WO2011034128A1 - コラーゲンネオエピトープ抗体 - Google Patents
コラーゲンネオエピトープ抗体 Download PDFInfo
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- WO2011034128A1 WO2011034128A1 PCT/JP2010/066029 JP2010066029W WO2011034128A1 WO 2011034128 A1 WO2011034128 A1 WO 2011034128A1 JP 2010066029 W JP2010066029 W JP 2010066029W WO 2011034128 A1 WO2011034128 A1 WO 2011034128A1
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- monoclonal antibody
- seq
- collagen
- neoepitope
- amino acid
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Images
Classifications
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- 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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Definitions
- the present invention relates to a monoclonal antibody capable of recognizing a neo-epitope of collagen, an immunoassay based thereon, a measurement method based thereon, a screening method based thereon, a patient selection method based thereon, and a kit based thereon.
- Cartilage degradation is a major feature of joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA).
- OA osteoarthritis
- RA rheumatoid arthritis
- Monitoring the progression of cartilage loss in such diseases provides useful information for disease management (including prognosis, diagnosis, and treatment).
- cartilage loss is monitored by detecting that the joint space is narrowed by X-rays.
- X-ray diagnosis is not satisfactory in terms of detection sensitivity and accuracy.
- the X-ray diagnosis merely indicates that cartilage loss has occurred in the past, and does not indicate the current state of cartilage degradation.
- alternative methods for monitoring the progression of cartilage breakdown in various joint diseases are very useful.
- MMPs matrix metalloproteinases
- SF synovial fluid
- proline is a major component that accounts for about 30% of collagen protein, and since proline hydroxide is also present in the vicinity of the collagenase cleavage site, it is known to greatly affect the binding affinity.
- an antibody for measuring type II collagen fragment is preferably one whose binding affinity is not affected by the presence or absence of proline hydroxylation.
- Non-patent Document 2 the detection sensitivity with respect to the type II collagen fragment which is mostly hydroxylated decreases.
- the problem to be solved by the present invention is to accurately measure the amount of collagen fragment (collagen neoepitope) resulting from collagenase digestion in a biological sample.
- the present inventors have developed the present invention by producing a novel monoclonal antibody whose binding ability does not change even when the proline of the neoepitope changes to a hydroxy form. completed.
- the present invention (1) An antibody that specifically binds to a collagen neoepitope fragment and binds to positions 962 to 975 of the amino acid sequence represented by SEQ ID NO: 20, wherein proline contained in the epitope is non-hydroxylated A monoclonal antibody whose binding affinity is substantially the same as that in the case of a hydroxylated form, (2) The monoclonal antibody according to (1), wherein the proline according to (1) is proline at position 971 in the amino acid sequence represented by SEQ ID NO: 20, (3) The monoclonal antibody according to (1), wherein the epitope is present in the region from the 957th position to the 975th position of the amino acid sequence represented by SEQ ID NO: 20, (4) The antibody according to (2), which is represented by SEQ ID NO: 14, 17 or 18 so that the immune reaction of the antibody to the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is inhibited An immunoassay for competing with a peptide comprising an amino acid sequence, wherein pro
- the monoclonal antibody of the present invention can specifically recognize the neoepitope terminal structure and is not affected by proline hydroxylation modification, so that the amount of collagen neoepitope fragment in a living body can be accurately detected and quantified.
- a collagenase cleavage site is shown at a position 2/3 from the amino terminus in the schematic diagram of fibrous collagen (type I, type II and type III) having a trimeric helix structure.
- the lower part shows collagenase cleavage sites of type II collagen of various animals and the amino acid sequences before and after. From the top, the order is human, cow, dog, rat, and mouse. It corresponds to 954-980 part of human type II collagen, and the 975-976 amino acid residues that are cleavage sites are indicated by asterisks.
- the result of the competitive immunoassay by the peptide fragment which has various C terminal structure of 20A10 is shown.
- the upper panel shows the results of a competitive immunoassay with collagenase digested and undigested 20A10.
- the lower table shows the change in specificity due to collagenase digestion and the cross-reactivity between type I, type II and type III collagen fragments.
- the result of the sandwich immunoassay by the combination with a type II collagen specific monoclonal antibody is shown.
- the measured values of collagen neoepitope fragment concentration when various concentrations of MMP inhibitor were added in bovine cartilage explant culture in the presence of 1 ng / ml human interleukin 1 are shown.
- the amino acid sequence of the variable region of 20A10 is shown.
- the upper row is the variable region for the heavy chain and the lower row is the variable region for the light chain.
- Each underlined sequence indicates the position of the
- I antibody type I, type II and type III collagen which are fibrous collagens, are composed of three peptide chains twisted in a spiral, and collagenase (for example, MMP-1, MMP-8, and MMP-13) ) Cleaves these fibrous collagen triple chains in a native state at a 3: 1 position (between 975-976 amino acid residues) from the N-terminus.
- the terminal structure in which the C-terminal of the N-terminal three-fourth fragment and the N-terminal of the C-terminal one-fourth fragment are newly called “neo-epitope” (FIG. 1).
- a collagenase fragment generated by cleavage is referred to as a “collagen neoepitope fragment”.
- C-terminal neoepitope 7 amino acid residues of the C-terminal neoepitope portion (corresponding to positions 969-975; hereinafter also referred to as C-terminal neoepitope) of human type II collagen (Accession No. NP_001835) Gly-Pro-Pro-Gly- Pro-Gln-Gly (SEQ ID NO: 1) is known to be conserved among animal species from humans to mice.
- Collagen is one of proteins characterized by a high hydroxyproline content, and the fifth proline residue from the C-terminal side (or 971st from the N-terminal side) of this sequence is often hydroxylated.
- the monoclonal antibody of the present invention is characterized by that its binding affinity does not change even when proline contained in a type II collagen neoepitope fragment (SEQ ID NO: 20) having a neoepitope on the C-terminal side is changed to a hydroxy form. It is done.
- “the binding affinity does not change” means that the binding affinity in the case of proline (non-hydroxylated) of the amino acid residue constituting the neoepitope and in the case of hydroxyproline (hydroxylated) is substantially reduced. It also means that they are the same.
