WO2011159000A1 - Human monoclonal antibody that binds specifically with oxidized low-density lipoprotein and carbamylated low-density lipoprotein, and antigen-binding fragment thereof - Google Patents

Abstract

The present invention relates to a human monoclonal antibody that binds specifically with oxidized low-density lipoprotein (ox-LDL) and carbamylated LDL (cLDL), and an antigen binding fragment (Fab) thereof, and more specifically relates to a human monoclonal antibody and the Fab thereof which bind specifically with human or rat atheromatous plaque lesions and to plaque 15, 16-46. The monoclonal antibody and the Fab thereof of the present invention include a heavy chain variable region (V_H) and a light chain variable region (V_L) specific to ox-LDL and cLDL, and are thus useful in diagnosis, prevention, and treatment of arteriosclerosis and other atheromatous vascular diseases. Also, the present invention is highly economical because detection of lesions in both humans and rats are possible, providing a novel antibody that can be used throughout a new drug development from the pre-clinical stage (animal testing stage) to the clinical stage.

Description

Human monoclonal antibodies and antigen binding fragments thereof that specifically bind to oxidized low density lipoproteins and carbamylated low density lipoproteins

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). 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). Examples of 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. In contrast, since 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.

In addition, 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. ).

In addition, according to the conventional studies, cyanate levels and protein carbamylation caused by cyanate, especially LDL carbamylation level (cLDL level), are increased in chronic kidney disease, thereby increasing the risk of atherosclerosis. This is because 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. 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). On the other hand, when cLDL is treated with EC, expression of LOX-1, a scavenger receptor of EC, is increased, and the increased LOX-1 shows an important atogenic effect on the EC. This is because 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).

The production of human antibodies specific for oxLDL or modified LDL not only has important implications for understanding the genetic structure of antibodies and the pathogenesis of atherosclerosis, but also can be used as diagnostic antibodies for diseases related to atherosclerosis. While useful, only a few anti-oxLDL monoclonal antibodies have been developed to date. Some of the antibodies are IgG or Fab that recognizes atherosclerotic lesions in humans and rabbits (Itabe et al. 1994; Palinski et al., J. Clin. Invest ., 98: 800-814, 1996; Shaw et. al., Arterioscler. Thromb. Vasc. Biol. , 21: 1333-1339, 2001), human antibodies that cross react in humans and mice (Schiopu et al., Circulation , 110 (14): 2047-2052, 2004) Is present but it is in IgG form. No human antibody Fab has been reported to recognize atherosclerotic lesions in humans and mice at the same time.

Antibodies that are available only to humans or mice (mouses) 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.

Therefore, 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. Thus, using a combination antibody library and phage display technology, 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.

Summary of the Invention

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.

In order to achieve the above object, 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).

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 Provided are a human monoclonal antibody (mAb) or an antigen binding fragment thereof (Fab) that specifically binds thereto.

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.

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.

In the present invention, 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.

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.

Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

In the present invention, 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. In addition, it was confirmed that the cross-reaction with cLDL.

In one aspect, 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 The present invention relates to a human monoclonal antibody (mAb) or an antigen binding fragment thereof (Fab) that specifically binds to LDL; cLDL).

In the present invention, 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.

In another aspect, 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 The present invention relates to a human monoclonal antibody (mAb) or an antigen binding fragment thereof (Fab) that specifically binds to LDL; cLDL).

In the present invention, 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 .

In the present invention, 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. In addition, 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.

In the present invention, the sequence is represented as an amino acid sequence. The term "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).

In the present invention, 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. Examples of the L chain include human κ chain or human λ chain. In addition, 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.

In the present invention, 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. Alternatively, using a recombinant technique, 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.

In another aspect, the present invention relates to a DNA sequence encoding the human monoclonal antibody or antigen-binding fragment thereof. In the present invention, 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, and SEQ ID NO: 24 is a DNA sequence encoding a light chain variable region (V _L ) of SEQ ID NO: 22.

In the present invention, the DNA sequence can be obtained by phagemid DNA sequencing, the method may be by a method commonly known in the art.

In one embodiment of the present invention, 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.

In another aspect, 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.

In 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. By wide variety of 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.

In another embodiment, 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. As a result of immunofluorescence or immunohistochemical analysis on the atherosclerotic plaque, it was confirmed that the plaque component present in each tissue lesion and the antigen-binding fragment (Fab) of the present invention specifically bind.

