Monoclonal antibody for recognizing platelet membrane glycoprotein and application of monoclonal antibody in antithrombotic treatment

Abstract

The invention relates to the field of human immunity, and particularly discloses a monoclonal antibody for recognizing platelet membrane glycoprotein and application of the monoclonal antibody in antithrombotic treatment. Monoclonal antibodies R813 and Y262 acting on different sites of a platelet receptor GP IIb-IIIa are developed, the monoclonal antibodies can well inhibit platelet aggregation, the two antibodies are combined to form the cocktail, the functions of the platelet receptor GP IIb-IIIa can be completely blocked, the platelet aggregation function is thoroughly inhibited, and the cocktail can be used for preparing the cocktail. The cocktail biological activity of the monoclonal antibody exceeds that of an antiplatelet drug Reopro, and the dosage is small; the antibody is prepared into an F (ab ') 2 fragment by an enzymolysis method, so that the molecular weight of the antibody is reduced while the biological activity is preserved, the amount of the antibody reaching an action target site is increased, the Fc terminal is removed, the HAMA reaction is reduced, and the platelet destruction caused by combination with a reticuloendothelial system is reduced. In addition, the enzymolysis method is simple to operate, low in cost and convenient for batch preparation and production of antibody fragments.

Description

Technical Field

[0001] The invention belongs to the field of human immune technology, and specifically relates to a monoclonal antibody for recognizing platelet membrane glycoprotein and its application in anti-thrombotic treatment. Background Art

[0002] Thrombi are small pieces of blood formed on the surface of the peeling or repairing of the inner surface of the cardiovascular system. In the variable fluid-dependent type, thrombi are composed of insoluble, deposited platelets, accumulated white blood cells and trapped red blood cells. The lesions caused or complicated by thromboembolism are about three times that of cancer, and are one of the diseases that seriously endanger human health. Therefore, preventing thrombosis is a focus and hot spot in contemporary medical research. Platelets, as the main component of thrombi, play a key role in the formation of thrombi. Platelets will adhere to the subendothelial tissue at the damaged part of the blood vessel through their surface membrane glycoproteins directly binding to plasma proteins. Adherent platelets or when platelets are affected by platelet activators produced during the coagulation process or tissue damage, they will deform, extend pseudopodia, and then release the contents of intracellular particles. At the same time, the platelet membrane glycoprotein GP IIb-IIIa complex is activated to form an adhesion molecule receptor. The binding of this receptor with fibrinogen can promote the adhesion and aggregation of platelets, ultimately leading to the formation of platelet thrombus at the damaged blood vessel.

[0003] The formation of platelet thrombi plays an important role in the pathogenesis of arterial thromboembolic diseases, including acute myocardial infarction and other coronary thrombosis. Therefore, a class of antiplatelet drugs has become an important treatment for arterial thromboembolic diseases including coronary heart disease. Currently, the commonly used antiplatelet drugs in clinical practice include aspirin, dipyridamole, thienopyridine, etc., but these drugs only act on a certain link of platelet activation, and their efficacy is not as good as platelet receptor GPIIb-Ⅲa antagonists. Platelet receptor antagonists directly inhibit the final common pathway of platelet aggregation, blocking the binding of fibrinogen to platelet GPIIb-Ⅲa, which can inhibit platelet aggregation, thereby preventing platelet thrombi from forming, thus becoming a strong, efficient and specific antiplatelet drug in clinical practice, showing good application prospects. There are three types of platelet receptor GPIIb-Ⅲa antagonists: monoclonal antibodies, such as c7E3, synthetic peptides, such as Eptifibatide, and non-peptide small molecules, such as Tirofiban.

