Ovarian cancer risk prediction

Abstract

Ovarian cancer risk in a female human subject is predicted by determining the amount of STK1 substance in a serum or plasma sample from the female human subject using first and second monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the serum form of human TK1. The amount of CA 125 or HE4 is determined in the serum or plasma sample or another serum or plasma sample from the female human subject. The risk of ovarian cancer in the female human subject is then predicted based on the amount of STK1 substance and the amount of one but not both of CA 125 and HE4.

Description

Technical Field

[0001] The present invention relates generally to the measurement of thymidine kinase 1 (TK1), and in particular to the prediction of ovarian cancer risk based on the measured amount of serum TK1 substance. Background Art

[0002] Ovarian cancer is the eighth most common cancer among women worldwide. The Global Cancer Observatory reports that in 2020, there were more than 30,000 new cases of ovarian cancer and more than 20,000 ovarian cancer-related deaths worldwide. More than two-thirds of ovarian cancer patients are diagnosed in the late stage of the disease (stage III or IV), which is associated with a 27% 5-year survival rate for patients with stage III cancer and a 13% 5-year survival rate for patients with stage IV cancer. In contrast, if ovarian cancer is detected in stage I, the 5-year survival rate will be significantly improved, with a 90% 5-year survival rate and an 84% 10-year survival rate for these cases.

[0003] The most commonly used serum biomarker for ovarian cancer is cancer antigen 125 (CA 125), also known as mucin 16 (MUC16). Early studies have shown that CA 125, combined with pelvic ultrasound or alone, can be used to screen large cohorts of the population for ovarian cancer. However, CA 125 has significant limitations in terms of sensitivity and specificity for the detection of early ovarian cancer [1]. In addition, CA 125 levels are also elevated in various other pathological conditions, such as endometriosis and non-malignant gynecological diseases [2].

[0004] To improve ovarian cancer detection, other biomarkers have been developed, such as human epididymis protein 4 (HE4), also known as WAP tetradisulfide core domain protein 2 (WFDC2). HE4 is a glycoprotein belonging to the whey acid tetradisulfide core protein family and is overexpressed in serous and endometrioid ovarian cancers. Studies have been conducted to evaluate individual serum biomarkers and combinations of these for the detection of ovarian cancer in women with pelvic masses. The combination of CA125 and HE4 has been shown to be more effective in predicting malignancy than either alone [3].

[0005] In addition, a dual marker algorithm based on CA 125 and HE4 was developed as the Risk of Ovarian Malignancy Algorithm (ROMA), which enhanced the clinical utility of these biomarkers in distinguishing benign from malignant ovarian cancer.[3-6] However, another study showed that there was no clinical benefit in using the ROMA index instead of CA 125 or HE4 alone in the detection of epithelial ovarian cancer.[7]

[0006] A new ovarian cancer diagnostic index, the Ovarian Malignancy Risk Index (ROMI), has been developed based on ROMA but combines thymidine kinase 1 (TK1), HE4, and CA 125[8]. Compared with ROMA, ROMI has better sensitivity and specificity in diagnosing all-stage or stage I+II ovarian cancer. In addition,

[12] concluded that the combined detection of CA 125, HE4, and TK1 can significantly improve the sensitivity of ovarian cancer diagnosis.

[0007] There remains a need to improve the sensitivity and specificity for early detection and for differentiating benign pelvic masses from malignant ovarian tumors. Summary of the Invention

[0008] The overall goal is to provide a risk profile for ovarian cancer.

[0009] The specific goal was to provide an ovarian cancer risk predictor that could differentiate benign pelvic masses from malignant ovarian tumors.

[0010] Another specific goal is to provide an ovarian cancer risk profile that can differentiate early-stage ovarian cancer (stages I and II) from late-stage ovarian cancer (stages III and IV).

[0011] These and other objectives are met by the embodiments as described herein.

[0012] The invention is defined by the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0013] One aspect of the present invention relates to a method for predicting the risk of ovarian cancer in a female human subject. The method includes using a kit to determine the amount of serum thymidine kinase 1 (STK1) substance in a serum or plasma sample from the female human subject, the kit comprising a first monoclonal antibody or a first antigen-binding fragment thereof that specifically binds to the serum form of human TK1 and a second monoclonal antibody or a second antigen-binding fragment thereof that specifically binds to the serum form of human TK1. The method also includes determining the amount of cancer antigen 125 (CA 125) or human epididymis protein 4 (HE4) in the serum or plasma sample or another serum or plasma sample from the female human subject. The method further includes predicting the risk of ovarian cancer in the female human subject based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4.

[0014] The combination of STK1 and either CA125 or HE4 showed better performance in distinguishing early-stage ovarian cancer from healthy controls than either of these alone, but also significantly outperformed STK1 combined with the ROMA index (i.e., STK1 combined with CA125 and HE4). However, this was not observed when the TK1 activity test was combined with other markers. In addition, the combination of TK1 protein with CA 125 or HE4 could more effectively distinguish early-stage disease (stages I and II) from late-stage disease (stages III and IV). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which:

[0016] Figure 1 TK1 levels in different groups, (A, B, C) serum TK1 protein (STK1p), (D, E, F) serum TK1 activity (STK1a), (A, D) all women, (B, E) premenopausal women, (C, F) postmenopausal women;

[0017] FIG2 ROC curves for (A) the biomarkers STK1p, CA 125, HE4 alone and as dual markers with STK1p, and for (B) the biomarkers STK1a, CA 125, HE4 alone and as dual markers with STK1a;

[0018] Figure 3 Diagnostic performance of STK1p alone, STK1p+CA125, STK1p+HE4, and STK1p+ROMA indices for (A) premenopausal women, (B) postmenopausal women, (C) benign masses versus healthy controls, (D) malignant ovarian cancer versus healthy controls, and (E) benign masses versus malignant ovarian cancer;

[0019] Figure 4 (A) STK1p+CA 125, (B) STK1a+CA 125, (D) STK1p+HE4, and (E) STK1a+HE4 combinations, and (C, F) ROC curve analysis in distinguishing early (I+II) from late (III+IV) malignant ovarian cancer;

[0020] Figure 5 (A, B, C) STK1p and (D, E, F) STK1a levels before and after surgery for (A, D) all women, (B, E) premenopausal women, and (C, F) postmenopausal women; and

[0021] Figure 6(A) STK1p / STK1p ratio and days at diagnosis, (B) STK1p levels at diagnosis and after chemotherapy, (C) STK1p+CA 125, (D) STK1p+HE4, (E) STK1a+CA 125, and (F) STK1a+HE4 in the presence or absence of recurrence after chemotherapy. DETAILED DESCRIPTION

[0022] The present invention relates generally to the measurement of thymidine kinase 1 (TK1), and in particular to the prediction of ovarian cancer risk based on the measured amount of serum TK1 (STK1) substance.

[0023] The success of ovarian cancer treatment is highly dependent on the timing of disease detection, as patients with early-stage disease have the best chance of survival. Studies have shown that only <25% of patients with ovarian cancer can be diagnosed in the early stages when there are no symptoms, while 70% of patients are diagnosed in the late stages. Despite considerable efforts for early detection, no cost-effective screening test has been developed to date. Therefore, early detection with the help of tumor-specific biomarkers can improve the clinical outcomes of patients with ovarian cancer. This is especially important for patients with vague or no symptoms. In 2008, Moore et al. established a mathematical algorithm for determining ovarian cancer risk (ROMA), which depends on the women's menopausal status and the preoperative levels of human epididymis protein 4 (HE4) and cancer antigen 125 (CA 125) in the serum [6]. Therefore, the ROMA index was designed to improve the effectiveness of the tumor marker CA 125 in the diagnosis and monitoring of epithelial ovarian cancer. As shown in the Examples section, the sensitivity and specificity of the ROMA index are 63% and 95%, respectively. Despite this, this is still insufficient for effective early detection of ovarian cancer.

[0024] Therefore, additional biomarkers are needed, and the results presented here suggest that STK1 may be such a new diagnostic tool. Adding STK1 to CA 125 or HE4 demonstrated greater than 70% sensitivity and 95% specificity. Therefore, by combining STK1 with CA 125 or HE4, the sensitivity for early detection of ovarian cancer may be increased.

[0025] The results showed that the combination of STK1 and CA 125 and STK1 and HE4 had higher sensitivity and 95% specificity, which can provide early detection of ovarian cancer and thus improve patient prognosis. When distinguishing malignant ovarian cancer from healthy controls, the dual biomarkers STK1 and CA 125 and STK1 and HE4 showed higher sensitivity compared to STK1 alone. When distinguishing benign ovarian cancer from malignant ovarian cancer, the combination of STK1 and HE4 had a sensitivity of 58%, followed by the combination of STK1 and CA 125 (44%), while the sensitivity of STK1 alone was 28%. These results indicate that the combination of STK1p and CA 125 provides better diagnostic performance in the detection of early ovarian cancer. In addition, the combination of STK1 and CA 125 and the combination of STK1 and HE4 significantly distinguished ovarian cancer patients based on stage and identified the probability of tumor recurrence in patients.

[0026] In addition, the combination of STK1 and CA 125 showed a higher positive predictive value (PPV) and negative predictive value (NPV) than the combination of STK1 and ROMA index (96% vs. 95% and 80% vs. 73%). The combination of STK1 and CA 125 had the highest sensitivity (64%) in distinguishing benign masses from healthy controls compared with other combinations.

