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WO2011159000A1 - Anticorps monoclonal humain qui se lie spécifiquement à une lipoprotéine de faible densité oxydée et à une lipoprotéine de faible densité carbamylée, et son fragment de liaison à un antigène - Google Patents

Anticorps monoclonal humain qui se lie spécifiquement à une lipoprotéine de faible densité oxydée et à une lipoprotéine de faible densité carbamylée, et son fragment de liaison à un antigène Download PDF

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WO2011159000A1
WO2011159000A1 PCT/KR2010/008219 KR2010008219W WO2011159000A1 WO 2011159000 A1 WO2011159000 A1 WO 2011159000A1 KR 2010008219 W KR2010008219 W KR 2010008219W WO 2011159000 A1 WO2011159000 A1 WO 2011159000A1
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seq
fab
monoclonal antibody
human monoclonal
ldl
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Korean (ko)
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장영주
서창원
이철주
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Ajou University Industry Academic Cooperation Foundation
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Ajou University Industry Academic Cooperation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the present invention provides a human monoclonal antibody or antigen-binding fragment thereof (Antigen-) that specifically binds to oxidized low-density lipoprotein (oxLDL) and carbamylated low-density lipoprotein (carLDylated LDL; cLDL).
  • Antigen- oxidized low-density lipoprotein
  • cLDL carbamylated low-density lipoprotein
  • Binding Fragment (Fab) and more specifically, specifically binds to MDA-LDL, Cu-LDL and cLDL, prepared using combinatorial antibody library and phage display technology. It relates to a human monoclonal antibody and antigen-binding fragment plaques 15, 16-46 (plaque 15, 16-46).
  • Atherosclerosis is a chronic disease in which soft masses, called aatheromas or plaques, accumulate inside the arteries (Hansson, 2005). During the plaque formation process, cell debris, smooth muscle cells and macrophage accumulate, and the oxidation of LDL is known to be involved in early plaque formation (Goncalves et al., Atherosclerosis , 205: 96-100, Lewis et al., Circulation , 120: 417-426, 2009, Jeon et al., Molecular Immunology , 44: 827-836, 2007).
  • Atherovascular vascular diseases include aneurysms, cerebral atherosclerosis, cerebral infarction, cerebral hemorrhage, angina pectoris, myocardial infarction, kidney sclerosis, retinal arteriosclerosis, and arterial occlusion.
  • the circulating cholesterol is carried by plasma lipoproteins, which are particles of complex lipids and protein compositions that transport blood lipids.
  • Low density lipoprotein (LDL) and high density lipoprotein (HDL) are major cholesterol carriers.
  • LDL is believed to be responsible for delivering cholesterol from the liver (where cholesterol is synthesized or obtained from a dietary source) to extrahepatic tissue in the body.
  • LDL has been popularly known as "bad" cholesterol because convincing evidence has been provided to support the notion that lipids deposited on atherosclerotic lesions are primarily derived from plasma LDL.
  • plasma HDL levels are inversely correlated with coronary heart disease, high plasma HDL levels are considered as negative risk factors.
  • Oxidative Low Density Lipoprotein (OXLL), etc., promotes the formation of atheromatous masses through inflammatory and immune responses in the vascular wall membrane mattress.
  • the cells are filled with bubbles and are sometimes called foam cells (Osto et al., Circulation, 118: 2174-2182, 2008; Jeon et al ., 2007).
  • Most of the cholesterol found in plaques is a result of foam cells ingesting oxLDL, so high plasma LDL levels are associated with a high incidence of disease. Therefore, high-fat diet, smoking, stress, lack of exercise, etc. are considered as risk factors of these diseases, and are emerging as one of serious diseases in modern society.
  • oxLDL has been reported to induce cardiovascular diseases in vivo (Faviou et al., Free Radic. Res., 39 (4): 419-29, 2005; Hiki et al., Journal of Cardiology , 53: 108-116, 2009). Increased oxLDL in plaques and plasma is also associated with the risk of atherosclerotic lesions (Nishi et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 22: 1649, 2002). The cause of atherosclerotic vascular disease has not been determined, but recent studies have shown that macrophages found in atherosclerotic plaques have LDL receptors with high levels of affinity for oxLDL.
  • oxLDL produces lipid peroxidation products such as reactive aldehydes, phospholipids, and malondialdehyde-modified LDL (MDA-LDL) that are chemically modified by malondialdehyde.
