Antibodies or fragments thereof, and their uses

Abstract

The present invention provides antibody, or a fragment thereof, that recognizes an antigen specific to colon cancer or oral cancer. Specifically, the present invention provides antibody, or a fragment thereof, that recognizes the N-linked sugar chain of annexin A2.

Description

[Technical Field]

[0001] The present disclosure relates to an antibody or a fragment thereof, etc. In particular, the present disclosure relates to an antibody or a fragment thereof that recognizes a colorectal cancer or oral cancer-specific antigen, and uses thereof, etc. [Background technology]

[0002] Antibodies (especially monoclonal antibodies) are widely used for early diagnosis, specific diagnosis, and screening of cancer, as well as for identifying molecular targets for cancer therapy. Recently, humanization of monoclonal antibodies has become feasible, and various humanized monoclonal antibodies are used in the treatment of malignant tumors and autoimmune diseases (e.g., CD3 (Orthoclone OKT3), glycoprotein IIb / IIIa receptor (ReoPro), ERBB2 (Herceptin), CD20 (Rituxan), CD25 (Zenapax and Simulect), RSVgpF (Synagis), TNF-α (Remicade), IL-6R, etc.).

[0003] For the application of antibodies, it is necessary to establish antibodies that specifically recognize cancer-specific antigens of various tumors, and antibody specificity is an important issue for the clinical application of antibodies. Although many studies have been conducted to identify novel molecules that are specifically expressed on the surface of cancer cells or in the serum of cancer patients, most of these studies have not efficiently identified molecules or epitopes that are thought to be cancer-specific markers. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 119180 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present disclosure is to provide an antibody or a fragment thereof that recognizes a colorectal cancer or oral cancer-specific antigen. [Means for solving the problem]

[0006] The inventors discovered that antibody 12G5A reacts with HT-29 cells derived from human colon adenocarcinoma, but does not react with differentiated HT-29 cells (normal intestinal epithelial cells) treated with sodium butyrate, and further improved the antibody.

[0007] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. An antibody or a fragment thereof that recognizes the N-linked sugar chain of annexin A2. Section 2. Item 1, wherein the antibody or fragment thereof is the antibody or fragment thereof of (I) or (II). (I) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1, and an antibody or a fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (II) a light chain variable region consisting of an amino acid sequence having an identity of 90% or more to the amino acid sequence set forth in SEQ ID NO: 1; and An antibody or fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 Section 3. Item 3. The antibody or fragment thereof according to Item 1 or 2, wherein the annexin A2 is a protein comprising the amino acid sequence shown in SEQ ID NO:3. Section 4. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of Items 1 to 3. Section 5. Item 5. The pharmaceutical composition according to Item 4, which is used for cancer diagnosis or cancer treatment. Section 6. Item 6. The pharmaceutical composition according to Item 5, wherein the cancer is colon cancer or oral cancer. Section 7. Use of annexin A2 having an N-linked glycan as a marker for colon cancer or oral cancer. Section 8. Item 8. The use according to Item 7, wherein the annexin A2 is a protein comprising the amino acid sequence shown in SEQ ID NO:3. [Effects of the Invention]

[0008] Provided are antibodies or fragments thereof that recognize colorectal cancer or oral cancer-specific antigens (specifically, N-linked glycans of annexin A2). Also provided are pharmaceutical compositions containing the antibodies or fragments thereof. Also provided are methods for using annexin A2 with N-linked glycans as a colorectal cancer or oral cancer marker. [Brief explanation of the drawings]

[0009] [Figure 1] The results of immunofluorescence staining are shown, as well as the amino acid sequences of the analyzed light chain variable region (SEQ ID NO: 1) and heavy chain variable region (SEQ ID NO: 2). [Figure 2] The amino acid sequence of human annexin A2 and the results of Western blotting are shown. [Figure 3] 1 shows overall survival and progression-free survival (PFS) curves based on 12G5A immunoreactivity in colorectal cancer. [Figure 4] 1 shows a comparison of the immunoreactivity of 12G5A with that of a commercially available antibody against annexin A2. [Figure 5] Comparison of 12G5A immunoreactivity in oral non-tumor stratified squamous epithelium (A), dysplastic epithelium (B), and oral squamous cell carcinoma (C) is shown. [Figure 6] The results of Western blotting and immunofluorescence staining are shown. [Figure 7] 1 shows the results of measuring the volume of colon cancer tumors when the 12G5A antibody was administered. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses antibodies or fragments thereof that recognize N-linked glycans of annexin A2. In this specification, such antibodies or fragments thereof may be referred to as "antibodies of the present disclosure," "antibody fragments of the present disclosure," "antibodies of the present disclosure or fragments thereof," etc.