- Binding affinity generally refers to the strength or affinity of the type of non-covalent interaction between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific, often the dissociation constant ( Kd).
- substantially the same means that the binding affinity of the hydroxylated form (hydroxyproline) is within the range of 80% to 120% of the binding affinity value of the non-hydroxylated form, It means that it is in the range of 90% to 110%, more preferably in the range of 95% to 105%. Further, it means that the cross-reactivity between a non-hydroxylated form (proline) and a hydroxylated form is 80% or more, preferably 90% or more, and more preferably 95% or more.
- the binding affinity of an antibody can be determined by known methods, for example, by Scatchard analysis using an ELISA assay (eg, Campbell, 1991; Segel, 1976).
- a typical example of such a monoclonal antibody is 20A10.
- the amino acid sequence of the variable region of 20A10 is shown in FIG.
- the upper row shows the heavy chain (SEQ ID NO: 3) and the lower row shows the light chain (SEQ ID NO: 4).
- the underlined portion indicates a complementarity determining region (CDR). (SEQ ID NOs: 5 to 10)
- the immunogen used for the production of the monoclonal antibody of the present invention can be produced by a method described in, for example, Antibodies: A Laboratory Manual (1989, Cold Spring Harbor Laboratory Press).
- the immunization method can be performed by a general method, for example, by administering an immunogen to a mammal by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. More specifically, for example, the immunogen is diluted to an appropriate concentration with a physiological saline-containing phosphate buffer (PBS), physiological saline, or the like, and is used in combination with a normal adjuvant as required. Give several doses at weekly intervals. When mice are used, a single dose is about 50 to 100 ⁇ g per mouse.
- the adjuvant refers to a substance that nonspecifically enhances the immune response to the antigen when administered together with the antigen. Examples of commonly used adjuvants include pertussis vaccine and Freund's adjuvant. Antisera can be obtained by collecting blood from mammals 3 to 10 days after the final immunization.
- a monoclonal antibody is produced by preparing a fused cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with an immunogen and a mammalian plasmacytoma cell (myeloma cell), and thereby producing a desired 5 ′. It can be carried out by selecting a clone producing a monoclonal antibody that recognizes deoxy-5′-methylthioadenosine and culturing the clone. The production of this monoclonal antibody can basically follow conventional methods.
- the mammal immunized with the immunogen is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and mice, rats and the like are used.
- the immunization method is the same as in the production of polyclonal antibodies. However, spleen cells are collected from the immunized animal 3 to 10 days after the final immunization.
- a cell that can be subcultured can be obtained by a method described in "Basic method of molecular cell biology" (issued by Takeichi Minamiedo, Takeichi Horie et al., 1994).
- a hybridoma can be obtained by fusing a plasmacytoma cell and an immune cell producing an antibody in the presence of Sendai virus or polyethylene glycol.
- the plasmacytoma cells used here are preferably plasmacytoma cells derived from the same isothermal animal even in the same isothermal animal.
- a mouse myeloma cell is used. It is preferable to use it.
- Plasmacytoma cells are p3x63-Ag8. A known item such as a UI can be used.
- the hybridoma is selected by HAT medium (medium supplemented with hypoxanthine, aminopterin, thymidine), and when the colonies are confirmed, the binding between the antibody secreted into the culture supernatant and the antigen is examined (screened). A hybridoma producing the antibody can be obtained.
- Examples of the screening method include various methods generally used for antibody detection such as spot method, agglutination reaction method, Western blot method, ELISA method, etc.
- the hybridoma culture supernatant is carried out according to the ELISA method using the reactivity with the neoepitope peptide as an index.
- a target antibody-producing strain that specifically reacts with the neoepitope peptide can be screened.
- clone 20A10 is exemplified as the obtained clone.
- the cloning of the strain capable of producing the target antibody obtained as a result of screening can be carried out by the usual limiting dilution method, soft agar method or the like.
- the cloned hybridoma can be cultured in a large amount in a serum medium or a serum-free medium as necessary. According to this culture, a relatively high purity desired antibody can be obtained as a culture supernatant.
- the hybridoma can be inoculated into the abdominal cavity of a mammal that is compatible with the hybridoma, such as a mouse, and the desired antibody can be collected in a large amount as mouse ascites.
- the culture supernatant containing the hybridoma producing the monoclonal antibody of the present invention and the ascites of the mouse can be used as a crude antibody solution without purification or modification.
- the above culture supernatant or ascites should be subjected to affinity ammonium chromatography such as saturated ammonium sulfate, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column. Etc.
- a recombinant antibody produced by cloning an antibody gene, incorporating it into an appropriate vector, introducing it into a host and producing it using a gene recombination technique can be used (for example, Carl et al., THERAPEUTIC MONOCLONAL ANTIBODIES, 1990).
- cDNA encoding the variable region of the antibody of interest (eg, 20A10) (eg, SEQ ID NOs: 3 and 4 for 20A10) is synthesized.
- 5'-Ampli FINDER RACEKit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA, et al., Proc. Natl. Acad. Sci. USA (1988) 85, page 8998, etc.).
- the target DNA fragment is purified from the obtained PCR product and ligated with vector DNA.
- a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector.
- the base sequence of the target DNA is confirmed by a known method such as the dideoxy method.
- DNA encoding the desired antibody V region is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
- DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region.
- the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer / promoter.
- host cells can be transformed with this expression vector to express the antibody.
- the expression of the antibody gene may be carried out by co-transforming the host by separately incorporating the heavy chain (H chain) or light chain (L chain) of the antibody into an expression vector, or DNA encoding the H chain and L chain. May be incorporated into a single expression vector to transform the host (see WO94 / 11523).
- the antibody When performing an immunoassay (immunological assay) described below using an antibody, the antibody itself can usually be labeled with various substances so that the behavior of the antibody can be detected.
- a preferred embodiment of the monoclonal antibody of the present invention includes a labeled monoclonal antibody.
- the antibody can be labeled by using a conventional method described in, for example, “Molecular Cell Biology Basic Experimental Method” (Takeichi Horie et al., 1994).
- various substances include chemiluminescent substances, enzymes, fluorescent substances, colored beads, radioisotopes, elements, metals, and biotin. Specific examples are shown below, but are not limited thereto.