[0004] The monoclonal antibody (McAb) technology was first created in 1975 and has played a great role in basic research in medicine and biology as well as in the diagnosis and treatment of diseases. However, with the widespread clinical application of monoclonal antibodies, people have found that there are some problems with mouse-derived monoclonal antibody drugs: the human anti-mouse antibody (HAMA) reaction produced after mouse-derived antibodies are used in the human body; the antibody molecular weight is large, and the amount of antibody molecules reaching the target site is insufficient; the antibody itself has weak effector function, etc., which makes the antibody treatment effect unsatisfactory. In response to these problems, people have carried out humanization transformation of antibodies, such as the common human-mouse chimeric antibodies, the preparation of small molecular weight antibody fragments such as single-chain antibodies, and the preparation of bifunctional antibodies. 7E3 is a mouse-derived anti-membrane glycoprotein IIb-IIIa monoclonal antibody prepared by American researchers in 1983. It was transformed into a human-mouse chimeric antibody fragment c7E3 in 1992. Large-scale clinical observations have shown that it has good anti-thrombotic function. It has been approved by the FDA and is widely used in the treatment of ischemic heart disease abroad, showing a good application prospect for this type of drug. However, the shortcoming of the monoclonal antibody c7E3 is that it only acts on an antigenic determinant cluster on the surface of platelets. It only blocks the receptor from one angle, so it is difficult to completely inhibit platelet aggregation. Therefore, clinical personnel have been looking for new monoclonal antibodies that can more effectively inhibit platelet aggregation. Summary of the invention

[0005] The purpose of the present invention is to provide a monoclonal antibody that recognizes platelet membrane glycoprotein and its application in anti-thrombotic therapy, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Monoclonal antibodies that recognize platelet membrane glycoproteins, including:

[0008] A monoclonal antibody against the platelet receptor GP IIIa, produced by the hybridoma cell line R813;

[0009] Monoclonal antibody against the platelet receptor GP IIb-IIIa complex, produced by the hybrid cell line Y262.

[0010] Hybridoma cell lines producing specific anti-platelet receptor monoclonal antibodies include:

[0011] Hybridoma cell lines producing monoclonal antibodies that recognize platelet GP IIIa: R813, Y88, Y128, and Y474;

[0012] Hybridoma cell lines producing monoclonal antibodies recognizing platelet GP IIb-IIIa complex: Y8, Y262, Y291, Y295, Y321, Y342, Y458, Y585 and Y700.

[0013] Preferably, the hybridoma cell line producing a monoclonal antibody recognizing platelet GP IIIa is preferably R813, and the hybridoma cell line producing a monoclonal antibody recognizing platelet GP IIb-IIIa complex is preferably Y262.

[0014] The method for preparing a monoclonal antibody against a specific antigenic determinant of a platelet surface glycoprotein comprises the following steps: using human platelets as an immunogen to immunize Balb / c mice, taking spleen cells and fusing them with SP2 / 0-Ag14 myeloma cells, developing hybridoma cells that produce a monoclonal antibody that recognizes platelet GPIIIa and a monoclonal antibody against the platelet receptor GP IIb-IIIa complex, using ELISA, FCM, Western blotting, affinity chromatography and other methods to identify the antigenic determinant of the monoclonal antibody and observe the binding activity of the monoclonal antibody to platelets, and preparing ascites and purifying the monoclonal antibody.

[0015] Preferably, monoclonal antibody fragments against specific antigenic determinants of platelet surface glycoproteins are prepared, and monoclonal antibody R813 and Y262F(ab')2 fragments are prepared by papain digestion and identified by SDS-PAGE.

[0016] A pharmaceutical composition for inhibiting platelet aggregation comprises a monoclonal antibody against platelet receptor GP IIIa, a monoclonal antibody against platelet receptor GP IIb-IIIa complex or a combination thereof, and a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the monoclonal antibody is IgG fragments Fab and F(ab')2 of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody or a genetically engineered antibody.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention has developed monoclonal antibodies R813 and Y262 that act on different sites of platelet receptor GP IIb-IIIa, both of which can well inhibit platelet aggregation function. Combining these two antibodies to form a "cocktail" can completely block the function of platelet receptor GP IIb-IIIa and thoroughly inhibit platelet aggregation function. The biological activity of the monoclonal antibody "cocktail" exceeds that of the antiplatelet drug Reopro and the dosage is small. The antibody is prepared into F(ab')2 fragments by enzymatic hydrolysis, which preserves the biological activity while also reducing the antibody molecular weight, thereby increasing the amount of antibody reaching the target site, and while removing the Fc end, the HAMA reaction is also reduced, reducing the platelet damage caused by binding to the reticuloendothelial system. In addition, the enzymatic hydrolysis method is simple to operate, low in cost, and convenient for batch preparation and production of antibody fragments. Therefore, the combined antibody "cocktail" with both strong and efficient biological activity and low cost has huge antithrombotic therapeutic potential and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of hybridoma cells produced by the present invention for producing monoclonal antibodies R813 and Y262;

