Monoclonal antibodies and their uses

Abstract

a monoclonal antibody or a fragment thereof, wherein heavy chain complementarity-determining regions (CDRs) 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDRs 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively; A monoclonal antibody or a fragment thereof, wherein heavy chain CDR1 to CDR3 consist of the amino acid sequences of SEQ ID NOS: 2 to 4, respectively, in which one or several amino acids have been deleted, substituted or added, and light chain CDR1 to CDR3 consist of the amino acid sequences of SEQ ID NOS: 5 to 7, respectively, in which one or several amino acids have been deleted, substituted or added, and wherein the monoclonal antibody has the ability to bind to KK-LC-1 protein; or A monoclonal antibody or fragment thereof that competes with a monoclonal antibody or fragment thereof having heavy chain CDR1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDR1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, and has binding affinity to KK-LC-1 protein.

Description

[Technical field]

[0001] The present invention relates to a monoclonal antibody and its use. More specifically, the present invention relates to a monoclonal antibody or a fragment thereof, a cancer detection kit, a cancer detection method, and a hybridoma cell line. This application claims priority based on Japanese Patent Application No. 2017-218872 filed on November 14, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] It is becoming clear that Kitakyushu lung cancer antigen-1 (KK-LC-1) can be used as a marker expressed in various cancers (see, for example, Patent Document 1). Therefore, it is important to develop a technique that can easily detect KK-LC-1. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6028253 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a technique for easily detecting KK-LC-1. [Means for solving the problem]

[0005] The present invention includes the following aspects. [1] Any one of the following monoclonal antibodies (i) to (iii) or a fragment thereof: (i) A monoclonal antibody or a fragment thereof, in which heavy chain complementarity determining regions (CDRs) 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDRs 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively. (ii) A monoclonal antibody or a fragment thereof, in which heavy chain CDR1 to 3 consist of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, in which one or several amino acids have been deleted, substituted or added, and light chain CDR1 to 3 consist of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, in which one or several amino acids have been deleted, substituted or added, and which has binding ability to KK-LC-1 protein. (iii) A monoclonal antibody or a fragment thereof, which competes with a monoclonal antibody or a fragment thereof having heavy chain CDR1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDR1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, and has binding affinity to KK-LC-1 protein. [2] The monoclonal antibody or fragment thereof described in [1], which has binding ability to KK-LC-1 protein in a fixed biological sample. [3] The monoclonal antibody or fragment thereof described in [2], wherein the biological sample is fixed with a cross-linking fixative. [4] The monoclonal antibody or fragment thereof described in [3], wherein the cross-linking fixative is formaldehyde, paraformaldehyde or glutaraldehyde. [5] The monoclonal antibody or a fragment thereof according to any one of [1] to [4], which has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:1. [6] The monoclonal antibody or a fragment thereof according to any one of [1] to [5], which has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:12. [7] The monoclonal antibody or a fragment thereof according to any one of [1] to [6], which is produced by a hybridoma cell line having the accession number NITE BP-02527. [8] A cancer detection kit comprising the monoclonal antibody or a fragment thereof described in any one of [1] to [7]. [9] A nucleic acid encoding the monoclonal antibody or a fragment thereof according to any one of [1] to [7].

[10] A method for detecting cancer, comprising the step of reacting a fixed biological sample with the monoclonal antibody or a fragment thereof described in any one of [1] to [7].

[11] A hybridoma cell line with accession number NITE BP-02527.

[12] A method for detecting KK-LC-1 protein in a biological sample, comprising the step of detecting a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a partial peptide thereof.