[0011] Annexin A2 is a calcium ion (Ca 2+ Annexin A2 is a phospholipid-binding protein. It is known that its expression is increased in cancer cells and is believed to be expressed on the cell membrane. SEQ ID NO: 3 shows the amino acid sequence of human annexin A2.

[0012] The N-linked sugar chain is bound to an asparagine residue in the amino acid sequence constituting annexin A2. In the amino acid sequence constituting annexin A2, the asparagine residue to which the sugar chain is bound may be, for example, an asparagine residue constituting an amino acid sequence represented by Asn-X-Ser or Asn-X-Thr (wherein X is any amino acid residue). For example, the N-linked sugar chain may be bound to the asparagine residue at position 62 in the amino acid sequence shown in SEQ ID NO: 3.

[0013] The N-linked sugar chain contains N-acetylglucosamine (GlcNAc). More specifically, examples include a high-mannose type having a structure in which a mannose oligomer is bound to diacetylchitobiose (GlcNAc-GlcNAc), a complex type having a structure in which mannose and at least one selected from the group consisting of N-acetylglucosamine (GlcNAc), galactose, and sialic acid are bound to diacetylchitobiose (GlcNAc-GlcNAc), and a mixed (hybrid) type having a structure in which a high-mannose type and a complex type are mixed to diacetylchitobiose (GlcNAc-GlcNAc).

[0014] The antibody of the present disclosure may be a polyclonal antibody or a monoclonal antibody, with a monoclonal antibody being preferred.

[0015] Examples of isotypes of antibodies (immunoglobulins) of the present disclosure include IgA, IgD, IgE, IgG (IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4), and IgM.

[0016] The origin of the antibodies of the present disclosure includes, for example, human-derived antibodies, mouse-derived antibodies, rat-derived antibodies, rabbit-derived antibodies, monkey-derived antibodies, chimpanzee-derived antibodies, and the like.

[0017] The antibody of the present disclosure may also be a chimeric antibody. A chimeric antibody refers to an antibody whose constant region is an amino acid sequence derived from a human and whose variable region is an amino acid sequence derived from a species other than a human (e.g., a mouse). The antibody of the present disclosure may also be a humanized antibody.

[0018] Antibody fragments of the present disclosure include, for example, Fab, Fab', F(ab')2, Fv, scFv, and the like.

[0019] The antibody or fragment thereof of the present disclosure may be (I) an antibody or fragment thereof comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2. In this specification, such an antibody or fragment thereof may be referred to as "the antibody or fragment thereof (I) of the present disclosure."

[0020] The antibody or fragment thereof of the present disclosure may be (II) an antibody or fragment thereof comprising a light chain variable region consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1, and a heavy chain variable region consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2. In this specification, such an antibody or fragment thereof may be referred to as "the antibody or fragment thereof (II) of the present disclosure."

[0021] In the antibody or fragment thereof (II) of the present disclosure, the identity with the amino acid sequence shown in SEQ ID NO: 1 is, for example, preferably 90% or more, more preferably 95% or more.

[0022] In the antibody or fragment thereof (II) of the present disclosure, the identity with the amino acid sequence shown in SEQ ID NO: 2 is, for example, preferably 90% or more, more preferably 95% or more.

[0023] Amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / using default parameters.

[0024] The antibody or fragment thereof (II) of the present disclosure may comprise a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 in which at least one amino acid has been deleted, substituted, or added, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 in which at least one amino acid has been deleted, substituted, or added.