[0027] Thus, combining STK1 with one of CA 125 and HE4, but not both, resulted in improved ovarian cancer risk prediction compared to any of these biomarkers (STK1, CA 125, or HE4) alone, or indeed compared to the combination of all three biomarkers (i.e., STK1, CA 125, and HE4) (STK1p+ROMA). This was quite surprising, as the combination of STK1, CA 125, and HE4 was previously considered a new ovarian malignancy risk index (ROMI), which was reported to have better sensitivity and specificity than ROMA [8]. Therefore, it was completely unexpected that by omitting one of CA 125 and HE4 from the prediction, the combination of STK1 and CA 125 or the combination of STK1 and HE4 achieved improved predictive power, and even better ovarian cancer risk prediction, compared to the combination of STK1, CA 125, and HE4.

[0028] One aspect of the present invention relates to a method for predicting the risk of ovarian cancer in a female human subject. The method includes determining the amount of STK1 substance in a serum or plasma sample from the female human subject using a kit comprising a first monoclonal antibody or a first antigen-binding fragment thereof that specifically binds to a serum form of human TK1 and a second monoclonal antibody or a second antigen-binding fragment thereof that specifically binds to a serum form of human TK1. The method also includes determining the amount of CA 125 or HE4 in the serum or plasma sample or another serum or plasma sample from the female human subject. The method further includes predicting the risk of ovarian cancer in the female subject based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4.

[0029] The TK1 protein in humans exists in various forms, depending on the presence of certain molecules, for example, the presence or absence of adenosine triphosphate (ATP); on the concentration of the protein, i.e., high or low concentration; on the type of protein, i.e., native or recombinant TK1; and on the site of the protein, i.e., in the serum or the cytosol.

[0030] Generally speaking, cytoplasmic and recombinant human TK1 exist as tetramers in the presence of ATP or at high concentrations, and as dimers in the absence of ATP or at low concentrations. The tetrameric forms of cytoplasmic and recombinant human TK1 have high TK1 activity, while the dimer forms have lower TK1 activity. Cytoplasmic TK1, also known as cellular TK1, is TK1 present inside cells and can be isolated from such cells.

[0031] In stark contrast, human STK1 can exist in the form of high-molecular-weight complexes, such as oligomers that possess or contain TK1 activity, as well as dimers and tetramers that have very low or even no TK1 activity. Oligomerization appears to be associated with the formation of disulfide cross-links in the blood. STK1 is present in the blood of patients and can therefore be detected in plasma or serum samples.

[0032] As used herein, STK1 substances refer to STK1 in its various forms, such as dimers, tetramers, oligomers, and complexes comprising STK1. STK1 substances are present in the blood, plasma, or serum of a subject. STK1 substances may then comprise STK1 in the aforementioned forms, such as dimers, tetramers, oligomers, and complexes comprising STK1. STK1 substances also include complexes comprising at least one STK1 protein unit and other molecules and / or macromolecules.

[0033] Various gene expression arrays have been proposed in the art to determine TK1 messenger RNA (mRNA) transcripts in cancer cell samples and biopsies. As mentioned above, TK1 exists in multiple forms in a subject, including cytoplasmic TK1 and serum TK1. Gene expression arrays that determine TK1 mRNA transcripts in such biopsy samples primarily measure TK1 mRNA transcripts of cytoplasmic TK1 present in cancer cells. Therefore, such gene expression arrays cannot be used to determine the level of STK1 substances in female human subjects.

[0034] In one embodiment, determining the amount of the STK1 substance comprises contacting a serum or plasma sample with a first monoclonal antibody or a first antigen-binding fragment thereof and a second monoclonal antibody or a second antigen-binding fragment thereof. This embodiment further comprises measuring the amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment thereof that is bound to the STK1 substance.

[0035] Contacting the serum or plasma sample with the first and second monoclonal antibodies, or their first and second antigen-binding fragments, can be achieved by adding the first and second antibodies, or their first and second antigen-binding fragments, to the serum or plasma sample and incubating the serum or plasma sample with the first and second monoclonal antibodies, or their first and second antigen-binding fragments. The first and second monoclonal antibodies, or their first and second antigen-binding fragments, thereby bind to the STK1 substance, thereby forming a complex between the first and second monoclonal antibodies, or their first and second antigen-binding fragments, and the STK1 substance. In such an embodiment, measuring the amount of the first or second monoclonal antibody, or their first or second antigen-binding fragment, bound to the STK1 substance can include measuring or quantifying the complex between the first and second monoclonal antibodies, or their first and second antigen-binding fragments, and the STK1 substance, thereby measuring or quantifying the amount of the first or second monoclonal antibody, or their first or second antigen-binding fragment, bound to the STK1 substance.

[0036] In one embodiment, the method further comprises correlating the measured amount of the first or second monoclonal antibody, or its first or second antigen-binding fragment, bound to the STK1 substance with the amount of the STK1 substance. This can be performed using a predefined correlation between the measured amount of the first or second monoclonal antibody, or its first or second antigen-binding fragment, bound to a reference TK1 substance and the concentration of the reference TK1 substance. An exemplary reference TK1 substance that can be used when generating such a predefined correlation is recombinant human TK1.

[0037] Therefore, a predefined correlation can be generated by adding the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, to different samples containing different concentrations of a reference TK1 substance (preferably recombinant human TK1), or contacting the two. The amount of the first or second monoclonal antibody, or the first or second antigen-binding fragment thereof, that binds to the reference TK1 substance (preferably recombinant human TK1) is then measured in the different samples, thereby obtaining a standard curve, function, or relationship between the concentration of the reference TK1 substance (preferably recombinant human TK1) and the measured amount of the first or second monoclonal antibody, or the first or second antigen-binding fragment thereof, that binds to the reference TK1 substance (preferably recombinant human TK1).

[0038] The measured amount of the first or second monoclonal antibody or first and second antigen-binding fragments thereof bound to the STK1 substance in the serum or plasma sample can then be mapped or converted to the concentration of the STK1 substance in the serum or plasma sample using this predefined correlation (such as a standard curve, function or relationship).

[0039] It is generally preferred to use the same kit and the same type of first and second monoclonal antibodies, or first and second antigen-binding fragments thereof, to generate a predefined correlation, such as for determining the amount of STK1 substance in a serum or plasma sample from a female human subject. Thus, in a preferred embodiment, the first and second monoclonal antibodies, or first and second antigen-binding fragments thereof, are capable of specifically binding not only to a serum form of human TK1, but also to a reference TK1 substance (preferably recombinant human TK1).

[0040] In one embodiment, the serum or plasma sample is treated before or during incubation with the first and second monoclonal antibodies or first and second antigen-binding fragments thereof. Such sample treatment can be used to stabilize selected STK1 forms in the serum or plasma sample and / or to break up larger STK1 complexes or oligomers into smaller complexes or multimers.

[0041] Thus, in one embodiment, a sample diluent or pretreatment buffer is added to the serum or plasma sample, preferably prior to or in connection with the addition of the first and second antibodies or first and second antigen-binding fragments thereof to the serum or plasma sample, preferably prior to the addition of the antibodies or antigen-binding fragments thereof to the serum or plasma sample.

[0042] In one embodiment, the sample dilution buffer comprises ATP, preferably at a concentration selected in the interval of 0.5 mM to 50 mM, such as 0.5 mM to 20 mM or 1.5 mM to 50 mM. As previously described herein, ATP stabilizes the tetrameric form of TK1, which has high enzymatic TK1 activity.

[0043] In another embodiment, the sample dilution buffer contains a reducing agent. The reducing agent can then disrupt disulfide crosslinks in larger STK1 complexes and oligomers to obtain smaller STK1 forms, such as tetramers. Various reducing agents capable of disrupting disulfide bonds can be used according to the embodiment, including but not limited to dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamine (DTBA), tris(2-carboxyethyl)phosphine) (TCEP), and combinations thereof. The amount of reducing agent is generally selected in the range of 0.1 mM to 10 mM.

[0044] In one embodiment, the sample dilution buffer may contain both ATP and a reducing agent.

[0045] In one embodiment, the method comprises adding a sample dilution buffer to a serum or plasma sample. In this embodiment, the sample dilution buffer comprises ATP and a reducing agent. In a specific embodiment, the sample dilution buffer comprises ATP, and its concentration is selected in the interval of 0.5mM to 50mM, preferably selected in the interval of 0.5mM to 20mM, and more preferably selected in the interval of 1.5mM to 50mM. In a specific embodiment, the reducing agent is selected from the group consisting of: dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamine (DTBA), tris (2-carboxyethyl) phosphine) (TCEP) and any combination thereof. In a specific embodiment, the sample dilution buffer comprises the reducing agent selected in the interval of 0.1mM to 10mM.

[0046] In one embodiment, the amount of STK1 substance is determined using first and second monoclonal antibodies, or first and second antigen-binding fragments thereof, that specifically bind to a serum form of human TK1 in a serum or plasma sample taken from a human female subject after treatment for ovarian cancer. In this embodiment, the amount of CA 125 or HE4 is determined in a serum or plasma sample, or another serum or plasma sample, taken from the female subject after treatment for ovarian cancer. In this embodiment, the method includes predicting ovarian cancer recurrence in the female subject based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4.

[0047] Thus, in this embodiment, ovarian cancer risk prediction includes predicting ovarian cancer recurrence, i.e., predicting the risk of ovarian cancer recurrence in a female subject undergoing ovarian cancer treatment. Treatment of ovarian cancer typically involves surgery, and in particular, removal of the uterus, i.e., hysterectomy, sometimes along with removal of one or both of the ovaries and fallopian tubes, i.e., bilateral salpingo-oophorectomy (BSO). Alternatively or additionally, treatment of ovarian cancer may involve chemotherapy.

[0048] As shown herein, patients who relapse after treatment (such as chemotherapy) have significantly higher levels of STK1+CA 125 and STK1+HE4 ( Figure 6 C and 6D).