  • MDA-LDL and Cu-LDL As a representative example of oxLDL, oxidation occurs mainly on the free amino group of apoB.
  • Atherosclerotic lesions include substances with physical and immunochemical properties similar to oxLDL or MDA-LDL (Itabe et al., J. Biol. Chem, 269 (21): 15274-15279, 1994).
  • the concentration of antibody against MDA-LDL is high in plasma of cardiovascular disease, atherosclerosis patients, hypercholesterolemic rabbits, and apoE-/-or LDL receptor deficient mice, and is related to the progression of atherosclerosis.
  • Autoantibodies to oxLDL are also present in patients with preeclampsia, a maternal disease (Fialova et al. ).
  • cyanate levels and protein carbamylation caused by cyanate are increased in chronic kidney disease, thereby increasing the risk of atherosclerosis.
  • cLDL causes vascular cell injury.
  • Vascular cell injury involves the death of endothelial cells (EC) and the proliferation of vascular smooth muscle cells. It is well known that the death of endothelial cells (EC) is an early symptom of atherosclerosis. EC injury is also associated with the degree of LDL carbamylation. In patients with advanced kidney disease receiving hemodialysis, plasma cLDL levels are many times higher than in healthy individuals. (Ok et al., Kidney International , 68: 173, 2005).
  • cLDL also contributes to the pathogenesis of atherosclerosis in patients with uremic disorders (urea-increased urea levels in patients with renal disease, which causes protein carbamylation of urea to cyanate).
  • Treatment of cLDL to human aortic vascular smooth muscle cells (VSMC) increases morphological changes and proliferation, and expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (Asci et al., Nephrology 13: 480, 2008). (Apostolov et al., Arterioscler. Thromb. Vasc. Biol. 27: 826, 2007).
  • cLDL induces EC proliferation, which is via mitogen-activated protein kinases (MAPKs), which induces EC killing.
  • MAPKs mitogen-activated protein kinases
  • the combination of cell proliferation and death leads to high turnover of vascular EC (Apostolov et al., J. Physiol. Heart. Circ. Physiol. 292: 1836, 2007).
  • LOX-1 a scavenger receptor of EC
  • cLDL binds to EC's LOX-1 and shows cytotoxicity and monocyte adhesion to accelerated EC (Apostolov et al., Arterioscler. Thromb. Vasc. Biol . 29: 1622, 2009).
  • Antibodies that are available only to humans or mice are effective only in their respective species, for example, they cannot use one antibody from the experimental animal phase to the clinical trial phase during drug development, Humanization was essential for the step application. This requires a large cost economically, the development of new drugs and the study of diseases have been required to develop antibodies that can be used consistently in both humans and mice, but the research is insufficient.
  • the present inventors have tried to isolate human monoclonal antibodies that specifically bind to substances present in lesions of atherosclerotic diseases such as oxLDL and cLDL.
  • oxLDL and cLDL substances present in lesions of atherosclerotic diseases
  • cLDL atherosclerotic diseases
  • arteries in both humans and mice By specifically binding to curable plaques, isolated human monoclonal antibodies and antigen-binding fragments thereof, plaques 15 and 16-46 (plaque 15, 16-46), which are highly industrially available, such as drug development, are characterized and characterized.
  • the present invention has been completed.
  • An object of the present invention is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to oxidized LDL (oxLDL) and carbamylated LDL (Parque 15, 16-46). 46).
  • Another object of the present invention is to provide a composition for diagnosing atherosclerosis containing the human monoclonal antibody or antigen-binding fragment thereof.
  • the present invention provides a heavy chain variable region comprising heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, oxidized LDL (oxLDL) and carbamylated low density lipoprotein (provided are a human monoclonal antibody (mAb) or an antigen binding fragment (Fab) thereof which specifically binds to carbamylated LDL; cLDL).
  • mAb monoclonal antibody
  • Fab antigen binding fragment
  • the invention also relates to oxidized LDL (oxLDL) and carbamylated low density lipoprotein (cLDL), comprising a light chain variable region comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6
  • oxLDL oxidized LDL
  • cLDL carbamylated low density lipoprotein
  • mAb human monoclonal antibody
  • Fab antigen binding fragment thereof
  • the present invention also provides a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and a light chain variable region comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, Human Monoclonal Antibody (mAb) or Antigen Binding Fragment (Fab) thereof that specifically binds to Oxidized LDL (oxLDL) and Carbamylated Low Density Lipoprotein (cLDL) To provide.
