Methods for detecting and recovering melanoma cells derived from the yellow race, and reagents and carrier-immobilized molecules used in these methods

Abstract

To provide methods capable of detecting and / or recovering melanoma cells derived from the yellow-skinned races included in samples by discriminating them from contaminating cells, and provide reagents and carrier-immobilized molecules for use in those methods.SOLUTION: A problem is solved by methods for detecting melanoma cells derived from the yellow-skinned races contained in a sample, by detecting any gene expression in the sample as follows: (i) a gene for encoding polypeptide containing at least any amino acid sequence of 163 kinds containing MCAM(GenBank No.NP_006491.2); (ii) the gene for encoding the polypeptide containing at least the amino acid sequence having the homology of 70% or more with the amino acid sequence of the (i); and (iii) the gene for encoding the polypeptide containing at least the amino acid sequence where 1 or multiple amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of the (i).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting and recovering melanoma (malignant melanoma) cells contained in a sample, and a reagent for detecting melanoma cells, a reagent for recovering melanoma cells, and a carrier-immobilized molecule used therefor. In particular, the present invention relates to a method for detecting and recovering melanoma cells derived from yellow race among the melanoma cells, and a reagent for detecting melanoma cells derived from yellow race, a reagent for recovering melanoma cells, and a carrier-immobilized molecule used therefor. More specifically, the present invention relates to a method for detecting and recovering melanoma cells derived from yellow race contained in a sample by distinguishing them from contaminating cells contained in the same sample using a protein expressed by the melanoma cells derived from yellow race or a gene thereof, and a reagent and a carrier-immobilized molecule used in these methods. [Background technology]

[0002] When tumor cells leave the primary focus, they penetrate into blood vessels or lymphatic vessels, circulate in the blood or lymph, and finally invade other organs or tissues to form metastatic focus. Here, tumor cells circulating in the blood are also called CTCs (circulating tumor cells), and many clinical trials and studies have been conducted on them. For example, it is known that by measuring the number of CTCs contained in blood collected from a patient (Patent Document 1) and examining protein expression and gene mutations or translocations in the CTCs, information can be obtained on early detection of metastatic malignant tumors, prediction of the patient's condition after tumor detection or treatment, and prediction of recurrence. However, the number of CTCs contained in blood is very small, and blood contains a wide variety of impurity cells such as red blood cells and white blood cells, so that when detecting and analyzing CTCs, a technology is required to distinguish and collect / detect a small number of cells from a large number of cells.

[0003] Conventionally, proteins (tumor markers) expressed in CTCs, such as cytokeratin and EpCAM (Epithelial Cell Adhesion Molecule) (Patent Document 1), have been used to detect CTCs. However, cytokeratin and EpCAM are not expressed in melanoma (malignant melanoma) cells, and it is not possible to detect or recover melanoma-derived CTCs using these markers as indicators. Therefore, there is a demand for the development of a melanoma cell marker capable of detecting melanoma-derived CTCs. Furthermore, the distribution of melanoma disease types differs between Caucasians and Asians, with Caucasians tending to have superficial spreading types, whereas Asians tending to have acral lentigo types. Therefore, in order to detect melanoma-derived CTCs in Asians, such as Japanese, a marker different from the marker for Caucasian melanoma-derived CTCs may be required. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2008-533487 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide methods for detecting and / or recovering melanoma cells derived from yellow race contained in a sample while distinguishing them from contaminating cells, as well as reagents and carrier-immobilized molecules to be used in these methods. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors performed comparative expression analysis using a next-generation sequencer between two Japanese-derived melanoma cell lines as examples of melanoma cell lines derived from yellow race, three Caucasian-derived melanoma cell lines, and white blood cells, which are samples from healthy subjects and are an example of contaminating cells contained in blood samples, and as a result, found genes specifically expressed in melanoma cells derived from yellow race.Furthermore, they found that melanoma cells derived from yellow race contained in a sample can be detected and / or recovered by distinguishing them from contaminating cells using such genes as markers, thereby completing the present invention.

[0007] That is, the present invention is as follows.

[0008] [1] A method for detecting melanoma cells derived from the Asian race, comprising the step of detecting, in a sample, the expression of at least one gene selected from the group consisting of the following (i) to (iii): (i) a gene encoding a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; (ii) a gene encoding a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 163; (iii) A gene encoding a polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163.

[0009] [2] The method for detecting melanoma cells derived from the yellow race described in [1], characterized in that the detection of expression of the gene is the detection of a polypeptide encoded by the gene, and the detection of the polypeptide is detection using an antibody or an aptamer that specifically recognizes the polypeptide.

[0010] [3] The method for detecting melanoma cells derived from the yellow race described in [1] or [2], characterized in that the sample is a blood sample.

