange column and fractionated into 14 protein peaks; The protein peak XIII could produce the rat isolated Langendorffs preparations to decrease their contracture heights, ultimately leading to complete cardiac arrest. Peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each and a single protein peak be obtained. It migrated as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains which showed that it was homogeneous with one band. Its mol. wt was calculated to be 7.381 kDa orderly by Image Master VDS. The results showed that the protein peak XIII was a CTX and then we named it CTX in this paper.2. Gel filtration chromatography of immunotoxin: The reaction products of CTX and SPDP were loaded on Superdex30 gel column and three major protein peaks were obtained, in which the first was CTX-PDP with large molecular weight. The reaction products of MAIL4R and SPDP were loaded on Superdex30 gel column, and 2 peaks were obtained, in which the first peak was MAIL4R-PDP; After reduction of MAIL4R-PDP by DTT, two main peaks were obtained after filtration on the column, in which the first peak was MAIL4R-SH. Finally, filtration of excessive reaction products of CTX-PDP and MAIL4R-SH were carried out on Superdex30 gel column with two protein peaks obtained, in which the first peak was the immunotoxin MAIL4R-CTX, and the second peak was unbound CTX-PDP.3. In reduced SDS-PAGE polyacrylamide gel electrophoresis, MAIL4R-CTX showed two zones of minor molecular weight protein. Then antivenomous serum made immunoprecipitation among MAIL4R-CTX, CTX, CTX-PDP and peakⅡof MAIL4R-CTX, and eventually the first three samples formed the lines of sedimentation. It is found in Dotimmunobindingassay of the immunotoxin that, immunotoxin MAIL4R-CTX not only had strong antigen-antibody response with anti-cobra venom antibody, but also could directly react with goat anti-mouse antibody, suggesting that MAIL4R-CTX contained both the cobra venom cytotoxin and mouse antibody (ie MAIL4R)4. Normal pancreas tissue samples did not stain or showed weak positive staining for IL-4R. In contrast, all 26 pancreatic cancer specimens overexpress IL-4R; Both human pancreatic cancer cell lines BxPC-3and PANC-1 were stained brown, while human lung cancer cells H1299 who did not express IL-4R were not stained.5. BxPC-3 and PANC-1 cells have high expression of IL-4R were stained brown and H1299 cells that do not express IL-4R were not stained in DAB method.6. By MTT method, we observed that MAIL4R-CTX significantly inhibited growth of BxPC-3, PANC-1 cells that have high expression of IL-4R at the concentration of 1.2 ug / ml, while for H1299 cells with no or low expression of IL-4R, inhibitory effect appeared when the concentration was more than 5 ug / ml. At the concentration of 18.75 ug / ml, the cell killing rate of MAIL4R-CTX on PANC-1 and BxPC-3 cells was 86.4% and 95.2% respectively, while it was only 26.8% on H1299 cells; CTX had rapid killing effects on PANC-1, BxPC-3 and H1299 cells. After 4h's action,the inhibition rate of CTX in the concentration of 8 ug / ml on three cell strains was 88.5%, 87.2% and 90% respectively; Monoclonal antibody of interleukin-4 receptor had no obvious killing effect on the above cells. 4. Conclusions1. A purified CTX isoforms from the Naja atra venom was obtained by SP-Sephadex C-25 cation-exchange chromatography, Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography step by step.2. With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX can be successful connected together to be a novel immunotoxin MAIL4R-CTX.3. IL-4R overexpress in pancreatic cancer specimens and cells but not in normal pancreas. Immunotoxin MAIL4R-CTX had selective killing effect on pancreatic cancer cells which have high expression of IL-4R.