The Humanization Of A Novel Monoclonal Antibody3A4Targeting Leukemia Stem Cells

BackgroundLeukemia is the major cause of pediatric cancer-related morbidity and mortality. There are about2million children with leukemia in China and the number of patients are increasing every year. Although the anti-neoplastic protocols have been optimized in recent years, some patients are still experiencing treatment failure due to incomplete eradication of the leukemia stem cells (LSC). Monoclonal antibody (mAb) can specifically and effectively kill tumor cells, which has become a hotspot of leukemia treatment studies. Therefore, the identification of targets existing on leukemia cells especially on LSC is extremely important for improving the outcomes.CD45RA has been found to be expressed brightly on hematopoietic malignant cells including B and myeloid lineage leukemia and LSC. while it is not expressed on neutrophils, platelets, erythrocytes and memory T cells. Due to these properties, CD45RA may be an attractive target for the design of antibody-based therapies.Previously, we have generated a new anti-CD45RA murine mAb3A4and a human mouse chimeric antibody scFv3A4-Fc in our laboratory. However, as the3A4and scFv3A4-Fc are foreign antigens, they may trigger human anti-mouse or anti-chimeric antibody response. To reduce the immunogenecity of3A4murine antibody, humanization of Fab fragment via genetic engineering approach has been proposed. It is a time consuming and labor intensive process to screen a humanized antibody with high affinity by conventional methods. Therefore, the aim of this study was to establish a rapid screening approach for antibody humanization. and to obtain a humanized antibody Hu3A4with high affinity. Furthermore, we constructed a novel immunotoxin to enhance the clinical potential of antibody therapy.MethodsThis project included three parts:1. Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells;2. Study on the preparation and function of a humanized antibody with high affinity.3. Study on the preparation and identification of scFv3A4-FcRap (ranpirnase, Rap) immunotoxin.1. Successful construction and stable expression of an anti-CD45RA scFv-EGFPfusion protein in Chinese hamster ovary cells1.1Construction of pHMCH3/scFv3A4-EGFP expression vector:The EGFP gene were inserted into pcDNA3.1(+) to construct a plasmid named pcDNA3.1/EGFP, then the EGFP gene replaced the Fc fragment of pHMCH3/scFv3A4-Fc to construct a eukaryotic expression vector pHMCH3/scFv3A4-EGFP.1.2The expression and detection of scFv3A4-EGFP fusion protein1) The plasmid of pHMCH3/scFv3A4-EGFP was transfected into CHO cells by using lipofectamineTM2000. Transfected cells were selected in the presence of G418(600μg/ml).2) Two weeks after selection, the total RNA from the scFv3A4-EGFP gene transfected CHO cells (CHO/scFv3A4-EGFP) was extracted and analyzed by RT-PCR.3) CHO/scFv3A4-EGFP was incubated with fluorescein isothiocyanante-conjugated goat anti-mouse IgG Fab antibody (GAM-Fab-FITC) and observed under a multicolor fluorescence microscope.4) The affinity and antigen recognizing ability of scFv3A4-EGFP fusion protein was detected by flow cytometric analysis.5) The positive clone was isolated by limiting dilution, the highest-producing and stable expression clone was chosen.6) The supernatant was harvested, and then purified by Ni-IDA Sepharose columns. The purified fusion protein scFv3A4-EGFP was identified by SDS-PAGE and Western-Blot assav.7) The secretory pathway of scFv3A4-EGFP/CHO cells was detected by fluorescence microscopy and brefeldin A (BFA) blocking experiments.8) The photostable character between scFv3A4-EGFP and3A4-FITC was detected by fluorescence microscopy.2. Study on the preparation and function of a humanized antibody with high affinity2.1Humanization design and construction of recombinant fusion protein expression vectors1) Humanization design of3A4antibodyThe human germline consensus templates for3A4variable fragments of heavy chain (VH) and light chain (VL) were identified using the IMGT/V-QUEST online tool. Then two homology models of the3A4VH and3A4VL were built separately with the assistance of Discovery Studio2.5software to find the key residues in their frameworks. The CDRs of3A4Fv were grafted into the chosen human FR templates to construct3A4VHa and3A4VLa genes, then the key residues were mutated back to construct a mutated3A4VHb and3A4VLb genes.2). Construction of recombinant fusion protein expression vectorsIn order to elucidate the feasibility of shortening the screening time for the humanized antibody, one murine and two humanized expression vectors were constructed by PCR combined with restriction enzyme digestion, including pHMCH3/scFv3A4-EGFP,pHMCH3/scFv3A4a-EGFP, and pHMCH3/scFv3A4b-EGFP. To further confirm the affinity of humanized scFv. we constructed the pHMCH3/scFv3A4-FcHis,pHMCH3/scFv3A4a-FcHis, and pHMCH3/scFv3A4b-FcHis expression vectors.2.2The expression and screening of a humanized antibody with high affinity1) The plasmids of pHMCH3/scFv3A4-FcHis, pHMCH3/scFv3A4a-FcHis, pHMCH3/scFv3A4b-FcHis, pHMCH3/scFv3A4-EGFP. pHMCH3/scFv3A4a-EGFP and pHMCH3/scFv3A4b-EGFP were transfected into CHO cells by using lipofectamineTM2000. Transfected cells were observed by a fluorescence microscope and were selected in the presence of G418(600μg/ml). 