Treatment stratification based on STK1

Abstract

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

Description

[0001] TREATMENT STRATIFICATION BASED ON STK1

[0002] TECHNICAL FIELD

[0003] The present invention generally relates to measurement of thymidine kinase 1 (TK1) and in particular to treatment stratification of prostate cancer patients based on measured serum TK1 (STK1) levels.

[0004] BACKGROUND

[0005] Thymidine kinase 1 (TK1) (EC 2.7.1.21), also referred to as 2’-deoxythymidine kinase or ATP-thymidine 5’-phosphotransferase, is an enzyme involved in deoxyribonucleic acid (DNA) precursor synthesis. TK1 phosphorylates thymidine to allow incorporation into DNA. Expression of TK1 is a marker of active cellular proliferation with low intracellular concentration during G0 / G1 phase of the cell cycle and increasing during the S / G2 phases.

[0006] A form of TK1 is present at high levels also in sera and plasma from humans and animals with malignant tumors. Therefore, serum TK1 activity measurements have been used for monitoring and for prognostic purpose in several different malignant diseases, but primarily in case of leukemia and lymphoma.

[0007] Furthermore, TK1 is the only proliferation marker that can be determined in blood, and it is likely to provide a large clinical benefit if available as a routine laboratory test.

[0008] The serum TK1 activity has been measured using a radioactive substrate125l-dUrd (the PROLIFIGEN® TK-REA, DiaSorin Inc) for several decades, but this radio-enzymatic assay has had limited use and preferentially in case of malignant hematologic malignancies. A non-radiometric TK1 activity assay (TK LIAISON® assay, DiaSorin Inc.) has been available in recent years. This is a sensitive and robust assay and has provided clinically valuable information in humans mainly in hematologic malignancies, particularly for monitoring therapy and predicting relapse.

[0009] During the last 15 years antibodies against human TK1 have been available and enabled the determination of the TK1 protein levels, in contrast to TK1 activity, both in hematologic as well as in solid tumor diseases, such as prostate cancer, and several other forms of solid and hematologic tumors.

[0010] Jagarlamudi et al., Breast and prostate cancer patients differ significantly 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, BMC Cancer (2015), 15: 66 discloses that there are differences in the specific activities and the composition of active and inactive subunits of TK1 in sera from patients with hematological malignancies compared to those with breast and prostate cancer.

[0011] Li et al., Serum thymidine kinase 1 is associated with Gleason score of patients with prostate carcinoma, Oncology Letters (2018), 16(5): 6171-6180 discloses that serum TK1 concentration is a more reliable prognostic biomarker than total prostate-specific antigen (PSA) according to Gleason scores in screening of benign prostatic hyperplasia (BPH) or prostate malignancy.

[0012] Jagarlamudi et al., 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 discloses that TK 210 ELISA analysis of sera from prostate and breast cancer patients had significantly higher TK1 protein levels compared to those in sera from healthy blood donors.

[0013] Jagarlamudi et al., Analytical and clinical characterization of an optimized dual monoclonal sandwich ELISA for the quantification of thymidine kinase 1 (TK1) protein in human blood samples, PLoS One (2022) 17(10): e027544 discloses that receiver operator characteristic (ROC) curve analysis indicated that measurement of STK1 protein levels had higher sensitivity than enzyme activity measurements in prostate patients.

[0014] Jagarlamudi et al., The combination of AroCell TK 210 ELISA with Prostate Health Index of prostatespecific antigen density can improve the ability to differentiate prostate cancer from noncancerous conditions, The Prostate (2019), 79(8): 856-863 discloses that serum TK1 as measured by AroCell TK 210 ELISA is significantly higher in patients with prostate cancer than with benign urological conditions. The serum TK1 was correlated to Prostate Health Index but not to the Gleason score.

[0015] SUMMARY

[0016] It is a general objective of the invention to achieve a treatment stratification for patients diagnosed with prostate cancer.

[0017] This and other objectives are met by embodiments as disclosed herein.

[0018] The present invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims. An aspect of the invention relates to a treatment stratification method. The method comprises determining a level of serum thymidine kinase 1 (STK1) material in a body fluid sample from a patient diagnosed with prostate cancer using an antibody, or an antigen-binding fragment thereof, specifically binding to a serum form of human TK1 . The method also comprises comparing the level of STK1 material with a threshold level and selecting chemotherapy for the patient if the level of STK1 material is equal to or above the threshold value and selecting an androgen receptor signaling inhibitor (ARSI) therapy for the patient if the level of STK1 material is below the threshold value.

[0019] The present invention enables a treatment stratification for patients diagnosed with prostate cancer. The present invention can thereby assist the physician in selecting the most appropriate treatment for the prostate cancer patient in terms of reducing the hazardous ratio (HR) for the patient, and thereby improving the survival of the patient.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

[0022] Fig. 1 illustrates Kaplan-Meier survival plots for mHSPC patients in analyses adjusted for age (A) and PSA at diagnosis (B).

[0023] Fig. 2 illustrates prostate cancer death random forest classification error estimates for mean prediction error and class-specific prediction errors for alive and prostate cancer death. The error bars represent empirically estimated 95% confidence intervals for Monte Carlo error. The classifiers used in each model are written on the right-hand side of the image, on the y-axis.

[0024] Fig. 3 illustrates Kaplan-Meier survival plots for mCRPC patients dichotomized for STK1 levels using a cut-off at 0.61 g / L. Data represent overall survival. (A) all 197 mCRPC patients, no stratification for treatment; (B) 99 mCRPC patients treated with abiraterone or enzalutamide; (C) 98 mCRPC patients treated with docetaxel; (D) all 197 mCRPC patients, stratified for treatment groups.

[0025] DETAILED DESCRIPTION

[0026] The present invention generally relates to measurement of thymidine kinase 1 (TK1) and in particular to treatment stratification of prostate cancer patients based on measured serum TK1 (STK1) levels. Prostate cancer is the most common non-cutaneous malignancy among men in developed countries and second to third most common cause of cancer-related death. Localized prostate cancer can often be managed effectively with surgery or radiation therapy. The cornerstone of management for advanced hormone-sensitive prostate cancer (HSPC) is androgen deprivation therapy (ADT). ADT is often combined with taxane-based chemotherapy or androgen-receptor signaling inhibitors (ARSIs), such as enzalutamide or the CYP17 inhibitor abiraterone. Eventually the prostate cancer develops the ability to grow despite ADT and combination treatments, giving rise to castration-resistant prostate cancer (CRPC). After development of CRPC, treatment often involves ARSIs and also taxane-based chemotherapy. Still, clinical responses to these treatments vary, and adverse effects are common. Procedures that can guide decisions concerning different types of available therapy options are particularly desirable in order to improve the effectiveness of the prostate cancer treatment while minimizing any adverse effects.