According to an embodiment of the present invention, since 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. In addition, by using a method known in the art it can be used for the treatment and prevention of atherosclerosis by combining the antibody or Fab with drugs and the like. In addition, it was confirmed that it has specificity not only in humans but also in mice (mouses) which are widely used as experimental animals, and thus may be particularly useful in developing new drugs.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Antibody Isolation of oxLDL Using Phage Display Technology

1-1: human sample acquisition

Plaque samples were obtained after consent was obtained from patients with atherosclerosis who visited Ajou University Hospital. The agreement and study plan were certified by the Institutional Review Board of Ajou University Medical Center.

1-2: Obtaining Antibodies Using Phage Display Technology

Antigen-specific antibody Fab isolation by human combinatorial IgG library construction and phage display is described in Jeon et al ,. (2007). For library construction, total RNA was obtained from atherosclerotic plaques isolated during surgery of two patients with atherosclerosis. Plaques were stored at -20 degrees in RNAlate (Ambion, US). Plaque tissue was cut with scissors and tissue was digested with a tissue mill (Tissue-Tearor, Biospec Product, US). RNA was isolated using TRIzol solution (Invitrogen, New Zealand). During phage display, antibody clones that specifically bind to MDA-LDL were obtained by comparing the reactivity to MDA-LDL and native LDL in phage supernatant. And it was named plaque 15, 16-46 (plaque 15, 16-46).

Example 2 Genetic Structure Analysis of Plaques 15, 16-46 Fab

2-1: cDNA sequence analysis of plaque 15, 16-46 Fab

In this example, 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). Acylated LDL or HDL are described in Basu et al. ( Proc. Natl. Acad. Sci., 73: 3178-3182, 1976). Carbamylate LDL or HDL was prepared by the method described in Ok et al . ( Kidney International , 68: 173, 2005).

Then, in order to analyze the specific gene structure of the Fab selected by the above experimental procedure, V _H and V _L cDNA sequences were determined using an automated sequencing analyzer (CoreBio Technology, Korea). Thus, primers represented by the following SEQ ID NOs: 15 and 16 were used.

SEQ ID NO: 15 V _H , 5'-ACCTATTGCCTACGGCAGCCG-3 '

SEQ ID NO: 16 V _L , 5'- AAGACAGCTATCGCGATTGCAG-3 '

The sequence results were used by BLAST (Basic Local Alignment Search Tool) and DNAPLOT (V BASE) to analyze homology with germline, genes used, and mutations.

As a result, as shown in Figure 1, the determined V _H and V _L amino acid sequence was confirmed. The analysis of their sequences features is shown in Table 1. On the other hand, 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). On the other hand, 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, and 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). In particular, 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.

Table 1

TABLE 2

2-2: Confirmation of Somatic Hypermutation in Plaques by Analysis of AID mRNA Expression

Evidence of somatic hypermutation and class switch recombination can be confirmed by analyzing local expression of activation-induced cytidine deaminase (AID) mRNA transcription. To amplify AID mRNA transcripts by RT-PCR, RNA was obtained from atherosclerotic patient PBMCs and plaque samples from which cDNA was obtained. PCR is described by Coker et al. (2003). Primers shown below SEQ ID NOs: 17-20 were used for PCR.

SEQ ID NOs: 17-20

AID1P-5’-GAG GCA AGA AGA CAC TCT GG-3 ’

AID2P-5’-GTG ACA TTC CTG GAA GTT GC-3 ’

AID3P-5’-TAG ACC CTG GCC GCT GCT ACC-3 ’

AID4P-5’-CAA AAG GAT GCG CCG AAG CTG TCT GGA G-3 ’

As a result, as shown in Figure 2, the plaque 15, 16-46 (plaque 15, 16-46) 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.

Example 3 Biochemical Characterization of Fabs

3-1: Analysis by immunoblotting

In order to analyze the binding characteristics of mAb Fab, in this experiment, non-suppressor family of bacteria TOP10F 'cells were modified with phage Fab DNA. After inducing protein expression in modified cells, 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.

As a result, as shown in Figure 3, specifically, it was confirmed that the response to the anti-human IgG (Fab-specific) mAb, the approximate molecular size of this Fab was ~ 45 Kdal.

3-2: Analysis of Fab binding by ELISA

Binding properties of Fabs were determined by ELISA. In a standard assay, 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. Wells were washed twice with PBST and once with TBS containing 0.05% Tween. Finally, bound alkaline phosphatase activity was measured using paranitrophenyl phosphate as substrate. The results were optically measured for absorbance at 405 nm wavelength with an ELISA reader (DI Biotech, Model ELx808).