[0049] In one embodiment, the amount of STK1 substance is determined using a first and a second monoclonal antibody, or a first and a second antigen-binding fragment thereof, that specifically binds to a serum form of human TK1 in a serum or plasma sample taken from a female subject prior to treatment for ovarian cancer (such as in connection with the diagnosis of ovarian cancer). In this embodiment, the amount of CA 125 or HE4 is determined in a serum or plasma sample or another serum or plasma sample taken from the female subject prior to treatment for ovarian cancer (such as in connection with the diagnosis of ovarian cancer). Ovarian cancer recurrence in the female subject is then preferably predicted based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4 determined in the serum or plasma sample taken from the female subject prior to treatment for ovarian cancer, and the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4 determined in the serum or plasma sample taken from the female subject after treatment for ovarian cancer.

[0050] In one embodiment, predicting the risk of ovarian cancer comprises predicting the stage of ovarian cancer in the female human subject based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4.

[0051] For example, the method can include predicting whether a female human subject has FIGO stage I or II ovarian cancer or FIGO stage III or IV ovarian cancer based on the amount of STK1 substance and the amount of one, but not both, of CA 125 and HE4.

[0052] In general, FIGO stage I means that cancer is confined to one or both ovaries. Sometimes FIGO stage I is divided into stage IA (growth is confined to one ovary and there is no tumor on the outer surface), stage IB (growth is confined to both ovaries and there is no tumor on the outer surface) and stage IC (tumor is stage IA or stage IB but there is tumor on the surface of one or both ovaries). FIGO stage II means that cancer has spread to the uterus or other adjacent organs. Sometimes FIGO stage II is divided into stage IIA (cancer extends and / or metastasizes to the uterus and / or fallopian tubes), stage IIB (cancer extends to other pelvic tissues) and stage IIC (tumor is in stage IIA or IIB but there is tumor on the surface of one or both ovaries). FIGO stage III means that cancer has spread to lymph nodes or the inner wall of the abdomen. Sometimes FIGO stage III is divided into stage IIIA (tumor is confined to the true pelvis), stage IIIB (peritoneal surface metastasis diameter≤2cm) and stage IIIC (pelvic extraperitoneal metastasis diameter>2cm). FIGO stage IV means that cancer has spread to distant organs, such as lungs or liver.

[0053] As shown herein, the combination of STK1 and CA 125 was significantly higher in FIGO stage III+IV patients compared to FIGO stage I+II patients ( Figure 4 A). Similar results were obtained with the combination of STK1 and HE4 ( Figure 4 D).

[0054] In a specific embodiment, the female human subject is in a premenopausal state. Thus, in one embodiment, when the serum or plasma sample is collected from the female human subject, the female subject is in a premenopausal state.

[0055] The experimental data used herein indicate that, for female human subjects in the premenopausal state, the amount of STK1 substance in combination with the amount of one, but not both, of CA 125 and HE4 is particularly useful for detecting all stages of cancer, detecting benign tumor masses from healthy controls, distinguishing malignant ovarian cancer from healthy controls, and distinguishing benign masses from malignant ovarian cancer, compared to the postmenopausal state.

[0056] In a specific embodiment, the method comprises determining the amount of CA 125 in a serum or plasma sample or another serum or plasma sample from a female human subject. In this specific embodiment, the method comprises predicting the risk of ovarian cancer in a female human subject based on the amount of STK1 substance and the amount of CA 125 but not the amount of HE4.

[0057] Thus, in a specific embodiment, the risk prediction for ovarian cancer in a female human subject is based solely on the amount of STK1 substance and the amount of CA 125.

[0058] As shown herein, the combination of STK1 and CA 125 gave the best diagnostic performance in the detection of early-stage ovarian cancer compared to a healthy control group. For premenopausal women, the combination of STK1 and CA 125 showed higher PPV and NPV than the combination of STK1 and ROMA index (96% vs. 95% and 80% vs. 73%). The results strongly suggest that the dual biomarker STK1 and CA 125 has the best diagnostic performance in detecting both benign and malignant ovarian cancer compared to a healthy control group.

[0059] In another specific embodiment, the method comprises determining the amount of HE4 in a serum or plasma sample or another serum or plasma sample from a female human subject. In this specific embodiment, the method comprises predicting the risk of ovarian cancer in a female human subject based on the amount of STK1 substance and the amount of HE4, but not the amount of CA 125.

[0060] Thus, in a specific embodiment, the prediction of ovarian cancer risk in a female human subject is based solely on the amount of STK1 substance and the amount of HE4.

[0061] In one embodiment, a dual biomarker index can be calculated based on the determined amount of STK1 substance and the amount of CA 125, or based on the determined amount of STK1 and the amount of HE4. The dual biomarker index can be calculated according to various embodiments, i.e., dual biomarker index 1 (ln 1) = f (STK1, CA 125) and dual biomarker index 2 (ln 2) = g (STK1, HE4), where STK1 represents the amount of STK1 substance, CA 125 represents the amount of CA 125, HE4 represents the amount of HE4, and f() is a function and g() is a function. For example, the dual biomarker indices ln 1 and ln 2 can be calculated using logistic regression of STK1 and CA 125 or STK1 and HE4. For example, such logistic regression can be performed using MedCalc version 17.6.

[0062] Typically, logistic regression produces coefficients (and their standard errors and significance levels) of the formula to predict the logit transformation of the probability that the characteristic of interest (COI) is present:

[0063] Logit(p)=b0+b1X 1+ b2X 2+ b3X3+……b k X k

[0064] Where p is the probability of the COI being present. The logit transformation is defined as the log odds, odds = p / (1-p), which represents the probability of the COI being present divided by the probability of the COI not being present, such that logit(p) = ln(p / 1-p).

[0065] This logistic regression was used to calculate ln 1 and ln 2 for the serum samples in the Examples. The ln 1 and ln 2 values ​​ranged from 0 to 1.0. The median ln 1 value for the healthy group was 0.23, while the median ln 1 value for patients with ovarian cancer (including both benign and malignant) was 0.98. The corresponding median ln 2 values ​​were 0.26 for the healthy group and 0.95 for the ovarian cancer patients.

[0066] The ln 1 and ln 2 values ​​can be used as described herein, such as for early diagnosis of ovarian cancer. For example, ln 1 and ln 2 values ​​between 0 and 0.70 indicate a healthy (95% CI), i.e., a low risk of ovarian cancer, while ln 1 and ln 2 values ​​between 0.70 and 1.00 indicate an ovarian cancer patient, i.e., a high risk of ovarian cancer.

[0067] For the differentiation of benign from malignant, ln 1 and ln 2 values ​​below 0.60 indicated benignity (95% CI), and ln 1 and ln 2 values ​​between 0.60 and 1.00 indicated malignant ovarian cancer.

[0068] Accordingly, to distinguish between early stage (stage I+II) and late stage (stage III+IV) ovarian cancer, values ​​of 0 to 0.40 of ln1 and ln2 indicate early stage ovarian cancer, and values ​​above 0.40 to 1.00 indicate late stage ovarian cancer.

[0069] The first and second monoclonal antibodies, or first and second antigen-binding fragments thereof, specifically bind to STK1 species, and in particular specifically bind to the serum form of the TK1 protein.

[0070] The specificity of an antibody or antigen-binding fragment thereof can be determined based on affinity and / or avidity. The equilibrium constant (K) for the dissociation of the antigen from the antibody or antigen-binding fragment thereof is d Affinity, expressed as K, is a measure of the strength of binding between an antigenic determinant and an antigen-binding site on an antibody or its antigen-binding fragment. d The smaller the value of , the stronger the binding strength between the antigenic determinant and the antibody or its antigen-binding fragment. Alternatively, affinity can also be expressed as an affinity constant (K a ), which is 1 / K d As will be clear to the skilled person, affinity can be determined in a manner known per se depending on the specific antigen of interest.

[0071] Avidity is a measure of the strength of binding between an antibody or its antigen-binding fragment and its associated antigen. Avidity is related to the affinity between an antigenic determinant and an antigen-binding site on an antibody or its antigen-binding fragment, as well as the number of associated binding sites present on the antibody or its antigen-binding fragment.

[0072] Typically, the antibody or antigen-binding fragment thereof will be expressed as 10 -5 to 10 -12 mol / L (M) or less, and preferably 10 -7 to 10 -12 M or less, and more preferably 10 -8 to 10 -12 The dissociation constant (K d ), that is, with 10 5 to 10 12 M -1 or greater, and preferably 10 7 to 10 12 M -1 or greater, and more preferably 10 8 to 10 12 M -1 The association constant (K a ) binds to its antigen.

[0073] Generally speaking, more than 10 -4 Any K of M d Value (or less than 10 4 M -1 Any K a Preferably, the antibody or antigen-binding fragment thereof will bind to the STK1 substance with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM or even lower, such as 1 nM or lower.

[0074] Specific binding of an antibody or antigen-binding fragment thereof to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, and their different variants known per se in the art.

[0075] In one embodiment, at least one of the first and second antibodies or antigen-binding fragments thereof is specific for an epitope or peptide consisting of an amino acid sequence from the C-terminal region of human TK1.

[0076] The peptide is preferably selected from a portion of TK1 ranging from amino acid position 200 to the end of TK1, i.e., amino acid position 234 in humans (SEQ ID NO: 28). In a specific embodiment, the peptide is selected from a portion of the TK1 protein ranging from amino acid position 205 (preferably 210) to amino acid position 230 (preferably 225).

[0077] The peptide is preferably an N-mer, wherein N is an integer in the range of 8 to 20, preferably an integer in the range of 10 to 15. The peptide preferably consists of N consecutive amino acids in the C-terminal region of the TK1 protein.

