Study On The Biological Characteristics Of A Novel Leukemia Stem Cell Recognizing Monoclonal Antibody 3A4 And Its Genetic Engineering

Blackground and aimsBeing the sixth frequent tumor in China, acute leukemia is a common hematopoietic malignancy in both children and adults. The morbidity and mortality are highest in patients with malignancies under the age of 35. In children, acute leukemia is the major life-threatening disease, the number of patients increase at the rate of more than 15,000 per year[2]. Although combined chemotherapy and hematopoietic stem cell transplantation (HSCT) have greatly improved the rate of complete remission (CR) and the rate of 5-year disease-free survival (DFS), there are still some patients experiencing 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. Molecular targeting therapy can specifically kill tumor cells, minimizing the side effects on non-targeting tissue cells. thus, it has become a hotspot of leukemia treatment studies. During the past decade, studies on clinical treatment with monoclonal antibody based targeting therapy has obtained promising results with great success, thus it has become an important research area of molecular targeting therapy[28.29]?In recent years,anti-CD20[15] and anti-CD33 [30] have been successfully used in clinical treatment on B-NHL and AML, respectively. In order to treat leukemia effectively, more monoclonal antibodies capable of identifying leukemic cells are needed. Many studies have showed that leukemia stem cells (LSCs) are the source of disease relapse. It is easy to expect that if an antibody could target and kill the LSCs specifically, the prognosis of leukemia would have a new breakthrough.Zhejiang Children's Hospital (ZCH)-6-3A4 or 3A4 was generated in our laboratory by the method described in the literature. It had been identified as a novel mouse anti-human CD45RA monoclonal antibody by the 7th International Workshop and Conference on Human Leucocyte Differentiation Antigens (HLDA7). Our preliminary data have showed the 3A4 can identify more leukema cells than the existing anti-CD45RA antibody (clone name:L48). Furthermore,3A4 can recognize over 99% leukemia stem cells (LSCs) of KG1a cell line. Therefore, we conducted a systematic studies on the biological characteristics and genetic engineering of 3A4 to discover the potential application of this new antibody in targeting therapy for human leukemias.Materials and MethodsThis project included two parts:1. Study on the biological characteristics and function of murine antibody 3A4;2. Study on the preparation and identification of 3A4 genetic antibody.1 Study on the characteristics and function of murine antibody 3A41.1 Preparation and identification of 3A4 mAb:The ascites containing 3A4 antibody (Ab) derived from Balb/c mice was purified by affinity chromatography using SPA Sepharose column. The targeting efficiency of 3A4 on KG1a cells and the immunoglobulin subclass of 3A4 were detected by flow cytometry (FCM). We identified the antigen 3A4 binding to by blocking experiment. The purity and the molecular weights (MW) of the heavy and light chains were identified by SDS-PAGE. 1.2 The expression pattern of 3A4:3A4-FITC was prepared according to modified Marshall's method and evaluated by flow cytometry (FCM). The reactivities of 3A4 and CD45RA(clone name:L48) with human leukemia cell lines, blast cells from leukemia patients and blast cells from patients with relapse or resistant disease were analyzed and compared by FCM. Meanwhile, the capacity of 3A4 recognizing CD34+CD38-CD123+ AML-LSCs was identified by FCM.1.3 The function of murine 3A4:The internalization of 3A4 was studied by the method applying papain digestion and flow cytometry. The capacity of 3A4Ab specifically binding to 3A4 Ag and activating complement was determined by complement dependent cytotoxicity (CDC).3A4-NCTD immunotoxin was prepared by active ester and the targeting killing was evaluated in vitro.2 Study on the antibody engineering of 3A42.1 Cloning and sequencing of the varible regions for both heavy (VH3A4) and light (VL3A4) chain genes from 3A4 hybridoma cell line:Total RNA was prepared from hybridoma cell line 3A4 by using the Trizole kit. The VH3A4 and VL3A4 genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) with family specific primer pairs, respectively. The PCR products were cloned into pGEM(?)