Antibody against MELK protein, use and method of production thereof, polynucleotide reagent, as well as in vitro method for detecting MELK protein in a sample.

Abstract

The present invention relates to a monoclonal antibody against MELK. Furthermore, the present invention provides a method for diagnosing a MELK-related disease, a method for detecting a MELK protein, a method for evaluating a drug effect after treatment with a MELK inhibitor, and a method for examining an individual for whom treatment with a MELK inhibitor is highly effective, each method comprising the use of the monoclonal antibody and a diagnostic reagent containing the aforementioned antibody.

Description

Antibody against MELK protein, use and method of production thereof, polynucleotide, reagent, as well as in vitro method for detecting MELK protein. IN A SAMPLE Technical field

[0001] The present invention relates to monoclonal antibodies against MELK, to methods for diagnosing MELK-associated diseases using said antibodies, to methods for detecting the MELK protein, to methods for determining the efficacy of the drug after treatment with a MELK inhibitor, to methods of examining patients for whom a MELK inhibitor has a high therapeutic effect, and to diagnostic reagents containing said antibodies. Antecedent Technique

[0002] MELK, that is, Embryonic Zipper Kinase Leucine Materna (reference sequence: GenBank Accession No.: NM_014791.3; SEQ ID NO: 21), was identified some time ago as a new member of the serine-threonine kinase snf1 / AMPK family that is involved in mammalian embryonic development (Heyer BS et al., Dev Dyn. 1999 Aug, 215(4): 344-51 (NPL 1). This gene has been shown to play an important role in stem cell regeneration (Nakano I et al., J Cell Biol. 2005 Aug 1, 170(3): 413-27 (NPL 2)), in cell cycle progression (Blot J et al., Dev Biol. 2002 Jan 15, 241(2): 327-38 (NPL 3); Seong HA et al., Biochem J. 2002 Feb 1, 361(Pt 3): 597-604 (NPL 4)), and in splicing of the mRNA precursor (Vulsteke V et al., J Biol Chem. 2004 Mar 5, 279(10): 8642-7. Epub 2003 Dec 29 (NPL 5)).

[0003] Furthermore, gene expression profiling using genome-wide cDNA microarrays, including 23,040 genes, showed that MELK is upregulated in breast cancer (Lin ML et Petition 870260046788, dated 05 / 18 / 2026, p. 7 / 141 2 / 55 al., Breast Cancer Res. 2007, 9 (1): R17 (NPL 6); WO 2006 / 016525 (PTL 1); WO 2006 / 016525 (PTL 1); and WO 2008 / 023841 (PTL 2)). In fact, MELK is upregulated in several cancer cells, for example, in lung cancer cells, bladder cancer cells, lymphoma cells, cervical cancer cells, and the like (see WO 2004 / 031413 (PTL 3); WO 2007 / 013665 (PTL 4); and WO 2006 / 085684 (PTL 5)). Northern blot analysis of multiple human tissues and cancer cell lines has shown that although MELK is overexpressed at a significantly high level in most breast cancers and breast cancer cell lines, it is not expressed in normal vital organs (heart, liver, lung and kidney) (WO 2006 / 016525 (PTL 1)).Furthermore, it has been shown that suppression of MELK expression by siRNA leads to a significant inhibition of human breast cancer cell proliferation (PTL 1), and that a small molecule inhibitor against MELK reduces the size of a mouse breast cancer xenograft (Chung S et al., Oncotarget. 2012 Dec, 3(12): 1629-1640 (NPL 7); Chung S et al., Oncotarget. 2016 Feb 24, 7(14): 18171-18182 (NPL 8)).

[0004] Therefore, MELK is believed to be a suitable target for anticancer agents, and a monoclonal antibody against MELK may be predicted to be useful as a diagnostic agent in therapy. In addition to examples of successful clinical applications of monoclonal antibodies, such as the diagnostic agents Trastuzumab, Rituximab, and Bevacizumab against breast cancer, malignant lymphoma, and colon cancer, several monoclonal antibodies against other molecular targets are being developed, and their diagnostic effects are being evaluated. From the point of view of effective patient selection for therapeutic agents, these diagnostic agents are anticipated to result in more effective treatment methods. Petition 870260046788, dated 05 / 18 / 2026, p. 8 / 141 3 / 55 List of Citations [Patent Literature] [PTL 1] WO 2006 / 016525 [PTL 2] WO 2008 / 023841 [PTL 3] WO 2004 / 031413 [PTL 4] WO 2007 / 013665 [PTL 5] WO 2006 / 085684 [Non-Patent Literature] [NPL 1] Heyer BS et al., Dev Dyn. 1999 Aug, 215(4): 344-51 [NPL 2] Nakano I et al., J Cell Biol. 2005 Aug 1, 170(3): 413-27 [NPL 3] Blot J et al., Dev Biol. 2002 Jan 15, 241(2): 327-38 [NPL 4] Seong HA et al., Biochem J. 2002 Feb 1,361(Pt 3): 597-604 [NPL 5] Vulsteke V et al., J Biol Chem. 2004 Mar 5, 279(10): 8642-7. Epub 2003 Dec 29 [NPL 6] Lin ML et al., Breast Cancer Res. 2007, 9(1): R17 [NPL 7] Chung S et al., Oncotarget. 2012 Dec, 3(12): 1629-1640 [NPL 8] Chung S et al., Oncotarget. 2016 Feb 24, 7(14): 18171-18182 Invention Disclosure [Problems to be Solved by Invention]

[0005] It is important for diagnostic agents in tumor treatment using molecularly targeted drugs to detect cellular proteins that are overexpressed in most parts of a target tumor, and are not expressed or are expressed only in a minimal proportion in normal tissues. However, it is difficult to detect proteins expressed in tumors specifically and with high sensitivity, and it is also difficult to obtain antibodies against such proteins. For example, regarding MELK, which is considered to be a target for anticancer agents, there are some antibodies on the market. However, when the present inventors stained cells expressing MELK using commercially available antibodies Petition 870260046788, dated 05 / 18 / 2026, page 9 / 141 4 / 55 of the results they obtained, a positive signal (false positive) was occasionally observed even in cells where the MELK expression level was low. With such antibodies having inadequate immunological specificity, there is a concern about not being able to clearly detect differences in MELK expression levels using antibody reaction concentration as an indicator. Therefore, a problem to be solved by the present invention is to provide antibodies that bind specifically to MELK and with high sensitivity. [Means to Solve the Problems]

[0006] The present inventors investigated an antibody that specifically binds to MELK among monoclonal antibodies obtained by immunizing mice with the MELK antigen, and were able to identify a clone that can specifically bind to the MELK protein forcibly expressed in cells, and can specifically detect the endogenous MELK protein expressed within cells and tissues, with good sensitivity.

[0007] Specifically, the present invention relates to the following: [1] an antibody or antigen-binding fragment thereof, which may bind to MELK protein or a partial peptide thereof, and comprising one or both of: a variable region of the heavy chain comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising: Petition 870260046788, dated 05 / 18 / 2026, page 10 / 141 5 / 55 a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; [2] the antibody or antigen-binding fragment thereof of [1], comprising one or both of a variable heavy chain region comprising amino acid sequence SEQ ID NO: 7 and a variable light chain region comprising amino acid sequence SEQ ID NO: 8; [3] the antibody or antigen-binding fragment of the same as [1] or [2], which specifically recognizes the polypeptide consisting of the amino acid sequence of SEQ ID NO: 9; [4] an antibody or the antigen-binding fragment thereof, which competes with the antibody of any of [1] to [3] for specific binding with respect to MELK; [5] the antibody or antigen-binding fragment thereof of any one of [1] to [4], which is conjugated with an affinity label, enzyme label, radioisotope label, or fluorescent label; [6] a polynucleotide encoding the antibody or its antigen-binding fragment from any of [1] to [5]; [7] a reagent comprising the antibody or antigen-binding fragment thereof of any one of [1] to [5], wherein the reagent is for diagnosing a disease associated with MELK, for determining the efficacy of the drug after treatment with a MELK inhibitor, or for examining whether a patient for whom a MELK inhibitor has a high therapeutic effect; [8] a method for diagnosing a disease associated with MELK or a predisposition to developing said disease in a patient, Petition 870260046788, dated 05 / 18 / 2026, page 11 / 141 6 / 55 comprising the steps of: (a) contacting an isolated sample from said patient with the antibody or antigen-binding fragment from any of [1] to [5]; (b) detect the MELK protein in said sample by detecting the binding of said sample to said antibody or antigen-binding fragment thereof; and (c) compare the level of MELK protein in said sample with a control, where it is indicated that said patient suffers from, or is at risk of developing, said disease when the level of MELK protein is high compared to the control; [9] the reagent of [7] or the method of [8], wherein the said disease associated with MELK is a cancer in which MELK is expressed or endometriosis;

[10] the reagent or method of [9], wherein said cancer is selected from the group consisting of breast cancer, bladder cancer, cervical cancer, cholangiocellular cancer, chronic myelocytic leukemia (CML), colorectal cancer, esophageal cancer, stomach cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, and small cell lung cancer (SCLC);

[11] a method for detecting MELK protein in a sample, comprising the steps of: (a) contacting a patient-isolated sample with the antibody or antigen-binding fragment of any of [1] to [5]; and (b) detecting the MELK protein in said sample by detecting the binding of said sample to said antibody or antigen-binding fragment; Petition 870260046788, dated 05 / 18 / 2026, page 12 / 141 7 / 55

[12] a method for determining the effectiveness of the drug after treatment with a MELK inhibitor in a patient, comprising the steps of: (a) contacting an isolated sample from a patient with the antibody or its antigen-binding fragment from any of [1] to [5]; (b) detect the MELK protein in said sample by detecting the binding of said sample to said antibody or antigen-binding fragment thereof; and (c) compare the level of MELK protein in said sample with the expression level before drug administration, whereby when said MELK protein level is low compared to before drug administration, it is indicated that the drug was effective in said patient;

[13] a method of examining whether a MELK inhibitor has a high therapeutic effect on a patient, comprising the steps of: (a) contacting an isolated sample from said patient with the antibody or antigen-binding fragment from any of [1] to [5]; (b) detect the MELK protein in said sample by detecting the binding of said sample to said antibody or antigen-binding fragment thereof; and (c) compare the MELK protein level in said sample with a control, wherein when the MELK protein level is comparable to, or greater than, the control, it is indicated that the therapeutic effect of the MELK inhibitor is elevated in said patient;

[14] the method of any of [8] to

[13] , wherein the said sample is a cell or tissue isolated from the said patient; and

[15] a method for producing an antibody that can bind to the protein Petition 870260046788, dated 05 / 18 / 2026, page 13 / 141 8 / 55 MELK, or a partial peptide thereof, comprising the steps of: (a) cultivate a cell comprising a vector inserted with the [6] polynucleotide; and (b) recover said antibody from the cell culture or culture medium.

