JP2010180212A - Pharmaceutical composition containing antibody for oxidized ldl receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-oxidized LDL receptor (LOX-1) antibody having potency to inhibit binding of a ligand by binding to the LOX-1 and able to be used as a pharmaceutical composition. <P>SOLUTION: The anti-LOX-1 antibody which is a human monoclonal antibody, and a fragment peptide having potency to bind to the LOX-1 and cells for producing the antibody are provided. The antibody can be used as a prophylactic-therapeutic composition for various inflammatory diseases, arteriosclerosis. post-operative blood vessel restenosis of PTCR and PTCA, and the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention comprises a substance capable of binding to an oxidized LDL receptor and inhibiting the binding of an in vivo ligand of the oxidized LDL receptor to the oxidized LDL receptor, and a pharmaceutically acceptable carrier, (1) A pharmaceutical composition for treating a disease caused by binding of platelets or activated platelets to the oxidized LDL receptor, or uptake of platelets or activated platelets by cells expressing the oxidized LDL receptor, (2) Leukocyte tissue infiltration caused by inflammatory reaction after arteriosclerosis, myocardial ischemia reperfusion injury, percutaneous coronary thrombolysis (PTCR), or after percutaneous coronary angioplasty (PTCA) (3) after arteriosclerosis, myocardial ischemia reperfusion injury, percutaneous coronary thrombolysis (PTCR), or percutaneous coronary angioplasty (PTCA) Cure inflammation Pharmaceutical compositions for, and (4) percutaneous coronary thrombolysis procedure (PTCR) or percutaneous coronary angioplasty pharmaceutical composition for the treatment of post-surgical vascular restenosis (PTCA), it relates.

  The various forms of cholesterol (free, long chain fatty acid, and ester forms) present in all tissues and blood are primarily derived from biosynthesis in the liver. Free-type cholesterol biosynthesized in the liver is taken up by very low density lipoprotein (VLDL), and in the blood, by the action of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL), intermediate density lipoprotein (IDL) ) And then metabolized to low density lipoprotein (LDL). This LDL plays an important role in the construction of the cell membrane of a living body by being taken into peripheral cells via the LDL receptor.

However, when this LDL is oxidatively modified by the action of cells such as vascular endothelial cells, various chemical and physical factors, or various factors such as heat, modified LDL called oxidized LDL (Oxidized LDL) It will occur in the blood.
Since there is a sufficient amount of antioxidant in the bloodstream, oxidized LDL is not likely to occur in the bloodstream, but even if it occurs, most of them are metabolized in the liver.

On the other hand, in the intravascular subcutaneous and vascular walls, oxidized LDL is produced by cell-dependent chemical modification of vascular endothelial cells and macrophages and cell-independent chemical modification by the action of Fe3 +, etc. In contrast, oxidized LDL generated in the subendothelial and vascular walls is accumulated in the cells of macrophages.
Accumulation of oxidized LDL in macrophage cells is due to the fact that the oxidized LDL thus generated is a receptor for various modified LDLs (oxidized LDL, acetyl LDL, succinyl LDL, malondialdehyde LDL). It is by being taken up into a cell through a scavenger receptor (Nature, Vol.343, p.531-535, 1990; Nature, Vol.343, p.570-572, 1990; Proc. Natl. Acad USA, Vol.87, p.9133-9137, 1990; Proc. Natl. Acad. Sci. USA, Vol.87, p.8810-8814, 1990; Curr. Opin. Lipodol., Vol.2, p.295-300, 1991; and J. Clin. Invest., Vol.90, p.1450-1457, 1992).

  This macrophage scavenger receptor, unlike the LDL receptor, does not undergo receptor down-regulation depending on the amount of intracellular cholesterol. Therefore, macrophages that have submerged in the vascular endodermis or vascular wall accumulate a large amount of cholesterol in the cells by taking in a large amount of denatured LDL, and become foam cells ("molecular arteriosclerosis", Chapter 4 “Inflammatory cells 1. Scavenger receptor”, pp. 249-258, 1995, published by Medical Review.

  On the other hand, the macrophages that have entered the vascular endoderm and the vascular wall are transferred from the bloodstream in response to the generated signals of oxidized LDL generated in various parts of the bloodstream, such as the vascular subendothelium or the vascular wall. Derived from macrophages. That is, oxidized LDL is chemotactic for macrophages and monocytes in the bloodstream, and has the property of concentrating monocytes and macrophages on vascular endothelial cells. In addition, it has the property of inducing retraction to the blood vessel wall, the property of inducing differentiation of the drawn monocytes into macrophages, and the property of suppressing the migration of the macrophages that have completed differentiation.

In order to collect monocytes and macrophages into vascular endothelial cells, an oxidized LDL receptor (Oxidized-LDL Receptor, Ox-LDL Receptor or LOX-) expressed on vascular endothelial cells recently identified by the present inventors was used. Nature, Vol.386, p.73-77, 1997, and lipid biochemistry research, Vol.39, p.83-84, 1997) are being revealed to be deeply involved. is there.
From previous studies, when oxidized LDL in the bloodstream is taken into vascular endothelial cells via oxidized LDL receptors, the production of nitric oxide (NO) in the cells is inhibited, resulting in vascular endothelium. It has been experimentally proved that expression of cell adhesion molecules is induced on the surface of cells. From this, it is considered that, as a result of the expression of cell adhesion molecules, macrophages and monocytes are trapped on vascular endothelial cells, and the trapped macrophages and monocytes are submerged in the blood vessel subendothelium and blood vessel wall. Thus, it is considered that macrophages that have submerged in the vascular endodermis and vascular wall become foamed cells by the uptake of oxidized LDL via macrophage scavenger receptors as described above.

Since the foam cell formation of macrophages in the blood vessel wall is the main cause of arteriosclerosis, the trigger of the onset of arteriosclerosis is the above-mentioned aggregation of monocytes and macrophages to vascular endothelial cells It is considered.
The oxidized LDL receptor, which is deeply involved in the aggregation of monocytes and macrophages into vascular endothelial cells, has been sought for many years by many researchers. As a result, the present inventors succeeded in identifying them for the first time in 1997. It is something that has been bathed. With regard to the oxidized LDL receptor, detailed studies other than the above-described properties and functions are currently being actively pursued.

  Aging cells and cells that are heading for cell death (such as apoptosis) are cells that are detrimental to the body, and phagocytosis by other cells of the cells is given normal healthy cells by such cells This is an essential process to protect against various adverse effects (Immunol. Today, Vol. 14, p. 131-136, 1993). It has been proposed that various changes in the surface of the aging cells and cells that are undergoing cell death (such as apoptosis) are the mechanism of recognition by phagocytic cells.

  Among these changes, trans-location of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the cell membrane is the earliest in the process to cell death (apoptosis) (J. Exp. Med., Vol.182, p.1545-1556, 1995). Thus, recognition of PS exposed to the surface of cells can be considered the most important mechanism for removal of aging cells and cells that are heading for cell death (such as apoptosis). In macrophages, it has been proposed that the binding of aging cells and cells towards cell death (such as apoptosis) occurs via various receptors, including CD36 and class A scavenger receptors (J Clin. Invest., Vol.90, p.1513-1522, 1989; Proc. Natl. Acad. Sci. USA, Vol.93, p.12456-12460, 1996). CD36 recognizes apoptotic photoreceptor outer segments by reticuloepithelial cells and monocyte-derived macrophage thymocytes and neutrophils in cooperation with αvβ3 integrin and thrombospondin It is involved in sphere recognition (J. Clin. Invest., Vol. 90, p. 1513-1522, 1989; J. Biol. Chem., Vol. 271, 20536-20539, 1996). In thymic macrophages, phagocytosis of thymocytes toward apoptosis is reduced by treatment with an anti-class A scavenger receptor antibody or treatment of the gene of the class A scavenger receptor (Proc. Natl. Acad. Sci. USA, Vol.93, p.12456-12460, 1996).

Macrophages are specialized in phagocytosis of cell abnormal substances and pathogenic organisms, but many of the neighboring cells are also involved in phagocytosis of the apoptotic cells (Immunol. Today, Vol. 14). , p. 131-136, 1993). The role of PS in phagocytosis by the molecule has not yet been elucidated, but it has been reported that PS and oxidized LDL compete in phagocytosis of aging cells and cells towards cell death (such as apoptosis) (Proc Natl. Acad. Sci. USA, Vol. 92, p.1396-1400, 1995). This finding provides an interest in examining whether the LOX-1 receptor on the vascular endothelium for oxidized LDL is involved in the phagocytic mechanism. Oka et al. Recently reported that LOX-1 mediates phagocytosis of senescent erythrocytes and apoptotic cells in vascular endothelial cells (Proc. Natl. Acad. Sci. USA. , Vol.95, p.9535-9540, 1998).
However, the interaction between other cells such as platelets and leukocytes and the oxidized LDL receptor LOX-1 has not been elucidated at all.

LOX-1 is a major receptor for oxidized LDL expressed in vascular endothelial cells and is predicted to mediate the effects of oxidized LDL on endothelial cells. LOX-1 expression is found not only in cultured endothelial cells, but also in the intact aortic athero-intima and in the human carotid artery. LOX-1 expression is induced by certain cytokines in vitro and increased in hypertensive rats. This indicates that LOX-1 expression is strongly regulated by stimuli associated with arteriosclerosis (Circulation, Vol. 96, p. 1-104, 1997; Biochem. Biophys. Res. Commun., Vol.237, p.496-498, 1997).
However, the causal relationship between LOX-1 and arteriosclerosis and intimal thickening has not been elucidated yet.

In acute myocardial infarction, coronary arteries that send blood to the myocardial tissue are blocked by arteriosclerosis or thrombus and the myocardial tissue is destroyed. In recent years, a wide range of treatments have been implemented to restore coronary blood flow and reduce myocardial necrosis by performing percutaneous coronary thrombolysis (PTCR) and percutaneous coronary angioplasty (PTCA) early in the onset. The improvement in survival rate and cardiac function is improved. However, if the blood flow disruption time exceeds a certain limit, the myocardial ischemia reperfusion injury accompanied by a serious inflammatory reaction is caused by newly causing myocardial injury by resuming the blood flow and reducing the amount of myocardial tissue that can be saved. Happens.
On the other hand, PTCR and PTCA involve injuries to the intima of blood vessels because a catheter or balloon is inserted into an arterial occlusion site and the stenosis site is physically removed to open the blood vessel. When vascular injury occurs, vascular smooth muscle proliferation and subintimal migration are induced, and restenosis with thickened PTCR and PTCA treatment sites occurs. In the case of PTCA, vascular restenosis occurs in about 40% of patients within several months after the procedure.
The relationship between LOX-1 and the development of arteriosclerosis has not been clarified, although there is a suggestion about its possibility. The relationship between inflammation in arteriosclerosis, inflammation in ischemic reperfusion injury after PTCR or PTCA treatment and LOX-1, and the relationship between vascular restenosis after PTCR or PTCA treatment and LOX-1 It has not been revealed yet.

