A monoclonal antibody that specifically reacts with the NCC-ST-439 antigen, and a method for producing the same.

Abstract

【assignment】 Antibodies that recognize carbohydrate antigens used as tumor markers do not have sufficient antigen specificity, and the detection of tumor markers using such antibodies has the problem of reduced detection specificity and reduced accuracy in cancer diagnosis. [Solution] In order to solve the above problems, the present invention provides an antibody that specifically reacts with the sugar chain of the NCC-ST-439 antigen used as a tumor marker, and a method for producing the antibody.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody that specifically reacts with the NCC-ST-439 antigen and a method for producing the same.

Background Art

[0002] In recent years, many antibodies against carbohydrate antigens used as tumor markers have been reported. The NCC-ST-439 antigen recognized by an antibody that recognizes esophageal cancer tissue is a carbohydrate antigen containing sialic acid, and is known to increase in gastric cancer, lung cancer, breast cancer, pancreatic cancer, etc. (Non-Patent Document 1).

[0003] In addition, the monoclonal antibody DUPAN-2 prepared using human pancreatic cancer cultured cells HPAF-1 as an immunizing antigen is known to react with a mucin-like protein produced at a higher rate in pancreatic cancer cells, and sialic acid is considered to be involved in its antigenic determinant (Non-Patent Documents 2 and 3). This antigen is called the DUPAN-2 antigen and is particularly used as a marker for digestive system cancers such as pancreatic cancer and biliary tract cancer.

[0004] On the other hand, antibodies that recognize carbohydrate antigens used as these tumor markers are generally isolated as those that recognize the entire specific cancer cells as an antigen, and their antigen specificity is not sufficient, and they can react with a plurality of carbohydrate antigens. In the detection of tumor markers using such antibodies, there is a problem that the detection specificity decreases and the accuracy of cancer diagnosis decreases.

[0005] Therefore, in order to perform more accurate diagnosis using tumor markers, it has been necessary to obtain an antibody that specifically reacts with a specific carbohydrate antigen.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] The present invention aims to provide an antibody that specifically reacts with the glycans of the NCC-ST-439 antigen, which is used as a tumor marker, and a method for producing the same. [Means for solving the problem]

[0008] The inventors of this invention conducted intensive research to solve the above problems and succeeded in obtaining an antibody that specifically recognizes the NCC-ST-439 antigen by using the sugar chain of the NCC-ST-439 antigen itself as the antigen. The antibody obtained by this method recognizes the NCC-ST-439 antigen with high specificity among mucin antigens used as various tumor markers and does not react with other tumor marker antigens such as DUPAN-2.

[0009] In other words, the present invention provides the following in one embodiment. [1] NCC-ST-439 antigen having the following glycosylation structure It responded with TIFF0007893544000001.tif1973 and DUPAN-2 antigen having the following sugar chain structure Antibodies and their antigen-binding fragments that do not react with TIFF0007893544000002.tif867. [2] The antibody or antigen-binding fragment described in [1] above, wherein the reactivity to the DUPAN-2 antigen is less than 10% of the reactivity to the NCC-ST-439 antigen. [3] The antibody or antigen-binding fragment described in [1] above, wherein the reactivity to the DUPAN-2 antigen is less than 1% of the reactivity to the NCC-ST-439 antigen. [4] A rat antibody, the antibody or antigen-binding fragment described in any one of the above [1] to [3]. [5] A method for producing an antibody or antigen-binding fragment according to any one of the above items [1] to [4], The method comprising the step of immunizing an animal with a polymer compound to which the NCC-ST-439 antigen is conjugated. [6] A method for producing cells that produce an antibody or antigen-binding fragment according to any one of the above items [1] to [4], The method comprising the step of immunizing an animal with a polymer compound to which the NCC-ST-439 antigen is conjugated. [7] The method according to [5] or [6], 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. [8] An immunoassay method characterized by using an antibody or antigen-binding fragment described in any one of the above items [1] to [3]. [9] An immunoassay reagent comprising the antibody or antigen-binding fragment described in any one of the above items [1] to [3]. [Effects of the Invention]

[0010] The antibody of the present invention highly specifically recognizes the NCC-ST-439 antigen among mucin antigens used as tumor markers, and does not react with other tumor marker antigens such as DUPAN-2. Therefore, it is possible to specifically detect cancer cells expressing the NCC-ST-439 antigen, such as pancreatic cancer cells, enabling more accurate cancer diagnosis.