  • mAb Monoclonal Antibody
  • Fab Antigen Binding Fragment
  • the present invention also provides oxidized LDL (oxLDL) and cLDL specific human monoclonal antibodies or antigen-binding fragments thereof containing the heavy chain variable region represented by SEQ ID NO: 21.
  • the present invention also provides an oxLDL and cLDL specific human monoclonal antibody or antigen-binding fragment thereof containing the light chain variable region represented by SEQ ID NO: 22.
  • the present invention also provides an oxLDL and cLDL specific human monoclonal antibody or antigen-binding fragment thereof containing the heavy chain variable region represented by SEQ ID NO: 21 and the light chain variable region represented by SEQ ID NO: 22.
  • the present invention also provides a DNA sequence encoding the human monoclonal antibody or antigen-binding fragment thereof.
  • the present invention also provides a composition for diagnosing atherosclerosis containing the human monoclonal antibody or antigen-binding fragment thereof.
  • the atherosclerotic vascular disease may be selected from the group consisting of aneurysms, cerebral arteriosclerosis, cerebral infarction, cerebral hemorrhage, angina pectoris, myocardial infarction, nephropathy, retinal arteriosclerosis, and arterial obstruction.
  • Figure 1 shows the amino acid sequence of the V H and V L genes of the plaque 15, 16-46 (plaque 15, 16-46) Fab, DNA sequence is GenBank accession No. AY957524 and AAY957525 were used. The same amino acid sequence was denoted by-and the amino acid substituted by mutation was indicated in red as compared to the Germline gene.
  • Figure 2 is a photo of the result of amplifying the AID mRNA transcribed from the plaque and PBMC by RT-PCR column 1 DNA 100bp ladder marker, column 2 patient plaque results, column 3 electrophoresis of the PBMC results.
  • Figure 3 is an immunoblot photograph of purified plaque 15, 16-46 (plaque 15, 16-46) Fab, showing the passage of whole bacterial pellets (column 1), soluble extract (column 2), soluble extracts through the column.
  • Fab (4 rows), which lost bound material when passing through the column (3 columns) and soluble extracts, were separated by SDS-PAGE under non-reducing conditions and detected by anti-human IgG mAb (Fab specific). It is a photograph.
  • FIG. 4 is plaque 15, 16-46 Fab ELISA results for LDL and HDL chemically modified in different forms.
  • FIG. 5 is a photograph of IF and IHC analysis of the recognition of sclerotic lesions of human, rat, control, etc. by plaque 15, 16-46 (plaque 15, 16-46) Fab.
  • a fragment that binds to the MDA-LDL antigen is newly isolated using phage display technology, and the separated antigen-binding fragment (Fab) is specifically bound to MDA-LDL and Cu-LDL, which are representative forms of oxLDL.
  • Fab antigen-binding fragment
  • the present invention provides an oxidized low density lipoprotein (oxLDL) and a carbamylated low density lipoprotein (Carbamylated) containing a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3
  • oxLDL oxidized low density lipoprotein
  • carbamylated low density lipoprotein carbamylated low density lipoprotein
  • the present invention relates to a human monoclonal antibody (mAb) or an antigen binding fragment thereof (Fab) that specifically binds to LDL; cLDL).
  • the light chain variable region of the antibody or Fab preferably contains the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, but may be a known light chain variable region.
  • the present invention provides an oxidized low density lipoprotein (oxLDL) and a carbamylated low density lipoprotein (Carbamylated) containing a light chain variable region comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6
  • oxLDL oxidized low density lipoprotein
  • carbamylated low density lipoprotein carbamylated containing a light chain variable region comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6
  • mAb human monoclonal antibody
  • Fab antigen binding fragment thereof
  • the heavy chain variable region of the antibody or Fab preferably contains the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but may be a known heavy chain variable region.
  • a preferred embodiment of the present invention comprises a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and a light chain variable region comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 .
  • the heavy chain variable region it may be characterized in that it comprises a heavy chain FR region of SEQ ID NO: 7 to SEQ ID NO: 10.
  • the light chain variable region may be characterized in that it comprises a light chain FR region of SEQ ID NO: 11 to SEQ ID NO: 14.
  • Another preferred embodiment of the present invention contains the heavy chain variable region represented by SEQ ID NO: 21.
  • the heavy chain variable region represented by SEQ ID NO: 21 includes the CDR and FR regions of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 7 to 10.
  • Another preferred embodiment of the present invention contains a light chain variable region represented by SEQ ID NO: 22.
