JP7272627B2 - Biomarkers for evaluation of ovarian tumors - Google Patents

Description

The present invention relates to biomarkers for assessing ovarian tumors. More particularly, it relates to a biomarker for assessing the presence and malignancy of ovarian tumors consisting of C-mannosylated tryptophan, which biomarker is used to aid in the assessment of the presence and malignancy of ovarian tumors. related to methods, etc.

Ovarian cancer has the poorest prognosis among gynecologic malignant tumors, and both the morbidity and mortality rates are steadily increasing. In 2010, approximately 160,000 people worldwide died from ovarian cancer. According to statistical data in 2014, the cumulative risk of contracting all cancers in women is approximately 46% (1 in 2 women will be diagnosed with cancer), of which ovarian cancer is approximately 1% (87%). 1 out of 10 people will get cancer). In addition, the cumulative mortality risk for women is about 16% for all cancer types (1 in 6 women die from cancer), while ovarian cancer is about 0.5% (1 in 188 women die from cancer). death). Thus, ovarian cancer is classified as a cancer with a poor prognosis after being diagnosed, and the importance of early detection is recognized. Early ovarian cancer often has few subjective symptoms, and even when the tumor is small, it is likely to metastasize to distant or lymph nodes, which is one of the reasons for the poor prognosis of ovarian cancer.

Therefore, establishment of an effective early diagnosis marker is required as a therapeutic strategy for ovarian cancer. At present, benign or malignant tumors are determined by diagnostic imaging and blood tumor marker measurement after being detected by pelvic examination and echo examination. CA125 is a typical ovarian cancer test marker that is used, but it is said to have a detection sensitivity of about 50% and a specificity of about 70% in the early stage of ovarian cancer, and the marker accuracy is sufficient. hard to say. In particular, serum CA125 is used to help predict the development of ovarian cancer in patients with pelvic masses, but many benign gynecologic and non-gynecologic conditions also raise CA125, CA125 shows a normal value in about half of early stage ovarian cancers (eg, Non-Patent Documents 1 to 4).

Einhorn et al., Hematol Oncol Clin North Am, 6(4):843-850 (1992) Campbell et al., Br J Obstet Gynaecol., 97(4):304-11 (1990) Jacobs et al., BMJ, 306(6884):1030-1034 (1993) DePriest et al., Gynecol Oncol, 51(2):205-209 (1993)

The present invention provides a biomarker that can accurately detect and evaluate the presence of ovarian tumors, and a method for evaluating the presence of ovarian tumors using the biomarkers, and the malignancy of the tumor if present. is the subject.

C-mannosylated tryptophan in which mannose is carbon-carbon bonded (C-C bond) to the C2 atom of tryptophan (hereinafter also referred to as C2-α-C-mannosylpyranosyl-L-tryptophan or C-Man-Trp) was first reported by Horiuchi et al. in 1994 (J. Biochem, 115:362-366 (1994)) to be contained in urine. It has been reported to correlate with renal function (Takahira et al., Am. J. Med., 110:192-197 (2001)). In the 2010s, C-Man-Trp was once again reported as a metabolite correlated with renal function, aging, lung function, etc. through serum metabolome analysis by multiple overseas groups (e.g., Menni et al., Int J Epidemiol, 42:1111-1119 (2013), Niewczas et al., Kidney Int., 85:12214-12224, Solini et al., J. Clin Edocrinol Metab, 101(2):696 -704 (2016)).

In order to further study the relationship between C-Man-Trp and renal function, etc., the present inventors first proceeded with the development of a method capable of isolating native C-Man-Trp at a high yield. In the above report, extraction with strong acid was used to extract C-Man-Trp from the sample, but preliminary experiments by the present inventors confirmed that C-Man-Trp is denatured by strong acid. Therefore, there was concern that strong acid extraction might affect the quantification of C-Man-Trp. Therefore, in order to prevent the denaturation of C-Man-Trp, we came up with the idea of using an organic solvent instead of a strong acid solvent. We found that denatured C-Man-Trp can be isolated with higher yield. Moreover, C-Man-Trp isolated by this method can be used not only by expensive liquid chromatography/mass spectrometer (LC-MS/MS) systems, but also by inexpensive high-speed chromatography ( HPLC) or ultra high performance chromatography (UPLC). By using HPLC or UPLC, simple and inexpensive measurement of C-Man-Trp concentration becomes possible. When C-Man-Trp was extracted from various tissues of mice by the above method and its concentration was measured, it was unexpectedly found that C-Man-Trp was present at high concentrations in the ovary and uterus. From this finding, the present inventors got the idea that ovarian diseases such as ovarian tumors and C-Man-Trp concentration may be related, and as a result of further research, ovarian cancer patients In, the concentration of C-Man-Trp in the blood is significantly higher than in healthy subjects, and in the non-malignant ovarian tumor (i.e., benign ovarian tumor and borderline malignant ovarian tumor) patient group and the malignant ovarian tumor patient group also found that the concentration of C-Man-Trp in blood was significantly different. And by using C-Man-Trp as a biomarker, it is possible to detect the presence of ovarian tumors more accurately than when using known ovarian tumor markers, and if the tumor exists, its malignancy can be determined. The inventors have found that ovarian tumors can be detected with higher accuracy by using them in combination with other ovarian tumor markers, and have completed the present invention.