[0011] Furthermore, by utilizing the method for producing antibodies that specifically recognize mucin antigens used as tumor markers in the present invention, it becomes possible to obtain antibodies that react with a high degree of specificity to specific tumor markers. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a diagram showing an outline of a method for producing an antibody that specifically reacts with the NCC-ST-439 antigen. [Figure 2] Figure 2 is a diagram showing the test results of the antigen immobilized ELISA method. [Figure 3] Figure 3 is a diagram showing the results of epitope analysis of the monoclonal antibody of the present invention. [Figure 4a] Figure 4a is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention. [Figure 4b] Figure 4b is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention. [Figure 4c] Figure 4c is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention. [Figure 4d] Figure 4d is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention. [Figure 4e] Figure 4e is a diagram showing the results of specificity analysis of the monoclonal antibody of the present invention. [Figure 5] Figure 5 is a schematic diagram of the sugar chain used for the specificity evaluation of the monoclonal antibody of the present invention. [Figure 6a] Figure 6a is a diagram showing the results of specificity analysis of the monoclonal antibody S18201R of the present invention using free purified sugar chains. [Figure 6b] Figure 6b is a diagram showing the results of specificity analysis of the monoclonal antibody S18202R of the present invention using free purified sugar chains. [Figure 6c] Figure 6c is a diagram showing the results of specificity analysis of the monoclonal antibody S18203R of the present invention using free purified sugar chains. [Figure 6d] Figure 6d is a diagram showing the results of specificity analysis of the monoclonal antibody S18204R of the present invention using free purified sugar chains. [Figure 7a] Figure 7a is a diagram showing the reactivity of the monoclonal antibody S18201R of the present invention with the NCC-ST-439 standard product. [Figure 7b] Figure 7b shows the reactivity of the monoclonal antibody S18202R of the present invention with respect to the NCC-ST-439 standard. [Figure 7c] Figure 7c shows the reactivity of the monoclonal antibody S18203R of the present invention with respect to the NCC-ST-439 standard. [Figure 7d] Figure 7d shows the reactivity of the monoclonal antibody S18204R of the present invention to the NCC-ST-439 standard. [Figure 7e] Figure 7e shows the correlation between absorbance measured using the monoclonal antibody S18201R of the present invention in a test using serum from cancer patients and the NCC-ST-439 value measured using a commercially available ELISA kit. [Modes for carrying out the invention]

[0013] Embodiments of the present invention will be described below. The following description is merely illustrative, and the scope of the present invention is not limited to this description. The invention may be modified and implemented as appropriate without impairing its spirit.

[0014] (definition) Unless otherwise specified, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which the present invention belongs.

[0015] In this specification, when multiple numerical ranges are given, a range consisting of any combination of the lower and upper limits of those ranges is also meant.

[0016] In this specification, when an antibody and a certain compound are described as "reacting," "showing reactivity," "having reactivity," "binding," or when an antibody "recognizes" a certain compound, these terms include the meanings commonly used in the field of this invention, and are all used synonymously. Whether or not an antibody "reacts" with a certain compound can be confirmed by methods well known to those skilled in the art, such as antigen-immobilized ELISA, competitive ELISA, or sandwich ELISA, as well as by methods utilizing the principle of surface plasmon resonance (SPR method). The SPR method can be performed using equipment, sensors, and reagents commercially available under the name Biacore®.

[0017] In this specification, "not reacting" between the antibody of the present invention and a certain compound means that the antibody of the present invention and the certain compound do not react substantially. "Substantially not reacting" means, for example, in an antigen-immobilized ELISA method, that the addition of the compound does not substantially affect the binding of the antibody to the immobilized antigen. "Substantially not reacting" can also be confirmed by methods and means well known to those skilled in the art other than the antigen-immobilized ELISA method described above.

[0018] In this specification, "specific reaction" of an antibody, or "specificity" of an antibody, means the ability of the antibody to react to an epitope detected on an antigen, while having relatively little or virtually no detectable reactivity with other antigens. For example, when an antibody "specifically reacts" to a particular antigen, the antibody reacts to that antigen but not to other antigens. In a preferred embodiment, when an antibody "specifically reacts" to a particular antigen, for example, in an antigen-immobilized ELISA, the interaction between the antibody and the immobilized antigen is inhibited by the free antigen but not by other free antigens. For example, the inhibition by the antigen-immobilized ELISA is controlled by the IC of a free antigen. 50 When expressed as such, the IC of the specific antigen 50 In contrast, IC for nonspecific antigens 50The dilution ratio may be 10x, 100x, 200x, 300x, 400x, 500x, 1000x, or 10000x. Also, the IC of the specific antigen may be... 50 If the reactivity of the specific antigen is 1 / X of that of other antigens, the reactivity of the specific antigen can also be expressed as X times the reactivity to other antigens. Preferably, the reactivity of other antigens is 20%, 15%, 10%, 5%, 4%, 3%, 2%, or less than 1% of that of the specific antigen. In a preferred embodiment of the present invention, the antibody of the present invention reacts with the NCC-ST-439 antigen but does not react with the DUPAN-2 antigen.