[0021] Figure 2 The electrophoresis analysis of the purified monoclonal antibodies R813 and Y262 IgG of the present invention;

[0022] Figure 3 It is the Western blot transfer electrophoresis pattern of the present invention;

[0023] Figure 4 This is the electrophoresis analysis of the affinity chromatography products of the monoclonal antibodies R813 and Y262 of the present invention, wherein A is a protein marker, B is a non-reduced product of R813, C is a protein marker, and D is a non-reduced product of Y262;

[0024] Figure 5 The binding activity of the monoclonal antibodies R813 and Y262 of the present invention to platelets, 1 is a negative control, 2 is a positive control SZ-22, 3 is the monoclonal antibody R813, and 4 is the monoclonal antibody Y262;

[0025] Figure 6 For the present invention 125 I-labeled monoclonal antibodies R813 and Y262 radioimmunoassay results;

[0026] Figure 7 The electrophoresis analysis diagram of the monoclonal antibodies R813 and Y262 F(ab')2 prepared in the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] See also Figure 1 - Figure 7 As shown, monoclonal antibodies that recognize platelet membrane glycoproteins include:

[0029] A monoclonal antibody against the platelet receptor GP IIIa, produced by the hybridoma cell line R813;

[0030] Monoclonal antibody against the platelet receptor GP IIb-IIIa complex, produced by the hybrid cell line Y262.

[0031] Hybridoma cell lines producing specific anti-platelet receptor monoclonal antibodies include:

[0032] Hybridoma cell lines producing monoclonal antibodies that recognize platelet GP IIIa: R813, Y88, Y128, and Y474;

[0033] Hybridoma cell lines producing monoclonal antibodies recognizing platelet GP IIb-IIIa complex: Y8, Y262, Y291, Y295, Y321, Y342, Y458, Y585 and Y700.

[0034] Human platelets can be used to immunize Balb / c mice, and a number of monoclonal antibodies (about 20 clones) against platelet GP IIIa or platelet GP IIb-IIIa have been successfully developed. After repeated screening and testing, it was proved that Y262, Y321, Y474 and R813 can effectively inhibit platelet aggregation. 125 The radioimmunoassay competitive inhibition assay of I-labeled monoclonal antibodies and the mean fluorescence intensity overlay method of flow cytometry revealed that the monoclonal antibodies recognized different antigenic determinants of platelet surface glycoproteins. The first group of monoclonal antibodies R813, Y88, Y128 and Y474 recognized platelet GP IIIa, and the second group of monoclonal antibodies Y8, Y262, Y291, Y295, Y321, Y342, Y458, Y585 and Y700 recognized platelet GP IIb-IIIa complex.

[0035] The hybridoma cell line producing the monoclonal antibody recognizing platelet GP IIIa is preferably R813, and the hybridoma cell line producing the monoclonal antibody recognizing platelet GP IIb-IIIa complex is preferably Y262.

[0036] The invention discloses a method for preparing a monoclonal antibody against a specific antigenic determinant of a platelet surface glycoprotein. The method comprises the following steps: using human platelets as immunogens to immunize Balb / c mice, taking spleen cells and fusing them with SP2 / 0-Ag14 myeloma cells, developing hybridoma cells that produce a monoclonal antibody that recognizes platelet GPIIIa and a monoclonal antibody against the platelet receptor GP IIb-IIIa complex, identifying the antigenic determinant of the monoclonal antibody and observing the binding activity of the monoclonal antibody to platelets by using methods such as ELISA, FCM, Western blotting and affinity chromatography, preparing ascites and purifying the monoclonal antibody, preparing monoclonal antibody fragments against a specific antigenic determinant of a platelet surface glycoprotein, preparing monoclonal antibody R813 and Y262F(ab')2 fragments by papain digestion and identifying them by SDS-PAGE, and observing the platelet aggregation inhibitory activity of the monoclonal antibody R813 and Y262F(ab')2 fragments and their "cocktail".