That is, the present invention is as follows.
[1] A biomarker for assessing the presence and grade of ovarian tumors consisting of C-mannosylated tryptophan.
[2] Whether or not an ovarian tumor exists in the test animal, or the malignancy of the existing ovarian tumor, including the step of detecting or quantifying the biomarker of [1] in a sample derived from the test animal method to assist in the evaluation of
[3] A method for analyzing the probability of presence of an ovarian tumor or malignant ovarian tumor in a test animal, comprising the step of detecting or quantifying the biomarker of [1] in a sample derived from the test animal.
[4] The method according to [2] or [3], which includes the following steps (i) to (iii).
(i) a step of quantifying the biomarker according to [1] in a sample derived from a test animal;
(ii) comparing the amount of the biomarker quantified in (i) with a reference value; and (iii) based on the results of (ii), if the amount of the biomarker quantified in (i) is greater than or equal to the reference value. and assessing the presence of an ovarian tumor in the subject animal or the presence of a malignant ovarian tumor in the subject animal [5] Furthermore, in a sample derived from the subject animal, at least one other ovarian tumor marker The method of [4], comprising the step of quantifying the amount of
[6] The method of [5], wherein the other ovarian tumor marker is selected from the group consisting of CA125, CEA and CA19-9.
[7] The method according to any one of [2] to [6], wherein the sample is plasma.
[8] The method according to any one of [2] to [7], wherein the subject animal is human.
[9] The method of [2] to [8], wherein the biomarker of [1] is extracted from the sample using an organic solvent.
[10] A test kit for evaluating the presence and malignancy of an ovarian tumor, comprising an antibody that specifically recognizes C-mannosylated tryptophan.
[11] The kit of [10], further comprising an antibody that specifically recognizes another ovarian tumor marker.
[12] A method for treating an ovarian tumor, comprising the following steps (i) to (iv).
(i) a step of quantifying the biomarker according to [1] in a sample derived from a test animal;
(ii) comparing the amount of biomarker quantified in (i) to a reference value;
(iii) Based on the results of (ii), if the amount of the biomarker quantified in (i) is equal to or higher than the reference value, the subject animal has an ovarian tumor, or the subject animal has a malignant ovarian tumor and (iv) administering a therapeutic agent for ovarian tumor to the test animal evaluated to have an ovarian tumor based on the result of (iii) [13] said therapeutic agent for ovarian tumor is selected from the group consisting of paclitaxel, carboplatin, bevacizumab, olapanib, nivolumab, pembrolizumab, avelumab, atezolizumab, cisplatin, etoposide, and bleomycin.
[14] The method of [12] or [13], further comprising the step of quantifying the amount of at least one other ovarian tumor marker in the sample derived from the subject animal.
[15] The method of [14], wherein the other ovarian tumor marker is selected from the group consisting of CA125, CEA and CA19-9.

C-mannosylated tryptophan, optionally in combination with other ovarian tumor markers, can detect the presence of ovarian tumors with higher accuracy than known ovarian tumor markers, and if the tumor is present, its It can be a biomarker that can evaluate the degree of malignancy.

FIG. 1 shows a schematic diagram of the method for extracting, separating, detecting and quantifying C-Man-Trp from samples. FIG. 2 shows the detection and quantification results of C-Man-Trp. Samples were prepared by organic solvent extraction, then centrifuged and subjected to UPLC, as described in the Examples below. (a) HILIC assay profile for determining C-Man-Trp levels. Absorbance at 280 nm (upper panel), fluorescence intensity (excitation at 302 nm/emission at 350 nm) (middle panel), and mass abundance at m/z value of 367.15 [M+H] ⁺ Chromatogram of chemically synthesized C-Man-Trp monitored by measuring the abundance (below). Arrows indicate retention times corresponding to C-Man-Trp. (b) C-Man-Trp level calibration curve based on chemically synthesized C-Man-Trp. C-Man-Trp levels were quantified by measuring fluorescence intensity (FLR) as described in the Examples below. Mouse tissue samples were prepared and subjected to UPLC. C-Man-Trp was measured by HILIC in mouse brain (c), liver (d), plasma (e) and urine (f) samples. Figure 3 shows C-Man-Trp levels in mouse tissues and plasma. C-Man-Trp concentrations were measured in tissue samples from male (n = 6) and female (n = 6) mice (6-week-old C57BL/6 mice) as described in the Examples below. The levels of C-Man-Trp concentration in Table 1 are illustrated. Data represent mean ± standard deviation (SD). FIG. 4 shows C-Man-Trp levels in plasma and CA125 levels in serum from ovarian tumor patients. C-Man-Trp is represented as CMW in the figure. C-Man-Trp concentrations were measured in the plasma of 7 healthy subjects and 49 ovarian tumor patients (8 benign, 8 borderline, 33 malignant) as described in the Examples below. The median value was 148.1 nM in healthy subjects, 174.9 nM in benign ovarian tumor patients, 208.5 nM in borderline malignant tumor patients, and 333.6 nM in malignant ovarian cancer patients. Since there is a significant difference in C-Man-Trp in plasma between healthy subjects and patients with ovarian tumor, the presence or absence of ovarian tumor can be determined from the quantitative value of C-Man-Trp in plasma. Furthermore, since C-Man-Trp in the plasma of malignant ovarian cancer is significantly high, it is possible to diagnose malignancy of ovarian tumor from the quantitative value of C-Man-Trp in plasma. CA125, a conventional ovarian cancer marker, also showed a significant difference in each group, but some cases showed high values in benign and borderline malignant groups, and some cases showed low values in malignant groups. FIG. 5 shows ROC (Receiver Operating Characteristic) curves created using C-Man-Trp levels in plasma and CA125 levels in serum of ovarian tumor patients. C-Man-Trp is represented as CMW in the figure. Using the data of the ovarian tumor patient group described above, a ROC curve was created to compare C-Man-Trp as a diagnostic marker for malignant ovarian cancer with CA125, a conventional ovarian cancer marker. In diagnosing malignant ovarian cancer, the sensitivity of CA125 was 72.4%, the specificity was 86.7%, and the AUC (Area Under the Curve) was 0.831 (cutoff value 190.3 U/mL), whereas C-Man-Trp Sensitivity was 93.1%, specificity was 86.7%, and AUC was 0.915 (cutoff value 223.5 nM), making it a more sensitive marker. Furthermore, in diagnosing malignant ovarian cancer, using a formula combining C-Man-Trp and CA125 (CMW+CA125; 0.022×CMW+0.0013×CA125-5.38, cutoff value 0.645), the sensitivity was 93.1% and the specificity was 93.1%. was 86.7% and AUC was 0.931, which was even better. Accuracy as a marker capable of diagnosing malignancy of ovarian tumors was confirmed from the quantitative value of C-Man-Trp in plasma.

