Effects Of Immuotoxin CCL25-PE38 On Controlling T Lineage Acute Lymphoblastic Leukemia

Objective Acute lymphoblastic leukemia (ALL) is one of the highest morbidity and mortality of cancers. Most patients are dead of its metastasis and relapse. Our previous reports demonstrated that the chemokine receptor 9 (CCR9) highly expressed on CD4+T cells in T lineage acute lymphoblastic leukemia (T-ALL), but rarely expressed on normal T cells and other tissues. The studies showed that CCR9 played a pivotal role in the chemotherapy drug resistant and invasion in T-ALL. It showed that blocking the function of chemokine/chemokine receptor may inhibit the progress of malignant tumors. Moreover, CCR9 may be one of the potential strategies for targeted therapy of T-ALL. In this study, we investigated whether the MOLT4 cells which naturally highly express CCR9 can be successfully killed by delivering Pseudomonas exotoxin 38 (PE38) toxin fused with the specific ligand CC chemokine ,25(CCL25).Methods The first, we use Gene splicing by overlap PCR (SOE-PCR) to clone the cDNA of CCL25 and use PCR to clone the cDNA of PE38 from pRB391. Secondly, the PE38 moiety was fused to the C-termini of CCL25 by SOE-PCR. Then the CCL25-PE38 was inserted into pET32a(+)、pET30a(+) and pcDNA3.1 plasmids to construct the expression vectors. The expression of interest proteins were induced by IPTG in prokaryotic expression system and identified by SDS-PAGE and Western Blot. For eukaryotic expression, CCL25-PE38 possessed leader sequence which mediated its secretion into the 293T cells culture supernatants. After transfection, the transfection efficiency was monitored by using indirectly immunofluorescense, the supernatants were preliminarily purified by Millipore ultrafilittration and concentrated supernatants were analyzed by Western Blot. The chemotattractant activity was detected by transwell migration assay. The binding and internalization assays were detected by flow cytometry and confocal laser scanning microscopy. Cell viability was assessed by WST-1 assay and Annexin V-FITC/PI staining. The growth of xenograft CCR9+ tumors established by injection of MOLT4 cells subsutaneously into SCID mice was investigated to analyse the antitumor effects of CCL25-PE38.Results We demonstrated that CCL25, PE38 and CCL25-PE38 can be successfully cloned by SOE-PCR and/or PCR. The expression vectors were constructed by directed cloning CCL25-PE38 into pET32a(+)、pET30a(+) and pcDNA3.1 plasmids. The immunotoxin CCL25-PE38 can be induced by IPTG in prokaryotic expression system, but it mainly presented as protein of inclusion bodies. The immunotoxin CCL25-PE38 can effectively express in 293T cells and secrete into medium. CCL25-PE38 was obtained through premilinary purification. CCL25-PE38, but not PE38, chemoattracted CCR9-positive MOLT4 cells, bound its specific receptor and internalized into the cell cytosol. Moreover, CCL25-PE38 was able to kill MOLT4 cells specifically in vitro via apoptosis inducing effect, but did not affect the viability of CCR9 negative L929 cells. Furthermore, xenograft CCR9+ tumors were established by injection of MOLT4 cells subcutaneously into SCID mice. Tumor suppression was detected in the mice treated with CCL25-PE38.Conclusion This work shows that CCL25-PE38 can be successfully constructed by gene engineering. CCR9 high expressing MOLT4 cells can be successfully killed by delivering PE38 toxin fused with the ligand CCL25. Moreover, CCL25-PE38 can suppress the growth of xenograft CCR9+ tumors. The strategy may have significant therapeutic effect on T-ALL.