Platelets, which are one of the blood cell components along with leukocytes and red blood cells, play an important role in the hemostatic mechanism (blood coagulation system), and when in contact with damaged vascular endothelial cells, various blood coagulation factors (platelet factor) Etc.) and cause blood coagulation (hemostasis) by causing an adhesion and agglutination reaction at the site of injury. Platelets form aggregates when they come into contact with blood coagulation inducers such as collagen, ADP, or thrombin. Furthermore, platelets are greatly involved in immune responses, particularly inflammatory responses. That is, the cosmetic plate contacts and adheres to injured vascular endothelial cells, and releases vascular permeability factors and factors involved in activation of complement components that migrate leukocytes. Regarding the relationship between platelets and inflammation, for example, glomerulonephritis is well known. Many lesions in the kidney, such as glomerulonephritis, develop based on immunological mechanisms. Inflammation released by antibodies that bind to immune complexes and glomeruli in the bloodstream activate various inflammatory cells such as complement, neutrophils, monocytes / macrophages, and platelets It is thought to cause various lesions via mediators.
The following facts have been clarified as evidence that platelets are involved in the development of glomerulonephritis. (1) Platelet membrane antigen, platelet factor-4 (PF-4), β-thromboglobulin (β-TG) and PDGF (platelet-) by electron microscopy and immunohistochemical techniques Derived growth factor) and other granular substances in platelets can be seen in glomerular lesions. (2) Decrease in platelet concentration of platelets and increase in blood flow. (3) Shortening of platelet life. (4) Increased platelet aggregation with ADP, (5) Positive rate of platelet aggregation test representing IC in the bloodstream is high in some nephritis, (6) Antiplatelet drugs Decreased proteinuria and improved kidney function.

  The involvement of platelets and the coagulation system in primary and persistent glomerulonephritis is also evident from experimental nephritis and clinical findings. When glomerular tissue damage occurs due to infiltration of inflammatory cells due to nephritis, collagen in the glomerular basement membrane is exposed and the intrinsic coagulation system is activated. On the other hand, the exogenous coagulation system is activated when tissue thromboplastin is released due to damage to the vascular wall and the coagulation activator is released from infiltrated monocytes. When platelets are added to this and fibrin is deposited to form a thrombus, a sclerotic lesion such as arteriosclerosis progresses, and at the same time, the fibrinolytic system is activated. Platelets are involved in thrombus formation resulting from the activation of the coagulation system, resulting in glomerular damage from circulatory disturbances.

  Thus, platelet activation that is deeply involved in the onset of nephritis has been analyzed by analysis of serum sick nephritis. In the analysis, when an antigen binds to sensitized basophils, platelet-activating factor (PAF) is released, and a release reaction occurs in response to the receptor of platelets. That is, platelet activation involves not only IC and complement, but also mechanisms such as thromboxane A2, PAF, and vascular wall ADPase.

In relation to the above-described properties of platelet coagulation and involvement in various inflammations, for example, DIC (disseminated intravascular coagulation syndrome; disseminated intravascular coagulation syndrome; dissemination) Intravascular coagulation syndrome, defibrin syndrome or consumable coagulation injury), platelet purpura and idiopathic thrombocytopenia.
In DIC, a substance that activates the intrinsic coagulation process flows into blood vessels by some mechanism, and thrombus is formed extensively in small blood vessels throughout the body, and blood coagulation factor V, blood coagulation factor VIII and platelets Decreased when consumed. Diseases that are likely to cause this disease include septicemia caused by bacteria (eg, Gram-negative bacilli), acute promyelocytic leukemia, hemolytic uremic syndrome, amniotic fluid emboli, pre-placental early detachment, adenocarcinoma metastasis, snake venom Known are bite bites, lung surgery, and prostate surgery.
Platelet purpura is a disease in which blood cells such as platelets and red blood cells cause allergic reactions and autoimmune reactions due to drugs, resulting in destruction of platelets and thrombocytopenia. Sudden thrombocytopenia is a condition in which platelets are rapidly removed by spleen macrophages, and frequently occurs due to infections caused by bacteria or viruses or autoimmune diseases such as SLE.
On the other hand, the interaction between LOX-1 and platelets and activated platelets, the relationship between LOX-1 and various thrombocytopenia, and the relationship between LOX-1 and various inflammations have not been clarified yet. .

In a series of inflammatory reaction processes, adhesion of leukocytes to vascular endothelial cells and subsequent infiltration of leukocytes into extravascular tissues occur at an early stage of the inflammatory reaction. That is, when some damage is caused to a local tissue due to an external or internal factor, normally white blood cells flowing at high speed in the bloodstream of blood quickly migrate to vascular endothelial cells (“leukocyte migration”). Then, it is tethered to vascular endothelial cells via various adhesion molecules (selectin, etc.) (“initial adhesion”) and rolls on the vascular endothelial cell membrane (“rolling”). The leukocytes then bind tightly to the vascular endothelial cells through adhesion with other adhesion molecules (such as integrins) (“strong binding”), and then extravasate from the gap between the vascular endothelial cells and the vascular endothelial cells. Infiltrate the tissue (“infiltration”). After leukocytes infiltrate the tissue, they migrate to the site of tissue injury ("transition"), and a series of inflammatory reactions are initiated.
The relationship between LOX-1 and the adhesion of such leukocytes to vascular endothelial cells, rolling on vascular endothelial cells, and infiltration of leukocytes into tissues has not yet been clarified.

As for the ligand that binds to the oxidized LDL receptor (LOX-1) and is taken into the cell via the receptor, the oxidized LDL in the blood (the progress of foaming of macrophages on the blood vessel wall causing arteriosclerosis etc.) Triggers the aggregation of monocytes and macrophages into vascular endothelial cells, which are early biological reactions in the process), some artificially prepared senescent erythrocytes, and some artificially prepared apoptotic progression It is not known except for sex cells.
Identifying and elucidating the function of the oxidized LDL receptor, as well as various biological ligands of the oxidized LDL receptor, and finding a substance capable of inhibiting the binding of the in vivo ligand to the oxidized LDL receptor It has extremely important significance in the development of drugs useful for the prevention and treatment of various diseases caused by binding of a ligand to an oxidized LDL receptor and / or incorporation of the ligand into an oxidized LDL receptor-expressing cell.

  That is, the present invention identifies an in vivo ligand of an oxidized LDL receptor, finds a substance having an ability to inhibit the binding of the in vivo ligand to the oxidized LDL receptor, and uses the substance to oxidize the ligand. It is intended to provide a pharmaceutical composition for preventing and treating various diseases by suppressing or inhibiting binding to LDL receptor and / or uptake of the ligand into cells.

As a result of diligent research on ligands that bind to mammalian oxidized LDL receptor (LOX-1) and are taken into cells such as vascular endothelial cells via the receptor, the present inventors found the following and found the present invention. It came to complete.
(1) Oxidized LDL receptor binds to platelets and activated platelets and takes them into cells (phagocytosis), and further, such binding and uptake (phagocytosis) binds to oxidized LDL receptor For example, it is inhibited by an antibody that binds to oxidized LDL receptor.
(2) Substances that bind to oxidized LDL receptor, such as antibodies that bind to oxidized LDL, significantly inhibit thrombocytopenia in diseases associated with thrombocytopenia.
(3) A substance that binds to oxidized LDL receptor, for example, an antibody that binds to oxidized LDL, significantly inhibits the infiltration of leukocytes into tissues, which is the initial stage of the inflammatory reaction in various inflammatory diseases.
(4) A substance that binds to oxidized LDL receptor, for example, an antibody that binds to oxidized LDL, significantly inhibits vascular restenosis after percutaneous coronary angioplasty (PTCA).

These completely novel findings indicate that oxidized LDL receptors are profound in various inflammations, thrombocytopenia, vascular stenosis and postoperative restenosis in percutaneous coronary angioplasty, and sclerotic lesions such as arteriosclerosis. Involved and those diseases can be treated with substances that bind to oxidized LDL receptor and inhibit the binding of oxidized LDL in vivo ligand to oxidized LDL receptor (for example, antibodies that bind to oxidized LDL) For the first time.
That is, based on these findings, by using a substance that binds to oxidized LDL receptor and inhibits the binding of oxidized LDL in vivo to oxidized LDL receptor (for example, an antibody that binds to oxidized LDL), It is possible to develop drugs for preventing and treating various diseases.

That is, the present invention is an invention as described in the following (1) to (12).
(1) a substance capable of binding to an oxidized LDL receptor and inhibiting the binding of an in vivo ligand of the oxidized LDL receptor to the oxidized LDL receptor, and a pharmaceutically acceptable carrier, A pharmaceutical composition for treating a disease caused by binding of platelets or activated platelets to an oxidized LDL receptor, or uptake of platelets or activated platelets by cells expressing the oxidized LDL receptor.
(2) The pharmaceutical composition according to (1) above, wherein the disease is a disease accompanied by thrombocytopenia.
(3) The pharmaceutical composition according to (1), wherein the disease is a kidney disease.
(4) The pharmaceutical composition according to any one of (1) to (3) above, wherein the substance is an antibody that binds to an oxidized LDL receptor or a part of the antibody.
(5) A leukocyte tissue comprising a substance capable of binding to an oxidized LDL receptor and inhibiting the binding of an in vivo ligand of the oxidized LDL receptor to the oxidized LDL receptor, and a pharmaceutically acceptable carrier Infiltration inhibitor.
(6) The leukocyte tissue infiltration is inflammation after arteriosclerosis, myocardial ischemia reperfusion injury, percutaneous coronary thrombolysis (PTCR), or percutaneous coronary angioplasty (PTCA). The leukocyte tissue infiltration inhibitor as described in (5) above, which is leukocyte tissue infiltration caused by reaction.
(7) The leukocyte tissue infiltration inhibitor according to (5) or (6) above, wherein the substance is an antibody that binds to oxidized LDL receptor or a part of the antibody.
(8) An anti-inflammatory comprising a substance capable of binding to an oxidized LDL receptor and inhibiting the binding of an in vivo ligand of the oxidized LDL receptor to the oxidized LDL receptor, and a pharmaceutically acceptable carrier Agent.
(9) The inflammation is inflammation after arteriosclerosis, myocardial ischemia reperfusion injury, percutaneous coronary thrombolysis (PTCR), or percutaneous coronary angioplasty (PTCA). The anti-inflammatory agent according to (8) above, wherein
(10) The anti-inflammatory agent according to (8) or (9) above, wherein the substance is an antibody that binds to an oxidized LDL receptor or a part of the antibody.
(11) Transdermal comprising a substance capable of binding to an oxidized LDL receptor and inhibiting the binding of an in vivo ligand of the oxidized LDL receptor to the oxidized LDL receptor, and a pharmaceutically acceptable carrier Agent for post-operative vascular restenosis of coronary artery thrombolysis (PTCR) or percutaneous coronary angioplasty (PTCA).
(12) The therapeutic agent for vascular restenosis according to (11), wherein the substance is an antibody that binds to an oxidized LDL receptor or a part of the antibody.

The pharmaceutical composition of the present invention results from abnormal kinetics of oxidized LDL receptor, binding of platelets and activated platelets to oxidized LDL receptor, and / or their incorporation into oxidative LDL-expressing cells (phagocytosis). Prevention of various diseases such as various diseases, various diseases accompanied by leukocyte infiltration in the lesion process (eg inflammation), various inflammations, arteriosclerosis, postoperative vascular restenosis such as PTCR and PTCA, and the like Useful in therapy.
In addition, the pharmaceutical composition comprising a human antibody against the human oxidized LDL receptor included in the present invention is extremely useful as a pharmaceutical because it does not cause any side effects such as allergies when a mouse-derived antibody is administered to humans. It is.