  • the heavy chain variable region represented by SEQ ID NO: 22 includes the CDR and FR regions of SEQ ID NOs: 4 to 6 and SEQ ID NOs: 11 to 14.
  • the most preferable aspect of this invention contains the heavy chain variable region represented by SEQ ID NO: 21, and the light chain variable region represented by SEQ ID NO: 22.
  • amino acid used in the present invention means all amino acids existing in nature, and preferably, amino acids represented as follows according to the three-letter or one-letter notation of the alphabet used to denote amino acids, respectively. it means.
  • Glycine (Gly / G), Alanine (Ala / A), Valine (Val / V), Leucine (Leu / L), Isoleucine (Ile / I), Serine (Ser / S), Threonine (Thr / T), Aspartic Acid (Asp / D), glutamic acid (Glu / E), asparagine (Asn / N), glutamine (Gln / Q), lysine (Lys / K), arginine (Arg / R), cysteine (Cys / C), methionine ( Met / M), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), histidine (His / H), proline (Pro / P).
  • the human monoclonal antibody means a human immunoglobulin in which all regions including the variable and normal regions of the H chain and the L chain are derived from a gene encoding human immunoglobulin.
  • the L chain include human ⁇ chain or human ⁇ chain.
  • the human monoclonal antibody of the present invention may be a human monoclonal antibody having an isotype of any one of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE.
  • the human monoclonal antibody can be prepared according to the existing general monoclonal antibody production method using human antibody-producing transgenic non-human mammals such as human antibody-producing transgenic mice which are generally used.
  • a recombinant E. coli cell producing a recombinant human monoclonal antibody obtained by transforming a host cell by cDNA encoding each of the heavy and light chains of such a human monoclonal antibody of the present invention. It may be a method obtained from the culture broth by culturing.
  • the present invention relates to a DNA sequence encoding the human monoclonal antibody or antigen-binding fragment thereof.
  • the DNA sequence may include a DNA sequence represented by SEQ ID NOs: 23 and 24.
  • SEQ ID NO: 23 is a DNA sequence encoding a heavy chain variable region (V H ) of SEQ ID NO: 21
  • SEQ ID NO: 24 is a DNA sequence encoding a light chain variable region (V L ) of SEQ ID NO: 22.
  • the DNA sequence can be obtained by phagemid DNA sequencing, the method may be by a method commonly known in the art.
  • the antigen-binding fragment (Fab) isolated in the present invention is immunofluorescence on the ApoE-/-rat arterial tissue fragments and atherosclerotic plaques isolated from placental tissue of preeclampsia patients, and arterial plaques of atherosclerotic patients Through analysis (IF) or immunohistochemistry (IHC) analysis, it was confirmed that each tissue atherologic lesion and the antigen-binding fragment (Fab) of the present invention were specifically bound to diagnose the lesion.
  • IF analysis
  • IHC immunohistochemistry
  • the present invention relates to a composition for diagnosing atherosclerotic disease comprising a human monoclonal antibody or an antigen-binding fragment thereof according to the present invention.
  • Atherosclerosis may be selected from the group consisting of aneurysms, cerebral arteriosclerosis, cerebral infarction, cerebral hemorrhage, angina pectoris, myocardial infarction, nephropathy, retinal arteriosclerosis, and arterial occlusion.
  • the diagnostic composition of the present invention contains a human monoclonal antibody or an antigen-binding fragment thereof according to the present invention, and a sample or a sample sample can be processed into a diagnostic composition to obtain a result.
  • the diagnostic composition may be included in the form of a kit or nucleic acid chip commonly used in the art.
  • samples including all biological samples obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of assay that performs the "sample” or "sample sample”. Methods of obtaining bodily fluids and tissue biopsies from mammals are commonly known.
  • a preferred source is biopsy of atherosclerotic disease lesions.
  • the antigen-binding fragment (Fab) of the present invention is isolated from ApoE-/-rat arterial tissue fragment and placental tissue of human atherosclerosis patient or preeclampsia patient using phage display technology.
  • Fab antigen-binding fragment
  • the human monoclonal antibody or antigen-binding fragment thereof (Fab) of the present invention specifically binds to an atherologic lesion site, it can be used for diagnosis of atherovascular disease.
  • Fab antigen-binding fragment thereof
  • it can be used for the treatment and prevention of atherosclerosis by combining the antibody or Fab with drugs and the like.