[0025] In an amino acid sequence in which at least one amino acid has been deleted, substituted, or added, the upper limit of the number of deleted, substituted, or added amino acids is, for example, 25, and preferably about 10. More specifically, in the amino acid sequence shown in SEQ ID NO: 1, the number may be, for example, 1 to 18, 1 to 15, 1 to 10, 1 to 5, or 1 to 3. More specifically, in the amino acid sequence shown in SEQ ID NO: 2, the number may be, for example, 1 to 24, 1 to 20, 1 to 18, 1 to 15, 1 to 10, 1 to 5, or 1 to 3.

[0026] Techniques for introducing mutations such as deletion, substitution, or addition of at least one amino acid into a specific amino acid sequence are known in the art and can be carried out using any method, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, or random mutagenesis.

[0027] When at least one amino acid is substituted, the substitution may be a conservative substitution. As used herein, the term "conservative substitution" refers to substituting an amino acid with an amino acid having a side chain with properties similar to those of the amino acid. Specific examples of conservative substitutions include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0028] In the antibody or fragment thereof (II) of the present disclosure, the amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 may, for example, conserve the amino acid sequence of positions 1 to 10, the amino acid sequence of positions 100 to 107, or the amino acid sequence of positions 125 to 136 of the amino acid sequence shown in SEQ ID NO: 1. Of these amino acid sequences, any one amino acid sequence may be conserved, any two amino acid sequences may be conserved, or all three amino acid sequences may be conserved. Preferably, the amino acid sequence of positions 100 to 107 of the amino acid sequence shown in SEQ ID NO: 1 is conserved.

[0029] In the antibody or fragment thereof (II) of the present disclosure, the amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2 may, for example, conserve the amino acid sequence of positions 1 to 6, the amino acid sequence of positions 47 to 54, the amino acid sequence of positions 71 to 77, or the amino acid sequence of positions 118 to 144 of the amino acid sequence shown in SEQ ID NO: 2. Of these amino acid sequences, any one amino acid sequence may be conserved, any two amino acid sequences may be conserved, any three amino acid sequences may be conserved, or all four amino acid sequences may be conserved. Preferably, the amino acid sequence of positions 47 to 54 of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of positions 71 to 77 of the amino acid sequence shown in SEQ ID NO: 2 is conserved.

[0030] The antibodies of the present disclosure can be prepared by known methods. For example, polyclonal antibodies can be prepared by immunizing a mammal with a lysate of cells (e.g., colon cancer cells) that express annexin A2 having an N-linked sugar chain as an immunogen, or monoclonal antibodies can be prepared by preparing hybridomas. The antibody fragments of the present disclosure can also be prepared by known methods, for example, by treating the antibodies obtained by the above-described methods with an enzyme.

[0031] The present disclosure also encompasses a pharmaceutical composition comprising the above-described antibody or fragment thereof. In this specification, such a pharmaceutical composition may be referred to as the "pharmaceutical composition of the present disclosure."

[0032] The pharmaceutical composition of the present disclosure may contain one or more of the above-mentioned antibodies or fragments thereof in combination.

[0033] The content of the antibody or fragment thereof in the pharmaceutical composition of the present disclosure is not particularly limited and can be set appropriately up to 100% by mass, for example, about 0.0001 to 99.9% by mass.

[0034] The pharmaceutical composition of the present disclosure comprises the antibody or fragment thereof described above, and may further comprise other ingredients. Examples of such other ingredients include pharmaceutically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, antioxidants, preservatives, coating agents, colorants, and other drugs such as gastric mucosa protectants. These ingredients may be used singly or in combination of two or more.

[0035] The dosage form of the pharmaceutical composition of the present disclosure is not particularly limited, and examples include tablets, pills, capsules, powders, fine granules, granules, liquids, troches, jellies, injections, plasters, extracts, suppositories, suspensions, tinctures, ointments, poultices, nasal drops, inhalants, liniments, lotions, aerosols, and the like.

[0036] The pharmaceutical composition of the present disclosure can be prepared by a conventional method by combining the above-described antibody or fragment thereof with other ingredients as needed.

