Study On Humanized Anti-Platelet GPⅡ B/ⅢA Phage Single Chain FV Antibody

In recent years, with the changing of people's living style, thrombotic disease is becoming the "the first killer" to people's health. The incidence ranks number one among all diseases and it has a tendency to be increasing, thereby becomes one of the emphasis and hot points in current medicial research.Vascular endothelium injure, stasis of blood stream and fibrinolysis activity depressed take different part in the thrombosis, of which platelet activation plays an important role in the pathogenesis. Anti-platelet therapy becomes an important part in the prevention and treatment of thrombotic disease.Platelet surface contain many glycoproteins including GPⅡ bⅢa , GP Ⅰ b/Ⅸ , GP Ⅵ .. GP Ⅴ which respectively take part in the adhension ,aggregation,release reaction of platelet. GP Ⅱ b/Ⅲa acts as an important part in platelet aggregation reaction by mediating the connection between platelet and fibrinogen. Genetic engineering antibodies of anti-platelet glycoprotein which have been explored recently can inhibit the adhension, aggregation and release reaction by inhibiting the different platelet glycoprotein functions in order to prevent thrombotic diseases. A new platelet aggregation inhibitor-Abciximab (7E3 ) belongs to anti- GP Ⅱ b/Ⅲa monoclonal mouse-human chimerized monoclonal antibody and has been used to prevent thrombosis. By now, it has been proved to be effective inPCI (percutaneous coronary intervention) therapy by inhibiting platelet aggregation. But it is still a mouse-human chimerized monoclonal antibody produced by hybridoma technology, which is composed of mouse Fv and huaman Fc.Though its antigenicity is weaker than all pure mouse-derived monoclonal antibody, it still contains mouse Fv fragment and has antigenity to human. It can also cause HAMA (human anti-mouse antibody) reaction. To resolve this problem, phage surface display technology has been used to produce completely humanized monoclonal antibody. In this study it was used to develop completely humanized anti-GP Ⅱ b/Ⅲa ScFv phage antibody.It has been proved that ITP is an autoimmune disease. Anti-platelet antibodies can be detected in most ITP patients' plasma or platelet elute, which bind to specific GP by Fab and then activate mononuclear-macrophage or complement system. The antibodies can distroy platelet or cause megakaryocyte dysmaturity by ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) and result in thrombocytopenia or dysfunction.Most Anti-platelet antibodies act on GP Ⅱ b/Ⅲa.Most anti-GP Ⅱ b/Ⅲa antibodies cause platelet destruction and very few of them can also cause platelet aggregation defect. Enlightened by this,we screened out the ITP patients ' plasma and only choose plasma containing anti-GP Ⅱ b/Ⅲa antibodies that inhibit platelet aggregation. A new type anti-platelet agent— humanized anti- GP Ⅱ b/Ⅲa phage ScFv antibody can then be developed by phage surface display technology ,which can be used to inhibit platelet aggregation for the management of thrombotic diseases. Other anti-platelet glycoprotein(such as GPⅥ, GP Ⅰ a-Ⅱ a, GP Ⅰ b/Ⅸ) phage ScFv antibodies are also being developed by this technology now.In this study, the autoantibody which binds GP Ⅱ b/Ⅲa and inhibits platelet aggregation has been screened out successfully. A humanized phage antibody library which contains anti-GP Ⅱ b/Ⅲa ScFv antibody has been constructed. A good platform has been set up for further sreening and manufacturing humanized anti-GP Ⅱ b/Ⅲa phage ScFv antibody.PART ONE: SCREENING GPⅡB/ⅢA AUTOANTIBODYPOSITIVE PLASMA WHICH INHIBITS THE AGGREGATION FUNCTION OF PLATELETObjective: To screen out the ITP patients whose plasma contains GP Ⅱ b/Ⅲa specific autoantibody and inhibits the aggregation function of normal platelets for further preparation and construction of humanized anti-platelet GP Ⅱ b/Ⅲa single chain Fv library.Materials and Methods: 1. 95 chronic ITP patients (37males, 58 females, median age 42.6 years, range: 15-68 years) in the inpatient and outpatient department of QiLu Hospital of Shandong University from 2003.7 to 2004.3 were studied. All the patients fulfilled the creteria of ITP. The platelet counts of all patients were lower than 100×109/L (range: 19-62×109/L). Twelve normal subjects were used as control, who had no blood transfusion or preganant history in recent 3 months before blood sampling. Methods: The plasma of 95 chronic ITP patients was measured by modified MAIPA assay for the GP Ⅱ b/Ⅲa specific autoantibodies and the influence of the autoantibodies on the platelet aggregation induced by ADP and collagen was investigated in platelet aggregation test(turbidity method).Results: 1. MAIPA assay: GP Ⅱ b/Ⅲa specific autoantibodies were detected in 41 of 95(43.2%) ITP patients'plasma ,5 (5.3%) were markedly positive which was then measured