[0078] In one embodiment, the peptide consists of the following amino acid sequence: GEAVAARKLF (SEQ ID NO: 1). In another embodiment, the peptide consists of the following amino acid sequence: NCPVPGKPGE (SEQ ID NO: 2). In another embodiment, the peptide consists of the following amino acid sequence: PVPGKPGEAV (SEQ ID NO: 3). In another embodiment, the peptide consists of the following amino acid sequence: NCPVPGKPGEAV (SEQ ID NO: 4).

[0079] The monoclonal antibody specific for the epitope consisting of GEAVAARKLF (SEQ ID NO: 1) has: a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence of DYEMH (SEQ ID NO: 5), a VH domain CDR2 having the amino acid sequence of AIHPGYG GTAYNQKFKG (SEQ ID NO: 6), a VH domain CDR3 having the amino acid sequence of FITKFDY (SEQ ID NO: 7), a variable light (VL) domain CDR1 having the amino acid sequence of KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a VL domain CDR2 having the amino acid sequence of LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 having the amino acid sequence of WQGTHFPWT (SEQ ID NO: 10).

[0080] Monoclonal antibodies specific for the epitopes NCPVPGKPGE (SEQ ID NO:2), PVPGKPGEAV (SEQ ID NO:3), and NCPVPGKPGEAV (SEQ ID NO:4) have a VH domain CDR1 having the amino acid sequence of DYEMH (SEQ ID NO:5), a VH domain CDR2 having the amino acid sequence of AILPGSGGTAYNQKFKG (SEQ ID NO:11), a VH domain CDR3 having the amino acid sequence of LITTFDY (SEQ ID NO:12), a VL domain CDR1 having the amino acid sequence of KSSQSLLDSDGKTYLN (SEQ ID NO:13), a VL domain CDR2 having the amino acid sequence of LVSKLDS (SEQ ID NO:9), and a VL domain CDR3 having the amino acid sequence of WQGTHFPWT (SEQ ID NO:10).

[0081] In another embodiment, one of the first and second antibodies or the first and second antigen-binding fragments thereof is specific for a conformation-dependent epitope of human TK1. The monoclonal antibody specific for such a conformation-dependent epitope has a VH domain CDR1 having the amino acid sequence SGYSWH (SEQ ID NO: 14), a VH domain CDR2 having the amino acid sequence YIHYSGSTTYNPSLKG (SEQ ID NO: 15), a VH domain CDR3 having the amino acid sequence WGTGHWYFDV (SEQ ID NO: 16), a VL domain CDR1 having the amino acid sequence RSSTGAVTTTNYAN (SEQ ID NO: 17), a VL domain CDR2 having the amino acid sequence GTNNRVP (SEQ ID NO: 18), and a VL domain CDR3 having the amino acid sequence ALWYSNHWV (SEQ ID NO: 19).

[0082] The above three proposed examples of monoclonal anti-TK1 antibodies that may be used in accordance with the embodiments are further disclosed in WO 2015 / 094106, which is incorporated herein by reference for its teachings regarding monoclonal anti-TK1 antibodies.

[0083] Thus, in one embodiment, at least one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is selected from the group consisting of: a monoclonal antibody, or an antigen-binding fragment thereof, that is specific to GEAVAARKLF (SEQ ID NO: 1) of human TK1; a monoclonal antibody, or an antigen-binding fragment thereof, that is specific to at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and a monoclonal antibody, or an antigen-binding fragment thereof, that is specific to a conformation-dependent epitope of human TK1.

[0084] In another embodiment, at least one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for an epitope or peptide consisting of KPGEAVAARKLFAPQ (SEQ ID NO: 20). At least one additional amino acid, such as a cysteine ​​residue, can be added to the N-terminus or C-terminus of the peptide, preferably the N-terminus, for coupling to other molecules, such as carrier proteins.

[0085] Antibodies specific for this epitope are further disclosed in WO 95 / 29192, which is incorporated herein by reference for its teachings regarding anti-TK1 antibodies.

[0086] In another embodiment, at least one of the first and second monoclonal antibodies or their first and second antigen-binding fragments is specific for an epitope or peptide consisting of an amino acid sequence from the active site of TK1. The peptide is preferably selected from a portion of human TK1 ranging from amino acid position 150 to amino acid position 190. In a specific embodiment, the peptide is selected from a portion of TK1 ranging from amino acid position 155 (preferably 160 and more preferably 161) to amino acid position 185 (preferably 183).

[0087] The peptide is preferably an M-mer, wherein M is an integer in the range of 10 to 40, preferably an integer in the range of 20 to 30 and more preferably 23 or 24. The peptide preferably consists of M consecutive amino acids in the active site of the TK1 protein.

[0088] At least one additional amino acid, such as a cysteine ​​residue, may be added to the N-terminus or C-terminus of the peptide, preferably the N-terminus, to serve as a coupling to other molecules, such as a carrier protein.

[0089] In one embodiment, the peptide consisting of the amino acid sequence from the active site of TK1 has an amino acid sequence corresponding to amino acid positions 161 to 183 in human TK1, ie, has an amino acid sequence of AYTKRLGTEKEVEVIGGADKYHS (SEQ ID NO: 21).

[0090] Antibodies specific for this epitope are further disclosed in WO 2008 / 142664, which is incorporated herein by reference for its teachings regarding anti-TK1 antibodies.

[0091] In further embodiments, at least one of the first and second monoclonal antibodies or first and second antigen-binding fragments thereof is a monoclonal antibody or fragment thereof as disclosed in WO 2019 / 201901, which is incorporated herein by reference for its teachings regarding monoclonal anti-TK1 antibodies.

[0092] For example, the monoclonal antibody can be mAb 6C6, mAb 4H4, or mAb 23C11.

[0093] mAb 6C6 VH Domain (SEQ ID NO:22):

[0094] METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFSFSSSYWICWVRQAPGKGLEWIACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG

[0095] mAb 6C6 VL domain (SEQ ID NO:23):

[0096] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPKLLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISDSPYVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0097] mAb 4H4 VH domain (SEQ ID NO:24):

[0098] METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWIACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG

[0099] mAb 4H4 VL domain (SEQ ID NO:25):

[0100] MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSSTSGGGVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0101] VH domain of mAb 23C11 (SEQ ID NO:26):

[0102] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGIIYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG

[0103] VL domain of mAv 23C11 (SEQ ID NO:27):

[0104] MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSTFSSYGNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0105] In one embodiment, one of the first monoclonal antibody, or its first antigen-binding fragment, and the second monoclonal antibody, or its second antigen-binding fragment, is specific for a peptide consisting of an amino acid sequence from the C-terminal region of TK1. In this embodiment, the other of the first monoclonal antibody, or its first antigen-binding fragment, and the second monoclonal antibody, or its second antigen-binding fragment, is specific for a peptide selected from the group consisting of: a peptide consisting of an amino acid sequence from the C-terminal region of TK1; a peptide consisting of another amino acid sequence from the C-terminal region of TK1; and a peptide consisting of an amino acid sequence from the active site of TK1. In a specific embodiment, the other of the first monoclonal antibody, or its first antigen-binding fragment, and the second monoclonal antibody, or its second antigen-binding fragment, is specific for a peptide consisting of another amino acid sequence from the C-terminal region of TK1.

[0106] According to the present invention, a kit is used to determine the amount of STK1 substance in a serum or plasma sample. The kit comprises a first monoclonal antibody or a first antigen-binding fragment thereof and a second monoclonal antibody or a second antigen-binding fragment thereof. The first and second monoclonal antibodies can be selected from the illustrative examples of monoclonal anti-TK1 antibodies described above.

[0107] In a specific embodiment, the kit comprises a first monoclonal antibody or a first antigen-binding fragment thereof, which is specific for an epitope selected from the group consisting of: i) GEAVAARKLF (SEQ ID NO: 1) of human TK1; ii) at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and iii) a conformation-dependent epitope of human TK1. The kit further comprises a second monoclonal antibody or a second antigen-binding fragment thereof, which is specific for an epitope selected from the group consisting of: i) GEAVAARKLF (SEQ ID NO: 1) of human TK1; ii) at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and iii) a conformation-dependent epitope of human TK1.

[0108] In one embodiment, the first monoclonal antibody or its first antigen-binding fragment is a so-called capture antibody that is fixed to a support or intended to be fixed to a support, and the second monoclonal antibody or its second antigen-binding fragment is a so-called detection antibody. In another embodiment, the second monoclonal antibody or its second antigen-binding fragment is a capture antibody that is fixed to a support or intended to be fixed to a support, and the first monoclonal antibody or its first antigen-binding fragment serves as a detection antibody.

[0109] In one embodiment, the first and second monoclonal antibodies, or first and second antigen-binding fragments thereof, are specific for different epitopes in the STK1 species.

[0110] In another embodiment, the first and second monoclonal antibodies, or their first and second antigen-binding fragments, are specific for the same epitope in the STK1 substance. This is possible because the same epitope can be present in multiple copies within a high-molecular-weight complex of multiple TK1 protein units. Thus, the STK1 substance can be a multivalent complex of multiple (i.e., at least two) TK1 protein units. Indeed, the same type of monoclonal antibody or antigen-binding fragment can be used as the first and second monoclonal antibodies, or their first and second antigen-binding fragments.

[0111] In one embodiment, one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of an amino acid sequence from the active site of TK1, and the other of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of an amino acid sequence from the C-terminal region of TK1.

[0112] In another embodiment, one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of a first amino acid sequence from the C-terminal region of TK1, and the other of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of a first amino acid sequence from the C-terminal region of TK1 or a second, different amino acid sequence from the C-terminal region of TK1.

[0113] In further embodiments, one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of an amino acid sequence from the C-terminal region of TK1, and the other of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a conformation-dependent epitope of human TK1.

