WO2020179953A1 - Marqueur composite radioactif pour le diagnostic d'un mélanome malin et utilisation de ce dernier - Google Patents

Marqueur composite radioactif pour le diagnostic d'un mélanome malin et utilisation de ce dernier Download PDF

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WO2020179953A1
WO2020179953A1 PCT/KR2019/002649 KR2019002649W WO2020179953A1 WO 2020179953 A1 WO2020179953 A1 WO 2020179953A1 KR 2019002649 W KR2019002649 W KR 2019002649W WO 2020179953 A1 WO2020179953 A1 WO 2020179953A1
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protein
diabetic retinopathy
diagnosis
analysis
marker
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Korean (ko)
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박성준
이철주
우세준
주신영
이선정
정현교
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(주)레티마크
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a composite marker for diabetic retinopathy diagnosis and a use thereof, and more particularly, to a composite for diabetic retinopathy diagnosis with increased diagnostic performance consisting of two or more blood markers specific for diabetic retinopathy diagnosis. It's about the marker.
  • it relates to a composition for diagnosing diabetic retinopathy using the composite marker, a diagnostic kit, and a method of providing information necessary for diabetic retinopathy diagnosis.
  • Diabetic retinopathy is a typical complication of diabetes that occurs when microvessels of the retina are damaged, and is one of the three major blind diseases in ophthalmology along with age-related macular degeneration and glaucoma.
  • DR Diabetic retinopathy
  • the number of diabetic patients increased by 21% from about 2 million in 2012 to about 2.45 million in 2016, while the number of diabetic retinopathy was about 260,000 in 2012, which was 38% to 336,000 in 2016. Increased to, and the increase was greater than that of diabetes.
  • Diabetic retinopathy occurs because abnormally new blood vessels are formed in this area, causing bleeding without normal blood vessel function.
  • Diabetic retinopathy is classified into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) according to its severity. In addition, NPDR is classified into mild, moderative and severe NPDR depending on the degree. Nonproliferative diabetic retinopathy shows retinal bleeding, microvascular perfusion, and cotton patches, but maintains good vision until late. In proliferative diabetic retinopathy, new blood vessels are formed in the retina, and if these blood vessels rupture, it causes severe bleeding in the vitreous cavity. It is absorbed over time, but becomes a fibrous tissue, which in turn causes tractional retinal detachment and rebleeding. Leads to blindness.
  • NPDR non-proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • Diabetic retinopathy is difficult to diagnose early because there are few initial symptoms, and when symptoms (vision loss, loss of focus, glare) are in progress, the disease has already progressed, and despite treatment (laser, vitreous surgery), it leads to blindness. There are many patients. However, the need for early detection and suppression of diabetic retinopathy and early treatment for high-risk groups is emerging, since it is possible to maintain visual acuity if appropriate treatment is received at the beginning of the onset and blood sugar management is thorough. However, the exact etiology has not yet been identified, and biomarkers that determine the progression of retinopathy have very limited problems.
  • the present inventors discovered blood protein markers with high sensitivity and specificity, and tried to improve the early diagnosis ability of diabetic retinopathy by using them in combination.
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • An object of the present invention is to provide a complex marker for diabetic retinopathy diagnosis composed of blood markers specific for diabetic retinopathy diagnosis.
  • Another object of the present invention is to provide a composition or a diagnostic kit for diabetic retinopathy using the complex marker for diabetic retinopathy diagnosis.
  • Another object of the present invention is to provide a method of providing information necessary for diabetic retinopathy diagnosis using the complex marker for diabetic retinopathy diagnosis.
  • the present invention provides a complex marker for diabetic retinopathy diagnosis composed of mannose-binding protein C (MBL2), pancreatic triacylglycerol lipase (PNLIP), and galectin-3-binding protein (LGALS3BP).
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • the complex marker is SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding). protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin), and FCN2 (Ficolin 2).It may further include at least one marker selected from the group consisting of.
