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Objective@#To develop the chimeric antibodies against neuraminidase (NA) of H7N9 and to identify their biological activity and function.@*Methods@#The genes of variable regions of the light chain (VL) and heavy chain (VH) obtained by mouse hybridoma technology were cloned respectively into the expression VH and VL vectors bearing human-derived Cγ1, and Cκ1 and co-transfected into 293T cells. The chimeric antibodies were purified and their functions were investigated.@*Results@#Two chimeric antibodies, 1E2 and 3E3 against neuraminidase (NA) of H7N9 were obtained. Both antibodies recognized similar antigenic epitopes. MAb 1E2 and 3E3 could prevent the infectivity with H7N9 and H11N9 virus and reduce their size of viral plaque.@*Conclusions@#The chimeric antibodies specific for N9 could prevent the infection of N9 subtype influenza virus as well as the NAI-resistant mutants and could be a potential immunotherapy approach for H7N9 treatment.
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Objective To investigate the effects of ch1D1, an anti-CD86 chimeric antibody, on autoreactive B lymphocytes isolated from patients with systemic lupus erythematosus ( SLE) . Methods Flow cytometry analysis was performed to measure the expression of CD86 on the surface of B cells isolated from patients with SLE and to analyze the effects of ch1D1 on the activation of CD4+T cells. The method of magnetic bead sorting was used to separate B cells, NK cells and CD4+T cells from PBMC collected from healthy subjects and patients with SLE for subsequent experiments. Antibody-dependent cell-mediated cyto-toxicity (ADCC) and complement-dependent cytotoxicity (CDC) that were mediated by ch1D1 were meas-ured with LDH release assay. Effects of ch1D1 on the secretion of auto-antibodies and the proliferation of CD 4+ T were detected by ELISA and 3 H -thymidine ( 3 H-TdR) incorporation assay, respectively. Results The levels of CD80 (68. 08±14. 28 vs 46. 10±12. 14, n=24, P<0. 000 1) and CD86 (44. 72±14. 90 vs 13. 99±10. 74, n=24, P<0. 000 1) expressed on the surface of B cells isolated from patients with SLE were significantly higher than those from the healthy subjects, suggesting the abnormal activation of B cells. Com-pared with the negative control group and the murine monoclonal antibody 1D1, ch1D1 was more effective in mediating the ADCC and CDC responses (P=0. 017 2, P=0. 038 8). Activated T cells significantly en-hanced the secretion of auto-antibodies by B cells isolated from patients with SLE. Compared with the nega-tive control group, the enhanced secretion of auto-antibodies was significantly inhibited by treatment with ch1D1 (P=0. 001 9). Moreover, ch1D1 significantly inhibited the proliferation and activation of CD4+T cells induced in patients with SLE (P=0. 002 4, P=0. 049 5). Conclusion ch1D1, the anti-CD86 chim-eric antibody, could effectively mediate the ADCC and CDC responses against autoreactive B cells isolated from patients with SLE, inhibit the secretion of auto-antibodies and suppress the proliferation and activation of auto-reactive CD4+T cells. It might be a potential immunotherapy agent for the treatment of SLE.
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Objective:On the basis of the use of chemical methods to establish mouse model of lupus nephritis and its biological identification , we investigate the reverse effect of pathological lesions of B 7-1 human-mouse chimeric antibody blockade against B7/D28 signaling pathway in mice with lupus nephritis model.Methods:Pristane was injected intraperitoneally to 6-week-old female C57BL/6J mice at dose of 0.5 ml per mouse in one go,and urine protein,ANA and renal pathological changes were detected on a monthly basis.Mice whose urine protein content reached ++and ANA fluorescence intensity reached ++were randomly devided into three groups ,five each.Antibody intervention group was sequentially injected with B 7-1-mouse chimeric antibody by orbital venous , positive control group was injected with immunosuppressant CTX , negative control group was injected with isotype control IgG.Urine protein and ANA were also detected on a monthly basis.Mice were sacrificed three months after intervention was executed.Kidney was used for H&E dying , IC detection and electric microscope observation.Results: After four-month Pristane induction , urine protein content of 80%mice reached +-+++,meanwhile,serum ANA fluorescence intensity reached ++-+++.Glomerulonephritis infiltrating cells were observed Mice with urine protein and ANA , glomerular inflammatory cell infiltration , tubular epithelial cell degeneration visible edema ,vascular congestion significantly ,fibrosis.After antibody intervention ,urine protein content in antibody intervention group gradually reduced from ++-+++to ±-+++,ANA ++-+++to +-++,and were significantly different from that in the negative control group ( P<0.01 ).Analysis of kidney H&E dying showed that antibody glomerular infiltration of inflammatory cells in the intervention group and tubular congestion and other symptoms were improved significantly.Immunofluorescence staining indicated that fluorescence intensity of IC was significantly reduced in the antibody intervention group.Electron dense deposits reduction and glomerular basement membrane uniformity were observed in antibody intervention group by electric microscope when compared with the negative control group.Conclusion:B7-1 antibodies could downregulate immune response through inhibiting B 7-1/CD28 signaling pathway , reducing the production of autoantibodies and reversing pathological damage caused by autoimmune response .