[13] The method according to

[12] , wherein the partial peptide is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12. Effect of the Invention

[0006] According to the present invention, a technique for easily detecting KK-LC-1 can be provided. According to the present invention, for example, it becomes possible to detect the expression of KK-LC-1 by immunohistochemical staining. Furthermore, anti-KK-LC-1 monoclonal antibodies can be supplied semi-permanently and can be suitably used as diagnostic agents. [Brief description of the drawings]

[0007] [Figure 1] 1 is a graph showing the results of Experimental Example 2. [Diagram 2] 6(a) to 6(d) are photographs showing the results of immunohistochemical staining in Experimental Example 3. [Diagram 3] 6(a) to 6(d) are photographs showing the results of immunohistochemical staining in Experimental Example 4. [Figure 4] 6(a) to 6(c) are photographs showing the results of immunohistochemical staining in Experimental Example 5. [Diagram 5] 6(a) and (b) are photographs showing the results of immunohistochemical staining in Experimental Example 6. [Figure 6] 1 is a graph showing the results of epitope mapping in Experimental Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] [Monoclonal antibody or its fragment] In one embodiment, the present invention provides a monoclonal antibody or a fragment thereof having binding ability to KK-LC-1 protein in a fixed biological sample. Although it was difficult to obtain such a hybridoma cell line, the inventors succeeded in establishing a hybridoma cell line producing a monoclonal antibody capable of reacting with KK-LC-1 protein in a fixed biological sample, as described later in the Examples. The NCBI accession number of the KK-LC-1 protein is NP_001017978.1, and its amino acid sequence is shown in SEQ ID NO:86.

[0009] In this specification, examples of monoclonal antibody fragments include conventionally known antibody fragments, more specifically, F(ab')2, Fab', Fab, Fv, scFv and the like.

[0010] The monoclonal antibody or a fragment thereof of this embodiment may be obtained by immunizing an animal, or may be obtained by screening using a phage library or the like. The monoclonal antibody or a fragment thereof of this embodiment may be produced from a hybridoma strain described below, or may be a genetic recombinant. The host of the genetic recombinant is not particularly limited, and may be, for example, a microorganism such as Escherichia coli or yeast, an insect cell, a plant cell, an animal cell, or the like.

[0011] The monoclonal antibody or a fragment thereof of this embodiment can be suitably used, for example, in immunohistochemical staining. Conventionally, there has been no anti-KK-LC-1 monoclonal antibody that can be used in immunohistochemical staining. The monoclonal antibody or a fragment thereof of this embodiment can be supplied semi-permanently and is easy to control the quality of, so that it can be suitably used, for example, as a diagnostic agent.

[0012] In this embodiment, the fixed biological sample is preferably a biological sample fixed with a cross-linking fixative. That is, the KK-LC-1 protein to be detected by the monoclonal antibody or a fragment thereof of this embodiment is preferably present in a biological sample fixed with a cross-linking fixative. The monoclonal antibody or a fragment thereof of this embodiment can react well with the KK-LC-1 protein in such a biological sample. As described above, there has been no anti-KK-LC-1 monoclonal antibody that can be used for immunohistochemical staining of a fixed biological sample. Examples of the cross-linking fixative include formaldehyde, paraformaldehyde, and glutaraldehyde.

[0013] The monoclonal antibody or a fragment thereof of this embodiment is preferably produced by a hybridoma cell line having the accession number NITE BP-02527. The monoclonal antibody or a fragment thereof produced by the hybridoma cell line having the accession number NITE BP-02527 has binding affinity not only to KK-LC-1 protein in a fixed biological sample, but also to KK-LC-1 protein in an unfixed biological sample.

[0014] The monoclonal antibody or a fragment thereof of this embodiment preferably has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, a monoclonal antibody produced by a hybridoma cell line having the accession number NITE BP-02527 has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0015] It is more preferable that the monoclonal antibody or a fragment thereof of this embodiment has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12. As described later in the Examples, the inventors identified the epitope recognized by the monoclonal antibody by epitope mapping. As a result, it was revealed that the monoclonal antibody produced by the hybridoma cell line with the accession number NITE BP-02527 has binding affinity to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12. As described later, this monoclonal antibody may also have binding affinity to a peptide in which the C-terminal serine residue, the N-terminal asparagine residue, the N-terminal asparagine residue, and the second lysine residue from the N-terminus are deleted in the amino acid sequence set forth in SEQ ID NO: 12.