1. Biomarkers of the present invention The present invention uses C-mannosylated tryptophan (hereinafter "C-Man-Trp") (the structural formula is shown in Formula 1) (hereinafter sometimes referred to as the "biomarker of the present invention"). In the present specification, benign ovarian tumors, borderline malignant ovarian tumors and malignant ovarian tumors are collectively referred to as "ovarian tumors", and benign ovarian tumors and borderline malignant ovarian tumors are collectively referred to as "non-malignant ovarian tumors". Malignant ovarian tumors and malignant ovarian tumors are sometimes collectively referred to as "ovarian cancer".

As shown in the examples below, the combined use of the biomarker of the present invention and a known ovarian tumor marker to determine the presence of malignant ovarian tumors makes it possible to more accurately determine the presence of malignant ovarian tumors. Proven. Such known ovarian tumor markers include, for example, CA125, CEA, CA19-9, and the like.

These known ovarian tumor biomarkers can be produced according to known protein synthesis methods such as solid phase synthesis method, liquid phase synthesis method and the like. The resulting protein can be purified and isolated by known purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof. Alternatively, it may be isolated and purified from a biological sample by a method known per se.

Ovarian tumors determined or evaluated by the biomarkers of the present invention include superficial epithelial stromal tumors, sex cord stromal tumors, germ cell tumors, and other tumors. Non-malignant ovarian tumors of superficial epithelial stromal tumors include serous cystadenoma, mucinous cystadenoma, endometrioid adenoma, clear cell adenoma, clear fibroma, superficial papilloma, and Brenner tumor. , Malignant ovarian tumors of surface epithelial stromal tumors include serous (cystic) adenocarcinoma, mucinous (cystic) adenocarcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma, adenocarcinoma fibroma, adenosarcoma, Mixed mesodermal tumor (carcinosarcoma), malignant Brenner's tumor, transitional cell carcinoma, undifferentiated carcinoma and the like. Benign ovarian tumors of sex cord stromal tumors include capsular cell carcinoma, fibroma, sclerosing stromal tumor, Sertoli-stromal cell carcinoma (well-differentiated), Leydig cell carcinoma (phylum cell carcinoma), ring-shaped Sex cord tumors with tubules, etc., and borderline malignant ovarian tumors of sex cord-stromal tumors include granulosa cell gland, Sertoli/stromal cell tumor (moderately differentiated), steroid (lipid) cell tumor, and gynecomastia. Nandroblastoma and the like are mentioned, and malignant ovarian tumors of sex cord stromal tumors include fibrosarcoma, Sertoli stromal cell tumor (poorly differentiated type) and the like. Benign ovarian germ cell tumors include mature cystic teratoma (dermoid cystoma), mature solid teratoma, and ovarian goiter. Borderline malignant germ cell tumors of the ovary include immature teratoma. (G1, G2), carcinoid, goiter carcinoid, etc. Malignant ovarian tumors of germ cell tumors include undifferentiated germ cell tumors, yolk sac tumor (endodermal sinus tumor), embryonal carcinoma (embryonal carcinoma) ), polyembryoma, choriocarcinoma, mature cystic teratoma with malignant transformation, and immature teratoma (G3). Other benign ovarian tumors include non-specific soft tissue tumors and adenomatous tumors. Other borderline malignant ovarian tumors include gonadal blastoma (pure type). Other malignant ovarian tumors include , carcinoma, sarcoma, malignant lymphoma (primary), secondary (metastatic) tumor, and the like. Evaluation of the degree of malignancy of ovarian tumors, for example, taking a test animal with serous cystadenoma as an example, determines whether the serous cystadenoma is benign or borderline malignant depending on the concentration of the biomarker of the present invention in the animal. or malignant (ie, serous (cystic) adenocarcinoma).

Animals that can be tested or evaluated using the biomarkers of the present invention (that is, test animals) are not particularly limited, but preferably include animals suspected of having ovarian tumors. Examples of animal types include mammals (e.g., humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, rats, etc.) and birds (e.g., chickens, etc.). etc. Preferably mammals, more preferably humans.

