The Construction And Function Study On Bivalent And Tetravalent Anti-human CD19 Antibody

Background and aims:According to the reports in the literature, the children with new malignant tumors in China have reached 30,000 cases every year, of which 1/3 is leukemia. Acute leukemia is the most common malignant tumor which threatens the life and health of children. With the improvement diagnostic approaches and the treatment options, the complete remission (CR) rate of Chinese children with acute lymphoblastic leukemia (ALL) has reached more than 95% at present, and the 5 year survival rate has increased to more than 80%[1]. However, the multi-drug resistance of leukemia and recurrence is still an important factor affecting the prognosis of patients with leukemia. So, looking for new targets at the molecular and genetic levels and designing the specific targeted drugs will provide unique means for individualized ALL treatment.Tumor targeting therapy is directed to the target cells for specific killing, through specific cell surface molecules and directly target into cells, so as not to affect the normal tissue cell function. Among them, the most in-depth study is antibody targeted therapy.CD19 is a promising molecular target for immunotherapy of B cell malignancies after CD20.The specificity and stability make it an ideal target for targeting purpose, because the antigens are only expressed on normal or malignant B lymphocyte membrane; and will not be lost in the process of malignant transformation of B cells.ZCH-4-2E8 (an anti-human CD 19) antibody which was generated by our group is a murine antibody IgM. Previous studies showed that it can directly target B leukemia cells.Our laboratory had successfully constructed human-mouse chimeric antibody scFv2E8-Fc based on the parental 2E8 antibody. Functional tests have showed that the affinity to its antigen of the modified scFv2E8-Fc antibody has significantly reduced as compared to that of its parental murine 2E8 antibody which will obviously affect the targeting efficacy when it is to be used in patients. Therefore, higher affinity antibody is desired when we intend to develop this antibody further for clinical application purposes. According the report from the literature for other antibodies, the affinity of the antibody will be significantly improved after the increase of antigen binding sites and the molecular weight of the antibody. However, the multi-valency antibody of our 2E8 has not been studied. In this study,we try to construct the divalent and tetravalent anti-human CD 19 antibodies on the basis of the preliminary study by genetic engineering technology, and perform subsequent function researches in vitro and in vivo.The project mainly consisted of the following three parts:1. Construction of eukaryotic expression vector of anti-human CD 19 divalent and tetravalent antibody.2. Expression and function study of the divalent and tetravalent multi-meric proteins in vitro.3. Through the animal model of hematopoietic malignancies, the therapeutic effects of the multi-valent antibodies generated were studied in vivo.Methods:1 Construct eukaryotic expression vector of scFv2E8 and diascFv2E8 antibody 1.1 Construct eukaryotic expression vector of single chain Fv 2E8 (scFv2E8)pcDNA3.1-scFv2E8-Fc transfected CHO cells capable of secreting scFv2E8-Fc fusion protein were generated by our laboratory in previous studies.Diabody-scFv2E8 gene was amplified by RT-PCR from the mRNA of the scFv2E8-Fc-CHO cells and cloned into pcDNA3.1 plasmid to construct the eukaryotic expression vector of diascFv2E8 antibody gene.1.2 Construction eukaryotic expression vector of pcDNA3.1/Fc-6×hisThe pcDNA3.1/Fc plasmid successfully constructed in our laboratory previously was as template. Primers were designed according to Fc gene fragment for PCR. The 6×his tag was introduced into downstream of the Fc segment by primers. EcoR I and Xba I restriction sites were introduced into the end of 5 ’and 3’ gene sequences. Intermediate vector of pcDNA3.1/Fc-6xhis was constructed.1.3 Eukaryotic expression vector construction of divalent antibody pcDNA3.1/ diascFv2E8Plasmid pcDNA3.1/scFv2E8 was made as template.Design primer to amplify the VH2E8-linker5-VL2E8 gene fragment by SOE-PCR.The connecting peptide (linker) between VH2E8 and VL2E8 was shorted to five amino