The figure which shows the state of the binding | bonding and uptake | capture (phagocytosis) to the bovine aortic vascular endothelial cell (BAE) of an aging erythrocyte and an apoptotic cell. The fraction (a) shows the state of binding of aging erythrocytes to BAE. The fraction (b) shows the results of the assay using natural erythrocytes. The fraction (c) shows the state of binding and uptake of apoptotic HL60 cells to BAE. The part (d) shows the result of the assay using HL60 cells not subjected to treatment for inducing apoptosis. The figure which shows the state of the binding | bonding and uptake (phagocytosis) to aging erythrocytes and apoptotic cells to BLOX1-CHO. The fraction (a) shows the state of binding of aged erythrocytes to BLOX1-CHO. The fraction (b) shows the results of the assay using natural erythrocytes. The fraction (c) shows the state of uptake of senescent erythrocytes into BLOX1-CHO after 6 hours of incubation. The partial diagram (d) shows the state of binding of apoptotic HL-60 cells to BLOX1-CHO. The part (e) shows the result of the assay using HL60 cells not subjected to treatment for inducing apoptosis. The figure which shows the effect of the density | concentration dependent inhibition by the oxidation LDL or the acetylation LDL of the binding | bonding of aging erythrocytes to BLOX1-CHO. Black circles indicate competitive inhibition by oxidized LDL. Open circles indicate inhibition by acetylated LDL. White squares indicate the results when using LDL. The figure which shows the effect of inhibition by various LOX-1 ligands on the binding of senescent erythrocytes to BLOX1-CHO or BAE. The diagonal line shows the result of the assay using BLOX1-CHO, and the black line shows the result of the assay using BAE. The figure which shows the effect of inhibition by the anti- LOX-1 antibody of the binding | bonding of the aging erythrocyte to BLOX1-CHO or BAE. The diagonal line shows the result of the assay using BLOX1-CHO, and the black line shows the result of the assay using BAE. The figure which shows the effect of inhibition by various LOX-1 ligands on the binding of Apoptotic HL-60 cells to BLOX1-CHO or BAE. The diagonal line shows the result of the assay using BLOX1-CHO, and the black line shows the result of the assay using BAE. The figure which shows the effect of inhibition by LOX-Fc of the binding | bonding to aging erythrocyte and apoptoticHL-60 cell to BLOX1-CHO or BAE. The fraction (a) shows the results of the assay using aged erythrocytes. Figure (b) shows the results of an assay using apoptotic HL-60 cells. The diagonal line shows the result of the assay using BLOX1-CHO, and the black line shows the result of the assay using BAE. The figure which shows the state of appearance of PS over time to the erythrocyte surface accompanying aging of erythrocytes. The figure which shows the state of the binding | bonding of the aging erythrocyte to BLOX1-CHO depending on the aging degree of erythrocyte. White circles show the results when erythrocytes are cultured in a phosphate buffer, and black circles show the results when erythrocytes are cultured in a phosphate buffer containing 0.1% glucose. The figure which shows the effect of inhibition with the PS liposome or PC liposome of the binding | bonding of the aging erythrocyte to BLOX1-CHO. A black circle shows the inhibitory effect by PS liposome. White circles show the results when PC liposomes are used. The figure which shows the effect of competitive inhibition by the PS liposome and PC liposome of the binding | bonding of the aging erythrocyte to BAE. The figure which shows the effect of inhibition by the PS liposome of the binding to ApOXoticHL-60 cell to BLOX1-CHO or BAE. The hatched lines indicate the results of the assay using BLOX1-CHO. The black line shows the result of the assay using BAE. The figure which shows the effect of the inhibition by the various phospholipid liposome of the binding | bonding of aging erythrocyte to BLOX1-CHO. The figure which shows the grade of the coupling | bonding to LOX-1 of the platelet measured using the flow cytometer, or activated platelet. The figure which shows the correlation with the coupling | bonding of activated platelet to LOX-1, and the density | concentration of oxidized LDL. The figure which shows the inhibitory effect by the anti- LOX-1 antibody of the coupling | bonding of activated platelet to LOX-1. The figure which shows the grade of the coupling | bonding to the wild-type CHO cell or LOX-1-CHO of the platelet or activated platelet measured using the flow cytometer. The figure which shows the correlation with the coupling | bonding of activated platelet to LOX-1, and the density | concentration of oxidized LDL, and the coupling | bonding of activated platelet to LOX-1 at the time of activating platelet with collagen. The figure which shows the state of the coupling | bonding and / or uptake | capture to platelets of BLOX1-CHO or CHO-K1. The fraction (a) shows the state of binding to BLOX1-CHO. The fraction (b) shows the results of the assay using CHO-K1. The figure which shows the state of uptake (phagocytosis) of platelets into BLOX1-CHO. The figure which shows the effect of inhibition by the anti- LOX-1 antibody of the coupling | bonding of platelet to BLOX1-CHO. The figure which shows the effect of inhibition by the anti- LOX-1 antibody of the coupling | bonding of platelet to BAE. The figure which shows the state of the increase in the coupling | bonding of the activated platelet to BLOX1-CHO accompanying the activation by the thrombin or collagen of platelet. The figure which shows the effect of inhibition by the PS liposome or PC liposome of the binding to platelets BLOX1-CHO. The figure which shows the effect of inhibition by the oxidized LDL or natural LDL of the binding to platelets BLOX1-CHO. White circles show the inhibitory effect when oxidized LDL is used. Black circles show the results of the assay using natural LDL. The figure which shows the grade of the discharge | release of ET-1 from BAE accompanying the coupling | bonding of activated platelets activated by thrombin and collagen, or resting platelets to BAE. The figure which shows the change of the amount of thrombin production in PRP accompanying the coupling | bonding of platelets in platelet rich plasma (PRP) to BLOX1-CHO. The figure which shows the therapeutic effect of the anti-LOX-1 antibody with respect to thrombocytopenia. The figure which shows the inhibitory effect of the anti-LOX-1 antibody with respect to the infiltration | swelling to the structure | tissue of leukocytes. The figure which shows the inhibitory effect of the anti-LOX-1 antibody with respect to the infiltration | swelling to the structure | tissue of leukocytes. The figure which shows the inhibitory effect of the anti- LOX-1 antibody with respect to the protein leakage which is a parameter of the progression of the inflammatory reaction accompanying the infiltration of the leukocyte to the tissue. The figure which shows the inhibitory effect of the anti-LOX-1 antibody with respect to the vascular restenosis after PTCA operation.

Hereinafter, the present invention will be described in detail by clarifying the general production method of the antibody used in the present invention and the meanings of the terms used in the present invention.
The “mammal” in the present invention means human, cow, goat, rabbit, mouse, rat, hamster, guinea pig and the like, preferably human, cow, rat, mouse or hamster, particularly preferably Human.
The “oxidized LDL receptor” in the present invention is an oxidized LDL receptor derived from the “mammal”, and preferably is an oxidized LDL receptor of human, bovine, rabbit, rat or mouse as described in the previous report. Body (Oxidized-LDL Receptor, Ox-LDL Receptor or LOX-1) (Nature, Vol.386, p.73-77, 1997; Lipid Biochemical Research, Vol.39, p.83-84, 1997; JP-A-9-98787; GenBank Accession No. BAA81912; Biochem. J., Vol. 330 (Pt. 3), p. 1417-1422, 1998).
Specifically, it is a human oxidized LDL receptor having the amino acid sequence set forth in SEQ ID NO: 1 or a bovine oxidized LDL receptor having the amino acid sequence set forth in SEQ ID NO: 2.

  The “oxidized LDL receptor” referred to in the present invention includes the amino acid sequence (SEQ ID NO: 1) of the mammalian oxidized LDL receptor, particularly preferably the human oxidized LDL receptor described in the aforementioned literature. And a polypeptide having substantially the same amino acid sequence as the amino acid sequence of the bovine oxidized LDL receptor (SEQ ID NO: 2).

  Here, “having substantially the same amino acid sequence” means that a plurality of amino acids in the amino acid sequence, preferably, as long as it has substantially the same biological properties as the polypeptide containing the previously reported amino acid sequence, Is a polypeptide having an amino acid sequence in which 1 to 10 amino acids, particularly preferably 1 to 5 amino acids are substituted, deleted and / or modified, and a plurality of amino acids, preferably 1 It means that a polypeptide having an amino acid sequence to which 10 to 10 amino acids, particularly preferably 1 to 5 amino acids are added is also included in the scope of the “oxidized LDL receptor” of the present invention.

  The oxidized LDL receptor in the present invention is a transmembrane protein and has an extracellular region. “Extracellular region” means the extracellular region of an “oxidized LDL receptor” defined as described above. Here, the “extracellular region” has the following meaning. That is, not only the oxidized LDL receptor (Oxidized-LDL Receptor, Ox-LDL Reseptor or LOX-1) as described above, but almost all receptors or cell membrane surface molecules belong to the transmembrane protein, Is connected to the membrane by a hydrophobic peptide region that penetrates the lipid bilayer once or several times, and as a whole has three main components: an extracellular region, a transmembrane region, and a cytoplasmic region. It has a structure composed of regions. Furthermore, such transmembrane proteins may be homodimers, heterodimers, as monomers, or together with another chain having the same amino acid sequence or a chain having a different amino acid sequence, respectively. Alternatively, it exists in the form of an oligomer.

  The “extracellular region” used in the present invention refers to the entire partial structure (partial region) existing on the outside of the membrane to which the membrane protein is linked, among the entire structure of the transmembrane protein as described above. Or a part, in other words, all or a region other than the region taken into the membrane (transmembrane region) and the region in the membrane following the region in the membrane (intracellular region) or Mean part.

The “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, preferably a monoclonal antibody.
Specifically, it is an antibody that binds to the aforementioned oxidized LDL receptor (preferably human oxidized LDL receptor) or a part thereof.
The “antibody” of the present invention comprises the above-mentioned mammalian oxidized LDL receptor, preferably human oxidized LDL receptor (including natural, recombinant, synthetic, and cell culture supernatant) or a part thereof as an antigen ( Natural antibodies obtained by immunizing mammals such as mice, rats, hamsters, guinea pigs and rabbits, chimeric antibodies and human antibodies (CDR-grafted antibodies) that can be produced using genetic recombination technology ), And human antibodies that can be produced using human antibody-producing transgenic animals and the like.
In the case of a monoclonal antibody, a monoclonal antibody having any isotype such as IgG, IgM, IgA, IgD, or IgE is also included. Preferably, it is IgG or IgM.

  The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method. That is, for example, an antigen, optionally together with Freund's Adjuvant, a mammal, preferably a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse or cow, more preferably Immunize mice, rats, hamsters, guinea pigs or rabbits. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. Monoclonal antibodies are prepared from the antibody-producing cells obtained from the immunized animal and myeloma cells (myeloma cells) having no autoantibody-producing ability, and the hybridomas are cloned and used for immunization of mammals. It is produced by selecting clones that produce monoclonal antibodies that show specific affinity for the antigen.

  Specifically, the monoclonal antibody can be produced as follows. That is, the above-mentioned human-derived connective tissue growth factor (including natural, recombinant, synthetic, and cell culture supernatant) or a part thereof is used as an immunogen, and the immunogen is optionally added to Freund's Adjuvant. ) In addition to mice, rats, hamsters, guinea pigs or rabbits, preferably mice, rats or hamsters (transgenic animals produced to produce antibodies from other animals such as human antibody-producing transgenic mice). Immunization is carried out by injection or transplantation into the subcutaneous, intramuscular, intravenous, food pad, or abdominal cavity of (including). Usually, immunization is carried out 1 to 4 times about every 1 to 14 days from the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of times of immunization and the time interval can be appropriately changed depending on the nature of the immunogen used.