[0008] The present invention also relates to:

[16] a method for detecting a marker for diagnosing a disease associated with MELK, or a predisposition to developing said disease, comprising the steps of: (a) contacting an isolated sample from a patient with the antibody or antigen-binding fragment thereof from any of [1] to [5]; and (b) detecting the MELK protein in said sample as said marker by detecting the binding of said antibody or antigen-binding fragment thereof to said sample;

[17] the antibody or antigen-binding fragment of any of [1] to [5] for use in the diagnosis of a disease associated with MELK, or a predisposition to develop said disease;

[18] use of the antibody or antigen-binding fragment thereof of any of [1] to [5] in the manufacture of a reagent for the diagnosis of a disease associated with MELK, or a predisposition to develop said disease;

[19] a method for detecting a marker of drug efficacy of a MELK inhibitor, comprising the steps of: (a) contact a patient-isolated sample containing the antibody or antigen-binding fragment from any of [1] to [5]; and Petition 870260046788, dated 05 / 18 / 2026, page 14 / 141 9 / 55 (b) detect the MELK protein in said sample as said marker by detecting the binding of said antibody or antigen-binding fragment thereof to said sample;

[20] the antibody or antigen-binding fragment thereof of any of [1] to [5] for use in determining the efficacy of the drug after treatment with a MELK inhibitor;

[21] use of the antibody or antigen-binding fragment thereof of any of [1] to [5] in the manufacture of a reagent for determining the efficacy of the drug after treatment with a MELK inhibitor;

[22] a method for detecting a marker of sensitivity to treatment with the MELK inhibitor, comprising the steps of: (a) contacting a patient-isolated sample with the antibody or antigen-binding fragment thereof from any of [1] to [5]; and (b) detecting the MELK protein in said sample as said marker by detecting the binding of said antibody or antigen-binding fragment thereof to said sample;

[23] the antibody or antigen-binding fragment thereof of any of [1] to [5] for use in testing as to a patient for whom a MELK inhibitor has a high therapeutic effect; and

[24] use of the antibody or its antigen-binding fragment from any of [1] to [5] in the manufacture of a reagent for testing as to a patient for whom a MELK inhibitor has a high therapeutic effect.

[0009] In addition to the above, other objectives and features of the present invention will become more fully apparent when the detailed description that follows is read in conjunction with the accompanying figures and examples. However, it should be understood that both the preceding summary of the present invention and the detailed description Petition 870260046788, dated 05 / 18 / 2026, page 15 / 141 The following 10 / 55 embodiments are illustrative, and not restrictive, of the present invention or other alternative embodiments of the present invention. In particular, although the present invention is described in this document with reference to several specific embodiments, it will be appreciated that the description is illustrative of the present invention and is not to be interpreted as limiting the present invention. Various modifications and applications may occur for those skilled in the art, without departing from the spirit and scope of the present invention, as described by the appended claims. Similarly, other objectives, features, benefits and advantages of the present invention will be evident from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art.These objectives, characteristics, benefits, and advantages will become evident from the above, in combination with the accompanying examples, data, and figures, and any reasonable conclusions that can be drawn from them, either alone or in consideration of the references incorporated herein. Brief Description of the Drawings

[00010] Figure 1 is a photomicrograph showing the specificity of the anti-MELK antibody (OTSMAb01) in immunohistochemical staining using a cell line for a forced expression system. As a result of immunohistochemical staining using an anti-MELK antibody (OTSMAb01), specific staining was observed in a paraffin section prepared from a cell line with forced MELK expression (MELK / 293T), but no staining was observed in the cell line introduced with an empty vector (Mock / 293T) that was used as the negative control. On the other hand, even with commercially available antibodies (MELK (N449)pAb (Bioworld Technology) and MELK(HPA017214)pAb (SigmaAldrich)), immunohistochemical staining showed that, although it was Petition 870260046788, dated 05 / 18 / 2026, page 16 / 141 11 / 55 showed a specific staining in MELK / 293T, there was no staining in the Mock. Furthermore, the graph below the photographs is a graphical representation of the percentage (%) of MELK-positive cells within the number of cells with forced expression, as seen with the immunohistochemical staining results.

[00011] Fig. 2A: Figs. 2A and B are photographs showing the result of western blotting using OTSMAb01, which shows MELK expression in various cell line types with different levels of MELK expression. PK-45P, BT-549, MDA-MB231, and A549 are cell lines with high MELK expression, and MCF7, Hep G2, and HT-29 are cell lines with low MELK expression.

[00012] Fig. 2B is a continuation of Fig. 2A.

[00013] Fig. 3A: Figs. 3A and 3B are photomicrographs showing the specificity of an anti-MELK antibody (OTSMAb01) in immunohistochemical staining using cell lines expressing MELK. The upper panel shows the results of immunohistochemical staining with the anti-MELK antibody of the present invention (OTSMAb01), where staining was detected in paraffin sections prepared from high MELK expression cell lines such as PK-45P, BT-549, MDA-MB-231 and A549, while almost no staining was observed in low MELK expression cell lines such as MCF-7, Hep G2, and HT-29. On the other hand, in the middle panel and the bottom panel showing the results for staining with commercially available antibodies (aMELK (N449)pAb and MELK(HPA017214)pAb), staining was observed in both high MELK expression cell lines and low MELK expression cell lines.Analysis using cell lines revealed that, compared to antibodies... Petition 870260046788, dated 05 / 18 / 2026, page 17 / 141 12 / 55 commercially available, the anti-MELK antibody of the present invention (OTSMAb01) had high specificity in immunohistochemical staining. Furthermore, the graphs below the photographs are graphs graphically representing the percentage (%) of MELK-positive cells within each of the cell lines, as seen with the immunohistochemical staining results.

[00014] Fig. 3B is a continuation of Fig. 3A.

[00015] Fig. 4 shows the correlation between expressions in clinical breast cancer samples 1 to 3 in immunohistochemical staining and semi-quantitative RT-PCR. A: A positive finding for MELK was observed for all breast cancer samples by immunohistochemical staining using the anti-MELK antibody of the present invention (OTSMAb01). On the other hand, normal breast samples were hardly stained. B: From the immunohistochemical staining results, the rate of MELK-positive cells within tumor cells was calculated and graphically represented as a graph. C: Results obtained using RT-PCR expression analysis of the same clinical cases correlated with immunohistochemical staining. The analysis using clinical samples clarified the high specificity of the anti-MELK antibody of the present invention (OTSMAb01) in immunohistochemical staining. [Method for Implementing the Invention] Detailed Description

[00016] Although any methods and materials similar or equivalent to those described in this document may be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited in terms of sizes, shapes, dimensions, materials, methodologies, protocols, etc. Petition 870260046788, dated 05 / 18 / 2026, p. 18 / 141 13 / 55 particulars described in this document, as these may vary according to experimentation and routine optimization. It is also to be understood that the terminology used in the description is for the purpose of describing only particular versions or embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[00017] The present invention provides anti-MELK monoclonal antibodies that can specifically bind to the MELK protein or to partial peptides thereof. The present invention provides evidence that the anti-MELK monoclonal antibodies of the present invention have high specificity in detecting the MELK protein by immunohistochemical staining.

[00018] The anti-MELK monoclonal antibody of the present invention (OTSMAb01) has at least the following amino acid sequences in the variable regions: OTSMAb01, amino acid sequence of the variable region of the heavy chain (excluding the signal sequence): EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLE WIGWIDPENGNTIYDPKFQGKASVTADTSSNTAYLQLSSLTSEDTAVY YCTSHHYSAMDYWGQGTSVTVSS (SEQ ID NO: 7). OTSMAb01, amino acid sequence of the variable region of the light chain (excluding the signal sequence): DVVMTQTPLSLPVSLRDQASISCRSSQSLVHSNGNTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLIISRVEAEDLGVYFCS QSTYVPLTFGAGTKLELKRAD (SEQ ID NO: 8).

[00019] The antibodies of the present invention can also be produced by recombinant techniques, using DNAs that encode the aforementioned amino acid sequences.

[00020] The antibodies of the present invention were obtained from multiple antibody-producing hybridomas, obtained by immunization Petition 870260046788, dated 05 / 18 / 2026, page 19 / 141 14 / 55 mice were tested and a selection process was conducted to identify antibodies with MELK binding capacity that were positive on forced expression cells (positive control cells) and negative on negative control cells using the immunostaining method. Among the selected antibodies, one antibody was also chosen that was positive on cells expressing endogenous MELK (positive control cells) and negative on negative control cells. When the interaction with MELK was not strong enough, antibodies with weak binding capacity would remain in the background. Therefore, by conducting immunostaining using cell lines with previously quantified endogenous MELK expression, and then conducting the test, it was possible to select an antibody with a strong binding capacity to the target MELK.

