WO2016194363A1

WO2016194363A1 - Anti-human vap-1 monoclonal antibody

Info

Publication number: WO2016194363A1
Application number: PCT/JP2016/002618
Authority: WO
Inventors: Kohei Matsumoto; Akiko Kawasaki; Takahito Imagawa; Akiko Nishimura
Original assignee: R-Tech Ueno, Ltd.
Priority date: 2015-05-29
Filing date: 2016-05-30
Publication date: 2016-12-08
Also published as: JP2018080114A

Abstract

Provided is an anti-human VAP-1 monoclonal antibody having high specificity. The monoclonal antibody may have a combination of a heavy chain variable region of SEQ ID NO. 1 and a light chain variable region of SEQ ID NO. 2; a combination of a heavy chain variable region of SEQ ID NO. 3 and a light chain variable region of SEQ ID NO. 4; a combination of a heavy chain variable region of SEQ ID NO. 5 and a light chain variable region of SEQ ID NO. 6; or an antibody binding fragment thereof. An immunoassay method using an anti-human VAP-1 monoclonal antibody according to the invention and a kit for use in the immunoassay are also provided.

Description

ANTI-HUMAN VAP-1 MONOCLONAL ANTIBODY

The present invention relates to an anti-human VAP-1 monoclonal antibody and a kit comprising the antibody for the detection of human VAP-1.

Vascular adhesion protein-1 (VAP-1), which is also referred to as semicarbazide-sensitive amine oxidase (SSAO), is a membrane-bound and circulating enzyme involved in inflammation, detoxication of amines, etc. Specifically, the enzyme bound to the surface of vascular endothelium can work as an adhesive molecule to leukocytes such as granulocytes, and lymphocytes or monocytes, which play a role in inflammation and/or immunity. In addition, the circulating enzyme is involved in detoxication of amines through its amine oxidase activity. High VAP-1/SSAO activity has been found in plasmas and/or various tissues of patients of diseases such as diabetes, atopic dermatitis, psoriasis, obesity, arteriosclerosis, and cardiac diseases.

A method for detecting VAP-1 with high sensitivity is a key technology for studying VAP-1. An enzyme capture assay (ECA) may be a candidate for the method. Under the ECA, VAP-1 is captured by an antibody and then the VAP-1/SSAO activity is measured. We had investigated existing anti-VAP-1 antibodies and identified merely one polyclonal antibody that could be used in the ECA among those antibodies. Polyclonal antibodies, however, are inherently accompanied with major problems in practical use such as lot-to-lot variation, instable supply and enormous costs for the continuous production.

An object of the present invention is to provide a monoclonal antibody having a high specificity to human VAP-1, for overcoming the problems as mentioned above. A further object is to provide a method for detecting human-VAP-1 that is able to measure human VAP-1 stably, precisely and effectively.

In one aspect, the present invention provides an anti-human VAP-1 monoclonal antibody comprising a combination of heavy and light chain variable regions selected from the following (1) to (3), and an antigen-binding fragment thereof:
(1)
1G3 Heavy chain
MGWSCIILFLVATATGVHSQVQLQQPGAELVRPGSSVKLSCKASGHTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDTATLTVDKSSSSAYMQLSSPTSEDSAVYYCTRSHYYSSSPDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLG (SEQ ID NO: 1), and
1G3 Light chain
MESQTQVFVYMLLWLSGVDGDIVMTQSREFMSTSVGDRVSVTCKASQNVGANVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCLQYNSFPLTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV (SEQ ID NO: 2);

(2)
3F10 Heavy chain
MECNWILPFILSVTSGVYSEVQLQQSGTVLARPGASVKMSCKASGYTFNTFWMHWVKQRPGKGLEWIGAIYPGNSDTTYYQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTIYYASSHFDSWGQGATLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLG (SEQ ID NO: 3), and
3F10 Light chain
MDFHVQIFSFMLISVTVILSSGEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSTYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV (SEQ ID NO: 4);

(3)
3G6 Heavy chain
MEWPCIFLFLLSVTEGVHSQVQLQQSGAELVRPGSSVKISCKASGYTFSSYWVNWVKQRPGQGLEWIGQIYPGDGDTNQNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSDYDYDGSYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLG (SEQ ID NO: 5), and
3G6 Light chain
MVFTPQILGLMLFWISASRGDIVLIQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSLETEDFGMYFCQQSYSWPHTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV (SEQ ID NO: 6).

The present invention also provides an anti-human VAP-1 monoclonal antibody having an amino acid sequence that has amino acid deletion, substitution or addition of one to several amino acids, such as one, two or three amino acids, in the amino acid sequence(s) of the heavy chain variable region and/or the light chain variable region or an antigen-binding fragment thereof that can bind specifically to human VAP-1.

The present invention also provides an anti-human VAP-1 monoclonal antibody comprising an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of the heavy chain variable region and/or the light chain variable region, or an antigen-binding fragment thereof that can bind specifically to human VAP-1.

Further, a DNA having a nucleic acid sequence that encodes the amino acid sequence of the above-described anti-human VAP-1 monoclonal antibody or an antigen-binding fragment thereof is also provided.