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Objective To study the effects of chimeric B7 antibodies on mononuclear cells from patients with ankylosing spondylitis (AS).Methods Chimeric antibodies of ch4E5 and chlD1 against B7 with a final concentration of 5 μg/ml were respectively added into culture media of mononuclear cells from patients with AS.Two other groups including huIgG-treated and untreated culture medium of mononuclear cells from patients with AS were set up as the controls.The culture medium of mononuclear cells from healthy subjects were prepared as normal control group.The disparities of mononuclear cell death in culture among each group were analyzed by using micro-lymphocyte cytotoxicity test (MLCT).The expression of membrane molecules were analyzed by FCM.The concentrations of cytokines in culture media were measured by ELISA.Results Both groups treated with the chimeric antibody showed lower scores for cell death than untreated group as indicated by MLCT (P＜0.01).FCM analysis demonstrated that both CD4/CD8 ratio (P ＜0.05) and membrane expression of CD154 (P＜0.01) decreased in chimeric antibody treated groups,but the number of CD4+CD25high Treg cells (P＜0.01) increased as compared with those in untreated group.The concentrations of IL-2 and TNF-α were down-regulated (P＜0.05) in chimeric antibody treated groups,while IL-4 and TGF-β were up-regulated (P＜0.02) as compared with those in untreated group.Conclusion Chimeric B7 antibodies might affect the expression of T cell membrane molecules and the cytokine production by mononuclear cells through B7/CD28 and CD4O/CD154 signaling pathways in patients with AS.
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Objective To express human-mouse chimeric antibody against anthrax protective anti-gen and to analyze its biological activities. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reduetase(DHFR) gene as the selection and complication marker. First, the light and heavy chain variable region gene of the monoclonal antibody 5E1 were cloned by RT-PCR, at the same time the human IgG1 heavy chain constant region gene and kappa type constant region gene were cloned. Next, the human-mouse chimeric antibody genes were synthesized by fusion PCR. Then, the hu-man-mouse chimeric antibody gene were inserted into MCS of pSecTag and B1 to construct pSecTag-5E1L and B1-5E1H, respectively. Finally, heavy chain expression cassette excised from the B1-5E1H with Bgl Ⅱ/BamH Ⅰ was further cloned into the Bgl Ⅱ site of the pSecTag-5E1L to construct pSecTag-5E1. Plasmid pSecTag-5E1 was transfected into CHO(dhfr) engineering cells and high production cell clones that were screened by enhancing MTX concentration. After collecting medium and purifying chimeric antibody with af-finity chromatogram, purified chimeric antibody was analyzed by SDS-PAGE, Western blot. Results A sta-ble and high production cell line was acquired at MTX concentration 5×10~(-8) mol/L. Conclusion The hu-man-mouse chimeric antibodies were successfully expressed in CHO cells.
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The Her-2 proto-oncogene encodes a 185kDa transmenbrane glycoprotein p185 which has intrinsic tyrosine kinase activity. It is overexpressed in several malignant human tumors like breast cancer. A chimeric antibody by assembling a single-chain Fv antibody and a human IgG1 Fc fragment was constructed. This chimeric antibody reacts with tumor surface antigen p185c-erbB-2 specifically. In order to put the antibody into clinical application, two steps purification method was used to attain the antibody’s purity more than 95%. Both the lyophilized pharmaceutical formulations of the antibody were found. The formulations can keep the stability and activity of the antibody for at least one year. These results were the foundation of the chimeric antibody for cancer therapy.
