WO2019189882A1

WO2019189882A1 - Monoclonal antibody specifically reacting with ncc-st-439 antigen and method for producing same

Info

Publication number: WO2019189882A1
Application number: PCT/JP2019/014335
Authority: WO
Inventors: 知啓三浦; 宮崎　修
Original assignee: 積水メディカル株式会社
Priority date: 2018-03-30
Filing date: 2019-03-29
Publication date: 2019-10-03

Abstract

A conventional antibody recognizing a sugar chain antigen, said sugar chain antigen being to be used as a tumor marker, shows an insufficient antigen specificity. When such an antibody is used in detecting a tumor marker, there arises a problem that the detection specificity is lowered and thus cancer diagnosis accuracy is also lowered. To solve this problem, the present invention provides an antibody that specifically reacts with the sugar chain of NCC-ST-439 antigen to be used as a tumor marker and a method for producing the antibody.

Description

Monoclonal antibody specifically reacting with NCC-ST-439 antigen and method for producing the same.

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

In recent years, many antibodies against sugar chain antigens used as tumor markers have been reported.
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). .

In addition, 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.

On the other hand, 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.

Therefore, in order to perform a more accurate diagnosis using a tumor marker, it has been necessary to obtain an antibody that specifically reacts with a specific sugar chain antigen.

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.

As a result of intensive studies to solve the above-mentioned problems, 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.

That is, this invention provides the following in one aspect | mode.
[1] NCC-ST-439 antigen having the following sugar chain structure

And DUPAN-2 antigen having the following sugar chain structure

Antibodies and antigen-binding fragments thereof that do not react with.
[2] The antibody or antigen-binding fragment thereof according to [1], wherein reactivity to DUPAN-2 antigen is less than 10% of reactivity to NCC-ST-439 antigen.
[3] The antibody or antigen-binding fragment thereof according to [1], wherein reactivity to DUPAN-2 antigen is less than 1% of reactivity to NCC-ST-439 antigen.
[4] The antibody or antigen-binding fragment thereof according to any one of [1] to [3], which is a rat antibody.
[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.
[8] An immunoassay method comprising using the antibody or antigen-binding fragment thereof according to any one of [1] to [3].
[9] 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.

Furthermore, by using a method for producing an antibody that specifically recognizes a mucin antigen used as a tumor marker of the present invention, it is possible to obtain an antibody that reacts highly specifically with a specific tumor marker.

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. 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. .

Hereinafter, embodiments of the present invention will be described. The following description is merely an example, and the scope of the present invention is not limited to these descriptions, and can be appropriately modified and implemented without departing from the spirit of the present invention.

(Definition)
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, when a range of a plurality of numerical values is indicated, a range composed of a combination of an arbitrary lower limit value and upper limit value of the plurality of ranges is also meant in the same manner.

In this specification, when a compound with an antibody “reacts”, “shows reactivity”, “reactivity”, “binds”, or “recognizes” a compound with an antibody, The meanings commonly used in the field of the invention are included, and all are used synonymously. Whether or not 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).

In the present specification, “the antibody does not react” with the antibody of the present invention means that the antibody of the present invention and the compound do not substantially react. “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.

As used herein, that 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. For example, 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. In addition, 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. Preferably, the reactivity of the other antigen is less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% to the specific antigen. In a preferred embodiment of the invention, the antibody of the invention reacts with the NCC-ST-439 antigen and does not react with the DUPAN-2 antigen.

As used herein, “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). 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.

Also, Padlan (1995 FASEB J. 9: 133-139), Vajdos et al. As shown in 2002 J Mol Biol 320: 415-428, it is known that only a part of the CDR residues actually contact the antigen, and the CDR residues that do not contact the antigen are those of the Chothia CDR. It can be identified by molecular modeling or empirically from the outer region of Kabat CDRs. When a CDR or one or more residues thereof is removed, it is usually replaced with another human antibody sequence or an amino acid that occupies the corresponding position in the consensus of such a sequence. Positions for substitution within CDRs and amino acids can also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

As used herein, 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). Non-limiting examples of 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. 5; (iii) an Fd fragment consisting of VH and CH domains; (iv) a single arm VL of an antibody And an Fv fragment consisting of the VH domain; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); (Vii) a synthetic linker, optionally bifurcated Includes further combinations of isolated CDRs. Alternatively, as known as single chain Fv (scFv), it can also be made as a single protein chain that pairs VL and VH regions with 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). 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. These antibody fragments are obtained using conventional techniques known to those skilled in the art.