[0037] As shown in the Examples below, the antibody or fragment thereof of the present disclosure reacts with cancer cells or cancer tissues but does not react with normal cells or tissues. In other words, since the antibody or fragment thereof of the present disclosure reacts specifically with cancer cells or cancer tissues, the pharmaceutical composition of the present disclosure can be suitably used for cancer diagnosis. In this specification, this pharmaceutical composition for cancer diagnosis may be referred to as the "pharmaceutical composition for cancer diagnosis of the present disclosure."

[0038] When the pharmaceutical composition of the present disclosure is used for cancer diagnosis, the above-mentioned antibody or fragment thereof is preferably labeled with a labeling substance such as an enzyme, e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), or β-D-galactosidase; or a fluorescent dye.

[0039] The pharmaceutical composition for cancer diagnosis of the present disclosure can be used to diagnose the presence or absence and degree of cancer by reacting it with a biological sample and confirming the presence or absence and degree of an immune reaction.

[0040] Examples of cancers targeted by the pharmaceutical composition for cancer diagnosis of the present disclosure include colorectal cancers such as colon cancer and rectal cancer; and oral cancers such as squamous cell carcinoma. Therefore, the pharmaceutical composition for cancer diagnosis of the present disclosure is suitable as a composition for diagnosing colorectal cancer. Furthermore, the pharmaceutical composition for cancer diagnosis of the present disclosure is suitable as a composition for diagnosing oral cancer.

[0041] Examples of biological samples include body fluids, tissues, or cells collected from a subject. Examples of body fluids include urine, serum, plasma, blood (whole blood), large intestine fluid, saliva, cerebrospinal fluid, synovial fluid, lymphatic fluid, amniotic fluid, ascites, pleural effusion, milk, bile, and various tissue fluids. Examples of tissues include large intestine tissues such as colon tissue and rectal tissue; oral tissues; and examples of cells include epithelial cells and mucosal cells.

[0042] The subject from which the biological sample is collected is preferably a mammal. It may be not only a human but also a non-human mammal. Examples of the human subject include cancer patients or suspected cancer patients. More specific examples of cancer include colorectal cancers such as colon cancer and rectal cancer; and oral cancers such as squamous cell carcinoma. Examples of non-human mammals include mammals kept as pets, livestock, laboratory animals, etc. Examples of such non-human mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

[0043] The method for collecting the biological sample is not particularly limited, and any method known in the art can be used.

[0044] The method for reacting the pharmaceutical composition for cancer diagnosis of the present disclosure with a biological sample is not particularly limited, and any method known in the technical field can be used.

[0045] The presence or absence and the degree of an immune reaction can be confirmed, for example, by detecting a signal from the labeling substance described above. The method for detecting the signal is not particularly limited, and methods known in the art can be used.

[0046] As shown in the Examples below, it has been confirmed that high immunoreactivity to the antibody or fragment thereof of the present disclosure is associated with a worse overall survival rate and progression-free survival (PFS) in humans diagnosed with colorectal cancer. Therefore, by comparing the immunoreactivity to the antibody or fragment thereof of the present disclosure with a set reference value, the prognosis of colorectal cancer can be diagnosed (predicted). In other words, the pharmaceutical composition for cancer diagnosis of the present disclosure (more specifically, the pharmaceutical composition for colon cancer diagnosis) can be more suitably used for the prognosis (prediction) of colorectal cancer.

[0047] Immunoreactivity can be evaluated, for example, by the proportion of cells in which a signal from the labeling substance is detected (in other words, the proportion of cells expressing annexin A2 having an N-linked sugar chain relative to the total number of cells). For example, a ratio of 50% or more can be used as the reference value. Alternatively, the reference value can be the immunoreactivity to a biological sample previously collected from the same subject.

[0048] When the immunoreactivity in the biological sample is high compared to the reference value, it is predicted that the prognosis of colorectal cancer in the subject from whom the biological sample was collected may be worse, whereas when the immunoreactivity in the biological sample is low compared to the reference value, it is predicted that the prognosis of colorectal cancer in the subject from whom the biological sample was collected may be better.

[0049] As shown in the Examples below, the antibody or fragment thereof of the present disclosure reacts not only with oral squamous cell carcinoma but also with dysplastic epithelium, and therefore the pharmaceutical composition for cancer diagnosis of the present disclosure (more specifically, the pharmaceutical composition for oral cancer diagnosis) can be more suitably used to diagnose precancerous lesions of oral cancer, such as epithelial dysplasia.