[0019] In this specification, “antibody” means 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 modifiable region (“HCVR” or “VH”) and a constant region (containing CH1, CH2, and CH3 domains). Each light chain comprises a modifiable region (“LCVR” or “VL”) and a constant region (CL). The VH and VL regions may be further divided into hypervariable regions designated as complementarity-determining regions (CDRs) and scattered within highly conservable regions designated as frameworks (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amine-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The modifiable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "antibody" also includes all genetically modified antibodies, such as antibodies expressed in prokaryotes and non-glycosylated antibodies.

[0020] Furthermore, as shown in Padlan (1995 FASEB J. 9:133-139) and Vajdos et al. 2002 J Mol Biol 320:415-428, it is known that only a portion of the CDR residues actually come into contact with the antigen, and the CDR residues that do not come into contact with the antigen can be identified by molecular modeling or empirically from the region of the Kabat CDR located outside the Chothia CDR. When a CDR or one or more residues thereof are removed, they are usually replaced by an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such a sequence. The CDR and the position of substitution within the amino acid can also be selected empirically. The empirical substitution may be conserved or non-conserved.

[0021] In this specification, the “antigen-binding fragment” of an antibody means one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., NCC-ST-439). Non-limiting examples of binding fragments contained within the “antigen-binding fragment” of an antibody include: (i) a monovalent fragment consisting of VL, VH, CL, and CH domains, the Fab fragment; (ii) a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, the F(ab′)2 fragment; (iii) an Fd fragment consisting of VH and CH domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs that may optionally be linked to a synthetic linker. Furthermore, as known as single-chain Fv (scFv), it can also be produced as a single protein chain in which the VL and VH regions are paired by a synthetic linker using genetic recombination (Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). In addition, the "antigen-binding fragment" may be a binding domain immunoglobulin fusion protein containing (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) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. These antibody fragments can be obtained using prior art known to those skilled in the art.

[0022] The antibodies or antigen-binding fragments usable in the present invention may be of any animal origin, including birds and mammals. Preferably, the antibodies or fragments are of human, chimpanzee, rodent (e.g., mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel, cattle, horse, donkey, cattle, or dog origin. The antibodies of the present invention include a chimeric molecule in which the constant region of an antibody derived from one species is combined with an antigen-binding site derived from another species. Furthermore, the antibodies of the present invention include a humanized molecule in which the antigen-binding site of an antibody derived from a non-human species (e.g., mouse origin) is combined with a constant region and framework region of human origin.

[0023] The antibody of the present invention can be obtained from a hybridoma expressing the antibody, or from a host cell expressing the antibody through genetic recombination. As host cells, for example, CHO cells, lymphocytes, bacterial cells such as Escherichia coli, and fungal cells such as yeast can be used.

[0024] Furthermore, the antibodies of the present invention can be produced in non-human animals or plants that have been genetically modified using genetic engineering technology.

[0025] In the present invention, monoclonal antibodies produced by hybridoma S18201R, hybridoma S18202R, hybridoma S18203R, or hybridoma S18204R are preferred as antibodies. The above hybridomas are internationally deposited under the Budapest Convention as described below.

[0026] Name of depositary institution: National Institute of Technology and Evaluation, Patent Microbial Depository Center; Address of depositary institution: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818); Date of deposit: May 17, 2018. Accession number: NITE BP-02717 (Hybridoma S18201R) Accession number: NITE BP-02718 (Hybridoma S18202R) Accession number: NITE BP-02719 (Hybridoma S18203R) Accession number: NITE BP-02720 (Hybridoma S18204R)

[0027] In this specification, "alkyl or alkyl group" may be any aliphatic hydrocarbon group consisting of a linear, branched, cyclic, or combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but for example, it may have 1 to 20 carbon atoms (C1 to 20), 1 to 15 carbon atoms (C1 to 15), or 1 to 10 carbon atoms (C1 to 10).

[0028] The alkyl group may have one or more substituents. For example, C1-8 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, etc. Examples of substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or disubstituted amino groups, substituted silyl groups, or acyls. If the alkyl group has two or more substituents, they may be the same or different.