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. Preferably, 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. In addition, 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. As host cells, for example, CHO cells, lymphocyte cells, bacterial cells such as E. coli, and fungal cells such as yeast can be used.

In addition, 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.

As the antibody in the present invention, for example, 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.

Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center, Address of depositary institution: Room 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture (Zip 292-0818), Date of deposit: 2018 May 17th.
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)

In the present specification, the “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. For example, 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. Examples of the substituent 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.

In the present specification, “alkylene” means a divalent group consisting of a linear or branched saturated hydrocarbon.

In the present specification, 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. Examples of 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.

In the present specification, the “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.

In the present specification, “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

In the present specification, the “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.

(Method for producing antibody specifically reacting with NCC-ST-439 antigen)
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. In a preferred embodiment, 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. As 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. For example, a monoclonal antibody that specifically reacts with the above DUPAN-2 antigen could be prepared by the same method.

(Use of the antibody of the present invention)
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. Specifically, 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). In one embodiment, 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. For example, 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). As the 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.

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. As a method for binding the labeling substance and the antibody, 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. There are no particular limitations on the type of immobilized antibody or labeled antibody and the method for producing them, and for example, an enzyme such as peroxidase or alkaline phosphatase (hereinafter sometimes referred to as ALP) can be used as the labeling substance. . In this case, 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 In the case of ', 5,5'-tetramethylbenzidine or ALP, the enzyme activity can be measured using p-nitrophenyl phosphate or the like. When 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. In addition to the antibody of the present invention, the reagent may contain other components necessary for carrying out the immunoassay method, such as a buffer solution and a preservative.

In one embodiment, for example, when 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 the specific substrate.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Test Example 1] Production of monoclonal antibody specific for NCC-ST-433 antigen
1. Materials (1) NCC-ST-439 modified maleimide, NCC-ST-439 glycopeptide, various sugar chain-related compounds (2) Freund's complete adjuvant: Wako Pure Chemical Industries, 014-09541
(3) Myeloma cells (SP2 / O)
(4) RPMI 1640, GlutaMAX: GIBCO, 61870-036
(5) Fetal Bovine Serum (FBS): manufactured by BIOLOGICAL INDUSTRIES, 04-001-1A
(6) HAT medium: Cosmo Bio, 16231004
(7) 96-well plate: NUNC, 167008
(8) HRP-labeled goat anti-rat IgG (H & L) antibody: manufactured by Southern Biotech, 3050-05
(9) “Ranazyme (registered trademark) ST-439 plate”: Kainos / Nippon Kayaku; HRP-labeled NCC-ST-439 antibody (existing antibody) and standard solution (NCC-ST-439 standard product) are included as constituent reagents

2. Preparation of 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. Mixed in PBS at a ratio of 4: 1, reacted at room temperature for 2 hours and left at 4 ° C. overnight. Thereafter, the buffer was replaced with PBS by dialysis.
For cross-linking of glycopeptide, 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.

3. Preparation of hybridoma producing anti-NCC-ST-439 monoclonal antibody (3-1) Immunization to animals Prepared by mixing equal amounts of the immunization conjugate solution (0.2-2 mg / ml) and Freund's complete adjuvant. Using the emulsion, F344 rats were injected with 10-40 μg per animal. In addition, the emulsion injection was repeated 7-8 times at weekly intervals. The antibody titer in the antiserum obtained by collecting blood from the tail vein was measured by the antigen-immobilized ELISA method described later.

(3-2) Primary screening (antigen-immobilized ELISA method)
The presence of the anti-NCC-ST-439 antibody in the immunized animal antiserum was prepared by the same method as the conjugate solution for immunization, and the ELISA method in which the conjugate solution of NCC-ST-439 and BSA was immobilized This was confirmed by (antigen-immobilized ELISA method). Details of the antigen-immobilized ELISA method are as follows.
(3-2-1) Preparation of ELISA-immobilized ELISA plate 20 mM phosphate buffer containing 150 mM sodium chloride so that the conjugate solution of NCC-ST-439 glycopeptide and BSA is 0.1 μg / mL. (PH 7.2; hereinafter referred to as PBS), 50 μL 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 μL of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), and then 100 μL 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.
(3-2-2) Antigen-immobilized ELISA method Each well of the ELISA plate was washed three times with 400 μL of PBST, and then diluted with BSA-PBST from 1500 times to 13500 times. 50 μL was added to each well and allowed to stand at room temperature for 1 hour. Each well was washed 3 times with 400 μL PBST, and then 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. Each well was washed with 400 μL of PBST three times, 50 μL 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. The enzyme reaction was stopped by adding 50 μL of 5N sulfuric acid, and the absorbance at a wavelength of 492 nm was measured. As a result of the measurement, spleen or lymph node was extracted from a mouse having a high antibody titer, and spleen-derived cells or lymph node-derived cells were prepared and used for cell fusion.