  Hybridomas that secrete monoclonal antibodies can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, 495-497, 1975) and modifications according thereto. That is, antibody-producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably from the spleen obtained from the immunized mammal as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human It is prepared by cell fusion with a myeloma cell having no autoantibody producing ability derived from a mammal such as mouse, rat or human.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), and P3 / X63-Ag8.U1 (P3U1). , SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM -AGR, D1R11 or CEM-T15 can be used.
The screening of hybridoma clones producing monoclonal antibodies is carried out by cultivating the hybridomas in, for example, a microtiter plate, and determining the reactivity of the culture supernatant of the wells in which proliferation has been observed with the immunizing antigen used in the aforementioned mouse immunization. For example, it can be carried out by measuring by an enzyme immunoassay such as RIA or ELISA.

Production of monoclonal antibodies from hybridomas is carried out in vitro or in vivo in mice, rats, guinea pigs, hamsters or rabbits, preferably mice or rats, more preferably mice ascites, and the resulting culture supernatant. Or by isolation from ascites of mammals.
When cultivating in vitro, in order to grow, maintain and preserve the hybridoma according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the research and the culture method, etc., to produce a monoclonal antibody in the culture supernatant It is possible to carry out using any nutrient medium that is derived from a known nutrient medium or a known basic medium as used in the above.

Examples of the basic medium include a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. The basic medium can contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances depending on the purpose.
Isolation and purification of the monoclonal antibody can be carried out by using the above-mentioned culture supernatant or ascites, saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52 etc.), anti-immunoglobulin column or It can be performed by subjecting to affinity column chromatography such as a protein A column.

The “chimeric antibody” in the present invention is a monoclonal antibody produced by genetic engineering. Specifically, for example, the variable region is a variable region derived from mouse immunoglobulin, and the constant region is human immunoglobulin. It means a chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody characterized by being a constant region derived from globulin.
The constant region derived from human immunoglobulin has a unique amino acid sequence depending on isotypes such as IgG, IgM, IgA, IgD, and IgE, but the constant region of the recombinant chimeric monoclonal antibody in the present invention belongs to any isotype. It may be a constant region of groggrin. Preferably, it is a constant region of human IgG.
The chimeric monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.

  For example, a mouse / human chimeric monoclonal antibody can be prepared with reference to experimental medicine (special issue), Vol. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-73280. That is, a human immunoglobulin downstream of an active VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from a DNA encoding the mouse monoclonal antibody isolated from a hybridoma producing a mouse monoclonal antibody. A CH gene (C gene encoding the heavy chain constant region) obtained from the DNA encoding, and an active VL gene (coding the light chain variable region) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma The CL gene obtained from DNA encoding human immunoglobulin (C gene encoding the light chain constant region) downstream of the rearranged VJ gene) is arranged so that it can be expressed individually or separately. Inserting into an expression vector, transforming a host cell with the expression vector, the transformation It can be produced by culturing cells.

  Specifically, first, DNA is extracted from a mouse monoclonal antibody-producing hybridoma by a conventional method, and then the DNA is digested with an appropriate restriction enzyme (for example, EcoRI, HindIII, etc.) and subjected to electrophoresis (for example, 0 (Use 7% agarose gel) Perform Southern blotting. The electrophoresed gel is stained with, for example, ethidium bromide, and after taking a photograph, the marker is marked, the gel is washed twice and immersed in a 0.25M HCl solution for 15 minutes. Then, immerse in 0.4N NaOH solution for 10 minutes while gently shaking. Transfer to a filter by a conventional method, collect the filter after 4 hours, and wash twice with 2 × SSC. After the filter is sufficiently dried, baking (75 ° C., 3 hours) is performed. After baking, the filter is placed in a 0.1 × SSC / 0.1% SDS solution and treated at 65 ° C. for 30 minutes. Then, immerse in 3 × SSC / 0.1% SDS solution. The obtained filter is put into a plastic bag together with the prehybridization solution and treated at 65 ° C. for 3 to 4 hours.

Next, ³² P-labeled probe DNA and a hybridization solution are put into this and reacted at 65 ° C. for about 12 hours. After completion of hybridization, the filter is washed under an appropriate salt concentration, reaction temperature and time (for example, 2 × SSC-0.1% SDS solution, room temperature, 10 minutes). Place the filter in a plastic bag, add a small amount of 2 × SSC, seal, and perform autoradiography.
By the Southern blotting method, rearranged VDJ gene and VJ gene encoding the H chain and L chain of mouse monoclonal antibody, respectively, are identified. The region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation and incorporated into a phage vector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.), and E. coli (for example, LE392, NM539, etc.) is used with the phage vector. ) To produce a genomic library. For example, the Benton Davis method (Science, Vol. 196, pp. 180-182, 1977) can be obtained by using an appropriate probe (H chain J gene, L chain (κ) J gene, etc.). ) To obtain positive clones each containing the rearranged VDJ gene or VJ gene. A restriction enzyme map of the obtained clone is prepared, the nucleotide sequence is determined, and it is confirmed that the gene containing the target rearranged V _H (VDJ) gene or _VL (VJ) gene is obtained. .

On the other hand, isolated Separately, human C _H gene and human C _L gene used for chimerization. For example, when a chimeric antibody with human IgG1 is produced, isolating the C _kappa gene is C gamma ₁ gene and the C _L gene is C _H gene. These genes using mouse C gamma ₁ gene and mouse C _kappa gene corresponding to the human C gamma ₁ gene and human Cκ gene by utilizing high homology in nucleotide sequence of the mouse immunoglobulin gene and the human immunoglobulin genes as a probe, the human genome It can be obtained by isolating from a library.

Specifically, for example, a 3 kb HindIII-BamHI fragment from clone Ig146 (Procedural National Academy of Science (Proc. Natl. Acad. Sci. USA), 75, 4709-4713, 1978). And a 6.8 kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 474-478, 1981) as a probe. lambda Charon 4A of HaeIII-AluI genomic library (cell (cell), Vol. 15, No. 1157～ pp 1174, 1978) from within includes a human C _kappa gene, DNA fragments that retain the enhancer region Is isolated. Moreover, the human C gamma ₁ gene, for example, a human fetal hepatocyte DNA is digested with HindIII, after fractionated by agarose gel electrophoresis, and inserted into a band of 5.9kb in Ramuda788, isolated with the probes .

Thus the mouse V _H gene was to isolated and mouse V _L gene, and human C _H gene and using human C _L gene, human downstream of the mouse V _H gene while considering the promoter region and enhancer region The C _H gene and the human C _L gene downstream of the mouse V _L gene are incorporated into an expression vector such as pSV2gpt or pSV2neo using an appropriate restriction enzyme and DNA ligase according to a conventional method. At this time, the mouse V _H gene / human C _H gene and the mouse V _L gene / human C _L gene chimeric gene may be arranged simultaneously in one expression vector, or may be arranged in separate expression vectors. it can.

The chimeric gene insertion expression vector thus prepared was used for the protoplast fusion method, the DEAE-dextran method, the calcium phosphate method or the electrolysis method to myeloma cells that did not produce antibodies themselves, such as P3X63 / Ag8 / 653 cells or SP210 cells. Introduced by drilling method. The transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain the desired chimeric monoclonal antibody-producing cells.
The target chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.

  The “human antibody (CDR-grafted antibody)” in the present invention is a monoclonal antibody produced by genetic engineering, and specifically, for example, part or all of the complementarity determining region of the hypervariable region. Is the complementarity determining region of the hypervariable region derived from a mouse monoclonal antibody, the framework region of the variable region is the framework region of the variable region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin It means a human monoclonal antibody characterized by

Here, the complementarity-determining regions of the hypervariable regions are the three regions (Complementarity-determining residues; CDR1, CDR2) that exist in the hypervariable regions in the variable region of the antibody and directly bind to the antigen in a complementary manner. , CDR3), and the framework region of the variable region refers to four relatively conserved regions (Framework: FR1, FR2, FR3, FR4) interposed before and after the three complementarity determining regions.
In other words, for example, a monoclonal antibody in which all regions other than part or all of the complementarity determining region of the hypervariable region of a mouse monoclonal antibody are replaced with the corresponding region of human immunoglobulin.
The constant region derived from human immunoglobulin has a unique amino acid sequence depending on the isotype such as IgG, IgM, IgA, IgD, and IgE. However, the constant region of the human monoclonal antibody in the present invention belongs to any isotype. It may be a constant region of glycine. Preferably, it is a constant region of human IgG. Further, the framework region of the variable region derived from human immunoglobulin is not limited.

The human monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
For example, a recombinant human monoclonal antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to JP-T-4-506458 and JP-A-62-2296890. That is, from a hybridoma producing a mouse monoclonal antibody, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated, and the mouse H chain CDR gene is isolated from a human immunoglobulin gene. A human H chain gene encoding the entire region other than the human H chain CDR corresponding to the chain CDR and a human L chain gene encoding the entire region other than the human L chain CDR corresponding to the pre-mouse L chain CDR are isolated.

  The isolated mouse H chain CDR gene and the human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse L chain CDR gene and the human L chain gene are also expressed appropriately. Into another expression vector. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can also be introduced so that they can be expressed in the same expression vector. By transforming host cells with the expression vector thus prepared, human-type monoclonal antibody-producing transformed cells are obtained, and by culturing the transformed cells, the desired human-type monoclonal antibody is obtained from the culture supernatant. obtain.

The term “human antibody” in the present invention means that all regions including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain encode human immunoglobulin. It is an immunoglobulin derived from a gene.
The human antibody can be obtained by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into a locus of a mammal other than human, such as a mouse, with an antigen according to a conventional method. It can be produced in the same manner as the polyclonal antibody or monoclonal antibody production method.
For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 15, 146-156, 1997; Nature Genetics, Vol. 7, pp. 13-21, 1994. National Publication No. 4-504365; International Application Publication No. WO94 / 25585; Nikkei Science, June, 40 to 50, 1995; Nature, 368, 856- 859, 1994; and JP-A-6-500363.
It is also possible to apply a method for producing human-derived protein from milk of transgenic cows and pigs, which is a recently developed technology (Nikkei Science, April 1997, pages 78 to 84). .

The “part of the antibody” in the present invention means a region of a part of the above-described antibody in the present invention, preferably a monoclonal antibody. Specifically, F (ab ′) ₂ , Fab ′, Fab, Fv (variable Fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody) (Expert Opin. Ther. Patents), Vol. 6, 5 No., pp. 441-456, 1996).

Here, “F (ab ′) ₂ ” and “Fab ′” are produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, and the like. An antibody fragment produced by digestion before and after the disulfide bond existing between the heavy chains. For example, when IgG is treated with papain, it is cleaved upstream of a disulfide bond existing between two H chains in the hinge region, and _L consisting of V _L (L chain variable region) and C _L (L chain constant region). To produce two homologous antibody fragments in which the H chain fragment consisting of a chain and V _H (H chain variable region) and CHγ ₁ (γ1 region in the H chain constant region) is linked by a disulfide bond in the C-terminal region Can do. Each of these two homologous antibody fragments is called Fab ′. In addition, when IgG is treated with pepsin, an antibody fragment that is cleaved downstream of the disulfide bond existing between the two heavy chains in the hinge region to produce a slightly larger antibody fragment than the two Fab's connected in the hinge region is produced. Can do. This antibody fragment is called F (ab ′) ₂ .