[0037] A pharmaceutical composition for inhibiting platelet aggregation comprises a monoclonal antibody against platelet receptor GP IIIa, a monoclonal antibody against platelet receptor GP IIb-IIIa complex or a combination thereof, and a pharmaceutically acceptable carrier or excipient.

[0038] The monoclonal antibody is IgG fragment Fab and F(ab')2 of the monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody or genetically engineered antibody.

[0039] In a preferred embodiment of the present invention, antibodies R813 and Y262 targeting different antigenic determinants were screened using I-labeled monoclonal antibody radioimmunoassay and flow cytometry average fluorescence intensity superposition method. Y262 and R813 were used in combination on platelets and it was found that the combined action of these two monoclonal antibodies can completely inhibit the ADP-induced platelet aggregation reaction, and the amount of antibody used is small (total amount is 6-7 μg / mL). The test results show that monoclonal antibodies R813 and Y262 act on different sites of the platelet receptor GPIIb-IIIa, and the monoclonal antibody "cocktail" composed of the two completely blocks the binding of fibrinogen to the platelet receptor GPIIb-IIIa, and completely inhibits platelet aggregation activity. And its F(ab')2 fragment can still completely inhibit platelet aggregation activity, and its biological activity exceeds that of c7E3 (trade name Reopro). The use of two monoclonal antibodies to form a monoclonal antibody "cocktail" 100% blocked the receptor function of platelet membrane glycoprotein IIb-IIIa, which is a first in the world and its biological activity exceeds Reopro (c7E3). Reopro only acts on one site of the platelet receptor GP IIb-IIIa to block the receptor from one angle, and the amount of antibody required to achieve a 50% platelet aggregation inhibition rate is 5.5μg / mL. The "cocktail" we have successfully developed acts on two different sites on the platelet receptor GP IIb-IIIa, completely blocking the binding of fibrinogen and the receptor in terms of spatial configuration. The amount of antibody required to achieve a 50% inhibition rate is only 3.3μg / mL, which not only significantly reduces the amount of monoclonal antibodies used, but also greatly enhances the platelet aggregation function inhibition effect, exceeding Reopro (c7E3). In order to reduce the HAMA response of monoclonal antibodies, reduce the molecular weight and increase the antibody concentration at the target site, we transformed monoclonal antibodies R813 and Y262 into F(ab')2 fragments by enzymatic hydrolysis (studies have shown that the HAMA response of pure antibody F(ab')2 fragments is not greater than that of chimeric antibodies, and its affinity is high and can be used directly for treatment). In vitro experiments show that the combined action of the F(ab')2 fragments of this antibody can still completely block the function of the platelet receptor GPIIb-IIIa and completely inhibit platelet aggregation.

[0040] Example 1: Preparation of hybridoma cells producing monoclonal antibodies R813 and Y262

[0041] 1.1 Materials:

[0042] Balb / c mice were purchased from Charles River Laboratories Inc. Canada, SP2 / 0-Ag14 mouse myeloma cells, chronic myeloma leukemia cells (K562), acute lymphoblastic leukemia cells (MOLF-4), and human acute T-cell leukemia cells (JurkatB6-1) were all purchased from ATCC, USA. D-MEM / F, RPMI1640, L-Glutamine, MEM non-Essential, HT Supplement, HAT Suplement, and Antibiotic-Antimycotic were all products of GIBCO, 8-Azaguanie was a product of Sigma, 50% PEG1500 Solution was a product of ROCH, and the mouse antibody subclass typing kit was a product of Biorad. The remaining reagents were of domestic analytical grade.