2) After transfection for24h,48h and72h, the supernatants of transfected CHO cells were harvested and detected by flow cytometric analysis.3) Two weeks after selection, the total RNA from the CHO/scFv3A4-EGFP and CHO/scFv3A4a-FcHis were extracted and analyzed by RT-PCR.4) The affinity of scFv3A4b-EGFP and scFv3A4b-FcHis were measured by the method based on antibody blocking and competitive binding assays.2.3Construction of the expression vectors for humanized antibody Hu3A4with high affinityThe constant regions of human IgG were obtained from PBMC by PCR, then we constructed the pHMCH3/Hu3A4VH-CH and pHMCH3/Hu3A4VL-CL by PCR combined with restriction enzyme digestion.2.4Study on the expression and function of humanized antibody Hu3A4with high affinity1) The plasmids of pHMCH3/Hu3A4VH-CH and pHMCH3/Hu3A4VL-CL were transfected into CHO cells by using lipofectamineTM2000. Transfected cells were selected in the presence of G418(600μg/ml).2) Two weeks after selection, the total RNA from the Hu3A4gene transfected CHO cells (CHO/Hu3A4) was extracted and analyzed by RT-PCR.3) CHO/Hu3A4was incubated with phycoerythrin-conjugated goat anti-human IgG κ-chain antibody (GAH-κ-PE) and fluorescein isothiocyanate-conjugated goat anti-human IgG Fc antibody (GAH-Fc-FITC), then they were observed under a multicolor fluorescence microscope.4) The positive clone was isolated by limiting dilution, then the highest-producing clone was chosen.5) The supernatant was harvested, and then purified by SPA sepharose columns. The purified Hu3A4antibody was identified by SDS-PAGE and Western-blotting.6) The affinity of Hu3A4and scFv3A4b-FcHis was measured by the method based on an antibody titration and competitive binding assays.7) The capacity of Hu3A4and scFv3A4b-FcHis specifically killing target cells were tested by complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC) assays. 8) The effectiveness of Hu3A4and scFv3A4b-FcHis were compared in the treatment of NOD/SCID mice with leukemia.3. Study on the preparation and identification of scFv3A4-FcRap immunotoxin.3.1Construction of the expression vectors for scFv3A4-FcRap immunotoxinThe gene of Rap was artificially synthesized first, then the pHMCH3/scFv3A4-Fc Rap was constructed by PCR combined with restriction enzyme digestion.3.2Study on the expression and function of scFv3A4-FcRap immunotoxin1) The plasmid of pHMCH3/scFv3A4-FcRap was transfected into CHO cells by using lipofectamineTM2000. Transfected cells were selected in the presence of G418(600μg/ml).2) Two weeks after selection, the total RNA from the scFv3A4-FcRap immunotoxin gene transfected CHO cells (CHO/scFv3A4-FcRap) was extracted and analysed by RT-PCR.3) CHO/scFv3A4-FcRap was incubated with PE-conjugated goat anti-mouse IgG Fab antibody (GAM-Fab-PE) and GAH-Fc-FITC. then they were observed under a multicolor fluorescence microscope.4) The positive clone was isolated by limiting dilution, then the highest-producing clone was chosen.5) The supernatant was harvested, and then purified by Ni-IDA sepharose columns. The purified scFv3A4-FcRap immunotoxin was identified by SDS-PAGE and Western-blotting.6) The affinity of scFv3A4-FcRap was measured by the method based on antibody titration and competitive binding assays.7) The characters of internalization and cytotoxicity of scFv3A4-FcRap were detected by FCM.8) The capacity of scFv3A4-FcRap and scFv3A4-FcHis specifically killing target cells were tested by CDC and ADCC assays.9) The effectiveness of scFv3A4-FcRap and scFv3A4-FcHis were compared in the treatment of NOD/SCID mice with leukemia. Results1. Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells:The plasmids of pcDNA3.1/EGFP and pHMCH3/scFv3A4-EGFP were successfully constructed. Then they were transfected into CHO cells and selected by G418for two weeks. The scFv3A4-EGFP gene was amplified from the CHO/scFv3A4-EGFP cells by RT-PCR. Under multicolor fluorescence microscope, the fluorescence could be clearly observed in the cytoplasm of CHO/scFv3A4-EGFP cells. The supernatant of CHO/scFv3A4-EGFP was detected by flow cytometry. The positive rate of scFv3A4-EGFP on CD45RA+KG1a cells was85.82%before subcloning. The scFv3A4-EGFP fusion protein could be blocked by its parental murine antibody3A4on KG1a cells and they had a similar pattern of antigen recognition, indicating that the binding activity and specificity of the engineered fusion protein scFv3A4-EGFP were