The Cloning And Expression Of Anti-MRP3scFv And STRAIL Fusion Protein And Study Of Its Targeted Apoptosis-inducing Effect On Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and most malignantprimary brain tumor in adults, classified as grade IV astrocytoma by the WHO. Despitetechnological advances in surgical management, combined regimens of radiotherapywith new generation chemotherapy, the median survival for these patients is15monthsand there is an urgent need for new treatments. Antibody-based therapy shows enhancedspecificity for killing tumor cells and minimizes the toxicity to normal tissues,represents new development of tumor immunotherapy.Overexpression of multidrug resistance protein3(MRP3) in GBM solid tumorsand cell lines and relative lack of expression in normal brain, along with its localizationto the cell membrane, make MRP3an excellent molecular target for antibody-basedtherapy. In2010, Kuan’s laboratory had isolated3recombinant, fully humansingle-chain antibody fragment (scFv) antibodies, which specifically react with theextracellular N-terminus of human MRP3. These scFvs are valuable antibodies forGBM antibody-based therapy.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also designatedas APO-2ligand, is a member of the TNF family. TRAIL activates the extrinsicpathway of apoptosis by binding to TRAIL-R1(DR4) or TRAIL-R2(DR5). TRAIL isprobably one of the most promising anti-tumor agents based on the pronouncedselective activity towards malignant cells and its lack of activity towards normal cells.A soluble form of TRAIL (sTRAIL) can be generated due to proteolytic cleavage of theextracellular domain of TRAIL or alternative mRNA splicing and thereby still retainingtumour selective pro-apoptotic activity.In this study, by recombinant DNA technology, we for the first time constructed a novel fusion protein, designated antiMRP3(scFv)-sTRAIL, comprising the humanantiMRP3scFv fragment genetically linked to the N-terminus of human sTRAIL withlinker (Gly4-Ser)3. The antiMRP3(scFv)-sTRAIL fusion protein is designed to induceapoptosis in GBM cells only after specific binding of antiMRP3(scFv)-sTRAIL to theGBM cells surface antigen MRP3. The paper discourses the effects ofantiMRP3(scFv)-sTRAIL on apoptosis in GBM cells and its mechanism upon thefollowing aspects.PartⅠ The expression and purification of antiMRP3(scFv)-sTRAIL fusionprotein and identification of its biological functions.To express and purify antiMRP3(scFv)-sTRAIL fusion protein and to observe itsbiological functions. PCR was employed to amplify the gene sequences ofantiMRP3(scFv) and sTRAIL. The recombinant expression plasmidpMAL-antiMRP3(scFv)-sTRAIL was constructed by inserting antiMRP3(scFv) andsTRAIL gene sequences into pMAL-c2prokaryotic expression vector. Clonescontaining inserted sequences were checked by colony PCR, restriction digestion andDNA sequencing analysis. The partially purified antiMRP3(scFv)-sTRAIL with maltosebinding protein (MBP) tag was obtained by transforming the recombinant expressionplasmid pMAL-antiMRP3(scFv)-sTRAIL into the E.coli BL21with IPTG inductionand amylose resin chromatography. Found electroband of purified fusion protein at92kD by SDS-PAGE analysis.The expression rate of the fusion protein in E.coli BL21wasabout30%and purified fusion protein reached a purity of95%.MRP3-positive U251glioblastoma multiforme cells were treated with differentdose of antiMRP3(scFv)-sTRAIL fusion protein for24h, parallel MBP protein isshown as a control. It was found by MTT test that antiMRP3(scFv)-sTRAILsignificantly inhibited the proliferation of U251cells in a dose-dependently manner,whereas MBP showed none of these effect. Then U251cells were cultured for24h withIC50dose of recombinant antiMRP3(scFv)-sTRAIL. Afterwards, cells morphology wasobserved under microscope. U251cells treated with antiMRP3(scFv)-sTRAIL showedobvious apoptosis morphological changes, whereas