[0043] 1.2 Methods:

[0044] Platelets were separated from normal adult anticoagulated whole blood, washed 3 times with TEN, suspended in PBS and adjusted to a platelet count of 1x10 8 cells / ml, 0.2mL was injected into the peritoneal cavity of 8-week-old female Balb / C mice, and repeated 4 weeks later. 0.2mL of platelet suspension was injected into the tail vein of mice 4 weeks later. The mice were killed 3 days later, and the spleen was removed to prepare spleen cell suspension. g Splenocytes and 1x10 7 SP2 / 0-Ag14 cells in the logarithmic growth phase were fused using conventional methods and cultured in HAT medium and HT medium for 2 weeks respectively, and then switched to ordinary complete medium. During this period, ELISA was performed on platelet-coated ELISA plates for cell clones. The results showed that more than 20 hybridoma cell clones had significant binding reactions with platelets. For platelet-positive and screened hybridoma cell clones, they were subcloned three times using the limiting dilution method, and then gradually transferred to 24-well plates and culture flasks for culture, and ascites was prepared (see Figure 1). Thus, 12 cell lines that can stably secrete antiplatelet monoclonal antibodies were obtained, namely R813, Y8, Y88, Y128, Y262, Y291, Y295, Y321, Y458, Y474, Y585 and Y700. Flow cytometry was used to observe the reaction of these antibodies with human red blood cells, white blood cells, chronic myeloma leukemia cells (K562), acute lymphoblastic leukemia cells (MOLF-4) and human acute T-cell leukemia cells (Jurkat B6-1). The results showed that these 12 antibodies had no cross-reaction with these cells. At the same time, karyotype analysis of the cells producing these antibodies showed that they were indeed hybridoma cells. The monoclonal antibody subclass typing kit (Biorad) was used for analysis, and the results showed that monoclonal antibodies R813, Y128, Y321, Y458, Y474, Y585 and Y700 were IgG1, Kappa; monoclonal antibodies Y8 and Y295 were IgG 2a , Kappa; monoclonal antibodies Y88, Y262 and Y291 are IgG 2b , Kappa. Dynamic observations of hybridoma cell lines of two of the preferred monoclonal antibodies R813 and Y262 have been conducted for several consecutive years, proving that these two hybridoma cell lines can stably secrete high-titer monoclonal antibodies for a long time. Now the two cell lines have been established as production cell banks.

[0045] Example 2: Preparation and purification of antibodies R813 and Y262 IgG

[0046] 2.1 Materials:

[0047] Pristane was a product of Sigma, Balb / C mice were purchased from Shanghai Experimental Animal Center, Protein G 1 mL and pre-packed columns were products of Pharmacia, and the remaining reagents were of domestic analytical grade.

[0048] 2.2 Methods:

[0049] Male 10-week-old Balb / c mice were sensitized by intraperitoneal injection of 1 mL of pristane. One to two weeks later, 1 to 10 × 10 hybridoma cells of R813 and Y262 were respectively placed in the 6 Inject into the peritoneal cavity of sensitized mice, and observe and collect ascites after about a week. The mouse ascites was dialyzed against 0.02M PBS (pH7.0) at 4°C overnight, then centrifuged at 10,000 rpm x 15 min at 4°C to remove the precipitate, and filtered through a 0.2μm filter. 1mL of the filtrate was added to the Protein G column each time, 0.02M PBS (pH7.0) was used to balance and wash away impurities, and 0.1M Gly-HCl (pH2.7) was used to elute IgG and collect it. The collected product was dialyzed against PBS, concentrated and quantified, and identified by 10% SDS-PAGE. The results showed that the antibody content in the ascites was about 8mg / mL, and the purity of the purified antibody was above 90% (see Figure 2 ).

[0050] Example 3: Study on platelet binding activity of antibodies R813 and Y262

[0051] 3.1 Materials:

[0052] Platelets were from whole blood of normal adult volunteers, GAM-FITC was a product of Shanghai Huamei Company, GAM-HRP was a product of Sigma Company, CNBr-Sepharose4B was a product of Pharmacia, protein marker was a product of Shanghai Institute of Biochemistry, Sebastian Company, the protein compositions were 43KD, 66KD, 97KD, 130KD and 200KD, and the other reagents were domestic analytical grade reagents.