[0016] The monoclonal antibody or fragment thereof of this embodiment may be any one of the following (i) to (iii). (i) A monoclonal antibody or a fragment thereof, in which heavy chain complementarity determining regions (CDRs) 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDRs 1 to 3 consist of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively. (ii) A monoclonal antibody or a fragment thereof, wherein heavy chain CDR1 to 3 consist of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, in which one or several amino acids have been deleted, substituted or added, and light chain CDR1 to 3 consist of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, in which one or several amino acids have been deleted, substituted or added, and wherein the monoclonal antibody has binding affinity to KK-LC-1 protein. (iii) A monoclonal antibody or a fragment thereof which competes with a monoclonal antibody or a fragment thereof having heavy chain CDRs 1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, and light chain CDRs 1 to 3 consisting of the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, and has binding affinity to KK-LC-1 protein.

[0017] SEQ ID NOs: 2 to 4 are the amino acid sequences of heavy chain CDR1 to 3 of the monoclonal antibody produced by the hybridoma cell line having the accession number NITE BP-02527, respectively. SEQ ID NOs: 5 to 7 are the amino acid sequences of light chain CDR1 to 3 of the monoclonal antibody produced by the hybridoma cell line having the accession number NITE BP-02527, respectively.

[0018] That is, the monoclonal antibody or a fragment thereof of this embodiment may have the same heavy chain CDR1 to 3 and light chain CDR1 to 3 as those of a monoclonal antibody produced by a hybridoma cell line having the accession number NITE BP-02527.

[0019] Alternatively, the monoclonal antibody or a fragment thereof of this embodiment may have heavy chain CDR1-3 and light chain CDR1-3 having mutations compared to the heavy chain CDR1-3 and light chain CDR1-3 of the monoclonal antibody produced by the hybridoma cell line having the accession number NITE BP-02527, as long as it has binding ability to KK-LC-1 protein in a fixed biological sample.

[0020] That is, in the monoclonal antibody or fragment thereof of this embodiment, the heavy chain CDR1 to 3 may have the amino acid sequences of SEQ ID NOs: 2 to 4, respectively, in which one or several amino acids have been deleted, substituted or added, and the light chain CDR1 to 3 may have the amino acid sequences of SEQ ID NOs: 5 to 7, respectively, in which one or several amino acids have been deleted, substituted or added.

[0021] Here, "one or several" may mean, for example, 1 to 10, for example, 1 to 5, or for example, 1 to 3.

[0022] The monoclonal antibody or fragment thereof of this embodiment may have a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:9.

[0023] Alternatively, the monoclonal antibody or fragment thereof of this embodiment may be a monoclonal antibody or fragment thereof that competes with a monoclonal antibody or fragment thereof produced by a hybridoma cell line having accession number NITE BP-02527 and has binding affinity to the KK-LC-1 protein.

[0024] Here, the term "competing with the target antibody" means that, for example, when a monoclonal antibody produced by a hybridoma cell line having accession number NITE BP-02527 is reacted with the KK-LC-1 protein and then the target antibody is reacted, the monoclonal antibody produced by the hybridoma cell line having accession number NITE BP-02527 at least partially dissociates the bond between the KK-LC-1 protein and the monoclonal antibody, and binds to the KK-LC-1 protein.

[0025] Here, "at least a portion" may mean 10% or more, 30% or more, 50% or more, 70% or more, or 90% or more of the moles of the above-mentioned KK-LC-1 protein.

[0026] The monoclonal antibody or a fragment thereof of this embodiment may be humanized. Examples of human antibodies include chimeric antibodies, humanized antibodies, and fully human antibodies. Here, the term "chimeric antibody" refers to an antibody in which the variable region is an antibody derived from a non-human animal and at least a part of the constant region is an antibody derived from a human. The term "humanized antibody" refers to an antibody in which only the complementarity determining regions of the heavy and light chains are an antibody derived from a non-human animal and the constant region and framework region are an antibody derived from a human. The term "fully human antibody" refers to an antibody whose entirety, including the complementarity determining regions, is derived from a human. If the monoclonal antibody or a fragment thereof is a human antibody or a fragment thereof, it has low immunogenicity even when administered to a human, and side effects such as anaphylactic shock can be suppressed. Therefore, if the monoclonal antibody or a fragment thereof of this embodiment is a human antibody or a fragment thereof, it can be administered to a human.