[0029] In this specification, "alkylene" means a divalent group consisting of a linear or branched saturated hydrocarbon.

[0030] In this specification, when a functional group is defined as "may be substituted," the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and if there are two or more substituents, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may have further substituents. Examples of such substituents include, but are not limited to, alkyl halides.

[0031] In this specification, "NCC-ST-439 antigen" refers to a glycan having the following structure, which has been reported to be recognized by the NCC-ST-439 antibody (Kumamoto. K. et.al. Biochem. Biophys. Res. Commun. (1998), 247(2): 514-17). TIFF0007893544000003.tif1770

[0032] In this specification, "DUPAN-2 antigen" refers to a glycan having the following structure, which has been reported to be recognized by the DUPAN-2 antibody (Kawa. S. et.al. Pancreas (1994), 9(6): 692-697). In this specification, "NCC-ST-439 antigen-binding peptide" means a peptide to which the NCC-ST-439 antigen is bound, as found in tumor cells and other organisms in the body.

[0033] (Method for producing antibodies that specifically react with the NCC-ST-439 antigen) The antibody that specifically recognizes the NCC-ST-439 antigen according to the present invention is obtained by the method outlined in Figure 1. Specifically, while conventional tumor marker-reactive antibodies were isolated using tumor cells themselves as antigens (Figure 1A), the method for producing an antibody that specifically reacts with the NCC-ST-439 antigen according to the present invention (Figure 1B) involves supporting the glycans constituting the NCC-ST-439 antigen on a high-molecular-weight compound via a linker, and immunizing mammals such as mice with this compound. Using known methods described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988)), etc., spleen cells or lymph node cells of the animal are excised and hybridomas are produced by cell fusion with myeloma cells. From the produced hybridoma cell population, those that produce antibodies that specifically react with cancer cells are isolated. Conventional methods require epitope analysis only after the antibody has been obtained. In contrast, the present invention allows for efficient antibody production because the immunogen and epitope are matched, enabling the acquisition of antibodies that specifically react with particular glycosylation antigens. However, advanced techniques are required for glycosylation synthesis.

[0034] The linker structure is not particularly limited, but for example, C1-C12 substituted alkyl groups, alkylene groups, ethylene glycol, polyethylene glycol, amino acids, peptides, etc. can be used.

[0035] The polymer compounds are not particularly limited, but for example, proteins such as albumin, ovalbumin, Pseudomonas aeruginosa exotoxin, tetanus toxin, lysine toxin, diphtheria toxin, cholera toxin, heat-labile enterotoxin, keyhole limpet hemocyanin, epidermal growth factor, fibroblast growth factor, transferrin, platelet-derived growth factor, poly-L-lysine, and poly-L-glutamine can be used.

[0036] The polymer compound to which the NCC-ST-439 antigen is bound in the present invention may or may not contain a portion of the NCC-ST-439 antigen-binding peptide. In a preferred embodiment, the polymer compound to which the NCC-ST-439 antigen is bound in the present invention does not contain the NCC-ST-439 antigen-binding peptide portion.

[0037] Hybridomas can be prepared according to methods known in the field, such as the polyethylene glycol method, the Sendai virus method, or the electric current method. The obtained hybridomas can be amplified according to known methods, and the desired hybridomas can be selected while confirming the properties of the antibodies produced. Hybridoma cloning can be performed by known methods such as the limiting dilution method or the soft agar method.

[0038] In addition to being produced directly from the hybridoma mentioned above, the antibodies can also be obtained by preparing host cells that express the antibodies through genetic recombination and then obtaining them from those host cells. Examples of host cells that can be used include CHO cells, lymphocytes, bacterial cells such as Escherichia coli, and fungal cells such as yeast.

[0039] Furthermore, the method for producing the antibody described above is not limited to the NCC-ST-439 antigen, but can be used to produce antibodies specific to the glycosylation antigens of other known glycosylation proteins. For example, a monoclonal antibody that specifically reacts with the DUPAN-2 antigen was produced using a similar method.

[0040] (Uses of the antibody of the present invention) The antibody of the present invention can be used in immunoassays for detecting the NCC-ST-439 antigen in biological samples as a tumor marker. Specifically, various known methods for detecting mucin tumor markers in biological samples using the antibody can be utilized, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, and other techniques, but are not limited to these (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, DM, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston). In one embodiment, the antibody of the present invention can be used as an immobilized (solid-phase) antibody immobilized on an insoluble carrier, or as a labeled antibody labeled with a labeling substance. Both such immobilized antibodies and labeled antibodies are included in the scope of the present invention. For example, an immobilized antibody can be produced by physically adsorbing or chemically binding (may be via a suitable spacer) the antibody of the present invention to an insoluble carrier. As the insoluble carrier, an insoluble carrier consisting of a polymer substrate such as polystyrene resin, an inorganic substrate such as glass, or a polysaccharide substrate such as cellulose or agarose can be used. Its shape is not particularly limited, and any shape can be selected, such as plate-like (e.g., microplates or membranes), beads or fine particles (e.g., latex particles or magnetic particles), or cylindrical (e.g., test tubes).