[0053] 3.2 Methods:

[0054] (1) ELISA method for determination of platelet binding activity of antibodies R813 and Y262: healthy human whole blood was anticoagulated, platelet-rich plasma (PRP) was separated, washed three times with TEN solution, and platelet binding activity was determined by 5x10 5 Platelets / well were coated, overnight at 4℃, blocked with 2% BSA-PBS at 37℃ for 2 hours, fixed with 0.5% glutaraldehyde, washed with 0.01% Tween20-PBS, GAM-HRP as secondary antibody, OPD as color, and conventional ELISA to measure the fluorescence A492 value at 492nm. Mouse IgG was used as negative control, and SZ-22 (anti-platelet GPIIbα) was used as positive control. The results showed that R813 and Y262 had good platelet binding activity.

[0055] (2) Flow cytometry to determine the binding activity of antibodies R813 and Y262 to platelets: 5 μL PRP (platelet concentration 5x10 8 / mL) was added with 50 μL of 1:100 diluted antibody R813 and Y262 mouse ascites, left at room temperature for 30 minutes, washed, added with GAM-FITC, and analyzed by flow cytometry ( XL) detection. Mouse IgG was used as negative control and SZ-22 of the same dilution was used as positive control. The results showed that R813 and Y262 had platelet binding ability (see Figure 5 ).

[0056] (3) Western blot determination of the binding sites of antibodies R813 and Y262 to platelet membrane proteins: PRP was separated from whole blood anticoagulated with BDTA, washed three times with TEN solution, and the platelet count was adjusted to 2×10 8 / mL, add 1mmol / LPMSF, 1% Triton X-100 to lyse platelets, centrifuge and take supernatant, add 60μL / well, perform non-reducing 5%-15% logarithmic gradient SDS-PAGE, transfer to nitrocellulose membrane, block with 2% BSA-PBS, cut into different strips, add R813 and Y262 antibodies, incubate at 37℃ for 2 hours, GAM-HRP is used as secondary antibody, and finally add chloronaphthol for color development. At the same time, IgG is used as negative control and SZ-22 antibody is used as positive control. The results show that R813 acts on platelet GPIIIa, while Y262 has no reaction (see Figure 3 ).

[0057] (4) Affinity chromatography to determine the interaction sites of R813 and Y262 antibodies with platelet membrane proteins: CNBr-Sepharose4B gel was swollen with 1mmol / L hydrochloric acid for 4 hours, washed with coupling buffer 3 times, and purified R813 and Y262 were added to the gel after sufficient dialysis, and shaken at 4°C for 16 hours. Washed with coupling buffer in a sand core funnel, soaked in 1mmol / L ethanolamine twice, each time for 1 hour, filtered with coupling buffer, 25mmol / L Tris-HCl, and TBS in turn, and finally loaded onto the column after coupling. Prepare platelet disruption solution in the same way as in method (3), and add 2x10 9 The platelet fragments were circulated and then eluted with 0.05 mol / L diethylamine-5 mmol / L EDTA buffer. The eluate was dialyzed against PBS and concentrated and identified using 10% SDS-PAGE. The results showed that the antigen acting on R813 was GPIIIa, while the antigen acting on Y262 was GPIIb-IIIa complex (see Figure 4 ).

[0058] Example 4: Antibodies R813 and Y262 interact with different sites of the platelet receptor GPIIb-IIIa complex

[0059] 4.1 Materials

[0060] 125 I was purchased from China National Atomic Energy Corporation, Sephadex G-25 gel was a product of Phamacia, and the other reagents were all domestic analytical grade reagents.