[0050] As shown in the Examples below, the antibody or fragment thereof of the present disclosure reacts with cancer cells or cancer tissues but does not react with normal cells or tissues. Therefore, since the antibody or fragment thereof of the present disclosure reacts specifically with cancer cells or cancer tissues, the pharmaceutical composition of the present disclosure can be suitably used for cancer treatment. In this specification, this pharmaceutical composition for cancer treatment may be referred to as the "pharmaceutical composition for cancer treatment of the present disclosure."

[0051] For example, as shown in the Examples below, the antibody or fragment thereof of the present disclosure has the effect of suppressing tumor volume, and therefore, the pharmaceutical composition of the present disclosure containing the antibody or fragment thereof of the present disclosure can be suitably used for cancer treatment. Furthermore, for example, the pharmaceutical composition for cancer treatment of the present disclosure may further contain a compound having a therapeutic effect against cancer. The compound may be bound to the above-mentioned antibody or a fragment thereof. In other words, it may be, for example, an antibody-drug conjugate (ADC).

[0052] Examples of cancers targeted by the pharmaceutical composition for cancer treatment of the present disclosure include colorectal cancers such as colon cancer and rectal cancer; and oral cancers such as squamous cell carcinoma. Therefore, the pharmaceutical composition for cancer treatment of the present disclosure is suitable as a composition for treating colorectal cancer. Furthermore, the pharmaceutical composition for cancer treatment of the present disclosure is suitable as a composition for treating oral cancer.

[0053] The subject to be ingested with the pharmaceutical composition for cancer treatment of the present disclosure is preferably a mammal. It may be not only a human but also a non-human mammal. Examples of the human subject include cancer patients or those suspected of having cancer. More specific examples of cancer include colorectal cancers such as colon cancer and rectal cancer; and oral cancers such as squamous cell carcinoma. Examples of non-human mammals include mammals kept as pets, livestock, laboratory animals, etc. Examples of such non-human mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

[0054] Methods for administering the pharmaceutical composition for cancer treatment of the present disclosure include, for example, oral administration and parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, peritoneal, rectal, transdermal, topical, etc.) administration.

[0055] The dosage (intake) of the pharmaceutical composition for cancer treatment of the present disclosure is not particularly limited and is determined depending on the age, weight, sex, severity of symptoms, administration method, etc. of the subject to be administered.

[0056] The present disclosure also encompasses the use of annexin A2 having an N-linked glycan as a marker for colon cancer. The present disclosure also encompasses the use of annexin A2 having an N-linked glycan as a marker for oral cancer.

[0057] As described above, the antibody or fragment thereof of the present disclosure recognizes the N-linked glycan of annexin A2, and therefore cancer can be diagnosed by evaluating the immunoreactivity. Therefore, annexin A2 having an N-linked glycan is preferably used as a cancer marker. As described above, the use of the cancer marker, more specifically, the colon cancer marker, makes it possible to diagnose colon cancer, diagnose the prognosis of colon cancer, and the like. As mentioned above, the use of this cancer marker, more specifically, an oral cancer marker, makes it possible to diagnose oral cancer and precancerous lesions of oral cancer, which are currently difficult to determine even with pathological tissue diagnosis.

[0058] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0059] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure may be combined in any way to specify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0060] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were conducted under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass." Furthermore, the blending amount of each component listed in each table is also expressed in "% by mass" unless otherwise specified.

[0061] cell culture Human colon adenocarcinoma-derived HT-29 cells were cultured with 1 mM sodium butyrate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) for 48 hours to induce differentiation. As an indicator of cell differentiation, alkaline phosphatase activity was measured at 37°C for 15 minutes using p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) as a substrate. The reaction was stopped by adding NaOH. The amount of p-nitrophenol was determined by measuring the absorbance at 405 nm.

