STK1-based treatment stratification

Abstract

A treatment stratification method comprising determining the level of STK1 substance in a bodily fluid sample from a patient diagnosed with prostate cancer using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form. The method further comprises comparing the level of the STK1 substance to a threshold level, selecting chemotherapy for the patient if the level of the STK1 substance is equal to or above the threshold, and selecting androgen receptor signaling inhibitor (ARSI) therapy for the patient if the level of the STK1 substance is below the threshold.

Description

Technical Field

[0001] This invention generally relates to the measurement of thymidine kinase 1 (TK1), and more particularly to treatment stratification of prostate cancer patients based on measured serum TK1 (STK1) levels. Background Technology

[0002] Thymidine kinase 1 (TK1) (EC 2.7.1.21), also known as 2'-deoxythymidine kinase or ATP-thymidine 5'-phosphotransferase, is an enzyme involved in the synthesis of deoxyribonucleic acid (DNA) precursors. TK1 phosphorylates thymidine to allow incorporation into DNA. TK1 expression is a marker of active cell proliferation, with low intracellular concentrations during the G0 / G1 phase of the cell cycle and increased concentrations during the S / G2 phase.

[0003] TK1 is also present at high levels in the serum and plasma of people and animals with malignant tumors. Therefore, serum TK1 activity measurements have been used for monitoring and prognostic purposes for several different malignant diseases, but primarily for leukemia and lymphoma.

[0004] Furthermore, TK1 is the only proliferation marker that can be measured in blood, and if used as a routine laboratory test, it could provide significant clinical benefits.

[0005] Using radioactive substrates 125 The I-dUrd (PROLIFIGEN® TK-REA, DiaSorin Inc.) assay for serum TK1 activity has been available for decades, but its applications are limited, and it is preferred for hematologic malignancies. In recent years, a non-radioactive TK1 activity assay (TK LIAISON® assay, DiaSorin Inc.) has become available. This is a sensitive and stable detection method that provides clinically valuable information primarily in hematologic malignancies, particularly for monitoring treatment and predicting recurrence.

[0006] Over the past 15 years, antibodies against human TK1 have become available and have been able to identify TK1 protein levels that are opposite to TK1 activity in hematologic and solid tumor diseases such as prostate cancer and several other forms of solid tumors and hematologic malignancies.

[0007] Breast and prostate cancer patients differsignificantly in their serum thymidine kinase 1 (TK1) specific activities compared with those hematological malignancies and blood donors: implications of using serum TK1 as a biomarker, published by Jagarlamudi et al. BMC Cancer (2015), 15: 66 disclosed that there were differences in the specific activity and composition of active and inactive subunits of TK1 in the serum of patients with hematologic malignancies compared with patients with breast cancer and prostate cancer.

[0008] Li et al. published a study showing that Serum thymidine kinase 1 is associated with Gleason scores in patients with prostate carcinoma. Oncology Letters (2018), 16(5):6171-6180 disclosed that serum TK1 concentration is a more reliable prognostic biomarker than total prostate-specific antigen (PSA) in screening for benign prostatic hyperplasia (BPH) or prostate cancer, based on the Gleason score.

[0009] Jagarlamudi et al. published an article titled "AroCell TK 210 ELISA for determination of TK1 protein: age-related reference ranges and comparison with other TK1 assays." Biotechniques (2020) 68(6): 335-342 disclosed that serum from prostate cancer and breast cancer patients showed significantly higher TK1 protein levels compared to serum from healthy blood donors.

[0010] Analytical and clinical characterization of anoptimized dual monoclonal sandwich ELISA for the quantification of thymidinekinase 1 (TK1) protein in human blood samples published by Jagarlamudi et al. PLoS One (2022) 17(10): e027544 disclosed that receiver operating characteristic (ROC) curve analysis showed that in prostate patients, the measurement of STK1 protein levels had higher sensitivity than the measurement of enzyme activity.

[0011] The combination of AroCell TK 210 ELISA with Prostate Health Index of prostate-specific antigen density can improve theability to differentiate prostate cancer from noncancerous conditions, published by Jagarlamudi et al. The Prostate (2019), 79(8): 856-863 disclosed that serum TK1, measured by the AroCell TK 210 ELISA, was significantly higher in patients with prostate cancer than in patients with benign urinary tract diseases. Serum TK1 was associated with prostate health index but not with Gleason score. Summary of the Invention

[0012] The overall objective of this invention is to achieve treatment stratification for patients diagnosed with prostate cancer.

[0013] This and other objectives are achieved through the implementation plan disclosed here.

[0014] This invention is defined in the independent claims. Other embodiments of the invention are defined in the dependent claims.

[0015] One aspect of the invention relates to a treatment stratification method. The method includes determining the level of serum thymidine kinase 1 (STK1) substance in a bodily fluid sample from a patient diagnosed with prostate cancer, using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form. The method further includes comparing the STK1 substance level to a threshold level; if the STK1 substance level is equal to or higher than the threshold, chemotherapy is selected for the patient; if the STK1 substance level is lower than the threshold, and androgen receptor signaling inhibitor (ARSI) therapy is selected for the patient.

[0016] This invention enables treatment stratification for patients diagnosed with prostate cancer. Therefore, this invention can help physicians select the most appropriate treatment for prostate cancer patients in terms of reducing their risk ratio (HR), thereby improving patient survival. Attached Figure Description

[0017] The implementation scheme and its further objectives and advantages can be best understood by referring to the following description in conjunction with the accompanying drawings, wherein: Figure 1 shows the Kaplan-Meier survival curves of mHSPC patients in an analysis adjusted for age (A) and PSA at diagnosis (B).

[0018] Figure 2 The random forest classification error estimates for prostate cancer mortality are shown, representing the mean prediction error and category-specific prediction error for survival and prostate cancer mortality. The error bars represent the 95% confidence intervals of the empirical estimates of the Monte Carlo error. The classifiers used in each model are written on the right side of the image, on the y-axis.

[0019] Figure 3 shows the Kaplan-Meier survival curves for mCRPC patients after bisecting STK1 levels with a cutoff of 0.61 µg / L. Data represent overall survival. (A) All 197 mCRPC patients, unstratified by treatment; (B) 99 mCRPC patients treated with abiraterone or enzalutamide; (C) 98 mCRPC patients treated with docetaxel; (D) All 197 mCRPC patients, stratified by treatment group. Detailed Implementation

[0020] This invention generally relates to the measurement of thymidine kinase 1 (TK1), and more particularly to treatment stratification of prostate cancer patients based on measured serum TK1 (STK1) levels.

[0021] Prostate cancer is the most common non-cutaneous malignancy among men in developed countries and is the second to third leading cause of cancer-related death. Localized prostate cancer can often be effectively controlled with surgery or radiation therapy. The cornerstone of treatment for advanced hormone-sensitive prostate cancer (HSPC) is androgen deprivation therapy (ADT). ADT is usually used in combination with taxane-based chemotherapy or androgen receptor signaling inhibitors (ARSIs), such as enzalutamide or the CYP17 inhibitor abiraterone. Ultimately, despite ADT and combination therapy, prostate cancer retains its ability to grow, leading to castration-resistant prostate cancer (CRPC). Treatment after CRPC development typically involves ARSIs and taxane-based chemotherapy. Nevertheless, clinical responses to these treatments vary, and side effects are common. To improve the effectiveness of prostate cancer treatment while minimizing any side effects, procedures that guide decisions regarding available treatment options for different types of prostate cancer are particularly desirable.

