Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
2000-07-12
2002-06-25
Chan, Christina Y. (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C530S387300, C530S388100, C530S388250, C530S391300, C530S391100, C424S130100, C424S134100, C424S141100, C424S142100, C424S178100, C435S007920, C435S810000
Reexamination Certificate
active
06410020
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to hybridomas producing a monoclonal antibody reactive to human cholesterol ester transfer protein(CETP), a monoclonal antibody and its fragment reactive to human CETP, an immobilized monoclonal antibody and immobilized antibody fragment, a labeled monoclonal antibody and labeled antibody fragment, a kit for detection, assay, separation or purification of human CETP, a method for detection, assay, separation and purification of human CETP and a pharmaceutical composition containing said monoclonal antibody or said antibody fragment.
BACKGROUND OF THE INVENTION
There are three types of cholesterol, free type, long chain fatty acid type and ester type, in all the tissues and blood plasma in organisms. The former two play important roles in the composition of cell membranes. The latter is physiologically inactive and exists mainly in a storage form. Cholesterols in a body are derived from ingestion in the small intestine or from biosynthesis in various tissues, especially in the liver. Most cholesterols are derived from biosynthesis in the liver.
Free cholesterol biosynthesized and secreted from the liver is incorporated in very low density lipoprotein(VLDL). Then, by the actions of lipoprotein lipase(LPL) and hepatic triglyceride lipase(HTGL), it is metabolized to low density lipoprotein (LDL) through intermediate density lipoprotein (IDL). By the incorporation of LDL into LDL receptors of peripheral cells, free cholesterol is supplied to cells.
There is a pathway from the peripheral cells to the liver called the cholesterol reverse transfer system, which goes in reverse of the way from liver to peripheral cells as mentioned above. Surplus free cholesterol supplied in the peripheral cells from the liver is drawn by high density lipoprotein(HDL) in blood. Then, by the action of lecithin cholesterol acyl transferase(LCAT), it is converted to cholesterol ester and is stored in high density lipoprotein(HDL) in blood. By the action of cholesterol ester transfer protein(CETP), the cholesterol ester stored in HDL is transferred to VLDL, IDL or LDL in blood. By the incorporation of VLDL, IDL or LDL cholesterol ester received through LDL receptors in the liver, cholesterol is indirectly transferred to the liver.
Recently, the reverse cholesterol transfer system has drawn much attention as a mechanism for preventing the peripheral cells from accumulating cholesterol and thereby preventing atherosclerosis. In fact, as for HDL which plays an important role in the reverse cholesterol transfer system, many epidemiological surveys show that decrease of cholesterol ester in blood HDL is one of the risk factors of coronary artery disorders. It is now well recognized that HDL is a lipoprotein having anti-arteriosclerosis action.
In addition to the importance of blood HDL, it became recognized that CETP is also important because it mediates transfer of cholesterol ester in HDL into blood LDL. Therefore, it became an urgent matter to elucidate the relationship between CETP and various diseases such as CETP deficiency, hyperlipidemia, hyperalphalipoproteinemia, hypercholesterolemia, hypolipidemia, arteriosclerosis, diabetes and nephrotic syndrome.
For instance, it is experimentally demonstrated that several times higher CETP are secreted in blood of the patients with hyperlipidemia compared to those of healthy volunteers. The following findings have been made in relation to arteriosclerosis. When CETP activity is low, arteriosclerosis is not easily induced and the level of HDL cholesterol is high. In contrast, when CETP activity is high, arteriosclerosis is easily induced and the level of HDL cholesterol is low. Such relationships have been experimentally demonstrated(Current Therapy, vol. 7, 9:36-45(1989)).
In order to elucidate the relationship between various diseases and CETP, assay methods for CETP in body fluids such as blood plasma from a healthy person or patient having the above various diseases, especially immunoassay methods such as radio-immunoassay(RIA) by using a monoclonal antibody against CETP(anti-CETP monoclonal antibody), or enzyme-immunoassay(EIA, ELISA) are being developed together with an anti-CETP monoclonal antibody used for the assay methods.