[0062] Monoclonal antibody generation Five BALB / c mice were cultured at 1x10 induced with sodium butyrate. 7Mice were immunized intraperitoneally with HT-29 cells weekly. Serum was obtained from the mice after the third immunization. IgG was purified from the serum using a HiTrap Protein G HP column (GE Healthcare, Germany) and coupled to M-270 epoxy magnetic beads according to the manufacturer's protocol. Cell lysate proteins were also isolated from HT-29 cells (without sodium butyrate treatment) using cell lysis buffer. The solubilized protein mixture was adsorbed onto M-270 epoxy magnetic beads containing antibodies against sodium butyrate-induced HT-29 cells and subsequently used for immunization to generate monoclonal antibodies.

[0063] Monoclonal antibodies were produced according to the hybridoma technique of Kohler and Milstein. Hybridoma clones were screened and isolated by limiting dilution using the following two-step process.

[0064] By immunofluorescence staining, clones producing antibodies that reacted with HT-29 cells but not with differentiated HT-29 cells treated with sodium butyrate were first selected.

[0065] We then examined whether the candidate antibodies could be used to immunostain formalin-fixed, paraffin-embedded colon cancer tissue sections and oral cancer tissue sections. The antibody subclasses were determined using IsoQuick (EnviroLogix Inc., Portland, ME, USA). The antibodies were purified from the culture supernatant using ImmunoAssist MG-PP (Kanto Chemical Co., Tokyo, Japan).

[0066] Immunofluorescence staining Cells were fixed with 4% (m / v) paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum. The cells were then incubated with culture supernatant containing 12G5A, followed by incubation with an Alexa Fluor 488-conjugated anti-mouse antibody. Images were acquired using a confocal laser scanning microscope (Leica TCS SP8, Germany). 12G5A immunoreactivity was visualized with green fluorescence, and nuclei were stained with DAPI. The results of immunofluorescence staining of the resulting monoclonal antibody (12G5A) are shown in Figure 1.

[0067] The reactivity of the 12G5A antibody was greater in HT-29 colon cancer cells (Fig. 1A) than in sodium butyrate-induced HT-29 cells (Fig. 1B), suggesting that the 12G5A antibody is cancer cell-specific. As shown in Figure 1C, alkaline phosphatase activity was enhanced when sodium butyrate was added, confirming that the cells used in Figure 1B had undergone differentiation.

[0068] The amino acid sequence of the light chain variable region (SEQ ID NO: 1) and the heavy chain variable region (SEQ ID NO: 2) of the monoclonal antibody (12G5A) were analyzed by sequence analysis (FIG. 1D).

[0069] Peptide Mass Fingerprinting and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometer Purified 12G5A antibody was coupled to M-270 epoxy magnetic beads according to the manufacturer's protocol. 12G5A antigen was isolated from SW480 cell lysates by affinity chromatography using 12G5A antibody-coupled M-270 epoxy magnetic beads. The approximately 40 kDa protein band was excised from the SDS-PAGE gel, stained with a Coomassie Brilliant Blue staining kit (Integrale, Tokushima, Japan), digested with trypsin, and analyzed by MALDI-TOFMASS SPEC ANALYSIS (Genomine, Seoul, Korea).

[0070] MALDI-TOF mass spectrometry indicated that the approximately 40 kDa protein was human annexin A2.

[0071] siRNA-mediated gene silencing Annexin A2 was silenced using Thermo Fisher Scientific siRNA (Waltham, MA, USA); Cat. No. 4390824: s1383 ​​and s9548. Trilencer-27 Universal scrambled negative control siRNA (Origene Technologies, Rockville, MD, USA) was used as a non-silencing control. siRNA was transfected into cells using Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were used for subsequent studies 48 hours after transfection.

[0072] Immunoblotting Proteins were electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with Block Ace (blocking milk; Yukijirusi, Sapporo, Japan) and then incubated with 0.5 μg / ml of 12G5A or anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was assessed using a Western blotting detection kit (Promega, Madison, WI, USA). The Western blotting results are shown in Figure 2B (lane 1: mock siRNA, lane 2: s1383 ​​siRNA, lane 3: s9548 siRNA).

[0073] The results of siRNA targeting and immunoblotting confirmed that 12G5A reacts with annexin A2.

[0074] Patients and histopathological classification After receiving research approval from the Review Committee of Gifu University Graduate School of Medicine (specific approval numbers: 2019-202, 2019-0444, 28-421, 2019-157, 28-524), 55 specimens were collected from patients who had undergone surgical treatment and were diagnosed with colorectal cancer.