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Aim To prepare sterically stabilized liposomes modified with chimeric TNT-3 monoclonal antibody (chTNT-3) and investigate their immunoreactivity and in vitro targeting. Methods An endgroup functionalized polyethylene glycol-lipid derivative (pyridylthiopropionoylamino-polyethylene glycolhydrogenated soy phosphatidylethanolamine, PDP-PEG-HSPE ) was synthesized and incorporated to sterically stabilized liposomes. After mild thiolysis of the PDP groups by dithiothreitol, liposomes were covalently linked with maleimide-derivatized chTNT-3 and formed sterically stabilized immunoliposomes.Coupling efficiency, antibody density, size distribution, immunoreactivity of chTNT-3-modified sterically stabilized liposomes (chTNT-3-SLs) and specific binding properties of the chTNT-3-SLs to fixed Raji cells were determined, separately. Results Higher initial Ab/PDP-PEG-HSPE molar ratios resulted in higher antibody density on the surface of liposomes but lower coupling efficiency. The optimal coupling efficiency of 71% was obtained while antibody density in liposome was 106 μg antibody/μmol phospholipids (as initial antibody/PDP-PEG-HSPE = 1: 10). The chTNT-3-SLs had a narrow size distribution after extrusion and the mean size of this immunoliposomes was (115 ± 33) nm. The immunoreactivity of chTNT-3 can be preserved after efficient attachment of maleimide-derivatized chTNT-3 to the surface of liposomes. But calculated per antibody concentration, the immunoreactivity of chTNT-3-SLs would obviously decrease compared to that of chTNT-3 or chTNT-3 derivatives. Significantly higher binding of chTNT-3-SLs to fixed Raji cells directed by chTNT-3 was obtained compared to other preparations in serial dilutions (P＜0.01). Conclusion chTNT-3-SLs prepared by PDP-PEG-HSPE method remained most immunoreactivity of chTNT-3 and was able to bind nuclear antigens in vitro.
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The purpose of this article is to review the current status and future perspectives of antibody therapy against cancer. Eight antibody drugs against cancer are now commercially and clinically available for treatment of cancer in the United States and two of them are also available in Japan. Current data suggest that antibodies or their genes against cancer can be used in order to increase the tumor specificity of various new immunotherapeutic or gene therapeutic approaches against cancer, thereby enhancing the tumoricidal effect of each treatment while reducing the side effects.<br>
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Aim To study the inhibitory effect of anti-ErbB2 chimeric antibody chA21 on proliferation of human malignant breast cancer cell lines SKBR3 and explore its possible mechanisms through studying its effects on ErbB2 distribution and PI3K/Akt,Erk1/2 signal transduction.Methods The inhibitory effect of chA21 on SKBR3 was assessed by MTT assay.The effect of chA21 on distribution of ErbB2 on cell plasma membrane was determined by FACS(Fluorescence-activated cell sorting)analysis;p-Akt and p-Erk1/2 were detected by Western blot.Results The inhibitory rate of chA21 on SKBR3 cells in vitro increased in a dose and time dependent manner.Furthermore,it could down-regulate the distribution of ErbB2 on plasma membrane and decrease the activation of p-Akt and p-Erk1/2.Conclusions ChA21 can inhibit the proliferation of SKBR3 cell in vitro.Down-regulation ErbB2 on cell plasma membrane resulting in decreasing the activation of p-Akt and p-Erk1/2 may be one of the mechanisms of the inhibitory effect.
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Aim To explore the effects of anti-HER-2 chimeric antibody chA 21 on proliferation and apoptosis of SKBR3 cells.Methods MTT colorometric assay,HE staining,transmission electron microscopy,flow cytometry,and TUNEL were used to study the proliferation inhibition and apoptosis induction of SKBR3 cells by chA 21 in vitro.Results Proliferative inhibition rates and apoptotic index of SKBR3 cells were increased in a dose and time dependent manner after exposure to chA21(0.2～5.4 mg?L~(-1)).Conclusion chA 21 could remarkably inhibit proliferation of SKBR3 cells in vitro and apoptosis induction may be one of its main mechanisms.