[0027] [Nucleic acid] In one embodiment, the invention provides a nucleic acid encoding the monoclonal antibody or fragment thereof described above.

[0028] The nucleic acid of this embodiment preferably encodes a monoclonal antibody or a fragment thereof having binding ability to KK-LC-1 protein in a fixed biological sample, wherein heavy chain CDR1 to 3 have the amino acid sequences of SEQ ID NOs: 2 to 4, or the amino acid sequences of SEQ ID NOs: 2 to 4 in which one or several amino acids have been deleted, substituted or added, and light chain CDR1 to 3 have the amino acid sequences of SEQ ID NOs: 5 to 7, or the amino acid sequences of SEQ ID NOs: 5 to 7 in which one or several amino acids have been deleted, substituted or added, respectively.

[0029] The nucleic acid of this embodiment may have a heavy chain variable region gene nucleotide sequence of SEQ ID NO: 10 and a light chain variable region gene nucleotide sequence of SEQ ID NO: 11. Alternatively, the nucleic acid of this embodiment may have a mutation as long as the encoded monoclonal antibody or a fragment thereof has the ability to bind to the KK-LC-1 protein in a biological sample to which it is fixed.

[0030] That is, the nucleic acid of this embodiment may be one in which the base sequence of the heavy chain variable region gene has a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the base sequence of SEQ ID NO: 10, and the base sequence of the light chain variable region gene has a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the base sequence of SEQ ID NO: 11.

[0031] Here, the sequence identity of the subject base sequence to the reference base sequence can be determined, for example, as follows. First, the reference base sequence and the subject base sequence are aligned. Here, each base sequence may contain gaps so as to maximize sequence identity. Next, the number of matching bases in the reference base sequence and the subject base sequence is calculated, and the sequence identity can be determined according to the following formula (1). Sequence identity (%) = number of matching bases / total number of bases in the target sequence × 100 (1)

[0032] The nucleic acid of this embodiment may be contained in a vector. The vector may be an expression vector capable of expressing the above-mentioned monoclonal antibody or a fragment thereof. The above-mentioned vector may be introduced into a host, such as a microorganism such as E. coli or yeast, an insect cell, a plant cell, or an animal cell. That is, in one embodiment, the present invention provides a host into which the above-mentioned vector has been introduced.

[0033] [Cancer detection kit] In one embodiment, the present invention provides a cancer detection kit comprising the above-mentioned monoclonal antibody or a fragment thereof. According to the kit of this embodiment, cancer can be easily detected by immunohistochemical staining of a fixed biological sample. The fixed biological sample may be the same as those described above, for example, a fixed tissue slice.

[0034] Examples of cancers that can be detected by the kit of this embodiment include gastric cancer, lung cancer, breast cancer, colon cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colorectal cancer, liver cancer, brain tumors, uterine cancer, ovarian cancer, leukemia, osteosarcoma, mesothelioma, testicular tumors, and gastrointestinal stromal tumors.

[0035] [Cancer detection methods] In one embodiment, the present invention provides a method for detecting cancer, comprising a step of reacting the above-mentioned monoclonal antibody or a fragment thereof with a fixed biological sample. The detection method of this embodiment can also be said to be a data collection method for diagnosing whether or not a subject is affected by cancer. Note that the data collection method does not include a step of a doctor making a judgment.

[0036] When KK-LC-1 protein is detected in a fixed biological sample using the detection method of this embodiment, it can be determined that the application of cancer treatment targeting KK-LC-1 would be effective for the patient from whom the biological sample was derived.