[0075] All 55 primary tumors were examined macroscopically and microscopically to determine the depth of invasion, lymph node status, and distant metastasis according to the TNM classification. Lymphatic and vascular invasion was determined according to the 8th Japanese Classification of Colorectal Cancer. In this classification, the status of cancer invasion into lymphatic vessels (ly) and blood vessels (v) was histopathologically determined as ly0 and v0 (no invasion), ly1 and v1 (mild invasion), ly2 and v2 (moderate invasion), and ly3 and v3 (severe invasion). The cancer invasion pattern was histopathologically classified as INFa (expansive proliferation), INFb (intermediate), and INFc (invasive proliferation).

[0076] Immunohistochemical staining A rabbit monoclonal antibody against annexin A2 was purchased from CST (D11G2, Cambridge, MA, USA). All tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Tissues were immunostained as previously described. Briefly, deparaffinized tissue slices were incubated in 10% normal goat serum for 30 minutes. Slides were then incubated with 2 μg / ml of 12G5A or D11G2 antibody for 1 hour at room temperature or overnight at 4°C, respectively. For 12G5A, tissue specimens were incubated with goat anti-murin immunoglobulin μ chain antibody conjugated with horseradish peroxidase (1:200) (Abcam, cat # ab98679; Cambridge, MA, USA). For D11G2, tissues were immunostained with antibody conjugated to the ImmPRESS™ Polymerization Reporter Enzyme Staining System (Vector Laboratories, Burlingame, CA, USA). Finally, the reaction was developed with 3,3′-diaminobenzidine and counterstained with hematoxylin.

[0077] Evaluation of immunohistochemical staining and statistical analysis Results from immunohistochemical staining were expressed as a percentage calculated as the proportion of immunoreactive colon cancer cells relative to the total cell count. The proportion of 12G5A-immunoreactive cells was determined by scoring 10 high-power fields for each sample. Staining was considered "low" if less than 50% of cancer cells showed immunoreactivity and "high" if 50% or more showed immunoreactivity.

[0078] The relationship between 12G5A immunoreactivity and clinicopathological features for 24 cases with low 12G5A immunoreactivity and 31 cases with high 12G5A immunoreactivity is shown in Table 1 .

[0079] [Table 1]

[0080] Overall survival (OS) and progression-free survival (PFS) curves were plotted using the Kaplan-Meier method, and differences in survival rates were compared using the log-rank test in univariate survival analyses. Multivariate Cox proportional hazards regression analysis was also performed to calculate the hazard ratio for death according to 12G5A epitope expression. The results are shown in Figure 3 and Table 2.

[0081] [Table 2]

[0082] As shown in Figure 3, the overall survival and progression-free survival (PFS) of patients with high 12G5A immunoreactivity were significantly worse than those of patients with low 12G5A immunoreactivity (overall survival: P = 0.039, PFS: P = 0.033).

[0083] Furthermore, as shown in Table 2 , multivariate analysis of survival risk showed that 12G5A immunoreactivity was not independently associated with prognosis (it was correlated with T factors in colorectal cancer and therefore not an independent prognostic factor).

[0084] The results of immunohistochemical staining using the 12G5A or D11G2 antibody are shown in Figure 4. The scale bar represents 50 μm.

[0085] Figure 4A shows the results of staining with D11G2, and Figure 4B shows the results of staining with 12G5A. A commercially available antibody specific for annexin A2 (D11G2) was confirmed to immunostain both noncancerous glandular cells (indicated by arrowheads) and invasive colorectal adenocarcinoma cells (indicated by arrows) (Figure 4A). On the other hand, when the specimen used in Figure 4A was stained with 12G5A, the tissue specimen was found to have low 12G5A immunoreactivity (Figure 4B), suggesting that the immunoreactivity of antibody 12G5A is not necessarily identical to that of the commercially available antibody D11G2. Figure 4C shows a case in which immunoreactivity with 12G5A was higher than that with D11G2. Strong annexin A2 immunoreactivity was observed on the cell surface, in the cytoplasm, and in the nucleus. 12G5A immunoreactivity was almost exclusively observed in the cytoplasm. Figure 4D shows a case in which immunoreactivity with 12G5A was lower.