acids of G4S,6×his tag and the terminator TAG were introduced into the end of the VL2E8 gene and cloned into pcDNA3.1 vector.Construction eukaryotic expression vector of divalent antibody pcDNA3.1/diascFv2E8.2 Eukaryotic expression vector construction of two type tetravalent antibody pcDNA3.1/diascFv2E8-Fc and pcDNA3.1/scFv2E8-P532.1 Eukaryotic expression vector construction of tetravalent antibody pcDNA3.1/ diascFv2E8-FcThe gene fragment VH2E8-linker5-VL2E8 of divalent antibody eukaryotic expression vector pcDNA3.1/diascFv2E8 was cloned into the intermediate vector pcDNA3.1/Fc-6×his.Eukaryotic expression vector of tetravalent antibody pcDNA3.1/diascFv2E8-Fc were constructed through connecting two disulfide bonds between the Fc segments.2.2 Eukaryotic expression vector construction of tetravalent antibody pcDNA3.1/ scFv2E8-P53Plasmid pcDNA3.1/scFv2E8 was used as template.Primers to amplify the Hind III-scFv2E8-BamH I gene fragment by PCR were designed.The full length of P53-hinge-his×6 gene fragment was synthesized, which was linked to scFv2E8 gene by a connecting peptide (human IgG3 upstream hinge region).The scFv2E8 protein was automatically assembled into tetramer by P53 tetramerization domain.Eukaryotic expression vector of tetravalent antibody pcDNA3.1/scFv2E8-P53 was constructed.3 The expression and function study of divalent antibody diascFv2E8, tetravalent antibody diascFv2E8-Fc and scFv2E8-P533.1 The expression of divalent antibody diascFv2E8, tetravalent antibodydiascFv2E8-Fc and scFv2E8-P53 in eukaryotic cells CHO cells were transfected with transfection grade plasmidpcDNA3.1/diascFv2E8, pcDNA3.1/diascFv2E8-Fc and pcDNA3.1/scFv2E8-P53. Thetransfected CHO cells were maintained in an appropriate concentration of G418. Thedivalent and tetravalent antibody activity in the supernatant was identified by flowcytometry. The cells secreting the antibodies interested were subcloned by limitingdilution method.Multivalent proteins in the supernatant were collected and concentratedthrough centrifugation as 20:1 ratio by Millipore tube. Recombinant protein with 6*histag was purified by Ni-IDA affinity chromatography. Modified Marshall method wasused to prepare the fluorescent-labeled antibody 2E8-FITC.3.2 The function study of divalent antibody diascFv2E8, tetravalent antibodydiascFv2E8-Fc and scFv2E8-P53 The function studies on concentrated and purified divalent, tetravalent antibodieswere as follows:The concentrations of the recombinant proteins were measured byBCA method;Titration experiments of the recombinant proteins diascFv2E8,diascFv2E8-Fc and scFv2E8-P53 were conducted by flow cytometry; Blockingexperiment of recombinant proteins diascFv2E8, diascFv2E8-Fc and scFv2E8-P53 to2E8-FITC were performed by flow cytometry; RT-PCR was used to detect theexpression of target gene of transfected cells; Identification of recombinant proteinsscFv2E8-P53,diascFv2E8-Fc and diascFv2E8 was carried out by immunofluorescenceassay; The relative affinity of scFv2E8-P53, diascFv2E8, diascFv2E8-Fc and scFv2E8was identified by competitive binding experiments;The internalization characteristics ofscFv2E8-P53, diascFv2E8, diascFv2E8-Fc and scFv2E8 were detected by flowcytometry; The dissociation experiment of antibodies scFv2E8, diascFv2E8,diascFv2E8-Fc and scFv2E8-P53 were conducted by flow cytometry; Apoptosis studiesinduced by antibody scFv2E8, diascFv2E8, diascFv2E8-Fc and scFv2E8-P53 wereconducted; The ADCC and CDC effects of human-mouse chimeric antibodyscFv2E8-Fc with tetravalent antibody diascFv2E8-Fc were compared; The expressionof recombinant proteins in the supernatant was detected by SDS-PAGE andWestern-Blot.4 Study on the therapeutic effect of multivalent antibodies on animal models of hematopoietic malignanciesBurkitt’s lymphoma animal model was established by tail vein injection of Raji cells in NOD/SCID mice. Mice were randomly divided into 6 groups:PBS blank control group, model group, scFv2E8 antibody treatment group, diascFv2E8 antibody treatment group, diascFv2E8-Fc antibody treatment group and scFv2E8-P53 antibody treatment group. After one week injection of Raji cells, each mouse in antibody treatment group received the injection of 50μg of the purified antibody each for 3 consecutive days. The situations of mice were closely observed. Limb paralysis was considered as the