In another aspect, the present invention provides a hybridoma deposited under the accession number NITE BP-02034, NITE BP-02035 or NITE BP-02036, and a monoclonal antibody produced by any of the hybridomas.

In another aspect, the present invention provides an immunoassay method for detecting human VAP-1 with one or two of the above described monoclonal antibodies or fragments thereof.

In another aspect, the present invention provides an immunoassay kit for detecting VAP-1, comprising a plate and at least one reagent for immunoassay in combination with one or two of the above described monoclonal antibodies or fragments thereof. The kit may be the one suitable for conducting Enzyme Capture Assay (ECA) or Enzyme-Linked Immunosorbent Assay (ELISA).

In another aspect, the present invention provides a composition for immunohistochemically detecting human VAP-1, comprising the above described monoclonal antibody. The present invention also provides a kit for the immunohistochemical analysis comprising the composition.

The monoclonal antibody according to the present invention has a high specificity and a high binding ability to human VAP-1, and is preferably used for immunoassay, especially for ECA or ELISA such as sandwich ELISA.

Fig. 1-1 shows antibody titers determined by means of antigen-ELISA in which the antigen HTG-VAP-1 was immobilized on the ELISA plate. Culture supernatants from the hybridomas established in Example 1 were examined. Fig. 1-2 shows antibody titers determined by means of antigen-ELISA in which the antigen HTG-VAP-1 was immobilized on the ELISA plate. Monoclonal antibodies purified from the culture supernatants of the hybridomas established in Example 1 were examined. Fig. 2-1 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized with goat anti-human VAP-1 polyclonal antibody (comparative example). Fig. 2-2 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized with mouse anti-human VAP-1 monoclonal antibody 1G3 (Example 2). Fig. 2-3 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized with mouse anti-human VAP-1 monoclonal antibody 3F10 (Example 2). Fig. 2-4 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized with mouse anti-human VAP-1 monoclonal antibody 3G6 (Example 2). Fig. 3-1 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized by using 5 μg/ml of mouse anti-human VAP-1 monoclonal antibody 1G3. Fig. 3-2 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized by using 10 μg/ml of mouse anti-human VAP-1 monoclonal antibody 1G3. Fig. 3-3 shows the result of ECA for detecting VAP-1 in serum with an ECA plate sensitized by using 20 μg/ml of mouse anti-human VAP-1 monoclonal antibody 1G3. Fig. 4-1 shows the results of the sandwich ELISA to detect VAP-1 in serum or HTG-VAP-1. In the assay, plates sensitized with anti-human VAP-1 monoclonal antibody 1G3 as the capture antibody were used. Biotinylated antibodies 1G3, 3F10, 3G6 and TK8-14 were used as detection antibodies. Fig. 4-2 shows the results of the sandwich ELISA to detect VAP-1 in serum or HTG-VAP-1. In the assay, plates sensitized with the anti-human VAP-1 monoclonal antibody 3F10 as a capture antibody were used. Biotinylated antibodies 1G3, 3F10, 3G6 and TK8-14 were used as detection antibodies. Fig. 4-3 shows the results of the sandwich ELISA to detect VAP-1 in serum or HTG-VAP-1. In the assay, plates sensitized with anti-human VAP-1 monoclonal antibody 3G6 as a capture antibody were used. Biotinylated antibodies 1G3, 3F10, 3G6 and TK8-14 were used as detection antibodies. Fig. 4-4 shows the results of the sandwich ELISA to detect VAP-1 in serum or HTG-VAP-1. In the assay, plates sensitized with the anti-His tag monoclonal antibody as a capture antibody was used. Biotinylated antibodies 1G3, 3F10, 3G6 and TK8-14 were used as detection antibodies. Fig. 5-1 shows the results of the sandwich ELISA to detect HTG-VAP-1. In the assay, plates sensitized with the anti-human VAP-1 monoclonal antibody 1G3 or 3G6 as a capture antibody and biotin-labeled monoclonal antibody 3F10 as a detection antibody were used. Fig. 5-2 shows the results of the sandwich ELISA to detect VAP-1 in serum. In the assay, plates sensitized with anti-human VAP-1 monoclonal antibody 1G3 or 3G6 as a capture antibody and the biotin-labeled monoclonal antibody 3F10 as a detection antibody were used.

In one aspect, the present invention provides an anti-human VAP-1 monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody according to the present application includes an antibody comprising a combination of a heavy chain variable region and a light chain variable region of any of the following (1) to (3):
(1)
a heavy chain variable region having the amino acid sequence of SEQ ID NO. 1; a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 1; or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 1, and
a light chain variable region having the amino acid sequence of SEQ ID NO. 2; a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 2; or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 2;

(2)
a heavy chain variable region having the sequence of SEQ ID NO. 3; a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 3; or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 3, and
a light chain variable region having the sequence of SEQ ID NO. 4, a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 4;

(3)
a heavy chain variable region having the sequence of SEQ ID NO. 5, a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 5, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 5, and
a light chain variable region having the sequence of SEQ ID NO. 6, a sequence having deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO. 6
wherein the monoclonal antibody binds specifically to human VAP-1.

The terms used in the specification and claims are explained in detail below. Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise.