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Objective: Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185 erbB2 is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185 erbB2 monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185 erbB2 chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr - cell. Methods: The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr - cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185 erbB2 chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185 erbB2 , were measured with MTT assay in vitro. Results: The anti-p185 erbB2 chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr - was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185 erbB2 specifically, but did not react with that non-overexpressing p185 erbB2 . Immunoprecipitation test confirmed that the chimeric antibody could bind to p185 erbB2 specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185 erbB2 . Conclusion: The anti-p185 erbB2 mouse/human chimeric antibody that was expressed in CHO-dhfr - cells can bind to p185 erbB2 specifically and inhibit proliferation of SKBR3 cells overexpressing p185 erbB2 . It has a potential application in biotherapy of cancer.
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In order to reduce its immunogenicity and increase its therapeutic usefullness, the murine anti-gastric cancer McAb 3H11 was genetically engineered into a human-mouse chimeric antibody. The 3H11 light and heavy chain variable genes were inserted into chimeric antibody expression vectors respectively. The chimeric light chain expression vector was first transfected into murine Sp2/0 myeloma cells using lipofectin. Transfectants that stably expressed human-mouse chimeric light chain were isolated by mycophenolic acid selection. Then the chimeric heavy chain expression vector was transfected into the transfectomas. Histidinol resistent cells were obtained and among these, clones stably secreting human-mouse chimeric antibodies were selected. The expression of human-mouse chimeric light and heavy chain mRNAs were proved by RT-PCR and the expressed chimeric antibody was demonstrated capable of binding to human gastric cancer cell MGC803
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For construction of anti-CD3 human/murine chimeric antibody genes, a selective first-strand cDNA synthesis from mRNA or RNA of murine McAb HIT3a was performed using murine Ig constant region primers, and then cDNA of heavy and light chain variable domains of murine immunoglobulin were amplified by PCR using a set of degenerated oligonucleotide primers. Using these cDNA fragments as probes, the L and H chain V region exons encoding the murine McAb anti-CD3 were isolated from the gene library of HIT3a DNA and inserted into mammalian expression vectors containing the human ? and yl constant region exons for construction of human/murine chimeric antibody genes.
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Exons encoding the variable regions of the light and heavy chain of the murine McAb HIT3a against CD3 antigen were isolated from HIT3a gene library and inserted into mammalian expression vectors containing the human K and y1 region exons. The chimeric genes were transfected into murine SP2/0 hybridoma cells by Lipofectin. Antibody levels of culture supernatants from transfectomas ranged 21~32 ?g/ml. The chimeric antibodies bound specifically to human T cells and competed effectivelly with the parental anti-CD3 murine McAb for binding to CD3 antigen on human T cells. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced with the chimeric antibodies as compared with anti-CD3 murine McAb. The mitogenic activity of human T cells can be suppressed and enhanced by the anti-CD3 chimeric antibodies. The cytotoxicity to tumor cells and proliferation of human T cells mediated by the anti-CD3 chimeric antibodies were much higher than IL-2 alone. Chimeric HIT3a antibody is a clinically relevant, genetically engineering antibody with potential use in treatment of graft-versus-host disease in tranplantation, autoimmune diseases and some tumors.
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Objective:To construct eukaryotic expression vector for anti-human melanoma chimeric antibody,and express the antibody in eukaryotic cells.Methods:Variable regions of the murine antibody were cloned from the anti-human melanoma hybridoma cell line HB8459,HB8760,Mel3,and was inserted them into the chimeric antibody eukaryotic expression vector pMH-CA.The constructed vector was transfected into 293T,the chimeric antibody was expressed,as detected by RT-PCR,ELISA,and Western blot.Results:Eukaryotic expression vectors of the chimeric antibody against human melanoma were successfully constructed,and expressed in eukaryotic cells.The expression of chimeric gene was confirmed by RT-PCR,specificity of the chimeric antibody was proved by ELISA and Western blot.Conclusion:Anti-human melanoma chimeric antibody was expressed successfully.