[0041] Examples of labeling substances for producing labeled antibodies include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, or radioisotopes, gold colloid particles, and colored latex. Methods for conjugating the labeling substance to the antibody include the glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid method, which are available to those skilled in the art. The types of immobilized antibodies and labeled antibodies, and their production methods, are not particularly limited; for example, enzymes such as peroxidase and alkaline phosphatase (hereinafter sometimes referred to as ALP) can be used as labeling substances. In this case, enzyme activity can be measured using a specific substrate of the enzyme (for example, if the enzyme is horseradish peroxidase (hereinafter sometimes referred to as HRP), for example, 1,2-phenylenediamine (hereinafter sometimes referred to as OPD) or 3,3',5,5'-tetramethylbenzidine; in the case of ALP, for example, p-nitrophenyl phosphate). When biotin is used as a labeling agent, it is common to react it with avidin or enzyme-modified avidin.

[0042] The antibody of the present invention may be provided as an immunoassay reagent for use in the immunoassay method described above. In addition to the antibody of the present invention, the reagent may also include other components necessary for carrying out the immunoassay method, such as a buffer solution and a preservative.

[0043] In one embodiment, for example, if the antibody of the present invention contains an enzyme as a labeling substance, it may be provided in the form of a kit together with a reagent containing its specific substrate. [Examples]

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0045] [Test Example 1] Production of NCC-ST-433 antigen-specific monoclonal antibody 1.Material (1) NCC-ST-439 modified maleimide, NCC-ST-439 glycopeptide, and various glycan-related compounds (2) Freund's complete adjuvant: Manufactured by Wako Pure Chemical Industries, Ltd., 014-09541 (3) Myeloma cells (SP2 / O) (4) RPMI1640, GlutaMAX: GIBCO, 61870-036 (5) Fetal Bovine Serum (FBS): Manufactured by Biological Industries, 04-001-1A (6) HAT culture medium: Cosmo Bio Co., Ltd., 16213004 (7) 96-hole plate: NUNC, 167008 (8) HRP-labeled goat anti-rat IgG (H & L) antibody: Southern Biotech, 3050-05 (9) "Ranazyme (registered trademark) ST-439 Plate": Kainos / Nippon Kayaku; contains HRP-labeled NCC-ST-439 antibody (existing antibody) and standard solution (NCC-ST-439 standard) as constituent reagents.

[0046] 2. Preparation of NCC-ST-439 modified maleimide or NCC-ST-439 glycosylated peptide crosslinked protein for immunogen use The crosslinking of glycosylated maleimide was performed by reducing human transferrin, mixing the reduced human transferrin with the glycosylated maleimide in a weight ratio of 4:1 in PBS, reacting at room temperature for 2 hours, and leaving it overnight at 4°C. Subsequently, the buffer was replaced with PBS by dialysis. For crosslinking of glycopeptides, maleimide-activated OVA and glycopeptides were mixed in PBS in a weight ratio of 4:1, reacted at room temperature for 2 hours, and the buffer was replaced with PBS by dialysis to obtain the immunoconjugate solution.

[0047] 3. Creation of hybridomas producing anti-NCC-ST-439 monoclonal antibodies. (3-1) Immunity to animals An emulsion prepared by mixing equal volumes of the aforementioned immunization conjugate solution (0.2-2 mg / ml) and Freund's complete adjuvant was injected into F344 rats at a dose of 10-40 μg per rat. This emulsion was then repeated 7-8 times at one-week intervals. Antibody titers in antiserum obtained from tail vein blood samples were measured using the antigen-immobilized ELISA method described later.