The term "anti-human VAP-1 monoclonal antibody" as used herein refers to a monoclonal antibody that binds to human VAP-1. The origin of the antibody may be, but is not particularly limited to, mouse, rat, rabbit, goat, or human. The "monoclonal antibody" may include a chimeric antibody and a humanized antibody. The immunoglobulin class of the monoclonal antibody according to the present application is not particularly limited.

The term "sequence identity" is determined by comparing two sequences aligned under optimal conditions over the sequence to be compared. Here, the sequence to be compared may have an addition or deletion (e.g., gap and the like) in the optimum alignment of the two sequences. A sequence identity can be calculated with a program such as FASTA, BLAST, CLUSTAL W and the like which are available from a public database, for example, DDBJ (http://www.ddbj.nig.ac.jp). Alternatively, commercially available sequence analysis software, for example Vector NTI^(R) software and GENETYX^(R) ver. 12, may also be used to determine the sequence identity.

The term "antigen-binding fragment" is a fragment of an antibody molecule that specifically binds to the original antigen. Examples of antigen-binding fragments may include Fab (fragment of antigen binding), F(ab')₂, Fab', single chain Fv, disulfide stabilized Fv, dimerized V region fragment, peptide containing CDRs and the like (Expert Opinion on Therapeutic Patents, 6(5), 441-456, 1996). Antibodies and antigen-binding fragments may be prepared by any method well known in the art. (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988, http://www.gene.mie-u.ac.jp/Protocol/Original/Antibody.html, US 6,331,415, US 5,693,761, US 5,225,539, US 5,981,175, US 5,612,205, US 5,814318, US 5,545806, US 7,145,056, US 6,492,160, US 5,871,907, and US5,733,743).

In one aspect, the present invention also provides a hybridoma that secrets any one of the above-described monoclonal antibodies. The hybridomas were deposited on April 17, 2015 (date of the original deposit), with National Institute of Technology and Evaluation (NITE), Patent Microorganisms Depositary, an International depositary authority, which is located at the address of #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan. In particular, the hybridomas produce monoclonal antibodies 3F10, 1G3 and 3G6 and they were named VAP-1 Cl.AF-3F10H7A11H9, VAP-1 Cl.AF-1G3G5H2H1 and VAP-1 Cl.AF-3G6G7A5G8, respectively. They were accepted under the Accession Numbers NITE BP-02034, NITE BP-02035 and NITE BP-02036, respectively.

The monoclonal antibodies of the present invention may be obtained by culturing the hybridoma in a conventional manner. The antibody is secreted into the culture supernatant and may be isolated and purified from the culture supernatant according to a conventional purification method, for example, by means of protein A affinity chromatography.

In one embodiment, the monoclonal antibody or an antigen-binding fragment thereof according to the present invention may be obtained by introducing an expression vector containing a polynucleotide sequence that encodes the antibody comprising the particular heavy chain variable region and the light chain variable region into cells and expressing the polynucleotide in the cells. In particular, an expression vector is constructed so that the sequence encoding the antibody is located downstream of an expression-controlling region such as an enhancer or a promoter to produce the antibody under the control of the region. Host cells are transformed with the expression vector to produce the antibody.

Host cells may be, for example, eukaryotic cells such as animal cells, plant cells, and fungus cells. Animal cells may include mammalian cells, e.g., CHO, COS, NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero cells, amphibian cells, e.g. Xenopus oocyte, insect cells, e.g. Sf9, Sf21 and Tn5. Fungal cells may include yeasts, e.g. Saccharomyces, such as Saccharomyces cerevisiae, and filamentous fungi, e.g. Aspergillus, such as Aspergillus niger. Prokaryotic cells such as Escherichia coli (E. coli), e.g. JM109, DH5α and HB101 and Bacillus subtilis may be used as host cells. Introduction of the expression vector into the host cells may be carried out by a known method such as calcium phosphate method, a DEAE dextran method, lipofection and the like.

The monoclonal antibody or an antigen-binding fragment thereof according to the present invention binds to human VAP-1 with a high specificity. The monoclonal antibody or the fragment thereof according to the present invention is preferably used in immunoassay to detect human VAP-1 in a biological sample. Examples of immunoassay may encompass all types of assays utilizing the antigen-antibody reaction. Enzyme Capture Assay (ECA), Enzyme-Linked Immunosorbent Assay (ELISA), radioimmunoassay, fluorescent antibody method and the like are exemplified. In the description and the claims, the term "Enzyme Capture Assay (ECA)" refers to a method for the detection and/or quantification of an enzyme of interest by capturing the enzyme by an antibody immobilized on a substrate and measuring the activity of the captured enzyme.

In one aspect, the present invention provides a kit for the detection and/or measurement of human VAP-1 comprising one or more of the antibodies or fragments according to the present invention and at least one reagent for immunoassay. The antibodies may be labeled if needed. Various immunoassay reagents are known to the art. The immunoassay reagent to be included in the kit may be appropriately selected from the known reagents depending on the type of the assay employed.

In one embodiment, the present invention provides an ECA kit comprising the monoclonal antibody 1G3 or 3G6 or an antigen-binding fragment thereof. In addition, the kit may comprise a plate, a blocking solution such as a solution containing bovine serum albumin (BSA), a milk protein solution and the like; a washing solution such as a phosphate buffered saline solution containing a detergent, e.g. a phosphate buffered saline (PBS) solution containing Tween 20; a VAP-1 substrate such as benzylamine, and a colorimetric substrate such as horseradish peroxidase (HRP). The monoclonal antibody or an antigen-binding fragment thereof according to the present invention may be provided under conditions in which the antibody or the fragment is immobilized on the plate.

