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WO2019189882A1 - Anticorps monoclonal réagissant de manière spécifique avec un antigène ncc-st-439 et procédé de production associé - Google Patents

Anticorps monoclonal réagissant de manière spécifique avec un antigène ncc-st-439 et procédé de production associé Download PDF

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WO2019189882A1
WO2019189882A1 PCT/JP2019/014335 JP2019014335W WO2019189882A1 WO 2019189882 A1 WO2019189882 A1 WO 2019189882A1 JP 2019014335 W JP2019014335 W JP 2019014335W WO 2019189882 A1 WO2019189882 A1 WO 2019189882A1
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Prior art keywords
antigen
antibody
ncc
sugar chain
present
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Japanese (ja)
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知啓 三浦
宮崎　修
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Sekisui Medical Co Ltd
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Sekisui Medical Co Ltd
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Priority claimed from CN201811031188.0A external-priority patent/CN110317274A/zh
Application filed by Sekisui Medical Co Ltd filed Critical Sekisui Medical Co Ltd
Priority to CN201980022654.5A priority Critical patent/CN112424232A/zh
Priority to US17/043,287 priority patent/US20210017290A1/en
Priority to JP2020509355A priority patent/JPWO2019189882A1/ja
Priority to EP19776519.1A priority patent/EP3778645A4/fr
Publication of WO2019189882A1 publication Critical patent/WO2019189882A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a monoclonal antibody that specifically reacts with the NCC-ST-439 antigen and a method for producing the same.
  • NCC-ST-439 antigen recognized by an antibody recognizing esophageal cancer tissue is a sugar chain antigen containing sialic acid, and is known to be elevated in gastric cancer, lung cancer, breast cancer, pancreatic cancer and the like (Non-patent Document 1). .
  • the monoclonal antibody DUPAN-2 prepared using human pancreatic cancer cell culture HPAF-1 as an immunizing antigen is known to react with a mucin-like protein produced at a higher rate than pancreatic adenocarcinoma cells. Is considered to be related to sialic acid (Non-Patent Documents 2 and 3).
  • This antigen is called DUPAN-2 antigen and is used as a marker for digestive system cancer such as pancreatic cancer and biliary tract cancer.
  • antibodies that recognize sugar chain antigens used as tumor markers are generally isolated as those that recognize specific cancer cells as antigens, and their antigen specificity is not sufficient. Can react with a plurality of sugar chain antigens. Detection of a tumor marker using such an antibody has a problem that detection specificity is lowered and accuracy of cancer diagnosis is lowered.
  • An object of the present invention is to provide an antibody that specifically reacts with the sugar chain of NCC-ST-439 antigen used as a tumor marker and a method for producing the same.
  • the present inventors succeeded in obtaining an antibody that specifically recognizes the NCC-ST-439 antigen sugar chain itself as an antigen.
  • the antibody obtained by this method recognizes the NCC-ST-439 antigen highly specifically among mucin antigens used as various tumor markers and does not react with other tumor marker antigens such as DUPAN-2.
  • this invention provides the following in one aspect
  • [5] A method for producing the antibody or antigen-binding fragment thereof according to any one of [1] to [4], The method comprising the step of immunizing an animal with the polymer compound bound with the NCC-ST-439 antigen.
  • [6] A method for producing a cell that produces the antibody or antigen-binding fragment thereof according to any one of [1] to [4], The method comprising the step of immunizing an animal with the polymer compound bound with the NCC-ST-439 antigen.
  • [7] The method described in [5] or [6] above, wherein the polymer compound bound to the NCC-ST-439 antigen does not contain the peptide portion of the NCC-ST-439 antigen-binding peptide.
  • An immunoassay method comprising using the antibody or antigen-binding fragment thereof according to any one of [1] to [3].
  • An immunoassay reagent comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [3].
  • the antibody of the present invention recognizes the NCC-ST-439 antigen highly specifically among mucin antigens used as tumor markers and does not react with other tumor marker antigens such as DUPAN-2. Therefore, cancer cells expressing the NCC-ST-439 antigen, such as pancreatic cancer cells, can be specifically detected, and a more accurate cancer diagnosis is possible.
  • FIG. 1 is a diagram showing an outline of a method for producing an antibody that specifically reacts with the NCC-ST-439 antigen.
  • FIG. 2 is a diagram showing the test results of the antigen-immobilized ELISA method.
