Targeting Killing Of The B-lineage Leukemia Stem Cells And Mechanism With Norcantharidin Encapsulated Liposomes Modified With A Novel CD19 Monoclonal Antibody 2E8 In Vitro

Background and aimsB lineage lymphocytic leukemia(B-lineage ALL) is a common hematopoietic malignancy in children.Chemotherapy is currently the principal treatment modality of the disease.Despite the treatment outcome has recently been improved significantly with this type of therapy,about 20%of the patients remain treatment failure due to either the systemic nonspecific cytotoxicities caused by the therapeutic agents or the disease relapse with incomplete eradication of all the leukemia cells.A recent study has demonstrated that when purified CD34+ CD38 + CD19+ and CD34+CD38-CD19+ bone marrow(BM) or peripheral blood(PB) cells from pediatric B-lineage ALL patients were intravenously injected into lethally irradiated newborn non obese diabetes/severe combined immunodeficiency(NOD/SCID/IL2rc) null mice, CD34+CD38+CD19+ and CD34+CD38-CD19+ cells were able to initiate B-lineage ALL in primary recipients,whereas purified CD34+CD38-CD19-CD10- cells showed normal human hematopoietic repopulation.Furthermore,transplantation of CD34+CD38+CD19+ cells resulted in the development of B lineage ALL in secondary recipients.Clearly,the CD34+CD19+CD38+/CD38- cell populations possess the capacity for indefinite proliferation and self-renewal which are currently named a new class of leukemia stem cells(LSC),B-lineage LSCs.Therefore,the CD19 antigen is a specific marker of B lineage ALL which can be an excellent target to both B-lineage LSCs and their progeny.Monoclonal antibody(mAb)-based targeted therapy is one of the most promising methods for leukemia.CD20 and CD33 monoclonal antibody have been widely used for the treatment of B lineage non-Hodgkin's lymphoma(B-NHL) and of acute myeloid leukemia(AML),respectively.The immunoliposome is one branch of targeting therapies with double features of monoclonal antibody-oriented and liposome drug delivery.Immunoliposomes encapsulating anticancer drugs not only can specifically transfer a large number of drug molecules to tumor cells for tumor kill,but also significantly reduce the non-specific cytotoxicity of the drugs to normal tissues.It has been reported that immunoliposomes can even overcome the resistance of anticancer drugs.Currently,the encouraging results via immunoliposomes have been used in studying the treatments of hematologic malignancies.For instances,anti-CD 19 targeted liposomal anticancer drugs,i.e.doxorubicin(DOX) or vincristine(VCR) have been seen the association with increased specific cyotoxicity to B lineage leukemia cells relative to non-targeted liposomes in vitro,they can also prolong the survival in mice compared to non-targeted liposomes or free DXR in vivo.Anti-CD19 targeted liposomal imatinib can transfer a large number of imatinib molecules to SUP-15 cells,so it overcomes the resistance of the leukemia cells to imatinib which are not inhibited by lower concentration of the drug.However,these reported immunoliposomes can not effectively kill the B-lineage LSCs because the chemotherapeutic agents encapsulated in the liposomes,such as DOX,VCR and imatinib are unable to exert the cytotoxicity to LSCs in quiescent state.Norcantharidin(NCTD) is a stem cell-specific agent capable of inducing apoptosis in AML-LSCs by inhibiting the stem cell-related anti-apoptotic axis - the HLF/SLUG activity.But,NCTD killing effect on normal HSCs nonspecifically attenuated the advantages of its treatment to LSCs.ZCH-4-2E8(2E8) is a novel anti-human CD19 monoclonal antibody generated in our laboratory.In previous studies,we have confirmed that 2E8-NCTD immunotoxin confers a clear role in targeting killing of CD19+ leukemic cells.In this study,we intended to develop 2E8-NCTD-liposomes by encapsulating NCTD into liposomes modified with the 2E8 monoclonal antibody;to observe the targeting recognition and killing of B lineage LSCs and their progeny;to elucidate the targeting killing mechanism of 2E8-NCTD-liposomes to build the foundation for further development of the therapeutic agent 2E8-NCTD-liposomes in vivo.Methods(1) Preparation of 2E8 monoclonal antibody and identification:The ascites containing 2E8 antibody(Ab) derived from Balb/c mice were purified by gel filtration chromatography-SephacryL S-300-HR column.The targeting efficiency of 2E8 on CD19+ Nalm-6 cells was detected by flow cytometry(FCM).The purity and molecular weight(MW) of the heavy and light chains were identified by SDS-PAGE.