[0048] (3-2) Primary screening (antigen-immobilized ELISA method) The presence of anti-NCC-ST-439 antibodies in the antiserum of the immunized animals was confirmed by an ELISA method (antigen-immobilized ELISA) using a conjugate solution of NCC-ST-439 and BSA, prepared in the same manner as the immunization conjugate solution. Details of the antigen-immobilized ELISA method are as follows. (3-2-1) Preparation of antigen-immobilized ELISA plates The conjugate solution of NCC-ST-439 glycopeptide and BSA was dissolved in 20 mM phosphate buffer (pH 7.2; hereafter referred to as PBS) containing 150 mM sodium chloride to a concentration of 0.1 μg / mL. 50 μL of this solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours or overnight at 4°C. Each of the aforementioned wells was washed three times with 400 μL of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), then 100 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added, and the mixture was blocked at room temperature for 1 hour or overnight at 4°C. This was used as an ELISA plate. (3-2-2) Antigen-immobilized ELISA method After washing each well of the ELISA plate three times with 400 μL of PBST, 50 μL of immunized animal antiserum and non-immunized animal serum, diluted 1500 to 13500 times with BSA-PBST, were added to each well and allowed to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 50 μL of HRP-labeled rat IgG (H&L), diluted 5000 times with BSA-PBST, was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured. Based on the measurement results, the spleen or lymph nodes were removed from mice with high antibody titers, and spleen-derived cells or lymph node-derived cells were prepared and used for cell fusion.

[0049] (3-2) Test Results The results are shown in Figure 2. Compared to non-immunized animal serum, immunized animal antiserum consistently showed higher absorbance at lower dilution ratios. In this measurement system, absorbance depends on the concentration of antibodies against NCC-ST-439 contained in the animal serum. In other words, the results above indicate that immunized animal antiserum contains a higher concentration of antibodies against NCC-ST-439 and has a higher antibody titer compared to non-immunized animal serum. From rats with high antibody titers as a result of the measurement, the spleen or lymph nodes were excised, and spleen-derived cells or lymph node-derived cells were prepared and used for cell fusion.

[0050] (4)Cell fusion Either the spleen-derived cells or lymph node-derived cells were mixed with myeloma cells in a 1:1 ratio and electrofused. The fused cells were suspended in HAT medium and cultured in a CO2 incubator at 37°C and 5% CO2 for 8 days to obtain fused cells (hybridomas).

[0051] (5) Selection of hybridomas (antigen-immobilized ELISA method) The same procedure was followed as in the antigen-immobilized ELISA method described above, except that the culture supernatant of fusion cells was used instead of antiserum from immunized animals. Based on the measurement results, wells with high absorbance were selected as wells containing anti-NCC-ST-439 antibody-producing hybridomas (positive wells).

[0052] (6) Cloning Using the anti-NCC-ST-439 antibody-producing hybridoma strains selected in the primary and secondary screenings described above, we performed monoclonalization of the hybridomas and purification of the monoclonal antibodies. Monoclonalization was performed using a standard method (limiting dilution method), and positive wells were selected in the same manner as the antigen-immobilized ELISA method described above, ultimately yielding four types of anti-NCC-ST-439 monoclonal antibody-producing hybridomas. The monoclonal antibodies produced by these four hybridomas, S18201R, S18202R, S18203R, and S18204R, are referred to as S18201R antibody, S18202R antibody, S18203R antibody, and S18204R antibody, respectively.

[0053] [Test Example 2] Epitope analysis of the monoclonal antibody of the present invention 1. Test Method The epitopes of NCC-ST-439 that react with S18201R, S18202R, S18203R, and S18204R antibodies were confirmed to be similar to those of existing antibodies by competitive ELISA as described below. First, ELISA plates were prepared in the same manner as the antigen-immobilized ELISA method described above. Then, using BSA-PBST as the solvent, the existing antibodies diluted 2-fold were mixed with the culture supernatants of hybridomas producing S18201R, S18202R, S18203R, and S18204R antibodies diluted 2-8-fold, as well as the culture supernatants of hybridomas producing a control antibody that does not react with NCC-ST-439. These were used as the mixture. After washing each well of the ELISA plate three times with 400 μL of PBST, 50 μL / well of the above mixture was dispensed and allowed to stand at room temperature for 1 hour. Next, each well was washed three times with 400 μL of PBST, then 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured.

[0054] 2. Test Results The results are shown in Figure 3. When the control antibody that does not react with NCC-ST-439 was mixed with the existing antibody, no decrease in absorbance was observed. On the other hand, when the S18201R, S18202R, S18203R, and S18204R antibodies were mixed with the existing antibody, a decrease in absorbance was observed. In this measurement system, absorbance depends on the amount of the existing antibody bound to the conjugate of NCC-ST-439 and BSA immobilized on the plate. In other words, the decrease in absorbance upon addition of S18201R, S18202R, S18203R, and S18204R antibodies indicates that the S18201R, S18202R, S18203R, and S18204R antibodies in the solution bind near the binding site between the existing antibody and the conjugate, inhibiting the binding of the existing antibody to the conjugate. Therefore, it was found that the S18201R, S18202R, S18203R, and S18204R antibodies recognize epitopes similar to those of existing antibodies.

