US-12618840-B2 - Antibody for detecting acetylation of COX2 protein, and uses thereof

Abstract

The present invention relates to an antibody for detecting acetylation of COX2 protein, and uses thereof, and more specifically, to an antibody that specifically recognizes the acetylation of S565 residue of the COX2 protein; and uses thereof for diagnosing neurodegenerative diseases or inflammatory diseases. An antibody or a functional fragment thereof according to the present invention specifically binds to an acetylated residue of COX2 protein, and can thus be very effectively used for diagnosing neurodegenerative diseases, inflammatory diseases, and the like in which the degree of acetylation of S565 residue of the COX2 protein is reduced.

Inventors

• Jae-sung Bae
• Hee Kyung Jin
• Ju Youn Lee

Assignees

• KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION

Dates

Publication Date

20260505

Application Date

20200918

Priority Date

20190920

Claims (17)

1. 1 . An antibody or a functional fragment thereof that specifically recognizes the acetylation of cyclooxygenase 2 (COX2) protein; wherein the antibody or the functional fragment thereof is an antibody or a functional fragment thereof comprising an antibody light chain variable region (VL) having a complementarity determining region (CDR) L1 including an amino acid sequence having SEQ ID NO: 5, a complementarity determining region (CDR) L2 including an amino acid sequence having SEQ ID NO: 6, and a complementarity determining region (CDR) L3 including an amino acid sequence having SEQ ID NO: 7 and an antibody heavy chain variable region (VH) having a complementarity determining region (CDR) H1 including an amino acid sequence having SEQ ID NO: 8, a complementarity determining region (CDR) H2 including an amino acid sequence having SEQ ID NO: 9, and a complementarity determining region (CDR) H3 including an amino acid sequence having SEQ ID NO: 10; or an antibody or a functional fragment thereof comprising an antibody light chain variable region (VL) having a complementarity determining region (CDR) L1 including an amino acid sequence having SEQ ID NO: 21, a complementarity determining region (CDR) L2 including an amino acid sequence having SEQ ID NO: 22, and a complementarity determining region (CDR) L3 including an amino acid sequence having SEQ ID NO: 23 and an antibody heavy chain variable region (VH) having a complementarity determining region (CDR) H1 including an amino acid sequence having SEQ ID NO: 24, a complementarity determining region (CDR) H2 including an amino acid sequence having SEQ ID NO: 25, and a complementarity determining region (CDR) H3 including an amino acid sequence having SEQ ID NO: 26; or wherein the antibody or the functional fragment thereof is an antibody or a functional fragment thereof comprising a light chain variable region (VL) including an amino acid sequence having SEQ ID NO: 11 and a heavy chain variable region (VH) including an amino acid sequence having SEQ ID NO: 12; or an antibody or a functional fragment thereof comprising a light chain variable region (VL) including an amino acid sequence having SEQ ID NO: 27 and a heavy chain variable region (VH) including an amino acid sequence having SEQ ID NO: 28.
2. 2 . The antibody or the functional fragment thereof of claim 1 , wherein the acetylation is acetylation in S565 residue of cyclooxygenase 2 (COX2) protein defined by SEQ ID NO: 1.
3. 3 . The antibody or the functional fragment thereof of claim 1 , wherein the epitope of the antibody is a peptide including an amino acid sequence represented by SEQ ID NO: 2 and consisting of 9 to 50 amino acids.
4. 4 . The antibody or the functional fragment thereof of claim 1 , wherein the epitope of the antibody is a peptide consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
5. 5 . The antibody or the functional fragment thereof of claim 1 , wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD.
6. 6 . The antibody or the functional fragment thereof of claim 1 , wherein the functional fragment of the antibody is selected from the group consisting of a diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv and scFv.
7. 7 . A polynucleotide encoding the antibody or the functional fragment thereof of claim 1 .
8. 8 . A vector comprising the polynucleotide of claim 7 .
9. 9 . A host cell transformed with the vector of claim 8 .
10. 10 . A method for preparing an antibody or a functional fragment thereof that specifically recognizes acetylation of cyclooxygenase 2 (COX2) protein, comprising steps of producing a polypeptide including light chain and heavy chain variable regions by culturing the cell of claim 9 under a condition in which the polynucleotide is expressed, and recovering the polypeptide from the cell or a culture medium culturing the cell.
11. 11 . A composition for diagnosing neurodegenerative diseases comprising the antibody or the functional fragment thereof of claim 1 .
12. 12 . The composition of claim 11 , wherein the neurodegenerative diseases are one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine-pony-cerebellar atrophy (OPCA), Shay-Drager syndrome, striatal-nigular degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Nieman-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction.
13. 13 . A kit for diagnosing neurodegenerative diseases comprising the antibody or the functional fragment thereof of claim 1 .
14. 14 . A composition for diagnosing inflammatory diseases comprising the antibody or the functional fragment thereof of claim 1 .
15. 15 . The composition of claim 14 , wherein the inflammatory diseases are one or more selected from the group consisting of dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, inflammatory bowel disease, lupus, hepatitis, cystitis, nephritis, sjogren's syndrome, uveitis, ankylosing spondylitis, endometritis, multiple sclerosis, sepsis, septic shock, chronic obstructive pulmonary disease and arthritis.
16. 16 . A method for diagnosing neurodegenerative diseases comprising steps of: a) obtaining a sample from a subject; b) measuring an acetylation level of COX2 protein by adding the antibody or the functional fragment thereof of claim 1 to the sample; and c) comparing the acetylation level of the COX2 protein with that of a normal subject, and determining that a subject having a reduced acetylation level of the COX2 protein compared to the normal subject suffers from neurodegenerative diseases.
17. 17 . A method for diagnosing inflammatory diseases comprising steps of: a) obtaining a sample from a subject; b) measuring an acetylation level of COX2 protein by adding the antibody or the functional fragment thereof of claim 1 to the sample; and c) comparing the acetylation level of the COX2 protein with that of a normal subject, and determining that a subject having a reduced acetylation level of the COX2 protein compared to the normal subject suffers from inflammatory diseases.