[0011] [4] A method for recovering melanoma cells derived from the yellow race, comprising the steps of: detecting or capturing at least one polypeptide selected from the group consisting of the following (iv) to (vi) in a sample, thereby detecting or capturing melanoma cells derived from the yellow race; and recovering the detected or captured melanoma cells derived from the yellow race: (iv) a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43; (vi) A polypeptide comprising at least an amino acid sequence having one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43.

[0012] [5] The method for recovering melanoma cells derived from the yellow race described in [4], characterized in that the detection or capture of the polypeptide is detection or capture using an antibody or aptamer that specifically recognizes the polypeptide.

[0013] [6] The method for recovering melanoma cells derived from the yellow race described in [4] or [5], characterized in that the sample is a blood sample.

[0014] [7] A reagent for detecting melanoma cells derived from the Asian race, comprising as an active ingredient a molecule that binds to the expression product of at least one gene selected from the group consisting of the following (i) to (iii): (i) a gene encoding a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; (ii) a gene encoding a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 163; (iii) A gene encoding a polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163.

[0015] [8] The reagent described in [7], characterized in that the molecule that binds to the expression product of the gene is an antibody or aptamer that specifically recognizes the polypeptide encoded by the gene.

[0016] [9] A reagent for detecting or capturing melanoma cells derived from the yellow race, comprising as an active ingredient a molecule that binds to at least one polypeptide selected from the group consisting of the following (iv) to (vi): (iv) a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43; (vi) A polypeptide comprising at least an amino acid sequence having one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43.

[0017]

[10] The reagent according to [9], characterized in that the molecule that binds to the polypeptide is an antibody or an aptamer that specifically recognizes the polypeptide.

[0018]

[11] A carrier-immobilized molecule for capturing melanoma cells derived from the yellow race, comprising a carrier and a molecule that binds to at least one polypeptide selected from the group consisting of the following (iv) to (vi) supported on the carrier: (iv) a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43; (vi) A polypeptide comprising at least an amino acid sequence having one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43.

[0019]

[12] The carrier-immobilized molecule according to

[11] , characterized in that the molecule that binds to the polypeptide is an antibody or an aptamer that specifically recognizes the polypeptide. Effect of the Invention

[0020] Effects of the Invention The present invention makes it possible to provide a method for detecting and recovering melanoma cells derived from yellow race, which can distinguish melanoma cells derived from yellow race in a sample from contaminating cells and detect and / or recover them, as well as a reagent and a carrier-immobilized molecule for use in these methods. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of MCAM gene expression analysis in leukocytes and melanoma cells derived from Japanese and Caucasians. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be described in detail below based on preferred embodiments thereof.

[0023] <Method for detecting and recovering melanoma cells derived from yellow race> The method for detecting melanoma cells derived from the yellow race of the present invention is a method comprising the step of detecting the expression of at least one gene selected from the group consisting of the following (i) to (iii) (hereinafter sometimes referred to as "yellow race-derived melanoma cell-specific genes") in a sample: (i) a gene encoding a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; (ii) a gene encoding a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 163; (iii) A gene encoding a polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163.

[0024] Furthermore, the method for recovering melanoma cells derived from the yellow race of the present invention comprises the steps of detecting or capturing at least one or more polypeptides selected from the group consisting of (iv) to (vi) below (hereinafter sometimes referred to as "yellow race-derived melanoma cell-specific transmembrane polypeptides") in a sample to detect or capture melanoma cells, and recovering the detected or captured melanoma cells derived from the yellow race: (iv) a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43; (vi) A polypeptide comprising at least an amino acid sequence having one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43.

[0025] According to the present invention, melanoma cells derived from yellow race contained in a sample can be detected using as an index the detection of the expression of a gene specific to melanoma cells derived from yellow race. Also, according to the present invention, melanoma cells derived from yellow race contained in a sample can be detected or captured and recovered using as an index the detection of a transmembrane polypeptide specific to melanoma cells derived from yellow race among the expression products of the gene specific to melanoma cells derived from yellow race.

[0026] The "sample" in the present invention is not particularly limited as long as it is a sample in which melanoma cells derived from the yellow race, which are the target of detection and recovery, may be present, and examples thereof include biological tissues (cells, tissues, organs, body fluids, whole blood, ascites, etc.) collected from the yellow race, such as Japanese people. The yellow race is not limited to cancer patients, but may also be healthy individuals. The sample may be the biological tissue itself; a dilution solution obtained by diluting whole blood, ascites, etc. with an appropriate buffer; a component derived from whole blood, such as serum, plasma, umbilical cord blood, or apheresis blood; or a suspension in which a piece of blood-containing tissue, such as liver, lung, spleen, kidney, tumor, or lymph node, is suspended in an appropriate buffer. Furthermore, the sample may be a fraction containing melanoma cells derived from the yellow race, which is obtained by separating and recovering these by centrifugation or the like. Among these, the sample according to the present invention is preferably a blood sample, such as whole blood, a dilution solution of whole blood, a component derived from whole blood (serum, plasma, umbilical cord blood, apheresis blood, etc.), and a suspension of tissue containing blood, as well as a fraction containing melanoma cells derived from the yellow race obtained from these.