[0027] Previous studies as referred to in the background section have suggested that serum TK1 expression could be used as biomarker for presence of prostate cancer. Experimental data as presented herein shows that STK1 levels can be used for treatment stratification of prostate cancer patients to thereby be used as a tool in assisting clinicians in selecting the most appropriate treatment for a prostate cancer patient. This means that STK1 levels measured in a body fluid sample of a subject or patient diagnosed with prostate cancer, in particular metastatic prostate cancer, can be used in the selection of prostate cancer treatment for the patient.

[0028] This means that STK1 levels measured in a body fluid sample of a patient diagnosed with prostate cancer can be used in prostate cancer risk stratification when selecting optimal treatment for prostate cancer patients. Hence, prostate cancer patients with high levels of STK1 levels will have a lower hazard ratio if treated with a first anti-cancer treatment (chemotherapy), whereas prostate cancer patients with comparatively lower STK1 levels will instead have a lower hazard ratio if treated with a second, different anti-cancer treatment (an androgen receptor signaling inhibitor (ARSI) therapy).

[0029] An aspect of the invention therefore relates to a treatment stratification method. The method comprises determining a level of STK1 material in a body fluid sample from a patient diagnosed with prostate cancer using an antibody, or an antigen-binding fragment thereof, specifically binding to a serum form of human TK1. The method also comprises comparing the level of STK1 material with a threshold value and selecting chemotherapy for the patient if the level of STK1 material is equal to or above the threshold value and selecting an ARSI therapy for the patient if the level of STK1 material is below the threshold value. Thus, the invention is based on the unexpected finding that STK1 levels as measured from patients suffering from and diagnosed with prostate cancer correlates with survival of the prostate cancer patients as represented by hazard ratio (HR) for the prostate cancer patients. Experimental data as presented herein shows that that prostate cancer patients having elevated STK1 levels, i.e., equal to or above the threshold value, benefit from a first prostate cancer treatment in terms of chemotherapy, whereas prostate cancer patients having a lower level of STK1 material, i.e., below the threshold, instead benefit from a second prostate cancer treatment in terms of ARSI therapy.

[0030] In fact, for the prostate cancer patients having lower STK1 levels, ARSI therapy led to a higher survival probability as compared to chemotherapy as shown in Fig. 3D. This was highly surprising since it was believed that the more aggressive chemotherapy would be beneficial also for this group of prostate cancer patients. Hence, for this group of prostate cancer patients with comparatively low STK1 levels, the survival probability increased and the HR decreased if the patients were treated with ARSIs rather than with chemotherapy. However, in the group of prostate cancer patients with STK1 levels equal to or above the threshold value, chemotherapy was associated with a significantly higher survival probability as compared to ARSI therapy as shown in Fig. 3D.

[0031] Accordingly, prostate cancer patients having a determined level of STK1 material below the threshold value benefit from the ARSI therapy, whereas prostate cancer patients having a determined level of STK1 material equal to or above the threshold value benefit from the chemotherapy.

[0032] In an embodiment, the patient is diagnosed with metastatic prostate cancer. In such an embodiment, the method comprises determining the level of STK1 material in the body fluid sample from a patient diagnosed with metastatic prostate cancer using the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1 .

[0033] Such prostate cancer patients with diagnosed metastases are typically diagnosed as suffering from either metastatic hormone-sensitive prostate cancer (mHSPC) or metastatic castrate-resistance prostate cancer (mCRPC). mHSPC patients are typically treated with ADT either alone or in combination with chemotherapy or ARSI therapy. Eventually the prostate cancer develops the ability to grow despite ADT, giving rise to castration-resistant prostate cancer (CRPC). After development of CRPC, treatment often involves ARSI therapy or chemotherapy. In an embodiment, the prostate cancer patient is diagnosed with metastatic castration-resistant prostate cancer. In such an embodiment, the method comprises determining the level of STK1 material in the body fluid sample from a patient diagnosed with metastatic castration-resistant prostate cancer using the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1 .

[0034] ARSIs include androgen synthesis inhibitors (ASIs), such as abiraterone, and androgen receptor antagonists (ARAs), such as enzalutamide, apalutamide, and darolutamide.

[0035] Abiraterone acetate is an androgen synthesis inhibitor derived from pregnenolone and is an irreversible inhibitor of 17, 20-lyase and 17-alpha hydroxylase, which are products of the CYP17 gene. Abiraterone inhibits the production of androgen in the testes, adrenal glands, and tumor cells.

[0036] ARAs block the androgen binding site of androgen receptor (AR) and inhibit the nuclear translocation of AR and subsequent association of AR with nuclear DNA. This results in attenuation of coactivator mobilization leading to cellular apoptosis and decreased prostate tumor volume. The first-generation ARAs including bicalutamide, nilutamide, and flutamide do not completely block AR activity. Enzalutamide, apalutamide, and darolutamide are the currently utilized second-generation ARAs and have no agonistic activity on AR compared to first-generation ARAs.

[0037] Chemotherapy for prostate cancer patients include taxel-based chemotherapy, such as docetaxel and cabazitaxel, mitoxantrone and estramustine.

[0038] In an embodiment, the method comprises selecting chemotherapy for the patient if the level of STK1 material is equal to or above the threshold value and selecting ARSI therapy for the patient if the level of STK1 material is below the threshold value.

[0039] In an embodiment, the chemotherapy is selected from the group consisting of docetaxel, cabazitaxel, mitoxantrone, estramustine and any combination thereof, preferably docetaxel. Chemotherapy treatment as used herein, also encompass other types of anti-cancer treatments in addition to the chemotherapy, such as a combination of chemotherapy and ADT, a combination of chemotherapy and a steroid drug, such as prednisone, and a combination of chemotherapy and ARSI. For instance, docetaxel treatment is often combined with the steroid drug prednisone. In an embodiment, the ARSI is selected from the group consisting of ASI treatment, ARA treatment and any combination thereof. In the case of ASI treatment, a single ASI or a combination of multiple, i.e., at least two, ASIs could be administered to the prostate cancer patient. Correspondingly, in the case of ARA treatment, a single ARA or a combination of multiple ARAs could be administered to the prostate cancer patient. Also, a combination of at least one ASI and at least one ARA could be administered to the prostate cancer patient as an example of an ARSI therapy. ARSI therapy as used herein also encompass a combination of ARSI therapy and at least one other anti-cancer treatment with the proviso that the at least one other anti-cancer treatment is not chemotherapy. For instance, a combination of ARSI therapy and ADT could be used.