-T easy vector, then transfected into DH5?Ecoli and positive recombinants were identified and purified. After sequencing with automatic DNA sequencer, the sequences of the genes interested were analyzed online.2.2 The construction and expression of single-chain 3A4 (ScFv3A4) eukaryotic expression vector:ScFv3A4 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase and verified through sequencing. The target genes were inserted into eukaryotic expreisson vector pcDNA3.1. Then, the CHO cells were transfected by the recombinated vector to express the single chain antibody ScFv3A4. The activity of the protein was identified by FCM. The affinity constant of ScFv3A4 was measured by the method based on competitive antibody/antigen binding. Whether the ScFv3A4 gene had integrated into the DNA of host cell was determined by RT-PCR.2.3 The construction and expression of human-mouse chimeric 3A4 (Hm3A4) eukaryotic expression vector:The ScFv3A4 gene were inserted into a eukaryotic expreisson vector named pHMCH3 which was generated in our laboratory. Then, the CHO cells were transfected by the recombinated vector pHMCH3/Hm3A4 to express the chimeric antibody Hm3A4. The activity of the protein was identified by FCM. The affinity constant of ScFv3A4 was measured by the method based on competitive antibody/antigen binding. Whether the Hm3A4 gene had been integrated into the DNA of the host cell CHO was determined by RT-PCR analysis.2.4 Study on the function of Hm3A4 antibody:The capacity of Hm3A4 Ab specifically binding with 3A4 Ag and complement activation was determined by complement dependent cytotoxicity (CDC) assay. The capacity of antibody dependent cell mediated cytotoxicity of Hm3A4 was analyzed through Annixin v-PI staining following by FCM analysis.Results1 The characteristics and function of murine antibody 3A41.1 Preparation and identification of 3A4 mAb:Murine monoclonal antibody 3A4 was prepared through intraperitoneally inoculating 1063A4 hybridoma cells into the Balb/C mice to generate ascites. The antibody containing ascites were harvested and purified through SPA Sepharose chromatography. Mouse subclass of the 3A4 antibody was determined using a murine antibody subclass kit and FCM analysis. The results showed that the positive rates of 3A4 reactivity with goat anti-mouse IgGl and goat anti-mouse?were 99.48% and 99.02%, respectively, indicating the subclass of the antibody 3A4 was mouse IgGlK. Blocking experiment showed that the positive rates of CD45RA, CD45 and CD45RO before and after blocking by 3A4 were 98.18% vs. 8.15%,99.95% vs.99.77%,99.95% vs.99.96%, respectively, indicating that 3A4 belonged to the CD45RA category. Electrophoretic analysis after reduction with DTT showed that the heavy and light chain molecular weights of antibody 3A4 were approximately 57.9 kDa and 30.0 kDa, respectively.1.2 The reactive patterns of 3A4 with leukemia cells:In order to analyze the reactivity of 3A4 antibody by FCM analysis, fluorochrome-conjugated 3A4 antibody is needed. Fluorescein Isothiocyanate conjugated 3A4 antibody (3A4-FITC) was successfully generated according to Marshall's method. The absorbency of the 3A4-FITC at A280nm and A495nm were 0.31 and 0.19, respectively. We calculated F/P value according to the formula F/P= (2.87×A495)/(A280-0.35×A495). The F/P value of generated 3A4-FITC was 2.24. close to the ideal ratio (1-2) of FITC labeled antibody. The titration test showed that 0.5?g of 3A4-FITC was able to saturate 106 KGla cells The reactivities of 3A4 with leukemia cell lines of T-ALL patients were similar to those of a standard CD45RA antibody (clone name:L48), P values were 0.313. In leukiama cell lines, B lineage ALL and AML patients, the positive rates of 3A4 were significantly higher than those of CD45RA (P=0.045, P=0.009 and P=0.000, respectively).3A4 antigen was highly expressed on leukemia cells of 9 patients with relapse or refractory disease, and the positive rates of 3A4 were over 90%, significantly higher than those of CD45RA (P= 0.016).3A4 antigen was also highly expressed on LSCs of KGla and 8 AML patients with positive rates ranged from 83.33% to 100%(median:97.83%).1.3 The internalization function of murine 3A4 Ab:When the antibody was incubated with targeting cell KG1a at 37?, the degrees of internalization at different time points were 5.3%(0.25h),7.3%(0.5h),36.9%(1h),69.2%(2h) and 72.6%(3h). 