2. Methods for Detection and Evaluation of Ovarian Tumor As shown in the Examples described later, the inventors found that the concentration of C-Man-Trp in plasma correlates with the degree of malignancy of ovarian tumor. Specifically, the plasma concentration of C-Man-Trp is significantly different between the healthy subject group and the ovarian tumor patient group, and the plasma concentration of C-Man-Trp is significantly different from that of non-malignant ovarian cancer. A significant difference was found between the tumor patient group and the malignant ovarian tumor patient group. Therefore, the present invention includes detecting or quantifying the biomarker of the present invention in a sample derived from a test animal (that is, a sample collected from the test animal). or a method for evaluating the malignancy of an existing ovarian tumor (e.g., evaluation, analysis, diagnosis, judgment, judgment, detection), or a method for assisting the evaluation (hereinafter collectively referred to as "this (sometimes referred to as "methods for evaluating inventions"). Alternatively, for a sample derived from a test animal, including detecting or quantifying the biomarker of the present invention, analysis of the probability of the presence of ovarian tumor or malignant ovarian tumor in the test animal (e.g., analysis, evaluation, detection) (hereinafter sometimes referred to as the "analytical method of the present invention"). Hereinafter, the evaluation method of the present invention and the analysis method of the present invention may be collectively referred to as the "method of the present invention".

Specifically, the method of the present invention includes (i) the step of quantifying the biomarker of the present invention in a sample derived from a test animal, (ii) the step of comparing the amount of the biomarker quantified in (i), and (iii) Based on the result of (ii), it may include a step of evaluating that the subject animal has an ovarian tumor or that the subject animal has a malignant ovarian tumor. In the present invention, "evaluating that it exists" includes evaluating that there is a high probability that it exists. In addition, step (iii), more specifically, evaluates that an ovarian tumor exists when the amount of the biomarker of the present invention is equal to or greater than a preset reference value, and the amount of the biomarker is and a step of evaluating the ovarian tumor to be a malignant ovarian tumor if it is equal to or greater than a reference value different from the reference value.

In the method of the present invention, the biomarker of the present invention may be used alone, or one or more of the above 1. may be used in combination with known ovarian tumor markers from For example, when using two types of biomarkers (i.e., the biomarker of the present invention and one known ovarian tumor marker) in combination, for example, two types of biomarkers are detected or quantified, and based on the results (Applying to the formula if necessary), the above step (iii) may be performed. Alternatively, first, the first type of biomarker is detected or quantified, and based on the results, the presence of ovarian tumors or malignant ovarian tumors in the test animal is evaluated. The biomarkers may be detected or quantified, and the results may be used to perform step (iii) above. Similar determination can be made when three or more types are used. A preferred combination when two or more are used in combination is a combination of the biomarker of the present invention and CA125. In the present specification, the biomarker of the present invention and the known ovarian tumor marker are sometimes collectively abbreviated as "biomarker".

The types of test animals for the method of the present invention are as described in 1. above. as described in The sample derived from the test animal is not particularly limited as long as it can contain the biomarker of the present invention. Examples include fluid, cerebrospinal fluid, interstitial fluid, and lymph, preferably serum or plasma, more preferably plasma. These samples can be obtained by a method known per se. For example, serum and plasma can be prepared by collecting blood from a subject animal according to a conventional method and separating the liquid component, and cerebrospinal fluid is , spinal tap, or other known means.

Extraction of C-Man-Trp from a sample can be performed by a method known per se. Examples include the method of extracting C-Man-Trp from a sample with a strong acid solvent containing an acid (eg, hydrochloric acid, perchloric acid, etc.) and a reducing agent treatment solution (eg, ascorbic acid) as described. However, since it was confirmed that C-Man-Trp is denatured by a strong acid as described above, it is preferable to use an organic solvent from the viewpoint of highly accurate detection and quantification. Such organic solvents include, for example, methanol, ethanol, propanol, benzene, acetonitrile, acetone, chloroform, combinations thereof, and the like. The organic solvent may contain other components such as weak acids (eg, formic acid, carbonic acid, hydrogen sulfide, acetic acid, etc.). In a preferred embodiment, the organic solvent is a solvent containing methanol, acetonitrile and formic acid (eg methanol:acetonitrile:formic acid=80:19.9:0.1).

C-Man-Trp thus extracted can be detected or measured by a method known per se. For example, Takahira et al., Am. J. Med., 110:192-197 (2001), a method of quantitative analysis by high performance liquid chromatography (HPLC) using a reversed-phase column and fluorescence detection, A HPLC method using reversed-phase chromatography and fluorescence detection described in WO99/09411, a quarantine assay method using an antibody that specifically recognizes C-Man-Trp, and a migration method described in WO2014/046163. Determination of C-Man-Trp by Reversed-Phase Chromatography with Sulfonic Acid-Based Ion-Pairing Reagents in Phase, Separation by HPLC and Electrospray, as described by Sekula et al., Sci Rep, 7:17400 (2017) detection and quantification methods using an ionization triple quadrupole mass spectrometer; As long as it can be measured, it is not limited to these methods.

Detection or quantification of biomarkers in a sample derived from a test animal can be performed in addition to the above methods, for example, by preparing an RNA (e.g., total RNA, mRNA) fraction from the sample, and measuring the biomarkers contained in the fraction. It can be carried out by detecting or quantifying marker transcripts. Accordingly, in one embodiment, the methods of the invention comprise detecting or quantifying using nucleic acid probes or nucleic acid primers each capable of specifically recognizing a biomarker transcript.