  • the present invention comprises an agent for measuring the mRNA or protein level of a complex marker for diabetic retinopathy diagnosis composed of MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase) and LGALS3BP (galectin-3-binding protein). It provides a composition for diabetic retinopathy diagnosis.
  • the complex marker is SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding). protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin), and FCN2 (Ficolin 2).It may further include at least one marker selected from the group consisting of.
  • the agent for measuring the mRNA level of the complex marker may be a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene of the marker.
  • the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment. (aptamer) may be included.
  • the present invention provides a diagnostic kit for diabetic retinopathy comprising the composition for diabetic retinopathy diagnosis.
  • the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an ELISA (Enzymelinked immunosorbent assay) kit, a protein chip kit, a rapid kit, or a MRM (Multiple reaction monitoring) kit.
  • RT-PCR reverse transcription polymerase chain reaction
  • DNA chip kit a DNA chip kit
  • ELISA Enzymelinked immunosorbent assay
  • protein chip kit a protein chip kit
  • a rapid kit or a MRM (Multiple reaction monitoring) kit.
  • MRM Multiple reaction monitoring
  • the present invention is the mRNA or protein of a complex marker for diabetic retinopathy diagnosis consisting of (a) manose-binding protein C (MBL2), pancreatic triacylglycerol lipase (PNLIP), and galectin-3-binding protein (LGALS3BP) from a biological sample of a patient. Measuring the level; And
  • It provides a method for providing information for diabetic retinopathy diagnosis comprising; (b) comparing the mRNA or protein expression level with a control sample.
  • the complex marker is SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding). protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin), and FCN2 (Ficolin 2).It may further include at least one marker selected from the group consisting of.
  • the method of providing information for diabetic retinopathy is the patient's age, body mass index (BMI), Hb1Ac test result, insulin treatment, hypertension, hyperlipidemia, and cardiovascular disease.
  • BMI body mass index
  • Hb1Ac test result insulin treatment
  • hypertension hypertension
  • hyperlipidemia hyperlipidemia
  • cardiovascular disease At least one clinical information selected from the group consisting of can be additionally included and compared with the control group.
  • the mRNA expression level is measured using reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. You can do it.
  • the measurement of the protein expression level is performed by using an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to a protein or peptide fragment. Can be done.
  • the protein expression level measurement is protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced).
  • Laser Desorption/Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioactive immunity diffusion method, octeroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis method, 2D electrophoresis analysis, liquid chromatography- Liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/ Mass Spectrometry (LCMS/MS), Western blot, enzyme linked immunosorbent assay (ELISA), multiple reaction monitoring (MRM), Parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM), or immune multiple reaction monitoring (iMRM) It can be done using.
  • LC-MS liquid chromatography- Liquid chromatography-Mass Spectrometry
  • LCMS/MS liquid chromatography-Mass Spectrometry/ Mass Spectrometry
  • Western blot enzyme linked immunosorbent assay
  • the method of providing information for diabetic retinopathy further comprises determining that the gene expression level or the protein expression level of the complex marker is increased compared to the control group, determining that it is diabetic retinopathy. can do.
  • the analysis in step (b) may be performed by a statistical analysis method.
  • the statistical analysis method may include a linear or nonlinear regression analysis method, a linear or nonlinear classification analysis method, a logistic regression method, and an Analysis of Variance; ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm using decision tree or kernel principal component analysis method, Markov Blanket analysis method , Recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis method, and a combination thereof.
  • ANOVA Analysis of Variance
  • the complex marker for diabetic retinopathy diagnosis of the present invention has superior sensitivity and diagnostic performance compared to the combination of other markers, and when the protein quantitative value of the complex marker and basic clinical information are combined and analyzed, it is highly diagnosed in early diabetic retinopathy. It was confirmed that it showed the ability.
  • the composite marker of the present invention can be conveniently used for early diagnosis of diabetic retinopathy because it can be conveniently analyzed using patient plasma without using a biopsy as a blood protein.