In one embodiment, the present invention provides a kit for sandwich ELISA utilizing one of the monoclonal antibodies or antigen-binding fragments thereof according to the present invention as a capture antibody, and one of the monoclonal antibodies or antigen-binding fragments thereof according to the present invention other than the capture antibody as a labeled or detection antibody. A kit that includes a plate, a blocking solution, a washing solution, and a colorimetric substrate in addition to the antibodies is exemplified. Labelling of antibody may be carried out with, for example, HRP, biotin and the like. When biotin is used, avidin that specifically binds to biotin may be labeled with a substance emitting a signal.

When antibody 1G3 or 3G6 is used as a capture antibody in combination with antibody 3F10 as a detection antibody for a sandwich ELISA, VAP-1 can be detected and measured with a good sensitivity. The combination of antibody 3G6 as a capture antibody with antibody 3F10 as a detection antibody is more preferably used.

In another embodiment, the anti-human VAP-1 monoclonal antibody according to the present invention is also useful for immunohistochemical detection of human VAP-1. Immunohistochemical detection is an established technique and can be performed according to known procedures by using the monoclonal antibody according to the present invention.

In particular, biological tissue section may be prepared from a frozen tissue or from a paraffin-embedded tissue. An anti-human VAP-1 monoclonal antibody according to the present invention is applied on the tissue section and allowed to react with VAP-1 in the section. The VAP-1 expressed in the tissue section can be detected and visualized when the monoclonal antibody is labeled preliminary with a signal emitting material. Alternatively, an unlabeled anti VAP-1 monoclonal antibody bound to VAP-1 in the tissue section may be visualized with a secondary antibody to the anti VAP-1 monoclonal antibody that is labelled with a signal emitting material.

A variety of fluorescent dyes and elements can be used to label the antibodies. These include peroxidase, colloidal gold, alkaline phosphatase and radioactive elements. Diaminobenzidine (DAB) staining may also be employed.

Therefore, in one embodiment, the present invention provides an immunohistochemistry kit for the detection of human VAP-1, comprising the above described anti-human VAP-1 monoclonal antibody or an antigen-binding fragment thereof, and at least one reagent for immunohistochemistry. The kit may be that comprising a labeled anti-human VAP-1 monoclonal antibody or an antigen-binding fragment thereof according to the present invention and a colorimetric substrate, or that comprising an anti-human VAP-1 monoclonal antibody, a labeled secondary antibody and a colorimetric reagent.

Establishment of hybridomas producing the monoclonal antibodies
A His-tagged recombinant human VAP-1 protein (HTG-VAP-1) purified with a TALON column was used as the immunogen. Four mice (Balb/c, female) were immunized with the VAP-1 protein mixed with Freund’s adjuvant. After three immunizations with intervals of two weeks, blood cells were isolated from the spleen or lymph node of the animals. The isolated blood cells were fused to mouse myeloma cells P3U1 according to the PEG method. The fused cells were distributed on 96-well plates and incubated until colony formation was observed.
Culture supernatant in the wells in which colony was observed was subjected to screening by means of ECA and sandwich ELISA.

Screening by ECA
- Antibody immobilized on the plate: anti-mouse IgG antibody
- Capture antibodies: cell culture supernatant from the wells in which colony formation were observed (expected to contain an antibody)
- Samples to be measured: HTG-VAP-1 and human serum from a healthy individual
- Preparation of ECA plate: the anti-mouse IgG antibody was immobilized on the 96-well plate. The culture supernatants were subsequently added to the plate so that the antibodies contained in the supernatants bound to the antibody immobilized on the plate. After the antibodies in the culture supernatants were immobilized on the plate via the anti-mouse IgG antibody, the plate was washed with PBS-Tween 20 (0.13%) to remove the unbound antibodies to give ECA plate for screening.

Measurement by ECA
Two fold diluted serum of a healthy individual (100 μl/well) was added to the ECA plate for screening and the plate was left to stand at room temperature for one hour to allow VAP-1 in the serum was captured by the capture antibodies immobilized on the plate. The plate was washed with a buffer solution, and then, a solution containing benzylamine (2 mM) which is a substrate for VAP-1, a fluorescence precursor ADHP (400 μM) and horseradish peroxidase (HRP; 2 U/ml) was added thereto. The plate was incubated at room temperature for three hours. In the presence of VAP-1, benzylamine will be oxidized to generate hydrogen peroxide. In the presence of hydrogen peroxide and horseradish peroxidase (HRP), non-fluorescent ADHP is oxidized to the highly fluorescent Resorufin. The fluorescence intensity was measured with a fluorescence plate reader to determine the amount of Resorufin. The amount of Resorufin was divided by the reaction time to determine the enzyme activity of VAP-1. The cell cultures whose culture supernatant contained an antibody that could bind to VAP-1 were identified.