[00021] The antibodies of the present invention bind specifically to MELK. Therefore, the antibodies of the present invention are useful as a tool for detecting MELK or the cells or tissues in which MELK is expressed. Furthermore, the antibodies of the present invention can be used as a labeled body conjugated to a label that is capable of detecting the antibody, and said labeled body is more preferable, for example, in the detection of cancer cells and cancer tissues that express MELK, such as colon cancer. As labels that are conjugated to the antibody of the present invention, it is sufficient provided that the label can detect an antibody bound to MELK, and the labels include affinity labels (e.g., biotin, avidin, and so on), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, and so on), fluorescent labels (e.g., FITC, rhodamine, and so on), etc. Petition 870260046788, dated 05 / 18 / 2026, page 20 / 141 15 / 55

[00022] When the antibody of the present invention is used as a diagnostic agent to select patients for cancer treatment, the antibody of the present invention can be used as is, or it can be transformed into a composition suitable for various types of use. I. Definitions

[00023] The words “a”, “an”, “the” and “the”, as used in this document, mean “at least one”, unless specifically indicated otherwise.

[00024] The terms “isolated” and “purified,” used in relation to a substance (e.g., peptide, antibody, polynucleotide, or similar), indicate that the substance does not substantially contain at least one substance that might otherwise be included in a natural source. Thus, an isolated or purified antibody refers to an antibody that does not substantially contain other cellular material, e.g., carbohydrate, lipid, and other contaminating proteins, from the cell or tissue source from which the antibody is derived. In a preferred embodiment, the antibodies of the present invention are isolated or purified.

[00025] The terms polypeptide and protein are used interchangeably in this document, and refer to polymers of amino acid residues. These terms are also applied to non-naturally occurring amino acid polymers comprising one or more non-naturally occurring amino acid residues, in addition to naturally occurring amino acid polymers. Non-naturally occurring amino acids include amino acid analogs, amino acid mimetics, and so forth.

[00026] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably in this document and Petition 870260046788, dated 05 / 18 / 2026, p. 21 / 141 16 / 55 refers to a nucleotide polymer.

[00027] Unless otherwise specified, the term “MELK-associated disease” refers to a cancer expressing MELK or endometriosis.

[00028] Unless otherwise specified, the term “cancer” refers to cancer that overexpresses the MELK gene, and examples include, but are not limited to, breast cancer, bladder cancer, cervical cancer, cholangiocellular cancer, chronic myelocytic leukemia (CML), colorectal cancer, esophageal cancer, stomach cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung cancer (SCLC), and so forth.

[00029] The term antibody, as used in this document, is intended to include immunoglobulins and fragments thereof having a specific reactivity to a designated protein or peptides thereof. Antibodies may include those fused with other proteins or markers and antibody fragments. Furthermore, in this document, “antibody” is used in its broadest sense and, specifically, provided it has the desired biological activity, includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies composed of at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments. The term antibody refers to all classes (e.g., IgA, IgD, IgE, IgG, and IgM).

[00030] An antibody fragment is a part of an intact antibody and generally comprises one or more antigen-binding regions or variable regions of an intact antibody. Therefore, in the present invention, an antibody fragment may include one or more Petition 870260046788, dated 05 / 18 / 2026, p. 22 / 141 17 / 55 antigen-binding portions of an intact antibody. The terms “antigen-binding portion” or “antigen-binding fragment of an antibody,” as used in this document, refer to one or more immunologically active fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., MELK). The antigen-binding function of an antibody has been shown to be performed by full-size antibody fragments. Examples of antibody fragments include the Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and also single-chain antibody molecules. Regardless of structure, antibody fragments bind to the same antigen as the antigen recognized by an intact antibody.The term “antibody fragment” also includes synthetic polypeptides that bind to the specific antigen or genetically engineered polypeptides, for example, polypeptides consisting of a variable region of the light chain, “Fv” fragments consisting of variable regions of a heavy chain and a light chain, recombinant single-chain polypeptide molecules where the variable regions of a light chain and a heavy chain are linked by a peptide linker (“scFv proteins”), as well as minimal recognition units consisting of amino acid residues mimicking the hypervariable regions.

[00031] Unless otherwise specified, all technical and scientific terms used in this document have the same meanings as the terms commonly understood by one skilled in the art to which the present invention pertains.

[00032] In the present invention, the specific binding between the MELK protein and an antibody can be evaluated, for example, by competition between the antibodies. Specifically, by using an antibody of the present invention as a reference antibody, for example, an antibody comprising the variable region of the heavy chain that Petition 870260046788, dated 05 / 18 / 2026, page 23 / 141 The specificity of a candidate antibody can be evaluated by comparing the amino acid sequence SEQ ID NO: 7 with the variable region of the light chain consisting of the amino acid sequence SEQ ID NO: 8. A representative reference antibody is OTSMAb01. When a candidate antibody competes with the antigen-antibody reaction between the reference antibody and the human MELK protein, it can be seen that the candidate antibody has equivalent specificity to the reference antibody. For example, when a reference antibody and the MELK protein react in the presence of a candidate antibody, and when the binding of the reference antibody with respect to the amount of reference antibody that binds to the MELK protein in the absence of the candidate antibody is inhibited by 10%, 20%, 30%, or 40%, more preferably 50% or more, it can be judged that there is competition between the antibodies.To assess competition between antibodies, not only the MELK protein, but also partial peptides thereof can be used, provided that the reference antibody binds to the peptide. Preferred partial peptides are, for example, the partial peptide consisting of the amino acid sequence SEQ ID NO: 9. II. Antibody Production

[00033] The present invention uses anti-MELK monoclonal antibodies. The antibodies are provided by methods well known in the art.

[00034] The illustrative antibody production techniques used by the present invention are described below. (i) Monoclonal antibodies

[00035] Monoclonal antibodies are obtained from a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are the same, except for natural mutations that may exist in varying quantities. Petition 870260046788, dated 05 / 18 / 2026, p. 24 / 141 19 / 55 minimum. Thus, the "monoclonal modifier" indicates the antibody's characteristic of not being a mixture with other antibodies.

[00036] For example, monoclonal antibodies can be produced using the hybridoma method which was first described by Kohler et al., Nature, 256: 495 (1975), or by the recombinant DNA method (US 4,816,567).

[00037] In the hybridoma method, mice or other available host animals, for example, hamsters and so forth, are immunized with the MELK polypeptide (MELK protein or a partial polypeptide thereof), and lymphocytes that produce, or can produce, antibodies that bind specifically to the MELK polypeptide are induced. Alternatively, lymphocytes can be immunized in vitro with the MELK polypeptide. After that, the lymphocytes are fused with myeloma cells using suitable fusion agents, such as polyethylene glycol, to produce hybridomas (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[00038] Prepared hybridomas are seeded in a suitable culture medium that preferably contains one or more substances that inhibit the proliferation or survival of myeloma cells of origin that have not fused and grown in that medium. For example, when the myeloma cells of origin lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas may typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that inhibit the proliferation of HGPRT-deficient cells.

[00039] The preferred myeloma cells are those that efficiently fuse, assist in stable and high-level antibody production by the selected antibody-producing cells, and are also sensitive to the medium, such as HAT medium. The cell lines Petition 870260046788, dated 05 / 18 / 2026, page 25 / 141 Preferred 20 / 55 myeloma cell lines include mouse myeloma cell lines, for example, those derived from MOPC-21 and MPC-11 mouse tumors, which are available from the Salk Institute Cell Distribution Center, San Diego, California, USA, as well as SP-2 cells and X63-Ag8-653 cells which are available from the American Type Culture Collection, Manassas, Virginia, USA. Regarding the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heterochromia cell lines have been described (Kozbor, J. Immunol., 133: 300 1 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[00040] The culture medium in which hybridomas are proliferating is analyzed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridomas is determined by immunoprecipitation methods or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[00041] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard 3D analysis by Munson et al., Anal Biochem. 107: 220-39 (1980).

[00042] After identifying hybridomas that produce an antibody with the desired specificity, affinity, and / or activity, this clone can be subcloned using limiting dilution and expanded through standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Examples of suitable culture media for this purpose include D-MEM medium and RPMI1640 medium. Furthermore, hybridomas can also be developed in vivo, such as ascites neoplasms within animals. Petition 870260046788, dated 05 / 18 / 2026, page 26 / 141 21 / 55

[00043] Monoclonal antibodies secreted by subclones can be purified to homogeneity. For example, the separation and purification of antibodies can be performed according to the separation and purification methods used for general proteins. For example, an antibody can be appropriately separated and isolated from the medium, ascites, or serum by appropriately selecting and combining the use of column chromatographies, such as affinity chromatography, filtration, ultrafiltration, coagulation, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing electrophoresis (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited to these. Protein A columns and protein G columns can be used as affinity columns. Illustrative protein A columns to be used include, for example, Hyper D, POROS, and Sepharose FF (Pharmacia).

[00044] In addition to affinity chromatography, illustrative chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography, and so on (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatography procedures can be performed by liquid-phase chromatography, such as HPLC and FPLC.

[00045] Monoclonal antibodies encoding DNAs can be easily isolated using conventional procedures (e.g., using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of a mouse antibody) and sequenced. Hybridomas are useful as a preferred source of such DNAs. After the DNAs are isolated, the synthesis of the isolated monoclonal antibody Petition 870260046788, dated 05 / 18 / 2026, page 27 / 141 22 / 55 within recombinant host cells can be achieved by placing the DNAs within (an) expression vector(s) and transfecting them into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, myeloma cells, and so on, which do not produce immunoglobulin proteins by other methods. A review on recombinant expression of antibody-encoding DNAs within bacterial cells includes Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

[00046] Another method for producing a specific antibody or antibody fragment that shows reactivity to MELK is to examine an expression library encoding immunoglobulin genes or portions thereof expressed within bacteria, using a MELK protein or a partial peptide thereof. For example, it is possible to express within bacteria a complete Fab fragment, VH region, and Fv region using a phage expression library. See, for example, Ward et al., Nature 341: 544-546 (1989); Huse et al., Science 246: 1275-1281 (1989); and McCafferty et al., Nature 348: 552-554 (1990). For example, by examining such a library using a MELK peptide, it is possible to identify immunoglobulin fragments that have reactivity to MELK. Alternatively, SCIDhu mice (available from GenPharm) can also be used to produce antibodies or fragments thereof.