  • a mixed human atherosclerotic plaque obtained from two patients with atherosclerosis was used to generate an Anti-oxLDL monoclonal antibody Fab.
  • the Fab was used to determine the relative reactivity of the phage antibody against MDA-LDL and native LDL by the monoclonal antibody Fab clone, plaques 15, 16-46 through the phage display procedure described in Example 1. Selected by comparison. Purchase of Native LDL and HDL was made by Chemicon International. MDA modifications for the production of MDA-LDL are also described in Fogelman et al. ( Proc. Natl. Acad. Sci., 77 (4I): 2214-2218, 1980).
  • V H and V L cDNA sequences were determined using an automated sequencing analyzer (CoreBio Technology, Korea).
  • primers represented by the following SEQ ID NOs: 15 and 16 were used.
  • BLAST Basic Local Alignment Search Tool
  • DNAPLOT V BASE
  • V H and V L amino acid sequence was confirmed.
  • the analysis of their sequences features is shown in Table 1.
  • the genes used by Fab were found to be V H 3-48 and V k 3-20, respectively.
  • the region D of V H used the D3-16 gene segment. Compared with the germline sequence, which is most similar to V H and V L of Fab, they have 87.8% and 96.8% similarity, respectively.
  • the replacement (R) variation ratio for silent (S) in CDRs of V H is very high (14/0).
  • the ratio of FRs in V H is 8/5
  • the CDRs in V L are 4/0
  • the FRs in V L are 1/3
  • many mutations in which amino acids such as Arg, Lys, and His occur are measured in quantity. It was found in the regions of H and V L (6 of 14 and 3 of 5 amino acids, respectively) (FIG. 1 and Table 2).
  • higher R / S ratios of CDRs of V H and V L than 2.9 indicate that the mutation in that region was under selection pressure through interaction with the antigen during B-cell development.
  • AID activation-induced cytidine deaminase
  • plaque 15, 16-46 plaque 15, 16-466 was confirmed that the local expression of mRNA transcription in the atherosclerotic plaque (atherosclerotic plaque) of the patient was made.
  • the Correct 335-bp AID RT-PCR product was observed in both patient plaque and PBMC samples.
  • non-suppressor family of bacteria TOP10F 'cells were modified with phage Fab DNA.
  • the soluble Fabs present in the culture supernatant or plasma membrane space extract are described in Jeon et al. Purification was carried out by using Ni ++ -NTA chromatography by the (2007) method. Purified Fabs were separated by SDS-PAGE under non-reducing conditions and analyzed by immunoblotting.
  • Binding properties of Fabs were determined by ELISA.
  • 100 microliters of PBS containing 1 microgram of antigen were placed in each well of a 96-well microtiter dish and coated at 4 degrees overnight. Each well was washed three times with PBS, and then reacted with PBS containing 5% BSA for 2 hours at room temperature. Thereafter, the wells were washed three times with PBS containing 0.05% Tween (PBST), and then the Fab was reacted at room temperature for 2 hours, followed by alkaline phosphatase-conjugated goth anti-human IgG antibody (1: And diluted with PBS containing 1% BSA at 5000) for 2 hours at room temperature.
  • PBST 0.05% Tween
  • Arterial tissue sections from frozen atherosclerosis were analyzed by immunofluorescence (IF Analysis). Human tissues were reacted with 10 ug / ml Fab for 2 hours at room temperature. Thereafter, the reaction was performed with 20 ug / ml FITC-labeled anti human IgG (Vector Labs) at room temperature for 1 hour. Frozen samples of atherosclerotic plaques were purchased from BioChain (Cat # T1236012Hd-4). Fluorescence images were obtained using Olympus fluorescence microscope model BX-61.
  • FIG. 5A positively-stained sclerosis plaques could be observed in FIGS. 5A-2 (40-fold magnification) and 5A-3 (200-fold magnification), and negative controls (stained without primary antibody).
  • Photograph; Figure 5A-1) (40 times magnified) was clearly seen.
  • FIG. 5B foam cells in the sclerosis plaques reacted specifically with Fab and appeared to be stained red.
  • FIGS. 5B-3 show a photograph magnified 100 times at the other two points of B-3 (40 times magnification). In the enlarged picture, sclerosis plaques (dark arrows) were seen more clearly.
  • Sclerosis plaque sections obtained from placental decidual tissue from preeclampsia patients were analyzed by the IF method using Fab.
  • the analysis method is the same as used in Example 4-1.
  • Tissue sections were obtained from placental tissues of patients with preeclampsia who visited Obstetrics and Gynecology.