[0061] 4.2 Methods

[0062] 125 I-labeled R813 radioimmunoassay: 30 μg R813 (1 mg / mL) was added to 10 μL 0.1 mol / LPB pH 7.5; 1 mg 125I and 10μL of 5mg / mL ChT, let stand at room temperature for 1 minute, then add 20mg / mL Na2S20820μL and let stand at room temperature for 1 minute, then add 100μL 0.5% BSA-PBS, add the reactants to the Sephadex G-25 gel column pre-equilibrated with 0.5% BSA-PBS, and elute with the same solution, collecting 10 drops in each tube. Measure the radioactivity count with a γ counter and collect the first radioactivity peak. As before, platelet coating was performed with different concentrations of mouse IgG, R813, Y262 and fixed concentration 125 After mixing I-R813, the coated plate was added and incubated at 37°C for 2 hours, then washed 3 times and the radioactive counts were detected by a γ counter. The results showed that as the concentration of R813 increased, the radioactive counts per well of the coated plate decreased, while the radioactive counts did not change with the concentrations of Y262 and mouse IgG, indicating that Y262 and R813 act on different antigenic sites (see Figure 6 ).

[0063] Example 5: Antibodies R813 and Y262 synergistically block platelet GPIIb-IIIa complexes to fully inhibit platelet aggregation function

[0064] 5.1 Materials:

[0065] ADP is a product of Shanghai Livzon Dongfeng Biological Products Co., Ltd., GAM-FITC is a product of Shanghai Huamei Co., Ltd., and the other reagents are all domestic analytical grade reagents.

[0066] 5.2 Methods:

[0067] (1) Flow cytometry mean fluorescence intensity superposition test: 5 μL PRP was reacted with antibodies R813, Y262 and the mixture of the two at room temperature for 30 minutes, then washed and GAM-FITC was added and detected by flow cytometry. Mouse IgG was used as a negative control. The results showed that the mean fluorescence intensity of positive cells when R813 and Y262 acted together was the sum of the two antibodies acting alone, indicating that R813 and Y262 can act on their respective antigenic determinants on the platelet GPIIb-IIIa complex, thereby producing a synergistic blocking effect on the complex molecules (see Table 1).

[0068] Table 1. Results of flow cytometry mean fluorescence intensity superposition method for monoclonal antibodies R813 and Y262

[0069] Antibody Positive cell percentage (%) Mean fluorescence intensity PBS 3.22 2.61 Mouse-IgG 6.48 3.26 SZ-22 90.2 83.1 R813 87.7 185.7 Y262 73.6 130.9 R813+mouse-IgG 86.4 174.8 R813+SZ-22 92.3 255.4 R813+R813 87.6 180.1 R813+Y262 88.1 338.5

[0070] (2) Test on the synergistic inhibition of ADP-induced platelet aggregation by R813, Y262 and the two: Citrate anticoagulated whole blood of healthy adults, separated PRP, added with different concentrations of antibodies R813, Y262 and the mixture of the two, incubated at 37°C for 5 minutes, then added with ADP (final concentration 5 μmol / L), and the aggregation results were detected. Mouse IgG was used as a negative control, and Reopro was used as a positive control. The results showed that both R813 and Y262 could effectively inhibit platelet aggregation. When the two acted together, the inhibitory effect on platelet aggregation was enhanced at the same antibody concentration, and the platelet aggregation function could be completely blocked. Its antithrombotic activity exceeded that of Reopro. It can also be calculated that the dosage of 50% inhibitor is lower than that of Reopro (see Tables 2, 3, and 4).

[0071] Table 2. Effects of different concentrations of monoclonal antibodies on platelet aggregation. First experiment

[0072]

[0073]

[0074] Second trial

[0075]

[0076] The third test

[0077]

[0078] Table 3. Effects of monoclonal antibodies R813, Y262 and their cocktail (DiMab) on platelet aggregation First trial

[0079]

[0080] Second trial

[0081]

[0082] The third test

[0083]

[0084]

[0085] Table 4. Dosage of 50% platelet aggregation inhibitor of monoclonal antibody cocktail (DiMab) and 7B3

[0086]

[0087] Example 6: Preparation and identification of antibody R813 and Y262 F(ab')2 fragments

[0088] 6.1 Materials

[0089] Papain was a product of Shanghai Livzon Dongfeng Biotechnology Co., Ltd., DBAB-52 gel was purchased from Westbas Co., Ltd., Shanghai Institute of Biochemistry, Chinese Academy of Sciences, DTT was a product of Fluka, and the remaining reagents were all domestic analytical grade reagents.