MBP and untreated group showedno such changes.The results indicate that recombinant antiMRP3(scFv)-sTRAIL fusion protein is successfully expressed in Escherichia coli and exhibites a potent apoptosis-inducingeffect toward U251glioblastoma multiforme cells, laying a solid foundation for furtherinvestigation of its targeted apoptosis induction activity toward U251glioblastomamultiforme cells.PartⅡ The targeted apoptosis-inducing effect of antiMRP3(scFv)-sTRAILfusion protein on U251glioblastoma multiforme cells.Target antigen-restricted binding of antiMRP3(scFv)-sTRAIL to the cell surfacewas assessed by flow cytometry using cell lines U251(MRP3-positive) and Jurkat(MRP3-negative). antiMRP3(scFv)-sTRAIL showed specific binding to theMRP3-positive tumor cell line U251, which could be specifically inhibited bypreincubation with parental antiMRP3(scFv), whereas antiMRP3(scFv)-sTRAILshowed minimal binding to MRP3-negative Jurkat cells, indicated that the targetedbinding of antiMRP3(scFv)-sTRAIL to U251cells was MRP3antigen-restricted.U251cells were treated with different dose of antiMRP3(scFv)-sTRAIL for24hin the presence or absence of parental antiMRP3(scFv) and TRAIL-neutralizing mAb2E5, then viability was examined by MTT assay. When the dose ofantiMRP3(scFv)-sTRAIL increased, the viability of U251cells became lowersynchronously in24h, whereas MBP protein showed no killing effect towards U251cells. The killing effect of antiMRP3(scFv)-sTRAIL towards U251cells was stronglyinhibited in the presence of parental antiMRP3(scFv) or TRAIL-neutralizing mAb2E5.IC50dose (62.5nmol/L) of antiMRP3(scFv)-sTRAIL was added into media of U251cells for8,12and24h, then apoptotic percentage was examined by flow cytometry. Itwas found by flow cytometry analysis that there were significantly apoptosis inantiMRP3(scFv)-sTRAIL treated U251cells at8h and increased in24h as compared toparental antiMRP3(scFv) and untreated U251cells. Consistented with the MTT assay,the apoptosis of U251cells can be significant inhibited by preincubation with parentalantiMRP3(scFv) or TRAIL-neutralizing mAb2E5.The results indicate that treatment with antiMRP3(scFv)-sTRAIL results in thespecific binding to the cell surface of MRP3-positive cells only. The pro-apoptoticactivity of antiMRP3(scFv)-sTRAIL can be inhibited by preincubation with parentalantiMRP3(scFv) or TRAIL-neutralizing antibody, indicated that antiMRP3(scFv) is the targeting moiety and sTRAIL is the cytotoxic moiety. Targeted antigen-restrictedbinding increases the pro-apoptotic activity of antiMRP3(scFv)-sTRAIL fusion protein.Importantly, off-target binding by antiMRP3(scFv)-sTRAIL shows no sign of toxicity.Part Ⅲ TRAIL-R1and TRAIL-R2activation by antiMRP3(scFv)-sTRAIL fusion proteinApoptosis can be mediated via binding of TRAIL to TRAIL-R1(DR4) orTRAIL-R2(DR5). Many tumors express higher levels of TRAIL-R2compared withTRAIL-R1, whereas TRAIL-R2signaling is only poorly activated by sTRAIL. U251cells showed clear TRAIL-R1and TRAIL-R2expression and a higher levels ofTRAIL-R2compared with TRAIL-R1by real-time PCR analysis. To analyze theinvolvement of TRAIL-R1and TRAIL-R2in apoptosis in U251cells we usedanti-TRAIL-R1and anti-TRAIL-R2blocking antibodies. U251cells were treated withIC50dose of antiMRP3(scFv)-sTRAIL for24h after incubation of the antibodies for1hand specific apoptosis was examined by flow cytometry. Blocking of TRAIL-R1reduced apoptosis induction by antiMRP3(scFv)-sTRAIL by28%, blocking ofTRAIL-R2and both receptors resulted in35%and46%apoptosis reduction. Westernblot analysis revealed that recombinant antiMRP3(scFv)-sTRAIL activated the extrinsicpathway, resulting in caspase-dependent apoptosis.From these experimental data, we conclude that antigen-restricted bindingconverts antiMRP3(scFv)-sTRAIL into a membrane-bound form of TRAIL that iscapable of signaling extrinsic apoptosis pathway not only through TRAIL-R1but alsothrough TRAIL-R2.