  • FIG. 3 is a figure which shows the result of the epitope analysis of the monoclonal antibody of this invention.
  • FIG. 4a is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention.
  • FIG. 4b is a diagram showing the result of specificity analysis of the monoclonal antibody of the present invention.
  • FIG. 4c is a diagram showing the results of the specificity analysis of the monoclonal antibody of the present invention.
  • FIG. 1 is a diagram showing an outline of a method for producing an antibody that specifically reacts with the NCC-ST-439 antigen.
  • FIG. 2 is a diagram showing the test results of the antigen-immobilized ELISA method.
  • FIG. 3 is a figure which shows the result of the epi
  • FIG. 4d is a diagram showing the result of specificity analysis of the monoclonal antibody of the present invention.
  • FIG. 4e shows the results of specificity analysis of the monoclonal antibody of the present invention.
  • FIG. 5 is a diagram showing a schematic diagram of the sugar chain used for the specificity evaluation of the monoclonal antibody of the present invention.
  • FIG. 6a is a view showing the result of a specificity analysis of a monoclonal antibody S18201R of the present invention using a free purified sugar chain.
  • FIG. 6b is a view showing the result of a specificity analysis of the monoclonal antibody S1822R of the present invention using a free purified sugar chain.
  • FIG. 6c is a view showing the results of a specificity analysis of the monoclonal antibody S18203R of the present invention using a free purified sugar chain.
  • FIG. 6d is a view showing the result of a specificity analysis of a monoclonal antibody S18204R of the present invention using a free purified sugar chain.
  • FIG. 7a is a graph showing the reactivity of the monoclonal antibody S18201R of the present invention against NCC-ST-439 standard products.
  • FIG. 7b shows the reactivity of the monoclonal antibody S1822R of the present invention against NCC-ST-439 standard.
  • FIG. 7c is a graph showing the reactivity of the monoclonal antibody S18203R of the present invention with respect to NCC-ST-439 standard products.
  • FIG. 7d shows the reactivity of the monoclonal antibody S18204R of the present invention against NCC-ST-439 standard.
  • FIG. 7e is a graph showing the correlation between the absorbance measured using the monoclonal antibody S18201R of the present invention and the value of NCC-ST-439 measured with a commercially available ELISA kit in a test using cancer patient serum. .
  • an antibody and a compound “react” can be confirmed by an antigen-immobilized ELISA method, a competitive ELISA method, a sandwich ELISA method or the like well-known to those skilled in the art, as well as surface plasmon resonance (surface plasmon resonance). It can be performed by a method using the principle of resonance (SPR method).
  • the SPR method can be performed using an apparatus, a sensor, and reagents that are commercially available under the name Biacore (registered trademark).
  • “Substantially does not react” means that, for example, in the antigen-immobilized ELISA method, the binding between the antibody and the immobilized antigen is not substantially affected by the addition of the compound. It can be confirmed that "substantially does not react” by methods and means well known to those skilled in the art other than the above-described antigen-immobilized ELISA method.
  • an antibody “reacts specifically”, or “specificity” of an antibody is the ability of the antibody to detectably react with an epitope presented on an antigen, while other antigens The detectable reactivity with is relatively small or substantially no reactivity is detected. For example, when an antibody “reacts specifically” with a particular antigen, the antibody reacts with the antigen but does not react with other antigens. In a preferred embodiment, when an antibody “reacts specifically” with a specific antigen, for example, the interaction between the antibody immobilized on the antigen-immobilized ELISA method and the antibody is inhibited by the free antigen. It is not inhibited by other free antigens.
  • IC 50 of nonspecific antigen when showing the inhibition by the antigen-immobilized ELISA method with an IC 50 of free antigen for IC 50 of the specific antigen, IC 50 of nonspecific antigen, 10-fold, 100-fold, It may be 200 times, 300 times, 400 times, 500 times, 1000 times, or 10,000 times.
  • the IC 50 of a specific antigen when the IC 50 of a specific antigen is 1 / X of that of another antigen, the reactivity of the specific antigen can be expressed as X times the reactivity with respect to the other antigen.
  • the reactivity of the other antigen is less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% to the specific antigen.
  • the antibody of the invention reacts with the NCC-ST-439 antigen and does not react with the DUPAN-2 antigen.
  • antibody refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (H) and two light chains (L) linked together by disulfide bonds.
  • Each heavy chain comprises a changeable region of the heavy chain ( "HCVR” or “VH”) and a heavy chain constant region (including CH 1, CH 2 and CH 3 domains).