[0027] According to the present invention, circulating tumor cells derived from melanoma originating from yellow race (i.e., melanoma cells originating from yellow race in the form of circulating tumor cells), which are present in only small amounts in a sample, can be detected or captured and recovered while being distinguished from contaminating cells. Here, "circulating tumor cells" refers to tumor cells that have infiltrated into blood vessels or lymphatic vessels from a primary tumor (primary focus) or a metastatic tumor (metastatic focus), and among these, circulating tumor cells circulating in the blood are referred to as "circulating tumor cells in blood (CTCs)". Circulating tumor cells in blood exist, for example, as tumor cells circulating in the peripheral bloodstream of a melanoma patient.

[0028] In the present invention, the "contaminating cells" contained in a sample refer to cells other than melanoma cells. When the sample is a blood sample, examples of the contaminating cells include red blood cells, white blood cells, and platelets. Among them, the method for detecting and recovering melanoma cells derived from yellow race of the present invention is a method excellent for distinguishing melanoma cells from white blood cells contained in a blood sample, for example.

[0029] In the present invention, the term "transmembrane polypeptide" refers to a polypeptide that includes at least one transmembrane domain. In the present invention, by targeting the transmembrane polypeptide among melanoma cell markers, melanoma cells derived from yellow race that express the transmembrane polypeptide on the cell membrane can be detected, and the cells can be captured and recovered via the transmembrane polypeptide.

[0030] In the present invention, the gene to be detected for expression (hereinafter sometimes referred to as "target gene") is typically (i) a gene encoding a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; However, in the present invention, a homologue of the gene shown in (i) above may be targeted (detected). In addition, since the DNA sequence of a gene may be mutated in nature (i.e., non-artificially) due to mutation or the like, in the present invention, such a natural mutant may be targeted.

[0031] The amino acid sequence of the homologue is (ii) an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; More preferably, the amino acid sequence may have a homology of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the entire amino acid sequence. The homology of the sequence can be determined, for example, by using the BLASTP program (Altschul et al., J. Mol. Biol., 215:403-410 (1990)).

[0032] Furthermore, the amino acid sequence of the mutant is (iii) an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in any of the amino acid sequences set forth in SEQ ID NOs: 1 to 163; Here, "multiple" means an integer of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. One or multiple amino acid residues preferably means 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, even more preferably 1 to 3 amino acid residues, and particularly preferably 2 or less.

[0033] In the present invention, the melanoma cell-specific gene derived from a yellow race has higher specificity for melanoma cells derived from a yellow race than for contaminating cells. Here, "high specificity" means that the melanoma cells derived from a yellow race have higher expression at the transcriptional or translational level than at least one contaminating cell (e.g., leukocytes of a healthy individual). Preferably, the melanoma cells derived from a yellow race have a melanoma cell-specific gene expression 1.5 times or more higher than that of the contaminating cells, more preferably 2 times or more higher, even more preferably 3 times or more higher, and particularly preferably 5 times or more higher (e.g., 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more).

[0034] Furthermore, the yellow race-derived melanoma cell-specific gene has higher specificity for yellow race-derived melanoma cells than for white race-derived melanoma cells. Here, "high specificity" means that the yellow race-derived melanoma cells have higher expression at the transcription or translation level than white race-derived melanoma cells. Preferably, the yellow race-derived melanoma cells have 1.5-fold or more higher expression of the yellow race-derived melanoma cell-specific gene than white race-derived melanoma cells. More preferably, the yellow race-derived melanoma cells have 2-fold or more higher expression, even more preferably, 3-fold or more higher expression, and particularly preferably, 5-fold or more higher (e.g., 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more higher) than white race-derived melanoma cells.

[0035] Tables 1 to 8 below show, for the amino acid sequences of SEQ ID NOs: 1 to 163, the SEQ ID NOs of each amino acid sequence, the name of the protein corresponding to the polypeptide consisting of each amino acid sequence, the GenBank accession number (GenBank No.) of each amino acid sequence, the SEQ ID NOs of the genes encoding the polypeptide consisting of each amino acid sequence and their open reading frame positions (ORF positions), the GenBank accession numbers (GenBank No.) of each gene, and the results of gene expression analysis obtained in the Examples below. Tables 9 to 23 below also show the formal name (full name) and aliases for each protein name listed in Tables 1 to 8, respectively.