[0055] [Test Example 3] Specificity Analysis of the Monoclonal Antibody of the Present Invention 1. Test Method The specificity of the S18201R, S18202R, S18203R, and S18204R antibodies was confirmed by competitive ELISA as described below. First, ELISA plates were prepared using the same method as the antigen-immobilized ELISA method described above. Then, using BSA-PBST as the solvent, the supernatant of S18201R, S18202R, S18203R, and S18204R antibody-producing hybridomas diluted 80-125 times, and previously used HRP-labeled NCC-ST-439 antibodies diluted 30 times were mixed with NCC-ST-439 sugar maleimide and BSA conjugates, DUPAN-2 sugar maleimide and BSA conjugates, and purified glycan Sialyl Lewis X(sLex) prepared to 0.1-10 μg / mL. These were used as the mixture. After washing each well of the ELISA plate three times with 400 μL of PBST, 50 μL / well of a mixture of the culture supernatant of S18201R, S18202R, S18203R, and S18204R antibody-producing hybridomas and each glycan-related compound was dispensed into each well and allowed to stand at room temperature for 1 hour. 50 μL / well of BSA-PBST was dispensed into the remaining wells and allowed to stand at room temperature for 1 hour. Of the aforementioned wells, the wells containing the mixture of the culture supernatant of S18201R, S18202R, S18203R, and S18204R antibody-producing hybridomas and each glycan-related compound were washed three times with 400 μL of PBST, and 50 μL / well of HRP-labeled rat IgG (H&L) diluted 5000-fold with BSA-PBST was dispensed into each well and allowed to stand at room temperature for 1 hour. In each of the aforementioned wells, 50 μL / well of BSA-PBST was dispensed, and in the wells, a mixture of the existing antibody and each glycan-related compound was dispensed, and the mixture was left to stand at room temperature for 1 hour. Next, each well was washed three times with 400 μL of PBST, then 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured.

[0056] 2. Test Results The results are shown in Figures 4a, b, c, d, and e. When using existing antibodies, a decrease in absorbance was observed when the NCC-ST-439 conjugate was mixed. Furthermore, a milder decrease in absorbance was observed when the DUPAN-2 conjugate was mixed at a high concentration compared to the case of NCC-ST-439 (Figure 4a). On the other hand, when using S18201R, S18202R, S18203R, and S18204R antibodies, a decrease in absorbance was observed only with NCC-ST-439 (Figures 4b, c, d, and e). In this measurement system, absorbance depends on the amount of antibody bound to the NCC-ST-439 and BSA conjugate immobilized on the plate. In other words, the decrease in absorbance with the addition of each glycan-related compound indicates that free glycan-related compounds in the solution react with the antibody in the solution, inhibiting the binding of the antibody to the immobilized conjugate. Therefore, the S18201R, S18202R, S18203R, and S18204R antibodies do not react with DUPAN-2, but specifically with NCC-ST-439.

[0057] [Test Example 4] Evaluation of specificity of purified glycans by competitive ELISA 1.Material Various Glycans

[0058] 2. Test Method The specificity of the S18201R, S18202R, S18203R, and S18204R antibodies was confirmed by competitive ELISA as described below. First, ELISA plates were prepared using the same method as the antigen-immobilized ELISA method described above. Then, S18201R, S18202R, S18203R, and S18204R antibodies, diluted to 225 to 500 ng / mL using BSA-PBST as the solvent, were mixed with glycans (Figure 5) serially diluted with BSA-PBST. These were used as the mixture. After washing each well of the ELISA plate three times with 400 μL of PBST, 50 μL / well each of the mixtures of the culture supernatant of the S18201R, S18202R, S18203R, and S18204R antibody-producing hybridomas and each glycan was dispensed and allowed to stand at room temperature for 1 hour. After washing three times with 400 μL of PBST, 50 μL / well of HRP-labeled rat IgG (H&L) diluted 5000-fold with BSA-PBST was dispensed and allowed to stand at room temperature for 1 hour. Next, each well was washed three times with 400 μL of PBST, then 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added. After standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured. The reaction rate was calculated by setting the absorbance without added sugar chains as 100%.