[0086] Figure 5 shows the results of immunohistochemical staining comparing 12G5A immunoreactivity in intraoral non-tumor stratified squamous epithelium (A), dysplastic epithelium (B), and oral squamous cell carcinoma (C).

[0087] No staining was observed in non-tumor stratified squamous epithelium in the oral cavity (A), whereas staining was observed in dysplastic epithelium (B) or oral squamous cell carcinoma (C). Dysplastic epithelium is a precancerous state, and it was confirmed that 12G5A exhibits immunoreactivity not only with cancer cells but also with precancerous cells.

[0088] Glycosidase treatment Glycopeptidase F was purchased from Takara Bio Inc. O-glycosidase and α2-3,6,8 neuraminidase were obtained from New England BioLabs (Beverly, MA, USA). Enzymatic deglycosylation of cell lysate proteins was performed according to the manufacturer's protocol. Western blotting results are shown in Figure 6A (lane 1: no glycopeptidase F treatment (12G5A); lane 2: O-glycosidase and α2-3,6,8 neuraminidase treatment (12G5A); lane 3: glycopeptidase F treatment (12G5A); lane 4: glycopeptidase F treatment (D11G2)). Glycopeptidase F specifically cleaves the bond between N-glycosidic glycans and proteins (GlcNAc-Asn bond), but does not act on glycans in which Fucα1-3 is linked to GlcNAc linked to Asn. O-glycosidases cleave O-linked disaccharides from glycoproteins at Core1 and Core3. α2-3,6,8 neuraminidase cleaves α2-3, α2-6, and α2-8 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides with high specificity, but the efficiency of cleaving α2-8 linkages is lower than that of α2-3 and α2-6 linkages.

[0089] As shown in Figure 6A, glycopeptidase F treatment reduced 12G5A immunoreactivity, whereas treatment with O-glycosidase or α2-3,6,8-neuraminidase did not. Furthermore, glycopeptidase F treatment did not reduce D11G2 immunoreactivity.

[0090] SW480 cells were also incubated for 48 hours in the presence or absence of 5 μg / ml tunicamycin (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan) to assess the effect of N-linked glycosylation inhibition on immunofluorescence staining. The results are shown in Figure 6B and C. Note that blue indicates nuclear staining with DAPI.

[0091] Inhibition of N-linked glycosylation by tunicamycin abolished 12G5A immunoreactivity, shown in green, in SW480 cells (Fig. 6B) compared with SW480 cells without tunicamycin treatment (Fig. 6C).

[0092] These results suggest that 12G5A recognizes an N-linked glycosylation-modified tumor-associated epitope of annexin A2.

[0093] Colon cancer tumor suppression effect 0.93x10 on Day 0 7 SW480 colon cancer cells were subcutaneously implanted into BALB / c nu / nu mice, and the antibody (12G5A 0.9 mg / mouse) was administered on days 11 and 37. Tumor volume was measured over time (6 mice). Tumor volume was also measured in a group that did not receive the antibody (MOCK, 5 mice). Tumor volume (mm 2 ) = (longest diameter of tumor; mm) × (shortest diameter of tumor; mm)2 × 0.5236. The results are shown in Figure 7.

[0094] As shown in Figure 7, tumor volume suppression was confirmed in the 12G5A administration group. In particular, tumor volume suppression in the 12G5A administration group was significantly confirmed on Day 39 (P < 0.05, Student's t-test).

Claims

1. An antibody or a fragment thereof of the following (I) that recognizes the N-linked sugar chain of annexin A2: (I) An antibody or fragment thereof comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

2.

2. (delete)

3. The antibody or fragment thereof according to claim 1, wherein the annexin A2 is a protein comprising the amino acid sequence shown in SEQ ID NO:

3.

4. A pharmaceutical composition comprising the antibody or fragment thereof described in claim 1 or 3.

5. The pharmaceutical composition according to claim 4, which is for cancer diagnosis or cancer treatment.

6. The pharmaceutical composition according to claim 5, wherein the cancer is colon cancer or oral cancer.

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