Preparation, Identification, Biological Characterization Of A Novel Monoclonal Antibody ZCH-2F10Recognizing B-lineage Leukemia Cell Surface Antigen And Its Clinical Prognostic Significance

ObjectivesLeukemia stem cells (LSCs) are defined as a small subpopulation having self-renewal ability, and unlimited repopulating and differentiation potential, and are maintained by asymmetric cell division. It is believed that LSCs are at least partially responsible for refractory and relapsing tumor resistance to therapy, leading to recurrence and metastasis. Acute lymphoblastic leukemia (ALL) is the most common type of pediatric hematological malignancy. As a heterogeneous group of diseases, B-cell acute lymphoblastic leukemia (B-ALL) is considered to represent the clonal proliferation of malignant-transformed B-lymphoid progenitors. A growing number of studies seem to confirm the leukemic stem cell hypothesis in B-ALL yet the data about the phenotype of LSC fraction are contradictory. Recently, Nishida et al. reported that CD9might be a useful marker for the identification of LSCs in B-ALL, and targeted therapies against CD9and its downstream signals would be novel therapeutic approaches. Therefore, in order to further explore the biological and clinical significance of CD9in B-ALL, we conducted the study including the following three parts:the first part is the preparation and identification of ZCH (Zhejiang Children’s Hospital)-2F10antibody (Ab); the second part is the study on the biological characteristics of antibody2F10; the third part is the study on the correlation between percentages of CD19±D34±2F10±cells at diagnosis and level of minimal residual disease in childhood B-ALL. Methods1Preparation and identification of2F10Ab1.1Generation of2F10hybridoma:The hybridoma was generated by the fusion of SP2/0myeloma cells with spleen cells obtained from mice immunized with Nalm-6cells. A steadily producing monoclonal antibody cell line was established after subcloning using limiting dilution method. Flow cytometry (FCM) was used for isotype determination of2F10Ab.1.2Sequencing of the variable regions for both heavy (VH2F10) and light (VL2F10) chain genes from2F10hybridoma cell line:Total RNA was prepared from2F10hybridoma cell line using the Trizol kit. The VH2F10and VL2F10genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) with family specific primer pairs, respectively. After sequencing with automatic DNA sequencer, the sequences of the genes interested were analyzed online.1.3Preparation of2F10-FITC:The ascites containing2F10Ab derived from BALB/c mice was purified by affinity chromatography using SPA Sepharose column. The purity and molecular weight (MW) of the heavy and light chains were identified by SDS-PAGE.2F10-FITC was prepared according to Marshall’s method and evaluated by FCM.1.4Identification of2F10antigen (Ag) protein:The MW of2F10Ag was evaluated using SDS-PAGE followed by western blot.2F10Ag was separated and purified from Nalm-6cell lysate by immunoprecipitation (IP) and assessed using SDS-PAGE. The Ag band on the gel was harvested and delivered for sequencing by tandem mass spectrometry (MS). Additionally, blocking experiment was used for further identification of the Ag2F10Ab binding to.1.5The expression pattern of2F10Ag:The reactivities of2F10and CD9(clone name:M-L13) with human leukemia cell lines, normal peripheral blood cells, blast cells from childhood acute leukemia patients were analyzed and compared by FCM. 2Study on the biological characteristics of2F10Ab2.1Study on the biological characteristics of2F10antigen-antibody system:The internalization of2F10was studied by the method applying papain digestion and flow cytometry. The capacity of2F10Ab specifically binding to2F10Ag and activating complement was determined by complement dependent cytotoxicity assay (CDC). Bromodeoxyuridine (BrdU) was used to label Nalm-6cells to enable detection of cells in the S-phase of the cell cycle, and then the cell subpopulation proliferation was analyzed by the simultaneous detection of BrdU and membrane antigen by FCM.2.2The effect of2F10Ab on leukemia animal model:First, the nude mice were inoculated with Nalm-6cells via the tail veins to establish the B-cell acute lymphoblastic leukemia animal model, and then one (early treatment group) or two (late treatment group) weeks later were injected with2F10Ab via their tail veins. The living conditions of the mice were monitored daily, and the survival was calculated after inoculation with Nalm-6cells.3Study on the correlation between percentages of CD19±D34±2F10±cells at diagnosis and level of minimal residual disease in childhood B-ALL:At diagnosis, the characteristic immnophenotype of the leukemia cells was determined by FCM and percentages of CD34+CD38-, CD19±CD34±2F10±and CD19±CD34±CD9±cells were calculated. Then, level of minimal residual disease (MRD) at day22during therapy was studied. Spearman rank correlation coefficients were calculated for the analysis of statistical associations between parameters.Results1Preparation and identification of2F10Ab:1.1Generation of2F10hybridoma:2F10hybridoma clone was generated by cell fusion and screened with FCM. Further