RNA fractions can be prepared using known techniques such as guanidine-CsCl ultracentrifugation and AGPC. High-purity total RNA can also be prepared quickly and easily from a specimen. Methods for detecting or quantifying biomarker transcripts in RNA fractions include methods using hybridization (Northern blot, dot blot, etc.), or PCR (RT-PCR, competitive PCR, real-time PCR, etc.). methods to be used, and the like. Quantitative PCR methods such as competitive PCR and real-time PCR are preferable in that expression changes of each gene encoding a biomarker can be detected quickly, easily, and quantitatively from a very small amount of sample.

The above-mentioned nucleic acid probes or nucleic acid primers are of a desired length by PCR using a primer set capable of amplifying a part or all of the transcription product of the biomarker gene, using cDNA or genomic DNA derived from the cells of the subject animal as a template. Alternatively, from the cDNA or genomic DNA library, clone the above gene or cDNA by colony or plaque hybridization, etc., and if necessary, use restriction enzymes, etc. to make a fragment of an appropriate length. can be obtained by Alternatively, it can be obtained by chemical synthesis using a commercially available DNA/RNA automatic synthesizer or the like.

The nucleic acid probe or nucleic acid primer is preferably labeled with a labeling agent to enable detection and quantification of the target nucleic acid. Examples of labeling agents include radioactive isotopes, enzymes, fluorescent substances, and luminescent substances. Examples of radioisotopes include [ ³² P], [ ³ H], and [ ¹⁴ C]. Enzymes that are stable and have a high specific activity are preferred, and examples include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, or the like is used. Luminol, luminol derivatives, luciferin, lucigenin, and the like are used as the light-emitting substance, for example. In addition, biotin-(strept)avidin can be used to bind probes and labeling agents.

Alternatively, the detection or quantification of biomarkers in a sample derived from a test animal is performed by extracting (modified) amino acids or peptides (peptides include proteins and modified (e.g., glycosylated) peptides) fractions from the sample. It can be examined by preparing and detecting or quantifying the translation product of the gene contained in the fraction. These amino acids or peptides can be detected or quantified by immunoassays (e.g., CLEIA, ELISA, FIA, RIA, Western blot, etc.) using antibodies that specifically recognize each amino acid or peptide. can. Accordingly, in one embodiment, the method of the present invention comprises detecting or quantifying biomarkers using antibodies that can specifically recognize each biomarker.

Antibodies that can specifically recognize biomarkers can be produced by existing general production methods using biomarker proteins or partial peptides or amino acids having epitopes as immunogens. As used herein, antibodies include polyclonal antibodies, natural antibodies such as monoclonal antibodies (mAb), chimeric antibodies that can be produced using genetic recombination technology, humanized antibodies and single-chain antibodies, and their binding properties. Fragments and the like include, but are not limited to. Preferably, the antibody is a polyclonal antibody, monoclonal antibody or binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specific examples thereof include F(ab') ₂ , Fab', Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The antibody class is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD and IgE. IgG or IgM is preferred, and IgG is more preferred in consideration of ease of purification and the like. In addition, in the present invention, commercially available antibodies or kits containing antibodies (e.g., AIA-pack CL (registered trademark) (Tosoh), etc.), arrays, etc. can be used as antibodies that can specifically recognize each biomarker. is also preferred.

When applying individual immunoassay methods to the method of the present invention, no special conditions, operations, etc. are required. A biomarker measurement system may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method. For details of these general technical means, reference can be made to review articles, books, and the like. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Continued Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. 1953), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd ed.) (Igakushoin, 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd ed.) (Igakushoin, Showa published in 1962), "Methods in ENZYMOLOGY" Vol.70 (Immunochemical Techniques (Part A)), Vol.73 (Immunochemical Techniques (Part B)), Vol.74 (Immunochemical Techniques (Part C)), Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), Ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), Ibid. Vol. )) (above, published by Academic Press).

Alternatively, biomarkers may be detected or quantified using proteome analysis in combination with an iTRAQ ^™ reagent (ABI) capable of high-throughput peptide detection or quantitative analysis and a mass spectrometer.

Comparison of amounts of biomarkers can be performed, for example, on a sample from a control animal without ovarian tumors or a control animal with ovarian tumors (hereinafter sometimes referred to as a "control sample") and a subject of interest. It can be carried out by quantifying the biomarker concentration in the sample derived from the test animal and comparing the two concentrations.

Alternatively, it may be done by comparing the amount of the biomarker to a reference value.
As a "reference value" for use in the present invention, the amount of the biomarker of the present invention in a control sample may be used. Alternatively, preset values from quantified values of biomarkers in control samples may be used. In this case, as the reference value, for example, a median value, an average value, or a mode value of the measured values of a plurality of individuals can be used as a control group.

The reference value may be a cutoff value. A "cutoff value" is a value that satisfies both high diagnostic sensitivity (prevalence accuracy rate) and high diagnostic specificity (no disease accuracy rate) when a disease is determined based on that value. For example, a value that exhibits a high positive rate in a group of individuals with ovarian tumor and a high negative rate in a group of individuals without ovarian tumor can be set as the cutoff value.

Methods for calculating cutoff values are well known in the art. For example, the amount of the biomarker of the present invention in each control sample (e.g. plasma) is quantified, the diagnostic sensitivity and diagnostic specificity in the quantified values are determined, and based on these values, commercially available analysis software is used to create an ROC (Receiver Operating Characteristic) curve. Then, the value at which the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can be obtained and used as the cutoff value. When two or more types of biomarkers are used, the amount of each biomarker can be combined to create an ROC curve. Specifically, in diagnosing malignant ovarian cancer, CA125 had a sensitivity of 72.4%, a specificity of 86.7%, and an AUC (Area Under the Curve) of 0.831 (cutoff value of 190.3 U/mL), whereas C -Man-Trp was a more sensitive marker with sensitivity of 93.1%, specificity of 86.7%, and AUC of 0.915 (cutoff value 223.5 nM). Furthermore, in diagnosing malignant ovarian cancer, using a formula combining C-Man-Trp and CA125 (CMW+CA125; 0.022×CMW+0.0013×CA125-5.38, cutoff value 0.645), the sensitivity was 93.1% and the specificity was 93.1%. was 86.7% and AUC was 0.931, which was even better. CMW is an abbreviation for C-Man-Trp.