(2) Preparation and evaluation of 2E8-blank-liposomes:A standard curve of Stewart analysis was established using spectrophotometer(detection of the content of phospholipid in liposomes);2E8 antibody was connected with blank-liposomes by using post-incorporation technology;the successful link between 2E8 antibody and blank-liposomes was proved by using FCM.The effects of 2E8 and Mal-PEG₂₀₀₀-DSPE lipid molar ratio of the antibody density on the surface of liposomes were determined by using Stewart analysis and BCA protein quantification assay Kit.The mean particle size and stability of 2E8-blank-liposomes were determined by a laser particle size analyzer.The targeting efficiency of 2E8-blank-liposomes to different cell lines and normal T,B lymphocytes and the binding stability were evaluated by using FCM.The internalization of 2E8-blank-liposomes in Nalm-6 cells was observed by using FCM and Laser scanning confocal microscope. (3) Preparation and evaluation of 2E8-NCTD-liposomes:NCTD-liposomes were prepared by the method of thin-film dispersion.The rate of liposome encapsulation was quantified by high performance liquid chromatography(HPLC).The mean particle size of 2E8-NCTD-liposomes was determined by the laser particle size analyzer.The targeting efficiency of 2E8-NCTD-liposomes to different cell lines and B lineage LSCs was evaluated by using FCM.The targeting discrepancy of 2E8-NCTD-liposomes to different cell lines was observed by Laser scanning confocal microscope.The cytotoxicity of 2E8-NCTD-liposomes,NCTD- liposomes and free NCTD to different cell lines were assayed by MTT method.(4) The targeting killing mechanism of 2E8-NCTD-liposomes:The phenomenon of CD19 receptor-mediated internalization of 2E8-NCTD-liposomes was demonstrated by Laser scanning confocal microscope and HPLC.The apoptosis changes of Nalm-6 cells treated by 2E8-NCTD-liposomes were observed through HE staining and agarose gel electrophoresis.The early apoptosis of Nalm-6 cells treated by 2E8-NCTD-liposomes,NCTD-liposomes and free NCTD were quantitatively assessed by FCM.The relative expressions of anti-apoptosis factors-HLF related to B lineage LSCs and its downstream pro-apoptotic factor- NFIL 3 in Nalm-6 treated by 2E8-NCTD-liposomes at the transcriptional level was detected by real-time fluorescence quantitative PCR method.Results(1) Preparation and identification of 2E8 monoclonal antibody:The targeting efficiency of 2E8 antibody on CD19+ Nalm-6 was 99.93%,close to that(99.94%) of a positive control antibody CD19 PE.The heavy chain and light chain molecular weight of 2E8 were approximately 70 kDa and 23 kDa,respectively.(2) Preparation and evaluation of 2E8-blank-liposomes:As the molar ratio of 2E8/Mal-PEG2000-DSPE increase,the antibody density on the surface of liposomes gradually increased,but the coupling efficiency of antibodies gradually decreased. When the 2E8/Mal-PEG2000-DSPE molar ratio was 1:50,the site of Mal-PEG2000-DSPE connection with the 2E8 was saturated.2E8 antibody was successfully linked to liposomes with the evidence of one to one linear relationship between liposomes labeled with Rhodamine(red fluorescence) and 2E8 antibody labeled with GAM-FITC(green fluorescence) in the upper right quadrant in Nalm-6 cells co-cultured with 2E8-blank-liposomes.The average particle size of 2E8-blank-liposomes and blank-liposomes were 121.25 nm and 106.30nm, respectively;and after they were placed in the HBS buffer(containing 3%of Tween 80 or the HBS buffer containing 50%calf serum in 4℃) up to 24 days,no significant changes in their average sizes were observed.During the course of preparing 2E8-blank-liposomes,the 2E8 antibody molecules were incorporated into the surface of liposomes in their intact conformation.The targeting efficiencies of 2E8-blank-liposomes on the Nalm-6 or Raji cells in either direct or indirect method were almost close to 100%,significantly higher than that on Molt-3 or acute erythroleukemia cell line K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).And with the extension of the incubation time and changes in incubation medium(PBS,50%fetal calf serum,50%human plasma),the binding of 2E8-blank-liposomes to Nalm-6 cells was stable with no significant changes in the average fluorescence intensity. Free 2E8 can block the specific binding of 2E8-blank-liposomes to Nalm-6 cells. The targeting efficiency of 2E8-blank-liposomes on the peripheral blood of normal B lymphocytes was(97.65±1.30)%,significantly higher than that on normal T cells ((4.70±1.78)%,P＜0.01).2E8-blank-liposomes can be internalized into Nalm-6 cells in the incubation periods of 30mins-8hs.