[0022] Previous studies mentioned in the background section have shown that serum TK1 expression can be used as a biomarker for the presence of prostate cancer. The experimental data presented in this article show that STK1 levels can be used for treatment stratification in prostate cancer patients, thus serving as a tool to assist clinicians in selecting the most appropriate treatment for prostate cancer patients. This means that STK1 levels measured in bodily fluid samples from subjects or patients diagnosed with prostate cancer, particularly metastatic prostate cancer, can be used to select prostate cancer treatment for patients.

[0023] This means that when choosing the optimal treatment for prostate cancer patients, STK1 levels measured in bodily fluid samples from patients diagnosed with prostate cancer can be used for prostate cancer risk stratification. Therefore, prostate cancer patients with high STK1 levels will have a lower risk ratio if treated with the first type of anticancer therapy (chemotherapy), while those with relatively low STK1 levels will have a lower risk ratio if treated with a second, different type of anticancer therapy (androgen receptor signaling inhibitor (ARSI) therapy).

[0024] Therefore, one aspect of the present invention relates to a treatment stratification method. The method includes determining the level of STK1 substance in a bodily fluid sample from a patient diagnosed with prostate cancer using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form. The method further includes comparing the STK1 substance level to a threshold; if the STK1 substance level is equal to or higher than the threshold, chemotherapy is selected for the patient; if the STK1 substance level is lower than the threshold, ARSI therapy is selected for the patient.

[0025] Therefore, this invention is based on the unexpected finding that STK1 levels measured in patients with and diagnosed with prostate cancer are associated with prostate cancer patient survival, as indicated by the hazard ratio (HR) for prostate cancer patients. The experimental data presented herein show that prostate cancer patients with elevated STK1 levels (i.e., equal to or above the threshold) benefit from the first type of prostate cancer treatment (chemotherapy), while prostate cancer patients with lower STK1 levels (i.e., below the threshold) benefit from the second type of prostate cancer treatment (ARSI therapy).

[0026] In fact, for prostate cancer patients with low STK1 levels, ARSI therapy leads to a higher survival rate compared to chemotherapy, such as... Figure 3D As shown. This is quite surprising, because more aggressive chemotherapy is believed to be beneficial for this group of prostate cancer patients as well. Therefore, for this group of prostate cancer patients with relatively low STK1 levels, if patients receive ARSI treatment instead of chemotherapy, the survival probability increases and the hazard ratio (HR) decreases. However, in the group of prostate cancer patients with STK1 levels equal to or above the threshold, as Figure 3D As shown, chemotherapy is associated with a significantly higher survival probability compared to ARSI therapy.

[0027] Therefore, prostate cancer patients with STK1 levels below the threshold benefit from ARSI therapy, while prostate cancer patients with STK1 levels equal to or above the threshold benefit from chemotherapy.

[0028] In one embodiment, the patient is diagnosed with metastatic prostate cancer. In such an embodiment, the method includes determining the level of STK1 substance in a bodily fluid sample from a patient diagnosed with metastatic prostate cancer using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form.

[0029] Patients diagnosed with metastatic prostate cancer are typically diagnosed with either metastatic hormone-sensitive prostate cancer (mHSPC) or metastatic castration-resistant prostate cancer (mCRPC). mHSPC patients are usually treated with adenosine monotherapy (ADT) or in combination with chemotherapy or an anti-inflammatory drug injection (ARSI). However, prostate cancer can eventually develop the ability to grow even under ADT treatment, leading to castration-resistant prostate cancer (CRPC). Once CRPC develops, treatment typically includes ARSI therapy or chemotherapy.

[0030] In one embodiment, a prostate cancer patient is diagnosed with metastatic castration-resistant prostate cancer. In such an embodiment, the method includes determining the level of STK1 substance in a bodily fluid sample from a patient diagnosed with metastatic castration-resistant prostate cancer using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form.

[0031] ARSIs include androgen synthesis inhibitors (ASIs), such as abiraterone, and androgen receptor antagonists (ARAs), such as enzalutamide, apatamide, and dalotamide.

[0032] Abiraterone acetate is an androgen synthesis inhibitor derived from pregnenolone. It is an irreversible inhibitor of CYP17 gene products 17,20-lyase and 17-α-hydroxylase. Abiraterone inhibits androgen production in the testes, adrenal glands, and tumor cells.

[0033] ARAs block the androgen-binding site of the androgen receptor (AR) and inhibit AR nuclear translocation and subsequent AR binding to nuclear DNA. This leads to reduced activation of coactivators, resulting in apoptosis and a decrease in prostate tumor volume. First-generation ARAs, including bicalutamide, nilutetide, and flutamide, do not completely block AR activity. Enzalutamide, apatamide, and dalotamide are currently used second-generation ARAs, which have no agonistic activity against AR compared to first-generation ARAs.

[0034] Chemotherapy for prostate cancer patients includes paclitaxel-based chemotherapy, such as docetaxel and cabazitaxel, mitoxantrone and estradiol.

[0035] In one implementation, the method includes selecting chemotherapy for the patient if the level of STK1 substance is equal to or higher than a threshold, and selecting ARSI therapy for the patient if the level of STK1 substance is lower than a threshold.

[0036] In one embodiment, chemotherapy is selected from the group consisting of docetaxel, cabazitaxel, mitoxantrone, estradiol, and any combination thereof, with docetaxel being preferred. The chemotherapy treatment described herein also includes other types of anticancer treatment besides chemotherapy, such as combinations of chemotherapy and ADT, combinations of chemotherapy and steroids such as prednisone, and combinations of chemotherapy and ARSIs. For example, docetaxel treatment is often used in combination with the steroid prednisone.

[0037] In one implementation, ARSI is selected from the group consisting of ASI therapy, ARA therapy, and any combination thereof. In the case of ASI therapy, a single ASI or a combination of multiple (i.e., at least two) ASIs can be administered to a prostate cancer patient. Correspondingly, in the case of ARA therapy, a single ARA or a combination of multiple ARAs can be administered to a prostate cancer patient. Furthermore, as an example of ARSI therapy, a combination of at least one ASI and at least one ARA can be administered to a prostate cancer patient. ARSI therapy as used herein also includes combinations of ARSI therapy and at least one other anticancer therapy, provided that said at least one other anticancer therapy is not chemotherapy. For example, a combination of ARSI therapy and ADT can be used.

[0038] In one implementation, ASI is abiraterone.

[0039] In one implementation, ARA is selected from the group consisting of enzalutamide, apalutamide, dalotamide, and any combination thereof.

[0040] Prostate cancer patients (especially mCRPC patients) with measured STK1 levels below a threshold showed a higher survival probability when receiving ARSI therapy, represented by ASI drugs such as abiraterone or ARA drugs such as enzalutamide, compared to mCRPC patients receiving chemotherapy, represented by docetaxel. Therefore, the treatment stratification method of the present invention preferably selects ARSI therapy for prostate cancer patients, particularly mCRPC patients, with measured STK1 levels below a threshold. This ARSI therapy is not only beneficial in terms of lower hazard ratios and increased survival probability, but also generally has fewer or at least less severe side effects compared to chemotherapy.