[0040] In an embodiment, the ASI is abiraterone.

[0041] In an embodiment, the ARA is selected from the group consisting of enzalutamide, apalutamide, darolutamide, and any combination thereof.

[0042] Prostate cancer patients, in particular mCRPC patients, having a determined level of STK1 material below the threshold value had a higher survival probability when treated with ARSI in terms of the ASI abiraterone or the ARA enzalutamide as compared to mCRPC patients treated with chemotherapy in terms of docetaxel. Accordingly, the treatment stratification method of the invention preferably selects ARSI therapy for prostate cancer patients, in particular mCRPC patients, having a determined level of STK1 material below the threshold value. Such ARSI therapy is not only beneficial in terms of being associated with a lower HR and increased survival probability but is generally marred by fewer or at least less severe side effects as compared to chemotherapy.

[0043] However, cancer patients, in particular mCRPC patients, having a determined level of STK1 material equal to or above the threshold value had a higher survival probability when treated with chemotherapy in terms of docetaxel as compared to mCRPC patients treated with ARSI in terms of the ASI abiraterone or the ARA enzalutamide. Accordingly, the treatment stratification method of the invention preferably selects chemotherapy for prostate cancer patients, in particular mCRPC patients, having a determined level of STK1 material equal to or above the threshold value.

[0044] In an embodiment, the threshold value is selected within an interval of from 0.5 up to 0.7 pg / L. In a preferred embodiment, the threshold value is selected within an interval of from 0.55 up to 0.65 pg / L, more preferably selected within an interval of from 0.57 up to 0.63 pg / L, and most preferably selected within an interval of from 0.59 up to 0.63 g / L, such as within an interval of from 0.60 up to 0.62 g / L. A currently preferred value of the threshold value is 0.61 pg / L.

[0045] The above-mentioned preferred interval for the threshold value was determined based on analysis of mHSPC patients but was also valid for mCRPC patients.

[0046] In an embodiment, the method also comprises obtaining the body fluid sample from the patient.

[0047] The body fluid sample is a body fluid sample comprising STK1 material. Non-limiting, but preferred, examples of such body fluid samples include a serum sample, a plasma sample, and a blood sample, preferably a serum sample or a plasma sample.

[0048] TK1 in humans are present in various forms depending on the presence of certain molecules, e.g., presence or absence of adenosine triphosphate (ATP); depending on the concentration of the protein, i.e., high or low concentration; depending on the type of the protein, i.e., native or recombinant TK1 ; and depending on the site of the protein, i.e., in serum or cytoplasm.

[0049] Generally, cytosolic and recombinant human TK1 occurs as tetramers in the presence of ATP or at high concentration, and as dimers in the absence of ATP or at low concentration. The tetramer form of cytosolic and recombinant human TK1 has high TK1 activity whereas the dimer form has lower TK1 activity. Cytosolic TK1 , also referred to as cellular TK1 , is TK1 present inside cells and can be isolated from such cells.

[0050] Human STK1 , in clear contrast, can be in the form of high molecular weight complexes, such as oligomers or comprising such oligomers, having TK1 activity and dimer and tetramer forms having very low or even lacking TK1 activity. The oligomerization seems to be related to the formation of disulfide cross linking occurring in the blood. STK1 is found in the blood of a patient and can thereby be determined in, among others, a blood sample, a plasma sample or a serum sample.

[0051] STK1 material as used herein refers to STK1 in its various forms, such as dimers, tetramers, oligomers and complexes comprising STK1. The STK1 material is a serum TK1 material, i.e., a STK1 material present in blood, blood plasma or serum in patient. The STK1 material may then comprise STK1 in the above-mentioned forms, such as dimers, tetramers, oligomers and complexes comprising STK1. STK1 material also includes complexes with at least one TK1 protein unit and other molecules and / or macromolecules.

[0052] In the art, various gene expressions arrays have been proposed to determine the TK1 mRNA transcripts in cancer cell samples and biopsies, including prostate cell samples and prostate biopsies. As mentioned above, TK1 is available in various forms in patients, including cytosolic TK1 and serum TK1. Gene expression arrays determining TK1 mRNA transcripts from such cell or biopsy samples are mainly assaying TK1 mRNA transcripts of cytosolic TK1 present in cancer cells. Hence, such gene expression arrays cannot be used to determine the level of STK1 material in a subject.

[0053] In an embodiment, determining the level of STK1 material comprises contacting the body fluid sample with the antibody, or the antigen-binding fragment thereof. This embodiment also comprises measuring an amount of antibody, or antigen-binding fragment thereof, bound to the STK1 material.

[0054] Contacting the body fluid sample with the antibody, or the antigen-binding fragment thereof, may be achieved by adding the antibody, or the antigen-binding fragment thereof, to the body fluid sample and incubating the body fluid sample with the antibody, or the antigen-binding fragment thereof. The antibody, or the antigen-binding fragment thereof, thereby binds to the STK1 material forming a complex between the antibody, or the antigen-binding fragment thereof, and the STK1 material. In such an embodiment, measuring the amount of antibody, or the antigen-binding fragment thereof, bound to the STK1 material can include measuring or quantifying the complex between the antibody, or the antigen-binding fragment thereof, and the STK1 material to thereby measure or quantify the amount of antibody, or the antigenbinding fragment thereof, bound to the STK1 material.

[0055] In an embodiment, the method also comprises correlating the measured amount of antibody, or the antigen-binding fragment thereof, bound to the STK1 material to a level of STK1 material. This may be performed using a pre-defined correlation between measured amount of antibody, or the antigen-binding fragment thereof, bound to a reference TK1 material and concentration of the reference TK1 material. A typical reference TK1 material that can be used when generating such a pre-defined correlation is recombinant human TK1.

[0056] The pre-defined correlation may, thus, be generated by adding the antibody, or the antigen-binding fragment thereof, to different samples comprising different concentrations of the reference TK1 material, preferably recombinant human TK1. The amount of antibody, or the antigen-binding fragment thereof, bound to the reference TK1 material, preferably recombinant human TK1 , is then measured in the different samples to thereby get a standard curve, function or relationship between concentration of reference TK1 material, preferably recombinant human TK1 , and the measured amount of antibody, or the antigen-binding fragment thereof, bound to the reference TK1 material, preferably recombinant human TK1.