Screening by sandwich ELISA
Screening was carried out in accordance with
- Capture antibody, i.e. antibody to be immobilized on the plate: goat anti-VAP-1 polyclonal antibody (Catalog No. AF3957, R&D Systems, Inc.)
- Antigens to be measured: HTG-VAP-1 and serum of a healthy individual (2-fold dilution)
- Primary antibody for detection: antibody contained in cell culture supernatant from the wells in which colony formation were observed (expected to contain an antibody)
- Secondary antibody: anti-mouse IgG antibody labeled with horseradish peroxidase (HRP)
- Measurement procedures: The capture antibody was immobilized on a 96-well plate. The antigen was added to the plate and allowed to be captured by the immobilized antibody. The primary antibody for detection, that is, the cell culture supernatant was added to the plate and the plate was left to stand to allow the antibody for detection in the culture supernatant bound to the antigen captured by the capture antibody. The plate was washed and then the secondary antibody was added to the plate. The signal from the secondary antibody was measured and the cell cultures whose culture supernatant contained an antibody that could bind to the antigen captured by the capture antibody were identified.

The cell cultures identified in either one of ECA and sandwich ELISA screenings were cloned by conducting limiting

dilution

3 or 4 times; and three monoclonal antibody-producing cell lines (hybridomas) were established (1G3, 3F10 and 3G6).

The inventors gave an ID to each clone as shown in table 1 and deposited the clones on April 17, 2015 (date of the original deposit) with National Institute of Technology and Evaluation (NITE), Patent Microorganisms Depositary, an International depositary authority, which is located at the address of #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan. They were accepted under the Accession Numbers shown in Table 1.

Purification of monoclonal antibodies from culture supernatants of the hybridomas
Three hybridomas established were proliferated and cell culture supernatant was collected. The anti-human VAP-1 monoclonal antibody was purified from 100ml of the culture supernatant by using protein A affinity column.

The results are shown in Table 2. The yields of purified monoclonal antibodies from 100ml culture supernatants were calculated based on their absorbance and were 2.3 mg for clone 1G3, 1.12 mg for clone 3F10, and 1.36 mg for clone 3G6.

The sequences of the heavy and light chain variable regions of the purified monoclonal antibodies were determined according to a conventional method. The sequence of each variable region is shown as follows:
1G3 heavy chain variable region: SEQ ID NO. 1
1G3 light chain variable region: SEQ ID NO. 2
3F10 heavy chain variable region: SEQ ID NO. 3
3F10 light chain variable region: SEQ ID NO. 4
3G6 heavy chain variable region: SEQ ID NO. 5
3G6 light chain variable region: SEQ ID NO. 6.
The class of each light chain variable region was kappa.

Titer of the monoclonal antibody
The titer of each monoclonal antibody to the antigen HTG-VAP-1 was determined by antigen ELISA using an ELISA plate on which the antigen HTG-VAP-1 was immobilized.
Firstly, a plate for antigen ELISA was prepared. HTG-VAP-1 was diluted in PBS to give a 10 μg/ml solution. The diluted solution (100 μl/well) was added to a 96-well plate and the plate was left to stand at 4°C overnight so as to the plate was sensitized by the antigen. The solution was removed and PBS containing 1% BSA (blocking solution) 200 μl was added to the plate. The plate was left to stand at 4°C overnight again, and the blocking solution was removed. The plate was left to stand at room temperature for 6 hours to dry and remove the blocking solution completely.

The culture supernatants of the hybridomas and the purified monoclonal antibodies were serially diluted and the diluted solution (100 μl/well) was added to the plate. Subsequently, a 1000-fold diluted HRP-labeled secondary antibody (100 μl/well) was added to the plate and the plate was left to stand at room temperature for one hour so that the secondary antibody bound to the antibody that bound to the antigen immobilized on the plate. After the one-hour reaction, unbound antibodies were removed and the plate was washed with PBS-Tween 20 (0.13%).

Then, a solution containing the substrate for HRP was added to the plate to induce the chromogenic reaction. After completion of the reaction, the absorbance at 450nm (A450) was measured. Results are shown in Fig. 1-1 (hybridoma culture supernatants) and Fig. 1-2 (anti-human VAP-1 monoclonal antibodies). As shown in Figs 1-1 and 1-2, the purified antibodies had almost the same reactivity as the culture supernatant of the hybridomas from which the antibodies were isolated. This result shows that the monoclonal antibodies can effectively be isolated and purified from the hybridoma culture supernatant by using the protein A affinity column without losing their activities.

Assessment of the antibodies for use in ECA
A 96-well plate was directly sensitized with each of the purified anti-human VAP-1 monoclonal antibodies obtained in Example 1 and it was assessed as to whether or not the antibodies were able to be used in the ECA. Serum from a healthy individual (2-fold dilution) that contains VAP-1 was used as sample. The detailed procedure of the assessment is described as follows:

Preparation of ECA plates sensitized with the anti-human VAP-1 monoclonal antibodies
Each of the anti-human VAP-1 monoclonal antibodies was diluted in PBS to give a 2 μg/ml solution. The solution (100 μl/well) was added to a 96-well plate and the plate was left to stand at 4°C overnight to allow the antibody bond directly to the plate. Then, the antibody solution was removed and a blocking solution PBS-BSA (1%) (200 μl/well) was added to the plate. The plate was left to stand at 4°C overnight and the blocking solution was removed. The plate was left to stand at room temperature for six hours so that the blocking solution was dried and removed completely to give ECA plates for assessment.