As to EIA(ELISA) assay with the anti-CETP monoclonal antibody, there are the examples by Imai et al.(Japanese Unexamined Patent publication No.HEI6-169793), Nakano, et al.,(Arteriosclerosis, vol. 19, 11:951, No. 22, (1991)), Takahama, et al.(Arteriosclerosis, vol. 20, 10:837, No. 135, (1992)), Sato, et al.(Arteriosclerosis, vol. 20, 10:836, No. 134, (1992)), H. Mezdur, et al.(Clinical Chemistry, vol. 40, 4:593-597(1994)). Clark et al.(Journal of Lipid Research, vol. 36,:876-889(1995)) reported the sandwich ELISA method by using two kinds of anti-CETP monoclonal antibodies or their Fab′ fragments.
However, these assay methods require complicated handling. Before assay, a plasma sample has to be heat-treated at 95-100° C. and/or pre-treated with surface active agents (surfactants, detergent) such as TWEEN 20 and TRITON X-100. Since heat treatment of a plasma sample causes denaturation of CETP in the sample, the assay result tells only the amount of denatured CETP. Therefore, it is impossible to assay intact CETP in a plasma sample accurately.
Also, R. Clark et al.(FASEB Journal, vol. 8, 7:A1343 No. 495(1994)), Takahashi et al.(Arteriosclerosis, vol. 20, 10:837 No. 136(1992) and ibid. vol. 21, 3:209 No. 97(1993)), Kanamitu et al.(ibid. vol. 21, 3:209 No. 98(1993)) and Waki et al.(ibid. vol. 22, 5:441 No. 194(1994)) reported assay results on CETP in plasma samples by ELISA, however, they did not describe in detail their preparation methods, properties of anti-CETP antibodies and specific assay procedures.
Regarding RIA assay methods with anti-CETP monoclonal antibody, Fukazawa et al.(Study on lipid biochemistry, vol. 34:163-166(1992)), Y. Marcel et al.(J. Clin. Invest., vol. 85:10-17(1990) and Adv. Exp. Med. Biol., vol. 243:225-230(1988)), Fukazawa et al.(J. Biochem., vol. 111:696-698(1992)), J.Koizumi et al. (Atherosclerosis, 90:189-196(1991), M. Brown et al.(Nature, vol. 342:448-451(1989)) and V. Dangremont et al.(Clinica Chimica Acta, vol. 231:147-160(1994)) published reports thereon.
However, these RIA methods also had the same defects as the EIA(ELISA) method had. The defects were complexity of handling (the methods required pre-treatment of a plasma sample with a detergent) and/or insufficient sensitivity for detection.
As described above, many researchers tried to establish an assay method for CETP. However, there is no report of a convenient and sensitive system which needs no pretreatment of a sample and which determines conveniently the amount of intact CETP with high sensitivity.
To elucidate the relationship between the diseases (such as arteriosclerosis, hyperlipidemia hyperalphalipoproteinmia and hypercholesterolemia) and CETP that plays an important role in the cholesterol reverse transfer system which may be highly related to the onset of the diseases, there is a strong reason for developing a simple and sensitive assay method that can be widely applied to clinical purposes to assay intact CETP in a body fluid including blood plasma from healthy persons or any patients, and for developing a monoclonal antibody useful for said assay method. However, these have not yet been established. The present invention provides an assay method widely applicable for clinical purposes and an anti-human CETP monoclonal antibody which is very useful not only for said assay method but also as a reagent for separation and purification of CETP and as a pharmaceutical product.
SUMMARY OF THE INVENTION
The inventors of the present invention extensively investigated assay methods for CETP in a human body fluid that can be widely applicable for clinical purposes and monoclonal antibodies against human CETP used for said assay methods. By using biologically active purified human CETP as an immunogen, the inventors succeeded in preparing three anti-human CETP monoclonal antibodies which resp
Kamada Masafumi
Okamoto Hiroshi
Tamatani Takuya
Chan Christina Y.
Huynh Phuong N.
Japan Tobacco Inc.
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