by platelet aggregation test- 2. platelet aggregation test:2 patients' plasma (2.1%) significantly inhibited the platelet aggregation function induced by ADP and collagen.After diluted by 2 times,the plasma(2.1%) still significantly inhibited the platelet aggregation function induced by ADP,but not by collagen.Conclusion: 1. ITP is an autoimmune disorder. GP Ⅱ b/Ⅲa specific antibodies can be detected in 43.2% ITP patients' plasma of the testing team. GP Ⅱ b/Ⅲa is animportant antigen which platelet specific antibodise act on in ITP. 2. Most GP Ⅱ b/Ⅲa specific antibodies cause platelet destruction and very few of them also cause platelet aggregation defects.2 plasma containing GP Ⅱ b/Ⅲa specific antibodies which can inhibit platelet aggregation function were screened out for the construction of a humanized phage antibody library of anti-platelet GP Ⅱ b/Ⅲa single chain Fv.PART TWO: CONSTRUCTION AND TEST OF A HUMANIZED PHAGE ANTIBODY LIBRARY OF ANTI-PLATELET GPⅡB/ⅢA SINGLE CHAIN FVObjective: To construct an anti-platelet GPⅡb/Ⅲa ScFv phage antibody library by phage surface display technology. To set up a platform for further sreening and manufacturing of humanized anti-platelet GP Ⅱ b/ Ⅲ a ScFv phage antibody.Materials and Methods: l.Objective: 2 plasma containing GP Ⅱb/Ⅲa specificautoantibody and significantly inhibiting platelet aggregation induced by ADPwas sreened out by modifed MAIPA assay. 2. Materials: pCANTAB-5Eplasmid , pHEN2 plasmid, helper phage M13K07, VH, ScFv Marker, Sfi Ⅰ /NotⅠ restricted digesting enzyme, E coli TG1, SephacrylTM S-400 HR resin,MicroSpinTM purifying column, TRIzol kit, cDNA synthesis kit, premiers. 3.Methods: The heavy chain and light chain variable region gene of human immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the patients screened out and randomly combined through a DNA linker encoding the peptide (Gly4Ser)3 to construct single chain Fv gene. ScFv gene was digested with Sfi I /Not I restricted digest enzyme to ligate the pCANTAB-5E cloning vector, then were electrically transformed to Ecoli.TG1.The TG1 containing ScFv-pCANTAB-5E was rescued by helper phage M13K07 to produce ScFv phage antibody and the phage antibody library was construted.The plasmid which was drew from the transformed Ecoli.TG1 was digested by Sfi Ⅰ /Not Ⅰ restricted digest enzyme for the identification of ScFv inserting rate. GP Ⅱ b/Ⅲa antigen used as antigen, anti-platelet GP Ⅱ b/Ⅲa ScFv inthe phage antibody library was detected by ELISA test.Results: 1 .Amplification of VH and VL:Using as cDNA as template and VH , 1-7CH pr VL1-6, CL as premiers,the size of VH is about 400bp and VL is about 380bp. 2. Amplification of linker from pHEN2 phagemid: pHEN2 phagemid contains linker gene fragments. Using linkerVL1~6 and linkerCH as premiers, linker genes complement with VH3' and VL5' terminatio and can be used to connect VH and VL were amplified from pHEN2 phagemid .The amplified linker's size is about 100bp. 3. VH and VL were connected with the amplified linker to form ScFv of 780bp in size. Amplifing ScFv with Sfi I - VH1-7 and Not I -CL as premiers, SfiI and Not I restricted digesting sites were added to ScFv two sites. 4.ScFv and pCANTAB-5E phagemid were digested by Sfi I and Not I restricted digest enzymes and were connected to form ScFv-pCANTAB-5E vector which was electrotransformed into Ecoli TG1. The transformed Ecoli TG1 was cultured in 2X YT culture base(100μg/ml ampicillin).Randomly select 4 clones of the cultured Ecoli.TG1,draw phagemid from them and digest the phagemid with Sfi I and NotI restricted digest enzymes. 780 bp size fragment can be seen in 3 Ecoli.TG1 clones after electrophoresis. The inserting rate is about 75%.5.Construction of the phage antibody library:After the transformed Ecoli TG1 was rescued by phage helper phage M13K07, the phage antibody library was constructed whose tite is about 1.62 × 1010cfu/ ml. 6. ELISA test: The mean optical density of the phage antibody library is siganificantly higher than that of the positive control (p<0.01).Conclusions: 1 .VH and VL gene were amplified and linker gene was amplified from phagemid successfully. The size of VH gene is about 400bp, VL is about 380bp and linker is about lOObp. 2. VH and VL were connected with linker to form ScFv whose size is about 780 bp successfully. 3. ScFv and pHEN2 phagemid were digested with Sfi Ⅰ / Not Ⅰ restricted digest enzymes and constructed ScFv-pHEN2 vector which was identified by Sfi Ⅰ / Not Ⅰ enzymes digesting that inserting rate is about75%. 4. After rescued by helper phage M13K07, the phage library was proved by ELISA assay that it contained the anti-GP Ⅱ b/Ⅲa ScFv phage antibody. A platform was found for further screening and manufacturing of the anti-GP Ⅱ b/Ⅲa ScFv phage antibody. 5. To improve the phage antibody library technology for researching more humanized phage antibody later.