(3-2) Test results The results are shown in FIG. As compared with non-immunized animal serum, the immunized animal antisera showed higher absorbance as the dilution factor was lower. In this measurement system, the absorbance depends on the antibody concentration against NCC-ST-439 contained in the animal serum. That is, the above-mentioned results indicate that the immunized animal antiserum contains a higher concentration of antibody against NCC-ST-439 and has a higher antibody titer than the sera of nonimmunized animals. As a result of the measurement, spleen or lymph node was extracted from a rat having a high antibody titer to prepare spleen-derived cells or lymph node-derived cells and used for cell fusion.

(4) Cell fusion One of the spleen-derived cells or lymph node-derived cells and myeloma cells were mixed at a cell number ratio of 1: 1, and electrofused. The fused cells were suspended in HAT medium and cultured in a CO ₂ incubator at 37 ° C. and 5% CO _{2 for} 8 days to obtain fused cells (hybridomas).

(5) Selection of hybridoma (antigen-immobilized ELISA method)
In the antigen-immobilized ELISA method described above, the same method was performed except that the culture supernatant of the fused cells was used instead of the immunized animal antiserum. As a result of the measurement, 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).

(6) Cloning Using the anti-NCC-ST-439 antibody-producing hybridoma selected in the primary screening and secondary screening described above, single cloning of the hybridoma and purification of the monoclonal antibody were performed.
Monocloning is performed by a standard method (limit dilution method), and positive wells are selected in the same manner as in the above-described antigen-immobilized ELISA method. Finally, four types of anti-NCC-ST-439 monoclonal antibody-producing hybridomas are obtained. It was. 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 Example 2] Epitope analysis of the monoclonal antibody of the present invention
1. 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. First, an ELISA plate was prepared in the same manner as the above-described antigen-immobilized ELISA method. In addition, with BSA-PBST as a solvent, existing antibodies diluted 2 times, hybridomas producing S18201R, S1822R, S1823R, S18204R antibody-producing hybridoma cultures diluted 2-8 times, and hybridomas producing a control antibody that does not react with NCC-ST-439 The culture supernatant was mixed. These are mixed liquids. Each well of the ELISA plate was washed 3 times with 400 μL of PBST, and then the above-mentioned mixed solution was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour.
Next, each well was washed 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 the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 μL of 1.5 N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

2. The test result is shown in FIG. When a control antibody that does not react with NCC-ST-439 was mixed with an existing antibody, no decrease in absorbance was observed. On the other hand, when the S18201R, S1822R, S1823R, and S18204R antibodies were mixed with existing antibodies, a decrease in absorbance was observed. In this measurement system, the absorbance depends on the amount of existing antibody bound to the conjugate of NCC-ST-439 and BSA immobilized on the plate. In other words, 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. Further, S18201R, S1822R, S1823R, S18204R antibody-producing hybridoma culture supernatant diluted with BSA-PBST as a solvent, and HRP-labeled NCC-ST-439 antibody diluted 30 times with 0.1-10 μg / NCC-ST-439 sugar maleimide / BSA conjugate, DUPAN-2 sugar maleimide / BSA conjugate and purified sugar chain Siaryl Lewis X (sLex) prepared in mL were mixed. These are mixed liquids. After washing each well of the ELISA plate with PBST 400 μL three times, 50 μL / well each of the above-mentioned S18201R, S1822R, S1823R, S18204R antibody-producing hybridoma culture supernatant and each sugar chain-related compound mixture was dispensed at room temperature. And left for 1 hour. To other wells, 50 μL / well of BSA-PBST was dispensed and allowed to stand at room temperature for 1 hour. Of the wells, 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 μL, and then HRP diluted 5000 times with BSA-PBST. Labeled rat IgG (H & L) was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. Of each well, 50 μL / 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 μL / well, and allowed to stand at room temperature for 1 hour.
Next, each well was washed 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 the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 μL of 1.5 N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