Preparation of ECA plate sensitized with goat anti-VAP-1 polyclonal antibody (comparative example)
The ECA plate of comparative example was prepared in the same manner as above except for using the goat anti-VAP-1 polyclonal antibody (Catalog No. AF3957: R&D Systems, Inc.). The inventors had previously assessed anti-VAP-1 antibodies available on the market and confirmed that only this polyclonal antibody among those antibodies could be used for the ECA.

Methods of Measurement
The inventors compared properties of each of the three monoclonal antibodies, with those of the goat anti-VAP-1 polyclonal antibody, to evaluate performance characteristics for the ECA kits. Specifically, the aforementioned ECA plates including the monoclonal antibody 1G3, 3F10 or 3G6; or the goat anti-human VAP-1 polyclonal antibody, were evaluated in the following manner. First, serum from a healthy individual was diluted by a reaction buffer. The diluted serum (100 μl/well) was added to each of the ECA plates; and the plates were left to stand at room temperature for one hour so that VAP-1 protein in the serum was captured by the antibodies immobilized on the plates. Then, the serum was removed and the plates were washed with PBS-Tween 20 (0.13%). The reaction buffer, which was used to dilute the serum, was used as the negative control.

A reaction solution containing 400 μM ADHP (10-acetyl-3, 7-dihydroxy phenoxazine), 2 U/ml HRP and 2 mM benzylamine was previously prepared for the detection reaction. The reaction solution (100 μl/well) was added to each of the above ECA plates; and the plates were incubated at 30°C. Then, fluorescence intensities were measured at 1, 2, 3, 4, and 5 hours of the reaction, for each of the ECA plates. Fig. 2-1 shows the fluorescence intensity over time measured at 595 nm at an excitation wavelength of 535 nm (hereafter, “F535/F595 over reaction time”), for the ECA plate including the goat anti-human VAP-1 polyclonal antibody. Further, Figs. 2-2, 2-3 and 2-4 show the F535/F595 over reaction time, for the ECA plates including the monoclonal antibodies obtained in Example 1, 1G3, 3F10 and 3G6, respectively. In Figs. 2-1 to 2-4, the results with the diluted serum are depicted as "Assay Control" and the results with the reaction buffer are depicted as "No Enzyme" in the figures.

As shown in Figs. 2-2 and 2-4, monoclonal antibodies 1G3 and 3G6 among the three antibodies established herein could preferably be used in the ECA. Monoclonal antibody 1G3 provided stronger signal intensities than the goat anti-VAP-1 polyclonal antibody and monoclonal antibody 3G6 provided signal intensities similar to that provided by the goat anti-VAP-1 polyclonal antibody (Fig. 2-1).

The ECA plate including the monoclonal antibody 1G3 provided stronger signal than the goat polyclonal antibody. Then, the inventors evaluated concentration dependent sensitivity of the monoclonal antibody 1G3. Monoclonal antibody 1G3 was diluted to give 5 μg/ml, 10 μg/ml, and 20 μg/ml solutions. A 96-well plate was directly sensitized with the diluted antibody solution to give the ECA plate. The ECA procedure was carried out in the same manner as described above. Results are shown in Figs. 3-1, 3-2 and 3-3. There was no difference in signal level depending on the concentrations used to sensitize the ECA plate.

Assessment of antibody combinations for sandwich ELISA
The three anti-human VAP-1 monoclonal antibodies 1G3, 3F10 and 3G6 were combined with each other and assessed for sandwich ELISA.

Preparation of ELISA plates
96-well plates were sensitized with each of the three antibodies in the same manner as described in Example 2 to prepare antibody plates. A control antibody plate was similarly prepared by sensitizing a 96-well plate with an anti-His tag antibody.

Each of the monoclonal antibodies was diluted in PBS to give a 5 μg/ml solution. The solution (100 μl/well) was added to a 96-well plate and the plate was left to stand at 4°C overnight so that the plate was sensitized with the antibody. The antibody solution was removed and a blocking solution PBS-BSA (1%) (200 μl/well) was added to the plate. The plate was left to stand at 4°C overnight again and then, the blocking solution was removed. The plate was left to stand at room temperature for six hours so that the blocking solution was removed and dried completely to give the sensitized ELISA plate.

Preparation of detection antibodies
Each of the monoclonal antibodies was labeled with biotin by a conventional method to give detection antibodies. A commercially available anti-VAP-1 monoclonal mouse antibody TK8-14 (sc-33670, Santa Cruz Biotechnology, Inc.) was labeled with biotin and used as a control detection antibody.