not significantly changed. The supernatant was purified, and then identified by SDS-PAGE and Western-blot analysis. The molecular weight of scFv3A4-EGFP fusion protein was approximately57kDa. It was stably expressed and secreted from the CHO/scFv3A4-EGFP cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was1350μg/L after purification. Furthermore, as compared to conventional labeled3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection.2. The expression and screening of a humanized antibody with high affinity:The highest identity to the3A4VH was X62106and X86355, while for the3A4VL was M23090and AF103571. The antibody7D11and5C8were chosen to model the structure of3A4with the assistance of Discovery Studio software. Fourteen murine FR residues were considered as the key residues. The plasmids of pHMCH3/scFv3A4-FcHis, pHMCH3/scFv3A4a-FcHis, pHMCH3/scFv3A4b-FcHis, pHMCH3/scFv3A4a-EGFP and pHMCH3/scFv3A4b-EGFP were constructed successfully. Then they were transfected into CHO cells and selected by G418for two weeks. Only18hours after transfection, the green fluorescence of GFP containing CHO cells could be observed by a fluorescent microscope. The binding activity of the fusion proteins interested could be easily detected by flow cytometry. The bands of the scFv3A4a-EGFP and scFv3A4a-FcHis gene were amplified from the CHO/scFv3A4-EGFP and CHO/scFv3A4a-FcHis cells by RT-PCR. The scFv3A4b-EGFP and scFv3A4b-FcHis had a similar affinity based on antibody blocking and competitive binding assays.3. Study on the preparation and function of humanized antibody Hu3A4with high affinity:The plasmids of pHMCH3/Hu3A4VH-CH and pHMCH3/Hu3A4VL-CL were successfully constructed. Then they were transfected into CHO cells and selected by G418for two weeks. The band of the Hu3A4gene was amplified from the CHO/Hu3A4cells by RT-PCR. Under multicolor fluorescence microscope, the fluorescence could be clearly observed in the cytoplasm of CHO/Hu3A4cells. The supernatant of CHO/Hu3A4was detected by flow cytometry. The positive rate of Hu3A4on CD45RA+KG1a cells was above98%after subcloning. The supernatant was purified. and then identified by SDS-PAGE and Western-Blot analysis. The molecular weight of Hu3A4was approximately160kDa. The Hu3A4antibody was soluble in the culture supernatant and the yield was2.5μg/ml after purification. The result of competitive binding assay showed that the affinity of Hu3A4on KG1a cells was3.7×1010M-1while that of scFv3A4b-FcHis antibody was4.7×109M-1. The CDC function of Hu3A4and scFv3A4b-FcHis antibodies was not obvious, but both of the antibodies could kill the target cells through the effects of ADCC. Both of the Hu3A4and scFv3A4b-FcHis antibodies could cure the leukemia mouse model effectively.4. Study on the preparation and function of scFv3A4-FcRap immunotoxin:The plasmids of pHMCH3/scFv3A4-FcRap was successfully constructed and transfected into CHO cells. The band of the scFv3A4-FcRap gene was amplified from the CHO/scFv3A4-FcRap cells by RT-PCR after two weeks’ selection by G418. Under the multicolor fluorescence microscope, the fluorescence could be clearly observed in the cytoplasm of CHO/scFv3A4-FcRap cells. The supernatant of CHO/scFv3A4-FcRap was detected by flow cytometric analysis. The positive rate of scFv3A4-FcRap on CD45RA+KG1a cells was above98%after subcloning. The supernatant was purified, and then identified by SDS-PAGE and Western-blot analysis. The molecular weight of scFv3A4-FcRap was approximately135kDa. The scFv3A4-FcRap immunotoxin was soluble in the culture supernatant and the yield was4-5μg/ml. The result of competitive binding assay showed that the affinity of scFv3A4-FcRap immunotoxin on KG1a cells was5.0×1O10M-1. The scFv3A4-FcRap immunotoxin had partial cytotoxic effect on KG1a cells. The CDC functions of scFv3A4-FcRap immunotoxin and scFv3A4-FcHis antibody were not obvious, but they could kill the target cells through the effects of ADCC, and the scFv3A4-FcRap immunotoxin was similar to the scFv3A4-FcHis antibody. Both of the scFv3A4-FcRap immunotoxin and scFv3A4-FcHis antibody could cure the leukemia mouse models effectively.Conclusion1. We have successfully established a method to prepare an anti-CD45RA scFv-EGFP fusion protein, which can be expressed and secreted stably in CHO cells and the system may be used for the expression of other scFv-EGFP fusion proteins.2. We have successfully established a rapid screening method for a humanized antibody, which can accelerate the process of antibody humanization when it combines with the modeling technology via a software.3. Successful construction and expression of Hu3A4and scFv3A4b-FcHis antibodies, which are suitable for further study on the treatment of leukemia.4. Successful construction and expression of scFv3A4-FcRap immunotoxin, which is suitable for the construction of other humanized scFv-FcRap immunotoxins.