[0037] Examples of cancers that can be detected by the method of this embodiment include gastric cancer, lung cancer, breast cancer, colon cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colorectal cancer, liver cancer, brain tumors, uterine cancer, ovarian cancer, leukemia, osteosarcoma, mesothelioma, testicular tumors, and gastrointestinal stromal tumors.

[0038] According to the method of the present embodiment, cancer can be detected simply and easily. The method of the present embodiment is made possible by the above-mentioned monoclonal antibody or a fragment thereof.

[0039] [Hybridoma cell line] In one embodiment, the present invention provides a hybridoma cell line having the accession number NITE BP-02527. The above-mentioned monoclonal antibody can be produced by culturing the hybridoma cell line of this embodiment. In addition, the produced monoclonal antibody can be cleaved with pepsin or papain to obtain an antibody fragment.

[0040] [Method for detecting KK-LC-1 protein in biological samples] In one embodiment, the present invention provides a method for detecting KK-LC-1 protein in a biological sample, the method comprising the step of detecting a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a partial peptide thereof. The partial peptide is preferably a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12. According to the method of this embodiment, even fixed KK-LC-1 protein in a biological sample can be detected.

[0041] Incidentally, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 corresponds to the 89th to 100th amino acids of the KK-LC-1 protein whose amino acid sequence is set forth in SEQ ID NO: 86. It is also known that the peptide consisting of the 80th to 101st amino acids of the KK-LC-1 protein is secreted into the blood. Therefore, by detecting the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12, it is possible to detect a partial peptide of the KK-LC-1 protein secreted into the blood.

[0042] [Other embodiments] In one embodiment, the present invention provides a method for treating cancer, comprising the steps of reacting a fixed biological sample derived from a patient with the above-mentioned monoclonal antibody or a fragment thereof, and administering a cancer treatment targeting KK-LC-1 to the patient if KK-LC-1 protein is detected in the biological sample.

[0043] Examples of cancer treatments targeting KK-LC-1 include cancer immunotherapy using KK-LC-1 protein as a cancer antigen, molecular targeted therapy targeting KK-LC-1 protein expressed on the cell membrane, etc. The molecular targeted therapy may be a treatment using an antibody drug that recognizes the KK-LC-1 protein. EXAMPLES

[0044] [Experimental Example 1] (Establishment of hybridoma cell lines) Mice were immunized with a peptide having the amino acid sequence set forth in SEQ ID NO:1, and hybridoma cell lines of about 30 clones were established.

[0045] [Experimental Example 2] (Confirmation of monoclonal antibody reactivity using ELISA method) The reactivity of the monoclonal antibody was confirmed by ELISA using the immunized peptide as an antigen. Specifically, a peptide having the amino acid sequence shown in SEQ ID NO: 1 was immobilized on a 96-well plate. As a control, a 96-well plate was used that had not been immobilized with the peptide and had only been blocked. A skim milk solution was used for blocking.

[0046] Next, the 96-well plate was blocked, and the culture supernatant of each established hybridoma cell line was added and reacted. For comparison, wells reacted with normal mouse serum were also prepared. Next, unreacted monoclonal antibodies were washed away, and anti-mouse secondary antibodies were reacted, and a color-developing substrate was added to develop color. The absorbance of each well was then measured using a plate reader.

[0047] Figure 1 is a graph showing the results of measuring the reactivity of monoclonal antibodies by ELISA. In Figure 1, the horizontal axis shows the clone names of hybridoma cell lines. The clone name "34B3" is the hybridoma cell line with the accession number NITE BP-02527.

[0048] As a result, it was confirmed that a large number of monoclonal antibodies showing reactivity with the KK-LC-1 protein were obtained by ELISA.

[0049] [Experimental Example 3] (Confirmation of monoclonal antibody reactivity by immunohistochemical staining 1) The monoclonal antibodies produced were used to stain formaldehyde-fixed tissue sections of normal and tumor regions from testicular cancer patients and normal and tumor regions from stomach tissues from gastric cancer patients. As a result, it was revealed that only the monoclonal antibody produced by one of the approximately 30 established hybridoma cell lines was able to stain KK-LC-1 protein in immunohistochemical staining after autoclaving.