[0041] However, cancer patients (especially mCRPC patients) with STK1 levels measured at or above a threshold showed a higher survival probability when receiving chemotherapy, such as docetaxel, compared to mCRPC patients receiving ARSI therapies, such as abiraterone (an ASI drug) or enzalutamide (an ARA drug). Therefore, the treatment stratification method of the present invention is preferably selected for chemotherapy in prostate cancer patients, particularly mCRPC patients, who have measured STK1 levels equal to or above a threshold.

[0042] In one embodiment, the threshold is selected within the range of 0.5 to 0.7 μg / L. In a preferred embodiment, the threshold is selected within the range of 0.55 to 0.65 μg / L, more preferably within the range of 0.57 to 0.63 μg / L, most preferably within the range of 0.59 to 0.63 μg / L, for example, within the range of 0.60 to 0.62 μg / L. Currently, a preferred threshold is 0.61 μg / L.

[0043] The preferred range of the above thresholds was determined based on the analysis of mHSPC patients, but it also applies to mCRPC patients.

[0044] In one implementation, the method further includes obtaining a body fluid sample from the patient.

[0045] A body fluid sample is a body fluid sample containing the substance STK1. Non-limiting but preferred examples of such body fluid samples include serum samples, plasma samples, and blood samples, with serum samples or plasma samples being preferred.

[0046] TK1 exists in humans in various forms, depending on the presence of certain molecules, such as the presence or absence of adenosine triphosphate (ATP); the concentration of the protein, i.e., high or low concentration; the type of protein, i.e., native or recombinant TK1; and the location of the protein, i.e., in serum or cytoplasm.

[0047] Typically, cytoplasmic and recombinant human TK1 exist as tetramers in the presence or at high concentrations of ATP, and as dimers in the absence or at low concentrations of ATP. The tetramer form of cytoplasmic and recombinant human TK1 exhibits high TK1 activity, while the dimer form has lower TK1 activity. Cytoplasmic TK1, also known as cellular TK1, is the TK1 present within cells and can be isolated from these cells.

[0048] In stark contrast, human STK1 can be in the form of high molecular weight complexes with TK1 activity, such as oligomers or containing such oligomers, as well as dimer and tetramer forms with very low or even no TK1 activity. Oligomerization appears to be associated with the formation of disulfide crosslinks that occur in the blood. STK1 is present in the blood of patients and can therefore be measured in blood samples, plasma samples, or serum samples, etc.

[0049] The STK1 substance used in this article refers to various forms of STK1, such as dimers, tetramers, oligomers, and STK1-containing complexes. STK1 substance is serum TK1 substance, i.e., STK1 substance present in a patient's blood, plasma, or serum. STK1 substance may then contain the aforementioned forms of STK1, such as dimers, tetramers, oligomers, and STK1-containing complexes. STK1 substance also includes complexes having at least one TK1 protein unit and other molecules and / or macromolecules.

[0050] In this field, various gene expression arrays have been proposed to determine TK1 mRNA transcripts in cancer cell samples and biopsy tissues (including prostate cell samples and prostate biopsy tissues). As mentioned above, TK1 exists in multiple forms in patients, including cytoplasmic TK1 and serum TK1. Gene expression arrays that measure TK1 mRNA transcripts from such cell or biopsy tissue samples primarily analyze TK1 mRNA transcripts of cytoplasmic TK1 present in cancer cells. Therefore, such gene expression arrays cannot be used to measure STK1 levels in subjects.

[0051] In one embodiment, determining the level of the STK1 substance involves contacting a body fluid sample with an antibody or an antigen-binding fragment thereof. This embodiment also includes measuring the amount of antibody or antigen-binding fragment thereof bound to the STK1 substance.

[0052] Contact between a bodily fluid sample and an antibody or its antigen-binding fragment can be achieved by adding the antibody or its antigen-binding fragment to the bodily fluid sample and incubating the sample together with the antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment then binds to STK1 substance, forming a complex between the antibody or its antigen-binding fragment and STK1 substance. In such an embodiment, measuring the amount of antibody or its antigen-binding fragment bound to STK1 substance can include measuring or quantifying the complex between the antibody or its antigen-binding fragment and STK1 substance, thereby measuring or quantifying the amount of antibody or its antigen-binding fragment bound to STK1 substance.

[0053] In one embodiment, the method further includes correlating the measured amount of antibody or antigen-binding fragment thereof binding to the STK1 substance with the level of the STK1 substance. This can be achieved by utilizing a predefined correlation between the measured amount of antibody or antigen-binding fragment thereof binding to a reference TK1 substance and the concentration of that reference TK1 substance. A typical reference TK1 material that can be used to generate such a predefined correlation is recombinant human TK1.

[0054] Therefore, a predefined correlation can be generated by adding antibodies or their antigen-binding fragments to different samples containing different concentrations of a reference TK1 substance (preferably recombinant human TK1). The amount of antibody or its antigen-binding fragment bound 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 antibody or its antigen-binding fragment bound to the reference TK1 substance (preferably recombinant human TK1).

[0055] Then, this predefined association, such as a standard curve, function, or relationship, can be used to map or convert the amount of antibody or its antigen-binding fragment that binds to STK1 substance in a measured body fluid sample into the concentration of STK1 substance in the body fluid sample.

[0056] Typically, it is preferred to use antibodies of the same type or antigen-binding fragments thereof to generate a predefined association to determine the level of STK1 substance in bodily fluid samples from patients diagnosed with prostate cancer. Therefore, in a preferred embodiment, the antibody or antigen-binding fragment thereof is capable not only of specifically binding to human TK1 in serum form but also of specifically binding to a reference TK1 substance, preferably recombinant human TK1.

[0057] In one embodiment, the body fluid sample is treated before or during incubation with an antibody or its antigen-binding fragment. This sample treatment can be used to stabilize selected STK1 forms in the body fluid sample and / or to break down larger STK1 complexes or oligomers into smaller complexes or polymers.

[0058] Therefore, in one embodiment, a sample diluent or pretreatment buffer is added to the body fluid sample before or simultaneously with the addition of the antibody or its antigen-binding fragment to the body fluid sample, preferably before the addition of the antibody or its antigen-binding fragment to the body fluid sample.

[0059] In one embodiment, the sample dilution buffer contains ATP, preferably selected at a concentration in the range of 0.5 mM to 50 mM, for example, 0.5 mM to 20 mM or 1.5 mM to 50 mM. As described earlier herein, ATP can stabilize the tetrameric form of TK1, which has high TK1 enzyme activity.

[0060] In another embodiment, the sample dilution buffer contains a reducing agent. The reducing agent can then break the disulfide bond crosslinks in larger STK1 complexes and oligomers to obtain smaller STK1 forms, such as tetramers. Depending on the embodiment, various reducing agents capable of breaking disulfide bonds can be used, 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 typically selected in the range of 0.1 mM to 10 mM.