  • FIG. 1 is data analyzed by statistically processing a logistic regression model by combining quantitative protein values of complex markers and basic clinical information of patients (DMR: diabetic retinopathy, Non DMR: non-diabetic retinopathy (diabetic patients), mild NPDR) : Early non-proliferative diabetic retinopathy, moderate NPDR: Mid-stage non-proliferative diabetic retinopathy, severe NPDR: End stage non-proliferative diabetic retinopathy, PDR: Proliferative diabetic retinopathy).
  • DMR diabetic retinopathy
  • Non DMR non-diabetic retinopathy (diabetic patients)
  • mild NPDR Early non-proliferative diabetic retinopathy
  • moderate NPDR Mid-stage non-proliferative diabetic retinopathy
  • severe NPDR End stage non-proliferative diabetic retinopathy
  • PDR Proliferative diabetic retinopathy
  • Figure 2 is a logistic regression model by combining the protein quantification values and basic clinical information of the patient according to the combination of MBL2, PNLIP and LGALS3BP (1) and ADAMTSL2, SELE and SIGLEC14 combination (2) among the six markers identified in the present invention. This is the data processed and analyzed.
  • the present inventors discovered a blood protein marker with high sensitivity and specificity, and tried to improve the early diagnosis ability of diabetic retinopathy by using them in combination.
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • the present invention relates to a complex marker for diabetic retinopathy diagnosis consisting of mannose-binding protein C (MBL2), pancreatic triacylglycerol lipase (PNLIP), and galectin-3-binding protein (LGALS3BP).
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • diagnosis means identifying the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether diabetic retinopathy has occurred.
  • diagnostic marker refers to a polypeptide or nucleic acid showing a significant increase or decrease in the gene expression level or the protein expression level in an individual with diabetic retinopathy compared to a normal control (individual non-diabetic retinopathy) ( Examples: mRNA, etc.), lipids, glycolipids, glycoproteins, and organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.).
  • Diabetic retinopathy is classified into early nonproliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR) according to the degree of progression.
  • Nonproliferative diabetic retinopathy is characterized by no blood vessel development, and proliferative diabetic retinopathy differs in its mechanism, such as blood vessel development, and nonproliferative diabetic retinopathy does not necessarily progress to proliferative diabetic retinopathy. Then, even a marker known as a diagnostic marker for proliferative diabetic retinopathy cannot necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.
  • the composite marker of the present invention can specifically diagnose both non-proliferative diabetic retinopathy and proliferative diabetic retinopathy, and in particular, can diagnose an early stage of non-proliferative diabetic retinopathy.
  • the complex markers are SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin) and FCN2 (Ficolin 2) It may be characterized in that it further comprises at least one marker selected from the group consisting of.
  • Mannose-binding protein C is also referred to as mannose-binding lectin (MBL) or mannan-binding protein (MBP).
  • MBL2 has an oligomer structure (400-700 kDa) and is composed of subunits containing three identical peptide chains consisting of approximately 30 kDa. It is produced by the liver in response to infection and is part of a number of other factors called acute stage proteins.
  • PNLIP pancreatic triacylglycerol lipase
  • PNLIP pancreatic triacylglycerol lipase
  • PNLIP is known to have a low serum concentration because it is secreted into the duodenum through the pancreatic duct system.However, when pancreatic functions such as pancreatitis or pancreatic adenocarcinoma are extremely destroyed, pancreatic enzymes including PNLIP are secreted into the serum. It is known that acute pancreatitis can be diagnosed by measuring.
  • LGALS3BP (galectin-3-binding protein) is a protein encoded by the LGALS3BP gene, and specifically binds to Mac-2 (human macrophage-associated lectin) and galactin 1 (galectin 1). LGALS3BP is known to increase in serum of cancer patients and HIV-infected patients, and is involved in immune responses associated with natural killer (NK) cells and lymphokine-activated killer (LAK) cytotoxicity.