[0114] In yet another embodiment, one of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a peptide consisting of an amino acid sequence from the active site of TK1, and the other of the first and second monoclonal antibodies, or the first and second antigen-binding fragments thereof, is specific for a conformation-dependent epitope of human TK1.

[0115] The antigen-binding fragment of an antibody as used herein can be selected from the group consisting of a single-chain antibody, an Fv fragment, a scFv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, a di-scFv fragment and a CDR region.

[0116] In one embodiment, the kit is a sandwich assay kit.In a specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit, preferably a sandwich ELISA.

[0117] In the following discussion, it is assumed that the first monoclonal antibody or its first antigen-binding fragment is a capture antibody and the second monoclonal antibody or its second antigen-binding fragment serves as a detection antibody. However, the embodiment is not limited thereto, and the capture antibody and the detection antibody may be switched.

[0118] Sandwich ELISA can be used to detect STK1 substances in serum or plasma samples by preparing the surface of a support (such as a solid support) to which a first monoclonal antibody or its first antigen-binding fragment, acting as a so-called capture antibody, is bound. In a preferred embodiment, a known amount of the first monoclonal antibody or its first antigen-binding fragment is bound to the surface of the support. Any non-specific binding sites on the surface are optionally but preferably blocked. The serum or plasma sample is then applied to the surface so that any STK1 substances present therein will be captured by the immobilized first monoclonal antibody or its first antigen-binding fragment. Unbound substances are preferably removed by one or more washing steps. A second monoclonal antibody or its second antigen-binding fragment, typically denoted as a detection antibody, is then added and allowed to bind to any STK1 substances captured by the first monoclonal antibody or its first antigen-binding fragment.

[0119] The amount of the second monoclonal antibody or its second Fab of combination is then determined by a direct or indirect detection method. For example, a mark or enzyme can be directly connected to the second monoclonal antibody or its second Fab, or indirectly connected to the second monoclonal antibody or its second Fab by linking (such as biotin-streptavidin or biotin-avidin linking). Alternatively, a secondary antibody or its secondary Fab that is marked or connected to an enzyme and specifically bound to the second monoclonal antibody or its second Fab can be used.

[0120] Thus, in one embodiment, the second monoclonal antibody or second antigen-binding fragment thereof has covalently attached biotin. Alternatively, the second monoclonal antibody or second antigen-binding fragment thereof has covalently attached streptavidin or avidin.

[0121] The kit preferably also contains horseradish peroxidase (HRP)-labeled streptavidin or HRP-labeled avidin. Alternatively, the kit also contains HRP-labeled biotin. The kit also contains an HRP substrate, such as 3,3',5,5'-tetramethylbenzidine (TMB) substrate, 3,3'-diaminobenzidine (DAB) substrate or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. In such a case, the level of STK1 substance in the sample can be determined by spectrophotometry, which detects the conversion of the chromogenic substrate by HRP into a detectable colored product.

[0122] In one embodiment, the kit further comprises a microtiter plate (MCP) as a support to which the first monoclonal antibody or the first antigen-binding fragment thereof is immobilized or intended to be immobilized.

[0123] In one embodiment, one of the first monoclonal antibody, or first antigen-binding fragment thereof, and the second monoclonal antibody, or second antigen-binding fragment thereof, is specific for an epitope consisting of GEAVAARKLF (SEQ ID NO: 1) and has: a VH domain CDR1 having the amino acid sequence of DYEMH (SEQ ID NO: 5), a VH domain CDR2 having the amino acid sequence of AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a VH domain CDR3 having the amino acid sequence of FITKFDY (SEQ ID NO: 7), a VL domain CDR1 having the amino acid sequence of KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a VL domain CDR2 having the amino acid sequence of LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 having the amino acid sequence of WQGTHFPWT (SEQ ID NO: 10).

[0124] In a specific embodiment, the other of the first monoclonal antibody, or first antigen-binding fragment thereof, and the second monoclonal antibody, or second antigen-binding fragment thereof, is specific for the epitopes NCPVPGKPGE (SEQ ID NO:2), PVPGKPGEAV (SEQ ID NO:3), and NCPVPGKPGEAV (SEQ ID NO:4), and has: a VH domain CDR1 having the amino acid sequence of DYEMH (SEQ ID NO:5), a VH domain CDR2 having the amino acid sequence of AILPGSGGTAYNQKFKG (SEQ ID NO:11), a VH domain CDR3 having the amino acid sequence of LITTFDY (SEQ ID NO:12), a VL domain CDR1 having the amino acid sequence of KSSQSLLDSDGKTYLN (SEQ ID NO:13), a VL domain CDR2 having the amino acid sequence of LVSKLDS (SEQ ID NO:9), and a VL domain CDR3 having the amino acid sequence of WQGTHFPWT (SEQ ID NO:10).

[0125] An example of a TK1 ELISA that can be used in accordance with the embodiments is the AroCell TK 210 ELISA. The kit does not necessarily have to be an ELISA kit. In another embodiment, the kit uses affinity chromatography in which the first monoclonal antibody or first antigen-binding fragment thereof is bound to a stationary phase, such as a gel matrix or beads in a column. For example, the gel matrix or beads can be made of agarose, such as In this case, STK1 species present in the serum or plasma sample will be retained in the column by binding to the immobilized first monoclonal antibody or its first antigen-binding fragment. After washing, the bound STK1 species can be eluted and detected using a second monoclonal antibody or its second antigen-binding fragment. For example, the amount of eluted STK1 species can be determined using Western blotting and the second monoclonal antibody or its second antigen-binding fragment for STK1 detection using direct or indirect detection methods.

[0126] Alternatively, the carrier may be a magnetic bead, such as Magnetic beads.

[0127] In another embodiment, the kit is a chemiluminescent immunoassay (CLIA) kit. CLIA is an immunoassay technique in which the label is a luminescent molecule. CLIA methods can be direct methods using luminophore labels or indirect methods using enzyme labels. Either method can be competitive or non-competitive. In direct CLIA methods, the luminophore labels used are typically acridinium esters and ruthenium esters, while the enzyme labels used in indirect methods are typically alkaline phosphatase (with adamantyl 1,2-dioxetane aryl phosphate (AMPPD) as a substrate) and HRP (with luminol or its derivatives as a substrate).

[0128] Furthermore, the kit does not necessarily have to contain a so-called capture antibody or antigen-binding fragment thereof. In contrast, multiple (i.e., at least two) different monoclonal antibodies or antigen-binding fragments thereof can be used to determine the level of STK1 substance without immobilizing at least one monoclonal antibody or antigen-binding fragment thereof.

[0129] Cancer antigen 125 (CA 125), also known as the ovarian cancer-associated tumor marker CA 125 and mucin-16 (MUC-16), is a protein that is encoded by the MUC16 gene in humans. CA 125 is a member of the mucin family of glycoproteins and has found application as a tumor marker or biomarker that may be elevated in the blood of some patients with certain types of cancer (most notably ovarian cancer) or other benign conditions.

[0130] Human epididymis protein 4 (HE4), also known as WAP tetradisulfide core domain protein 2 (WFDC2), is a protein encoded by the WFDC2 gene in humans. HE4 is a tumor marker for ovarian cancer.

[0131] In one embodiment, determining the amount of CA 125 or HE4 comprises determining the amount of CA 125 or HE4 in a serum or plasma sample or another serum or plasma sample from a female human subject using an electrochemiluminescence immunoassay (ECLIA) kit. ECLIA is a quantitative method for measuring antigens or antibodies based on changes in electrochemiluminescence (ECL) signals before and after an immune reaction.

[0132] For example, the amount of CA 125 in serum or plasma samples can be determined using Roche's CA 125II can be determined or measured, and the amount of HE4 in serum or plasma samples can be determined or measured using Roche's HE4, using e.g. E411 Analyzer to determine or measure.

[0133] Alternatively, the amount of CA 125 in serum or plasma samples can be determined or measured using a human CA 125 ELISA kit, which is commercially available from various suppliers such as Abcam, RayBiotech, R&D Systems, ThermoFisher Scientific, Abnova, etc. Correspondingly, the amount of HE4 in serum or plasma samples can be determined or measured using a HE4 ELISA kit, which is commercially available from various suppliers such as Abcam, ThermoFisher Scientific, Elabscience, R&D Systems, etc.

[0134] In one embodiment, the method further comprises selecting an anti-cancer treatment for the female human subject based on the predicted risk of ovarian cancer. Thus, based on the determined amount of STK1 substance and the determined amount of CA 125 or HE4 in the serum or plasma sample, the best or at least appropriate anti-cancer treatment is selected for the female human subject based on the predicted risk of ovarian cancer estimated for the female human subject. This means that female subjects with a higher predicted risk of ovarian cancer can be selected for more aggressive anti-cancer treatment compared to patients with a lower predicted risk of ovarian cancer. Examples of selectable anti-cancer treatments include one or more of surgery (hysterectomy, BSO) and chemotherapy. For example, female human subjects predicted to have a high risk of ovarian cancer can be selected for a first anti-cancer treatment, while other female human subjects with a lower risk of ovarian cancer can be selected for a second, different anti-cancer treatment.

[0135] In one embodiment, the method includes selecting a patient monitoring schedule for a female human subject based on the female human subject's predicted risk of ovarian cancer. Thus, based on the predicted risk of ovarian cancer for the female human subject, an optimal or at least appropriate patient monitoring schedule or regimen is selected for the female human subject. This means that, compared to female human subjects with a predicted high risk of ovarian cancer, female human subjects with a predicted lower risk of ovarian cancer can be selected for more frequent monitoring and follow-up (a first monitoring schedule), while female human subjects with a predicted lower risk of ovarian cancer can instead follow less frequent monitoring and follow-up (a second monitoring schedule).