[0057] This pre-defined correlation, such as standard curve, function or relationship, can then be used to map or convert the measured amount of antibody, or the antigen-binding fragment thereof, bound to the STK1 material in the body fluid sample to a concentration of the STK1 material in the body fluid sample.

[0058] It is generally preferred if the same type of antibody, or antigen-binding fragment thereof, is used for generating the pre-defined correlation as for determining a level of STK1 material in a body fluid sample from the patient diagnosed with prostate cancer. Hence, in a preferred embodiment, the antibody, or the antigen-binding fragment thereof, is capable of specifically binding to not only the serum form of human TK1 but also to the reference TK1 material, preferably recombinant human TK1.

[0059] In an embodiment, the body fluid sample is processed prior to or during the incubation of the body fluid sample with the antibody, or the antigen-binding fragment thereof. This sample processing may be used to stabilize selected STK1 forms in the body fluid sample and / or to break larger STK1 complexes or oligomers into smaller complexes or multimers.

[0060] Hence, in an embodiment, a sample dilution or pretreatment buffer is added to the body fluid sample prior to or in connection with adding the antibody or the antigen-binding fragment thereof to the body fluid sample, preferably prior to adding the antibody or the antigen-binding fragment thereof to the body fluid sample.

[0061] In an embodiment, the sample dilution buffer comprises ATP, preferably in a concentration selected within an interval of from 0.5 mM up to 50 mM, such as from 0.5 mM up to 20 mM or from 1 .5 mM up to 50 mM. As previously described herein, ATP stabilizes the tetramer form of TK1, which has high enzymatic TK1 activity.

[0062] In another embodiment, the sample dilution buffer comprises a reducing agent. The reducing agent may then break disulfide cross links in larger STK1 complexes and oligomers to obtain smaller STK1 forms, such as tetramers. Various reducing agents capable of breaking disulfide bonds can be used according to the embodiments including, but not limited to, dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamin (DTBA), tris(2-carboxyethyl)phosphine) (TCEP), and combinations thereof. The amount of the reducing agent is typically selected within an interval of from 0.1 mM up to 10 mM.

[0063] The sample dilution buffer may, in an embodiment, comprise both ATP and a reducing agent.

[0064] The antibody, or the antigen-binding fragment thereof, specifically binds to the STK1 material, and in particular binds specifically to the serum form of the TK1 protein.

[0065] The specificity of an antibody, or an antigen-binding fragment thereof, can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with the antibody, or the antigen-binding fragment thereof, (Kd), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody, or the antigen-binding fragment thereof. The lesser the value of Kd, the stronger the binding strength between the antigenic determinant and the antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1 / Kd. 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.

[0066] Avidity is the measure of the strength of binding between an antibody, or an antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody, or the antigen-binding fragment thereof, and the number of pertinent binding sites present on the antibody, or the antigen-binding fragment thereof.

[0067] Typically, antibodies will bind to their antigen with a dissociation constant (Kd) of 105to 1012moles / liter (M) or less, and preferably 107to 1012M or less and more preferably 108to 1012M, i.e. with an association constant (Ka) of 105to 1012M1or more, and preferably 107to 1012M1or more and more preferably 108to 1012M1.

[0068] Generally, any Kd value greater than 104M (or any Kavalue lower than 104M1) is generally considered to indicate non-specific binding. Preferably, an antibody, or an antigen-binding fragment thereof, will bind to the STK1 material with an affinity 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. Specific binding of an antibody, or an 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 the different variants thereof known per se in the art.

[0069] In an embodiment, the antibody is a monoclonal antibody, i.e., a monoclonal anti-TK1 antibody. In another embodiment, the antibody is a polyclonal antibody, i.e., a polyclonal anti-TK1 antibody.

[0070] In an embodiment, the antibody, or the antigen-binding fragment thereof, has specificity for an epitope or peptide consisting of an amino acid sequence from the C-terminal region of TK1 , preferably of human TK1.

[0071] The peptide is preferably selected from a portion of TK1 ranging from amino acid position 200 to the end of the TK1, i.e., amino acid position 234 in humans (SEQ ID NO: 28). In a particular 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.

[0072] The peptide is preferably an N-mer, wherein N is an integer within a range of from 8 up to 20, preferably within a range of from 10 up to 15. The peptide preferably consists of N consecutive amino acids in the C-terminal region of the TK1 protein.

[0073] In an 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 a further embodiment, the peptide consists of the following amino acid sequence PVPGKPGEAV (SEQ ID NO: 3). In yet another embodiment, the peptide consists of the following amino acid sequence NCPVPGKPGEAV (SEQ ID NO: 4).

[0074] A monoclonal antibody having specificity for an epitope consisting of GEAVAARKLF (SEQ ID NO: 1) has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence DYEMH (SEQ ID NO: 5), a VH domain CDR2 having amino acid sequence AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a VH domain CDR3 having amino acid sequence FITKFDY (SEQ ID NO: 7), a variable light (VL) domain CDR1 having amino acid sequence KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a VL domain CDR2 having amino acid sequence LVSKLDS (SEQ ID NO: 9) and a VL domain CDR3 having amino acid sequence WQGTHFPWT (SEQ ID NO: 10). A monoclonal antibody having specificity for the epitopes NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4) has a VH domain CDR1 having amino acid sequence DYEMH (SEQ ID NO: 5), a VH domain CDR2 having amino acid sequence AILPGSGGTAYNQKFKG (SEQ ID NO: 11), a VH domain CDR3 having amino acid sequence LITTFDY (SEQ ID NO: 12), a VL domain CDR1 having amino acid sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 13), a VL domain CDR2 having amino acid sequence LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 having amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

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

[0076] The above-three presented examples of monoclonal anti-TK 1 antibodies that can be used according to the embodiments are further disclosed in WO 2015 / 094106, the teaching of which regarding monoclonal anti-TK1 antibodies is incorporated herein by reference.

[0077] Hence, in an embodiment, the monoclonal antibody, or the antigen-binding fragment thereof, is selected from the group consisting of a monoclonal antibody or an antigen-binding fragment thereof having specificity for GEAVAARKLF (SEQ ID NO: 1) of human TK1 , a monoclonal antibody or an antigen-binding fragment thereof having specificity 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 an antigen-binding fragment thereof having specificity for a conformation dependent epitope of human TK1.

[0078] In another embodiment, the antibody, or the antigen-binding fragment thereof, has specificity for 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-terminal or C-terminal, preferably the N-terminal, of the peptide for use as coupling to other molecules, such as carrier proteins. An antibody having specificity for this epitope is further disclosed in WO 95 / 29192, the teaching of which regarding anti-TK1 antibodies is incorporated herein by reference.