subcloning was made to obtain the high purity monoclonal cell line. Mouse subclass of2F10Ab was determined using a murine antibody subclass kit and FCM analysis. The results showed that the positive rates of2F10reactivity with goat anti-mouse IgGl and goat anti-mouse κ were99.38%and99.61%, respectively, indicating the subclass of2F10Ab was murine IgGIκ.1.2Sequencing of the VH2F10and VL2F10genes from2F10hybridoma cell line:The VH2F10gene consisted of360bps encoding a peptide of120amino acid residues, while the VL2F10gene consisted of348bps encoding a peptide of116amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST, the VH2F10and VL2F10genes belonged to mouse IGVH and mouse IGVκ subclasses, respectively. Both VH and VL chains had4definitive frame regions (FR) and3complementary determinant regions (CDR).1.3Preparation of2F10-FITC:2F10Ab presented two bands of approximately54kDa and25kDa, respectively, when identified by SDS-PAGE. Fluorescein isothiocyanate conjugated2F10Ab (2F10-FITC) was successfully generated according to Marshall’s method. The titration test showed0.3μg2F10-FITC was able to saturate1×106Nalm-6cells.1.4Identification of2F10Ag:2F10Ag was identified as a protein with a MW of24kDa or so. The sequence of the Ag protein provided by tandem MS suggested that CD9Ag should be a possible candidate. Blocking experiment showed that the positive rates of CD9-FITC before and after blocking by2F10Ab were90.34%vs.0.83%, respectively, indicating that2F10Ag belonged to CD9protein.1.5The expression pattern of2F10Ag:2F10Ag and CD9Ag were both highly expressed on Nalm-6cells and MEG-1cells. Additionally,2F10Ag was also relatively highly expressed on HL-60cells. FCM analysis carried out with2F10-FITC, CD9-FITC and other fluorescent Abs specific to normal human peripheral blood cell antigens revealed that2F10Ag and CD9Ag were not expressed on the surface of red blood cells, whereas it was low expressed on B and T cells’, highly expressed on granulocytes’, monocytes’and platelets’surfaces. In the blasts of childhood acute leukemia,2F10Ag and CD9Ag were both heterogeneously expressed on the membrane.2Study on the biological characteristics of2F10Ab 2.1Study on the biological characteristics of2F10antigen-antibody system:2F10Ag could mediate the internalization of2F10-FITC rapidly after their binding both at4℃and37℃.2F10Ab could specifically bind to2F10Ag and activate complement to kill Nalm-6cells through CDC (0.1μg2F10Ab:10μl complement:5×104cells:the death rate of Nalm-6cells was78.88±5.23%). FCM showed that, for Nalm-6cells, the frequency of CD19+2F10+subpopulation increased from G1to S phase. Additionally, compared with the Nalm-6cells with high proliferative activity, the frequencies of CD19+2F10-, CD19-2F10-and CD19-2F10+subpopulations were significantly higher in those Nalm-6cells with low proliferative activity.2.2The effect of2F10Ab on leukemia animal model:Nude mice were used to establish the animal model of B-cell acute lymphoblastic leukemia. The mice became paralyzed rapidly seven weeks after inoculation followed by weight loss with bent spines, hogback, cachexia until death. In the late treatment group, the survival seemed to be similar to that in the controlled group (median survival time,63.5days compared with56days). However, in the early treatment group, the mice remain alive with no sign of disease by31March2013.3Study on the correlation between percentages of CD19±D34±2F10±cells at diagnosis and level of minimal residual disease in childhood B-ALL:The fraction of CD34+CD38-cells at diagnosis was not correlated with MRD level at day22of therapy (P=0.4759with Spearman rank correlation coefficient r=0.0848). Unexpectedly, both the initial frequency of CD19-CD34+2F10-cells and CD19-CD34+CD9-cells were significantly correlated with the logarithmic level of MRD at day22of therapy (P=0.0128, r=0.4566; P=0.0499, r=0.4440).Conclusions1. A novel B-lineage leukemia cell recognizing monoclonal antibody ZCH-2F10was successfully generated. The immunoglobulin subclass of2F10belongs to murine IgG1κ.2F10Ab could recognize CD9Ag. The heavy chain and light chain molecular weights of2F10Ab were approximately54kDa and25kDa. The VH2F10gene consists of360bps, while the VL2F10gene consists of348bps.2. The2F10-FITC fluorescent antibody is an excellent reagent with a very high sensitivity and specificity.2F10Ag was heterogeneously expressed on the membrane of the blasts of childhood acute leukemia.3.2F10Ag could mediate quick internalization of the binding antibody.2F10Ab could activate complement and kill Nalm-6cells expressing2F10Ag by CDC. Compared with the Nalm-6cells with high proliferative activity, the frequencies of CD19+2F10-, CD19-2F10-and CD19-2F10+subpopulations were significantly higher in those Nalm-6cells with low proliferative activity.4. B-cell acute lymphoblastic leukemia animal model had been successfully established in the nude mice.2F10Ab showed the excellent therapeutic efficacy in the early treatment group.5. Both the initial frequency of CD19-CD34+2F10-cells and CD19-CD34+CD9-cells were significantly correlated with the logarithmic level of MRD at day22of therapy in childhood B-cell acute lymphoblastic leukemia.