  • NK natural killer
  • LAK lymphokine-activated killer
  • SELE E-selectin
  • CD62E CD62 antigen-like family member E
  • LECAM2 leukocyte-endothelial cell adhesion molecule 2
  • interleukin 1 ⁇ tumor necrosis factor
  • lipopolysaccharide a cell adhesion molecule that is transiently expressed and induced in the vascular endothelial cells activated by 4-12 hours as the peak.
  • SELE is strongly expressed in vascular endothelial cells in inflammatory tissues and promotes invasion of these cells into the inflammatory site by mediating the phenomenon that neutrophils and monocytes roll over vascular endothelial cells. It is known to be involved in adhesion of cancer cells to vascular endothelial cells.
  • SIGLEC14 (Sialic acid-binding Ig-like lectin 14) is one of the subfamily of SIGLEC (Sialic acid-binding immunoglobulin-type lectins), and SIGLEC is a cell surface protein that binds to sialic acid and is mainly expressed on the surface of immune cells.
  • the protein interaction between SIGLEC and sialic acid acts as a switch to turn on and off the immune system, and cancer cells are also known to acquire resistance to the immune response by using the SIGLEC-sialic acid reaction.
  • ADAMTSL2 (ADAMTS-like protein 2) is a subfamily member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-like protein, and is a glycoprotein that binds to the cell surface and extracellular matrix.
  • ADAMTSL2 interacts with LTBP1 (latent transforming growth factor beta binding protein 1), and LTBP1 protein is involved in the storage of TGF- ⁇ 1, an important growth factor that regulates cell growth and division, so ADAMTSL2 has the possibility of using TGF- ⁇ 1. Adjust.
  • ADAMTSL2 is expressed in cancer tissues, it is used as a marker for cancer diagnosis.
  • IGFBP2 insulin like growth factor binding protein 2
  • IGFBP2 insulin like growth factor binding protein 2
  • DDI2 Protein DDI1 homolog2 is an internal peptide cleavage enzyme that activates nuclear respiratory factor 1 (Nrf1), which is involved in regulating cell growth and DNA replication, and compensates for protein degradation caused by proteasome abnormalities.
  • Nrf1 nuclear respiratory factor 1
  • ZG16B Zymogen granule protein 16 homolog B
  • PAUF pancreatic adenocarcinoma upregulated factor
  • Cp is the main protein that carries copper in the blood and plays an important role in iron metabolism. More than 95% of copper is present in the plasma of healthy people in the form of ceruloplasmin.
  • FCN2 (Ficolin 2) is a type of oligolectin and is composed of a short N-terminal partial-collagen-like domain and a fibrinogen-like domain. It is mainly expressed in the liver and is known to play an important role in the lectin pathway of the complement system by binding to N-acetylglucosamin in the bacterial cell wall and acting as an opsonin like a mannose binding protein.
  • the markers can be used for diabetic retinopathy, and in particular, it has been disclosed that the diagnostic performance of diabetic retinopathy can be increased using a composite marker of MBL2, PNLIP and LGALS3BP. None is known about the technology.
  • the present invention includes a preparation for measuring the mRNA or protein level of a complex marker for diabetic retinopathy diagnosis composed of MBL2 (mannose-binding protein C), PNLIP (pancreatic triacylglycerol lipase) and LGALS3BP (galectin-3-binding protein). It relates to a composition for diabetic retinopathy diagnosis.
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • the complex markers are SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin) and FCN2 (Ficolin 2) It may be characterized in that it further comprises at least one marker selected from the group consisting of.
  • the term "measurement of mRNA expression level" as used in the present invention is a process of confirming the presence and expression of mRNA of diabetic retinopathy diagnosis genes in a biological sample isolated from a patient suspected of diabetic retinopathy to diagnose diabetic retinopathy. Measure the amount. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), and RNase protection assay (RPA; RNaseprotection assay). ), Northern blotting, DNA chip, etc., but are not limited thereto.
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Real-time RT-PCR real-time reverse transcription polymerase reaction
  • RNase protection assay RNase protection assay
  • Northern blotting DNA chip, etc., but are not limited thereto.