Description

TECHNICAL FIELD This application is a U.S. National Stage application claiming priority to PCT/KR2020/012647 filed Sep. 18, 2020, which claims the priority of Korean Patent Application No. 10-2019-0116290, filed on Sep. 20, 2019, the entirety of which is a reference of the present application. The present invention relates to an antibody for detecting acetylation of COX2 protein, and a use thereof, and more specifically, to an antibody that specifically recognizes the acetylation of S565 residue of COX2 protein; and uses thereof for diagnosing neurodegenerative diseases or inflammatory diseases. CROSS-REFERENCE TO SEQUENCE LISTING This application contains a sequence listing filed in ST.25 format entitled “321901-1010 Sequence Listing_ST25.txt” created on Jul. 29, 2022, and having a file size of 26,650 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND ART Inflammatory diseases are closely associated with most of diseases, and as a result of basic research in molecular and cellular immunology, methods for diagnosing, treating and preventing diseases based on such immunology have been dramatically changed. One example thereof is the finding of an inducible form of a cyclooxygenase (COX) enzyme. COX protein was first purified in 1976, and constitutive cyclooxygenase (COX) cloned in 1988 was found to act in the synthesis of prostaglandin (PGs) from arachidonic acid (AA). After 3 years of such purification, an inducible enzyme having COX activity was identified and named as COX2, while constitutive COX was named as COX1. The expression of COX2 is under the regulation of pro-inflammatory cytokines and growth factors. Thus, it has been widely known up to now that COX2 acts on the regulation of both inflammation and cell growth. The COX2 is induced in many tissues and simultaneously shown structurally in the brain and spinal cord, wherein the COX2 acts on neural transmission for pain and fever. The two subtypes of COX are almost similar in structure, but have important differences in selectivity of a substrate and an inhibitor and intracellular positions thereof. Protective prostaglandin (PG), which preserves the shape of the gastric mucosa and maintains a normal renal function in the damaged kidney, is synthesized by COX1. On the other hand, PG synthesized by COX2 in immune cells plays a very important role in the inflammatory process. COX2 in a normal state is known to mediate various physiological phenomena such as immune responses, but it has been reported that abnormal overexpression or overactivation of COX2 is closely associated with the occurrence and development of various diseases. Specifically, COX2 is overexpressed in most acute or chronic inflammatory diseases and is very closely associated with the development of diseases (J Neuropathol Exp Neurol, Vol 63, September 2004 pp. 901 910). It has been reported that the expression of COX2 is increased in cancer tissues compared to normal tissues in most human cancers including bladder cancer, breast cancer, colorectal cancer, liver cancer, lung cancer, prostate cancer and stomach cancer. It has been reported that the expression of COX2 is increased in various diseases such as neuroinflammatory diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury, and ischemia. In particular, according to the research results of the present inventors, it was confirmed that in the case of neurodegenerative diseases including Alzheimer's disease, the brain expression of the COX2 protein was rapidly increased from a very early stage before observable symptoms of the diseases were expressed (KR10-2019-0068246). In addition, according to the results of previous studies by the present inventors, it was confirmed that the acetylation of COX2 protein, more specifically, the acetylation at S565 residue of human COX2 protein, was significantly reduced in biological samples from patients with neurodegenerative diseases including Alzheimer's disease (KR10-2018-0127656). Accordingly, if an antibody capable of specifically detecting the acetylation of S565 residue of the COX2 protein is developed in a biological sample, the antibody may very easily diagnose diseases such as neurodegenerative diseases and inflammatory diseases, in which the acetylation of S565 residue of the COX2 protein is reduced, and may be very effectively used in various research fields, but an antibody capable of specifically detecting the acetylation of the COX2 protein has not yet been developed. DISCLOSURE Technical Problem Therefore, the present inventors have repeated many studies to develop an antibody capable of specifically detecting the acetylation of S565 residue of COX2 protein which was reduced in neurodegenerative diseases, inflammatory diseases, and the like, and as a result, developed an antibody that recognized a specific peptide containing acetylated S565 residue of COX2 protein as an epitope, found that