[0059] 3. Test Results The results are shown in Figures 6a, b, c, and d. When using the S18201R, S18202R, S18203R, and S18204R antibodies, a decrease in absorbance was observed only with NCC-ST-439 (Figures 6a, b, c, and d). In this measurement system, absorbance depends on the amount of antibody bound to the conjugate of NCC-ST-439 and BSA immobilized on the plate. In other words, the decrease in absorbance with the addition of each sugar chain indicates that the free sugar chain in the solution reacts with the antibody in the solution, inhibiting the binding of the antibody to the immobilized conjugate. This indicates that the S18201R, S18202R, S18203R, and S18204R antibodies react specifically with NCC-ST-439.

[0060] [Test Example 5] Reactivity to specimens and NCC-ST-439 standard 1.Material HRP-labeled streptavidin (Thermo Fisher, 21126)

[0061] 2. Test Method We tested whether NCC-ST-439 could be measured in serum samples using sandwich ELISA with each antibody. Details of the sandwich ELISA are as follows. (2-1) Preparation of plates for sandwich ELISA The S18201R, S18202R, S18203R, and S18204R antibody-containing solutions were each dissolved in 20 mM phosphate buffer (pH 7.2; hereafter referred to as PBS) containing 150 mM sodium chloride to a concentration of 5 μg / mL. 50 μL of each solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours. Each of the aforementioned wells was washed three times with 400 μL of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), then 100 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added, and the mixture was blocked at room temperature for 1 hour. This was used as the ELISA plate. (2-2) Sandwich ELISA method After washing each well of the ELISA plate three times with 400 μL of PBST, 50 μL of serum from 10 cancer patients, serially diluted with BSA-PBST, and NCC-ST-439 standard, serially diluted with BSA-PBST, were added to each well, and the samples were left to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 50 μL of biotin-labeled S18201R, S18202R, S18203R, and S18204R antibodies, diluted to 2 μg / mL with BSA-PBST, were dispensed into each well of the plate on which the same antibodies were immobilized, and the plates were left to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 50 μL of HRP-labeled streptavidin, diluted to 0.2 μg / mL with BSA-PBST, was dispensed into each well, and the plates were left to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured. When serum from cancer patients was used, the absorbance measured using the NCC-ST-439 standard was used as a calibration curve to determine the NCC-ST-439 concentration.

[0062] 3. Test Results The results are shown in Figures 7a, b, c, d, and e. The absorbance increased in a concentration-dependent manner with the NCC-ST-439 standard (Figures 7a, b, c, and d). This indicates that the antibody reacted with the NCC-ST-439 antigen contained in the standard. Furthermore, the absorbance obtained using the serum of cancer patients in this study and the NCC-ST-439 value measured with a commercially available ELISA kit were proportional as an approximate straight line with a correlation coefficient of 0.99 or higher (Figure 7e). In other words, it was shown that the antibody of the present invention can measure NCC-ST-439 in the serum of cancer patients, similar to existing NCC-ST-439 antibodies.

[0063] Although the present invention has been described using the above specific examples, the scope of the present invention is not limited to these examples. Those skilled in the art will understand that various other configurations can be adopted without departing from the spirit of the present invention.

Claims

1. NCC-ST-439 antigen containing the following sugar chain structure And it reacted, DUPAN-2 antigen containing the following sugar chain structure An antibody that does not react with the antigen-binding fragment thereof, wherein the antibody (excluding CSLEX-1 antibody) and its antigen-binding fragment do not react with the peptide portion of the NCC-ST-439 antigen-binding peptide, and wherein, in a competitive ELISA method, the reactivity to the DUPAN-2 antigen is less than 10% of the reactivity to the NCC-ST-439 antigen.

2. The antibody or antigen-binding fragment according to claim 1, wherein in a competitive ELISA method, the reactivity to the DUPAN-2 antigen is less than 1% of the reactivity to the NCC-ST-439 antigen.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a rat antibody.

4. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 3, The method comprising the step of immunizing an animal with a polymer compound conjugated with the NCC-ST-439 antigen, wherein the polymer compound conjugated with the NCC-ST-439 antigen does not contain the peptide portion of the NCC-ST-439 antigen-binding peptide (except in the method in which the animal is a human).

5. A method for producing cells that produce an antibody or antigen-binding fragment according to any one of claims 1 to 3, The method comprising the step of immunizing an animal with a polymer compound conjugated with the NCC-ST-439 antigen, wherein the polymer compound conjugated with the NCC-ST-439 antigen does not contain the peptide portion of the NCC-ST-439 antigen-binding peptide (except in the method in which the animal is a human).

6. An immunoassay method characterized by using an antibody or antigen-binding fragment according to any one of claims 1 or 2.

7. An immunoassay reagent comprising the antibody or antigen-binding fragment described in claim 1 or 2.

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