  • Fab recognized specially damaged placental tissue vascular deletion arterial cells.
  • FIGS. 5C 1-3 200X
  • 400X FIGS. 5C4, 5
  • cross-sections of atherosclerotic vessels stained with Fab were clearly visible in rounded shapes (FIGS. 5C-2).
  • IF without primary antibody was used as a negative control (FIGS. 5C-6).
  • single cells were freshly isolated from a portion of tissue isolated for IF and analyzed by CD68 staining and FACS analysis.
  • the method of separating single cells is as follows. The membrane portion of the placental tissue isolated during surgery was cut out and washed with PBS until the stained blood was washed away. The cut placenta tissue was placed in petridish and the amount of PBS was added to the tissue. Thereafter, the collagenase was added and left for 3 hours in a CO 2 cell incubator. After filtration using a filter bag, the filtered cell solution was centrifuged for 10 min at 300 g at 4 ° C to quench single cells.
  • RBC buffer (NH 4 Cl 8.29g, KHCO 3 1g, EDTA disodium Salt 0.037 mg per 1L) was added to the separated cells 10 times or more and left for 2 minutes, and then centrifuged again at 4 °C and 300g. Separated. This procedure was repeated three times until RBC was completely removed, followed by two more washes with PBS containing 2% FBS. If the tissue remained fine, it was filtered once more using a nylon filter (Spectra / Mesh Nylon Filter, 53 ⁇ m).
  • FACS tubes In order to perform FACS analysis on the presence of macrophages using separated single cells using anti-CD68 antibody, put the separated single cells into FACS tubes to be 1 x 10 6 / tube. After the antibody was added, the reaction was carried out for 15 minutes in a 37 °C CO 2 incubator, and washed twice at 1,350rpm for 7 minutes with 2% FBS / PBS. Anti-mouse IgG-FITC was added to 2 ⁇ g / ml as Secondary Ab, and then left in 37 ° C. CO 2 incubator for 15 minutes, and washed twice at 1,350 rpm for 7 minutes with 2% FBS / PBS. Stained macrophages were analyzed by FACS (FACSVantage).
  • the ratio (M2) of CD68-positive macrophages among plaque cells was higher than the negative control (34.92% vs 5.16%).
  • the monoclonal antibody and antigen-binding fragment thereof include heavy chain variable region (V H ) and light chain variable region (V L ) specific for ox-LDL and cLDL, the diagnosis of atherosclerosis including atherosclerosis, It is useful for prevention and treatment. In addition, it is possible to detect lesions of both humans and rats, and since the antibody can be used in preclinical stages (experimental animal stages) and clinical stages when developing new drugs, it is excellent in economic efficiency.

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Abstract

La présente invention concerne un anticorps monoclonal humain qui se lie spécifiquement à une lipoprotéine de faible densité oxydée (ox-LDL) et à une LDL carbamylée (cLDL), et son fragment de liaison à un antigène (Fab), et plus spécifiquement un anticorps monoclonal humain et son Fab qui se lient spécifiquement à des lésions de la plaque athéromateuse chez le rat ou l'homme et à des plaques 15, 16-46. Ledit anticorps monoclonal et son Fab de la présente invention comportent une région variable de chaîne lourde (VH) et une région variable de chaîne légère (VL) spécifiques à ox-LDL et cLDL, et sont donc utiles dans le diagnostic, la prévention et le traitement de l'artériosclérose et d'autres maladies vasculaires athéromateuses. En outre, la présente invention est extrêmement économique, car elle permet la détection de lésions à la fois chez l'homme et chez le rat, en fournissant un nouvel anticorps qui peut être utilisé tout au long de la mise au point d'un nouveau médicament, depuis l'étape préclinique (étape d'expérimentation animale) jusqu'à l'étape clinique.
PCT/KR2010/008219 2010-06-14 2010-11-22 Anticorps monoclonal humain qui se lie spécifiquement à une lipoprotéine de faible densité oxydée et à une lipoprotéine de faible densité carbamylée, et son fragment de liaison à un antigène Ceased WO2011159000A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN118459581A (zh) * 2024-05-24 2024-08-09 东方海洋(北京)医学研究院有限公司 抗人氧化低密度脂蛋白的单克隆抗体及其应用
US12291579B2 (en) 2023-03-17 2025-05-06 Oxitope Pharma B.V. Anti-phosphocholine antibodies and methods of use thereof

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