71.8%(4h), respectively, showing that 3A4 could be efficiently internalized into the cells as the incubation time prolonged within the first three hours. However, when the antibody was incubated at 4?,3A4 could not be internalized into cells no matter how long the incubation time was.1.4 3A4-NCTD immunotoxin generation and targeting killing in vitro:The immunotoxin 3A4-NCTD was prepared by the modified active ester method. We detected the cytotoxicities of 3A4-NCTD to the targeting cell KGla cells. The results showed that the inhibition ratios at 24h,48h,72h and 96h were 1.73%,15.27%,25.79% and 61.09%, repectively.2 Preparation and identification of genetic engineering antibodies2.1 Cloning and sequencing of the VH3A4 and VL3A4 genes from 3A4 hybridoma cells:The results showed that the variable region gene of the 3A4 heavy chain (VH3A4) consists of 351 bps encoding a peptide of 117 amino acid residues, while the variable region gene of the 3A4 light chain gene (VL3A4) consists of 321 bps encoding a peptide of 107 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST, the VH3A4 and VL3A4 genes belonged to mouse IGVH and mouse IGV?subclasses, respectively. At the positions 22/96 of the heavy chain and 23/89 of the light chain genes, there were cysteine pairs, which played the key role in forming disulfo-bond between the two chains. Both VH and VL chains have 4 definitive frame regions (FR) and 3 complementary determinant regions (CDR).2.2 The construction and expression of single-chain 3A4 (ScFv3A4) eukaryotic expression vector:ScFv3A4 gene was amplified by SOE-PCR. The target genes were inserted into eukaryotic expreisson vector and trandfected into CHO cells. The transfected cells were selected with G418 and the culture supernatants of resistant clones were detected by FCM. After 2 weeks of culture, the reactivity of the ScFv3A4 on KG1a cells was detected wth a positive rate of 44.47%. The positive rates were increased to 97.80% and 99.78%, respectively after 4 and 5 weeks of continuous culture in the presence of G418. The result of competitive binding assay showed that the affinity of ScFv3A4 on 5×l05KG1a cells was l.2×1010M-1.The result of RT-PCR showed the ScFv3A4 gene could be amplified from the cDNA of transfected CHO cells. 2.3 The construction and expression of human-mouse chimeric 3A4 (Hm3A4) eukaryotic expression vector:The gene of Hm3A4 with 795 bps was inserted into an eukaryotic expression vector pHMCH3 and the recombinated expression vector pHMCH3-Hm3A4 was trandfected into CHO cells. The transfected cells were selected with G418 and the culture supernatants of G418 resistant clones were detected by FCM. The positive rates on KGla cells were 96.51% and 98.20% after 2 and 4 weeks of culture in the presence of G418 with MFI values of 86.60 and 198.23, respectively. The result of competitive binding assay showed that the affinity of Hm3A4 on 5×105KG1a cells was 1.1×1011M-1.The result of RT-PCR showed that the Hm3A4 genes could be amplified from the cDNA of the transfected CHO cells. The titration test of culture supernatants showed that the positive rate of 20?1 supernatants for 1×106 KG1a cells was 97.35% and the MFI was 47.41. When the supernatants increased to 2ml/106 KG1a cells, immunofluorescence was significantly increased with a MFI value to 360.71.2.4 The function of Hm3A4:The Hm3A4/CHO cells were gradually adapted to serum free medium (SFM) and the culture supernatants were harvested and purified by affinity chromatography using SPA Sepharose column. Western-blot analysis showed a single band of monomer size under reducing conditions and a band of dimer size under non-reducing conditions, the molecular weights were 43KDa and 100KDa. respectively.The activities of CDC on KGla cells were in a dose dependent manner and the inhibition rates of KG1a cells were 18.4%.48.6%.70.1% and 78.3% at the concentrations of 0.05?g/ml,0.1?g/ml, 1?g/ml,5?g/ml. respectively. In ADCC experiment, the inhibition rate of Hm3A4 on KG1a cells was 28.96%.Conclusion1.3A4 antibody could recognize CD45RA antigen of KGla cells. The immunoglobulin subclass of 3A4 belongs to murine IgG1?. The heavy chain and light chain molecular weights of 3A4 antibody were 57.9kDa and 30.3 kDa. respectively.2.3A4 could recognize more leukemic blasts of B lineage ALL and AML than a standard CD45RA antibody.3A4 could identify more than 90% of leukemia stem cells from KG1a cells and AML bone marrow cells, and recognize almost all of the leukemia cells from patients with relapsed or refractory diseases.3A4 could be internalized into the cytoplasm rapidly after binding to its antigen.3A4 could not activate complement and kill target cells. However,3A4-NCTD immunotoxin could effectively kill the target cells.3A4 antigen might be an appropriate drug target in in the targeting trhrapy.3. Eukaryotic expression vector pcDNA3.1/ScFv3A4 and the eukaryotic expression vector antibodies pHMCH3/Hm3A4 were constructed successfully. The single-chain antibody ScFv3A4 and the human-mouse chimeric antibody Hm3A4 were successfully expressed in CHO cells with excellent biological activity.