  • the agent for measuring the mRNA level of the complex marker is characterized in that it is a primer pair, probe or antisense nucleotide that specifically binds to the gene of the marker, and the nucleic acid information of the genes is known in GeneBank, etc. These primer pairs, probes or antisense nucleotides can be designed.
  • primer as used in the present invention is a fragment that recognizes a target gene sequence, and includes forward and reverse primer pairs, preferably, a primer pair that provides an analysis result having specificity and sensitivity.
  • a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can give high specificity. .
  • probe used in the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. do.
  • the type of probe is a material commonly used in the art and is not limited, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferred Hagi is PNA.
  • the probe is a biomaterial that includes an organism-derived or similar thing or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
  • antisense refers to a nucleotide base in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA: oligomeric heterodimer within the target sequence. It means an oligomer having a sequence of and a backbone between subunits. Oligomers may have exact sequence complementarity or approximate complementarity to the target sequence.
  • the agent for measuring the protein level of the complex marker is an antibody, interacting protein, ligand, nanoparticles, or aptamer that specifically binds to the protein or peptide fragment. can do.
  • protein expression level measurement is a process of checking the presence and expression of a protein expressed from a diabetic retinopathy diagnosis gene in a biological sample in order to diagnose diabetic retinopathy.
  • the amount of protein can be determined using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to the protein or peptide fragment of the gene.
  • the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time).
  • MALDI-TOF Microx Desorption/Ionization Time of Flight Mass Spectrometry
  • SELDI-TOF Surface Enhanced Laser Desorption/Ionization Time
  • Flight Mass Spectrometry analysis, radioimmunoassay, radioactive immunodiffusion method, octeroni immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation method, two-dimensional electrophoresis analysis, liquid chromatography-liquid chromatography -Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay), multiple reaction monitoring (MRM), parallel reaction Monitoring (parallel reaction monitoring: PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM), or immune multiple reaction monitoring (iMRM). It is not limited to this.
  • MRM multiple reaction monitoring
  • PRM parallel reaction Monitoring
  • PRM sequential windowed data independent acquisition of the total high-resolution
  • SWATH total high-resolution
  • SRM selected reaction monitoring
  • iMRM immune multiple reaction monitoring
  • an antibody refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
  • an antibody refers to an antibody that specifically binds to the complex biomarker for diabetic retinopathy of the present invention.
  • the antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • the antibody can be easily prepared using techniques well known in the art.
  • the polyclonal antibody can be produced by a method well known in the art, including the process of injecting the diabetic retinopathy marker protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody.
  • polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog.
  • the monoclonal antibody is a hybridoma method well known in the art (hybridoma method; see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol. Biol. , 222:58, 1-597, 1991).
  • the antibody prepared by the above method may be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules.
  • the functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.
  • the antibody of the present invention may be obtained commercially.
  • PNA Protein Nucleic Acid
  • DNA has a phosphate-ribose sugar backbone
  • PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by a peptide bond, which greatly increases the binding power and stability to DNA or RNA, resulting in molecular biology. , Diagnostic analysis and antisense therapy.
  • PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254(5037): 1497-500.
  • aptamer is an oligonucleotide or peptide molecule, and general information of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727(1998)].
  • the present invention relates to a diabetic retinopathy diagnosis kit comprising the diabetic retinopathy diagnosis composition.
  • the kit can be prepared by a conventional manufacturing method known in the art.
  • the kit may include, for example, an antibody in a freeze-dried form, a buffer, a stabilizer, an inactive protein, and the like.
  • the kit may further include a detectable label.
  • detectable label refers to an atom or molecule that specifically detects a molecule containing a label among molecules of the same type without a label.
  • the detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or fragment thereof.
  • the detectable label may include a radionuclide, a fluorophore, or an enzyme.
  • the kit can be used according to various immunoassays or immunostaining methods known in the art.
  • the immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining, and immunoaffinity purification.