[0079] In a further embodiment, the antibody, or antigen-binding fragment thereof, has specificity for an epitope or peptide consisting of an amino acid sequence from an active site of TK1. The peptide is preferably selected from a portion of TK1 ranging from amino acid position 150 to amino acid position 190 in human TK1. In a particular 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.

[0080] The peptide is preferably an M-mer, wherein M is an integer within a range of from 10 up to 40, preferably within a range from 20 up 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.

[0081] At least one additional amino acid, such as a cysteine residue, may be added to the N-terminal or C- terminal, preferably the N-terminal, of the peptide for use as coupling to other molecules, such as carrier proteins.

[0082] In an embodiment, the peptide consisting of an 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 , i.e., has amino acid sequence of AYTKRLGTEKEVEVIGGADKYHS (SEQ ID NO: 21).

[0083] An antibody having specificity for this epitope is further disclosed in WO 2008 / 142664, the teaching of which regarding anti-TK1 antibodies is incorporated herein by reference.

[0084] In a further embodiment, the antibody, or the antigen-binding fragment thereof, is a monoclonal antibody, or an antigen-binding fragment thereof, as disclosed in WO 2019 / 201901 , the teaching of which regarding monoclonal anti-TK1 antibodies is incorporated herein by reference.

[0085] For instance, the monoclonal antibody could be mAb 6C6, mAb 4H4 or mAb 23C11 . mAb 6C6 VH domain (SEQ ID NO: 22): METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFSFSSSYWICWVRQAPGKGLEW

[0086] IACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTL

[0087] VTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 6C6 VL domain (SEQ ID NO: 23):

[0088] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPK

[0089] LLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISDSPYVFGGGTEVWKGDPV

[0090] APTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLT STQYNSHKEYTCKVTQGTTSWQSFNRGDC mAb 4H4 VH domain (SEQ ID NO: 24):

[0091] METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWI

[0092] ACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPG

[0093] TLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 4H4 VL domain (SEQ ID NO: 25):

[0094] MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPK

[0095] LLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSSTSGGGVFGGGTEVWKGD

[0096] PVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLT

[0097] LTSTQYNSHKEYTCKVTQGTTSWQSFNRGDC mAb 23C11 VH domain (SEQ ID NO: 26):

[0098] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGI

[0099] IYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQP

[0100] KAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG mAb 23C11 VL domain (SEQ ID NO: 27):

[0101] MDTRAPTQLLGLLLLWLPGARCDWMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPK

[0102] LLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSTFSSYGNAFGGGTEVWKG

[0103] DPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSST LTLTSTQYNSHKEYTCKVTQGTTSWQSFNRGDC

[0104] In an embodiment, the level of STK1 material in the body fluid sample is determined using a kit. 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 can be selected from the above-described illustrative examples of monoclonal and polyclonal anti-TK1 antibodies.

[0105] In a particular embodiment, the kit comprises a first monoclonal antibody, or a first antigen-binding fragment thereof, having specificity 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 also comprises a second monoclonal antibody, or a second antigenbinding fragment thereof, having specificity 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 .

[0106] In an embodiment, the first antibody, or the first antigen-binding fragment thereof, is a so-called capture antibody immobilized to a support or intended to be immobilized to the support and the second antibody, or the second antigen-binding fragment thereof, is a so-called detection antibody. In another embodiment, the second antibody, or the second antigen-binding fragment thereof, is the capture antibody immobilized to the support or intended to be immobilized to the support, whereas the first antibody, or the first antigenbinding fragment thereof, is used as detection antibody.

[0107] In an embodiment, the first and second antibodies, or the first and second antigen-binding fragments thereof, have specificities for different epitopes in the STK1 material.

[0108] In another embodiment, the first and second antibodies, or the first and second antigen-binding fragments thereof, have specificities for the same epitope in the STK1 material. This is possible since the same epitope may be present at multiple copies in the high molecular weight complexes of multiple TK1 protein units. Thus, the STK1 material may be a multivalent complex of multiple, i.e. , at least two, TK1 protein units. In fact, the same type of antibody, or antigen-binding fragment thereof, could be used as the first and second antibody, or first and second antigen-binding fragment thereof.

[0109] In an embodiment, one of the first and the second antibody, or the first and second antigen-binding fragment thereof, has specificity for a peptide consisting of an amino acid sequence from the active site of TK1 and the other of the first and second antibody, or the first and second antigen-binding fragment thereof, has specificity for a peptide consisting of an amino acid sequence from the C-terminal region of TK1.

[0110] In another embodiment, one of the first and the second antibody, or the first and second antigen-binding fragment thereof, has specificity for a peptide consisting of a first amino acid sequence from the C- terminal region of TK1 and the other of the first and the second antibody, or the first and second antigenbinding fragment thereof, has specificity for a peptide consisting of the 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.

[0111] In a further embodiment, one of the first and the second antibody, or the first and second antigen-binding fragment thereof, has specificity for a peptide consisting of a first amino acid sequence from the C- terminal region of TK1 and the other of the first and the second antibody, or the first and second antigenbinding fragment thereof, has specificity for a conformation dependent epitope of human TK1 .

[0112] An antigen-binding fragment of an antibody as used herein can be selected from a group consisting of a single chain antibody, a 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, and a di-scFv fragment.

[0113] In an embodiment, the kit is a sandwich assay kit. In a particular embodiment, the kit is an Enzyme- Linked Immunosorbent Assay (ELISA) kit and preferably a sandwich ELISA.

[0114] In the discussion below, the first antibody, or first antigen-binding fragment thereof, is assumed to be the capture antibody with the second antibody, or second antigen-binding fragment thereof, acting as detection antibody. The embodiments are, however, not limited thereto but could switch capture and detection antibodies.

[0115] A sandwich ELISA can be used to detect STK1 material in a body fluid sample by preparing a surface of a support, such as a solid support, to which the first antibody, or the first antigen-binding fragment thereof, is bound as so-called capture antibody. In a preferred embodiment, a known quantity of the first antibody, or the first antigen-binding fragment thereof, is bound to the surface of the support. Any non-specific binding sites on the surface are optionally, but preferably, blocked. The body fluid sample is then applied to the surface so that any STK1 material present therein will be captured by the immobilized first antibodies, or first antigen-binding fragments thereof. Unbound material is preferably removed by one or multiple washing steps. The second antibody, or second antigen-binding fragment thereof, typically denoted detection antibody, is then added and is allowed to bind to any STK1 material captured by the first antibody, or the first antigen-binding fragment thereof.