  • Each light chain includes a light chain changeable region (“LCVR” or “VL”) and a light chain constant region (CL).
  • the VH and VL regions can be further divided into hypermutable regions termed complementarity determining regions (CDRs) and interspersed in many conserved regions termed frameworks (FR).
  • CDRs complementarity determining regions
  • Each VH and VL contains 3 CDRs and 4 FRs and is arranged from the amine terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the changeable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the term “antibody” also includes all genetically modified antibodies, eg, prokaryotic expressed antibodies, non-glycosylated antibodies.
  • an “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, NCC-ST-439).
  • binding fragments encompassed within an “antigen-binding fragment” of an antibody are: (i) a Fab fragment that is a monovalent fragment consisting of VL, VH, CL, and CH domains; (ii) hinge region A F (ab ′) 2 fragment, which is a divalent fragment comprising two Fab fragments joined by a disulfide bridge in FIG.
  • An “antigen-binding fragment” also includes (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) a CH2 constant.
  • a binding domain immunoglobulin fusion protein comprising an immunoglobulin heavy chain CH3 constant region fused to the region.
  • the antibody or antigen-binding fragment thereof that can be used in the present invention may be of any animal origin including birds and mammals.
  • the antibody or fragment is human, chimpanzee, rodent (eg, mouse, rat, guinea pig or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat or dog. Is the origin.
  • the antibodies of the present invention comprise chimeric molecules in which the constant region of an antibody derived from one species is combined with an antigen binding site derived from another species.
  • the antibodies of the invention include humanized molecules that combine antigen-binding sites of antibodies derived from non-human species (eg, mouse origin) and constant and framework regions of human origin.
  • the antibody of the present invention can be obtained from a hybridoma that expresses the antibody or a host cell that expresses the antibody by genetic recombination.
  • host cells for example, CHO cells, lymphocyte cells, bacterial cells such as E. coli, and fungal cells such as yeast can be used.
  • the antibody of the present invention can be produced in a non-human animal or plant that has been gene-transferred using a gene recombination technique.
  • hybridoma S18201R a monoclonal antibody produced by hybridoma S18201R, hybridoma S18202R, hybridoma S18203R or hybridoma S18204R is preferable.
  • the hybridoma is deposited internationally based on the Budapest Treaty as follows.
  • alkyl or alkyl group may be any of an aliphatic hydrocarbon group composed of a straight chain, a branched chain, a ring, or a combination thereof.
  • the number of carbon atoms of the alkyl group is not particularly limited, and examples thereof include 1 to 20 carbon atoms (C1 to 20), 1 to 15 carbon atoms (C1 to 15), and 1 to 10 carbon atoms (C1 to 10). obtain.
  • the alkyl group may have one or more arbitrary substituents.
  • C1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n -Heptyl, n-octyl and the like are included.
  • substituents examples include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or Although acyl etc. can be mentioned, it is not limited to these. When the alkyl group has two or more substituents, they may be the same or different.
  • alkylene means a divalent group consisting of a linear or branched saturated hydrocarbon.
  • a functional group when a functional group is defined as “may be substituted”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different.
  • the substituent group include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group.
  • NCC-ST-439 antigen is reported to be recognized by the NCC-ST-439 antibody (Kumamoto. K. et. Al. Biochem. Biophys. Res. Commun. (1998), 247. (2): 514-17) Means a sugar chain having the following structure.
  • DUPAN-2 antigen has been reported to be recognized by the DUPAN-2 antibody (Kawa. S. et al. Pancreas (1994), 9 (6): 692-697). It means a sugar chain having
  • NCC-ST-439 antigen-binding peptide means a peptide to which an NCC-ST-439 antigen is bound, which is found in a tumor cell or the like in a living body.
  • Antibodies that specifically recognize the NCC-ST-439 antigen of the present invention are obtained by the method outlined in FIG. Specifically, while the conventional tumor marker-reactive antibody was isolated using the tumor cell itself as an antigen (FIG. 1A), the NCC-ST-439 antigen according to the present invention was specifically used. In the method of producing a reactive antibody (FIG. 1B), a sugar chain constituting the NCC-ST-439 antigen is supported on a polymer compound via a linker, and this is immunized to a mammal such as a mouse.