[00047] In the additional embodiment, an antibody or its fragment can also be isolated from the antibody phage library prepared using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J Mol Biol, 222: 581-597 (1991), respectively, describe the isolation of mouse antibodies and human antibodies using phage libraries. Subsequent publications Petition 870260046788, dated 05 / 18 / 2026, p. 28 / 141 23 / 55 describe the production of high-affinity human antibodies (nM range) by chain scrambling (Marks et al., Biotechnology, 10: 779-783 (1992)), as well as combinatorial infection and recombination in vivo as a strategy to construct extremely large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Therefore, these techniques are means that can be performed as alternatives to conventional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[00048] The present invention provides suitable antibodies for diagnosing MELK-associated diseases, for determining drug efficacy after treatment with a MELK inhibitor, and for examining which patients a MELK inhibitor has a high therapeutic effect on. The present invention has successfully established a mouse monoclonal antibody clone (OTSMAb01) that can detect the MELK protein with high specificity in immunohistochemical staining. When this antibody clone was used in immunohistochemical staining of clinical breast cancer samples, it proved to have a positive finding in breast cancer samples, but almost no staining in normal breast samples.Furthermore, when commercially available anti-MELK antibodies were used, staining was observed in samples prepared from both high-expression and low-expression MELK cell lines, whereas when the anti-MELK antibody of the present invention was used, specific staining was observed in samples prepared from high-expression MELK cell lines. Therefore, the antibody of the present invention, with such high antigen specificity, is useful in screening patients with high levels of MELK expression, and also in patient screening. Petition 870260046788, dated 05 / 18 / 2026, page 29 / 141 24 / 55 in which treatment using MELK inhibitors is likely to be effective.

[00049] The amino acid sequences of the variable region of the heavy chain (V region of the H chain) and the variable region of the light chain (V region of the L chain) of the mouse anti-MELK monoclonal antibody clone of the present invention (OTSMAb01) are shown in SEQ ID NOs: 7 and 8, respectively.

[00050] CDRs (complementarity determining regions) comprising a variable region of the heavy chain and a variable region of the light chain can be determined according to well-known methods in the art. For example, the method described in Kabat et al. (Kabat EA et al., (1991) Sequence of Proteins of Immunological Interest. 5th Edition) or Chothia et al. (Chothia et al., J. Mol. Biol. (1987) 196; 901-917) is generally used for the determination of CDRs. The CDRs 1, 2, and 3 of the variable region of the heavy chain of the mouse anti-MELK monoclonal antibody clone (OTSMAb01) of the present invention, as determined according to Kabat's definition, are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the CDRs 1, 2, and 3 of the variable region of the light chain of the clone are shown in SEQ ID NOs: 4, 5, and 6, respectively.

[00051] Therefore, the present invention provides: an antibody or its antigen-binding fragment, comprising one or both of a variable heavy chain region and a variable light chain region, which can bind to a MELK protein or a partial peptide thereof, wherein the variable heavy chain region comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and Petition 870260046788, dated 05 / 18 / 2026, page 30 / 141 25 / 55 a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and where the variable region of the light chain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[00052] In the present invention, a partial peptide of the MELK protein to which an antibody of the present invention binds preferably comprises the amino acid sequence corresponding to positions 264-601 (SEQ ID NO: 9) of the MELK protein (SEQ ID NO: 22) and consists of an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 22. More preferably, the partial peptide of the MELK protein in the present invention may consist of the amino acid sequence of SEQ ID NO: 9.

[00053] An example of the variable region of the heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 described above is a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 7. An example of the variable region of the light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6 described above is a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 8. Petition 870260046788, dated 05 / 18 / 2026, page 31 / 141 26 / 55

[00054] Therefore, in one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising one or both of the variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and the variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 8.

[00055] The antibodies of the present invention can be prepared by conventional methods. For example, the antibody can be prepared according to conventional gene recombination techniques, inserting into a suitable vector a polynucleotide encoding an antibody polypeptide, introducing said vector into a host, and causing the host to produce the antibodies (for example, see Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 76775).

[00056] The nucleotide sequence of a polynucleotide encoding a variable region (V region) of the antibody of the present invention can be deduced from the amino acid sequence of a V region of the antibody of the present invention. As nucleotide sequences encoding the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of the antibody clone of the present invention, for example, the nucleotide sequences of SEQ ID NOs: 10 and 11, respectively, can be used. A polynucleotide encoding a V region of the antibody of the present invention can be synthesized based on sequence information using conventional methods, such as solid-phase synthesis techniques (Beaucage SL & Iyer RP, Tetrahedron (1992) 48, 2223-311; Matthes et al., EMBO J (1984) 3, 801-5) and oligonucleotide synthesis techniques (Jones et al., Nature (1986) 321, 522-5).

[00057] A polynucleotide encoding a V region of the antibody is inserted into an expression vector comprising a Petition 870260046788, dated 05 / 18 / 2026, page 32 / 141 27 / 55 polynucleotide that codes for a constant region (C) of the antibody.

[00058] To produce the antibody used in the present invention, a polynucleotide encoding said antibody (antibody gene) is inserted into the expression vector so as to express the antibody gene under the regulation of expression-regulating factors (e.g., enhancers, promoters). Host cells are transformed using said expression vector to express the antibody.

[00059] In antibody gene expression, the polynucleotide encoding the antibody H chain and the polynucleotide encoding the L chain can be inserted into different expression vectors, and then the resulting recombinant expression vectors are co-transfected into a host cell. Alternatively, the polynucleotide encoding the antibody H chain and the polynucleotide encoding the L chain can be inserted into the same expression vector together, and then the resulting recombinant expression vector is transfected into a host cell (e.g., WO 94 / 11523).

[00060] Antibody genes can be expressed by known methods. In the case of expression in mammalian cells, a conventional useful promoter, the antibody gene to be expressed, and a poly(A) signal (located downstream of the 3' end of the antibody gene) can be functionally linked. For example, the human cytomegalovirus immediate initial promoter / booster system can be used as a useful promoter / booster system.

[00061] Other promoter / booster systems may be used, for example, those derived from viruses (e.g., retroviruses, polyomavirus, adenovirus and simian virus 40 (SV 40)), as well as those derived from mammalian cells (e.g., human elongation factor 1α (HEF1a)), to express the antibody in the present invention. Petition 870260046788, dated 05 / 18 / 2026, page 33 / 141 28 / 55

[00062] When using the SV40 promoter / booster system, gene expression can be easily achieved using the method of Mulligan et al. (Nature (1979) 277, 108-14). When using the HEF1a promoter / booster system, gene expression can be easily achieved using the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).

[00063] When expressed in E. coli, a conventional useful promoter, a signal sequence to secrete the antibody of interest, and the antibody gene can be functionally linked. The lacZ promoter or the araB promoter can be used as promoters. When using the lacZ promoter, gene expression can be performed by the method of Ward et al. (Nature (1989) 341, 544-6.; FASBE J. (1992) 6, 2422-7), and when using the araB promoter, gene expression can be performed by the method of Better et al. (Science (1988) 240, 1041-3).

[00064] Regarding a signal sequence for secreting the antibody, when secretion of the antibody of interest into the periplasmic space of E. coli is desired, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379-83) can be used. The antibody secreted into the periplasmic space is isolated and then reduplicated so that the antibody adopts a suitable steric structure.

[00065] A virus-derived origin of replication (e.g., SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV)) or similar may be used. To increase the number of gene copies in a host cell system, the expression vector may additionally include selection marker genes, such as the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the E. coli hypoxanthine-guanine phosphoribosyltransferase (Ecogpt) gene, and the dihydrofolate reductase (dhfr) gene. Regarding the production of the antibody used in the present invention, a Petition 870260046788, dated 05 / 18 / 2026, page 34 / 141 29 / 55 arbitrary expression system, including eukaryotic and prokaryotic cell systems. Eukaryotic cells include established animal cell lines (e.g., mammals, insects, molds and fungi, and yeasts). Prokaryotic cells include bacterial cells, such as E. coli cells. Preferably, the antibody used in the present invention is expressed in mammalian cells, such as CHO cells, COS cells, myeloma cells, BHK cells, Vero cells, and HeLa cells.

[00066] Next, the transformed host cells are cultured in vitro or in vivo and the antibody of interest is produced. Host cells can be cultured using any known methods. The culture medium that can be used in this document may be DMEM, MEM, RPMI1640 or IMDM medium. The culture medium may contain serum supplements such as Fetal Calf Serum (FCS).

[00067] In addition to the host cells mentioned above, it is possible to use transgenic animals as hosts in the production of recombinant antibodies. For example, the antibody gene is inserted into a designated location in a gene encoding a protein that is naturally produced within the breast milk of an animal (e.g., β-casein); and a fusion gene is produced. A DNA fragment comprising the inserted fusion gene with the antibody gene is injected into a non-human animal embryo, and this embryo is then introduced into a female animal. The female animal that has the inserted embryo gives birth to a transgenic non-human animal. The antibody of interest is secreted in the milk of the mother of said transgenic non-human animal or her offspring. To increase the amount of breast milk containing said antibody, a suitable hormone may be administered to said transgenic animal (Ebert, KM et al., Bio / Technology (1994) 12, 699-702). Petition 870260046788, dated 05 / 18 / 2026, page 35 / 141 30 / 55

[00068] An antibody expressed and produced as described above can be isolated from the cells or body of a host animal and purified. The isolation and purification of the antibody, as used in the present invention, can be done using affinity columns. Other conventionally used methods can also be used for the isolation and purification of the antibody, and therefore the methods are not particularly limited. For example, various chromatographies, filtrations, ultrafiltrations, coagulations, and dialyses can be used alone or in combination to isolate and purify the antibody of interest (Antibodies A Laboratory Manual. Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). (ii) Antibody fragments

[00069] Several techniques have been developed for the production of antibody fragments. Conventionally, these fragments have been obtained via proteolytic digestion of intact antibodies (e.g., see Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, such fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, an F(ab')2 fragment can be formed by direct recovery of a Fab'-SH fragment from E. coli and chemical ligation (Carter et al., Bio / Technology 10: 163-167 (1992)). Another approach is to directly isolate an F(ab')2 fragment from a recombinant host cell culture. Other techniques for producing an antibody fragment would be obvious to experts in the field.In another embodiment, an optimal antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185; US 5,571,894; and US 5,587,458. For example, an antibody fragment could be a... Petition 870260046788, dated 05 / 18 / 2026, page 36 / 141 31 / 55 “linear antibody, as described, for example, in US 5,641,870. Such linear antibody fragments may have monospecificity or bispecificity. (iii) Labeled antibodies

[00070] The antibody of the present invention is optionally conjugated to an affinity label, an enzyme label, a radioisotope label, a fluorescent label, or a chemiluminescent label. For example, the presence of a label that is present within a MELK-expressing cancer, and which is detectable, allows the determination of the presence or absence of a cancer or tumor within the patient to be diagnosed. Furthermore, it is possible to determine the progression of the disease by the location of the label within the cancer.