[0116] The amount of bound second antibody, or second antigen-binding fragment thereof, is then determined by direct or indirect detection methods. For instance, a label or enzyme can be attached directly to the second antibody, or the second antigen-binding fragment thereof, or indirectly via a link, such as a biotinstreptavidin or a biotin-avidin link. It is, alternatively, possible to use a secondary antibody, or second antigen-binding fragment thereof, that is labeled or connected to an enzyme and binds specifically to the second antibody, or second antigen-binding fragment thereof.

[0117] Hence, in an embodiment the second antibody, or second antigen-binding fragment thereof, has a covalently attached biotin. Alternatively, the second antibody, or second antigen-binding fragment thereof, has a covalently attached streptavidin or avidin.

[0118] The kit preferably also comprises a horseradish peroxidase (HRP) labeled streptavidin or a HRP labeled avidin. Alternatively, the kit also comprises a HRP labeled biotin. The kit also comprises a HRP substrate, such as a 3, 3’, 5, 5’-tetramethylbenzidine (TMB) substrate, a 3,3’-diaminobenzidine (DAB) substrate or a 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate. In such a case, the level of STK1 material in the sample can be determined by spectrophotometric methods that detect the conversion of the chromogenic substrate by HRP into a colored product that is detectable.

[0119] In an embodiment, the kit also comprises a microtiter plate (MCP) as the support to which the first antibody, or first antigen-binding fragment thereof, is immobilized or is intended to be immobilized.

[0120] An example of an ELISA that could be used according to the invention is AroCell® TK 210 ELISA by AroCell AB, Bromma, Sweden.

[0121] The kit does not necessarily have to be an ELISA kit. In another embodiment, the kit uses affinity chromatography where the first antibody, or first antigen-binding fragment thereof, is bound to the stationary phase, such as to a gel matrix or beads in a column. For instance, the gel matrix or beads could be made of agarose, such as SEPHAROSE®. In such a case, STK1 material present in a body fluid sample will be entrapped in the column through binding to the immobilized first antibodies, or first antigen-binding fragments thereof. Following washing, the bound STK1 material can be eluted and detected using the second antibody, or second antigenbinding fragment thereof. For instance, the amount of eluted STK1 material can be determined using Western blotting and with the second antibody, or second antigen-binding fragment thereof, for STK1 detection using direct or indirect detection methods.

[0122] The support could alternatively be magnetic beads, such as DYNABEADS® magnetic beads.

[0123] In a further embodiment, the kit is a chemiluminescence immunoassay (CLIA) kit. CLIA is an immunoassay technique where the label is a luminescent molecule. CLIA methods can be direct, using luminophore markers, or indirect, using enzyme markers. Either method may be competitive or noncompetitive. In direct CLIA methods, the luminophore markers used are typically acridinium and ruthenium esters, while the enzymatic markers used in indirect methods are typically alkaline phosphatase with adamantyl 1 ,2-dioxetane aryl phosphate (AMPPD) substrate and HRP with luminol or its derivatives as substrate.

[0124] The kit does not necessarily have to include two antibodies, or antigen-binding fragments thereof, but could instead include only one type of antibodies, or antigen-binding fragments thereof.

[0125] Furthermore, the kit does not necessarily have to comprise a so-called capture antibody, or antigenbinding fragment thereof. In clear contrast, multiple, i.e., at least two, different antibodies, or antigenbinding fragments thereof, could be used to determine the level of STK1 material without the need for immobilizing at least one of the antibodies, or antigen-binding fragments thereof.

[0126] The invention also relates to use of an anti-thymidine kinase 1 (TK1) antibody, or an antigen-binding fragment thereof, specifically binding to a serum form of human TK1 in the preparation of a kit for treatment stratification. The use comprises determining a level of serum thymidine kinase 1 (STK1) material in a body fluid sample from a patient diagnosed with prostate cancer using the anti-TK 1 antibody, or the antigen-binding fragment thereof. The use also comprises comparing the level of STK1 material with a threshold level and selecting a chemotherapy for the patient if the level of STK1 material is equal to or above the threshold value and an ARSI therapy for the patient if the level of STK1 material is below the threshold value. EXAMPLE

[0127] The present Example evaluates whether serum TK1 levels could be used for treatment stratification of prostate cancer patients.

[0128] Materials and Methods

[0129] Patients

[0130] Cohort 1 consisted of 43 men with hormone-naive metastatic disease at diagnosis identified from the prostate cancer database of the Prostate Cancer Research Center of Tampere University, Finland. All men were diagnosed and treated at the urology outpatient clinic of Tampere University Hospital during 2000-2010. Metastatic disease was defined by the presence of distant metastases (stage M1) confirmed by bone scan imaging, regardless of tumor (T) stage or node (N) stage. Study participants were randomly selected from men satisfying the inclusion criteria and whom had their serum sample available from the time of diagnosis for STK1 measurement. Available information included date of diagnosis, biopsy Gleason score, clinical tumor, node and metastasis (TNM) stage, prostate-specific antigen (PSA) at the time of diagnosis, and the primary treatment method. Participants were followed and managed according to clinicians’ discretion and standard clinical practice. Furthermore, the data included the time of first disease recurrence (either biochemical or radiological) as well as the date and primary cause of death, obtained from the national death certificate registry maintained by Statistics Finland. Follow-up data was available until 31 August 2019, thus follow-up was available for minimum of nine years.

[0131] Cohort 2 and 3 included 200 patients with metastatic castrate-resistant prostate cancer (mCRPC) treated at the Department of Urology, Medical University of Vienna or at the Department of Urology, Semmelweis University in Budapest between 2011 and 2022. All patients had metastatic disease at the time of classification as being castrate-resistant. Serum samples were collected directly before initiation of systemic treatment. In this group, 102 patients had received androgen receptor signaling inhibitors (ARSIs) and 98 patients had been treated with docetaxel (DOC) chemotherapy for mCRPC. Follow-up was available for 197 of these patients.

[0132] STK1 measurements

[0133] Protein levels of TK1 in serum samples were determined using the AroCell® TK 210 ELISA (AroCell AB, Bromma, Sweden) according to the manufacturer’s instructions (www.arocell.com). In brief, serum samples, calibrators, and controls were pre-incubated with sample dilution buffer (SDB) for 1 hour at room temperature (20-25°C). After the pre-incubation, the samples were added to pre-washed anti-TK1 antibody coated plates and incubated for 2 more hours on a shaking platform. The plates were washed 4 times and biotinylated anti-TK1 antibody was added. After the incubation and four washes, enzyme labeled streptavidin horseradish peroxidase (HRP) was pipetted into the wells followed by an incubation step and four washes, a HRP substrate was added. Color development was subsequently stopped and the intensity of color was measured by a spectrophotometer at 450 nm. The color developed was in proportion to the concentration of STK1 protein in the sample. STK1 protein levels in the samples were calculated by using a calibrator curve and a four parameter logistic (4-PL) curve fitting program. Each sample was analyzed in duplicate and the mean value expressed as pg / L.