The “pharmaceutical composition” in the present invention comprises the fusion polypeptide of the present invention as an active ingredient, a pharmaceutically acceptable carrier, that is, an excipient, a diluent, a bulking agent, a disintegrant, a stabilizer, a preservative, A pharmaceutical composition together with one or more of a buffer, an emulsifier, a fragrance, a coloring agent, a sweetening agent, a thickening agent, a corrigent, a solubilizing agent or other additives, and the tablet, pill, powder, granule, It can be administered orally or parenterally in the form of injection, solution, capsule, trowel, elixir, suspension, emulsion or syrup.
In particular, in the case of injections, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. It can be produced by dissolving or suspending in the solution. The injection thus produced is 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg per day for a human patient in need of treatment. Can be administered several times to several times. Examples of administration forms include medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection. Intravenous injection is preferred.
In addition, the pharmaceutical composition of the present invention has abnormal kinetics of oxidized LDL receptor, binding of platelets and activated platelets to oxidized LDL receptor, and / or their incorporation into oxidized LDL-expressing cells (phagocytosis). Various diseases caused by the disease, various diseases accompanied by infiltration of leukocytes in the lesion process (for example, inflammation), various inflammations, arteriosclerosis, postoperative vascular restenosis such as PTCR and PTCA It is useful in the prevention and treatment.

“Inflammation” in the present invention refers to an internal factor or a biological tissue caused by various factors not limited to external factors such as bacterial infection, trauma, physical stimulation (eg, heat, cold, radiation, electricity, etc.) or chemical substances. In injury or dysfunction, it means a basic local pathological reaction involving leukocytes rolling and adhering on vascular endothelial cells and infiltrating extravascular tissues from the bloodstream.
Inflammation is generally divided into acute inflammation and chronic inflammation depending on the speed of its development and progression. In general, acute inflammation is an inflammation in which an inflammatory reaction develops relatively rapidly and progresses rapidly, and is clearly terminated. Chronic inflammation, on the other hand, is an inflammation in which an inflammatory response develops relatively slowly or gradually, or is manifested to an unclear degree of its presence, lasts for several weeks to several years, and its end is unclear. It is. The inflammation of the present invention includes both such acute inflammation and chronic inflammation.

  Inflammation in the present invention includes inflammation in tissues such as brain, eye, trachea, blood vessel, lung, liver, heart, pancreas, stomach, intestine, mesentery, kidney, skin, rhinitis membrane or joint. Specifically, for example, encephalitis, bronchitis, vasculitis, pneumonia, hepatitis, myocarditis, pancreatitis, enteritis, gastritis, peritonitis, nephritis (such as glomerulonephritis), arthritis (such as rheumatoid arthritis), post-ischemic reperfusion Inflammation in disorders (such as myocardial ischemia / reperfusion injury), inflammation resulting from post-transplant immune rejection, burns, inflammation in multiple organ disorders, postoperative inflammation of PTCA and PTCR, and inflammation associated with arteriosclerosis Can do.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited only to the embodiments described in the examples.
The “reference example” described below is an invention based on a viewpoint different from the present invention, but is referred to for technical comparison with the present invention.

<Reference example>
<1> Experimental Materials and Experimental Methods In the following examples, the following materials were used. Each test was performed by the following method.

(1) Cells Red blood cells obtained from healthy human blood were washed three times with a phosphate buffer, suspended in a phosphate buffer containing 10% glucose with 20% hematocrit, and used as natural erythrocytes.
Senescent erythrocytes were prepared by incubating the natural erythrocytes in phosphate buffer (20% hematocrit) at 37 ° C. for 4 days (except for use in time course experiments). Bovine aortic endothelial cells (BAE) were maintained according to a previously reported method (Nature, Vol. 385, p. 73-77, 1997). Jurkat and HL60 cells were maintained in RPMI 1640 medium (containing 10% fetal bovine serum, 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 250 ng / ml Fungizone (GIBCO)). Chinese hamster ovary (CHO) cells (BLOX-1-CHO) and wild-type CHO-K1 cells stably expressing bovine LOX-1 were maintained according to a previously reported method (Nature, Vol. 385, p. 73-). 77, 1997).

(2) Induction of apoptosis
Apoptosis of HL-60 cells was induced by incubating cells (1 × 10 ⁶ cells / ml) with 50 μM cycloheximide at 37 ° C. for 3 hours. Apoptosis of Jurkat cells was induced by incubating cells (1 × 10 ⁶ cells / ml) with 200 ng / ml anti-FasIgM antibody (clone CH-11, manufactured by Medical and Biological Laboratories) at 37 ° C. for 6 hours. . Apoptosis of bovine artery endothelial cells (BAE) was induced by incubating the cells with 50 ng / ml recombinant human tumor necrosis factor (R & D Systems) at 37 ° C. for 6 hours.

(3) Binding of erythrocytes to oxidized LDL receptor Erythrocytes (hematocrit value: 1%) were incubated with BAE, CHO-K1 cells or BLOX-1-CHO in a nutrient medium at 37 ° C. for 30 minutes. After removing erythrocytes that did not bind to each cell by washing multiple times with the nutrient medium, the cells were fixed with a phosphate buffer containing 4% formaldehyde and 0.2% glutaraldehyde. The number of red blood cells bound to each cell was counted (200-400 CHO cells or BAE cells were counted each time).

(4) Binding of cells (apoptotic cells) toward apoptosis to oxidized LDL receptor
Apoptotic cells (1 × 10 ⁶ cells / ml) were cultured with nutrient BAE, CHO-K1 cells or BLOX-1-CHO at 37 ° C. for 30 minutes. After removing apoptotic cells that did not bind to each cell by washing multiple times with the nutrient medium, the cells were fixed with a phosphate buffer containing 4% formaldehyde and 0.2% glutaraldehyde. After staining the cells with May-Giemsa, the percentage of bound cells or the uptake (phagocytosis) of one or more apoptotic cells was calculated (100-200 CHO cells or BAE cells were counted each time .)

(5) Lipoprotein Human LDL (d = 1.019 to 1.063) was isolated from fresh human plasma by gradient ultracentrifugation (Nature, Vol. 385, p. 73-77, 1997). Oxidative modification and acetylation of LDL were performed according to conventional methods (Biochem. Biophys. Res. Commun., Vol. 1091, p.63-67, 1991).

(6) Preparation of soluble LOX-1 (LOX-Fc) A cDNA (SEQ ID NO: 3) encoding the bovine oxidized LDL receptor LOX-1 (bLOX-1) was previously reported (Nature, Vol.386, p.73-). 77, 1997 and JP-A-9-98787).
The obtained cDNA was amplified by PCR using two primers (5'-GGGGATCCTGATCTCATAAAGAAACAG-3 '(SEQ ID NO: 5) and 5'-GCGGATCCTGTGCTCTCAATAGATTCGC-3' (SEQ ID NO: 6)). A cDNA fragment containing cDNA (base numbers 215 to 844 of SEQ ID NO: 3) encoding the extracellular region of bovine LOX-1 added to was prepared.

  Plasmid pCd5lneg1 containing genomic DNA containing exons encoding the human IgG1 hinge region, Cγ12, and Cγ13 (see DNA and Cell Biol., Vol. 9, p.347-353, 1990. Massachusetts General Hospital). Obtained from Dr. B. Seed (having the nucleotide sequence set forth in SEQ ID NO: 4) was digested with BamHI and linearized.

The cDNA encoding the extracellular region of bovine LOX-1 obtained as described above was ligated to the BamHI cleavage site (base number 169 of SEQ ID NO: 4) of this linearized plasmid using T4 DNA ligase. Plasmid pBLOX-Fc was constructed.
CHO-K1 cells cultured in a sub-confluent monolayer in HamF12 medium containing 10% FBS (fetal bovine serum) were added to pBLOX-Fc (1 μg) and expression plasmid vector pSVbsr (10 ng) using Lipofectamine (manufactured by GIBCO). Co-transformed with bsr (Blasticidin S-resistance) gene and SV40 virus-derived promoter).

After culturing for 48 hours, the medium was changed to HamF12 medium containing blasticidin-S (10 μg / ml, manufactured by Funakoshi), and further transformed cells were selected by cotransformation with pBLOX-Fc and pSVbsr. I got it.
The obtained transformed cells were maintained in HamF12 medium containing 10% FCS (fetal calf serum) and blasticidin-S (10 μg / ml, manufactured by Funakoshi).
In order to purify bLOX-1-Fc, the medium of transformant CHO-K1 cells cultured confluent in HamF12 medium containing blasticidin-S (10 μg / ml, manufactured by Funakoshi) was added to CHO-SFM-II (GIBCO / (BRL) and cultured for 3 days. After repeating this operation several times, 800 ml of culture supernatant was obtained. LOX-Fc in the culture supernatant was purified as follows using Affi-Gel Protein A MAPS-II kit (manufactured by Bio-rad).

The culture supernatant was added to a protein A agarose gel column previously equilibrated with a binding buffer. Subsequently, the column was washed with a binding buffer (15 bed volume) and then eluted with an elution buffer (5 bed volume). The eluate was collected and dialyzed by exchanging the external solution twice or more with a phosphate buffer to obtain purified LOX-Fc. The resulting purified LOX-Fc was ultrafiltered using Centriprep (Amicon) for concentration. Using BCA protein assay kit (PIERCE), it was confirmed that 866 μg / ml purified LOX-Fc was obtained.
The acquisition of the purified LOX-Fc was also confirmed by the following Western blotting.

Purified LOX-Fc was applied to a 12.5% SDS agarose gel (Daiichi Kagaku) and electrophoresed. After completion of the electrophoresis, blotting was performed on an Immobilon membrane (Millipore). The membrane was blocked overnight with Block Ace (Snow Brand). Reaction was performed using a biotin-labeled goat anti-human IgG antibody as a primary antibody and an ABC kit (manufactured by Vector), and color development was performed using a konica immunostain kit.
The amino acid sequence of LOX-Fc is shown in SEQ ID NO: 7, and the cDNA sequence of LOX-Fc is shown in SEQ ID NO: 8.

(7) Binding of soluble LOX-1 to red blood cells After fixing red blood cells with 4% formaldehyde and 0.2% glutaraldehyde, the cells were washed three times with a phosphate buffer. Red blood cells (hematocrit value: 1%) were then incubated with 50 μg / ml LOX-Fc or normal human IgG in Ham's F12 medium containing 10% fetal calf serum for 90 minutes at room temperature. After washing three times with the culture medium, the erythrocytes are further incubated with biotinylated anti-human IgG (Fc) at room temperature for 30 minutes to obtain a Tyramide signal amplification procedure (TSA-directed green, Dupont / NEN) according to the instruction manual of the product and subjected to fluorescence microscopy.

(8) Binding of soluble LOX-1 to Apoptotic cells Culture media of native Jurkat cells or apoptotic Jurkat cells (1 x 10 ⁶ cells / ml each) with 50 µg / ml LOX-Fc or normal human IgG Incubated for 60 minutes at 37 ° C. Vitotinylated anti-human IgG (1: 250 dilution) was added and the mixture was incubated at 37 ° C. for an additional 30 minutes. Cells are fixed with 4% formaldehyde and 0.2% glutaraldehyde, washed 3 times with phosphate buffer, and using the Tyramide signal amplification procedure (TSA-directed green, Dupont / NEN) Stained. Cells were then permeabilized with 0.1% Triton X-100, stained with 2.5 μg / ml propidium iodide, and analyzed with a confocal microscopy system (Bio-Rad). When not permeable, staining of cells with propidium iodide was undetectable.

(9) Externalization of phosphatidylserine (PS) onto senescent erythrocytes and apoptotic cells
Whether or not PS was exposed on the cell surface of senescent erythrocytes or apoptotic cells was detected by Annexin V-FITC using ApoAlert Annexin Vapoptosis kit (Clontech). Cells were analyzed by flow cytometry on a FACScalibur (Becton Dickinson).