- the chemiluminescent substance refers to, for example, luminol and acridinium ester.
- Examples of enzymes include ⁇ -galactosidase, alkaline phosphatase and peroxidase.
- Examples of fluorescent substances include europium cryptate, FITC, and RITC (and the like.
- Colored beads include protein A beads, wheat germ agglutinin (WGA) beads, streptavidin beads, and the like.
- Radioactive isotopes include, for example, 14C and 125I.
- the element refers to a lanthanide element such as europium, etc.
- the metal refers to ferritin, colloidal gold, etc. II Immunoassay
- the monoclonal antibody of the present invention can specifically recognize the C-terminal neoepitope structure of a collagen neoepitope fragment.
- This neoepitope is independent of collagen type. Therefore, for example, a collagen neoepitope fragment can be quantified for any collagen by performing a sandwich assay in combination with an antibody capable of recognizing epitopes specific to various collagens (see above for the production method).
- Specific sequences for each type of collagen are known to those skilled in the art. For example, the following sequences can be mentioned.
- Type I collagen-specific sequence Gly-Ser-Pro-Gly-Ala-Asp-Gly-Pro-Ala
- Type II collagen-specific sequence Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser
- Type III collagen specific Gly-Glu-Lys-Gly-Ser-Pro-Gly-Ala-Gln (SEQ ID NO: 13)
- the immunoassay using the monoclonal antibody of the present invention may be a competitive measurement or a non-competitive measurement.
- a 50% inhibitory concentration of an antibody immune reaction in a competitive immunoassay is 0.04 ⁇ M or less” means that when 0.04 ⁇ M of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 14, 17 or 18 is added, SEQ ID NO: 2 (Example 2) which means that the inhibition rate of the coupling
- the 50% inhibitory concentration is preferably 0.04 ⁇ M or less, more preferably 0.022 ⁇ M or less.
- a homogeneous assay method (measurement by a homogeneous system) or a heterogeneous assay method (measurement by a heterogeneous system) may be used.
- enzyme immunoassay enzyme immunoassay
- solid phase enzyme immunoassay ELISA
- fluorescence immunoassay FIA
- radioimmunoassay RIA
- time-resolved fluorescence immunoassay TR-FIA
- Chemiluminescence immunoassay immunoblot, Western blot, immunostaining, SPA, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and the like.
- a preferred embodiment of the immunoassay of the present invention is an ELISA method.
- the ELISA method is a method in which an antibody or antigen labeled with an enzyme is used and the amount of the antibody or antigen is quantified based on the activity of the labeled enzyme.
- An antigen-antibody conjugate labeled with an enzyme and a free-form labeled antigen, or an immobilized antibody or antigen is used to separate the antibody.
- As the solid phase agarose, the inner surface of a microtiter plate, latex particles, and the like can be used.
- Specific examples of the ELISA method include competitive immunoassay and sandwich immunoassay.
- the labeling enzyme include horseradish-derived peroxidase (hereinafter also referred to as HRP), alkaline phosphatase, and the like.
- the monoclonal antibody and immunoassay of the present invention are useful in various applications.
- the monoclonal antibody and immunoassay of the present invention are useful for measuring the activity of collagenase such as MMP.
- collagenase such as MMP.
- Three types of collagenases (Pendas AM et al., Genomics 1995; 26: 615-8. And Mitchell PG, etc.) can cleave the triple chains of fibrillar collagens of type I, II, and III in their native state. J Clin Invest 1996; 97: 761-8). Since the monoclonal antibody of the present invention can specifically recognize the neoepitope fragment generated by collagenase cleavage, its amount can be measured and the activity of collagenase can be evaluated.
- the monoclonal antibody and immunoassay of the present invention are useful for screening methods using the content of collagen neoepitope fragment as an index.
- Such screening is performed under conditions that allow the recombinant collagenase (purified or partially purified product) prepared by an expression vector or the like in the presence of a test substance to bind to the enzyme substrate (collagen) (for example, 0.1 M phosphate). Buffer pH 7.4, room temperature) to determine whether the test substance inhibits the binding of the enzyme substrate, ie, collagen neoepitope fragment is quantified.
- the enzyme substrate for example, 0.1 M phosphate
- the test substance may be any of peptides, proteins, non-peptide compounds, synthetic compounds (low molecular weight compounds, etc.), fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Moreover, the sample containing these may be sufficient.
- Candidate substances selected as collagenase inhibitors by screening are used to prevent diseases in which collagenase is known to be involved (for example, osteoarthritis, proliferative diseases such as cancer, osteoporosis, Alzheimer's disease, hypertension). ⁇ It can be a therapeutic agent.
- the monoclonal antibody and immunoassay of the present invention are useful for a method for selecting a disease patient including a step of measuring the content of a collagen neoepitope fragment contained in a biological sample.
- the immunoassay of the present invention can be used for biological samples from patients (any biological fluid commonly tested in clinical samples. For example, body fluids such as blood, urine, saliva, and sweat, and cells and / or tissues. The content of collagen epitope fragments in (including the extracts and supernatants) can be measured. Biological samples are also easily obtained without risk to the patient, and the measurement is simple and inexpensive.
- diseases in which the content of collagen neoepitope fragment is an indicator of progression eg, osteoporosis, osteoarthritis, rheumatoid arthritis, and other diseases that cause benign and malignant tumors of the bone or cartilage destruction
- the monoclonal antibodies and immunoassays of the present invention can be used to determine the extent of ongoing cartilage collagen degradation in patients with different types of osteoarthritis or rheumatoid arthritis.
- the kit of the present invention is characterized by including the monoclonal antibody of the present invention as a binding agent for detecting a collagen neoepitope fragment in a test sample.
- kits more generally include one or more components necessary to perform the assay.
- Components can be standards, reagents (diluents, buffers, etc.), containers and / or devices.
- one container in the kit may contain a monoclonal antibody that binds to a sequence specific for the type of collagen (eg, SEQ ID NOs: 11-13).
- Such antibodies can be provided attached to any support material known to those skilled in the art (eg, a well in a microtiter plate or a suitable membrane such as nitrocellulose).
- it may contain components to be used in the assay (eg, reagents or buffers).
- Such a kit can alternatively be labeled with a substance as described above suitable for direct or indirect detection of antibody binding.