[0134] Statistical analysis

[0135] Analyses were carried out by Statistikakademin AB, Uppsala, Sweden. The primary endpoint was overall survival (OS). The Cox proportional hazard regression method was used to estimate hazard ratios (HRs). The time metric was months since prostate cancer diagnosis. Follow-up ended at death, lost to follow-up (emigration), or the closing date of 31 June 2023 for cohort 1 , whichever came first. The follow-up was last updated in August 2022 for cohort 2 and 3. The analysis was adjusted for age, multivariable-adjusted analyses further adjusted for biopsy Gleason score, TNM stage, and PSA at diagnosis. Recurrence-free survival was evaluated in a separate Cox regression model with disease recurrence as the endpoint. In this analysis, the follow-up ended at the date of the first prostate cancer recurrence (PSA relapse or radiological progression of metastases in computed tomography despite castrate-level of serum testosterone), death, or the common closing date, whichever came first. Study participants were stratified by median and by quartiles of STK1 level. Participants with a STK1 level at or below the median were used as the reference group. Survival trends were analyzed by including the STK1 protein level in a Cox regression model as a continuous variable. Additionally, Kaplan-Meier curves were used to visualize the survival differences.

[0136] Results

[0137] The clinical characteristics of this multicenter study cohorts are described in Tables 1-3. The 43 men with de novo metastatic hormone-sensitive prostate cancer (mHSPC) in cohort 1 received gonadotropinreleasing hormone (GnRH) agonists during a period (2002-2011) when new generation antiandrogens were not yet available for mHSPC. Cohorts 2 and 3 consisted of 200 patients diagnosed with metastasis where survival information was available for 197 patients. The men in cohort 2 received enzalutamide or abiraterone (ENZA / ABI) between 2011 and 2022 and the men in cohort 3 received docetaxel treatment during the same period. Median age at diagnosis was similar in all three cohorts. The median PSA at diagnosis differed between cohorts: cohort 1 : 50 ng / mL, cohort 2: 93 ng / mL in, and cohort 3: 68 ng / mL. Table 1 - Population characteristics and prostate cancer clinical characteristics by STK1 levels of population of 43 men diagnosed with de novo M1 prostate cancer (cohort 1) Levels of STK1 in hormone-sensitive and castration-resistant prostate cancer

[0138] The median STK1 level in cohort 1 (mHSPC) at the time of diagnosis was 0.61 pg / L (Table 1). STK1 levels did not markedly vary by biopsy International Society of Urological Pathology (ISUP) Gleason score (median 0.23 vs. 0.21 pg / L for grade 3-5 vs grade 1 -2 tumors, respectively). PSA at diagnosis was higher in patients showing elevated STK1 levels (208 ng / mL) compared to the group with lower levels of STK1 (46 ng / mL). Elevated STK1 levels were associated with higher clinical T-stage at diagnosis (T1 / T2 versus T3 / T4; p = 0.021), whereas biopsy ISUP Gleason scores were similarly distributed (Table 1).

[0139] In the mCRPC cohorts, baseline median STK1 levels were 0.50 pg / L in patients subsequently treated with ARSIs (cohort 2) and 0.49 pg / L in docetaxel-treated patients (cohort 3). Median age at diagnosis did not markedly differ by STK1 level. The STK1 cut-off from mHSPC cohort 1 (0.61 pg / L) was used in the analyses of mCRPC patients of cohort 2 and 3. In this material, 123 patients showed STK1 levels below the cut-off and 74 patients showed higher STK1 levels. Median PSA levels in patients stratified for low and high levels of STK1 were 52.2 and 162 ng / mL, respectively (p = 0.0006) (Table 2). The median PSA levels at diagnosis was 93 (18-395) ng / mL and 68 (26-279) ng / mL in mCRPC cohorts 2 and 3, respectively.

[0140] Table 2 - Population characteristics and prostate cancer clinical characteristics by STK1 levels of patients with castrate-resistant prostate cancer, study population of 197 men (cohort 2 and 3)

[0141] Association between STK1 and overall survival in mHSPC patients

[0142] In cohort 1 , STK1 levels above the cut-off were associated with worse age-adjusted overall survival (HR 3.08 (1.49-6.36); p < 0.001) compared to men with STK1 levels below the cut-off (Table 3). The risk association remained unchanged after multivariable adjustment. A statistically significant increasing risk trend by STK1 level was observed both for prostate cancer-specific and overall risk of death (Table 3). In Kaplan-Meier analysis, stratification by STK1 showed distinct groups of differing disease-specific survival especially in analyses adjusted for age (Fig. 1A) and PSA at diagnosis (Fig. 1 B). Inclusion of STK1, ISUP Gleason score, and PSA as features in the random forest classification model produced most accurate prediction for prostate cancer death compared to any other model (Fig. 2). Notably, combination of these three variables displayed higher mean prediction accuracy compared to model using PSA and Gleason score alone, confirming independent additional prognostic value of STK1. All models with clinical T-stage as a classifier had worse mean prediction error compared to models without it. Therefore T-stage was left out of the models. Overall, the mean prediction error for prostate cancer death using STK1 , Gleason score, and PSA in combination is excellent (Fig. 2).

[0143] Table 3 - Overall survival of mHSPC patients after stratified by STK1 median at diagnosis

[0144] HR 1 - age-adjusted HR 2 - adjusted for ISUP Gleason score

[0145] HR 3 - adjusted for PSA at diagnosis HR 4 - adjusted for clinical T-stage

[0146] Association between STK1 and overall survival in mCRPC patients

[0147] Survival data was available for 197 of the patients diagnosed with mCRPC (cohort 2 and 3). STK1 levels above the cut-off were associated with worse overall survival (9.5 months versus 20.7 months) and a higher relative risk (age-adjusted HR 2.08 (1.47-2.97); p < 0.001) (Table 4). STK1 levels remained significantly associated with an increased relative hazard ratio after adjustment for age, PSA, and Eastern Cooperative Oncology Group (ECOG) performance status (HR 1.83 (1.34-2.86), p = 0.002). Gleason stage and T-stage were considered as irrelevant for this group of patients with advanced disease. Kaplan- Meier survival plots are shown in Figs. 3A-3D.