[0136] Example

[0137] Ovarian cancer is one of the most difficult tumors to detect and manage. It is often diagnosed in the late stages of the disease, which is associated with a poor prognosis. Therefore, detecting this cancer at an early stage is crucial for improving overall survival. In this example, TK1 protein and TK1 activity levels, as well as the biomarkers CA 125, HE4, and ROMA index, were determined. Elevated levels of TK1 protein were found in patients with both benign and malignant ovarian cancer. The combination of TK1 protein with CA 125 or HE4 showed higher sensitivity compared to the ROMA index. Therefore, TK1 protein is a promising serum biomarker that can complement CA 125 or HE4 in the early diagnosis of ovarian cancer.

[0138] Materials and methods

[0139] Study population and sample collection

[0140] This study included 134 serum samples from patients with ovarian tumors (72 benign tumors and 62 malignant ovarian cancers), as well as serum samples from 65 healthy women, who served as a control group. Between April 2018 and May 2021, serum samples were obtained from healthy women and women with ovarian tumors who visited the Department of Obstetrics and Gynecology, University Medical Centre Ljubljana. Blood samples were collected from all patients before surgery, and additional information about their lifestyle and gynecological and clinical status was obtained. For sample collection, strict standard operating procedures were followed, and serum was aliquoted and stored at -80°C until analysis. This study was approved by the National Medical Ethics Committee of the Republic of Slovenia (Nr.109 / 02 / 13). All patients gave written informed consent, agreeing to undergo diagnostic procedures and surgery and to participate in the study. Patient data were collected in the form of a prospective database design.

[0141] Measurement of CA 125 and HE4

[0142] In vitro quantitative fully automated electrochemiluminescence immunoassay (ECLIA) CA 125II and E411 immunoassay analyzer (Roche Diagnostics GmbH, Manheim, Germany) HE4 determines serum CA 125 and HE4 levels. The method is based on the electrochemiluminescence immunoassay (ECLIA) principle, combined with the sandwich immunoassay test principle. The reference ranges for serum HE4 and CA125 are <140 pmol / L and <35 kU / L, respectively[8].

[0143] Ovarian Malignancy Risk Algorithm (ROMA Index)

[0144] The ROMA index is calculated as follows before menopause: PI = -12.0 + (2.38 × LN [HE4]) + (0.0626 × LN [CA125]), and after menopause: PI = -8.09 + (1.04 × LN [HE4]) + (0.732 × LN [CA 125]), where LN = natural logarithm. As previously mentioned, premenopausal women have a higher risk of ovarian cancer with a ROMA value ≥ 11.4%, and postmenopausal women have a higher risk of ovarian cancer with a ROMA value ≥ 29.9% [8].

[0145] Serum TK activity (STK1a) and TK1 protein (STK1p) determination

[0146] As mentioned above, through All serum samples were analyzed for TK activity, and TK activity was expressed in U / L

[10] . TK1 protein levels in serum samples were measured using the AroCell TK 210 ELISA. The AroCell TK210 ELISA is a sandwich ELISA that utilizes two monoclonal anti-TK antibodies directed against the C-terminal region of human TK1. The assay was performed according to the manufacturer's instructions as previously described, and STK1p levels were expressed in ng / mL

[11] .

[0147] Statistical analysis

[0148] The normality of biomarker levels (including CA 125, HE4, STK1a, and STK1p levels) in healthy, benign, and malignant ovarian cancer serum samples was evaluated using the D'Agostino and Pearson comprehensive normality test. For continuous variables, the significance of differences was tested by the Mann-Whitney U test or the Wilcoxon signed rank test based on the comparison of independent samples or paired samples. Logistic regression analysis was performed to establish the best possible combination of biomarkers. The diagnostic performance of all possible combinations was evaluated by receiver operating characteristic (ROC) curves and area under the curve (AUC), with a confidence interval of 95% (95% CI). All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) and MedCalc 17.6. Statistical significance was achieved when P < 0.05.

[0149] result

[0150] Patient characteristics

[0151] A total of 199 women (134 patients with ovarian tumors and 65 healthy controls) were enrolled in this study between 2018 and 2021. Of the 134 women with ovarian tumors, 72 had benign tumors and 62 had malignant ovarian cancer. Serous tumors were the most common carcinoma (58%), followed by mucinous tumors (15%) and endometrial tumors (15%).

[0152] Among all patients, the mean ± SD age was 57.0 ± 3.7 years (range = 26.9-85.7). Twenty-one patients were premenopausal with an age range of 26.9-50.2 years (mean ± SD = 40.5 ± 3.3), while 41 patients were postmenopausal with an age range of 52.5-85.7 years (mean ± SD = 65.5 ± 2.9). In the benign ovarian disease group, 17% had ovarian endometriosis, 15% had serous cystadenoma, followed by 13% with serous cystadenofibroma and 10% with ovarian mucinous cystadenoma. The patient age ranged from 14.9 to 85.8 years, with a mean ± SD of 52.01 ± 3.8 years. Of the 72 patients with benign tumors, 33 were premenopausal with an age of 37.6 ± 3.4 years, and 39 were postmenopausal with an age of 54.3 ± 2.9 years. Of the 65 healthy controls, 43 were premenopausal with a mean ± SD age of 42.6 ± 2.5 years, and 21 were postmenopausal with a mean ± SD age of 55.1 ± 2.4 years. All patient characteristics are summarized in Table 1.

[0153] Table 1. Distribution of histologic type and disease stage among all patients and among premenopausal and postmenopausal women.

[0154]

[0155]

[0156] TK1 levels in different patient groups

[0157] AroCell TK 210 ELISA and The serum TK1 protein level (STK1p) and TK1 activity level (STK1a) were determined and compared with healthy controls (n=65), patients with benign ovarian cancer (n=72), and patients with malignant ovarian cancer (n=62). The STK1p concentration was significantly higher in the malignant ovarian cancer group compared with the benign and healthy controls (P<0.0001, Figure 1A). These results indicate that patients with malignant ovarian cancer have higher levels of STK1p compared to patients in benign and healthy controls. Comparison of STK1p between malignant and benign cancers showed significant differences between premenopausal and postmenopausal women ( Figure 1 B and 1C). For postmenopausal women, STK1p was significantly higher in the malignant group compared to the benign group, whereas in premenopausal women, there was no significant difference between the malignant and benign cancer groups (P = 0.34). STK1a values ​​showed another type of result, with no significant difference between the control group and either the malignant or benign ovarian cancer groups ( Figure 1 D). However, the group with malignant ovarian tumors had significantly higher STK1a levels compared to the group with benign cancer. In addition, no significant differences in STK1a levels were found among these groups in premenopausal women ( Figure 1 E). However, in postmenopausal women, significant differences in STK1a levels were observed between malignant and benign tumors and healthy controls ( Figure 1 F).

[0158] In all of these groups, CA 125 and HE4 values ​​were determined, and ROMA index values ​​were also calculated, as described in Materials and Methods. CA 125 levels were significantly higher in the malignant ovarian cancer group compared to the benign group and the healthy controls. Furthermore, CA 125 levels in the malignant group were significantly higher than those in the benign ovarian cancer group. However, both HE4 and ROMA index values ​​were significantly higher in the malignant ovarian cancer group compared to the healthy control group, but there were no differences between the benign and healthy control groups.

[0159] In the healthy control group, there was no significant difference in STK1p levels between premenopausal and postmenopausal women, nor in the benign and malignant ovarian cancer groups (Table 2). Similar results were observed with STK1a, CA 125, and HE4 in the healthy control group. In the benign cancer group, CA 125 levels were significantly higher in premenopausal women than in postmenopausal women. In contrast, HE4 levels were significantly higher in postmenopausal women than in premenopausal women in the benign cancer group (Table 2). In the malignant ovarian tumor group, STK1a, CA 125, and HE4 levels were significantly higher in postmenopausal women than in premenopausal women.

[0160] Table 2. STK1p, STK1a, CA 125 and HE4 levels in different groups.

[0161]

[0162]

[0163] The results of univariate and multivariate receiver operating characteristic (ROC) curve analysis are shown in Table 3. The area under the ROC curve (AUC) and standard error are based on the complete data set from 134 ovarian tumor serum samples and 64 healthy controls using STK1p, CA 125, HE4, STK1p+CA 125, STK1p+HE4, and STK1p+ROMA index biomarkers. Multivariate ROC analysis of the combination of STK1p, STK1p+CA 125, STK1p+HE4, and STK1p+ROMA index biomarkers was performed ( Figure 2A and 2B Among all possible combinations, the ROC AUC of three double combinations was higher than 0.90. The performance of these three combinations in distinguishing ovarian cancer samples from healthy controls was further evaluated.

[0164] Table 3. Univariate and multivariate ROC analysis of biomarkers.

[0165]

[0166]

[0167] All ovarian cancers versus all healthy controls

[0168] Figure 2A Shown are ROC curves for STK1p alone, CA 125 alone, HE4 alone, and dual markers (STK1p+CA 125, STK1p+HE4, and STK1p+ROMA index) when these combinations were calculated for 198 samples (134 ovarian tumors and 64 healthy controls). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each biomarker and combination were evaluated at the ROC curve cutoff point, with a specificity of approximately 95%.

[0169] Table 4 shows that STK1p combined with CA 125, HE4 and ROMA index achieved a sensitivity of more than 70% and a specificity of about 95%.

[0170] Table 4. ROC curve analysis of different biomarkers alone and in combination.