- a neoepitope peptide containing hydroxyproline represented by Gly-Pro-Hyp-Gly-Pro-Gln-Gly (SEQ ID NO: 2) was synthesized (manufactured by Greiner Bio-one). 10 mg of synthetic neoepitope peptide dissolved in 0.1 M phosphate buffer (pH 6.0) containing 1 ml of 5 mM EDTA and 10 mg of giant keyhole limpets hemocyanin (maleimidated KLH, manufactured by PIERCE) in 1 ml of purified water The dissolved product was mixed and reacted at room temperature for 4 hours and further at 4 ° C.
- peptide-KLH complex 0.1 mg together with Freund's complete adjuvant was intraperitoneally administered to 7 4-week-old A / J Jms Slc female mice for initial immunization. Then, after 21 days, 42 days and 63 days, 0.1 mg of peptide-KLH complex was boosted with Freund's incomplete adjuvant, and after 71 days, 0.1 mg of peptide-KLH complex was suspended in 0.1 ml of physiological saline. Was intraperitoneally administered for final immunization.
- Biotin labeling of neoepitope peptide 0.2ml of synthetic neoepitope peptide dissolved in 0.1M phosphate buffer (pH6.0) containing 0.4ml of 5mM EDTA and 0.6ml of PEO-maleimide activated biotin (PIERCE) 0.1ml What was dissolved in water was mixed, and after reacting at room temperature for 2 hours, biotin-labeled neoepitope peptide was purified by reverse phase HPLC.
- Tris buffer 50 mM Tris-HCl, pH 7.5
- Anti-mouse IgG antibody manufactured by Shiba Goat
- Nunk a 96-well microtiter plate
- Each well was washed once with 0.3 ml of a washing solution (saline containing 0.01% Tween 20), and then 0.3 ml of Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added and allowed to stand at room temperature for 2 hours for blocking.
- neoepitope antibody (20A10) by collagen type Human I, II or III collagen solution (Chondrex ) 10 ⁇ g / 10 ⁇ l in 2x enzyme reaction buffer (0.3M NaCl, 10mM CaCl2) 10 ⁇ l of 50 mM Tris buffer containing 0.005% Brij35, pH 7.6) was added to neutralize, and human activated MMP13 (human Pro-MMP13 (Calbiochem) was incubated with 1 mM APMA at 37 ° C. for 2 hours. Activated) 0.2 ⁇ g was added and reacted at 37 ° C. overnight. Thereafter, a stop solution (EDTA, final concentration 5 mM) was added to obtain a natural neoepitope solution.
- EDTA final concentration 5 mM
- Tris buffer 50 mM Tris-HCl, pH 7.5
- 0.35 ⁇ g anti-mouse IgG-Fc antibody Jackson Immuno Research
- 384 well microtiter plate Nunk Fixed overnight.
- Each well was washed once with 90 ⁇ l of a washing solution (physiological saline containing 0.01% Tween 20), and then 0.1 ml of Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added and allowed to stand at room temperature for 2 hours for blocking.
- the anti-neoepitope antibody 20A10 does not react with MMP13-undigested collagen, but reacts specifically with MMP13-digested collagen containing the end of the neoepitope. Moreover, it was confirmed that 20A10 binds with almost the same affinity to any neo-epitope of type I, type II or type III collagen. (FIG. 3) Accordingly, even when a large amount of undigested collagen coexists, the antibody does not become a background and can detect a digested fragment of collagen with high sensitivity, so that collagenase activity can be accurately quantified. In addition, degradation of type I and type III collagen can be measured by combining a sandwich antibody (capture antibody) with an antibody capable of recognizing a site specific to type I or type III collagen.
- a type II collagen-specific neoepitope measurement system In order to measure type II collagen, a part of a type II collagen neoepitope having a neoepitope on the C-terminal side (957-965 of the amino acid sequence shown in SEQ ID NO: 20)
- the synthetic peptide of Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser (SEQ ID NO: 12) corresponding to the position is used as an immunogen, and a cysteine linker is added to the amino terminus and amidation is performed to the carboxyl terminus. gave.
- the above mixture was dialyzed against distilled water and lyophilized to obtain 8 mg of type II collagen-specific internal sequence peptide-KLH complex.
- the peptide-KLH complex (0.004 mg) was administered intraperitoneally to each of four 4-week-old A / J Jms Slc and Balb / c female mice together with Freund's complete adjuvant for initial immunization. Then, after 21 days, 42 days and 63 days, 0.1 mg of peptide-KLH complex was boosted with Freund's incomplete adjuvant, and after 71 days, 0.1 mg of peptide-KLH complex was suspended in 0.1 ml of physiological saline. Was intraperitoneally administered for final immunization.
- biotin-labeled peptide was purified by reverse phase HPLC after overnight reaction at 4 ° C. Three days after the final immunization, the spleen was removed and spleen cells were collected.
- Spleen cells and mouse myeloma cells were fused with 50% polyethylene glycol 4000 and selected in a medium containing hypoxanthine, aminopterin, and thymidine.
- Screening of specific antibody-producing cells was performed using ELISA described below.
- Tris buffer 50 mM Tris-HCl, pH 7.5
- type II collagen internal sequence specific antibody 6G4 1.5 ⁇ g type II collagen internal sequence specific antibody 6G4
- buffer A containing 10-500 pM standard peptide or test sample (0.5 mM bovine serum albumin, 0.01% Tween80, 0.05% Proclin150, 50 mM containing 0.15 M NaCl) Tris buffer (pH 7.4) and 100 ⁇ l of buffer A containing 0.05 ng of HRP-labeled neoepitope antibody 20A10 were mixed and reacted at 4 ° C. for 16 hours.
- TMB-Substrate Chromogen (DAKO) was added for color development at room temperature for 30 minutes, and 100 ⁇ l of 0.05 M sulfuric acid was added to stop the reaction. The absorbance at 450 nm was measured.
- FIG. 4 Unlike neoepitope antibody 9A4, which has a low affinity for hydroxylated substances as described above, the antibody 20A10 binds with the same affinity for both non-hydroxylated and hydroxylated forms. It is not necessary to convert the ratio by the hydroxylated proline ratio, and the neoepitope concentration can be accurately quantified without being affected by hydroxylation modification.