A suitable cutoff value for the biomarker of the present invention in the collected plasma includes 223.5 (nM), and when combined with CA125, for example, 0.645 when using the following formula, These numerical values may vary depending on the sample size and other conditions, and are not limited to these numerical values.

Also, the reference value may be a branch point reference value set by decision tree analysis. The breakpoint is determined, for example, in each control sample, by determining the amount of each biomarker of the invention, performing a decision tree analysis, and measuring the impurity of the data expressed in a standard measure such as the Gini coefficient, entropy, chi-square statistic, etc. can be used to generate and determine a variable (eg, concentration of a biomarker of the invention in a sample) and its reference value. Decision tree analysis can be performed by a method known per se, such as a method using software for decision tree analysis (statistical analysis software R and rpart package).

As a result of comparing the amount of the biomarker of the present invention, for example, in a sample derived from a test animal, the biomarker of the present invention is measured as a statistically significantly higher value than the control sample, Or if it is above the reference value, it can be evaluated that the test animal has an ovarian tumor or malignant ovarian tumor, and the biomarker of the present invention shows a statistically significant difference compared to the control sample. is not observed, or is measured as a statistically significant low value, or if it is less than the above reference value, is there no ovarian tumor in the test animal (or there is a high probability that there is no ovarian tumor)? , or that a non-malignant ovarian tumor exists (or is likely to exist).

3. Method for treating or preventing ovarian tumor 2 above. As a result of the method of the present invention described in 1. above, if an ovarian tumor or a malignant ovarian tumor is evaluated in the test animal, based on the results of the evaluation, etc., the type of ovarian tumor to be administered to the test animal. A therapeutic agent or prophylactic agent (hereinafter collectively referred to as a "therapeutic agent") is selected (or determined), and a therapeutically or prophylactically effective amount of the therapeutic agent is administered to the subject animal to eliminate ovarian tumors. It can be treated or prevented. As used herein, the term "therapeutic drug" includes not only drugs intended for the radical treatment of ovarian tumors, but also drugs intended for the suppression of progression of these diseases or drugs intended for the alleviation of symptoms. shall be

Examples of such therapeutic agents include taxane preparations (e.g., paclitaxel, etc.), platinum preparations (e.g., carboplatin, etc.), molecular-targeted therapeutic agents (e.g., bevacizumab, olapanib, etc.), immune checkpoint inhibitors (e.g., nivolumab, pembrolizumab, etc.). , avelumab, atezolizumab, etc.), other anticancer agents (eg, cisplatin, etoposide, bleomycin, etc.), etc., but are not limited thereto. The above therapeutic agents may be used in appropriate combination according to the patient's symptoms.

The therapeutic agent is administered orally or parenterally as a pharmaceutical composition in an appropriate dosage form by mixing the active ingredient as it is or with a pharmaceutically acceptable carrier, excipient, diluent, etc. may Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. agents, emulsions, suspensions and the like. On the other hand, compositions for parenteral administration include, for example, injections and suppositories, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. It may include dosage forms. In addition, the dose of the therapeutic drug can be appropriately set according to various conditions such as the type of compound, symptoms, age, body weight, and drug acceptability of the subject to be administered.

4. Test Kit of the Present Invention Further, the present invention provides a test kit for evaluating the presence and malignancy of ovarian tumors (hereinafter referred to as the test kit of the present invention). The test kit of the present invention contains an antibody that specifically recognizes C-Man-Trp. Antibodies that specifically recognize C-Man-Trp include, for example, the antibodies described in WO99/09411. In addition, the test kit of the present invention includes the above 1. which specifically recognizes known ovarian tumor markers of 2. above. Specific examples thereof include antibodies that specifically recognize CEA, antibodies that specifically recognize CA19-9, antibodies that specifically recognize CA125, and the like.

In addition to the antibodies described above, the test kit of the present invention may contain other substances necessary in the reaction for detecting or quantifying the expression of the biomarker of the present invention. These other substances may be provided together with the antibody or the like, or may be provided together with separate reagents, as long as they do not adversely affect the reaction. Examples of the other substances include reaction buffer, competitor antibody, labeled secondary antibody (for example, when the primary antibody is a rabbit antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solutions, ELISA plates, and the like. In addition, the test kit of the present invention may include an instruction manual that describes how to use the kit and reagents, criteria for determining diseases, and the like. In addition, the test kit may contain the biomarker of the present invention and/or the known ovarian tumor marker, for use as a positive control, for example.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but it is clear that the present invention is not limited to these.

In Examples described later, experiments were conducted as follows.
<Reagent>
Reagents used in this study were purchased from Sigma (St. Louis, MO), Waters (Milford, MA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

<Synthesis of C2-α-C-mannosylpyranosyl-L-tryptophan>
Synthesis of C2-α-C-mannosylpyranosyl-L-tryptophan (C-Man-Trp) in a similar manner to a previously reported method (Manabe et al., J. Am Chem Soc, 121:9754 (1999)) bottom. The purity and identity of the final products were verified by ¹ H NMR spectroscopy and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Proton chemical shifts and coupling constants were consistent with previous reports. The MALDI mass spectrometry mass was consistent with the expected mass of the correct product.