HTG-VAP-1 500 ng/100 μl in PBS-BSA (1%) (100 μl/well), or 2- or 4-fold diluted serum of a healthy individual (100 μl/well) was added to each ELISA plate. The plate was left to stand at room temperature for one hour so that HTG-VAP-1 or VAP-1 in the serum was captured by the sensitized antibody (capture antibody). The antigen sample was then removed and the plate was washed with PBS-Tween 20 (0.13%). Each of the biotin-labeled antibodies was diluted in PBS to give a 5 μg/ml solution. The solution (100 μl/well) was added to the plate and the plate was left to stand at room temperature for one hour so that the biotin-labelled detection antibody bound to VAP-1 or HTG-VAP-1 captured by the capture antibody. Unbound biotin-labeled antibodies were removed, and then the wells washed with PBS-Tween 20 (0.13%). A 1000-fold diluted HRP-labeled streptavidin in PBS (100 μl/well) was subsequently added to the plate. The plate was then left to stand at room temperature for one hour, unbound HRP-labeled streptavidin was removed, and the plate was washed with PBS-Tween 20 (0.13%). A solution containing the substrate to HRP was added to induce the coloring reaction. After the termination of the coloring reaction, an absorbance at 450nm (A450) was measured.

Combinations of antibodies that effectively sandwich VAP-1 in the serum and the His-tagged VAP-1 (HTG-VAP-1) were investigated. Results are shown in Figs. 4-1, 4-2, 4-3 and 4-4.

Combination of 1G3 or 3G6 as a capture antibody and 3F10 as a detection antibody (or labeled antibody) provided high signal intensity when detecting human VAP-1 in the serum sample. The signal intensity with the 2-fold diluted serum (50 μl) reached a ceiling.
In the positive control plate sensitized with the anti-His tag antibody, all monoclonal antibodies bond to HTG-VAP-1 captured by the sensitized antigen. In contrast, only little or almost no signal was observed with serum that contains VAP-1 having no His tag. The biotinylated TK8-14 gave weak signal when used in the antibody plate sensitized with any one of 1G3, 3G6, and 3F10.

The combination of 1G3 as capture antibody and 3G6 as detection antibody, and the combination of 3G6 as capture antibody and 1G3 as detection antibody both gave relatively low signal intensities. Those results suggest that the epitopes on VAP-1 recognized by those two antibodies are close each other and the combination of 1G3 and 3G6 is not suitable for the sandwich ELISA. The same is applied to TK8-14. Monoclonal antibody 3F10 gave only a weak titer in Example 1 whereas the same antibody gave strong signal intensities when used as the detection antibody in the 1G3- and 3G6-sensitized plates. Accordingly, it is suggested that the epitope region recognized by 3F10 and the epitope region recognized by 1G3 or 3G6 are enough distant from each other. On the other hand, the signal intensities were generally weak when 3F10-sensitized plate was used. It is suggested that the titer of 3F10 is lower than that of the other two antibodies.

Further, in the anti-His tag sensitized plate, VAP-1 having no His tag in serum provided little or almost no signal when any of the monoclonal antibodies was used as a detection antibody. All antibodies could bind to HTG-VAP-1 captured by the anti-His tag antibody and provided strong signal intensity. This indicates that all the labeled antibodies used could recognize and bind to HTG-VAP-1 having the His tag and it is demonstrated that the specificities of the antibodies were not impaired by the biotinylation.

In view of the results of the above examples, monoclonal antibodies 1G3 and 3G6 were selected as candidates for sensitizing the plate (capture antibody). Monoclonal antibody 3F10 was selected as a candidate for detection antibody (labelled antibody) to be used for plates sensitized by 1G3 or 3G6. Although 3F10 has a relatively low titer, the antibody recognizes an epitope region separate from those recognized by the other two antibodies.

Optimal combination for sandwich ELISA
VAP-1 in the sequentially diluted serum and sequentially diluted HTG-VAP-1 were detected by sandwich ELISA using the biotin-labeled antibody 3F10 (detection antibody) in combination with the antibody 1G3-sensitized plate or the antibody 3G6-sensitized plate to determine the optimal combination.

Each of the monoclonal antibodies 1G3 and 3G6 was diluted in PBS to give an antibody solution. The solution (100 μl/well) was added to a 96-well plate and the plate was left to stand at 4°C overnight so that the plate was sensitized with the antibody. The antibody solution was removed and a blocking solution PBS-BSA (1%) (200 μl/well) was added to the plate. The plate was left to stand at 4°C overnight again and then, the blocking solution was removed. The plate was left to stand at room temperature for six hours so that the blocking solution was removed and dried completely to give the 1G3- and 3G6- sensitized ELISA plates.

HTG-VAP-1 and serum of a healthy individual were sequentially diluted by PBS-BSA (1%). The diluted sample (100 μl/well) was added to the plate and the plate was left to stand at room temperature for one hour so that HTG-VAP-1 or VAP-1 in the serum was captured by the sensitized antibody. The sample was then removed and the plate was washed with PBS-Tween 20 (0.13%). The biotin-labeled monoclonal antibody 3F10 was diluted in PBS to give a 5 μg/ml solution. The solution (100 μl/well) was added to the plate and the plate was left to stand at room temperature for one hour so that the biotin-labelled detection antibody bound to VAP-1 or HTG-VAP-1 captured by the capture antibody. Unbound biotin-labeled antibodies were removed, and then the plate was washed with PBS-Tween 20 (0.13%). A 1000-fold diluted HRP-labeled streptavidin in PBS (100 μl/well) was subsequently added to the plate. The plate was then left to stand at room temperature for one hour, unbound HRP-labeled streptavidin was removed, and the plate was washed with PBS-Tween 20 (0.13%). A solution containing the substrate to HRP was added to induce the coloring reaction. After the termination of the coloring reaction, an absorbance at 450nm (A450) was measured.
The results are shown in Figs. 5-1 and 5-2.