- Pre-incubation plates (Costar) with enzyme reaction buffer (50 mM Tris buffer, pH 7.6 containing 0.3 M NaCl, 10 mM CaCl2, 0.005% Brij35) and human activated MMP13 and MMP, which are types of human type II collagenase After adding the inhibitor and preincubating at room temperature for 30 minutes, the amount of collagen neoepitope in the enzyme reaction solution was measured by the sandwich ELISA system described in Example 4 above.
- enzyme reaction buffer 50 mM Tris buffer, pH 7.6 containing 0.3 M NaCl, 10 mM CaCl2, 0.005% Brij35
- human activated MMP13 and MMP which are types of human type II collagenase
- Collagenase activity measurement system in human chondrocyte culture system Add 10 ng / ml human type II collagen solution to 96-well culture plate (Sumitomo Bakelite), incubate overnight at 4 ° C, then culture medium (0.1 mg / ml BSA, ITS, containing 50 ⁇ M L-ascorbic acid) Wash once with DMEM medium) to make a collagen-coated plate.
- Culture medium 0.1 mg / ml BSA, ITS, containing 50 ⁇ M L-ascorbic acid
- DMEM medium Normal human chondrocytes
- the culture medium was changed, and 1 ng / ml human interleukin 1 ⁇ (Genzyme) and 10 ng / ml Oncostatin M (Sigma) and the MMP inhibitor to be tested were added at various concentrations, and further 2 Incubate for days.
- EDTA reaction stop solution
- the culture supernatant was recovered, and the collagen neoepitope concentration contained therein was measured by the sandwich ELISA system described in Example 4 above. The activity of MMP inhibitors was measured.
- the amplified fragment was cloned using TOPO TA Cloning Kit (manufactured by Invitrogen), and the base sequence was analyzed using Applied Biosystems 3130 Genetic Analyzer (manufactured by Applied Biosystems). Thereby, the amino acid sequence of the variable region was identified (FIG. 7).
- the neoepitope can be accurately detected and quantified without being affected by proline hydroxylation modification that changes depending on the health and nutritional state for each collagen type of type I, type II, and type III collagen.
- cleavage of type II collagen by collagenase serves as an index of cartilage metabolism, and is useful as a diagnostic method and kit for evaluating pathological progression and therapeutic effects in cartilage lesions such as osteoarthritis.
- collagenase cleavage of type I collagen serves as an indicator of extracellular matrix metabolism in connective tissues throughout the body, and is useful as a diagnostic method and kit for evaluating the progress of fibrosis in various organs and the effects of antifibrotic treatment.
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Abstract
Description
(1)コラーゲンネオエピトープ断片に特異的に結合し、配列番号20で示されるアミノ酸配列の962位から975位に結合する抗体であって、当該エピトープに含まれるプロリンが非水酸化体の場合における結合親和性と、水酸化体の場合における結合親和性が実質的に同一であるモノクローナル抗体、
(2)前記(1)記載のプロリンが、配列番号20で示されるアミノ酸配列における971位のプロリンである前記(1)に記載のモノクローナル抗体、
(3)エピトープが配列番号20で示されるアミノ酸配列の957位から975位の領域に存在する、前記(1)に記載のモノクローナル抗体、
(4)前記(2)に記載の抗体であって、配列番号2で示されるアミノ酸配列からなるペプチドへの該抗体の免疫反応が阻害されるように、配列番号14、17又は18で示されるアミノ酸配列からなるペプチドを競合させるイムノアッセイにおいて、該免疫反応の50%阻害濃度がいずれのペプチドにおいても0.04μM以下である抗体、
(5)1)相補性決定領域に下記アミノ酸配列を含む重鎖可変領域;KYGIN(配列番号5)、WINTYSGMTT YADDFKG(配列番号6)、およびSLGYDYGGFAY(配列番号7)、並びに2)相補性決定領域に下記アミノ酸配列を含む軽鎖可変領域;RSGQTLVHDNENTYFH(配列番号8)、KISNRFS(配列番号9)、およびSQNTHVPFT(配列番号10)を有するモノクローナル抗体、
(6)1)配列番号3のアミノ酸配列を有する重鎖可変領域;および 2)配列番号4のアミノ酸配列を有する軽鎖可変領域;を有するモノクローナル抗体、