[0090] 6.2 Methods

[0091] (1) Preparation and identification of R813 and Y262 F(ab')2 fragments: R813 and papain were mixed at a mass ratio of 5:1 and reacted at 4°C for 24 hours. The reaction was terminated with iodoacetamide (final concentration 80 mmol / L). After dialysis with 0.01 mol / L Tris-HCl pH 8.0, the column was loaded on a DEAB-52 ion exchange column and eluted with 0-0.3 mol / L NaCl 0.01 mol / L Tris-HCl pH 8.0. The first peak was collected. Y262 and papain were mixed at a mass ratio of 80:1 and reacted at 4°C for 35 minutes. Then papain and Y262 were added at a mass ratio of 1:160 and mixed at 4°C for 35 minutes. Next, R813 was terminated and purified. The difference was that the second elution peak was collected. The collected material was subjected to reduced and non-reduced 10% SDS-PAGE. The results showed that the F(ab')2 fragments of the two antibodies were obtained after papain digestion and purification. (See Figure 7 )

[0092] (2) R813 and Y262F(ab')2 fragments inhibit ADP-induced platelet aggregation test: The method is the same as before. The results show that R813 and Y262F(ab')2 fragments can still work synergistically to completely inhibit platelet aggregation function. (See Table 5)

[0093] Table 5. Inhibitory effect of antibody F(ab')2 fragment on platelet aggregation

[0094]

[0095] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A monoclonal antibody that recognizes platelet membrane glycoprotein, characterized in that: include: A monoclonal antibody against the platelet receptor GP IIIa, produced by the hybridoma cell line R813; Monoclonal antibody against the platelet receptor GP IIb-IIIa complex, produced by the hybrid cell line Y262.

2. A hybridoma cell line producing a specific anti-platelet receptor monoclonal antibody, characterized in that: include: Hybridoma cell lines producing monoclonal antibodies that recognize platelet GP IIIa: R813, Y88, Y128, and Y474; Hybridoma cell lines producing monoclonal antibodies recognizing platelet GP IIb-IIIa complex: Y8, Y262, Y291, Y295, Y321, Y342, Y458, Y585 and Y700.

3. The hybridoma cell line producing specific anti-platelet receptor monoclonal antibodies according to claim 2, characterized in that: The hybridoma cell line producing the monoclonal antibody recognizing platelet GP IIIa is preferably R813, and the hybridoma cell line producing the monoclonal antibody recognizing platelet GP IIb-IIIa complex is preferably Y262.

4. A method for preparing a monoclonal antibody against a specific antigenic determinant of a platelet surface glycoprotein, characterized in that: Human platelets were used as immunogens to immunize Balb / c mice, and spleen cells were fused with SP2 / 0-Ag14 myeloma cells to develop hybridoma cells that produce monoclonal antibodies that recognize platelet GP IIIa and monoclonal antibodies against platelet receptor GP IIb-IIIa complex. ELISA, FCM, Western blotting and affinity chromatography were used to identify the antigenic determinants of the monoclonal antibodies and observe the binding activity of the monoclonal antibodies to platelets. Ascites was prepared and the monoclonal antibodies were purified.

5. The method for preparing a monoclonal antibody against a specific antigenic determinant of a platelet surface glycoprotein according to claim 4, characterized in that: include: Monoclonal antibody fragments against specific antigenic determinants of platelet surface glycoprotein were prepared. Monoclonal antibody R813 and Y262F(ab')2 fragments were prepared by papain digestion and identified by SDS-PAGE.

6. A pharmaceutical composition for inhibiting platelet aggregation, characterized in that: The invention comprises a monoclonal antibody against platelet receptor GP IIIa, a monoclonal antibody against platelet receptor GP IIb-IIIa complex or a combination thereof, and a pharmaceutically acceptable carrier or excipient.

7. A pharmaceutical composition for inhibiting platelet aggregation according to claim 6, characterized in that: The monoclonal antibody is IgG fragment Fab and F(ab')2 of the monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody or genetically engineered antibody.

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