[0061] In one implementation, the sample dilution buffer may contain ATP and a reducing agent.

[0062] Antibodies or their antigen-binding fragments specifically bind to STK1 substances, particularly specifically to the serum form of TK1 protein.

[0063] The specificity of an antibody or its antigen-binding fragment can be determined based on affinity and / or cohesion. This is determined by the equilibrium constant (Ka) of the dissociation of the antigen from the antibody or its antigen-binding fragment. 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, the stronger the binding strength between the antigenic determinant and the antibody or its antigen-binding fragment. Alternatively, affinity can also be expressed as the affinity constant (K). a ), that is, 1 / K d Those skilled in the art will understand that affinity can be determined based on a specific target antigen using methods known in the art.

[0064] Affinity is a measure of the strength of binding between an antibody or its antigen-binding fragment and the corresponding antigen. Affinity is related to the affinity between the antigenic determinant and its antigen-binding site on the antibody or its antigen-binding fragment, as well as the number of corresponding binding sites present on the antibody or its antigen-binding fragment.

[0065] Typically, antibodies will be in the form of 10 -5 Up to 10 -12 moles per liter (M) or lower dissociation constant (K) d ) binds to its antigen, preferably 10 -7 Up to 10 -12 M or lower, preferably 10 -8 Up to 10 -12 M, which is 10 5 Up to 10 12 M -1 Or higher, preferably 10 7 Up to 10 12 M -1 Or higher, preferably 10 8 Up to 10 12 M -1 The binding constant (K) a ).

[0066] Typically, anything greater than 10 -4 M of K d Value (or any value below 104 M) -1 K a The value is generally considered to indicate nonspecific binding. Preferably, the antibody or its antigen-binding fragment 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, for example less than 5 nM or even lower, such as 1 nM or lower.

[0067] The specific binding of an antibody or its antigen-binding fragment to an antigen or antigenic determinant can be determined by any suitable means known per se, including, for example, Scatchard assays and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays, as well as various variants known per se in the art.

[0068] In one embodiment, the antibody is a monoclonal antibody, specifically a monoclonal anti-TK1 antibody. In another embodiment, the antibody is a polyclonal antibody, specifically a polyclonal anti-TK1 antibody.

[0069] In one embodiment, the antibody or its antigen-binding fragment is specific to an epitope or peptide consisting of an amino acid sequence from the C-terminal region of TK1, preferably human TK1.

[0070] The peptide is preferably selected from amino acid positions 200 to the terminal portion of TK1, i.e., amino acid position 234 in humans (SEQ ID NO: 28). In one specific embodiment, the peptide is selected from amino acid positions 205, preferably 210 to 230, preferably 225 of the TK1 protein.

[0071] The peptide is preferably an N-mer, wherein N is an integer in the range of 8 to 20, and more preferably an integer in the range of 10 to 15. The peptide is preferably composed of N consecutive amino acids in the C-terminal region of the TK1 protein.

[0072] 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 NCPPVPGKPGE (SEQ ID NO: 2). In another embodiment, the peptide consists of the following amino acid sequence PPVPGKPGEAV (SEQ ID NO: 3). In yet another embodiment, the peptide consists of the following amino acid sequence NCPPVPGKPGEAV (SEQ ID NO: 4).

[0073] A monoclonal antibody specific to the epitope composed of GEAVAARKLF (SEQ ID NO: 1) has the following components: a variable weight (VH) domain complementarity-determining region 1 (CDR1) with the amino acid sequence DYEMH (SEQ ID NO: 5), a VH domain CDR2 with the amino acid sequence AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a VH domain CDR3 with the amino acid sequence FITKFDY (SEQ ID NO: 7), a variable light (VL) domain CDR1 with the amino acid sequence KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a VL domain CDR2 with the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 with the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0074] Monoclonal antibodies specific to epitopes NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) have the following VH domains: CDR1 with amino acid sequence DYEMH (SEQ ID NO: 5), CDR2 with amino acid sequence AILPGSGGTAYNQKFKG (SEQ ID NO: 11), CDR3 with amino acid sequence LITTFDY (SEQ ID NO: 12), CDR1 with VL domain with amino acid sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 13), CDR2 with VL domain with amino acid sequence LVSKLDS (SEQ ID NO: 9), and CDR3 with VL domain with amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0075] In another embodiment, the antibody or its antigen-binding fragment is specific for a conformation-dependent epitope of human TK1. A monoclonal antibody specific for this conformation-dependent epitope has: a VH domain CDR1 with the amino acid sequence SGYSWH (SEQ ID NO: 14), a VH domain CDR2 with the amino acid sequence YIHYSGSTTYNPSLKG (SEQ ID NO: 15), a VH domain CDR3 with the amino acid sequence WGTGHWYFDV (SEQ ID NO: 16), a VL domain CDR1 with the amino acid sequence RSSTGAVTTTNYAN (SEQ ID NO: 17), a VL domain CDR2 with the amino acid sequence GTNNRVP (SEQ ID NO: 18), and a VL domain CDR3 with the amino acid sequence ALWYSNHWV (SEQ ID NO: 19).

[0076] Examples of the above three monoclonal anti-TK1 antibodies that can be used according to the implementation scheme are further disclosed in WO 2015 / 094106, and its teachings on monoclonal anti-TK1 antibodies are incorporated herein by reference.

[0077] Therefore, in one embodiment, the monoclonal antibody or its antigen-binding fragment thereof is selected from the group consisting of: a monoclonal antibody or its antigen-binding fragment that is specific to GEAVAARKLF (SEQ ID NO: 1) of human TK1, a monoclonal antibody or its antigen-binding fragment 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 its antigen-binding fragment that is specific to a conformation-dependent epitope of human TK1.

[0078] In another embodiment, the antibody or its antigen-binding fragment is specific to an epitope or peptide consisting of KPGEAVAARKLFAPQ (SEQ ID NO: 20). 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, for coupling with other molecules, such as carrier proteins.

[0079] WO 95 / 29192 further discloses antibodies specific to this epitope, and its teachings on anti-TK1 antibodies are incorporated herein by reference.

[0080] In another embodiment, the antibody or its antigen-binding fragment is specific to an epitope or peptide consisting of an amino acid sequence from the TK1 active site. The peptide is preferably selected from the TK1 moiety of human TK1 at amino acid positions 150 to 190. In a specific embodiment, the peptide is selected from the moiety of TK1 at amino acid positions 155, preferably 160, more preferably 161, to 185, preferably 183.

[0081] The peptide is preferably an M-mer, wherein M is an integer in the range of 10 to 40, more preferably an integer in the range of 20 to 30, and even more preferably 23 or 24. The peptide is preferably composed of M consecutive amino acids in the active site of the TK1 protein.

[0082] 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, for coupling with other molecules, such as carrier proteins.

[0083] In one embodiment, the peptide consisting of an amino acid sequence from the TK1 active site has an amino acid sequence corresponding to amino acid positions 161 to 183 in human TK1, namely, an amino acid sequence having AYTKRLGTEKEVEVIGGADKYHS (SEQ ID NO: 21).