  • the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
  • RT-PCR reverse transcription polymerase chain reaction
  • ELISA enzyme linked immunosorbent assay
  • MRM multiple reaction monitoring
  • the kit can be used for mass spectrometry.
  • the specific amino acid residues of the protein are myristoylation, isoprenylation, prenylation, glypiation, lipoylation, acylation, alkylation, methylation, demethylation, amidation, It may have modifications such as ubiquitination, phosphorylation, deamidation, glycosylation, oxidation, or acetylation.
  • the present invention provides a composite marker for diabetic retinopathy diagnosis consisting of (a) manose-binding protein C (MBL2), pancreatic triacylglycerol lipase (PNLIP), and galectin-3-binding protein (LGALS3BP) from a biological sample of a patient. measuring the mRNA or protein level; And
  • It relates to a method for providing information for diabetic retinopathy diagnosis comprising; (b) comparing the mRNA or protein expression level with a control sample.
  • the complex marker in step (a) is SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), ADAMTSL2 (ADAMTS-like protein 2), IGFBP2 (insulin like growth factor binding). Protein 2), DDI2 (Protein DDI1 homolog2), ZG16B (Zymogen granule protein 16 homolog B), Cp (Ceruloplasmin), and FCN2 (Ficolin 2).It is characterized in that it further comprises at least one marker selected from the group consisting of.
  • biological sample refers to a sample such as tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid or urine, in which the protein expression level or gene expression level differs due to the onset of diabetic retinopathy. And, preferably, it means blood, plasma, and serum.
  • the method of providing information for diabetic retinopathy includes at least one clinical information selected from the group consisting of the patient's age, body mass index (BMI), Hb1Ac test result, insulin treatment, hypertension, hyperlipidemia, and cardiovascular disease. It may contain additionally.
  • the method of providing information for diabetic retinopathy may further include determining that it is diabetic retinopathy when the gene expression level or protein expression level of the complex marker is increased compared to the control group.
  • the measurement of the mRNA expression level in step (a) is the measurement or comparison of the mRNA expression level: reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. Can be used, but the present invention is not limited thereto.
  • the mRNA expression level in the normal control group and the mRNA expression level in diabetic retinopathy patients can be checked, and the onset of diabetic retinopathy can be diagnosed or predicted by comparing the levels of these expression levels.
  • the measurement of the protein expression level in step (a) may be measured and compared using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to proteins or peptide fragments.
  • the protein expression level measurement or comparison analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced Laser Desorption/ Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioactive immunodiffusion method, octeroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation method, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry ( Liquid chromatography-Mass Spectrometry, LC-MS), LCMS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbentassay), but are not limited thereto.
  • MALDI-TOF Microx Desorption/Ionization Time of Flight Mass Spectrometry
  • MRM multiple reaction monitoring
  • PRM parallel reaction monitoring
  • SWATH sequential windowed data independent acquisition of the total high-resolution
  • SRM selected reaction monitoring
  • iMRM immune multiple reaction monitoring
  • MRM is a method of determining an exact fragment of a substance, breaking it in a mass spectrometer, selecting a specific ion from the broken ions once more, and obtaining the number using a detector connected in series.
  • the protein or fragment thereof can be quantified using a mass spectrometer in blood samples of normal individuals and individuals suspected of diabetic retinopathy.
  • the analysis in step (b) can be analyzed using a statistical method or an algorithm to improve the accuracy of the diagnosis, and a linear or nonlinear regression analysis method, an preceding or nonlinear classification analysis method, and a logistic regression analysis method (logistic regression), Analysis of Variance (ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm or kernel principal component analysis using decision trees Method, Markov Blanket analysis method, recursive feature elimination or entropy-basic regression feature elimination analysis method, forward floating search or rear floating search analysis method, and their An analysis method selected from the group consisting of combinations can be used.
  • the statistical method uses a logistic regression analysis method, but is not limited thereto.