[0050] A hybridoma cell line that produced a monoclonal antibody capable of staining KK-LC-1 protein in immunohistochemical staining was deposited domestically at the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (Deposit date: August 3, 2017, Deposit number: NITE P-02527, Cell name "277 34B3 20170123"). After that, the hybridoma cell line (Deposit number: NITE P-02527) was transferred to international deposit (International deposit authority: National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), Deposit date: October 10, 2018, Deposit number: NITE BP-02527, Cell name "277 34B3 20170123").

[0051] 2(a) to (d) are photographs showing the results of immunohistochemical staining using a monoclonal antibody obtained from a hybridoma cell line (accession number: NITE BP-02527). All images were taken at a magnification of 400x.

[0052] Figure 2(a) is a photograph showing the staining results of a tissue section from a normal region of a patient with testicular cancer. Figure 2(b) is a photograph showing the staining results of a tissue section from seminoma, a type of testicular cancer. Figure 2(c) is a photograph showing the staining results of a tissue section from a normal region of stomach tissue from a patient with gastric cancer. Figure 2(d) is a photograph showing the staining results of a tissue section from a tumor region of stomach tissue from a patient with gastric cancer.

[0053] As a result, it was confirmed that the KK-LC-1 protein was stained in all the stained tissue sections. This result demonstrated that the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527) has binding affinity to the KK-LC-1 protein in the fixed biological sample. In addition, the expression of the KK-LC-1 gene was positive in both the tumor and normal tissues of the stomach cancer patient used in this experimental example.

[0054] Furthermore, the above results clarified that even if a monoclonal antibody shows high reactivity in ELISA, it does not necessarily show reactivity in immunohistochemical staining.

[0055] [Experimental Example 4] (Confirmation of monoclonal antibody reactivity by immunohistochemical staining 2) The reactivity of the monoclonal antibody obtained from the hybridoma cell line (Accession Number: NITE BP-02527) was examined. Specifically, the monoclonal antibody obtained from the hybridoma cell line (Accession Number: NITE BP-02527) was absorbed with the peptide used for immunization of mice when the hybridoma cell line (Accession Number: NITE BP-02527) was established, and then immunohistochemical staining was performed in the same manner as in Experimental Example 3 to examine the reactivity. For comparison, immunohistochemical staining was also performed using a monoclonal antibody that had not been subjected to the absorption procedure.

[0056] For the monoclonal antibody absorption, a peptide having the amino acid sequence shown in SEQ ID NO: 1 was used at a final concentration of 0.05 mg / mL, and the absorption was carried out overnight at 4°C.

[0057] Figures 3(a) to (d) are photographs showing the results of immunohistochemical staining. All were taken at a magnification of 200x. Figures 3(a) and (b) are photographs showing the results of staining tissue sections of tumor regions in the stomach tissue of a patient with gastric cancer. Figure 3(a) shows the results of immunohistochemical staining using a monoclonal antibody that had not been subjected to an absorption procedure. Figure 3(b) shows the results of immunohistochemical staining using a monoclonal antibody that had been subjected to an absorption procedure.

[0058] Figures 3(c) and (d) are photographs showing the staining results of tissue sections of normal regions of stomach tissue from a gastric cancer patient. Figure 3(c) shows the results of immunostaining using a monoclonal antibody that has not been subjected to an absorption procedure, and Figure 3(d) shows the results of immunostaining using a monoclonal antibody that has been subjected to an absorption procedure.

[0059] As a result, it was found that the reactivity to KK-LC-1 disappeared in the monoclonal antibody that had been subjected to the absorption procedure. This result indicates that the prepared monoclonal antibody has a specific reactivity to KK-LC-1. In addition, the expression of the KK-LC-1 gene was positive in both the tumor area and normal area tissues of the stomach cancer patient used in this experiment.