[00071] Suitable markers for use include, for example, fluorescent markers such as fluorescein and rhodamine; and enzyme markers such as luciferase. The detectable marker / label used for detection can be selected based on the imaging mode used. A conjugate between such a marker and an antibody can be prepared using protocols and practices known in the art. In the present invention, the antibody of the invention can be conjugated to a desired marker just before use, or it can be provided as an antibody conjugated to a marker.

[00072] A conjugate between an antibody and a label can be prepared using various bifunctional protein-binding agents, such as N-succinimidyl 3-(2-Pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional imidoester derivatives (e.g., Dimethyl adipimidic acid HCl), active esters (e.g., Disuccinimidyl suberate), aldehydes (e.g., Glutaraldehyde), Petition 870260046788, dated 05 / 18 / 2026, p. 37 / 141 32 / 55 bis-azide compounds (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Alternatively, a fusion protein comprising the antibody and the label may be prepared, for example, by recombinant techniques or peptide synthesis. Suitable examples of such fusion proteins include fusion proteins between labeling proteins such as ECFP, EYFP, or EGFP and an antibody. III. Diagnosis of MELK-associated diseases, examination of patients for whom a MELK inhibitor has a high therapeutic effect (pre-treatment diagnosis), or determination of drug efficacy after treatment with a MELK inhibitor (post-treatment diagnosis)

[00073] MELK is useful as a diagnostic marker for MELK-associated diseases, and also as a marker to assess the sensitivity of diseases to a MELK inhibitor and the efficacy of said MELK inhibitor drug. Therefore, the antibodies of the present invention can be used as a reagent to detect a marker for the diagnosis of MELK-associated diseases, such as cancer, for the examination of patients for whom a MELK inhibitor has a high therapeutic effect, or for the determination of drug efficacy after treatment with a MELK inhibitor.

[00074] More specifically, the MELK protein in an isolated sample from a patient can be detected using the antibody of the present invention to perform the diagnosis of MELK-associated diseases, the examination of patients for whom a MELK inhibitor has a high therapeutic effect, or the determination of drug efficacy after treatment with a MELK inhibitor. Therefore, the Petition 870260046788, dated 05 / 18 / 2026, page 38 / 141 33 / 55 The present invention provides methods for diagnosing a disease associated with MELK or a predisposition to developing the disease in a patient, methods for examining whether a MELK inhibitor has a high therapeutic effect on a patient, and methods for determining the efficacy of the drug after treatment with a MELK inhibitor by detecting the MELK protein in a sample isolated from a patient using the antibody of the present invention. These methods comprise the following steps: (a) contacting an isolated sample from a patient with the antibody or antigen-binding fragment of the present invention; (b) detect MELK protein in the sample by detecting the binding of the sample to the antibody or antigen-binding fragment thereof; and (c) compare the level of MELK protein in the sample with a control.

[00075] In a typical embodiment, the aforementioned sample is a cell or tissue isolated from the aforementioned patient, preferably a tissue isolated from said patient. Therefore, normally each of the methods of the present invention is conducted in vitro on samples isolated from patients. Methods for isolating tissues and cells from patients using techniques such as biopsies and blood collection are known. It is also possible to use biological samples removed from patients through medical procedures conducted for treatment (surgeries and so on). Cells and tissues isolated from a patient can be appropriately treated before contact with the antibody. For example, tissue samples obtained from a patient can generally be transformed into sections after freezing, further fixed using alcohol, formalin, and so on, and used as samples for immunohistological analysis. Petition 870260046788, dated 05 / 18 / 2026, page 39 / 141 34 / 55 Alternatively, after fixing tissue samples, cultured cells, and so on with formalin and similar substances, it is possible to obtain sections for immunohistological analysis by paraffin fixation.

[00076] The binding of the antibody or antigen-binding fragment of the present invention to a sample, i.e., binding to the antigenic protein in the sample, can be detected by a method known to those skilled in the art. More specifically, after contact of the antibody of the present invention with said sample, removing the antibodies that were not bound to the MELK protein in the sample by washing, and detecting the antibody that remained in the sample, the binding of the antibody of the present invention and the MELK protein in the sample can be detected. At this point, when the antibody is directly labeled, the presence of the antibody of the present invention bound to the MELK protein can be detected by label detection. When the label is a detectable label, such as enzymes, fluorescent substances, luminescent substances and particles, such labels can be readily detected.In addition to these, when the antibody of the present invention is labeled (affinity-labeled) with an affinity substance (binding substance), such as biotin, the presence of the antibody can be captured using a binding pair, such as labeled avidin. Alternatively, when the antibody of the present invention is not directly labeled, the antibody of the present invention can be detected using an antibody-binding reagent. For example, protein A or an antibody against the antibody, after labeling, can be used as an antibody-binding reagent to detect the antibody of the present invention.

[00077] In the diagnosis of MELK-associated diseases, in step (c) above, when the MELK protein level is high in Petition 870260046788, dated 05 / 18 / 2026, pp. 40 / 141 35 / 55 comparison with a control level (normal control level, preferably the MELK protein expression level in a sample isolated from a healthy patient who does not suffer from a MELK-associated disease), this indicates that the patient suffers from a MELK-associated disease or is at risk of developing it.

[00078] Furthermore, when examining a patient for whom a MELK inhibitor has a high therapeutic effect, in step (c) above, when the MELK protein level is equivalent to or elevated compared to a control level (preferably, the MELK protein expression level in a tissue of a patient diagnosed with a MELK-associated disease), this indicates that the therapeutic effect of a MELK inhibitor is high in the patient.

[00079] On the other hand, when determining the drug efficacy after treatment with a MELK inhibitor, in step (c) mentioned above, when the MELK protein level is low compared to a control level (preferably, the MELK protein expression level in a sample isolated from the patient before drug administration), this indicates that the drug was effective in the patient.

[00080] Patients who have been shown to have a MELK-associated disease by a diagnostic method of the present invention are likely to become treatment patients with a MELK inhibitor. Therefore, following the diagnostic method of the present invention, a MELK inhibitor can be administered to patients who have been shown to have a MELK-associated disease. Alternatively, a MELK inhibitor can also be administered to patients who have shown a potential to receive an enhanced therapeutic effect from a MELK inhibitor after examination. Furthermore, when it is shown that a MELK inhibitor has a therapeutic effect in patients who have been administered the MELK inhibitor, the Petition 870260046788, dated 05 / 18 / 2026, pp. 41 / 141 36 / 55 MELK inhibitor can be administered continuously to the same patients.

[00081] More specifically, the present invention relates to methods of treating MELK-associated diseases, comprising the step of identifying one of the patients selected from the group below by the methods of the present invention and administering the MELK inhibitor to said patient: a patient who has been shown to have a disease associated with MELK using the diagnostic method of the present invention; A patient who has been shown to have a high chance of receiving an enhanced therapeutic effect from a MELK inhibitor; and a patient who was administered a MELK inhibitor and has been shown to have received a therapeutic effect from the inhibitor.

[00082] In the present invention, known compounds can be used as a MELK inhibitor administered to patients. For example, there are several known compounds that inhibit the enzymatic action of MELK (WO 2012 / 016082; WO 2013 / 109388; Oncotarget. 2012, 3: 1629-1640; Oncotarget. 2016; 7: 17652-17664).

[00083] In the context of the present invention, a control level measured using a biological sample known not to suffer from a MELK-associated disease (e.g., non-cancerous) is called the “normal control level.” When the MELK protein level in a sample isolated from a patient is high compared to the normal control level, the patient can be diagnosed as having a MELK-associated disease requiring treatment.

[00084] On the other hand, a measured control level of a biological sample known to suffer from a MELK-associated disease (e.g., cancer) is called a “disease control level” (e.g., cancer control level). When the level of MELK protein in a sample isolated from a patient before treatment Petition 870260046788, dated 05 / 18 / 2026, page 42 / 141 If a MELK inhibitor response of 37 / 55 is equivalent to, or high compared to, the disease control level, the patient may be diagnosed as receiving a high therapeutic effect from the MELK inhibitor.

[00085] Furthermore, when the MELK protein level in an isolated sample from a patient after treatment with a MELK inhibitor is lower than the disease control level of the same patient before drug administration, it can be diagnosed that there was drug efficacy in the treatment, that is, that the patient received a high therapeutic effect from the MELK inhibitor.

[00086] In one embodiment, normal cells (or tissues) obtained from an unaffected region (e.g., non-cancerous region) of an organ that has a MELK-associated disease (e.g., cancer) to be treated can be used as the normal control. In another embodiment, the control level can be determined by statistical methods based on results obtained through analysis of MELK protein levels previously measured in samples derived from patients whose disease status (e.g., cancerous or non-cancerous) is known. The control level can additionally be derived from a database of expression patterns derived from previously tested samples (cells or tissues). When the sample to be evaluated is a tissue sample, it is preferable to use a sample derived from the same tissue as the control sample.