(10) Preparation of phospholipid liposomes Multilamellar liposomes were prepared to contain phospholipids, phosphatidylcholine, and free cholesterol in a 1: 1: 1 molar ratio. The lipid was mixed in chloroform and dried with nitrogen gas. The dried lipid was resuspended in phosphate buffer to a final concentration of 10 mM. The resulting mixture was mixed and sonicated for 10 minutes and left at 4 ° C. until use.

<2> Results and Discussion As shown in FIG. 1, it was found that BAE cells have the ability to phagocytose senescent erythrocytes and apoptotic cells.
Fig. 1 (a) shows senescent erythrocytes, Fig. 1 (b) shows native erythrocytes, Fig. 1 (c) shows apoptotic HL60 cells, and Fig. 1 (d) shows untreated HL60 cells incubated with BAE cells. And BAE cells are not bound to native erythrocytes and untreated HL60 cells.
Since this phagocytic activity is clearly inhibited by oxidized LDL, we tested whether LOX-1, the major oxidized LDL receptor on BAE cells, mediates phagocytic activity.

As shown in FIGS. 2 (a) to 2 (e), a stable transformant BLOX1-CHO expressing BLOX-1 is induced in HL-60 cells in which apoptosis is induced by treatment with senescent erythrocytes and cycloheximide. They were bound (Fig. 2 (a) and (d)) and phagocytosed (Fig. 2 (c)). Since BLOX-1-CHO binds to apoptotic Jurkat cells treated with anti-Fas antibody and apoptotic BAE treated with tumor necrosisfactorα, phagocytosis of apoptotic cells by LOX-1 affects the cell type and initiation signal. It is possible not to depend on it. BLOX-1-CHO did not bind significantly to native erythrocytes or untreated HL-60 cells. Wild-type CHO cells did not bind to senescent erythrocytes and apoptotic cells. Thus, LOX-1 was found to mediate the binding and phagocytosis of aged erythrocytes and apoptotic cells. The binding of senescent erythrocytes was inhibited by oxidized LDL and acetyl LDL in a dose-dependent manner, but not by native LDL (FIG. 3). The half-maximum of inhibition by oxidized LDL was 0.1 μg · ml ⁻¹ . The reported concentrations of oxidized LDL are believed to cause other biological responses (Nature, 344, 160-162 (1990), J. Cell. Biol., 120, 1011-1019 (1993), J. Clin. Invest., 86, 75-79 (1990), J. Clin. Invest., 95, 1262-1270 (1995)), the effective concentration of oxidized LDL is very low in this study.

In order to investigate whether LOX-1 functions as a receptor for aging / apoptotic cells in BAE, the inhibitory effects of various LOX-1 ligands were compared between BAE and BLOX-1-CHO (FIG. 4). Black represents BAE, and diagonal lines represent BLOX-1-CHO). When fucoidan and poly [I] were added to oxidized LDL, most of the binding of aged erythrocytes to BLOX-1-CHO disappeared. The weak ligands of LOX-1, dextran sulfate (50 μg · ml ⁻¹ ), acetyl LDL (10 μg · ml ⁻¹ ) and heparin (50 μg · ml ⁻¹ ) also competed although they were weak. Poly (I), a LOX-1 ligand, competed well, but Poly (A), not a LOX-1 ligand, did not affect binding. LOX-1 is thought to mediate the binding of senescent erythrocytes in BAE since the ligand specificity and competition order in BAE were the same as in BLOX-1-CHO. In fact, the LOX-1 monoclonal antibody (# 19-1) inhibited the binding of senescent erythrocytes to BAE as well as the binding to BLOX-1-CHO (FIG. 5).

  The binding order of the compounds of apoptotic HL-60 to BLOX1-CHO and BAE was in the same order, suggesting that LOX-1 also mediates the binding of apoptotic cells in BAE (FIG. 6). The degree of inhibition was lower than that of aging erythrocytes. This result is thought to be due to the higher affinity of apoptotic cells to LOX-1 than that of senescent erythrocytes in vitro.

  To confirm that LOX-1 directly recognizes subtle surface changes in aging and apoptotic cells, LOX-Fc, a fusion protein of the extracellular region of LOX-1 and the Fc region of human IgG, was used. . Using LOX-Fc facilitates detection of LOX-1 and ligand binding. Addition of LOX-Fc to the medium did not affect the control human IgG, but significantly inhibited the binding of aging erythrocytes and apoptotic cells to BLOX-1-CHO and BAE (FIG. 7a and 7b). Next, we attempted to stain senescent cells and apoptotic cells using LOX-Fc. Staining with LOX-Fc could visualize putative ligands localized on the surface of aged erythrocytes. On the other hand, no distribution of ligand was observed in normal erythrocytes. When Jurkat cells treated with Fas antibody with Propidium iodide and LOX-Fc are double-stained, putative LOX-1 ligand is selectively expressed on the surface of Jurkat cells together with aggregated chromatin rather than non-aggregated chromatin It was shown that. This means that LOX-1 ligand expression on the cell surface occurs in the process of apoptosis.

  Anionic phospholipids in the plasma membrane of normal cells are distributed asymmetrically (Biochem. J., 294, 1-14 (1993)). Most phosphatidylinositol (PI) and virtually most PS are present in the inner leaflet of the plasma membrane. This asymmetry is maintained by a putative ATP-dependent translocase for the "flip-flop" or "scrambling" movement of negative phospholipids (Experientia 46, 644-656 (1990), Science 272, 1495-1497, 1996) It disappears under certain conditions including (Adv. Exp. Med. Biol., 406, 21-28 (1996)). Asymmetric loss, ie PS externalization, is considered the earliest sign of apoptosis (J. Exp. Med., 182, 1545-1556 (1995)). A phagocytic system in which Apoptotic cells and senescent cells compete with PS has been reported (Proc. Natl. Acad. Sci. USA 92, 1396-1400 (1995), J. Immunol. 149, 4029-4035 (1992), J Immunol. 148, 2207-2216 (1992), J. Immunol. 151, 4274-4285 (1993)). This system appears to recognize cell PS manifestations. In this connection, the relationship between the binding of senescent / apoptotic cells to LOX-1 and the binding to PS was investigated. Like apoptotic cells, it is detected by the binding of annexin V. The appearance of PS on the surface of aging erythrocytes increased with time (FIG. 8). This change was accompanied by increased binding to BLOX-1-CHO over time (FIG. 9). The fact that the physiological concentration of glucose inhibited the appearance of PS on the surface of aging erythrocytes (FIG. 8) indicates that an ATP depletion-dependent mechanism is involved in the appearance of PS, and further that BLOX-1-CHO binding during the aging process of erythrocytes This suggests that the increase in ability is also inhibited (FIG. 9).

  Furthermore, phospholipid liposomes containing PS inhibited the binding of aged erythrocytes and apoptotic HL-60 to BLOX-1-CHO and BAE, even though PC (phosphatidylcholine) liposomes were not affected (Fig. 10 to FIG. 13). Inhibition by PS liposomes was concentration dependent and complete inhibition was achieved with 1 μM total phospholipid. Inhibition was not only PS liposomes, but also PI-containing alonic liposomes, phosphatidylic acid, phosphatidylglycerol and cardiolipin (Fig. 12), while phosphatidylethanolamine and sphingo were also inhibited. Binding was not inhibited in liposomes composed of neutral phospholipids such as myelin (FIG. 13).

Liposomes containing these negative phospholipids not only competed, but were not taken up by BLOX-1-CHO. These results suggest that LOX-1 recognizes PS or other anionic phospholipids that appear on the plasma membrane during aging or apoptosis.
The above studies showed that most of the phagocytic activity was mediated by LOX-1 (estimated to be 3/4 from the effect of antibody inhibition). Several receptors including macrophage CD36 (J. Clin. Invest., 90, 1513-1522, 1989) and class A scavenger receptors (Proc. Natl. Acad. Sci. USA 93, 12456-12460 (1996)) Has been reported to bind to and / or phagocytose senescent / apoptotic cells. They are also thought to recognize different cell types at different stages of apoptosis and senescence of different cells. The lower molecular weight excess in endothelial cells as opposed to macrophages is ideal for LOX-1 to recognize changes to apoptotic cells around endothelial details such as blood cells, smooth muscle cells and endothelial cells themselves It is suggested that.

Interestingly, the receptor described above is known to bind to oxidized LDL (J. Bio. Chem., 268, 11811-11816 (1993), Proc. Natl. Acad. Sci. USA., 88, 4931-4935 (1991)). Oxidized LDL may be expressed specifically in apoptotic cells, for example, having a structure similar to PS. PS is a function of apoptotic cells by Peritoneal macrophages (Proc. Natl. Acad. Sci. USA., 92, 1396-1400 (1995)) and sertori cells (J. Biol. Chem., 272, 2354-2358 (1997)). Although reported as a competitive inhibitor of phagocytosis, inhibition by PS in phagocytosis mediated by CD36 and class A scavenger receptors is unclear.
There have been conflicting reports regarding CD36. Ryeom et al. (J. Biol. Chem., 271, 20536-20539 (1996)) showed that the binding of the apoptotic photoreceptor outer segment to CD36 in the retinal pigment epithelium was inhibited by PS. On the other hand, Fasok et al. (J. Immunol., 149, 4029-4035 (1992)) report that phagocytosis by monocyte-derived macrophages mediated by αvβA integrin / CD36 is not inhibited by PS. The latter phagocytosis is rather dependent on the RGDS sequence of thrombospondin (J. Immunol., 149, 4029-4035 (1992)). Macrosialin is considered a candidate molecule for phagocytosis because it binds to PS and oxidized LDL in macrophages. However, direct evidence using expression systems has not been reported so far (Proc. Natl. Acad. Sci. USA., 92, 9580-9584 (1995). From the above test, PS and oxidized LDL were , LOX-1 mediated phagocytosis was shown to be inhibited in transformed and native endothelial cells, and LOX-1 is expressed in several types of cells, including macrophages. It was suggested that -1 is part of the phagocytic system in other cells.

In terms of the procoagulant activity of PS on the cell surface, apoptotic cells are the focus of blood clotting (Lupus., 5, 480-487 (1996), Proc. Natl. Acad. Sci. USA., 93, 1624-1629 (1996)). In order to keep the blood non-coagulated, cells exposing PS to the cell surface must be removed from the circulatory system. Although this function is highly dependent on the reticuloendothelial cell system, the above studies suggest the importance of an in situ removal system on vascular endothelial cells. LOX-1 may have evolved as a molecule that mediates the non-coagulant activity of endothelial cells. It is considered that the noncoagulant activity of LOX-1 inhibited by oxidized LDL needs to be considered in promoting the pathogenic physiological thrombosis of arteriosclerosis. The circulation of senescent / apoptotic cells to be removed affects physiological homeostasis such as, for example, immune conditions that regulate the pathophysiology of atherosclerosis (Immunol. Lett., 43, 125-132 (1994)) Is also possible.
Lectin-like molecules are thought to be involved in the phagocytic system of senescent / apoptotic cells (Immunology 56, 351-358 (1985), FEBS Lett., 296, 174-178 (1992), J. immunol., 153, 3218 -3227 (1994)). Although it is necessary to further analyze the binding specificity for carbohydrates, LOX-1 with a lectin-like structure may be involved in phagocytosis of the phagocytic system.
That is, in the above test, LOX-1 was identified as a phagocytic receptor for aging / apoptotic cells in endothelial cells, and LOX-1 recognized the appearance of PS on the cell surface, which is the earliest stage in the apoptotic process Identified.