[0084] WO 2008 / 142664 further discloses antibodies specific to this epitope, and its teachings on anti-TK1 antibodies are incorporated herein by reference.

[0085] In another embodiment, the antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, as disclosed in WO 2019 / 201901, whose teachings on monoclonal anti-TK1 antibodies are incorporated herein by reference.

[0086] For example, a monoclonal antibody could be mAb 6C6, mAb 4H4, or mAb 23C11.

[0087] mAb 6C6 VH domain (SEQ ID NO: 22): METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFSFSSSYWICWVRQAPGKGLEWIACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 6C6 VL domain (SEQ ID NO: 23): MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPKLLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISDSPYVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC mAb 4H4 VH domain (SEQ ID NO: 24): METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWIACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 4H4 VL domain (SEQ ID NO: 25): MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSSTSGGGVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC mAb 23C11 VH domain (SEQ ID NO: 26): METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGIIYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 23C11 VL domain (SEQ ID NO: 27): MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSTF SSYGNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC In one embodiment, a kit is used to determine the level of STK1 substance in a body fluid sample. The kit preferably comprises a first antibody or a first antigen-binding fragment thereof, and a second antibody or a second antigen-binding fragment thereof. The first and second antibodies may be selected from the illustrative examples of monoclonal anti-TK1 antibodies and polyclonal anti-TK1 antibodies described above.

[0088] In one specific embodiment, the kit comprises a first monoclonal antibody or a first antigen-binding fragment thereof, which is specific for epitopes selected from the group consisting of: i) GEAVAARKLF of human TK1 (SEQ ID NO: 1), ii) at least one of NCPVPGKPGE of human TK1 (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4), and iii) a conformation-dependent epitope of human TK1. The kit also comprises a second monoclonal antibody or a second antigen-binding fragment thereof, which is specific for epitopes selected from the group consisting of: i) GEAVAARKLF of human TK1 (SEQ ID NO: 1), ii) at least one of NCPVPGKPGE of human TK1 (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4), and iii) a conformation-dependent epitope of human TK1.

[0089] In one embodiment, the first antibody or its first antigen-binding fragment is a so-called capture antibody immobilized on or intended to be immobilized on a vector, and the second antibody or its second antigen-binding fragment is a so-called detection antibody. In another embodiment, the second antibody or its second antigen-binding fragment is a capture antibody immobilized on or intended to be immobilized on a vector, while the first antibody or its first antigen-binding fragment is used as a detection antibody.

[0090] In one embodiment, the first and second antibodies or their first and second antigen-binding fragments are specific to different epitopes in the STK1 substance.

[0091] In another embodiment, the first and second 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 may exist in multiple copies in a high molecular weight complex of multiple TK1 protein units. Therefore, the STK1 substance can be a multivalent complex of multiple (i.e., at least two) TK1 protein units. In fact, antibodies of the same type, or their antigen-binding fragments, can be used as the first and second antibodies, or their first and second antigen-binding fragments.

[0092] In one embodiment, one of the first and second antibodies or its first and second antigen-binding fragments are specific to a peptide consisting of an amino acid sequence from the TK1 active site, while the other of the first and second antibodies or its first and second antigen-binding fragments are specific to a peptide consisting of an amino acid sequence from the C-terminal region of TK1.

[0093] In another embodiment, one of the first and second antibodies, or its first and second antigen-binding fragments, is specific to a peptide consisting of a first amino acid sequence from the C-terminal region of TK1, while the other of the first and second antibodies, or its first and second antigen-binding fragments, is specific to a peptide consisting of a first amino acid sequence from the C-terminal region of TK1 or a different second amino acid sequence from the C-terminal region of TK1.

[0094] In another embodiment, one of the first and second antibodies or its first and second antigen-binding fragments are specific to a peptide consisting of a first amino acid sequence from the C-terminal region of TK1, while the other of the first and second antibodies or its first and second antigen-binding fragments are specific to a conformation-dependent epitope of human TK1.

[0095] The antigen-binding fragments of the antibodies used in this article can be selected from the following groups: single-chain antibodies, Fv fragments, scFv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, single-domain antibodies (sdAb), scFv-Fc fragments, and di-scFv fragments.

[0096] 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.

[0097] In the following discussion, it is assumed that the first antibody or its first antigen-binding fragment is a capture antibody, and the second antibody or its second antigen-binding fragment acts as a detection antibody. However, the implementation is not limited to this, and the capture antibody and the detection antibody can be switched.

[0098] Sandwich ELISA can be used to detect STK1 substances in body fluid samples by preparing the surface of a carrier (such as a solid-phase carrier) to which a first antibody or its first antigen-binding fragment is bound as a so-called capture antibody. In a preferred embodiment, a known amount of the first antibody or its first antigen-binding fragment is bound to the surface of the carrier. Optionally, but preferably, any non-specific binding sites on the surface are blocked. The body fluid sample is then applied to the surface such that any STK1 substances present therein are captured by the immobilized first antibody or its first antigen-binding fragment. Unbound substances are preferably removed by one or more washing steps. A second antibody or its second antigen-binding fragment (commonly referred to as a detection antibody) is then added and allowed to bind to any STK1 substances captured by the first antibody or its first antigen-binding fragment.

[0099] The amount of the bound second antibody or its second antigen-binding fragment can then be determined by direct or indirect detection methods. For example, a label or enzyme can be directly attached to the second antibody or its second antigen-binding fragment, or indirectly attached via a linker, such as a biotin-streptavidin or biotin-avidin linker. Alternatively, a second antibody or its second antigen-binding fragment, labeled or linked to an enzyme, can be used to specifically bind to the second antibody or its second antigen-binding fragment.

[0100] Therefore, in one embodiment, the second antibody or its second antigen-binding fragment has a covalently linked biotin. Alternatively, the second antibody or its second antigen-binding fragment has a covalently linked streptavidin or avidin.

[0101] The kit preferably also contains horseradish peroxidase (HRP)-labeled streptavidin or HRP-labeled avidin. Alternatively, the kit may also contain HRP-labeled biotin. The kit may also contain an HRP substrate, such as 3,3',5,5'-tetramethylbenzidine (TMB) substrate, 3,3'-diaminobenzidine (DAB) substrate, or 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. In this case, the level of STK1 in the sample can be determined spectrophotometrically by detecting the conversion of the chromogenic substrate into a detectable colored product via HRP.

[0102] In one embodiment, the kit further comprises a microtiter plate (MCP) as a carrier, on which a first antibody or a first antigen-binding fragment thereof is immobilized or intended to be immobilized.

[0103] An example of an ELISA that can be used according to the present invention is the AroCell® TK 210 ELISA from AroCell AB of Bromma, Sweden.

[0104] The kit does not necessarily have to be an ELISA kit. In another embodiment, the kit uses affinity chromatography, in which a first antibody or its first antigen-binding fragment binds to a fixed substance, such as beads in a gel matrix or column. For example, the gel matrix or beads can be made of agarose, such as SEPHAROSE®.

[0105] In this scenario, STK1 substances present in the body fluid sample are trapped in the column by binding to a fixed first antibody or its first antigen-binding fragment. After washing, the bound STK1 substances can be eluted and detected using a second antibody or its second antigen-binding fragment. For example, for STK1 detection using direct or indirect detection methods, the amount of eluted STK1 substances can be determined using Western blotting and a second antibody or its second antigen-binding fragment.