(7)前記(1)~(6)のいずれかに記載のモノクローナル抗体であってラベルされたモノクローナル抗体、
(8)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いたイムノアッセイ、
(9)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いたコラーゲンネオエピトープ断片の含有量の測定方法、
(10)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いて測定したコラーゲンネオエピトープ断片の含有量を指標とする、コラゲナーゼ活性の測定方法、
(11)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いて測定したコラーゲンネオエピトープ断片の含有量を指標とする、コラゲナーゼ阻害剤のスクリーニング方法、
(12)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いて生体試料に含まれるコラーゲンネオエピトープ断片の含有量を測定する工程を含む、コラゲナーゼが関与する疾患の患者の選別方法、
(13)前記(1)~(6)のいずれかに記載のモノクローナル抗体を含むキット、
(14)前記(1)~(6)のいずれかに記載のモノクローナル抗体を用いて生体試料に含まれるコラーゲンネオエピトープ断片の含有量を測定する工程を含む、コラゲナーゼが関与する疾患の診断方法、および
(15)コラゲナーゼが関与する疾患の診断に使用するための前記(1)~(6)のいずれかに記載のモノクローナル抗体、
に関する。
線維性コラーゲンであるI型、II型およびIII型コラーゲンは3本のペプチド鎖がらせん状に撚り合わさって構成されており、コラゲナーゼ(例えば、MMP-1、MMP-8、およびMMP-13)は、これらの線維性コラーゲン3重鎖を未変性の状態でN末から3:1の位置(975-976アミノ酸残基間)で切断する。そしてN末側4分の3断片のC末端およびC末側4分の1断片のN末端が新たに生じる末端構造を「ネオエピトープ」という(図1)。また、切断により生じたコラゲナーゼ断片を「コラーゲンネオエピトープ断片」という。中でも、ヒトII型コラーゲン(Accession No.NP_001835)のC末端側のネオエピトープ部分(969-975位に該当;以下、C末端ネオエピトープともいう)の7アミノ酸残基 Gly-Pro-Pro-Gly-Pro-Gln-Gly(配列番号1)はヒトからマウスまで動物種間で保存されていることが知られている。また、コラーゲンは高いヒドロキシプロリン含有率で特徴付けられるタンパク質の一つであり、この配列のC末側から5番目(もしくは、N末側から971番目)のプロリン残基も多くの場合に水酸化体(以下、ヒドロキシ体もしくはヒドロキシプロリンともいう)となっており、(配列番号2、Gly-Pro-Hyp-Gly-Pro-Gln-Gly、Hypは水酸化プロリン)その割合はコラーゲン合成時の健康状態や栄養状態による影響を受けやすく一定ではないことから、コラーゲンネオエピトープ断片の正確な定量を妨害していると考えられる。したがって、コラーゲンの分解を免疫学的に測定する場合であれば、ネオエピトープを検出する抗体の特性としては水酸化体と非水酸化体(プロリン)を同程度に免疫特異的に認識できることが本来は望ましい。
モノクローナル抗体の単離、精製は、上述の培養上清あるいは腹水を、飽和硫酸アンモニウム、イオン交換クロマトグラフィー(DEAEまたはDE52等)、抗イムノグロブリンカラムあるいはプロテインAカラム等のアフィニティカラムクロマトグラフィーに供すること等により行うことができる。
II イムノアッセイ
I型コラーゲン特異的配列:Gly-Ser-Pro-Gly-Ala-Asp-Gly-Pro-Ala(配列番号11)
II型コラーゲン特異的配列:Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser(配列番号12)
III型コラーゲン特異的:Gly-Glu-Lys-Gly-Ser-Pro-Gly-Ala-Gln (配列番号13)
III 有用性
(1)抗原の免疫:
Gly-Pro-Hyp-Gly-Pro-Gln-Gly(配列番号:2)に示すヒドロキシプロリンを含むネオエピトープペプチドを合成した(Greiner Bio-one社製)。合成ネオエピトープペプチド10mgを1mlの5mM EDTAを含む0.1Mリン酸緩衝液(pH6.0)に溶解したものと、10mgのgiant keyhole limpetsヘモシアニン(マレイミド化KLH、PIERCE社製)を1mlの精製水に溶解したものとを混合し、室温にて4時間、さらに4℃で一晩反応させた。上記の混合液を蒸留水に対して透析後に、凍結乾燥し、ネオエピトープペプチド‐KLH複合体 13mgを得た。
このペプチド‐KLH複合体0.1mgをフロイント完全アジュバントと共に4週令A/J Jms Slc雌マウス7匹に腹腔内投与し、初回免疫とした。その後、21日後、42日後および63日後にペプチド‐KLH複合体0.1mgをフロイント不完全アジュバントと共に追加免疫し、さらに71日後にペプチド‐KLH複合体0.1mgを生理食塩水0.1mlに懸濁した溶液を腹腔内投与し、最終免疫とした。
合成ネオエピトープペプチド0.2mgを0.4mlの5mM EDTAを含む0.1Mリン酸緩衝液(pH6.0)に溶解したものと、0.60mgのPEO-マレイミド活性化ビオチン(PIERCE社製)を0.1mlの蒸留水に溶解したものとを混合し、室温で2時間反応後に逆相HPLCにてビオチン標識ネオエピトープペプチドを精製した。
最終免疫の3日後に脾臓を摘出し、脾臓細胞を回収した。脾臓細胞とマウスミエローマ細胞(p3×63-Ag8.U1、東京腫瘤研究所)を50%のポリエチレングリコール4000を用いて融合させ、ヒポキサンチン、アミノプテリン、およびチミジンを含む培地で選択した。
細胞融合10日後に特異抗体産生細胞のスクリーニングを以下に説明するELISAを用いて行なった。384穴マイクロタイタープレート(ヌンク社製)の各ウェルに0.35μgの抗マウスIgG抗体(シバヤギ社製)を含むトリス緩衝液(50mM Tris-HCl、pH7.5)35μlを加えて4℃で16時間固定した。これらのウェルを90μlの洗浄液(0.01% Tween20を含む生理食塩水)で1回洗浄した後、ブロックエース(大日本製薬社製)を90μl加えて室温で2時間放置して、ブロッキングを行なった(抗マウスIgG抗体固相化プレート)。各ウェルを90μlの洗浄液で1回洗浄した後、15μlのハイブリドーマ培養上清を含む10μlの緩衝液A(0.5% ウシ血清アルブミン、0.01% Tween80、0.05% Proclin150、0.15M NaClを含む50mM トリス緩衝液、pH7.4)および0.05ngのビオチン標識ネオエピトープベプチドと2ngのStreptavidin-HRP(PIERCE社製)を含む10μlの緩衝液Aを混合し、4℃で16時間反応させた。
ネオエピトープ抗体(20A10)のネオエピトープ認識特異性を調べるために以下のアミノ酸配列を有するネオエピトープペプチドを用いて以下の方法で行なった。
Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-HyP-Gly-Pro-Gln-Gly (配列番号20で示されるアミノ酸配列の962-975位に該当、配列番号:14)
Gly-Pro-Gln-Gly (972-975位に該当、配列番号:15)