[0171] Biomarkers Cutoff value AUC Sensitivity Specificity PPV NPV STK1p 0.50 0.88 62.9％ 95.3％ 96.6％ 55.1％ STK1a 9.10 0.60 27.6％ 95.3％ 90.2％ 38.2％ CA 125 26.4 0.77 54.5％ 93.8％ 94.8％ 49.6％ HE4 65.5 0.71 47.8％ 93.8％ 94.1％ 46.2％ ROMA Index 16.7 0.73 42.5％ 95.3％ 95.0％ 44.2％ STK1p+CA 125 0.78 0.93 75.3％ 95.3％ 97.1％ 64.9％ STK1p+HE4 0.80 0.91 73.8％ 93.8％ 96.1％ 63.2％ STK1p+ROMA index 0.83 0.91 70.2％ 95.3％ 96.9％ 60.4％ STK1a+CA 125 0.76 0.78 49.3％ 95.3％ 95.7％ 47.3％ STK1a+HE4 0.72 0.71 47.1％ 95.3％ 95.5％ 46.2％ STK1a+ROMA Index 0.73 0.73 44.1％ 95.3％ 95.2％ 44.9％

[0172] Figure 2BROC curves are shown for STK1a alone, CA 125 alone, HE4 alone, and dual markers (STK1a+CA125, STK1a+HE4, and STK1a+ROMA Index). Overall, STK1a and STK1a in combination with other assays showed lower sensitivity compared to STK1p. In addition, the combination of CA 125 and STK1p showed higher sensitivity and PPV compared to either assay alone (Table 4).

[0173] Based on these results, further analysis was performed using the STK1p, STK1p+CA125, STK1p+HE4, and STK1p+ROMA indices. Patient sera were divided into all premenopausal women, all postmenopausal women, benign cancer, and malignant cancer subgroups. As shown in Table 5, the assay performance for these subgroups was evaluated by ROC curve analysis.

[0174] Table 5. ROC curve analysis of STK1p combined with other biomarkers in different subgroups.

[0175]

[0176]

[0177] Performance evaluation of dual biomarkers in subgroups of ovarian cancer patients

[0178] The performance of the combination of biomarkers and STK1p was evaluated in a subgroup of ovarian cancer patients using ROC curve analysis. The diagnostic performance of STK1p alone, STK1p+CA 125, STK1p+HE4, and STK1p+ROMA index are shown in Figure 3 and Table 5. In the detection of all stages of cancer, for premenopausal women, the AUC of STK1p+CA125 was 0.945 (95% CI: 0.879-0.981) ( Figure 3A ), which was higher than the STK1p+ROMA index (AUC=0.877, 95% CI: 0.795-0.936), and for postmenopausal women, the AUC of the STK1p+ROMA index (AUC=0.936, 95% CI=0.87-0.97) was higher than the AUC of STK1p+CA125 (AUC=0.920, 95% CI=0.85-0.965) ( Figure 3BIn the detection of benign tumors from healthy controls, the AUC of STK1p+CA 125 (AUC=0.914, 95% CI=0.85-0.95) was higher than that of STK1p+HE4 (AUC=0.877, 95% CI=0.81-0.927) and STK1p+ROMA index (AUC=0.876, 95% CI=0.809-0.926) ( Figure 3C For the discrimination of malignant ovarian cancer from healthy controls, all three dual biomarkers (i.e., STK1p+CA 125, STK1p+HE4, and STK1p+ROMA index) showed higher AUCs compared with STK1p alone (Table 5) ( Figure 3D In the differentiation of benign masses from malignant ovarian cancer, the AUC of STK1p+ROMA index (AUC=0.819, 95% CI=0743-0.88), the AUC of STK1p+HE4 (AUC=0.817, 95% CI=0.741-0.878), and the AUC of STK1p+CA 125 (AUC=0.731, 95% CI=0.648-0.804) were significantly higher than that of STK1p alone (AUC=0.60, 95% CI=0.52-0.685) (Table 5) ( Figure 3E ). These results indicate that the combination of STK1p+CA 125 gave the best diagnostic performance in the detection of early-stage ovarian cancer compared with the benign tumor group.

[0179] For premenopausal women, STK1p + CA 125 showed higher PPV and NPV than STK1p + ROMA index (95.7% vs. 95.1% and 80.0% vs. 72.7%). In the case of postmenopausal women, the PPV values ​​of all three combinations were similar (100%), among which the NPV of STK1p + ROMA index was higher (56.4%), followed by STK1p + CA 125 (51.2%) and STK1p + HE4 (50.0%). These results strongly suggest that the dual biomarker STK1p + CA 125 has the best diagnostic performance in detecting benign as well as malignant ovarian cancer compared with healthy controls. However, in the distinction between benign and malignant ovarian cancer, the combination of STK1p + ROMA index gave a higher sensitivity (66.1%) compared with other combinations, as shown in Table 5.

[0180] Similar analyses were performed using STK1a in these subgroups. In this case, STK1a+CA 125 demonstrated the highest sensitivity for distinguishing premenopausal women, benign and malignant ovarian tumors, from healthy controls, compared to the STK1a+HE4 and STK1a+ROMA indices. For distinguishing postmenopausal women with cancer, the STK1a+ROMA index demonstrated higher sensitivity compared to the other combinations. These results are similar to the combination of STK1p and CA125, which demonstrated the highest sensitivity for early detection of ovarian cancer.

[0181] Based on these results, further analysis was performed using STK1p+CA 125, STK1p+HE4, STK1a+CA 125, and STK1a+HE4 combinations. Serum levels of malignant ovarian cancer were subclassified based on FIGO stage, and STK1p+CA 125 was significantly higher in stage III+IV patients compared with stage I+II patients (P<0.0001, Figure 4 A) and STK1a+CA 125 (P=0.0104, Figure 4 In addition, ROC analysis showed that STK1p+CA 125 (AUC=0.81, sensitivity=50%, specificity=92%) had a higher ability to distinguish stage I+II from stage III+IV compared with STK1a+CA 125 (AUC=0.69, sensitivity=35.7%, specificity=92%). Figure 4 C). Similar results were obtained with the combination of STK1p+HE4 and STK1a+HE4 ( Figure 4 D and 4E), and ROC curve analysis showed that the AUC of STK1p+HE4 was 0.82, and the AUC of STK1a+HE4 was 0.80.

[0182] STK1p and STK1a levels before and after surgery

[0183] Patients with benign and malignant ovarian cancer (n=123) were followed up after surgery, and all (P=0.0002, Figure 5 A), premenopausal women (P = 0.0014, Figure 5 B) and postmenopausal women (P = 0.021, Figure 5 C), STK1p levels were significantly reduced after surgery. Among the 123 patients, 81 patients (61%) had a decreasing trend in STK1p levels compared with preoperative levels. In contrast, there was no significant difference in STK1a levels between preoperative and postoperative patients with benign and malignant cancers ( Figure 5 D, 5E and 5F).

[0184] In addition, there was a significant correlation between the ratio of STK1p at diagnosis / STK1p after surgery and the number of days after surgery ( Figure 6 A)(rs=0.25, P=0.0072). In addition, STK1p was measured in 23 patients with malignant ovarian cancer after chemotherapy. Except for 4 patients, STK1p levels in the remaining patients were significantly decreased after chemotherapy (P=0.013, Figure 6 B). During the follow-up period, 9 of the 32 patients experienced disease recurrence. Patients who relapsed after chemotherapy had significantly higher levels of STK1p+CA125 and STK1p+HE4 ( compared to patients who did not relapse). Figure 6 C and 6D). Figure 6 Similar results were observed with STK1a, as shown in E and 6F.

[0185] The results of this study suggest that combining STK1p with either CA125 or HE4 can improve the sensitivity and specificity of early detection of ovarian cancer. Combining these biomarkers offers a way to improve detection of patients with ovarian cancer at earlier stages of the disease, leading to a higher chance of curative treatment.

[0186] The above embodiments are to be understood as some illustrative examples of the present invention. Those skilled in the art will appreciate that various modifications, combinations, and variations may be made to the embodiments without departing from the scope of the present invention. Specifically, where technically possible, different partial solutions in different embodiments may be combined in other configurations. However, the scope of the present invention is defined by the appended claims.

[0187] References

[0188] 1.Hellstrom, et al., The HE4(WFDC2)protein is a biomarker for ovariancarcinoma, Cancer research, (2003) 63(13): 3695-3700.

[0189] 2.Drapkin, et al., Human epididymis protein 4(HE4) is a secretedglycoprotein that is overexpressed by serous and endometrioid ovariancarcinomas, Cancer research, (2005) 65(6): 2162-2169.

[0190] 3.Hada et al.,A,Comparison of the predictive performance of risk ofmalignancy indexes 1-4,HE4 and risk of malignancy algorithm in the triage ofadnexal masses,Joumal of ovarian research,(2020)13(1):1-9.

[0191] 4.Li,et al.,Does risk for ovarian malignancy algorithm excel humanepididymis protein 4 and CA125 in predicting epithelial ovarian cancer:ameta-analysis,BMC Cancer,(2012)12(1):1-18.

[0192] 5.Molina et al.,HE4 a novel tumor marker for ovarian cancer:comparison with CA 125 and ROMA algorithm in patients with gynecologicaldiseases,Tumor Biology,(2011)32(6):1087-1095.

[0193] 6.Moore,et al.,The use of multiple novel tumor biomarkers for thedetection of ovarian carcinoma in patients with a pelvic mass,GynecologicOncology,(2008)108(2):402-408.

[0194] 7.Jacob et al.,No benefit from combining HE4 and CA125 as ovariantumor markers in a clinical setting,Gynecologic oncology,(2011)121(3):487-491.

[0195] 8.Zhu,et al.,A combined strategy of TK1,HE4 and CA125 shows betterdiagnostic performance than risk of ovarian malignancy algorithm(ROMA)inovarian carcinoma,Clinica Chimica Acta,(2022)524:43-50.

