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Antibodies specific for glycoprotein VI and methods of producing these antibodies

A specific, monoclonal antibody technology, applied in the field of research and immunotherapeutics, which can solve the problems of endangering the life and health of patients, stimulating aggregation, forming thrombus and embolus, etc.

Inactive Publication Date: 2007-05-16
OTSUKA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may activate platelets, stimulate aggregation and eventually lead to the formation of thrombi and emboli, further endangering the life and health of the patient

Method used

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  • Antibodies specific for glycoprotein VI and methods of producing these antibodies
  • Antibodies specific for glycoprotein VI and methods of producing these antibodies
  • Antibodies specific for glycoprotein VI and methods of producing these antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Preparation of monoclonal GPVI antibody

[0093] Normal mice (Balb / c, female), Armenian hamsters (male), and GPVI knockout mice (produced at Otsuka GEN institute) were immunized as described below to produce monoclonal antibodies.

[0094] GPVI knockout mice were generated as described above (Mori et al. Neurosci. Res. 43:251-7, 2002). As shown in Figure 1A, the target vector was constructed by replacing the GPVI gene genome fragment of the 129 / Sv mouse genome lambda clone (Clalsite) with a pMC1-neo-polyadenylic acid (Stratagene) cassette (Ezumi Y. et al., Biochem Biophys Res Comm 277: 27-36, 2000), the replaced genome fragment contains the last 5 bases of exon 2 and the first half of exon 3. Electroporation allowed linearized constructs into AB2.2 ES cells from 129 / Sv mice (Lexicon Genetics Inc., The Woodlands, TX) and selected cells in G-418. Screening of G-418 resistant ES cell clones for successful homologous recombination at exons 2 and 3 in G-418-resistant ES cell clo...

Embodiment 2

[0110] GPVI-specific antibodies are effective inhibitors of collagen-induced platelet aggregation and adhesion

[0111] The inhibitory potential of the Fab fragment prepared according to Example 1 on collagen-induced platelet function (including collagen-induced platelet aggregation and platelet adhesion to fixed collagen) was tested under static and flowing conditions.

[0112] The antibodies were tested for in vitro platelet aggregation as follows. Due to differences in collagen response between individuals, a collagen dose experiment was first performed to determine the amount of collagen that gave 70%-90% of platelet aggregation within 5 minutes after addition. In addition, the type of collagen used in the assay may significantly affect the response. From experience, acid-insoluble horse tendon collagen (Nycomed, Germany) provides the strongest platelet aggregation response. Nieswandt (J. Biol. Chem. 275: 23998-24002, 2000 and US Patent Publication No. 2002 / 0141992), Lecut et ...

Embodiment 3

[0133] GPVI specific antibody inhibits collagen-induced secretion and thromboxane A 2 form

[0134] The Fab fragment of the GPVI-specific antibody of the present invention was also tested for collagen-induced secretion and thromboxane A. 2 (TXA 2 ) The impact of formation. Secretion refers to the release of biologically active content from the alpha and dense granules of platelets induced by an agonist.

[0135] One way to quantify the release induced by an agonist is to measure the ATP content in the culture medium by a luciferase assay using chemiluminescence methods. The collagen-induced ATP secretion was tested on platelet-rich plasma (PRP) using a bioluminescence aggregation analyzer (Chronolog Corporation, PA) and luciferase-luciferin reagent. The bioluminescence aggregation analyzer simultaneously measures the platelet aggregation and ATP secretion induced by the agonist. In short, human blood was drawn directly into 3.8% sodium citrate (9:1 volume of blood: citrate) with a...

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Abstract

The present invention describes antibodies generated against platelet membrane glycoprotein VI (GPVI), methods of producing the anti-GPVI antibodies, and the use of these antibodies as research and immunotherapeutic agents, in particular, as antithrombotic therapeutic agents.

Description

[0001] This application claims the rights and interests of U.S. Provisional Application No. 60 / 566,171 filed on April 29, 2004, which is incorporated herein by reference. Technical field [0002] The present invention relates to antibodies produced against platelet membrane glycoprotein VI (GPVI), fragments thereof or naturally-occurring variants thereof, methods for producing anti-GPVI antibodies and these antibodies as research and immunotherapeutic agents (especially as the treatment of thrombosis and other The use of therapeutic agents for vascular diseases). Background technique [0003] Platelets are small non-nucleated blood cells necessary for hemostasis control and wound healing. Circulating platelets are quite quiet under normal conditions. However, when a blood vessel is torn or damaged, platelets come into contact with multiple factors to initiate complex and interconnected cellular procedures that cause blood clotting and clot formation. This is reviewed in "Mechanism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P7/02
CPCC07K16/2803C07K2317/565C07K2317/92C07K2317/54A61K2039/505C07K2317/55C07K2316/96C07K2317/76A61P7/02C07K2317/34C07K16/28A61K39/395
Inventor N·N·坦登松本丰泷泽久生亿山庆滋
Owner OTSUKA PHARM CO LTD
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