Gly-Pro-Pro-Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln-Arg (配列番号20で示されるアミノ酸配列の969-980位に該当、配列番号:16)
Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-HyP-Gly-Pro-Gln-Gly (配列番号20で示されるアミノ酸配列の957-975位に該当、配列番号:17)
Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-Pro-Gly-Pro-Gln-Gly (配列番号20で示されるアミノ酸配列の957-975位に該当、配列番号:18)
ヒトI型、II型あるいはIII型コラーゲン溶液(Chondrex社製)10μg/10μlに2×酵素反応バッファー(0.3M NaCl、10mM CaCl2、0.005% Brij35を含む50mMトリス緩衝液、pH7.6)を10μl添加して中和し、ヒト活性化MMP13(ヒトPro-MMP13(Calbiochem社製)を1mM APMAで37℃、2時間インキュベートして活性化したもの) 0.2μgを添加して37℃で一晩反応した。その後、停止液(EDTA、終濃度5mM)を加えて天然型ネオエピトープ溶液とした。
各ウェルを90μlの洗浄液で1回洗浄した後、6.4-250nMの天然型ネオエピトープ溶液を含む10μlの緩衝液Aと1ng/mlのビオチン標識ネオエピトープペプチド(配列番号:2)と200ng/mlのStreptavidin-HRPを含む10μlの緩衝液Aおよび、15ng/mlのネオエピトープ抗体(20A10)を含む10μlの緩衝液Aをそれぞれ混合し、4℃、16時間反応させた。
II型コラーゲンの測定を行うために、C末端側にネオエピトープを有するII型コラーゲンネオエピトープの一部(配列番号20で示されるアミノ酸配列の957-965位に相当)に該当するGly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser(配列番号12)の合成ペプチドを免疫原とし、アミノ末端にシステインリンカー付加、カルボキシル末端にアミド化を施した。この合成ペプチド2.1mgを1mlの5mM EDTAを含む0.1Mリン酸緩衝液(pH6.0)に溶解したものと、8mgのgiant keyhole limpetsヘモシアニン(マレイミド化KLH、PIERCE社製)を3mlの5mM EDTAを含む0.1Mリン酸緩衝液(pH6.0)に溶解したものと混合し、室温にて3時間反応させた。
その後、21日後、42日後および63日後にペプチド‐KLH複合体0.1mgをフロイント不完全アジュバントと共に追加免疫し、さらに71日後にペプチド‐KLH複合体0.1mgを生理食塩水0.1mlに懸濁した溶液を腹腔内投与し、最終免疫とした。合成ペプチド0.2mgを0.1mlの5mM EDTAを含む0.1Mリン酸緩衝液(pH6.0)に溶解したものと、0.25mgのHPDP-ビオチン(PIERCE社製)を0.1mlのジメチルホルムアミドに溶解したものとを混合し、4℃で一晩反応後に逆相HPLCにてビオチン標識ペプチドを精製した。
最終免疫の3日後に脾臓を摘出し、脾臓細胞を回収した。
ネオエピトープおよびコラーゲンタイプ特異的内部配列を含む22残基からなる合成ペプチドGly-Glu-Lys-Gly-Glu-Pro-Gly-Asp-Asp-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-Hyp-Gly-Pro-Gln-Gly(954-975位に相当、配列番号19)を検量標準ペプチドとして合成した。HRP標識20A10を調製した。すなわち、20A10のIgG画分1mgを含む5mM EDTA含有0.1Mリン酸緩衝液(pH6.0)0.5mlに0.1Mメルカプトエチルアミン水溶液0.05mlを加えて37℃で1.5時間反応したのち、PD-10カラム(GEヘルスケア社製)によるゲルろ過を行って還元IgG画分を分取した。Peroxidase(Horse radish由来、Roche社製、HRP)1mgを含む5mM EDTA含有0.1Mリン酸緩衝液(pH6.0)0.2mlにSulfo-SMCC(PIERCE社製)を加えて室温で2時間反応したのち、PD-10カラム(GEヘルスケア社製)によるゲルろ過を行ってマレイミド化HRP画分を分取した。これに前記の20A10の還元IgG画分を加えて4℃で一夜反応し、高速ゲルろ過(5mM EDTA含有0.1Mリン酸緩衝液、pH6.0で平衡化したTSK-GEL G3000カラム(東ソー社製)を付属したLC-6Aシステム(島津社製))を行ってHRP標識20A10画分約0.5mgを分取した。96穴マイクロタイタープレート(ヌンク社製)の各ウェルに1.5μgのII型コラーゲン内部配列特異的抗体6G4を含むトリス緩衝液(50mM Tris-HCl、pH7.5)150μlを加えて4℃で16時間固定した。これらのウェルを300μlの洗浄液(0.01% Tween20を含む生理食塩水)で1回洗浄した後、ブロックエース(大日本製薬社製)を150μl加えて室温で2時間放置して、ブロッキングを行なった。各ウェルを300μlの洗浄液で1回洗浄した後、10-500pM標準ペプチドあるいは被検試料を含む50μlの緩衝液A(0.5% ウシ血清アルブミン、0.01% Tween80、0.05% Proclin150、0.15M NaClを含む50mM トリス緩衝液、pH7.4)および0.05ngのHRP標識ネオエピトープ抗体20A10を含む100μlの緩衝液Aを混合し、4℃で16時間反応させた。次に各ウェルを300μlの洗浄液で3回洗浄した後に、100μlのTMB-Substrate Chromogen(DAKO社製)を添加して室温で30分間発色させ、100μlの0.05M硫酸を添加して反応を停止し、450nmにおける吸光度を測定した。
5 ng/mlのヒトII型コラーゲン溶液(Chondrex社製)を96ウェルマキシソーププレート(NUNC社製)に添加し、4℃で一晩インキュベート後、洗浄バッファー(0.05 M Tris-HCl、 pH7.6)で2回洗浄してコラーゲンコーティングプレートとする。プレインキュベート用プレート(Costar 社製)に酵素反応バッファー(0.3M NaCl、10mM CaCl2、0.005% Brij35を含む50mMトリス緩衝液、pH7.6)とヒトII型コラゲナーゼの一種であるヒト活性化MMP13およびMMP阻害剤を加えて室温で30分間プレインキュベートした後、酵素反応溶液中のコラーゲンネオエピトープ量を上記実施例4で説明したサンドイッチELISA系により測定した。
10ng/mlのヒトII型コラーゲン溶液を96ウェル培養プレート(住友ベークライト社製)に添加し、4℃で一晩インキュベート後、培養メディウム(0.1mg/ml BSA、ITS、50μM L-アスコルビン酸を含むDMEM培地)で1回洗浄してコラーゲンコーティングプレートとする。
正常ヒト由来軟骨細胞(Chondrex社)をコーティングプレートにウェルあたり4×104個播種し、培養メディウムを用いて37℃、5%CO2条件下で培養する。1日後に培養液を交換して、1ng/mlヒトインターロイキン1β(Genzyme社製)および10ng/ml Oncostatin M(Sigma社製)と被験対象のMMP阻害剤を様々な濃度で添加してさらに2日間培養する。4日後に反応停止液(EDTA、終濃度5mM)を添加した後培養上清を回収し、この中に含まれるコラーゲンネオエピトープ濃度を上記実施例4に記載したサンドイッチELISA系により測定することにより、MMP阻害剤の活性を測定した。