[00087] Furthermore, according to one aspect of the present invention, the MELK protein level in a biological sample can be compared with various control levels measured from various reference samples. It is preferable to use control levels measured from reference samples derived from a tissue type that is similar to the tissue type of the patient-derived biological sample. Additionally, it is preferable to use standard values ​​for the level of Petition 870260046788, dated 05 / 18 / 2026, page 43 / 141 38 / 55 MELK protein in a population whose disease status is known. The standard value can be obtained using any methods known in the art. For example, a range of a mean value of + / - 2 SD or a mean value of + / - 3 SD can be used as a standard value.

[00088] The MELK protein level in a sample is considered to be high when the level is, for example, 10%, 25%, or 50% higher than the control level, or is more than 1.1 times, more than 1.5 times, more than 2.0 times, more than 5.0 times, more than 10.0 times, or more, compared to the control level. The MELK protein level in a sample is considered to be low when the level is, for example, 10%, 25%, or 50% lower than the control level, or is more than 1.1 times, more than 1.5 times, more than 2.0 times, more than 5.0 times, more than 10.0 times, or lower than the control level.

[00089] In a typical embodiment, the disease associated with MELK is a cancer that expresses MELK or endometriosis. Cancers that express MELK include, for example, breast cancer, bladder cancer, cervical cancer, cholangiocellular carcinoma, chronic myelocytic leukemia (CML), colorectal cancer, esophageal cancer, stomach cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, and small cell lung cancer (SCLC), but are not limited to these.

[00090] In a further embodiment, the invention provides methods for detecting a diagnostic marker for a MELK-associated disease or a predisposition to developing said disease, including the step of detecting, as said diagnostic marker, a MELK protein in a sample using the antibody or antigen-binding fragment thereof of the present invention. It has been clarified that MELK expression is increased in certain types of Petition 870260046788, dated 05 / 18 / 2026, p. 44 / 141 39 / 55 cancer cells compared to normal tissues. Therefore, if it is possible to specifically detect the level of MELK expression, it is useful as a diagnostic marker for MELK-associated diseases. In the context of the present invention, a diagnostic marker for a MELK-associated disease or the predisposition to develop said disease is a MELK protein in an isolated sample from a patient, which is detected by binding between the antibody or antigen-binding fragment of the present invention and is characterized as showing that said patient suffers from said disease, or is at risk of developing the disease, when the MELK expression level is high compared to a control level. Here, said control level is the normal control level, preferably the MELK protein expression level in an isolated sample from a healthy patient who does not suffer from MELK-associated disease.In general, it is preferable that the control level be the expression level in the same tissue as the tissue from which the cancer cells that the diagnostic marker is trying to detect originate.

[00091] The present invention also provides antibodies or antigen-binding fragments thereof of the present invention for use in the diagnosis of a disease associated with MELK, or of a predisposition to developing said disease. Alternatively, the present invention provides the use of the antibodies or antigen-binding fragments thereof of the present invention in the manufacture of a reagent for the diagnosis of a disease associated with MELK, or of a predisposition to developing said disease.

[00092] Furthermore, the present invention provides methods for detecting a marker of sensitivity to treatment with a MELK inhibitor, the methods comprising the step of detecting the MELK protein in a sample as said sensitivity marker, using Petition 870260046788, dated 05 / 18 / 2026, page 45 / 141 40 / 55 the antibody or antigen-binding fragment thereof of the present invention. It has been elucidated that MELK expression is specifically increased in certain types of cancer cells, and that the proliferation of such cancer cells is suppressed by a MELK inhibitor (WO 2004 / 031413; WO 2006 / 016525; WO 2007 / 013665; WO 2008 / 023841). In other words, it is possible to predict sensitivity to a MELK inhibitor using MELK expression as an index. This is because if MELK is expressed at a high level, a cell growth suppression effect by the MELK inhibitor can be anticipated. Therefore, if it is possible to specifically detect the level of MELK expression, it will be useful as a marker of sensitivity to treatment with the MELK inhibitor.In the context of the present invention, a marker of sensitivity to treatment with the MELK inhibitor is a MELK protein in an isolated sample from a patient, which is detected by the binding between the antibody or the antigen-binding fragment of the present invention and is characterized in that it is shown that the therapeutic effect of the MELK inhibitor is elevated in the patient when the MELK expression level is equivalent to, or high, compared to a control level. Here, said control level is preferably a disease control level, in other words, the MELK protein expression level in an isolated sample from an affected part of the patient known to suffer from a MELK-associated disease, particularly and preferably the MELK protein expression level in an isolated sample before treatment of a patient for whom a MELK inhibitor has an elevated therapeutic effect.

[00093] The present invention further provides antibodies or antigen-binding fragments thereof of the present invention for use in testing a patient for whom a MELK inhibitor has a high therapeutic effect. Alternatively, the Petition 870260046788, dated 05 / 18 / 2026, page 46 / 141 41 / 55 The present invention provides for the use of the antibody or antigen-binding fragment thereof of the present invention in the manufacture of a reagent for testing whether a MELK inhibitor has a high therapeutic effect on a patient.

[00094] The present invention also provides methods for detecting a drug efficacy marker for a MELK inhibitor, the methods comprising the step of detecting a MELK protein in a sample using an antibody or the antigen-binding fragment thereof of the present invention as said drug efficacy marker. It has been clarified that MELK expression is specifically increased in certain types of cancer cells, and that the proliferation of such cancer cells is suppressed by a MELK inhibitor (WO 2004 / 031413; WO 2006 / 016525; WO 2007 / 013665; WO 2008 / 023841). Consequently, cancerous tissues possessing such cancer cells can be reduced or killed by a MELK inhibitor. In other words, it is possible to assess the drug efficacy of a MELK inhibitor in a patient having such cancer cells using the level of MELK expression as an index.This is because, if the MELK expression level in an isolated tissue sample containing MELK-positive cancer cells is reduced compared to that in an isolated sample before treatment with the MELK inhibitor, it is possible to determine that the MELK-positive cancer cells have decreased due to the MELK inhibitor. Therefore, if it is possible to specifically detect the MELK expression level, it would be useful as a marker of drug efficacy for a MELK inhibitor. In the context of the present invention, a marker of drug efficacy for a MELK inhibitor is a MELK protein in an isolated sample from a patient to whom a MELK inhibitor has been administered, which is detected by binding to an antibody or the antigen-binding fragment thereof of the present invention, and characterized. Petition 870260046788, dated 05 / 18 / 2026, page 47 / 141 42 / 55 since it is shown that there was efficacy of the drug by the MELK inhibitor in said patient when the MELK expression level is low compared to the control level. Here, said control level is preferably the MELK protein expression level in the isolated sample from an affected part of said patient before drug administration.

[00095] The present invention also provides antibodies or antigen-binding fragments thereof of the present invention for use in determining drug efficacy after treatment with a MELK inhibitor. Alternatively, the present invention provides the use of an antibody or antigen-binding fragment thereof of the present invention in the manufacture of a reagent for determining drug efficacy after treatment with a MELK inhibitor. IV. Reagents or kits for diagnosing a MELK-associated disease, for examining patients for whom a MELK inhibitor has a high therapeutic effect, or for determining drug efficacy after treatment with a MELK inhibitor

[00096] The present invention provides reagents or kits for diagnosing a MELK-associated disease, for examining patients for whom a MELK inhibitor has a high therapeutic effect, or for determining drug efficacy after treatment with a MELK inhibitor. Specifically, these kits include an antibody or the antigen-binding fragment thereof of the present invention as a reagent for detecting the MELK protein. In one embodiment, the antibody for the reagent or diagnostic kit of the present invention may be labeled with a fluorescent substance, a luminescent substance, or a radioisotope.The methods for labeling an antibody and for detecting a labeled antibody are well known in the art, and it is possible to use any labels and methods. Petition 870260046788, dated 05 / 18 / 2026, pp. 48 / 141 43 / 55 for the present invention.

[00097] The present kits may include a combination of an antibody or the antigen-binding fragment thereof of the present invention with another marker detection reagent. The present kits may also include positive and negative control reagents for MELK, and a secondary antibody to detect the antibody of the present invention. For example, culture sections or tissue samples from cell lines known to highly express MELK would be advantageous as useful positive control reagents. Furthermore, for example, tissue samples obtained from healthy patients or non-cancerous tissues would be advantageous as useful negative control reagents. The secondary antibodies to detect an antibody of the present invention are preferably labeled with a fluorescent substance, a luminescent substance, a radioisotope, or an enzyme.The kit of the present invention may additionally include other materials desirable from a user's perspective from a commercial standpoint, including buffers, diluents, filters, needles, syringes, and instruction leaflets (e.g., documents, tapes, CD-ROMs, and so forth). These reagents and so forth may be retained in a labeled container. Suitable containers include bottles, small vials, and test tubes. The containers may be made of various materials, such as glass and plastic.

[00098] The present invention is explained in detail in this document with reference to its specific embodiments. However, it should be understood that the above explanation is in fact an illustrative and explanatory explanation, and is intended to explain the present invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various alterations and modifications can be made to it without departing from the spirit and scope. Petition 870260046788, dated 05 / 18 / 2026, pp. 49 / 141 44 / 55 of the present invention. Thus, the present invention is not limited to the above-mentioned explanation, but is intended to be defined by the appended claims and their equivalents.