[0036] [Table 1]

[0037] [Table 2]

[0038] [Table 3]

[0039] [Table 4]

[0040] [Table 5]

[0041] [Table 6]

[0042] [Table 7]

[0043]

Table 8

[0044]

Table 9

[0045]

Table 10

[0046]

Table 11

[0047]

Table 12

[0048]

Table 13

[0049]

Table 14

[0050]

Table 15

[0051]

Table 16

[0052]

Table 17

[0053]

Table 18

[0054] [Table 19]

[0055] [Table 20]

[0056] [Table 21]

[0057] [Table 22]

[0058] [Table 23]

[0059] In the present invention, "detecting gene expression" includes both detection of the presence or absence of gene expression and detection of the degree of expression, and in the present invention, the expression of the target gene is detected. The expression amount of a gene can be grasped as an absolute amount or a relative amount. When grasping the relative amount, for example, it can be judged by comparing with the expression amount of a gene of a prepared standard sample. A "standard sample" is a sample in which the presence or absence of expression of a target gene or the degree of expression is specified in advance, and for example, red blood cells, white blood cells, and platelets, more preferably white blood cells, can be used as the standard sample. In addition, tissue (normal tissue) of an Asian person not affected with melanoma and melanoma tissue of a Caucasian person can also be used as the standard sample.

[0060] In the present invention, "gene expression" includes both gene transcription and translation. Therefore, "detection of gene expression" in the present invention includes detection at the transcription level (mRNA level) and detection at the translation level (protein level) (i.e., detection of the polypeptide encoded by the gene).

[0061] In eukaryotic cells, during the transcription process of a gene, a reaction (splicing) occurs in which introns are removed from the mRNA precursor and the preceding and following exons are recombined, but there may be diversity in the recombination of exons. This results in the production of various mature mRNAs, which in turn result in the translation of various proteins. Such diverse mRNAs and proteins produced by differences in splicing are called "splicing variants." Therefore, the detection of gene expression in the present invention includes the detection of the splicing variant, so long as it is specifically recognized by a molecule that binds to the expression product of the target gene described below.

[0062] In the present invention, the expression of a gene can be detected by a known method or a method similar thereto, as appropriate. For detection at the transcription level, for example, a probe corresponding to an appropriate position in the base sequence (polynucleotide) of the transcription product (mRNA) of the target gene may be designed, and then detection may be performed by Northern blotting, dot blot, RNase protection assay, DNA microarray analysis, in situ hybridization, etc., or a primer set corresponding to an appropriate position in the polynucleotide may be designed, and then the polynucleotide may be amplified and detected using, for example, PCR, RT-PCR, TRC (Transcription Reverse Transcription Concerted) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription-Mediated Amplification) method, etc., or a sample containing the polynucleotide may be directly subjected to a sequencer for detection.

[0063] Examples of detection at the translation level include detection methods using antibodies (immunological techniques), such as immune cell staining, imaging cytometry, flow cytometry, ELISA (Enzyme-Linked Immuno Sorbent Assay), radioimmunoassay, immunoprecipitation, immunoblotting (Western blotting, etc.), antibody arrays, and in vivo imaging; and detection methods using aptamers instead of the antibodies. The immunological techniques may be performed automatically using an enzyme immunoassay device such as AIA-900 (Tosoh) or a chemiluminescent enzyme immunoassay device such as AIA-CL2400 (Tosoh) after preparing the necessary reagents.

[0064] In the present invention, from the standpoint of simplicity, detection of the expression of the gene is preferably at the translation level (detection of the polypeptide encoded by the target gene), and in particular, a method of detecting the polypeptide using an antibody that specifically recognizes the polypeptide (hereinafter sometimes referred to as an "anti-polypeptide antibody") or an aptamer that specifically recognizes the polypeptide (hereinafter sometimes simply referred to as an "aptamer") is preferred, and a method of detecting the polypeptide using the anti-polypeptide antibody is more preferred.

[0065] In the present invention, the "antibody" may be a polyclonal antibody or a monoclonal antibody, or may be a functional fragment of an antibody. Furthermore, the "antibody" includes all classes and subclasses of immunoglobulin. The "functional fragment" of an antibody means a part (partial fragment) of an antibody, which in the present invention specifically recognizes a polypeptide encoded by the target gene. Specific examples include Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide-linked Fv, single chain Fv (scFv), sc(Fv)2, diabody, multispecific antibody, and polymers thereof.