[0060] [Experimental Example 5] (Confirmation of monoclonal antibody reactivity by immunohistochemical staining3) The reactivity of the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527) was further examined. Specifically, the reactivity was examined by immunohistochemical staining in the same manner as in Experimental Example 3 using the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527). Here, the tissue sections used were normal and tumor regions of the stomach tissue of a stomach cancer patient, and samples in which the expression of the KK-LC-1 gene was negative.

[0061] 4(a) to (c) are photographs showing the results of immunohistochemical staining using a monoclonal antibody obtained from a hybridoma cell line (accession number: NITE BP-02527). All images were taken at a magnification of 400x.

[0062] Fig. 4(a) is a photograph showing the results of staining a tissue section from a normal region of stomach tissue from a gastric cancer patient, where the expression of the KK-LC-1 gene was negative, and Fig. 2(b) and (c) are photographs showing the results of staining a tissue section from a tumor region of stomach tissue from a gastric cancer patient, where the expression of the KK-LC-1 gene was negative.

[0063] As a result, KK-LC-1 protein was not detected in any of the stained tissue sections, further supporting the fact that the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527) has specific reactivity with KK-LC-1 in fixed biological samples.

[0064] [Experimental Example 6] (Confirmation of monoclonal antibody reactivity by immunohistochemical staining4) The reactivity of the monoclonal antibody obtained from the hybridoma cell line (accession number: NITE BP-02527) was compared with that of a commercially available anti-KK-LC-1 polyclonal antibody (type number "HPA004773", Sigma-Aldrich).

[0065] Specifically, using a monoclonal antibody (culture supernatant diluted 1 / 80) obtained from a hybridoma cell line (accession number: NITE BP-02527) and a commercially available anti-KK-LC-1 polyclonal antibody (type "HPA004773", Sigma-Aldrich, diluted 1 / 50), immunohistochemical staining was performed in the same manner as in Experimental Example 3 to examine the reactivity. Here, testis samples in which the KK-LC-1 gene is strongly expressed were used as tissue sections.

[0066] Figures 5(a) and (b) are photographs showing the results of immunohistochemical staining. Both were taken at a magnification of 400x. Figure 5(a) is a photograph showing the results of staining using a monoclonal antibody obtained from a hybridoma cell line (accession number: NITE BP-02527). Figure 5(b) is a photograph showing the results of staining using a commercially available anti-KK-LC-1 polyclonal antibody (type number "HPA004773", Sigma-Aldrich).

[0067] As a result, it was found that the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527) was able to detect KK-LC-1 in the fixed biological sample well, whereas the commercially available anti-KK-LC-1 polyclonal antibody was unable to stain KK-LC-1 in the fixed biological sample. This result demonstrates the usefulness of the monoclonal antibody obtained from the hybridoma cell line (Accession No.: NITE BP-02527).

[0068] [Experimental Example 7] (Identification of the base and amino acid sequences of monoclonal antibodies) Total RNA was prepared from the hybridoma cell line (Accession Number: NITE BP-02527) and reverse transcribed to prepare cDNA. Subsequently, the nucleotide sequences of the antibody heavy chain variable region gene and the antibody light chain variable region gene were sequenced by a standard method.

[0069] The nucleotide sequence of the identified heavy chain variable region gene is shown in SEQ ID NO: 10, and the nucleotide sequence of the light chain variable region gene is shown in SEQ ID NO: 11. The amino acid sequence of the heavy chain variable region deduced from the nucleotide sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0070] The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO: 4. The amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 5, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO: 7.

[0071] [Experimental Example 8] (Analysis of epitopes recognized by monoclonal antibodies) The antigen of the monoclonal antibody produced by the hybridoma cell line (Accession No.: NITE BP-02527) was identified by epitope mapping. Specifically, based on the amino acid sequence of the KK-LC-1 protein, 73 types of 15 amino acid residue peptides were synthesized with the N-terminal position shifted by one amino acid residue each, and a peptide array was produced. The amino acid sequences of the 73 types of peptides are shown in SEQ ID NOs: 13 to 85, respectively.