[00099] The present invention is described in more detail herein with reference to the Examples. However, although the materials, method and Examples that follow may serve to assist one skilled in the art in the preparation and use of certain embodiments of the present invention, they are intended only to illustrate aspects of the present invention and thus in no way limit the scope of the present invention. One skilled in the art may use methods and materials similar or equivalent to those described herein in the practice or testing of the present invention. [000100] All prior art documents cited in this document are incorporated by reference in this descriptive report. Examples [000101] The present invention is described in more detail herewith reference to the Examples, but should not be construed as being limited to them. [Materials and methods] Cell culture [000102] A MELK-forced expression cell line (MELK / 293T) was prepared by introducing a MELK expression vector into the 293T human embryonic kidney cell line acquired from GenHunter, and a cell line introduced with an empty vector (Mock / 293T) was prepared as a negative control. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C under a humidified atmosphere with 5% CO2. The MCF-7 human breast cancer cell line acquired from ATCC was maintained in supplemented MEM. Petition 870260046788, dated 05 / 18 / 2026, page 50 / 141 45 / 55 with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, non-essential amino acids, sodium pyruvate, and insulin, at 37°C, under a humidified atmosphere with 5% CO2. The human hepatoma cell line Hep G2 acquired from JCRB was maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, at 37°C, under a humidified atmosphere with 5% CO2. The human pancreatic cancer cell line PK-45P, received from the Institute of Development, Ageing, and Cancer of the Tohoku University, and the human lung cancer cell line A549, acquired from Dainippon Pharma Co., Ltd., were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, at 37°C, under a humidified atmosphere with 5% CO2.The human breast cancer cell line BT-549, acquired from ATCC, was maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, HEPES, sodium pyruvate, and insulin, at 37°C, under a humidified atmosphere with 5% CO2. The human colon cancer cell line HT-29, acquired from ATCC, was maintained in McCoy's 5A supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, at 37°C, under a humidified atmosphere with 5% CO2. The MDA-MB-231 human breast cancer cell line, acquired from ATCC, was maintained in L-15 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, at 37°C, under a humidified atmosphere without CO2. Clinical Samples [000103] Tissue samples surgically removed from breast cancers (formalin-fixed paraffin sections, frozen tissues), as well as the corresponding clinical information, were obtained from the Kanagawa Cancer Center after obtaining written informed consent. Petition 870260046788, dated 05 / 18 / 2026, page 51 / 141 46 / 55 Immunohistochemical (IHC) staining [000104] Formalin-fixed paraffin sections of MELK / 293T, Mock / 293T, MCF-7, Hep G2, HT-29, PK-45P, BT-549, MDA-MB-231, A549 and clinical samples were immersed three times for three minutes in xylene for deparaffinization and then rehydrated by immersing them in 100% ethanol twice for 1 minute, and then for 1 minute each in 90%, 70% and 50% ethanol. For antigen activation, the sections immersed in the antigen activation solution, pH 9 (Nichirei Biosciences Inc.), were incubated at 125°C for 30 seconds. After incubation, they were left to stand for 20 minutes at room temperature and washed for 5 minutes under running water. After immersing them in 3.0% hydrogen peroxide for 10 minutes to block endogenous peroxidase, they were washed with Washing Buffer (TBS-T (Takara Bio)) three times for 5 minutes.Furthermore, in order to block non-specific reactions, an appropriate amount of Protein Blocking Solution (Dako) was dripped onto the sections and then allowed to stand for 10 minutes. The primary antibodies (OTSMAb01, MELK(N449)pAb, MELK(HPA017214)pAb) were, respectively, dripped in appropriate amounts into a moistened container, allowed to stand for 60 minutes, and then washed with Wash Buffer three times for 5 minutes. Histofine Simple Stain MAX PO (Nichirei Biosciences Inc.) was dripped in appropriate amounts, as the secondary antibody, into a moistened container and allowed to stand for 30 minutes. These were then washed with Wash Buffer three times for 5 minutes. The chromogenic reaction was performed using DAB substrate solution (Nichirei Biosciences Inc.). The slide sections were immersed in hematoxylin (Dako) for 20 seconds and washed under running water.Dehydration was performed by immersing them in 50% ethanol. Petition 870260046788, dated 05 / 18 / 2026, page 52 / 141 47 / 55 70% and 90%, for 1 minute, respectively, and then immersing them twice for 1 minute in 100% ethanol. Finally, the sections were immersed twice for 3 minutes in xylene for penetration and closed using Mount-Quick (DAIDO SANGYO). RT-PCR [000105] Clinical frozen tissues (breast cancer) were disrupted by immersing the frozen tissue sections in TRIzol Reagent (Invitrogen) and extracted with chloroform. An approximately equivalent volume of 70% ethanol was added to the extract, and total RNA was extracted using the RNeasy Mini kit (QIAGEN). cDNA was then synthesized from the total RNA using a reverse transcriptase, SuperScript II Reverse Transcriptase (Invitrogen). PCR was then performed using ExTaq (Takara Bio) and the cDNA as a template. MELK was used as the target gene for expression analysis, and β-actin as the maintenance gene. MELK was amplified using the primer set MELK-1F: 5'ATGATCACCTCACGGCTA -3' (SEQ ID NO: 12), MELK-1R: 5'AGGTGTTCTGCATAAGG -3' (SEQ ID NO: 13), and β-actin was amplified using the primer set Beta-actin Fw: 5'AGGATGCAGAAGGAGATCAC -3' (SEQ ID NO: 14), Beta-actin Re: 5'AGAAAGGGTGTAACGCAACT -3' (SEQ ID NO: 15).The PCR amplification product was subjected to electrophoresis with an agarose gel, and the MELK gene expression levels were compared using Western blotting. [000106] The protein was extracted from MCF-7, Hep G2, HT-29, PK-45P, BT-549, MDA-MB-231, and A549 using RIPA lysis buffer. The target proteins were separated using SDS-based polyacrylamide gel electrophoresis (SDS-PAGE). The separated target proteins were transferred to nitrocellulose (NC). After blocking non-specific binding on the membrane surface using the solution of Petition 870260046788, dated 05 / 18 / 2026, page 53 / 141 48 / 55 Block-Ace (DS Pharma Biomedical) was incubated with test antibodies capable of binding to target proteins. After washing off excess test antibodies from the membrane using TBST Wash Buffer, it was incubated with peroxidase-labeled secondary antibody (HRP) capable of binding to test antibodies, and then excess secondary antibodies on the membrane were washed off using Wash Buffer. Using the ECL Western blotting detection system (GE Healthcare), detectable chemiluminescence was generated and recorded using film. [Example 1] Preparation of anti-MELK monoclonal antibody (1) Obtaining hybridomas that produce the anti-MELK antibody [000107] Analysis of the immunogenic region of the human MELK protein (SEQ ID NO: 22) showed that the total antigenic marker shifts favorably in the amino acid region 264-601 (SEQ ID NO: 9) which adopts a long loop structure and that the sequence specificity is also favorable, predicting that the antigenicity was high (Medical & Biological Laboratories Co, Ltd). Therefore, in order to produce a specific antibody for MELK, a recombinant protein from the amino acid region 264-601 (SEQ ID NO: 9) of the human MELK protein (SEQ ID NO: 22) encoded by the human MELK gene (SEQ ID NO: 21) was used as an immunogen. Initial immunization was conducted by adding 50 pg of the antigenic peptide to Freund's Adjuvant, emulsifying it, and injecting it subcutaneously into a Balb / c mouse (Japan SLC, Inc.).Immunizations from the second time onwards were conducted by subcutaneously injecting a preparation corresponding to 25 pg of antigenic peptide, as similarly prepared. Three days after the final immunization, splenocytes were prepared from mice under sterile conditions and fused with the cells of... Petition 870260046788, dated 05 / 18 / 2026, page 54 / 141 49 / 55 mouse myeloma SP2 / 0, using the polyethylene glycol method, according to conventional methods. (2) Selection of hybridoma that produces anti-MELK antibody [000108] The anti-MELK antibody was selected by immunohistochemical staining using the 293T cell line expressing the natural-size MELK protein (SEQ ID NO: 22). That is, after the 293T cell line forcibly expressing the natural-size MELK protein was formalin-fixed and transformed into paraffin sections, hybridomas showing a strong reaction with the MELK-forcibly expressing cell line (MELK / 293T) were selected for immunostaining. [000109] The immunohistochemical staining results of the OTSMAb01 hybridoma clone, which was confirmed to produce a specific antibody for MELK at a high level among the hybridomas tested, are shown in Fig. 1. As shown in the photographs and graph in Fig. 1, when stained using OTSMAb01, staining was observed in the paraffin section prepared from the cell line forcibly expressing MELK (MELK / 293T), similar to when using the commercially available anti-MELK polyclonal antibodies MELK(N449)pAb and MELK(HPA017214)pAb, which, in other words, showed that the proportion of MELK-positive cells was high. On the other hand, staining was hardly seen in the paraffin sections prepared from the cell lines introduced with the empty vector (Mock / 293T), which was used as a negative control, showing that the proportion of MELK-positive cells was low.These results showed that the OTSMAb01 hybridoma clone is useful as a hybridoma that produces an antibody that specifically detects MELK in immunohistochemical staining. [000110] This OTSMAb01 hybridoma clone was selected as a hybridoma to produce the antibody for further experiments. The Petition 870260046788, dated 05 / 18 / 2026, p. 55 / 141 A 50 / 55 clone of the OTSMAbOI hybridoma was grown on a large scale, and the culture solution was collected 2 to 3 weeks later. The antibody was purified from the culture solution using a Protein A column (GE Healthcare, NJ). In this document, the antibody of the present invention is also referred to as clone OTSMAb01. [Example 2] Evaluation of the specificity of the anti-MELK monoclonal antibody [000111] Next, using several cell lines with different levels of MELK expression, the specificity of OTSMAb01 was evaluated. That is, by comparing the results of immunohistochemical staining and Western blotting using OTSMAb01, it was assessed whether a staining corresponding to the level of MELK protein expression was observed. [000112] First, the expression levels of endogenous MELK protein in several cell lines derived from human cancer tissue were checked by Western blotting using OTSMAb01. As shown in Fig. 2, the human pancreatic cancer-derived cell line PK-45P, the human breast cancer-derived cell lines BT-549 and MDA-MB-231, and the human lung cancer-derived cell line A549 were high MELK expression cell lines, and the human breast cancer-derived cell line MCF-7, the human hepatoma-derived cell line Hep G2, and the human colon cancer-derived cell line HT-29 were low MELK expression cell lines. [000113] Next, immunohistochemical staining was performed on paraffin sections prepared from these cell lines using OTSMAb01 and commercially available anti-MELK polyclonal antibodies. As shown in Fig.3, when stained using OTSMAb01, staining was observed in paraffin sections prepared from the PK-45P, BT-549, MDA-MB cell lines. Petition 870260046788, dated 05 / 18 / 2026, page 56 / 141. 51 / 55 231 and A549, which were found to highly express MELK. A positive signal was detected in a high proportion of cells in these paraffin sections. On the other hand, in paraffin sections prepared from MCF-7, Hep G2, and HT-29, which are cell lines having low levels of MELK expression, there was almost no staining and hardly any positive signal was detected. [000114] On the other hand, when stained using the commercially available anti-MELK antibodies MELK(N449)pAb (Bioworld Technology) and MELK(HPA017214)pAb (Sigma-Aldrich), a signal was detected in a high proportion of cells in all cell lines tested, regardless of the level of endogenous MELK protein expression. Therefore, it was suspected that the result of detection using these commercially available antibodies may include false-positive signals. [000115] The above results showed that the anti-MELK antibody of the present invention (OTSMAb01) is a useful tool for obtaining detection results that correlate with the level of MELK protein expression in a sample by the immunohistochemical staining method, and it was elucidated that it has the advantageous characteristic of binding to MELK with high specificity compared to commercially available anti-MELK antibodies. [Example 3] Detection of MELK protein in clinical samples [000116] The specificity of the anti-MELK antibody of the present invention (OTSMAb01) in immunohistochemical staining was evaluated using clinical breast cancer samples. Fig. 4 shows the result of detecting MELK protein and RNA expression in tumor and non-tumor regions in clinical breast cancer samples 1 to 3, using immunohistochemical staining and semi-quantitative RT-PCR, respectively. As shown in Fig. 4, cells stained with OTSMAb01, i.e., MELK-positive cells, were Petition 870260046788, dated 05 / 18 / 2026, page 57 / 141 52 / 55 were detected in tumor regions where MELK RNA expression was verified, however, almost no staining was observed in non-tumor regions where MELK RNA expression was not verified, that is, MELK-positive cells were not detected. This result demonstrates that OTSMAb01 can specifically detect the MELK protein even in clinical samples. [Example 4] Analysis of the amino acid sequences of the variable regions of the anti-MELK monoclonal antibody [000117] The amino acid sequences of the variable regions of the anti-MELK antibody of the present invention (OTSMAb01) were analyzed. [000118] Total RNA was extracted from the OTSMAb01 hybridoma using the RNeasy mini kit (QIAGEN). Then, cDNA was synthesized from the total RNA using Super Script II Reverse Transcriptase (Invitrogen). The primers for cDNA synthesis were as follows: 3' heavy chain primer mIGCUniRv: 5'- CTGGGAAGGTGTGCACAC -3' (SEQ ID NO: 16) -Light chain initiator 3' mIGKRv2: 5'- GTTGTTCAAGAAGCACACGAC -3' (SEQ ID NO: 17) [000119] The dC polymer was added to the cDNA end using the 5' RACE System for Rapid Amplification of cDNA Ends (Invitrogen), and the polynucleotides encoding the variable regions of the monoclonal antibody were amplified using Platinum Taq DNA Polymerase High Fidelity (Invitrogen). The primers for amplification are as follows. The nucleotide I within the primer sequence indicates Inosine. 5' Heavy Chain 5' - and 5' Light Chain RACE Abridged Anchor Primer: 5'- GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG -3' (SEQ ID NO: 18); Petition 870260046788, dated 05 / 18 / 2026, p. 58 / 141 53 / 55 Heavy chain initiator 3' mIGCUniRv2: 5'- TGGACAGGGATCCAGAGTTCC -3' (SEQ ID NO: 19); and Light Chain Initiator 3' mIGKNesRv2: 5'- CAGATGTTAACTGCTCACTGGATGG -3' (SEQ ID NO: 20). [000120] The PCR products were cloned into the pGEM-T Easy Vector (Promega). The insertion fragment regions were sequenced to determine the nucleotide sequences of the variable regions (excluding signal sequences) of OTSMAb01. [000121] The amino acid sequences and nucleotide sequences of the variable region of the heavy chain and the variable region of the light chain of the mouse monoclonal antibody were determined as follows: OTSMAb01, Amino acid sequence of the variable region of the heavy chain (excluding the signal sequence): EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLE WIGWIDPENGNTIYDPKFQGKASVTADTSSNTAYLQLSSLTSEDTAVY YCTSHHYSAMDYWGQGTSVTVSS (SEQ ID NO:7) (encoded by the nucleotide sequence of SEQ ID NO: 10) OTSMAb01, Nucleotide sequence of the variable region of the heavy chain: 5'GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGG GGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAA GACTACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCT GGAGTGGATTGGATGGATTGATCCTGAGAATGGTAATACTATATAT GACCCGAAGTTCCAGGGCAAGGCCAGTGTAACAGCAGACACATC CTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGA CACTGCCGTCTATTACTGTACTAGTCACCATTACTCTGCTATGGAC TACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA -3' (SEQ ID NO: 10) Petition 870260046788, dated 05 / 18 / 2026, p. 59 / 141 54 / 55 OTSMAbOI, Amino acid sequence of the variable region of the light chain (excluding the signal sequence): DVVMTQTPLSLPVSLRDQASISCRSSQSLVHSNGNTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLIISRVEAEDLGVYFCS QSTYVPLTFGAGTKLELKRAD (SEQ ID NO: 8) (encoded by the nucleotide sequence of SEQ ID NO: 11) OTSMAb01, Nucleotide sequence of the variable region of the light chain: 5'GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTA GAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGCCTTGTAC ACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAG GCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTC TGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATT TCACACTCATAATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTT ATTTCTGCTCTCAAAGTACATATGTTCCGCTCACGTTCGGTGCTGG GACCAAGCTGGAGCTGAAACGGGCTGAT -3' (SEQ ID NO: 11) [000122] The CDR sequences of the antibody of the present invention (OTSMAb01), determined according to the definition of Kabat's principles are as follows: Heavy chain CDR1 (CDR-H1): DYYMH (SEQ ID NO: 1); Heavy chain CDR2 (CDR-H2): WIDPENGNTIYDPKFQG (SEQ ID NO: 2); Heavy chain CDR3 (CDR-H3): HHYSAMDY (SEQ ID NO: 3); Light chain CDR1 (CDR-L1): RSSQSLVHSNGNTYLH (SEQ ID NO: 4); CDR2 light chain (CDR-L2): KVSNRFS (SEQ ID NO: 5); and CDR3 light chain (CDR-L3): SQSTYVPLT (SEQ ID NO: 6). Industrial Applicability Petition 870260046788, dated 05 / 18 / 2026, pp. 60 / 141 55 / 55 [000123] The present invention has achieved the production of anti-MELK antibodies capable of detecting the MELK protein in isolated patient samples, such as clinical samples, with high specificity. Using the anti-MELK antibodies of the present invention, it is possible to detect the MELK protein in a sample with high sensitivity and low background. Consequently, the antibodies of the present invention are useful in the diagnosis of MELK-associated diseases, such as MELK-expressing cancer. Furthermore, since the antibodies of the present invention can detect MELK according to the level of MELK expression in the samples, it is also useful in examining patients for whom a MELK inhibitor has a high therapeutic effect (pre-treatment diagnosis) and in determining the efficacy of the drug after treatment with a MELK inhibitor in patients (post-treatment diagnosis).