  • 81 plasma samples were analyzed for tactical verification of biomarkers for diabetic retinopathy among plasma proteins (Table 1), mannose-binding protein C (MBL2), and pancreatic triacylglycerol (PNLIP).
  • lipase LGALS3BP (galectin-3-binding protein), SELE (E-selectin), SIGLEC14 (Sialic acid-binding Ig-like lectin 14), and ADAMTSL2 (ADAMTS-like protein 2).
  • MRM multiple reaction monitoring
  • the biomarker expression level conversion information and the clinical information conversion level are input using a logistic regression model to confirm the diagnostic ability improvement effect.
  • the probability of classification as diabetic retinopathy was estimated.
  • Table 3 as a result of adding and analyzing the expression levels of SELE, SIGLEC14 and ADAMTSL2 one by one based on clinical information + MBL2 + PNLIP + LGALS3BP, the diagnostic ability of diabetic retinopathy increased as the number of biomarkers increased. Confirmed that.
  • stage 1 normal
  • stage 2 normal and mild NPDR
  • Example 1 Diabetic retinopathy patient selection and plasma collection
  • Plasma samples from diabetic retinopathy patients were collected with the approval of the Institutional Review Board of Seoul National University Bundang Hospital. A total of 81 plasma samples were analyzed for quantitative detection of biomarkers using plasma proteins, and the clinical characteristics of the analyzed normal group (Non DMR) and diabetic retinopathy (DMR) disease group are shown in Table 1 below.
  • MBL2 mannose-binding protein C
  • PNLIP pancreatic triacylglycerol lipase
  • LGALS3BP galectin-3-binding protein
  • SELE E-selectin
  • SIGLEC14 Sialic acid
  • IGFBP2 insulin like growth factor binding protein 2
  • DDI2 Protein DDI1 homolog2
  • ZG16B Zymogen granule protein 16 homolog B
  • Cp Ceruloplasmin
  • FCN2 Fecolin 2
  • a representative peptide having a specific charge-to-mass ratio (m/z) for the six biomarker proteins is selected (Q1), and the peptide is broken by electric shock.
  • the ion (Q3) with the highest strength was selected.
  • At least one peptide with high sensitivity per protein was measured and injected into a mass spectrometer based on this to obtain an optimum value of fragmentation energy per transition, and three or more upper fragmented ions were selected based on the intensity (Table 2).
  • Top fragmentation ions for 11 biomarkers Gene name Trypsin fragment Target ion Target m/z ADAMTSL2 NFNIAGTVVK (SEQ ID NO: 1) 2y8 531.8007/801.4829 LGALS3BP SDLAVPSELALLK (SEQ ID NO: 2) 2y8 678.3927/870.5295 MBL2 WLTFSLGK (SEQ ID NO: 3) 2y6 476.2686/652.3665 SELE NWAPGEPNNR (SEQ ID NO: 4) 2y8 577.7705/854.4115 SIGLEC14 EGGEFTCR (SEQ ID NO: 5) 2y3 478.2005/436.1973 PNLIP TGYTQASQNIR (SEQ ID NO: 6) 2y5 619.8098/617.3365 IGFBP2 LIQGAPTIR (SEQ ID NO: 7) 2y7 484.798/742.4206 DDI2 DGDVVILR (SEQ ID NO: 8) 2y4 443.7533/
  • SIS Stable-isotope labeled standard
  • SIS peptide is a peptide obtained by substituting 13C and 15N for 12C and 14N in the amino acids of lysine (Lys, K) or arginine (Arg, R) at the C-terminus of the peptide. This has a difference in mass value from the endogenous peptide present in the blood, but since it has the same sequence, the peptide hydrophobicity is the same, so it elutes at the same retention time (RT) on the chromatogram.
  • RT retention time
  • 2-carboxylethyltrisphosphine Tris(2-carboxyethyl)-phosphine, TECP
  • 2-chloro 2-chloroacetamide
  • Acetamide (2-chloroacetamide) was added and reacted at 25° C. for 1 hour to reduce and alkylate disulfide bonds.