[0066] The anti-polypeptide antibody according to the present invention can be obtained by immunizing an animal with an antigen (a polypeptide encoded by a target gene, a partial peptide thereof, or a cell expressing these, etc.) and purifying the antiserum by conventional means (salting out, centrifugation, dialysis, column chromatography, etc.) if it is a polyclonal antibody. Also, a monoclonal antibody can be produced by the hybridoma method or recombinant DNA method. An example of the hybridoma method is the method of Kohler and Milstein (Kohler & Milstein, Nature, 256:495-497 (1975)). An example of the recombinant DNA method is a method in which DNA encoding the anti-polypeptide antibody is cloned from a hybridoma or B cell, etc., incorporated into an appropriate vector, and then introduced into a host cell (such as a mammalian cell line, E. coli, yeast cell, insect cell, plant cell, etc.) to produce the anti-polypeptide antibody as a recombinant antibody (for example, PJ Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 192:767-775 (1990)).

[0067] In the present invention, the molecules that bind to the expression product of the target gene, such as the anti-polypeptide antibody and aptamer, can be those bound to a labeling substance. The amount of the molecule bound to the expression product encoded by the target gene can be directly measured by using the molecule bound to a labeling substance and detecting the labeling substance. The labeling substance is not particularly limited as long as it can bind to the molecule and can be detected by a chemical or optical method, and examples of the labeling substance include fluorescent substances such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), and rhodamine isothiocyanate (RITC), enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), and alkaline phosphatase, and radioactive substances.

[0068] In the present invention, the detection of the expression of the gene is the detection of the polypeptide encoded by the gene, and when the anti-polypeptide antibody is used, an indirect detection method such as a method using a secondary antibody bound to the labeling substance or a method using a polymer bound to the secondary antibody and the labeling substance can also be used. Here, the "secondary antibody" is an antibody that exhibits specific binding to the anti-polypeptide antibody. For example, when the anti-polypeptide antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used for antibodies derived from various species such as rabbits, goats, and mice are commercially available, and an appropriate secondary antibody can be selected and used depending on the species from which the anti-polypeptide antibody is derived. In place of the secondary antibody, protein G derived from Streptococcus, protein A derived from Staphylococcus aureus, protein L derived from anaerobic positive streptococci, or the like, to which a labeling substance is bound, may be used.

[0069] In the present invention, "detection of melanoma cells derived from yellow race" means both detection of the presence or absence (qualitative detection) of melanoma cells derived from yellow race in a sample, and detection of the extent of the amount of melanoma cells derived from yellow race (quantitative detection), using the expression of the gene specific to melanoma cells derived from yellow race as an indicator, and also includes detection of whether cells collected from a sample are melanoma cells derived from yellow race.

[0070] The criteria for the detection of melanoma cells derived from the yellow race can be appropriately set according to the purpose, and are not particularly limited. For example, if there is even a small amount of detection signal corresponding to the number of melanoma cells derived from the yellow race or the expression level of the target gene, it may be determined that melanoma cells derived from the yellow race are present in the sample, or if there is a detection signal of a certain threshold or more, the determination may be made. The amount of melanoma cells derived from the yellow race may be detected according to the intensity of the detection signal. Furthermore, when cells are collected from the sample and then detected, it may be determined that the collected cells are melanoma cells derived from the yellow race if there is even a small amount of detection signal, or if there is a detection signal of a certain threshold or more, or if there is a signal of a certain value or more, or 2SD or more, 3SD or more, or 4SD or more of the average value of the signal, relative to the signal possessed by the contaminating cells collected from the sample (for example, red blood cells, white blood cells, etc. in the case of the blood sample).

[0071] Among the above Tables 1 to 8, the amino acid sequences shown in SEQ ID NOs: 1 to 43 in Tables 1 and 2 are the amino acid sequences of melanoma cell-specific transmembrane polypeptides derived from yellow race identified by the present inventors. In the method for recovering melanoma cells derived from yellow race of the present invention, (iv) a polypeptide comprising at least an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) a polypeptide comprising at least an amino acid sequence having 70% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43 (i.e., a homolog of (iv)), and (vi) a polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43 (i.e., a mutant of (iv)); and recovering the detected or captured melanoma cells derived from the yellow race.

[0072] In the method for recovering melanoma cells derived from the yellow race of the present invention, the step of detecting melanoma cells derived from the yellow race (detection step) is as described above. Examples of the method for recovering the detected melanoma cells derived from the yellow race include a method using a cell sorter and a method for recovering the cells using a pipette under a microscope (e.g., a micro-pick system or a recovery device disclosed in JP 2016-142616 A).

[0073] In addition, in the step of capturing melanoma cells derived from yellow race (capturing step), a carrier-immobilized molecule for capturing melanoma cells derived from yellow race, which comprises a carrier and a molecule that binds to the melanoma cell-specific transmembrane polypeptide derived from yellow race carried on the carrier, can be used. In this case, the recovery method of the present invention is preferably a method including a capturing step of contacting a carrier-immobilized molecule with a sample and capturing melanoma cells with the carrier-immobilized molecule, and a recovery step of removing impurities such as impurity cells that are not bound to the carrier-immobilized molecule and recovering the melanoma cells captured by the carrier-immobilized molecule.