[0106] The carrier can also be magnetic beads, such as DYNABEADS® magnetic beads.

[0107] In another embodiment, the kit is a chemiluminescent immunoassay (CLIA) kit. CLIA is an immunoassay technique in which the label is a luminescent molecule. The CLIA method can be a direct method using luminescent markers or an indirect method using enzyme markers. These two methods may be competitive or non-competitive. In the direct CLIA method, the luminescent markers used are typically acridine and ruthenium esters, while the enzyme markers used in the indirect method are typically alkaline phosphatase with adamantyl 1,2-dioxane aryl phosphate (AMPPD) as a substrate and HRP with luminol or its derivatives as a substrate.

[0108] The kit does not necessarily have to include two antibodies or their antigen-binding fragments; it can include only one type of antibody or its antigen-binding fragment.

[0109] Furthermore, the kit does not necessarily have to contain so-called capture antibodies or their antigen-binding fragments. In stark contrast, multiple (i.e., at least two) different antibodies or their antigen-binding fragments can be used to determine the level of STK1 without immobilizing at least one antibody or its antigen-binding fragment.

[0110] This invention also relates to the use of an anti-thymidine kinase 1 (TK1) antibody or an antigen-binding fragment thereof that specifically binds to the serum form of human TK1 in the preparation of a kit for treatment stratification. This use includes determining the level of serum thymidine kinase 1 (STK1) substance in bodily fluid samples from patients diagnosed with prostate cancer using an anti-TK1 antibody or an antigen-binding fragment thereof. This use also includes comparing the STK1 substance level to a threshold level, selecting chemotherapy for the patient if the STK1 substance level is equal to or higher than the threshold, and selecting ARSI therapy for the patient if the STK1 substance level is lower than the threshold.

[0111] Example This implementation plan evaluated whether serum TK1 levels could be used for treatment stratification in prostate cancer patients.

[0112] Materials and methods patient Cohort 1 included 43 men diagnosed with hormone-negative metastatic disease in the prostate cancer database of the Prostate Cancer Research Center at the University of Tampere, Finland. Between 2000 and 2010, all men were diagnosed and treated at the Urology outpatient clinic of Tampere University Hospital. Metastatic disease was defined by the presence of distant metastases (M1 stage) confirmed by bone scan imaging, regardless of tumor (T) stage or lymph node (N) stage. Study participants were randomly selected from men who met the inclusion criteria, and their serum samples were available for STK1 measurement at diagnosis. Available information included date of diagnosis, biopsy Gleason score, clinical tumor, lymph node, and metastasis (TNM) stage, prostate-specific antigen (PSA) at diagnosis, and primary treatment. Participants were tracked and managed according to clinician judgment and standard clinical practice. In addition, data included the time of first disease recurrence (biochemical or radiographic recurrence) and the date and primary cause of death, obtained from the national death certificate registry maintained by Statistics Finland. Follow-up data ended on August 31, 2019, therefore the follow-up period was at least 9 years.

[0113] Cohorts 2 and 3 comprised 200 patients with metastatic castration-resistant prostate cancer (mCRPC) treated at the Department of Urology, Medical University of Vienna or Semewish University of Budapest between 2011 and 2022. All patients had metastatic disease at the time of classification as castration-resistant. Serum samples were collected directly before the start of systemic therapy. Of these patients, 102 received androgen receptor signaling inhibitors (ARSIs), and 98 received docetaxel (DOC) chemotherapy for mCRPC. 197 of these patients were followed up.

[0114] STK1 Measurement Following the manufacturer's instructions (www.arocell.com), the AroCell® TK 210 ELISA (AroCell AB, Bromma, Sweden) was used to determine the TK1 protein level in serum samples. In short, serum samples, calibrators, and controls were pre-incubated with sample dilution buffer (SDB) at room temperature (20–25°C) for 1 hour. After pre-incubation, the samples were added to pre-washed plates coated with anti-TK1 antibody and incubated on a shaking platform for at least 2 hours. The plates were washed four times, and biotinylated anti-TK1 antibody was added. After incubation and four washes, enzyme-labeled streptavidin-horseradish peroxidase (HRP) was pipetted into the wells, followed by the incubation step and four washes, and the addition of HRP substrate. Color development was then stopped, and the color intensity was measured at 450 nm using a spectrophotometer. The resulting color is proportional to the concentration of STK1 protein in the sample. STK1 protein levels in the samples were calculated using calibration curves and a four-parameter logic (4-PL) curve fitting procedure. Each sample was analyzed in duplicate, and the average value is expressed as µg / L.

[0115] Statistical analysis The analysis was conducted by Statika Kademin AB in Uppsala, Sweden. The primary endpoint was overall survival (OS). Cox proportional hazards regression was used to estimate hazard ratios (HR). The time measure was the number of months since the diagnosis of prostate cancer. For cohort 1, follow-up ended at death, loss to follow-up (emigration), or the cutoff date of June 31, 2023, whichever occurred first. Follow-up for cohorts 2 and 3 was last updated in August 2022. The analysis was adjusted for age, and multivariate adjustments were further made based on biopsy Gleason score, TNM stage, and PSA at diagnosis. Survival was assessed for recurrence-free survival in a separate Cox regression model with disease recurrence as the endpoint. In this analysis, follow-up ended at the first prostate cancer recurrence (PSA recurrence or radiation progression with metastasis on computed tomography despite castration of serum testosterone levels), death, or the co-cutoff date, whichever occurred first. Study participants were stratified according to the median and quartiles of STK1 levels. Participants with STK1 levels equal to or below the median were used as the control group. Survival trends were analyzed by incorporating STK1 protein levels as a continuous variable into a Cox regression model. Furthermore, Kaplan-Meier curves were used to visualize survival differences.

[0116] result Tables 1-3 describe the clinical characteristics of this multicenter study cohort. In Cohort 1, 43 men with newly diagnosed metastatic hormone-sensitive prostate cancer (mHSPC) received gonadotropin-releasing hormone (GnRH) agonist therapy during the period before next-generation anti-androgens were available for mHSPC (2002–2011). Cohorts 2 and 3 consisted of 200 patients diagnosed with metastasis, of whom survival information was available for 197. Men in Cohort 2 received enzalutamide or abiraterone (ENZA / ABI) between 2011 and 2022, while men in Cohort 3 received docetaxel during the same period. The median age at diagnosis was similar across all three cohorts. Median PSA at diagnosis varied between cohorts: Cohort 1: 50 ng / mL, Cohort 2: 93 ng / mL, Cohort 3: 68 ng / mL.

[0117] Table 1-43 men diagnosed with newly diagnosed M1 prostate cancer (cohort 1) based on STK1 levels and clinical characteristics of prostate cancer.