[0196] 9.Jagarlamudi et al.,Breast,and prostate cancer patients differsignificantly in their serum thymidine kinase 1(TK1)specific activitiescompared with those hematological malignancies and blood donors:implicationsof using serum TK1 as a biomarker,BMC Cancer(2015)15:66.

[0197] 10.Ohrvik et al.,Sensitive nonradiometric method for determiningthymidine kinase 1 activity,Clinical Chemistry,(2004)50(9):1597-1606.

[0198] 11.Jagarlamudi et al.,Analytical and clinical characterization of anoptimized dual monoclonal sandwich ELISA for the quantification of thymidinekinase 1(TK1)protein in human blood samples,PLoS One,(2022)17(10):e0275444.

[0199] 12.Xi et al.,Research on application value of combined detection ofserum CA125,HE4 and TK1 in the diagnosis of ovarian cancer,European Reviewfor Medical and Pharmacological Sciences(2017)21:4536-4541.

Claims

1. A method for predicting the risk of ovarian cancer in a female human subject, the method comprising: determining the amount of serum thymidine kinase 1 (STK1) substance in a serum or plasma sample from the female human subject using a kit comprising a first monoclonal antibody or a first antigen-binding fragment thereof that specifically binds to a serum form of human TK1 and a second monoclonal antibody or a second antigen-binding fragment thereof that specifically binds to the serum form of human TK1; determining the amount of cancer antigen 125 (CA 125) or human epididymis protein 4 (HE4) in the serum or plasma sample or another serum or plasma sample from the female human subject; and The risk of ovarian cancer in the female human subject is predicted based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4.

2. The method of claim 1, wherein the female human subject is in a premenopausal state. 3 . The method of claim 1 , wherein predicting the risk of ovarian cancer comprises predicting the ovarian cancer stage of the female human subject based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4.

4. The method of claim 3, wherein predicting the ovarian cancer stage comprises predicting whether the female human subject has FIGO stage I or II ovarian cancer or FIGO stage III or IV ovarian cancer based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4.

5. The method according to claim 1 or 2, wherein Determining the amount of the STK1 substance comprises determining the amount of the STK1 substance in a serum or plasma sample obtained from the female human subject after treatment, preferably chemotherapy, for ovarian cancer using the first monoclonal antibody or the first antigen-binding fragment thereof that specifically binds to the serum form of human TK1 and the second monoclonal antibody or the second antigen-binding fragment thereof that specifically binds to the serum form of human TK1; Determining the amount of CA 125 or HE4 comprises determining the amount of CA 125 or HE4 in said serum or plasma sample or another serum or plasma sample taken from said female human subject after ovarian cancer treatment, preferably chemotherapy; and Predicting the risk of ovarian cancer includes predicting ovarian cancer recurrence in the female human subject based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4. The method according to claim 1 or 2, wherein predicting the risk of ovarian cancer comprises predicting whether the female subject has ovarian cancer or does not have ovarian cancer based on the amount of the STK1 substance and the amount of one but not both of CA 125 and HE4. 7 . The method of claim 6 , wherein predicting the risk of ovarian cancer comprises predicting whether the female subject has benign ovarian cancer or does not have ovarian cancer based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4.

8. The method of claim 6, wherein predicting the risk of ovarian cancer comprises predicting whether the female subject has malignant ovarian cancer or does not have ovarian cancer based on the amount of the STK1 substance and the amount of one, but not both, of CA 125 and HE4.

9. The method according to any one of claims 1 to 8, wherein Determining the amount of CA 125 or HE4 comprises determining the amount of CA 125 in the serum or plasma sample or the further serum or plasma sample from the female human subject; and Predicting the risk of ovarian cancer includes predicting the risk of ovarian cancer in the female human subject based on the amount of the STK1 substance and the amount of CA 125 but not the amount of HE4. 10 . The method of claim 9 , wherein predicting the risk of ovarian cancer comprises predicting the risk of ovarian cancer in the female human subject based solely on the amount of the STK1 substance and the amount of CA 125.

11. The method according to any one of claims 1 to 10, wherein determining the amount of the STK1 substance in the serum or plasma sample comprises: contacting the serum or plasma sample with the first monoclonal antibody or the first antigen-binding fragment thereof and the second monoclonal antibody or the second antigen-binding fragment thereof; and The amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment bound to the STK1 substance is measured. 12 . The method of claim 11 , further comprising correlating the measured amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment thereof bound to the STK1 substance with the amount of the STK1 substance.

13. The method of claim 12, wherein associating the measured amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment thereof comprises correlating the measured amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment thereof with the amount of STK1 substance using a predefined correlation between the measured amount of the first monoclonal antibody or the first antigen-binding fragment thereof or the second monoclonal antibody or the second antigen-binding fragment thereof that binds to recombinant human TK1 and the concentration of recombinant human TK1.

14. The method according to any one of claims 1 to 13, further comprising adding a sample dilution buffer to the serum or plasma sample, wherein the sample dilution buffer comprises: Adenosine triphosphate, preferably, its concentration is selected in the interval of 0.5 mM to 50 mM, preferably in the interval of 0.5 mM to 20 mM, and more preferably in the interval of 1.5 mM to 50 mM; and The reducing agent is preferably selected from the group consisting of dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamine (DTBA), tris(2-carboxyethyl)phosphine) (TCEP) and any combination thereof, and preferably, its concentration is selected in the range of 0.1 mM to 10 mM.

15. The method according to any one of claims 1 to 14, wherein One of the first monoclonal antibody or the first antigen-binding fragment thereof and the second monoclonal antibody or the second antigen-binding fragment thereof is specific for a peptide consisting of an amino acid sequence derived from the C-terminal region of TK1; and The other of the first monoclonal antibody or the first antigen-binding fragment thereof and the second monoclonal antibody or the second antigen-binding fragment thereof has specificity for a peptide selected from the group consisting of: a peptide consisting of an amino acid sequence derived from the C-terminal region of TK1; a peptide consisting of another amino acid sequence derived from the C-terminal region of TK1; and a peptide consisting of an amino acid sequence derived from the active site of TK1, preferably a peptide consisting of another amino acid sequence derived from the C-terminal region of TK1.

16. The method according to any one of claims 1 to 15, wherein The first monoclonal antibody or the first antigen-binding fragment thereof is selected from the group consisting of: a monoclonal antibody or antigen-binding fragment thereof specific for GEAVAARKLF (SEQ ID NO: 1) of human TK1; A monoclonal antibody or antigen-binding fragment thereof specific for at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and A monoclonal antibody or antigen-binding fragment thereof that is specific for a conformation-dependent epitope of human TK1; and The second monoclonal antibody or the second antigen-binding fragment thereof is selected from the group consisting of: a monoclonal antibody or antigen-binding fragment thereof specific for GEAVAARKLF (SEQ ID NO: 1) of human TK1; A monoclonal antibody or antigen-binding fragment thereof specific for at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and Monoclonal antibodies or antigen-binding fragments thereof specific for a conformation-dependent epitope of human TK1 17. The method of claim 15 or 16, wherein the first monoclonal antibody or the first antigen-binding fragment thereof is selected from the group consisting of: A monoclonal antibody or antigen-binding fragment thereof having the following regions a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 6; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 7; a variable light (VL) domain CDR1 having the amino acid sequence of SEQ ID NO: 8; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 9; and a VL domain CDR3 having the amino acid sequence of SEQ ID NO: 10; A monoclonal antibody or antigen-binding fragment thereof having the following regions has a VH domain CDR1 having the amino acid sequence of SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 11; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 12; a VL domain CDR1 having the amino acid sequence of SEQ ID NO: 13; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 9; and a VL domain CDR3 having the amino acid sequence of SEQ ID NO: 10; or A monoclonal antibody or antigen-binding fragment thereof having the following regions has a VH domain CDR1 of the amino acid sequence of SEQ ID NO: 14; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 15; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 16; a VL domain CDR1 having the amino acid sequence of SEQ ID NO: 17; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 18; and The VL domain CDR3 has the amino acid sequence of SEQ ID NO:

19.

18. The method of any one of claims 15 to 17, wherein the second monoclonal antibody or the second antigen-binding fragment thereof is selected from the group consisting of: A monoclonal antibody or antigen-binding fragment thereof having the following regions a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 6; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 7; a variable light (VL) domain CDR1 having the amino acid sequence of SEQ ID NO: 8; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 9; and a VL domain CDR3 having the amino acid sequence of SEQ ID NO: 10; A monoclonal antibody or antigen-binding fragment thereof having the following regions a VH domain CDR1 having the amino acid sequence of SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 11; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 12; a VL domain CDR1 having the amino acid sequence of SEQ ID NO: 13; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 9; and a VL domain CDR3 having the amino acid sequence of SEQ ID NO: 10; or A monoclonal antibody or antigen-binding fragment thereof having the following regions a VH domain CDR1 having the amino acid sequence of SEQ ID NO: 14; a VH domain CDR2 having the amino acid sequence of SEQ ID NO: 15; a VH domain CDR3 having the amino acid sequence of SEQ ID NO: 16; a VL domain CDR1 having the amino acid sequence of SEQ ID NO: 17; a VL domain CDR2 having the amino acid sequence of SEQ ID NO: 18; and The VL domain CDR3 has the amino acid sequence of SEQ ID NO:

19.

19. The method of any one of claims 1 to 18, wherein one of the first monoclonal antibody or the first antigen-binding fragment thereof and the second monoclonal antibody or the second antigen-binding fragment thereof is immobilized or intended to be immobilized to a solid support.

20. The method according to any one of claims 1 to 19, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

21. The method of any one of claims 1 to 20, wherein determining the amount of CA 125 or HE4 comprises determining the amount of CA 125 or HE4 in the serum or plasma sample or the further serum or plasma sample from the female human subject using an electrochemiluminescence immunoassay (ECLIA) kit.

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