All combinations examined could effectively be used for the sandwich ELISA assay. In particular, strong signal intensities were observed with the combination of 3G6 (capture antibody) and 3F10 (detection/biotin-labeled antibody) even with highly diluted samples. It is considered that the combination of 3G6 and 3F10 is optimal.

Assessment of the antibodies for use in immunohistochemistry
The present inventors assessed the monoclonal antibodies obtained in Example 1 for immunohistochemistry in human skin tissue. In particular, the antibodies were assessed by the procedures shown below.

Preparation of specimens and staining of the specimens
Frozen human skin tissue and OCT compound-embedded human skin tissue were sliced to give sample specimens. OCT compound was purchased from Sakura Finetek Japan Co.,Ltd, Tokyo Japan.
Those two sample specimens were subjected to immunohistochemical staining by means of the DAB staining technique.

The monoclonal antibodies 1G3, 3G6 and 3F10 obtained in Example 1 were used as primary antibodies. The antibody was diluted to give from 0.4 to 4 μg/ml solutions. The specimens were incubated with the diluted antibody solution for one hour and then, washed. Subsequently, the specimen was incubated with the secondary antibody, HRP-labelled anti-mouse IgG antibody and then, washed. Then, the specimen was developed by using One-Step Polymer-HRP Kit (HK595-50K, BioGenex Laboratories Inc.) and micrographic image of the specimen was obtained.

Evaluation of staining
Each of the monoclonal antibodies for immunohistochemical staining was assessed according to the following criteria:
(a) vascular endothelium was stained, and (b) the stained region by the monoclonal antibody was the same as the region stained by mouse anti-VAP-1 monoclonal antibody TK8-14 (Santa Cruz, sc-33670). TK8-14 had been confirmed to be useful for immunohistochemical staining of human VAP-1. When both criteria were found positive, the monoclonal antibody was evaluated useful for immunohistochemical detection of human VAP-1.

Results
The obtained images were assessed according to the above described criteria. All the antibodies examined were confirmed to be useful for staining human VAP-1 in the specimens obtained from vascular endothelium.
Accession Numbers

NITE BP-02034
NITE BP-02035
NITE BP-02036

Claims

A monoclonal antibody having a combination of a heavy chain variable region and a light chain variable region of any of the following (1) to (3), or an antigen-binding fragment thereof:
(1)
a heavy chain variable region having the sequence of SEQ ID NO. 1, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1, and
a light chain variable region having the sequence of SEQ ID NO. 2, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 2, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 2;
(2)
a heavy chain variable region having the sequence of SEQ ID NO. 3, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 3, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 3, and
a light chain variable region having the sequence of SEQ ID NO. 4, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 4, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 4;
(3)
a heavy chain variable region having the sequence of SEQ ID NO. 5, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 5, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 5, and
a light chain variable region having the sequence of SEQ ID NO. 6, a sequence having amino acid deletion, substitution or addition of one to several amino acids, such as one to three amino acids, in the sequence of SEQ ID NO. 6, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 6,
wherein the monoclonal antibody or the antigen-binding fragment binds specifically to human VAP-1.
The anti-human VAP-1 monoclonal antibody according to claim 1, comprising the following heavy and light chain variable regions:
a heavy chain variable region consisting of SEQ ID NO. 1; and
a light chain variable region consisting of SEQ ID NO. 2.
The anti-human VAP-1 monoclonal antibody according to claim 1, comprising the following heavy and light chain variable regions:
a heavy chain variable region consisting of SEQ ID NO. 3; and
a light chain variable region consisting of SEQ ID NO. 4.
The anti-human VAP-1 monoclonal antibody according to claim 1, comprising the following heavy and light chain variable regions:
a heavy chain variable region consisting of SEQ ID NO. 5; and
a light chain variable region consisting of SEQ ID NO. 6.
The monoclonal antibody according to any one of claims 1-4, which is a mouse monoclonal antibody.
A monoclonal antibody produced by any one of hybridomas deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary under the accession numbers NITE BP-02034, NITE BP-02035 and NITE BP-02036.
A hybridoma selected from hybridomas deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary under the accession numbers NITE BP-02034, NITE BP-02035 and NITE BP-02036.
An immunoassay method for detecting human VAP-1, wherein at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 is used.
An immunoassay kit for detecting human VAP-1, comprising at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 and at least one reagent for immunoassay.
The kit according to claim 9, wherein the kit is for conducting ECA, and the at least one monoclonal antibody is the monoclonal antibody having the combination of (1) or (3) as defined in claim 1.
The kit according to claim 9, wherein the kit is for conducting sandwich ELISA and comprises a capture antibody and a detection antibody,
wherein the capture antibody is the monoclonal antibody having the combination of (1) or (3) as defined in claim 1, and the detection antibody is the monoclonal antibody having the combination of (2) as defined in claim 1.
An immunohistochemistry method, comprising a step of detecting human VAP-1 with at least one monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6.
An immunohistochemistry kit for the detection of human VAP-1, comprising at least one monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6 and at least one reagent for immunohistochemistry.