<Experimental mouse>
Unless otherwise stated, C57BL/6 mice were purchased from Kiwa Laboratory Animals Co., Ltd., Japan. All mice were housed in autoclaved cages and maintained with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Wakayama Medical University, and were carried out in accordance with Wakayama Medical University's regulations regarding the implementation of animal experiments.

<Biochemical and metabolic parameters>
Other biochemical parameters in plasma and urine were analyzed by Nagahama Life Science Laboratory (Nagahama, Japan). Creatinine levels in plasma or urine were measured using the corresponding enzyme kits (Wako Pure Chemical). Plasma glycated albumin levels were measured by an enzymatic method (LusicaGA-L, Sekisui Medical Co., Ltd., Tokyo, Japan). Albumin levels in urine were measured by an enzyme-linked immunosorbent assay kit AKRAL-121 (Shibayagi, Gunma, Japan).

<Sample preparation>
The day before sacrifice, mice were transferred to metabolic cages for 24 hours for urinalysis. Mice were anesthetized with isoflurane and blood was collected from the heart. After cervical dislocation procedure, tissue was excised, washed with ice-cold phosphate-buffered saline (PBS, pH 7.2) and minced. Samples were frozen in liquid nitrogen and stored at -80°C until use. For measurement of C-Man-Trp levels, frozen samples were homogenized in extraction solution (methanol:acetonitrile:formic acid = 80:19.9:0.1), centrifuged at 12,000 x g for 15 min at 4°C, Clear was collected. Liquid samples (urine or plasma) were diluted with extraction solution and supernatants were similarly collected. All supernatants were further filtered using PVDF syringe filters (0.45 μm) prior to liquid chromatography analysis.

<Preparation of C-Man-Trp sample from human plasma>
The collected human blood was centrifuged at 2,000×g for 10 minutes at room temperature, and the supernatant was collected as human plasma. Samples were frozen in liquid nitrogen and stored at -80°C until use. To measure C-Man-Trp levels, plasma was mixed with 5 volumes of extraction solution (methanol:acetonitrile:formic acid = 50:49.9:0.1) and centrifuged at 12,000 xg for 15 minutes at 4°C. and collected the supernatant. All supernatants were further filtered using PVDF syringe filters (0.45 μm) prior to liquid chromatography analysis.

<Conditions of UPLC>
Samples for the C-Man-Trp assay were analyzed using a single quadrupole mass spectrometer (Waters H-Class QDa system) with a photodiode array detector, fluorescence detector, isocratic solvent manager, and electrospray ionization (ESI). ) equipped with a Waters Acquity UPLC (Ultra Performance Liquid Chromatography) H-Class system (Waters Corp., Milford, Mass.). Separation was performed on an Aquity UPLC BEH amide column (2.1 x 100 mm, 1.7 μm) maintained at 40°C. Both solvent A (water:acetonitrile = 15:85) and solvent B (water:acetonitrile = 45:55) contained 0.025% formic acid. C-Man-Trp was eluted with a linear gradient of 100% solvent A in 1 min and 0-100% solvent B in 5 min at a flow rate of 0.5 ml/min and solvent A was used before the next injection. The column was equilibrated for 5 minutes. For verification of the C-Man-Trp peak, the isocratic solvent manager was used to split the eluate into two streams. One of the channels was connected to a photodiode array detector followed by a fluorescence detector and the other to a single quadrupole mass spectrometer. C-Man-Trp was monitored using fluorescence detection (excitation at 302 nm/emission at 350 nm) and absorbance detection at 280 nm. Compound mass detection was performed under the following conditions: positive ionization mode; nebulized ( _N2 ) gas flow, 20 L/min; probe temperature, 600°C; cone voltage, 15 V; Ion recording mode selected with an m/z value of 367.15 [M+H] ⁺ . Data were corrected and processed using Empower 3 software.

C-Man-Trp was chemically synthesized as described above and used as a standard compound in the assay. The limits of detection for C-Man-Trp based on the measured fluorescence intensity and mass abundance were 25 nM and 10 nM, respectively. Extraction recovery was assessed by comparing samples from tissues (brain and liver), plasma and urine with samples containing known concentrations of C-Man-Trp. Extraction recoveries of C-Man-Trp detected by fluorescence intensity ranged from 90.9% (plasma) to 100.2% (brain). Extraction recoveries were between 100.7% (brain) and 101.3% (liver) as detected by mass spectrometry.

<Statistical analysis>
Analytical data of mouse samples were expressed as mean±standard deviation. Statistical analysis was performed using unpaired Student's T-test (GraphPad Prism7; GraphPad software). P < 0.05 was considered significant. Analytical data for human plasma were expressed as medians. Statistical analysis was performed using the Mann-Whitney U test and ROC analysis (JMP Pro 13). P < 0.05 was considered significant.