  • Rice seed enzyme was added so that the mass ratio of plasma protein to rice seed (wako) enzyme was 100:1, and reacted at room temperature for 4 hours.
  • Example 2-1 a heavy-labeled peptide was added (spiking) as an internal standard material (SIS peptide) determined in Example 2-1 to perform MRM analysis.
  • SIS peptide an internal standard material
  • Nano ultra 2D plus (Eksigent), a triple quadrupole mass spectrometer, QTarp 5500 (SCIEX) was used to monitor the transition of each selected protein in a scheduled MRM mode.
  • the peak area of the transition was calculated by processing raw data using Skyline (Mccoss lab, University of Washington, USA). Relative concentrations were compared for the endogenous/heavy labeled peptide using the peak area. Using the measured results, T-test and Area under the receiver operating characteristic (AUROC) values were generated to measure the predictive ability of each protein peptide. In order to confirm the predictive power combined with clinical information, a logistic regression model was used to input the expression level conversion information and the degree of conversion of clinical records to estimate a probability value classified as diabetic retinopathy. All statistical analyzes were performed using MedCal ver 17.1 (MedCalc).
  • the conversion information of the biomarker expression level and the degree of conversion of the clinical information are input using a logistic regression model in order to confirm the effect of improving diagnostic ability.
  • the probability values to be classified were estimated.
  • Diabetic retinopathy diagnosis performance according to the number of combined biomarkers Basic (STAGE1) C1 C2 C3 C4 AUC Clinical information LGALS3BP MBL2 PNLIP 0.817 Clinical information LGALS3BP MBL2 PNLIP ADAMTSL2 0.821 Clinical information LGALS3BP MBL2 PNLIP ADAMTSL2 SELE 0.848 Clinical information LGALS3BP MBL2 PNLIP ADAMTSL2 SELE SIGLEC14 0.869
  • a clinical information
  • b quantitative protein information
  • c integrated clinical information and protein quantitative information
  • d Delong et al. , 1988
  • stage 1 normal group and diabetic retinopathy
  • stage 2 normal and early nonproliferative diabetic retinopathy
  • the complex marker for diagnosing diabetic retinopathy not only has superior sensitivity and diagnostic performance compared to combinations of other markers, but also has a high diagnosis in early diabetic retinopathy by combining the quantitative value of protein and basic clinical information of the complex marker. As it shows the ability, it can be usefully used for early diagnosis of diabetic retinopathy.

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Abstract

La présente invention concerne un marqueur composite pour le diagnostic de la rétinopathie diabétique et son utilisation et, plus particulièrement, un marqueur composite pour le diagnostic de la rétinopathie diabétique, le marqueur composite comprenant deux marqueurs sanguins ou plus spécifiques pour le diagnostic de la rétinopathie diabétique et ayant ainsi une performance de diagnostic améliorée. De plus, la présente invention concerne une composition pour le diagnostic de la rétinopathie diabétique, une kit de diagnostic et un procédé pour fournir des informations nécessaires dans le but de diagnostiquer une rétinopathie diabétique, chacun utilisant le marqueur composite. Le marqueur composite pour le diagnostic de la rétinopathie diabétique de la présente invention a s'est avéré être supérieur à des combinaisons d'autres marqueurs en termes de sensibilité et de performance de diagnostic et présenter un potentiel de diagnostic élevé pour la rétinopathie diabétique initiale, suivant les analyses pour des combinaisons entre des valeurs de quantification de protéines du marqueur composite ainsi que des informations cliniques fondamentales. En outre, le biomarqueur composite de la présente invention n'utilise pas de protéines sanguines pour une biopsie mais peut avantageusement effectuer une analyse avec du plasma d'un patient et, en tant que tel, peut être avantageusement appliqué au diagnostic précoce de la rétinopathie diabétique.
PCT/KR2019/002649 2019-03-07 2019-03-07 Marqueur composite radioactif pour le diagnostic d'un mélanome malin et utilisation de ce dernier WO2020179953A1 (fr)

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