[0074] The recovery step may further include a washing step of removing impurities that are not bound to the carrier-immobilized molecules by injecting and removing a washing solution, and a release step of liberating the melanoma cells from the carrier-immobilized molecules to obtain free melanoma cells.

[0075] Examples of the carrier constituting the carrier-immobilized molecule include a plate, a fiber, a membrane, a particle, and a microchannel chip. In the carrier-immobilized molecule, the molecule that binds to the yellow race-derived melanoma cell-specific transmembrane polypeptide is preferably an antibody or an aptamer that binds to the yellow race-derived melanoma cell-specific transmembrane polypeptide. In addition, the carrier or the molecule that binds to the yellow race-derived melanoma cell-specific transmembrane polypeptide may be labeled with the labeling substance described above. As such a carrier-immobilized molecule, a conventionally known molecule may be appropriately used, or a commercially available molecule may be appropriately used.

[0076] The method for recovering melanoma cells derived from the yellow race captured by the carrier-immobilized molecule is not particularly limited, and a conventionally known method or a method based thereon can be appropriately adopted. For example, when the carrier is a plate, the liquid phase (supernatant) may be removed from the plate after the above-mentioned capture step, and when the carrier is a particle, the particles may be recovered by centrifugation or magnetic collection after the above-mentioned capture step, and the liquid phase (supernatant) may be removed. A suitable example of the recovery means is a means including a substrate provided with a holding part capable of holding melanoma cells and a recovery part that recovers the melanoma cells by suction and discharge using a nozzle, and a specific example is the recovery device disclosed in JP 2016-142616 A.

[0077] The melanoma cells derived from yellow race obtained by the method of the present invention for recovering melanoma cells derived from yellow race can be counted and used in various analyses.

[0078] Furthermore, information obtained by carrying out the method for detecting and recovering melanoma cells derived from the yellow race of the present invention can be used, for example, to evaluate or understand the pathology of a yellow race suffering from melanoma, and to evaluate the effectiveness of treatment. For example, by carrying out the method of the present invention in parallel with the treatment of a yellow race suffering from melanoma, recurrence can be predicted and the effectiveness of treatment can be evaluated based on the information obtained as a result. Specifically, by carrying out the method for detecting and recovering melanoma cells derived from the yellow race of the present invention after drug administration, changes in the expression of genes specific to yellow race-derived melanoma cells in a patient can be examined, and the effectiveness of treatment can be determined from the increase / decrease and progression of the expression. In this way, the method of the present invention may be used to predict recurrence and monitor the effectiveness of treatment.

[0079] <Reagents for detecting melanoma cells derived from yellow race, and carrier-immobilized molecules for capturing them> The reagent for detecting melanoma cells derived from the yellow race of the present invention (hereinafter sometimes referred to as "detection reagent") is a detection reagent containing, as an active ingredient, a molecule that binds to the expression product of a gene specific to melanoma cells derived from the yellow race. By carrying out the detection method for melanoma cells derived from the yellow race of the present invention using the reagent, it is possible to detect melanoma cells derived from the yellow race in a sample.

[0080] In the present invention, "expression product of at least one gene selected from the group consisting of (i) to (iii)" refers to a transcription product (mRNA) or translation product (protein) of a gene specific to melanoma cells derived from yellow race (target gene). Examples of the "molecule that binds to the transcription product" of the target gene include a primer used in the above-mentioned RT-PCR method and a probe used in Northern blotting. The molecule is a polynucleotide containing a base sequence complementary to the base sequence of the transcription product of the target gene. Preferably, the polynucleotide contains a base sequence of at least 15 consecutive bases, more preferably 20 bases, complementary to the base sequence of the transcription product of the target gene. Here, "complementary" does not need to be 100% complementary as long as specific hybridization with the transcription product occurs. Preferably, the complementary has a complementarity of 90% or more, more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Furthermore, examples of the "molecule that binds to the translation product" of the target gene include the anti-polypeptide antibody and the aptamer. In the present invention, the molecule that binds to the expression product of the target gene is preferably the anti-polypeptide antibody and the aptamer, and more preferably the anti-polypeptide antibody.

[0081] The detection reagent of the present invention may contain, in addition to the molecule that binds to the expression product of the target gene, other components such as a buffer solution, physiological saline; fluorescent substances such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), etc.; secondary antibodies that recognize and bind to the molecule that binds to the expression product of the target gene; enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, etc. modified on the secondary antibody, and their substrates.