[0072] Next, the peptide array was reacted with monoclonal antibodies to identify reactive peptides. The monoclonal antibodies were diluted 1 / 100 and 1 / 500 and reacted with each other.

[0073] 6 is a graph showing the results of epitope mapping. As a result, it was revealed that the monoclonal antibody has binding affinity to a peptide in the vicinity of LSMVENKLVELEHTL (SEQ ID NO: 63) to VELEHTLLSKGFRGA (SEQ ID NO: 71).

[0074] As a result of identifying the amino acid sequence common to the peptides bound by the monoclonal antibody, it was revealed that the epitope of this monoclonal antibody is NKLVELEHTLLS (SEQ ID NO: 12). Further studies are required to determine whether the entire amino acid sequence described in SEQ ID NO: 12 is essential for binding to the monoclonal antibody. In other words, this monoclonal antibody may also have binding activity to a peptide in which the C-terminal serine residue is deleted, the N-terminal asparagine residue is deleted, and the N-terminal asparagine residue and the second lysine residue from the N-terminus are deleted in the amino acid sequence described in SEQ ID NO: 12. [Industrial Applicability]

[0075] According to the present invention, a technique for easily detecting KK-LC-1 can be provided. According to the present invention, for example, it becomes possible to detect the expression of KK-LC-1 by immunohistochemical staining. Furthermore, anti-KK-LC-1 monoclonal antibodies can be supplied semi-permanently and can be suitably used as diagnostic agents. [Accession number]

[0076] NITE BP-02527

Claims

1. A monoclonal antibody or a fragment thereof, wherein heavy chain complementarity determining regions (CDRs) 1 to 3 each have the amino acid sequences of SEQ ID NOs: 2 to 4, and light chain CDRs 1 to 3 each have the amino acid sequences of SEQ ID NOs: 5 to 7, The monoclonal antibody has a binding affinity to Kitakyushu lung cancer antigen-1 (KK-LC-1) protein, The fragment is F(ab') ２ , Fab', Fab, Fv or scFv; A monoclonal antibody or a fragment thereof.

2. The monoclonal antibody or a fragment thereof according to claim 1, which has binding affinity to KK-LC-1 protein in a fixed biological sample.

3. The monoclonal antibody or fragment thereof of claim 2 , wherein the biological sample is fixed with a cross-linking fixative.

4. The monoclonal antibody or fragment thereof of claim 3, wherein the cross-linking fixative is formaldehyde, paraformaldehyde or glutaraldehyde.

5. The monoclonal antibody or fragment thereof according to any one of claims 1 to 4, which has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:

1.

6. The monoclonal antibody or a fragment thereof according to any one of claims 1 to 5, which has binding affinity to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:

12.

7. The monoclonal antibody or fragment thereof according to any one of claims 1 to 6, which is produced by a hybridoma cell line having the accession number NITE BP-02527.

8. A kit for detecting cancer, comprising the monoclonal antibody or a fragment thereof according to any one of claims 1 to 7.

9. A nucleic acid encoding the monoclonal antibody or a fragment thereof according to any one of claims 1 to 7.

10. A method for detecting cancer, comprising the step of reacting a fixed biological sample with the monoclonal antibody or a fragment thereof according to any one of claims 1 to 7.

11. A hybridoma cell line having the accession number NITE BP-02527.

12. A method for detecting KK-LC-1 protein in a biological sample, the method comprising the step of detecting a peptide having the amino acid sequence set forth in SEQ ID NO: 12, a peptide having the amino acid sequence set forth in SEQ ID NO: 12 with a C-terminal serine residue deleted, a peptide having the amino acid sequence set forth in SEQ ID NO: 12 with an N-terminal asparagine residue deleted, a peptide having the amino acid sequence set forth in SEQ ID NO: 12 with an N-terminal asparagine residue deleted and a lysine residue second from the N-terminus deleted, or a peptide having the amino acid sequence set forth in any of SEQ ID NOs: 63 to 71, using an antibody or a fragment thereof that specifically binds to the peptide, wherein the fragment is F(ab')2, Fab', Fab, Fv or scFv.

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