Example 1 Binding to oxidized LDL receptor The binding of human platelets to bovine LOX-1-CHO was analyzed in the same manner as in the above Reference Example.
As shown in FIG. 14, it was found that the binding of thrombin activated platelets to LOX-1 was significantly higher than that of native platelets to LOX-1 (FIG. 14 (a)). . Further, the binding of activated platelets to LOX-1 by thrombin was inhibited by phosphatidylcholine liposomes (100 μg / ml), but was inhibited by about 81%. On the other hand, phosphatidylcholine liposomes (100 μg / ml) were only inhibited by about 12%.
As shown in FIG. 15, the binding of platelets to LOX-1 decreased in inverse proportion to the oxidized LDL concentration.

As shown in FIG. 17, in the wild-type CHO not expressing LOX-1, binding of platelets to cells was not confirmed regardless of the presence or absence of thrombin, but in the CHO cells expressing LOX-1, the presence of thrombin. Platelets were significantly bound to the cells.
As shown in FIG. 18, the binding of thrombin-activated activated platelets to LOX-1-CHO increased depending on the thrombin concentration. Activated platelets activated with collagen (20 μg / ml) also significantly bound to LOX-1-CHO as in the case of thrombin.

Next, whether the binding of the activated platelets to LOX-1-CHO was inhibited by the anti-LOX-1 antibody was analyzed in the same manner as in the above Example.
As shown in FIG. 16, the binding and phagocytosis of activated platelets to LOX-1-CHO was significantly inhibited by anti-LOX-1 antibody.
Further, in a test conducted using BAE cells in the same manner as in the above example, significant binding of activated platelets to BAE was confirmed (fluorescence intensity: 150.94 ± 10.21). On the other hand, this binding was significantly inhibited by the anti-LOX-1 antibody (fluorescence intensity: 56.44 ± 14.71, inhibition rate: 63.3%). When a control IgG antibody was used, the binding was hardly inhibited (fluorescence intensity: 144.89 ± 11.93, inhibition rate: 4.0%).

Example 2 Binding and / or uptake of platelets and activated platelets into vascular endothelial cells and BLOX1-CHO <I. Materials and Methods>
<1> Preparation of platelets
Platelets were isolated according to standard methods of Baenzinger and Majerus et al. (Methods Enzymol., Vol. 31, p. 149-155, 1974). Briefly, it is as follows. Healthy human blood was collected in 3.8% sodium citrate (9: 1). The blood was centrifuged (200 × g, 15 minutes) to obtain platelet-rich plasma (PRP). Centrifuge the acid / citric acid / dextrose (ACD: 2.5% trisodium citrate, 1.5% citric acid, and 2% glucose; PRP: ACD = 9: 1) into PRP to obtain washed PRP. It was. Hepes-Tyrode's buffer containing ₁ μg / ml PGE ₁ (Sigma) (10 mM Hepes, 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH ₂ PO ₄ , 1.7 mM MgCl ₂ , 11.9 mM NaHCO ₃ and 5 mM glucose). The pellet suspension was centrifuged (1000 × g, 15 minutes). The resulting pellet was resuspended in Hepes-Tyrode's buffer and used as washed platelets.

<2> cells
BLOX-CHO was prepared in the same manner as in the above Reference Example. BLOX1-CHO and the parent cell line CHO-K1 were maintained in Ham's F12 medium (Gibco) under conditions of humidity 95%, 5% CO ₂ , and 37 ° C. However, 100 U / ml penicillin G, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin-B (manufactured by Gibco), and 10% fetal bovine serum were added to the medium. Each cell was seeded in a 24-well microplate before the assay.
Bovine aortic endothelial cells (BAE) were prepared in the same manner as previously reported (Nature, Vol. 386, p. 73-77, 1997). BAE was maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) under conditions of 95% humidity, 5% CO2, and 37 ° C. However, 100 U / ml penicillin G, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin-B (manufactured by Gibco), and 20% fetal bovine serum were added to the medium. BAEs were plated in 24-well microplates 2 days before the assay.

<3> Platelet binding assay Platelets were fluorescently labeled with Calcein by incubating at 37 ° C for 30 minutes with 1 µM Calcein-AM (Molecular Probe). Subsequently, excess Calcein-AM was removed by washing the fluorescently labeled platelets twice with Hepes-Tyrode's buffer containing 0.3% BSA (manufactured by Sigma). Then, the density was adjusted platelet 1 × 10 ⁸ cells / ml with 10% newborn calf serum (manufactured by Gibco) Hepes-Tyrode's buffer containing.
Each of BLOX1-CHO and CHO-K1 was washed twice with nutrient medium and incubated with platelets at 37 ° C. for 60 minutes. Cells were washed with phosphate buffer to remove platelets that did not bind to the cells. Cells were harvested and analyzed with a flow cytometer (FACS Calibur; Becton-Dikinson). The degree of platelet binding to each cell was expressed as the fluorescence intensity (FL-1-H; excitation 515-545 nm) for 10,000 cells.
BAE was washed twice with nutrient medium and incubated with platelets at 37 ° C. for 180 minutes. Cells were washed with phosphate buffer to remove platelets that did not bind to the cells. Cells were harvested and analyzed with a flow cytometer (FACS Calibur; Becton-Dikinson). The degree of platelet binding to each cell was expressed as the fluorescence intensity (FL-1-H; excitation 515-545 nm) for 10,000 cells. However, in some assays described below, anti-LOX-1 antibody or control mouse antibody (Vector) was added 10 minutes before adding platelets.

<4> Platelet uptake into cells (phagocytosis) assay
BLOX1-CHO or CHO-K1 cells were fluorescently labeled by incubation with the same fluorescent material as described above. After removing platelets that did not bind to the cells, the cells were cooled to 4 ° C. and incubated with Dil-labeled oxidized LDL at 4 ° C. for 30 minutes to visualize the plasma membrane and immediately fixed with 4% formaldehyde. The cells were then subjected to a confocal laser microscope (Biorad).

<5> Binding Assay for Activated Platelets Washed platelets were stimulated with the reagents shown in the following description of the drawings for 10 minutes at room temperature, and then stained with FITC-labeled anti-CD41a monoclonal antibody (Pharmingen). The intensity of staining was not different between resting and activated platelets. BLOX1-CHO was incubated with platelets at 37 ° C. for 60 minutes. Platelet binding to cells was analyzed with a flow cytometer as described above. However, in some assays described below, phospholipid liposomes or lipoproteins were added 10 minutes before the addition of platelets.

<6> Endothelin 1 (ET-1) production assay with BAE The final concentration of washed platelets was adjusted to 5 × 10 ⁷ cells / ml in Hepes-Tyrode's buffer (resting platelets) containing 2% newborn calf serum. Thereafter, stimulation was performed with 1 U / ml thrombin or 20 μg / ml collagen (activated platelets). After 20 minutes, hirudin (Calibiochem) was added to inactivate thrombin to a final concentration of 1 U / ml. BAE was then incubated with platelets at 37 ° C. for 180 minutes. After washing with nutrient medium, BAE was incubated for 20 hours in DMEM medium containing 0.2% BSA. The culture supernatant was collected, and the concentration of ET-1 was measured by a signed switch ELISA according to a previous report (J. Immunol. Methods, Vil. 127, p.165-170, 1990). However, in some assays, anti-LOX-1 antibody or control mouse IgG (10 μg / ml) was added to the BAE 10 minutes prior to the addition of platelets.

<7> Activation of prothrombin
Each cell of BLOX1-CHO and CHO-K1 was pre-cultured for 30 minutes in the presence or absence of 10 μg / ml anti-LOX-1 antibody. Cells were washed 3 times with Ham's F12 medium and incubated with 250 μl PRP (Platelet-richplasma) diluted 10-fold with 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Next, 90 mM CaCl ₂ (50 μl) containing butyloxycarbonyl-Val-Pro-Arg-methylcoumarylamide (15 nmol Peptide Institute), which is a fluoregenic substrate for thrombin, was added to the cells. Thrombin activation was quantified by measuring the amount of aminomerylcoumarin released in 30 minutes.

<8> Lipoprotein Human LDL (d = 1.019-1.063) was isolated from fresh human plasma by concentration gradient ultracentrifugation according to a previous report (Natire, Vol. 386, p. 73-77, 1997). LDL was oxidized at a concentration of 3 mg protein / ml by exposure to 7.5 μM CuSO ₄ at 37 for 20 hours. The oxidation was carried out by measuring the amount of tiobarbituric acid reactive substance (10.7 nmol / mg protein) and monitoring the gel electrophoresis state (relative gel electrophoresis state: 3.25) in comparison with natural LDL. Oxidized LDL was labeled with 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probe).

<9> Preparation of phospholipid liposomes Phospholipids, phosphatidylcholine and free cholesterol (1: 1: 1) were mixed in chloroform and dried in nitrogen gas. The dried lipids were resuspended in phosphate buffer to a final concentration of 10 mM and sonicated for 10 minutes.

<II. Description of Drawing>
<FIGS. 19 and 20> Binding of platelets to LOX-1 expressing cells
Binding of platelets labeled with calcein (green) was observed with BLOX-CHO (left), but not with CHOK1 (right) (FIG. 19).
Under the confocal microscope, the disappearance of platelets associated with BLOX1-CHO was observed. Plasma membranes were visualized with Dil (red) and platelets were labeled with calcein (green). Platelets were detected not only on the plasma membrane but also in the cells (FIG. 20).
<FIGS. 21 and 22> Neutralizing antibody against LOX-1 inhibited the binding of platelets to BLOX1-CHO (FIG. 21) or BAE (FIG. 22).
Platelet binding was significantly inhibited with anti-LOX-1 antibody (p <0.01) but not with control IgG. Platelet binding to cells was expressed as fluorescence intensity for 10,000 cells. Data are the mean (and standard deviation) of three trials.

<FIG. 23> Platelet binding to LOX-1 occurs depending on the degree of platelet activation.
Platelet binding to BLOX1-CHO was observed after stimulation with agonists. In the case of thrombin, platelet binding was enhanced depending on the concentration of thrombin. It was also enhanced by collagen stimulation.
<FIG. 24> Effect of phospholipids on thrombin-enhanced platelet binding to cells.
Phospholipid liposomes significantly inhibited platelet binding to BLOX1-CHO, but p-phosphatidylcholine liposomes did not.
<FIG. 25> Oxidized LDL inhibited platelet binding to BLOX1-CHO in a dose-dependent manner, whereas native LDL did not. The value was expressed as the binding rate of platelets when no lipoprotein was added.

<FIG. 26> BAE was incubated with resting platelets or activated platelets stimulated with thrombin or collagen for 3 hours. The amount of endothelin-1 (ET-1) released from BAE during 20 hours was measured by ELISA. Anti-LOX-1 antibody significantly inhibited ET-1 production enhanced by activated platelets. The culture supernatant of activated platelets had a limited effect compared to platelets themselves. Data are the mean (and standard deviation) of three trials.
<FIG. 27> Effect of interaction between platelets and LOX-1 on the production of thrombin in plasma.
Thrombin activity in platelet rich plasma (PRP) was significantly enhanced by incubation with BLOX1-CHO. Thrombin activity was determined by measuring the degree of fluorogenic substrate dissociation (30 minutes). Data are average values (and standard deviations) from three trials.