2. The test results are shown in FIGS. 4a, b, c, d and e. When the existing antibody was used, a decrease in absorbance was observed when the NCC-ST-439 conjugate was mixed. In addition, when the DUPAN-2 conjugate was mixed at a high concentration, a slight decrease in absorbance was observed compared to NCC-ST-439 (FIG. 4a). On the other hand, when the S18201R, S1822R, S1823R, and S18204R antibodies were used, a decrease in absorbance was observed only with NCC-ST-439 (FIGS. 4b, c, d, and e). In this measurement system, the 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 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 Example 4] Specificity evaluation by competitive ELISA for purified sugar chain
1. Material various sugar chains

2. 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 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 μL of PBST, and 50 μL / 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 μL of PBST, HRP-labeled rat IgG (H & L) diluted 5000 times with BSA-PBST was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour.
Next, each well was washed 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 the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 50 μL 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%.

3. The test results are shown in FIGS. 6a, b, c and d. When the S18201R, S1822R, S1823R, and S18204R antibodies were used, a decrease in absorbance was observed only with NCC-ST-439 (FIGS. 6a, b, c, and d). In this measurement system, the absorbance depends on the amount of antibody bound to the conjugate of NCC-ST-439 and BSA immobilized on the plate. That is, the decrease in absorbance due to the addition of each sugar chain indicates that the free sugar chain in the solution reacts with the antibody in the solution and inhibits the binding between the antibody and the immobilized conjugate. ing. That is, the antibodies S18201R, S1822R, S18203R, and S18204R react specifically with NCC-ST-439.

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

2. Test Method It was tested whether NCC-ST-439 contained in serum samples could be measured by sandwich ELISA using each antibody. Details of the sandwich ELISA are as follows.
(2-1) Preparation of sandwich ELISA plate 20 mM phosphate buffer containing 150 mM sodium chloride (pH 7.2; hereinafter referred to as PBS) so that the S18201R, S1822R, S1823R, and S18204R antibody-containing solutions are each 5 μg / mL. And 50 μL of the lysate was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.
Each well was washed three times with 400 μL of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), and then 100 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added. Blocking was performed at room temperature for 1 hour. This was used as an ELISA plate.
(2-2) Sandwich ELISA Method Each well of the ELISA plate was washed 3 times with PBST 400 μL, and then serially diluted with BSA-PBST 50 cancer serums of 10 cases and BSA-PBST serially diluted NCC-ST- 439 standard was added to each well and allowed to stand at room temperature for 1 hour.
Each well was washed 3 times with 400 μL PBST, and then biotinylated S18201R, S1822R, S18203R, S18204R antibodies diluted to 2 μg / mL with BSA-PBST were dispensed into the wells on the same plate with 50 μL each well. And allowed to stand at room temperature for 1 hour. Each well was washed with 400 μL of PBST three times, and then 50 μL of HRP-labeled streptavidin diluted to 0.2 μg / mL with BSA-PBST was dispensed into each well and allowed to stand at room temperature for 1 hour. Each well was washed with 400 μL of PBST three times, 50 μL 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. The enzyme reaction was stopped by adding 50 μL of 5N sulfuric acid, and the absorbance at a wavelength of 492 nm was measured. When cancer patient serum was used, the NCC-ST-439 concentration was measured using the absorbance measured with the NCC-ST-439 standard as a calibration curve.

3. 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. Furthermore, 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.

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

Claims

NCC-ST-439 antigen having the following sugar chain structure

And DUPAN-2 antigen having the following sugar chain structure

Antibodies and antigen-binding fragments thereof that do not react with.
The antibody or antigen-binding fragment thereof according to claim 1, wherein the reactivity to DUPAN-2 antigen is less than 10% of the reactivity to NCC-ST-439 antigen.
The antibody or antigen-binding fragment thereof according to claim 1, wherein the reactivity to DUPAN-2 antigen is less than 1% of the reactivity to NCC-ST-439 antigen.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a rat antibody.
A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising
The method comprising the step of immunizing an animal with the polymer compound bound with the NCC-ST-439 antigen.
A method for producing a cell that produces the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:
The method comprising the step of immunizing an animal with the polymer compound bound with the NCC-ST-439 antigen.
The method according to claim 5 or 6, wherein the polymer compound bound to the NCC-ST-439 antigen does not contain a 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 claims 1 to 3.
An immunoassay reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.