(3) Preparation and evaluation of 2ES-NCTD-liposomes:The encapsulating efficiency of NCTD-liposomes was 46.51%.The average particle size of 2E8-NCTD-liposomes and NCTD-liposomes were 118.32 nm and 104.12 nm, respectively;Similar to 2E8-blank-liposomes,the targeting efficiency of 2E8-NCTD-liposomes on the Nalm-6 or Raji cells significantly higher than that on Molt-3 or K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).The targeting efficiency of 2E8-NCTD-liposomes on CD34+CD19+CD38+ or CD34+CD19+CD38-cells were 98.00%or 92.45%,while that of NCTD-liposomes on both the cell subpopulations were less than 5%(P＞0.05).Red Rhodamine and green GAM-FITC fluorescence signals co-existed on the surface of Nalm-6 cells,whereas similar results were not observed on the surface of Molt-3 cells.The killing effects of 2E8-NCTD-liposomes on the Nalm-6 cells for 24-72 hrs showed a time- and dose-dependent manner in 10-50μM range of concentration.When the Nalm-6 cells were treated with free NCTD and 2E8-NCTD-liposomes for 24-72 hrs,the inhibitory rate of Nalm-6 cells by 2E8-NCTD-liposomes was significantly higher than that by the same concentration of NCTD-liposomes or free NCTD;It was also significantly higher than that of Molt-3 cells inhibited by the 2E8-NCTD liposomes (P＜0.01);The IC50 value of 2E8-NCTD-liposomes was significantly lower that that of NCTD-liposomes and free NCTD on Nalm-6 cells;It also was significantly lower than that of 2E8-NCTD-liposomes on Molt-3 cells.(4) The targeting killing mechanism of 2E8-NCTD-liposomes: 2E8-NCTD-liposomes were internalized into Nalm-6 cells through the CD19 receptor with the evidence of gradual accumulation of red fluorescence in the cytoplasm of Nalm-6 cells.The transferring efficiency of 2E8-NCTD-lipsomes for NCTD in Nalm-6 cells was four times more than that in Molt-3 cells;It was also two times more than that of NCTD-liposomes for NCTD in Nalm-6 cells.There were typical apoptotic changes,including cell shrinkage,nuclear condensation and fragmentation,apoptotic body formation,in Nalm-6 cells treated by the IC50 concentration of 2E8-NCTD-liposomes.Agarose gel electrophoresis of 2E8-NCTD-liposome-treated Naml-6 cells revealed a ladder-like pattern of DNA fragments,indicating the apoptosis induction was involved in the process of cell kill. 2E8-NCTD-liposomes induce the apoptosis of Nalm-6 cells in a dose-dependent manner.The apoptotic percentage of Nalm-6 cells((47.55±0.02)%) induced by the IC50 concentration of 2Eg-NCTD-liposomes was significantly higher than that induced by the same concentrations of either free NCTD((24.22±0.02)%,P＜0.05) %or NCTD-liposomes((27.09±0.01)%,P＜0.05)%for the same period of incubation time;It was also significantly higher than that of Molt-3 cells induced by the same concentration of 2E8-NCTD-liposomes.The relative expression of HLF decreased while that of NFIL 3 increased in Nalm-6 treated by the IC50 concentration of 2E8-NCTD-liposomes for 48 hrs in mRNA level,compared to that in untreated Nalm-6 cells.Conclusions:(1) CD19 mAb modified immunoliposomes,i.e.2E8-blank-liposomes and 2E8-NCTD-liposomes are successfully prepared.(2) 2E8-blank-liposomes can be used as a useful drug carrier targeting B lineage LSC (CD34CD19 + CD38 +/-) and their progeny.(3) 2E8-NCTD-liposomes can exert targeted cytotoxicity to CD19+ leukemia cells.(4) The targeting killing mechanism of 2E8-NCTD-liposomes is that NCTD is transferred into CD19+ leukemia cells through CD19 mediating internalization of 2E8-NCTD-liposomes and induced apoptosis of the targeted cells.(5) 2ES-NCTD-liposomes-induced apoptosis of B lineage LSCs is closely associated with inhibiting the LSC-related anti-apoptotic axis,namely,reducing the expression of the LSC-related anti-apoptotic factor-HLF gene and increasing the expression of the downstream pro-apoptotic factor-NFIL3 gene.