standard of the disease onset. The disease onset and death dates of the mice were recorded, and the survival curves were drawn.The mice were killed by cervical dislocation before death, and the paraffin embedded sections were made from each organ. HE staining and immunohistochemical examination were performed.Results:1 Successful construction of eukaryotic expression vectors of scFv2E8 and diascFv2E8 antibody1.1 Successful construction of eukaryotic expression vector of single chain Fv scFv2E8RT-PCR method was performed to obtain the target gene scFv2E8 from the pcDNA3.1-scFv2E8-Fc transfected CHO cells. Target gene was purified by gel extraction and TA cloned into pGEM(?)-T easy vector. Then the target gene fragment was cloned into pcDNA3.1 vector through the enzyme digestion with EcoR I and BamH I from pGEM-T/scFv2E8 vector. The plasmid pcDNA3.1/scFv2E8 was successfully constructed and confirmed by DNA sequencing.1.2 Successful construction of the eukaryotic expression vector of pcDNA3.1/Fc-6×hisPCR method was applied to obtain the target gene Fc-6xhis which was purifired by gel extraction and TA cloned into pGEM(?)-T easy vector.Then the target gene fragment was cloned into pcDNA3.1 vector through the enzyme digestion with EcoR I and Xba I from pGEM-T/Fc-6xhis vector.Intermediate vector pcDNA3.1/Fc-6xhis was successfully constructed and confirmed by DNA sequencing.1.3 Successful construction of eukaryotic expression vector of divalent antibody pcDNA3.1/diascFv2E8Target gene VH2E8-linker5-VL2E8(810bp) was obtained successfully by SOE-PCR method. The target gene was purified by gel extraction and TA cloned into pGEM(?)-T easy vector.Then the target gene fragment was cloned into pcDNA3.1 vector through the enzyme digestion with BamH I and EcoR I from pGEM-T/VH2E8-linker5-VL2E8 vector.The plasmid pcDNA3.1/diascFv2E8 was successfully constructed and confirmed by DNA sequencing.2 Successful construction of the eukaryotic expression vectors of two type tetravalent antibody pcDNA3.1/diascFv2E8-Fc and pcDNA3.1/scFv2E8-P532.1 Successful construction of the eukaryotic expression vector of tetravalent antibody pcDNA3.1/diascFv2E8-FcPlasmids pcDNA3.1/diascFv2E8 and pcDNA3.1/Fc-6xhis were cleaved through the enzyme digestion of BamH I and EcoR I.The target gene diascFv2E8 and plasmid pcDNA3.1/Fc-6×his were purified by gel extraction, and the purified products were successfully connected by molar ratio 3:1.The plasmid pcDNA3.1/diascFv2E8-Fc was successfully constructed and confirmed by DNA sequencing.2.2 Successful construction of the eukaryotic expression vector of tetravalent antibody pcDNA3.1/scFv2E8-P53The target gene scFv2E8 was obtained successfully by PCR method and was purified by gel extraction and TA cloned into pGEM(?)-T easy vector. The whole length of gene P53-hinge-his×6 (213bp) was synthesized, and was successfully constructed the plasmid pcDNA3.1/P53-hinge-hisx6.Then the target gene scFv2E8 was cloned into pcDNA3.1/P53-hinge-his×6 vector through the enzyme digestion with Hind Ⅲ and BamH Ⅰ from pGEM-T/Hind Ⅲ-scFv2E8-BamH Ⅰ vector.The plasmid pcDNA3.1/scFv2E8-P53 was successfully constructed and was confirmed by DNA sequencing.3 Successful expression and function study of divalent antibody diascFv2E8, tetravalent antibody diascFv2E8-Fc and scFv2E8-P533.1 The divalent antibody diascFv2E8,tetravalent antibody diascFv2E8-Fc and scFv2E8-P53 were successfully expressed in CHO cells.Transfection grade plasmid of pcDNA3.1/diascFv2E8, pcDNA3.1/diascFv2E8-Fc and pcDNA3.1/scFv2E8-P53 were successfully extracted. After transfection, the cells were cultured under the appropriate concentration of G418 (600 μg/ml) after the CHO cells were transfected. Flow cytometry showed that active divalent and tetravalent antibodies which could recognize CD 19 antigen on Raji cells were presented in the supernatant of transfected CHO cells.The transfected cell lines including CHO/diascFv2E8, CHO/diascFv2E8-Fc and CHO/scFv2E8-P53 were successfully established after three times of subcloning. The target protein from culture supernatant was collected and purified. Fluorescence-labeled antibody 2E8-FITC was successfully generated by modified Marshall method.3.2 The function studies of divalent antibody diascFv2E8, tetravalent antibody diascFv2E8-Fc and scFv2E8-P53The concentrations of the recombinant