  • a hybridoma is prepared by extracting spleen cells or lymph node cells of the animal and fusing them with myeloma cells by a known method described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988)). To do. From the prepared hybridoma cell population, those producing antibodies that react specifically with cancer cells are isolated. In the conventional method, it was necessary to analyze the epitope after the antibody was finally obtained, whereas in the method of the present invention, since the immunogen and the epitope match, the antibody can be efficiently produced. An antibody that specifically reacts with a specific sugar chain antigen can be obtained. However, advanced techniques are required in sugar chain synthesis.
  • the structure of the linker is not particularly limited, and for example, a C1-C12 optionally substituted alkyl group, alkylene group, ethylene glycol, polyethylene glycol, amino acid, peptide and the like can be used.
  • the high molecular compound is not particularly limited. Proteins such as blast growth factor, transferrin, platelet-derived growth factor, poly-L-lysine, poly-L-glutamine can be used.
  • the polymer compound bound with the NCC-ST-439 antigen of the present invention may or may not contain a part of the NCC-ST-439 antigen-binding peptide.
  • the polymer compound to which the NCC-ST-439 antigen of the present invention is bound does not contain an NCC-ST-439 antigen-binding peptide moiety.
  • the hybridoma can be produced according to a method known in the art. For example, a polyethylene glycol method, a method using Sendai virus, a method using current, and the like can be employed.
  • the obtained hybridoma can be propagated according to a known method, and a desired hybridoma can be selected while confirming the properties of the produced antibody.
  • the hybridoma can be cloned by a known method such as a limiting dilution method or a soft agar method.
  • the obtained antibody can be obtained from the host cell by preparing a host cell that expresses the antibody by genetic recombination in addition to directly producing it from the hybridoma.
  • host cells for example, CHO cells, lymphocyte cells, bacterial cells such as E. coli, and fungal cells such as yeast can be used.
  • the above antibody production method is not limited to the NCC-ST-439 antigen, and can be used to produce an antibody specific to a sugar chain antigen of another known sugar chain protein.
  • a monoclonal antibody that specifically reacts with the above DUPAN-2 antigen could be prepared by the same method.
  • the antibody of the present invention can be used in an immunoassay method for detecting NCC-ST-439 antigen in a biological sample as a tumor marker.
  • various known methods for detecting mucin tumor markers in a biological sample using an antibody can be used, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Immunofluorescence, immunoprecipitation, equilibrium dialysis, immunodiffusion, and other techniques can be used, but are not limited to these (eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, DM, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston).
  • the antibody of the present invention can be used as an immobilized (solid phase) antibody immobilized on an insoluble carrier or a labeled antibody labeled with a labeling substance. Any of such immobilized antibodies and labeled antibodies are included in the scope of the present invention.
  • an immobilized antibody can be produced by physically adsorbing the antibody of the present invention to an insoluble carrier or by chemically binding it (may be via an appropriate spacer).
  • an insoluble carrier an insoluble carrier made of a polymer substrate such as polystyrene resin, an inorganic substrate such as glass, a polysaccharide substrate such as cellulose or agarose, or the like can be used.
  • the shape is not particularly limited, and any shape such as a plate shape (for example, a microplate or a membrane), a bead or fine particle shape (for example, latex particles or magnetic particles), or a tubular shape (for example, a test tube) can be selected.
  • a plate shape for example, a microplate or a membrane
  • a bead or fine particle shape for example, latex particles or magnetic particles
  • a tubular shape for example, a test tube
  • Examples of the labeling substance for producing the labeled antibody include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, or radioisotopes, colloidal gold particles, and colored latex.
  • methods such as a glutaraldehyde method, a maleimide method, a pyridyl disulfide method, or a periodic acid method that can be used by those skilled in the art can be used.
  • an enzyme such as peroxidase or alkaline phosphatase (hereinafter sometimes referred to as ALP) can be used as the labeling substance. .
  • the enzyme when the enzyme is a specific substrate (horseradish peroxidase (hereinafter sometimes referred to as HRP)), for example, 1,2-phenylenediamine (hereinafter sometimes referred to as OPD) or 3, 3
  • HRP horseradish peroxidase
  • OPD 1,2-phenylenediamine
  • 3 3
  • the enzyme activity can be measured using p-nitrophenyl phosphate or the like.
  • biotin is used as a labeling substance, avidin or enzyme-modified avidin is generally reacted.
  • the antibody of the present invention can be provided as an immunoassay reagent for use in the above immunoassay method.
  • the reagent may contain other components necessary for carrying out the immunoassay method, such as a buffer solution and a preservative.