[0118] STK1 levels in hormone-sensitive and castration-resistant prostate cancer At diagnosis, the median STK1 level in cohort 1 (mHSPC) was 0.61 μg / L (Table 1). STK1 levels did not change significantly with the International Society of Urological Pathology (ISUP) Gleason score on biopsy (median values ​​were 0.23 and 0.21 μg / L for grades 3–5 and 1–2, respectively). Patients with elevated STK1 levels (208 ng / mL) had higher PSA at diagnosis compared to those with lower STK1 levels (46 ng / mL). Elevated STK1 levels were associated with higher clinical T stage at diagnosis (T1 / T2 vs. T3 / T4; p = 0.021), while the distribution of biopsy ISUP Gleason scores was similar (Table 1).

[0119] In the mCRPC cohort, the median baseline STK1 level was 0.50 μg / L for patients subsequently treated with ARSI (cohort 2), while the median baseline STK1 level was 0.49 μg / L for patients treated with docetaxel (cohort 3). Median age at diagnosis did not significantly differ in STK1 levels. The STK1 cutoff value (0.61 μg / L) from mHSPC cohort 1 was used for the analysis of mCRPC patients in cohorts 2 and 3. In this material, 123 patients had STK1 levels below the cutoff value, and 74 patients had STK1 levels above the cutoff value. The median PSA levels for patients stratified according to low and high STK1 levels were 52.2 and 162 ng / mL, respectively (p=0.0006) (Table 2). In mCRPC cohorts 2 and 3, the median PSA levels at diagnosis were 93 (18–395) ng / mL and 68 (26–279) ng / mL, respectively.

[0120] Table 2 - Population characteristics of castration-resistant prostate cancer patients based on STK1 levels and clinical characteristics of prostate cancer. The study population consisted of 197 men (cohorts 2 and 3).

[0121] The relationship between STK1 and overall survival in mHSPC patients In cohort 1, STK1 levels above the cutoff were associated with poorer age-adjusted overall survival compared to men with STK1 levels below the cutoff (HR 3.08 (1.49–6.36); p < 0.001) (Table 3). The risk associations remained unchanged after multivariate adjustment. A statistically significant trend of increasing risk with STK1 levels was observed for both prostate cancer-specific mortality risk and overall mortality risk (Table 3). In the Kaplan-Meier analysis, STK1 stratification revealed different groups with varying disease-specific survival, particularly at different age levels (Table 3). Figure 1A ) and PSA at diagnosis ( Figure 1BIn the analysis that corrected for this, including STK1, ISUP Gleason score, and PSA as features in the random forest classification model produced the most accurate prediction of prostate cancer mortality compared to any other model. Figure 2 Notably, the combination of these three variables showed higher mean predictive accuracy compared to models using PSA and Gleason scores alone, confirming the independent additional prognostic value of STK1. All models using clinical T stage as a classifier had worse mean predictive error compared to models without T stage. Therefore, T stage was excluded from the model. Overall, the mean predictive error of using STK1, Gleason score, and PSA in combination for prostate cancer mortality was excellent. Figure 2 ).

[0122] Table 3 - Overall survival of mHSPC patients stratified by median STK1 at diagnosis

[0123] HR 1 - Age-adjusted; HR 2 - ISUP Gleason score-adjusted. HR 3 - PSA-corrected at diagnosis; HR 4 - Clinical T-staging-corrected. The relationship between STK1 and overall survival in mCRPC patients Survival data were available for 197 patients diagnosed with mCRPC (cohorts 2 and 3). STK1 levels above the cutoff were associated with poorer overall survival (9.5 months vs. 20.7 months) and a higher relative risk (age-adjusted HR 2.08 (1.47–2.97); p < 0.001) (Table 4). After adjusting for age, PSA, and Eastern Cooperative Oncology Group (ECOG) performance status, STK1 levels remained associated with a significantly increased relative risk ratio (HR 1.83 (1.34–2.86), p = 0.002). Gleason stage and T stage were considered irrelevant to this group of advanced-stage patients. Kaplan-Meier survival curves showed… Figures 3A-3D middle.

[0124] Cohort 2, treated with ARSI abiraterone or enzalutamide, and cohort 3, treated with docetaxel, were analyzed separately. In the 99 patients treated with abiraterone or enzalutamide, the age-adjusted hazard ratio for STK1 levels above the cutoff was 2.54 (1.57–4.06) (p<0.001) (Table 4). This association remained significant after adjusting for age, PSA, and ECOG performance status scores (Table 4). In the 98 mCRPC patients treated with docetaxel, the age-adjusted hazard ratio associated with STK1 levels above the cutoff was 1.62 (0.97–2.71) lower than that in the ARSI treatment group (p=0.066). After adjusting for age, PSA, and ECOG performance status scores, the hazard ratio was 1.52 (0.91–2.56) (p=0.11).

[0125] Figures 3A-3D The Kaplan-Meier survival curves show the relationship between the total survival of cohort 2 and cohort 3 according to the STK1 level dichotomy.

[0126] Table 4 - Overall survival after prostate cancer diagnosis stratified by median STK1 at diagnosis. The study population consisted of 197 men with castration-resistant prostate cancer.

[0127] HR 1 - Age-adjusted; HR 2 - Age at diagnosis and PSA adjusted. HR 3 - Age at diagnosis, PSA, and ECOG adjusted Three independent cohorts representing different clinical scenarios and treatment stages were used to investigate the potential prognostic value of STK1 in advanced prostate cancer. In cohort 1, among 43 patients newly diagnosed with mHSPC who subsequently received ADT, only STK1 had an independent prognostic value for overall survival. The prognostic value was independent of PSA, and its accuracy was further improved when combined with identified clinical factors. Similarly, in a contemporary cohort of mHSPC patients receiving ADT and either ARSI or docetaxel, detectable STK1 at baseline was associated with a shorter time to castration resistance. These two cohorts suggest that STK1 may be a valuable tool for prognostic assessment of mHSPC. The cutoff value defined in mHSPC patients (cohort 1) was found to be associated with an age-adjusted HR of 2.08 in 197 mCRPC patients receiving ARSI (abiraterone or enzalutamide) or docetaxel. Limiting the analysis to mCRPC patients receiving ARSI resulted in an age-adjusted relative risk of 2.53 associated with elevated STK1 levels. In patients with mCRPC, the relative risk of elevated STK1 remains after adjusting for differences in PSA levels.

[0128] In patients receiving docetaxel, the relative risk associated with elevated STK1 (HR: 1.52) was lower than that observed in the ARSI treatment group. This difference in relative risk was not observed between docetaxel and ARSI patients with low STK1 levels. Figure 3D These findings suggest that ARSIs may not be suitable for patients with elevated STK1 levels, but may be suitable for patients with lower STK1 levels. High STK1 is associated with aggressive disease, and ARSI treatment has been reported to be ineffective in patients with Gleason 9–10 disease. Interestingly, the clinical benefit of docetaxel was only observed in patients with high disease burden (defined as the presence of visceral metastases and / or multiple bone metastases). These results indicate that STK1 can be used as a biomarker to identify advanced prostate cancer patients who should receive intensive therapy. The response of prostate cancer to anti-androgen therapy has been reported to be complex, involving the upregulation of tumor suppressor factors and the inhibition of growth-promoting factors such as MYC and E2F1.