proteins including diascFv2E8, diascFv2E8-Fc and scFv2E8-P53 detected by BCA method were 0.210mg/ml, 0.231mg/ml and 0.177mg/ml, respectively.The results from titration experiments showed that 2.1μg,1.5μg,2.0μg and 1.5μg of diascFv2E8, diascFv2E8-Fc, scFv2E8-P53 and 2E8-FITC antibodies were respectively needed to saturate 1×106Raji cells.Multivalent antibodies,especially the tetravalent antibody and diascFv2E8-Fc performed better than that of monovalent antibody in blocking experiment with parental murine 2E8 antibody,RT-PCR experiment showed that the target gene was successfully transfected to CHO eukaryotic cells.Green fluorescence was clearly observed in the cytoplasm of transfected cell line CHO/diascFv2E8, CHO/diascFv2E8-Fc and CHO/scFv2E8-P53 by immunofluorescence assay, which showed that the fusion protein was successfully expressed in the above three kinds of transfected cells.The relative affinity constants of scFv2E8-P53, diascFv2E8, diascFv2E8-Fc and scFv2E8 identified by competitive binding experiments were 1×10M-1,4×1010M-1,1.6×1011M- and 4× 1011M-1 respectively.Divalent antibody diascFv2E8 was internalized faster than monovalent antibody scFv2E8, but the internalization rate of two tetravalent antibody diascFv2E8-Fc and scFv2E8-P53 was not significantly different as comparing to scFv2E8.The dissociation experiment showed that the dissociation rate of tetravalent antibody was significantly lower than that of bivalent and monovalent antibodies. The apoptotic effect induced by tetravalent antibody diascFv2E8-Fc was the strongest, better than bivalent antibody diascFv2E8, while the monovalent antibody scFv2E8 almost had no apoptotic effect. The ADCC effect of tetravalent antibody diascFv2E8-Fc was better than scFv2E8-Fc, while the CDC effect of tetravalent antibody diascFv2E8-Fc at high concentration (50μg/ml,25μg/ml) showed better performance than that of scFv2E8-Fc.The results of SDS-PAGE and Western-Blot showed that diascFv2E8, diascFv2E8-Fc and scFv2E8-P53 protein products existed in mixture forms.4 Study on the therapeutic effect of multivalent antibody on the animal model of hematopoietic malignanciesBurkitt’s lymphoma animal model was successfully established by injecting Raji cells into NOD/SCID mice via the tail vein.The mice with Burkitt’s lymphoma presented weight loss, hind limb paralysis, scoliosis, arch back and cachexia. The results of HE staining examination showed that the meninges of mice were infiltrated by tumor cells.The brown yellow metastatic cells were also found in the meninges of mice by immunohistochemistry (anti-human CD 19 antibody) staining. Survival analysis indicated that statistically significant difference in survival time between different treatment groups (p<0.05).Multivalent antibody,especially the tetravalent antibody diascFv2E8-Fc presented a better performance than bivalent and monovalent antibodies in vivo treatment study.Conclusion:1 Eukaryotic expression vectors of bivalent antibody pcDNA3.1/diascFv2E8, two types of tetravalent antibody pcDNA3.1/diascFv2E8-Fc and pcDNA3.1/scFv2E8-P53 are successfully constructed.2 Divalent antibody diascFv2E8,tetravalent antibody diascFv2E8-Fc and scFv2E8-P53 are successfully expressed in eukaryotic CHO cells.These multivalent antibodies are able to recognize the CD 19 antigen on Raji cells.3 The function experiments in vitro show that the affinity of multivalent antibodies, especially the tetravalent antibody diascFv2E8-Fc is significantly higher than those of bivalent and monovalent antibodies,the dissociation rate of tetravalent antibody is significantly lower than those of bivalent and monovalent antibodies.The tetravalent antibody can play a better performance in vivo due to its prolonged half-life. Multivalent antibody can induce significantly higher apoptotic effect on tumor cells. The ADCC and CDC effects of tetravalent antibody diascFv2E8-Fc are better than human-mouse chimeric single chain antibody scFv2E8-Fc,which indicates that multivalent antibody can better target tumor cells by inducing apoptosis and CDC, ADCC effects.4 Burkitt’s lymphoma animal model has been successfully established.The function experiments in vivo show that the multivalent antibody,especially the tetravalent antibody diascFv2E8-Fc presents better therapeutic effect than bivalent and monovalent antibodies.