  • the antibody of the present invention when it contains an enzyme as a labeling substance, it may be provided in the form of a kit together with a reagent containing the specific substrate.
  • NCC-ST-439 modified maleimide for immunogen or NCC-ST-439 glycosylated peptide cross-linked protein cross-linking of sugar chain-modified maleimide reduces human transferrin, and reduces reduced human transferrin and sugar chain maleimide by weight.
  • PBS PBS at a ratio of 4: 1
  • maleimide-activated OVA and glycopeptide were mixed at a weight ratio of 4: 1 in PBS, reacted at room temperature for 2 hours, and the buffer was replaced with PBS by dialysis to obtain a conjugate solution for immunization.
  • PBS PH 7.2; hereinafter referred to as PBS
  • 50 ⁇ L of the lysate was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours or at 4 ° C. overnight.
  • Each well was washed three times with 400 ⁇ L of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), and then 100 ⁇ L of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added. Blocking was performed for 1 hour at room temperature or overnight at 4 ° C. This was used as an ELISA plate.
  • PBST PBS containing 0.05% Tween® 20
  • BSA-PBST bovine serum albumin
  • hybridoma antigen-immobilized ELISA method
  • the same method was performed except that the culture supernatant of the fused cells was used instead of the immunized animal antiserum.
  • a well having a high absorbance was selected as a well in which an anti-NCC-ST-439 antibody-producing hybridoma was present (positive well).
  • S18201R The monoclonal antibodies produced by these four types of hybridomas S18201R, S1822R, S1823R, and S18204R are referred to as S18201R antibody, S1822R antibody, S18203R antibody, and S18204R antibody, respectively.
  • Test Method S18201R, S1822R, S18203R, S18204R It was confirmed by competitive ELISA described below whether the epitope of NCC-ST-439 that reacts with the antibody is similar to that of the existing antibody.
  • an ELISA plate was prepared in the same manner as the above-described antigen-immobilized ELISA method.
  • each well was washed three times with 400 ⁇ L of PBST, 50 ⁇ L of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 ⁇ L of 1.5 N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.
  • citrate buffer pH 5.0
  • the decrease in absorbance due to the addition of S18201R, S1822R, S1823R, and S18204R antibodies means that the S18201R, S1822R, S1823R, and S18204R antibodies in the solution are bound in the vicinity of the binding site between the existing antibody and the conjugate. It shows that the binding of the conjugated to the conjugate is inhibited. Therefore, it was found that the S18201R, S1822R, S1823R, and S18204R antibodies recognize an epitope similar to that of the existing antibody.
  • Test Example 3 Specificity analysis of the monoclonal antibody of the present invention 1. The specificity of the test methods S18201R, S1822R, S18203R, S18204R antibodies was confirmed by competitive ELISA described below. First, an ELISA plate was prepared in the same manner as the above-described antigen-immobilized ELISA method.
  • the wells into which the S18201R, S1822R, S1823R, S18204R antibody-producing hybridoma culture supernatant and each sugar chain-related compound mixture were dispensed were washed three times with PBST 400 ⁇ L, and then HRP diluted 5000 times with BSA-PBST.
  • Labeled rat IgG (H & L) was dispensed at 50 ⁇ L / well and allowed to stand at room temperature for 1 hour.
  • 50 ⁇ L / well of a mixture of the existing antibody and each sugar chain-related compound was dispensed into wells into which BSA-PBST was dispensed at 50 ⁇ L / well, and allowed to stand at room temperature for 1 hour.
  • each well was washed three times with 400 ⁇ L of PBST, 50 ⁇ L of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 ⁇ L of 1.5 N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.
  • citrate buffer pH 5.0
  • the absorbance depends on the amount of antibody bound to the conjugate of NCC-ST-439 and BSA immobilized on the plate.
  • the decrease in absorbance due to the addition of each sugar chain-related compound means that the free sugar chain-related compound in the solution reacts with the antibody in the solution and inhibits the binding between the antibody and the immobilized conjugate. It shows that Therefore, it is shown that the S18201R, S1822R, S1823R, and S18204R antibodies do not react with DUPAN-2 but react specifically with NCC-ST-439.
  • test methods S18201R, S1822R, S18203R, S18204R antibodies was confirmed by competitive ELISA described below.