As a result, as shown in Figure 4, it was confirmed that Fab reacts strongly with both MDA-LDL, Cu-LDL and carbamylated LDL (cLDL). That is, it strongly binds to oxLDL and cross-reacts to cLDL. No significant reactivity was observed for MDA-HDL, native LDL, Cu-HDL, cHDL, acetylated LDL, native HDL and acetylated HDL. The Fab also did not respond to double stranded DNA or histones.

Example 4 Recognition of atherosclerotic lesions by mAb

4-1: Recognition of Human Atherosclerosis Lesions

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.

As a result, as shown in 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.

4-2: Recognition of Mouse Atherosclerosis Lesions

On the other hand, immunohistochemical analysis of the atherosclerotic tissues of ApoE-/-mice induced with atherosclerosis through high fat diet was performed with Fab. ApoE − / − rat arterial tissue fragments were reacted with 10 μg / ml Fab and visualized using the VECTASTAIN ABC kit (Vector Labs).

As a result, as shown in Fig. 5B, foam cells in the sclerosis plaques reacted specifically with Fab and appeared to be stained red. As shown in 1 to 3 of FIG. 5B, when stained with a higher concentration of Fab (B-3) than the negative control group (B-1) or a low concentration of Fab (B-2), much darker staining was observed. The photograph in the thick box of FIGS. 5B-3 shows 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.

4-3: Recognition of Human Preeclampsia Lesions

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.

As a result, as shown in Fig. 5C, Fab recognized specially damaged placental tissue vascular deletion arterial cells. IF-positive staining results of several different parts were confirmed in the 200X (FIGS. 5C 1-3) and 400X (FIGS. 5C4, 5) magnified images. Interestingly, 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).

4-4: Identification of Macrophages in Human Preeclampsia Lesions with FACS

In order to confirm the presence of macrophages around the plaque lesions of preeclampsia, 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.

In order to remove red blood 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 ℃ 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 μm).

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 ℃ CO ₂ incubator, and washed twice at 1,350rpm for 7 minutes with 2% FBS / PBS. Anti-mouse IgG-FITC was added to 2µg / 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).

 As a result, as shown in the box of FIG. 5C, the ratio (M2) of CD68-positive macrophages among plaque cells was higher than the negative control (34.92% vs 5.16%).

As described above in detail the specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Since the monoclonal antibody and antigen-binding fragment thereof according to the present invention 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.

Electronic file attached.

Claims (13)

Oxidized low density lipoprotein (ox-LDL) and carbamylated low density lipoprotein (cLDL) containing heavy chain variable regions comprising heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 Human monoclonal antibody (mAb) which specifically binds, or antigen binding fragment (Fab) thereof. Oxidized low density lipoproteins (ox-LDL) and carbamylated low density lipoproteins (cLDL) containing light chain variable regions comprising the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 Human monoclonal antibody (mAb) which specifically binds, or antigen binding fragment (Fab) thereof. An oxidized low density lipoprotein comprising 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 antibodies (mAb) or antigen binding fragments thereof (Fab) that specifically bind to (ox-LDL) and carbamylated low density lipoprotein (Carbamylated LDL; cLDL). The human monoclonal antibody or antigen-binding fragment thereof (Fab) according to claim 1 or 3, wherein the heavy chain variable region is represented by SEQ ID NO: 21. The human monoclonal antibody or antigen-binding fragment thereof (Fab) according to claim 2 or 3, wherein the light chain variable region is represented by SEQ ID NO: 22. Specific for oxidized low density lipoproteins (ox-LDL) and carbamylated low density lipoproteins (cLDL) containing heavy chain variable region represented by SEQ ID NO: 21 and light chain variable region represented by SEQ ID NO: 22 Human monoclonal antibodies (mAb) or antigen binding fragments thereof (Fab). A DNA sequence encoding the human monoclonal antibody or antigen-binding fragment (Fab) of any one of claims 1 to 3. A DNA sequence encoding the human monoclonal antibody of claim 6 or an antigen binding fragment (Fab) thereof. The DNA sequence of claim 8, wherein the DNA sequence comprises a sequence represented by SEQ ID NO: 23 or SEQ ID NO: 24. 10. A composition for diagnosing atherosclerotic disease, comprising the human monoclonal antibody of claim 1 or an antigen-binding fragment thereof. A composition for diagnosing atherosclerotic disease, comprising the human monoclonal antibody of claim 6 or an antigen-binding fragment thereof. The diagnostic composition according to claim 10, wherein the atherosclerotic vascular disease is 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 according to claim 11, wherein the atherosclerotic vascular disease is selected from the group consisting of aneurysms, cerebral arteriosclerosis, cerebral infarction, cerebral hemorrhage, angina pectoris, myocardial infarction, nephropathy, retinal arteriosclerosis, and arterial occlusion.

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