[0082] When the expression product of the yellow-race-derived melanoma cell-specific gene is a yellow-race-derived melanoma cell-specific transmembrane polypeptide, a molecule that binds to the polypeptide targets yellow-race-derived melanoma cells on whose cell surface the polypeptide is present, and can capture cells expressing the polypeptide as a membrane protein, i.e., yellow-race-derived melanoma cells, through the binding between the molecule and the polypeptide. Thus, among the detection reagents of the present invention, a reagent for detecting yellow-race-derived melanoma cells, which contains as an active ingredient a molecule that binds to the yellow-race-derived melanoma cell-specific transmembrane polypeptide (specifically, a molecule that binds to at least one or more polypeptides selected from the group consisting of the above (iv) to (vi)), can also be used as a reagent for capturing yellow-race-derived melanoma cells (hereinafter referred to as a "capture reagent").

[0083] Furthermore, examples of the "molecule that binds to a transmembrane polypeptide specific to yellow race-derived melanoma cells" include the above-mentioned anti-polypeptide antibody and aptamer, with the above-mentioned anti-polypeptide antibody being preferred.

[0084] The capture reagent of the present invention, like the detection reagent, may contain, in addition to the active ingredient, a molecule that binds to the yellow-derived melanoma-specific transmembrane polypeptide, other components such as a buffer solution, physiological saline; fluorescent substances such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), etc.; secondary antibodies that recognize and bind to molecules that bind to the expression products of the target genes; enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, etc. modified on the secondary antibodies, and their substrates.

[0085] In capturing melanoma cells derived from the yellow race, it is convenient to use a solid-phase molecule that binds to a melanoma cell-specific transmembrane polypeptide derived from the yellow race. Therefore, the present invention also provides a carrier-immobilized molecule for capturing melanoma cells derived from the yellow race (hereinafter sometimes referred to as a "capture carrier"), which comprises a carrier and a molecule that binds to a melanoma cell-specific transmembrane polypeptide derived from the yellow race carried on the carrier. The carrier-immobilized molecule is as described above.

[0086] The detection reagent, capture reagent, and capture carrier of the present invention can be combined with other preparations and instructions to prepare a kit. Examples of other preparations include control reagents, sample dilutions, and washing solutions. When combined with a labeled preparation, a molecule required for detecting the label (for example, a substrate for the enzyme when the label is an enzyme) can be included. EXAMPLES

[0087] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0088] Example 1: Identification of genes specifically expressed in melanoma cells derived from Asians Two melanoma cell lines derived from Asians, MEL-2 and MEL-18, which are Japanese-derived melanoma cell lines, were selected as melanoma cell lines derived from Caucasians, and three melanoma cell lines, 501mel, 888mel, and 928mel, were selected as melanoma cell lines derived from Asians. Differences in gene expression levels between these melanoma cell lines derived from Asians and Caucasians and between them and leukocytes from healthy subjects were analyzed using a next-generation sequencer by the following method.

[0089] (1) Melanoma cell lines were cultured in RPMI-1640 medium containing 10% (v / v) FBS (fetal bovine serum) at 37°C in a 5% CO2 environment, and then the melanoma cells were harvested by detaching the cells from the medium using 0.25% (w / v) trypsin / 1 mM EDTA (n=3).

[0090] (2) Total RNA was extracted from the melanoma cells obtained in (1) above using RNeasy Mini Kit (QIAGEN), and cDNA was synthesized and amplified from 10 ng of total RNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). Total RNA was also extracted from leukocytes collected from the blood of four healthy subjects in the same manner, and cDNA was synthesized and amplified from 10 ng of total RNA.

[0091] (3) Using 1 ng of the cDNA obtained in (2) above, a library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina) and the Nextera XT v2 Index Kit Set A (Illumina). Sequence analysis was then performed using Next-seq500 (Illumina) under the conditions of "read length 75 bp, single end read," and more than 10 million reads were decoded per sample.

[0092] (4) The nucleotide sequence (sequence data) decoded in (3) above was mapped to the human genome sequence using TopHat2 (Johns Hopkins University) and Bowtie2 (Johns Hopkins University). The human genome sequence and human gene information were obtained using BUILD GRCh38, which is published by NCBI (National Center for Biological Information). The mapped nucleotide sequence was used to calculate the expression value of each gene in units of FPKM (Fragments Per Kilobase of exon per Million reads mapped) from the number of reads of each gene decoded using Cufflinks (Washington University).

[0093] (5) Expression comparison was performed for 4 samples of 4 healthy white blood cells, 6 samples of 2 types of melanoma cell lines derived from Japanese people (yellow race), and 9 samples of 3 types of melanoma cell lines derived from Caucasians. Tables 1 and 2 show genes that have an average expression value (FPKM value) in Japanese melanoma cell lines (2 types, 6 samples) that is 10.00 times or more the average expression value (FPKM value) in healthy white blood cells (4 samples), and an average expression value (FPKM value) in Caucasian melanoma cell lines (3 types, 9 samples) that is less than 10.00 times the average expression value (FPKM value) in healthy white blood cells (4 samples), and that code for transmembrane proteins. Among the above genes, the analysis results for the gene encoding MCAM (SEQ ID NO: 1) for each sample are shown in FIG. 1.