  • an ELISA plate was prepared in the same manner as the aforementioned antigen-immobilized ELISA method. Further, S18201R, S1822R, S1823R, and S18204R antibodies diluted to 225 to 500 ng / mL using BSA-PBST as a solvent were mixed with sugar chains (FIG. 5) serially diluted with BSA-PBST. These are mixed liquids.
  • Each well of the ELISA plate was washed 3 times with 400 ⁇ L of PBST, and 50 ⁇ L / well of each of the above-described hybridoma culture supernatants of S18201R, S1822R, S1823R, and S18204R antibody-producing hybridomas and each sugar chain was dispensed at room temperature. Let stand for hours. After washing 3 times with 400 ⁇ L of PBST, HRP-labeled rat IgG (H & L) diluted 5000 times with BSA-PBST was dispensed at 50 ⁇ L / well and allowed to stand at room temperature for 1 hour.
  • each well was washed three times with 400 ⁇ L of PBST, 50 ⁇ L of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 ⁇ L of 1.5 N sulfuric acid was added to stop the enzyme reaction, the absorbance at a wavelength of 492 nm was measured, and the reaction rate was calculated with the absorbance when no sugar chain was added as 100%.
  • citrate buffer pH 5.0
  • Each well was washed 3 times with 400 ⁇ L PBST, and then biotinylated S18201R, S1822R, S18203R, S18204R antibodies diluted to 2 ⁇ g / mL with BSA-PBST were dispensed into the wells on the same plate with 50 ⁇ L each well. And allowed to stand at room temperature for 1 hour.
  • Each well was washed with 400 ⁇ L of PBST three times, and then 50 ⁇ L of HRP-labeled streptavidin diluted to 0.2 ⁇ g / mL with BSA-PBST was dispensed into each well and allowed to stand at room temperature for 1 hour.
  • the test results are shown in FIGS. 7a, b, c, d, and e.
  • the absorbance increases depending on the NCC-ST-439 standard product concentration (FIGS. 7a, b, c and d). That is, this indicates that the antibody is reacting with the NCC-ST-439 antigen contained in the standard product.
  • the absorbance when using serum from cancer patients in this study was proportional to the value of NCC-ST-439 measured with a commercial ELISA kit as an approximate line with a correlation coefficient of 0.99 or more (FIG. 7e). That is, it was shown that the antibody of the present invention can measure NCC-ST-439 in cancer patient serum in the same manner as the existing NCC-ST-439 antibody.

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Abstract

L'objectif de la présente invention est de fournir un anticorps classique reconnaissant un antigène de chaîne saccharidique, ledit antigène de chaîne saccharidique devant être utilisé en tant que marqueur tumoral, présentant une spécificité antigénique insuffisante. Le problème à résoudre par la présente invention se rapporte audit anticorps et concerne la spécificité de détection qui est abaissée, lorsqu'un tel anticorps est utilisé dans la détection d'un marqueur tumoral réduisant ainsi la précision du diagnostic du cancer. La solution selon la présente invention porte sur un anticorps qui réagit de manière spécifique avec une chaîne saccharidique de l'antigène NCC-ST-439, destiné à être utilisé en tant que marqueur tumoral ainsi qu'un procédé de production de l'anticorps.
PCT/JP2019/014335 2018-03-30 2019-03-29 Anticorps monoclonal réagissant de manière spécifique avec un antigène ncc-st-439 et procédé de production associé Ceased WO2019189882A1 (fr)

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CN201980022654.5A CN112424232A (zh) 2018-03-30 2019-03-29 与ncc-st-439抗原特异性反应的单克隆抗体及其制造方法
US17/043,287 US20210017290A1 (en) 2018-03-30 2019-03-29 Monoclonal antibody specifically reacting with ncc-st-439 antigen and method for producing same
JP2020509355A JPWO2019189882A1 (ja) 2018-03-30 2019-03-29 Ｎｃｃ−ｓｔ−４３９抗原と特異的に反応するモノクローナル抗体およびその製造方法。
EP19776519.1A EP3778645A4 (fr) 2018-03-30 2019-03-29 Anticorps monoclonal réagissant de manière spécifique avec un antigène ncc-st-439 et procédé de production associé

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CN110317274A (zh) * 2018-03-30 2019-10-11 积水医疗株式会社 与ncc-st-439抗原特异性反应的单克隆抗体及其制造方法
CN112424232A (zh) * 2018-03-30 2021-02-26 积水医疗株式会社 与ncc-st-439抗原特异性反应的单克隆抗体及其制造方法

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