Example 1 Assay of C-Man-Trp by Hydrophilic Interaction Liquid Chromatography (HILIC) A novel assay system for C-Man-Trp was established using fluorescence intensity or mass abundance detection of . As shown in Figure 2a, chemically synthesized C-Man-Trp (arrows) was separated by HILIC, showing absorbance at 280 nm (upper panel) and fluorescence intensity (excitation at 302 nm/emission at 350 nm) (middle panel). Figure ) was detected by monitoring. The mass of the peak of interest, believed to be C-Man-Trp, was confirmed by mass spectrometry with an m/z value of 367.15 [M+H] ⁺ (corresponding to C-Man-Trp) and its Mass abundance was also measured (bottom). Fluorescence intensity (FLR) and mass abundance (QDa) of C-Man-Trp were measured using standard compounds of C-Man-Trp at various concentrations. In this example, C-Man-Trp was quantified for each sample using the calibration curve shown in FIG. 2b. A typical elution pattern of C-Man-Trp was observed for liver (Fig. 2c), brain (Fig. 2d), plasma (Fig. 2e) and urine (Fig. 2f). In brain and liver, C-Man-Trp was detected as the major fluorescence peak with a retention time of 3.9 minutes. In plasma and urine, C-Man-Trp was detected, but there were other peaks with retention times close to that of C-Man-Trp.

Example 2 Quantification of C-Man-Trp Levels in Plasma and Tissues of C57BL/6 Mice A number of tissue and organ samples were prepared from C57BL/6 mice (male and female, 6 weeks old) as described above. bottom. C-Man-Trp levels were examined and quantified for each sample by UPLC-FLR analysis. As shown in Figure 3 and Table 1, C-Man-Trp was detected in all samples examined, but its levels varied between tissues. C-Man-Trp levels were particularly high in ovary and uterus. Other organs with high C-Man-Trp levels included brain, spleen, lung, bladder and testis. Levels of C-Man-Trp were low in skeletal muscle. Furthermore, C-Man-Trp levels were similar between sexes in the tissues and organs examined, excluding the reproductive organs. These results suggest that the production or metabolism of C-Man-Trp is regulated differently in each tissue and organ of mice.

Example 3: Quantitation of C-Man-Trp Concentration in Human Plasma -Man-Trp concentration was quantified. The results are shown in FIG. C-Man-Trp is represented as CMW in the figure. As shown in Figure 4, the median plasma concentration of C-Man-Trp was 148.1 nM in healthy subjects, 174.9 nM in patients with benign ovarian tumors, 208.5 nM in patients with borderline malignant tumors, and 208.5 nM in patients with malignant ovarian tumors. Cancer patients showed a value of 333.6 nM. Since there is a significant difference in C-Man-Trp in plasma between healthy subjects and patients with ovarian tumor, the presence or absence of ovarian tumor can be determined from the quantitative value of C-Man-Trp in plasma. Furthermore, since the C-Man-Trp in the plasma of malignant ovarian cancer is significantly high, it is possible to diagnose the malignancy of the ovarian tumor from the quantitative value of C-Man-Trp in the plasma. For CA125, a conventional ovarian cancer marker, the manufacturer's Serum concentration was measured according to the instructions, and a significant difference was observed in each group, but some cases showed high values even in the benign and borderline malignant groups, and some cases showed low values in the malignant group.

Example 4: Accuracy evaluation of C-Man-Trp as a biomarker Using the data of the ovarian tumor patient group obtained in Example 3, the diagnosis of ovarian cancer was compared with CA125, a known ovarian tumor marker. To assess the accuracy of C-Man-Trp as a marker, ROC curves were generated as described above. The results are shown in FIG. C-Man-Trp is represented as CMW in the figure. CA125 had a sensitivity of 72.4%, a specificity of 86.7%, and an AUC (Area Under the Curve) of 0.831 (cutoff value of 190.3 U/mL), whereas C-Man-Trp had a sensitivity of 93.1% and a specificity of 93.1%. The degree was 86.7%, and the AUC was 0.915 (cutoff value 223.5 nM), making it a more sensitive marker. Furthermore, in diagnosing malignant ovarian cancer, using the formula combining C-Man-Trp and CA125 (CMW + CA125; 0.022 × CMW + 0.0013 × CA125-5.38, cutoff value 0.645), the sensitivity was 93.1% and the specificity was 93.1%. was 86.7% and AUC was 0.931, which was even better. Accuracy as a marker capable of diagnosing malignancy of ovarian tumor was confirmed from the quantitative value of C-Man-Trp in plasma.

The C-mannosylated tryptophans of the present invention are useful as highly accurate biomarkers for determining the presence of ovarian tumors and/or ovarian cancers in test animals.

Claims (9)

A biomarker for assessing the presence and grade of ovarian tumors consisting of C-mannosylated tryptophan. A method for determining whether or not an ovarian tumor is present in a test animal, or determining the malignancy of an existing ovarian tumor, comprising the step of quantifying the biomarker of claim 1 in blood, serum or plasma derived from the test animal. Methods to assist assessment. 3. The method according to claim 2, comprising the following steps (i) to (ii).
(i) the step of quantifying the biomarker of claim 1 in blood, serum or plasma derived from the test animal; and (ii) the step of comparing the amount of the biomarker quantified in (i) with a reference value. 4. The method of claim 3, further comprising quantifying the amount of at least one other ovarian tumor marker in blood, serum or plasma from the subject animal. 5. The method of claim 4, wherein said other ovarian tumor marker is selected from the group consisting of CA125, CEA and CA19-9. The method of any one of claims 2-5, wherein the subject animal is a human. Method according to any one of claims 2 to 6, characterized in that the biomarker according to claim 1 is extracted from said blood, serum or plasma using an organic solvent. A test kit for assessing the presence and malignancy of ovarian tumors, comprising an antibody that specifically recognizes C-mannosylated tryptophan. 9. The kit of claim 8, further comprising antibodies that specifically recognize other ovarian tumor markers.

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