Claims

CLAIMS 1. An antibody or antigen-binding fragment thereof, capable of binding to MELK protein or a partial peptide thereof comprising the amino acid sequence of SEQ ID NO: 9, characterized in that it comprises both of the following: a variable heavy chain region comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a variable light chain region comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

2. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that it comprises both a variable heavy chain region comprising the amino acid sequence SEQ ID NO: 7 and a variable light chain region comprising the amino acid sequence SEQ ID NO:

8.

3. Antibody or antigen-binding fragment thereof, according to claim 1 or 2, characterized in that it specifically recognizes the polypeptide consisting of the amino acid sequence SEQ ID NO:

9. Petition 870260046788, dated 05 / 18 / 2026, p. 62 / 141 2 / 3 4. Antibody or antigen-binding fragment thereof, according to any one of claims 1 to 3, characterized in that it is conjugated with an affinity label, enzymatic label, radioisotope label or fluorescent label.

5. Polynucleotide, characterized in that it encodes the antibody or the antigen-binding fragment thereof, as defined in claim 2, comprising the nucleotide sequences of SEQ ID NOs: 10 and 11.

6. Reagent, characterized in that it comprises the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 4, wherein the reagent is for use in the diagnosis of a disease associated with MELK.

7. Reagent according to claim 6, characterized in that the disease associated with MELK is a cancer in which MELK is expressed.

8. Reagent, according to claim 7, characterized in that said cancer is selected from the group consisting of breast cancer, bladder cancer, cervical cancer, cholangiocellular cancer, chronic myelocytic leukemia (CML), colorectal cancer, esophageal cancer, stomach cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer and small cell lung cancer (SCLC).

9. In vitro method for detecting MELK protein in a sample, characterized in that it comprises the steps of: (a) contacting an isolated sample from an individual with the antibody or antigen-binding fragment thereof, as defined in any of claims 1 to 4; and Petition 870260046788, dated 05 / 18 / 2026, p. 63 / 141 3 / 3 (b) detecting MELK protein in said sample by detecting the binding of said sample to said antibody or antigen-binding fragment thereof.

10. Method according to claim 9, characterized in that said sample is a cell or tissue isolated from said individual.

11. Method for producing an antibody that can bind to MELK protein, or to a partial peptide thereof, comprising the amino acid sequence of SEQ ID NO: 9, characterized in that it comprises the steps of: (a) culturing a cell comprising a vector inserted with the polynucleotide, as defined in claim 5; and (b) recovering said antibody from the cell culture or culture medium.

12. Use of an antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 4, characterized in that it is in the preparation of a reagent for diagnosing a disease associated with MELK.

Images