樹立されたハイブリドーマ細胞から、RNeasy Mini Kit(QIAGEN社製)を用いて、RNAの抽出を行った。抽出したRNAを1μgから、5’RACE Syatem for Rapid Amplification of cDNA Ends、Version 2.0(Invitrogen社製)を用いて、DNA断片の増幅を行った。増幅された断片は、TOPO TA Cloning Kit(Invitrogen社製)でクローニングされ、Applied Biosystems 3130 Genetic Analyzer(Applied Biosystems社製)で塩基配列を解析した。それにより、可変領域のアミノ酸配列を特定した(図7)。
Claims (15)
- コラーゲンネオエピトープ断片に特異的に結合し、配列番号20で示されるアミノ酸配列の962位から975位に結合する抗体であって、当該エピトープに含まれるプロリンが非水酸化体の場合における結合親和性と、水酸化体の場合における結合親和性が実質的に同一であるモノクローナル抗体。
- 請求項1記載のプロリンが、配列番号20で示されるアミノ酸配列における971位のプロリンである請求項1に記載のモノクローナル抗体。
- エピトープが、配列番号20で示されるアミノ酸配列の957位から975位の領域に存在する、請求項1に記載のモノクローナル抗体。
- 請求項2に記載の抗体であって、配列番号2で示されるアミノ酸配列からなるペプチドへの該抗体の免疫反応が阻害されるように配列番号14、17又は18で示されるアミノ酸配列からなるペプチドを競合させるイムノアッセイにおいて、該免疫反応の50%阻害濃度がいずれのペプチドにおいても0.04μM以下である抗体。
- 1)相補性決定領域に下記アミノ酸配列を含む重鎖可変領域;KYGIN(配列番号5)、WINTYSGMTT YADDFKG(配列番号6)、およびS LGYDYGGFAY(配列番号7)、並びに2)相補性決定領域に下記アミノ酸配列を含む軽鎖可変領域;RSGQTLVHDN ENTYFH(配列番号8)、KISNRFS(配列番号9)、およびSQNTHVPFT(配列番号10)
を有するモノクローナル抗体。 - 1)配列番号3のアミノ酸配列を有する重鎖可変領域;および 2)配列番号4のアミノ酸配列を有する軽鎖可変領域;を有するモノクローナル抗体。
- 請求項1~6のいずれかに記載のモノクローナル抗体であってラベルされたモノクローナル抗体。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いたイムノアッセイ。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いたコラーゲンネオエピトープ断片の含有量の測定方法。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いて測定したコラーゲンネオエピトープ断片の含有量を指標とする、コラゲナーゼ活性の測定方法。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いて測定したコラーゲンネオエピトープ断片の含有量を指標とする、コラゲナーゼ阻害剤のスクリーニング方法。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いて生体試料に含まれるコラーゲンネオエピトープ断片の含有量を測定する工程を含む、コラゲナーゼが関与する疾患の患者の選別方法。
- 請求項1~6のいずれかに記載のモノクローナル抗体を含むキット。
- 請求項1~6のいずれかに記載のモノクローナル抗体を用いて生体試料に含まれるコラーゲンネオエピトープ断片の含有量を測定する工程を含む、コラゲナーゼが関与する疾患の診断方法。
- コラゲナーゼが関与する疾患の診断に使用するための請求項1~6のいずれかに記載のモノクローナル抗体。
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WO2014034754A1 (ja) | 2012-08-31 | 2014-03-06 | 塩野義製薬株式会社 | Psf1由来ペプチド |
JP2017530092A (ja) * | 2014-07-28 | 2017-10-12 | フィロゲン エスピーエー | 治療及び診断のための抗コラーゲン抗体 |
JP2019507731A (ja) * | 2016-01-28 | 2019-03-22 | ノルディック バイオサイエンス エイ/エス | VII型コラーゲンα1アッセイ |
JP2020528901A (ja) * | 2017-07-27 | 2020-10-01 | ノルディック・ビオサイエンス・エー/エスNordic Bioscience A/S | X型コラーゲンアルファ−1アッセイ |
WO2020246563A1 (ja) | 2019-06-05 | 2020-12-10 | 中外製薬株式会社 | 抗体切断部位結合分子 |
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TWI457442B (zh) * | 2012-05-17 | 2014-10-21 | Univ Ishou | 用以生產第ii型膠原片段抗體的融合瘤細胞株、及以尿液檢測退化性關節炎之方法 |
CN108431606B (zh) * | 2016-02-03 | 2022-04-05 | 北欧生物科技公司 | 纤维化的联合生物标志物测量 |
WO2021180063A1 (en) * | 2020-03-09 | 2021-09-16 | I-Mab Biopharma Co., Ltd. | Methods for detecting and quantifying biological modifications in antibodies |
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Cited By (7)
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WO2014034754A1 (ja) | 2012-08-31 | 2014-03-06 | 塩野義製薬株式会社 | Psf1由来ペプチド |
JP2017530092A (ja) * | 2014-07-28 | 2017-10-12 | フィロゲン エスピーエー | 治療及び診断のための抗コラーゲン抗体 |
JP2019507731A (ja) * | 2016-01-28 | 2019-03-22 | ノルディック バイオサイエンス エイ/エス | VII型コラーゲンα1アッセイ |
JP7091246B2 (ja) | 2016-01-28 | 2022-06-27 | ノルディック バイオサイエンス エイ/エス | VII型コラーゲンα1アッセイ |
JP2020528901A (ja) * | 2017-07-27 | 2020-10-01 | ノルディック・ビオサイエンス・エー/エスNordic Bioscience A/S | X型コラーゲンアルファ−1アッセイ |
JP7165718B2 (ja) | 2017-07-27 | 2022-11-04 | ノルディック・ビオサイエンス・エー/エス | X型コラーゲンアルファ-1アッセイ |
WO2020246563A1 (ja) | 2019-06-05 | 2020-12-10 | 中外製薬株式会社 | 抗体切断部位結合分子 |
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