<III. Result>
Oxidized LDL is known to change vascular endothelial cells from an anti-coagulant state to a coagulant state and enhance the interaction between platelets and vascular endothelial cells ( Curr. Opin. Lipidol., Vol. 8, p.320-328, 1997). The present inventors tried to study the relationship of LOX-1 to the interaction between platelets and vascular endothelial cells, and the involvement of LOX-1 in the thrombus formation system. Platelets fluorescently labeled with Calceion were incubated with cells stably expressing LOX-1 (BLOX1-CHO) or the parent cell line CHO-K1. Measurement with a fluorescence microscope revealed that a large number of platelets bound to the cell surface of BLOX1-CHO, but not CHO-K1 (FIG. 19). Next, in order to examine the end of the platelets bound to the cells, the cells were incubated with platelets and then observed with a confocal microscope. After fixing the cells, the plasma membrane of the cells was visualized by labeling with Dil (FIG. 20, red). The fluorescent signal emitted from platelets (FIG. 20, green) was detected not only on the plasma membrane but also in the cells. This indicated that LOX-1 not only binds platelets but also phagocytoses platelets.

  Next, antibodies that inhibit the binding of platelets to LOX-1 were screened by quantifying the enhancement of the fluorescence signal caused by the binding of platelets to cells with a flow cytometer. As a result, the monoclonal antibody against LOX-1 inhibited the binding of platelets to BLOX1-CHO (FIG. 21). On the other hand, an antibody unrelated to LOX-1 (an antibody that does not react with LOX-1) had no effect on the binding between platelets and BLOX1-CHO. Similarly, inhibitory antibodies against LOX-1 significantly reduced platelet binding to vascular endothelial cells (FIG. 22). This indicates that LOX-1 plays an important role in vascular endothelial cells. Surprisingly, despite the presence of other molecules (eg, P-Selectin, ICAM-1, PECAM-1) involved in the binding of platelets to vascular endothelial cells on vascular endothelial cells The degree of inhibition by anti-LOX-1 antibody was 50% or more. Molecules other than LOX-1 are known to be involved in various steps of leukocyte adhesion and rolling on vascular endothelial cells, and leukocyte infiltration into tissues. Thus, LOX-1 is thought to play a role in collaboration with other molecules at specific steps in the interaction between platelets and vascular endothelial cells.

In order to analyze in detail the mechanism of platelet binding to LOX-1, we examined whether the binding depends on the degree of platelet activation (FIG. 23). Platelets were incubated with BLOX1-CHO after stimulation with thrombin. As a result, the binding of platelets to cells increased depending on the concentration of thrombin. Collagen, which is another agonist (platelet activator) for platelets, also enhanced platelet binding.
Thrombin, collagen and thromboxane A2, which are agonists for platelets, are known to induce externalization of phosphatidylserine (PS) to the cell surface (J. Biol. Chem., Vol.265, p.17420-17423, 1990). PS is usually located in the inner leaflet in the lipid bilayer of the plasma membrane of platelets. Therefore, it was examined whether PS competes for the binding of platelets to LOX-1. As shown in FIG. 24, when PS was added to the medium, the binding of platelets to cells was almost completely inhibited. On the other hand, phosphatidylcholine (PC) which is not a ligand of LOX-1 was not inhibited. When these results are combined, it can be considered that PS on the cell surface of activated platelets is a determinant of the binding of activated platelets to LOX-1.

Oxidized LDL, first found as an in vivo ligand for LOX-1, also partially and competitively inhibited platelet binding to LOX-1 (FIG. 25). This means that the affinity for LOX-1 is higher for platelets than for oxidized LDL. It was also shown that oxidized LDL and platelets share LOX-1 as their respective receptors in vascular endothelial cells.
Based on these findings, we investigated whether the interaction between LOX-1 and platelets induces intracellular activation and cell dysfunction. As shown in FIG. 26, incubation of BAE with activated platelets increases ET-1 release from BAE. On the other hand, no increase was observed in resting platelets. Increased release of ET-1 in activated platelets was significantly inhibited by anti-LOX-1 antibody. Regarding the effect on the production of ET-1, the effect of the activated platelet culture medium containing the mediator released from the platelets was much smaller than the effect of the activated platelets themselves. LOX-1 has the potential to be involved in the process of vascular endothelial cell activation mediated by direct contact of endothelial cells with platelets (Circ. Res., Vol. 69, p. 832). 841, 1991). Interestingly, in hypercholesterolemic mice, it has been reported that inhibition of ET-1 action weakens vascular endothelial function (Hypertension, Vol. 31, p. 499-504, 1998). Increased production of atheromatous molecules via LOX-1 may have a profound effect on the progression of arteriosclerosis, as LOX-1 expression is enhanced in hypercholesterolemia It is believed that there is.

  In order to investigate the effect of platelet-LOX-1 interaction on the blood coagulation system, thrombin activity in PRP was analyzed when platelet-rich plasma (PRP) was added directly to BLOX1-CHO. (Figure 27). Thrombin activity was significantly higher in BLOX1-CHO than in wild-type CHO-K1. This increased thrombin activity was significantly inhibited by anti-LOX-1 antibody. On the other hand, it was not inhibited by control IgG. From this result and the fact that PS is involved in the blood coagulation cascade, it is understood that LOX-1 not only binds to platelets, but is also involved in the activation of the blood coagulation system, probably through binding activity with PS. Thus, LOX-1 is suggested to play a role as a regulator of vascular hemostasis and thrombus formation systems.

Example 3 Therapeutic effect of anti-LOX-1 antibody on LPS-induced thrombocytopenia
LPS (lipopolysaccharide; manufactured by Sigma) adjusted to 1 mg / ml with physiological saline was intraperitoneally administered to Sprague-Dawley rats (male 5 to 7 weeks old, manufactured by JCL) at a concentration of 3 mg / kg. Immediately after administration of LPS, an anti-LOX-1 antibody adjusted to 1 mg / ml with physiological saline or a control antibody not reactive to LOX-1 was intravenously administered at a concentration of 5 mg / kg. Rats to which neither LPS nor antibody was administered were used as normal controls. Blood was collected before LPS administration and 2 hours after LPS administration, and the blood platelet count was measured with an automatic blood cell analyzer, Sysmex F800 (Nihon Kohden).
The results are shown in FIG. In the control antibody administration group, platelet reduction occurred 2 hours after LPS administration. On the other hand, in the anti-LOX-1 antibody administration group, the decrease in platelets was significantly suppressed.

Example 4 Therapeutic effect of anti-LOX-1 antibody on LPS-induced lung injury
Anti-LOX-1 antibody (2, 5, 10 mg / kg) or physiological saline (5 mg / kg) was intravenously administered to Sprague-Dawley rats (6 to 7 weeks old, 6 mice in each group, manufactured by SLC) . Next, anesthesia was performed with pentobarbital (30 to 50 mg / kg, ip), and 1 hour after intravenous administration of the antibody (or physiological saline), LPS (lipopolysaccharide; manufactured by Sigma) adjusted with physiological saline was 1 mg. Administered via airway at a concentration of / kg. Normal rats, the control group, were administered physiological saline via the respiratory tract.
Twenty-four hours after LPS administration, each rat was laparotomized under ether anesthesia, the abdominal aorta was cut, and the blood was killed. Next, the pharynx was incised, and a cut-down tube (manufactured by JMS) was inserted into the airway. A physiological saline containing 0.05 mM EDTA, that is, a BALF (alveolar fluid) collection solution (5 ml) was injected with a 5 ml syringe through the tube, and the syringe was reciprocated 15 times to collect BALF. The recovered BALF was stored on ice and then centrifuged (1000 rpm, 10 minutes, 4 ° C.). The supernatant was removed by decantation, and 0.5 ml of BALF recovery solution was added and suspended briefly. The number of white blood cells contained in this suspension was measured with an automatic blood cell measuring device Sysmex F800 (manufactured by Nihon Kohden).
The results are shown in FIG. In the anti-LOX-1 antibody administration group, the number of leukocytes infiltrating the tissue was significantly suppressed depending on the antibody concentration. Surprisingly, the antibody concentration of 10 mg / kg inhibited leukocyte tissue infiltration by about 50%.

Example 5 Therapeutic effect of anti-LOX-1 antibody on LPS-induced inflammation
Anti-LOX-1 antibody (10 mg / kg) or physiological saline (10 mg / kg) was intravenously administered to Sprague-Dawley rats (200 g, manufactured by SLC). Then, 1 hour after intravenous administration of the antibody (or physiological saline), LPS (lipopolysaccharide; manufactured by Sigma) adjusted with physiological saline was administered to the sole of the foot at a concentration of 1 mg / kg. Normal rats as a control group were administered physiological saline to the sole of the foot.
Twelve hours after LPS administration, blood was collected from each rat according to a conventional method, and the number of leukocytes infiltrating the anterior chamber of the eye was measured with an automatic blood cell measuring device Sysmex F800 (manufactured by Nihon Kohden). In addition, the total amount of protein leaked into the anterior chamber of the eye was measured.
The results are shown in FIG. 30 and FIG. In the anti-LOX-1 antibody administration group, the number of leukocytes infiltrating the anterior chamber of the eye and the amount of protein leaked were significantly suppressed.

Example 6 Therapeutic effect of PTCA with anti-LOX-1 antibody on postoperative restenosis
Sprague-Dawley rats (approximately 300 g male, manufactured by SLC) were anesthetized with pentobarbital (30-50 mg / kg, ip). The carotid artery and external neck were exposed by surgery, and both arteries were temporarily connected to temporarily stop blood flow. Next, a hole was made in the external carotid artery, a 2F balloon catheter (manufactured by Baxter) was inserted, 0.4 ml of air was sent, and the blood vessel was rubbed against the intima three times with that pressure. Next, the external carotid artery was tied, and the carotid artery and internal carotid artery that had been temporarily connected were released to resume blood flow. The surgical site was sewn up and immediately anti-LOX-1 antibody (10 mg / kg) was intravenously administered.
Thereafter, the antibody (10 mg / kg) was intravenously administered 4 times every 3 days. Two weeks later, the rats were again anesthetized with pentobarbital (30 to 50 mg / kg, ip), reflux fixed with 4% formaldehyde / phosphate buffer, and the carotid artery was removed. The carotid artery was embedded in paraffin, six sections were prepared from one sample, and stained with Elastica-Wangeson. The NIH analyze system was used to evaluate the thickness of the intima of each section. The ratio of the area of the intima to the media was calculated, two sections where significant thickening was observed were selected, and the average was taken as the thickening amount of the sample.
The results are shown in FIG. Anti-LOX-1 antibody significantly suppressed PTCA postoperative restenosis.

Claims (10)

  Anti-LOX-1 antibody which is a human monoclonal antibody. A portion of an anti-LOX-1 antibody selected from the group consisting of F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv and dAb.   The antibody according to claim 1, wherein the human monoclonal antibody has a constant region of human IgG.   The antibody according to claim 3, wherein the human IgG is human IgG1.   The antibody or a part thereof according to claim 1 or 2, which is produced using a human antibody-producing transgenic animal.   The antibody or a part thereof according to claim 5, which is obtained by immunizing a mammal using a human oxidized LDL receptor or a part thereof as an antigen.   The antibody or a part thereof according to claim 6, wherein the mammal is a mouse.   An antibody-producing cell that produces the antibody according to any one of claims 1 to 7 or a part thereof.   The antibody-producing cell according to claim 8, which is prepared by cell fusion of an antibody-producing cell and a myeloma cell having no autoantibody-producing ability derived from a mammal.   The antibody-producing cell according to claim 9, wherein the antibody-producing cell is produced using a gene recombination technique.