[0129] STK1 levels may reflect the number of actively proliferating malignant cells, and ARSI therapy may be insufficient to treat rapidly growing tumors. Rapidly proliferating prostate cancer cells are expected to be sensitive to cell cycle-activating drugs such as docetaxel, which is an inhibitor of microtubule dynamics and therefore an inhibitor of mitosis.

[0130] One advantage of this study is that the cutoff values ​​established in the initial study of mHSPC patients were used in the follow-up study of mCRPC. Long-term follow-up time after prostate cancer diagnosis can also be used for the three groups with overall survival as the endpoint.

[0131] Therefore, STK1 can serve as a biomarker for risk stratification in patients with mHSPC and mCRPC. This finding suggests that patients with elevated STK1 levels should not receive ARSI treatment. In routine clinical practice, patients with aggressive disease (including rapid PSA doubling time) are often treated with docetaxel. Therefore, STK1 could be a useful tool for mCRPC treatment decisions.

[0132] The above embodiments should be understood as several illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the invention. Specifically, where technically possible, different portions of the solutions in different embodiments can be combined into other configurations.

Claims

1. A treatment stratification method, comprising: The levels of serum thymidine kinase 1 (STK1) in bodily fluid samples from patients diagnosed with prostate cancer were determined using an antibody or antigen-binding fragment thereof that specifically binds to human TK1 in serum form. Compare the levels of STK1 substance with threshold levels; and If the STK1 level is equal to or higher than the threshold, chemotherapy is selected for the patient; if the STK1 level is lower than the threshold, androgen receptor signaling inhibitor (ARSI) therapy is selected for the patient.

2. The method of claim 1, wherein determining the level of substance STK1 comprises: The levels of STK1 in bodily fluid samples from patients diagnosed with metastatic prostate cancer are determined using antibodies that specifically bind to serum-form human TK1 or their antigen-binding fragments.

3. The method of claim 2, wherein determining the level of substance STK1 comprises: The levels of STK1 in bodily fluid samples from patients diagnosed with metastatic castration-resistant prostate cancer are determined using antibodies that specifically bind to serum-form human TK1 or their antigen-binding fragments.

4. The method according to any one of claims 1 to 3, wherein the chemotherapy is selected from the group consisting of: docetaxel, cabazitaxel, mitoxantrone, estradiol, and any combination thereof, preferably docetaxel.

5. The method according to any one of claims 1 to 4, wherein the ARSI therapy comprises androgen synthesis inhibitor (ASI) therapy, androgen receptor antagonist (ARA) therapy, and any combination thereof.

6. The method of claim 5, wherein ASI is abiraterone.

7. The method according to claim 5 or 6, wherein the ARA is selected from the group consisting of enzalutamide, apalutamide, darotamide, and any combination thereof.

8. The method according to any one of claims 1 to 7, wherein comparing the level of STK1 comprises comparing the level of STK1 with a threshold level selected from the range of 0.5 to 0.7 μg / L, preferably selected from the range of 0.55 to 0.65 μg / L, and more preferably 0.61 μg / L.

9. The method according to any one of claims 1 to 8, wherein determining the level of substance STK1 comprises: The level of STK1 in serum or plasma samples is determined using antibodies or antigen-binding fragments that specifically bind to human TK1 in serum form.

10. The method according to any one of claims 1 to 9, wherein determining the level of STK1 substance in the body fluid sample comprises: Contact body fluid samples with antibodies or antigen-binding fragments of human TK1 that specifically bind to serum form; and Measure the amount of antibody or its antigen-binding fragment that binds to STK1 substance.

11. The method of claim 10, further comprising correlating the measured amount of antibody or antigen-binding fragment thereof bound to the STK1 substance with the level of the STK1 substance.

12. The method of claim 11, wherein correlating the measured amount of antibody or antigen-binding fragment thereof comprises using a predefined correlation between the measured amount of antibody or antigen-binding fragment thereof binding to recombinant human TK1 and the concentration of recombinant human TK1 to correlate the measured amount of antibody or antigen-binding fragment thereof with the level of STK1 substance.

13. The method according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to serum-form human TK1.

14. The method of claim 13, wherein the monoclonal antibody or its antigen-binding fragment is selected from the group consisting of: A monoclonal antibody or its antigen-binding fragment that is specific to human TK1 GEAVAARKLF (SEQ ID NO: 1); A monoclonal antibody or antigen-binding fragment thereof specifically targeting at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPVPGKPGEAV (SEQ ID NO: 4) for human TK1; and Monoclonal antibodies or antigen-binding fragments thereof that are specific to the conformation-dependent epitope of human TK1.

15. The method of claim 14, wherein the monoclonal antibody or its antigen-binding fragment comprises: It has a complementarity-determining region 1 (CDR1) with a variable weight (VH) domain containing the amino acid sequence SEQ ID NO: 5; CDR2 has a VH domain containing the amino acid sequence SEQ ID NO: 6; CDR3 has a VH domain containing the amino acid sequence SEQ ID NO: 7; CDR1, which has a variable light (VL) domain with the amino acid sequence SEQ ID NO: 8; The VL domain CDR2 has the amino acid sequence SEQ ID NO: 9; and The VL domain CDR3 has the amino acid sequence SEQ ID NO:

10.

16. The method of claim 14, wherein the monoclonal antibody or its antigen-binding fragment comprises: It has a complementarity-determining region 1 (CDR1) with a variable weight (VH) domain containing the amino acid sequence SEQ ID NO: 5; CDR2 has a VH domain containing the amino acid sequence SEQ ID NO: 11; CDR3 has a VH domain containing the amino acid sequence SEQ ID NO: 12; CDR1, which has a variable light (VL) domain with the amino acid sequence SEQ ID NO: 13; The VL domain CDR2 has the amino acid sequence SEQ ID NO: 9; and The VL domain CDR3 has the amino acid sequence SEQ ID NO:

10.

17. The method of claim 14, wherein the monoclonal antibody or its antigen-binding fragment comprises: It has a complementarity-determining region 1 (CDR1) with a variable weight (VH) domain containing the amino acid sequence SEQ ID NO: 14; CDR2 has a VH domain containing the amino acid sequence SEQ ID NO: 15; CDR3 has a VH domain containing the amino acid sequence SEQ ID NO: 16; CDR1, which has a variable light (VL) domain with the amino acid sequence SEQ ID NO: 17; The VL domain CDR2 has the amino acid sequence SEQ ID NO: 18; and The VL domain CDR3 has the amino acid sequence SEQ ID NO:

19.

18. The method according to any one of claims 14 to 17, wherein determining the level of STK1 substance comprises using a kit for determining the level of STK1 substance in a body fluid sample, said kit comprising: The first monoclonal antibody or its first antigen-binding fragment is specific for epitopes selected from the group consisting of: GEAVAARKLF of human TK1 (SEQ ID NO: 1); At least one of NCPPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and Conformation-dependent epitopes of human TK1; and A second monoclonal antibody or a second antigen-binding fragment thereof that is specific for epitopes selected from the group consisting of: GEAVAARKLF of human TK1 (SEQ ID NO: 1); At least one of NCPPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3), and NCPPVPGKPGEAV (SEQ ID NO: 4) of human TK1; and Conformation-dependent epitopes of human TK1.

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

20. The method according to claim 18 or 19, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

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