[0094] In Figure 1, WBC1 to WBC4 are the results of expression analysis of white blood cells, 501mel1 to 501mel3 are the results of expression analysis of 501mel cells, 888mel1 to 888mel3 are the results of expression analysis of 888mel cells, 928mel1 to 928mel3 are the results of expression analysis of 928mel cells, MEL-2 1 to MEL-2 3 are the results of expression analysis of MEL-2 cells, and MEL-18 1 to MEL-18 3 are the results of expression analysis of MEL-18 cells.

[0095] The average expression value (FPKM value) of the MCAM gene (sequence number 1) in melanoma cells derived from Caucasians (three types, nine samples) was approximately three times the average FPKM value of white blood cells (four samples), whereas the average FPKM value in melanoma cells derived from Asian Japanese people (two types, six samples) was more than 200 times the average FPKM value of white blood cells (four samples), indicating that the MCAM gene is specifically and highly expressed in melanoma cells derived from Japanese people (Table 1).

[0096] Tables 3 to 8 show genes whose average expression value (FPKM value) in Japanese melanoma cells (2 types, 6 samples) is 10.00 times or more higher than the average expression value (FPKM value) in white blood cells (4 samples) and whose average expression value (FPKM value) in Caucasian melanoma cells (3 types, 9 samples) is less than 10.00 times higher than the average expression value (FPKM value) in white blood cells (4 samples), and which code for proteins other than transmembrane proteins. [Industrial Applicability]

[0097] As described above, the present invention makes it possible to provide methods for detecting and / or recovering melanoma cells derived from a melanoma cell line derived from an yellow race in a sample, while distinguishing them from contaminating cells, as well as reagents and carrier-immobilized molecules for use in these methods.

Claims

1. A method for detecting melanoma cells derived from Japanese people, comprising the step of detecting, in a sample, the expression of at least one gene selected from the group consisting of (i) to (ii) below: (i) a gene encoding a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; (ii) A gene encoding a polypeptide consisting of an amino acid sequence having 90% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 163.

2. The method for detecting melanoma cells derived from Japanese people as described in claim 1, characterized in that the detection of expression of the gene is the detection of a polypeptide encoded by the gene, and the detection of the polypeptide is detection using an antibody or aptamer that specifically recognizes the polypeptide.

3. 3. The method for detecting melanoma cells derived from Japanese people according to claim 1 or 2, wherein the sample is a blood sample.

4. A step of detecting or capturing Japanese-origin melanoma cells by detecting or capturing at least one or more polypeptides selected from the group consisting of the following (iv) to (v) in a sample, and a step of recovering the detected or captured Japanese-origin melanoma cells: A method for recovering melanoma cells derived from Japanese people, comprising: (iv) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) A polypeptide consisting of an amino acid sequence having 90% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43.

5. The method for recovering melanoma cells derived from Japanese people described in claim 4, characterized in that the detection or capture of the polypeptide is detection or capture using an antibody or aptamer that specifically recognizes the polypeptide.

6. 6. The method for recovering melanoma cells derived from Japanese subjects according to claim 4 or 5, wherein the sample is a blood sample.

7. A reagent for detecting melanoma cells derived from Japanese people, comprising as an active ingredient a molecule that binds to the expression product of at least one gene selected from the group consisting of (i) to (ii) below: (i) a gene encoding a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 163; (ii) A gene encoding a polypeptide consisting of an amino acid sequence having 90% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 163.

8. The reagent according to claim 7 , wherein the molecule that binds to the expression product of the gene is an antibody or an aptamer that specifically recognizes the polypeptide encoded by the gene.

9. A reagent for detecting or capturing melanoma cells derived from Japanese people, comprising as an active ingredient a molecule that binds to at least one or more polypeptides selected from the group consisting of (iv) to (v) below: (iv) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) A polypeptide consisting of an amino acid sequence having 90% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43.

10. The reagent according to claim 9 , wherein the molecule that binds to the polypeptide is an antibody or an aptamer that specifically recognizes the polypeptide.

11. A carrier-immobilized molecule for capturing melanoma cells derived from Japanese people, comprising a carrier and a molecule that binds to at least one or more polypeptides selected from the group consisting of (iv) to (v) below, which are supported on the carrier: (iv) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 43; (v) A polypeptide consisting of an amino acid sequence having 90% or more homology to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 43.

12. The carrier-immobilized molecule according to claim 11, wherein the molecule that binds to the polypeptide is an antibody or an aptamer that specifically recognizes the polypeptide.

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