[0148] Cohort 2, treated with ARSIs abiraterone or enzalutamide, and cohort 3, treated with docetaxel, were analyzed separately. The age-adjusted hazard ratio for STK1 above cut-off in the group of 99 patients treated with abiraterone or enzalutamide was 2.54 (1.57-4.06) (p < 0.001) (Table 4). This association remained significant after adjustment for age, PSA and ECOG performance status scale (Table 4). In the group of 98 mCRPC patients treated with docetaxel, the age-adjusted hazard ratio associated with STK1 levels above cut-off was lower compared to the ARSI-treated group 1.62 (0.97-2.71) (p = 0.066). Adjustment for age, PSA and ECOG performance status scale resulted in a hazard ratio of 1.52 (0.91- 2.56) (p = 0.11).

[0149] Kaplan-Meier survival plots showing the association between overall survival dichotomized for STK1 levels for cohort 2 and 3 are shown in Figs. 3A-3D.

[0150] Table 4 - Overall survival after prostate cancer diagnosis stratified by STK1 median at diagnosis. Study population of 197 men with castrate-resistant prostate cancer

[0151] HR 1 - age-adjusted HR 2 - adjusted for age and PSA at diagnosis

[0152] HR 3 - adjusted for age, PSA and ECOG at diagnosis

[0153] Three separate cohorts representing different clinical scenarios and treatment eras were used to investigate the potential prognostic value of STK1 in advanced prostate cancer. In cohort 1 of 43 patients with newly diagnosed mHSPC and subsequently treated with ADT, only STK1 showed independent prognostic value for overall survival. The prognostic value was independent of PSA and the accuracy was further improved when combined with established clinical factors. Similarly, having detectable STK1 at baseline was associated with shorter time to castration resistance in a contemporary cohort of mHSPC patients managed with a combination of ADT and ARSIs or docetaxel. Together these two cohorts suggest that STK1 could be a valuable tool for prognostic evaluation in mHSPC. The cut-off defined in mHSPC patients (cohort 1) was found to be associated with an age-adjusted HR of 2.08 in 197 patients with mCRPC treated with either ARSIs (abiraterone or enzalutamide) or docetaxel. Restricting the analysis to mCRPC patients treated with ARSIs resulted in an age-adjusted relative risk of 2.53 being associated with elevated STK1 levels. The increased relative risk associated with elevated STK1 remained after adjustment for differences in PSA levels also in the mCRPC patients.

[0154] The relative risk associated with elevated STK1 in patients treated with docetaxel (HR: 1.52) was lower compared to that observed for ARSI treatment. This difference with regard to relative risk was not observed between the docetaxel and ARSI patients with low STK1 levels (Fig. 3D). These findings indicate that ARSIs may not be adequate for patients with elevated STK1 levels but may be so for patients with lower STK1 levels. High STK1 correlates to aggressive disease and ARSI treatment has been reported not to be effective for patients with Gleason 9-10 disease. Interestingly, the clinical benefit of docetaxel was, however, only observed for patients with high-volume disease (defined as presence of visceral metastases and / or multiple bone metastases). The results indicate the possibility that STK1 can be used as a biomarker for identification of patients with advanced prostate cancer that should be subjected to treatment intensification. The response of prostate cancer to antiandrogen therapy has been reported to be complex, involving upregulation of tumor suppressors and suppression of growth promoting factors such as MYC and E2F1 .

[0155] STK1 levels presumably reflect the number of malignant cells in active proliferation and it is possible that ARSI therapy is inadequate for treatment of rapidly growing tumors. Rapidly proliferating prostate cancer cells are expected to be sensitive to cell-cycle active drugs such as docetaxel, an inhibitor of microtubule dynamics and therefore mitosis.

[0156] A strength of the present study is that the cut-off that was established in the initial study mHSPC patients was used in the follow-up study of mCRPC. Long follow-up time after prostate cancer diagnosis was also available for the three groups where overall survival was used as endpoint.

[0157] Hence, STK1 could be used as biomarker for risk stratification of mHSPC and mCRPC patients. The finding indicates that patients with elevated levels of STK1 should not be treated with ARSIs. Patients with aggressive disease, including rapid PSA doubling times, are often treated with docetaxel in daily clinical practice. STK1 would thereby be useful as a tool for treatment decisions in mCRPC.

[0158] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

CLAIMS1 . A treatment stratification method comprising: determining a level of serum thymidine kinase 1 (STK1) material in a body fluid sample from a patient diagnosed with prostate cancer using an antibody, or an antigen-binding fragment thereof, specifically binding to a serum form of human TK1 ; comparing the level of STK1 material with a threshold level; and selecting a chemotherapy for the patient if the level of STK1 material is equal to or above the threshold value and an androgen receptor signaling inhibitor (ARSI) therapy for the patient if the level of STK1 material is below the threshold value.

2. The method according to claim 1 , wherein determining the level of STK1 material comprises determining the level of STK1 material in the body fluid sample from a patient diagnosed with metastatic prostate cancer using the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1 .

3. The method according to claim 2, wherein determining the level of STK1 material comprises determining the level of STK1 material in the body fluid sample from a patient diagnosed with metastatic castration-resistant prostate cancer using the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1 .

4. The method according to any one of the claims 1 to 3, wherein the chemotherapy is selected from the group consisting of docetaxel, cabazitaxel, mitoxantrone, estramustine 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) treatment, androgen receptor antagonist (ARA) treatment and any combination thereof.

6. The method according to claim 5, wherein the ASI is abiraterone.

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

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

9. The method according to any one of claims 1 to 8, wherein determining the level of STK1 material comprises determining the level of STK1 material in a serum sample or a plasma sample using the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1.

10. The method according to any one of claims 1 to 9, wherein determining the level of STK1 material in the body fluid sample comprises: contacting the body fluid sample with the antibody, or the antigen-binding fragment thereof, specifically binding to the serum form of human TK1 ; and measuring an amount of antibody, or antigen-binding fragment thereof, bound to the STK1 material.

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

12. The method according to claim 11 , wherein correlating the measured amount of antibody, or antigen-binding fragment thereof, comprises correlating the measured amount of antibody, or antigenbinding fragment thereof, to a level of STK1 material using a pre-defined correlation between measured amount of antibody, or antigen-binding fragment thereof, bound to recombinant human TK1 and a concentration of recombinant human TK1 .

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

14. The method according to claim 13, wherein the monoclonal antibody, or the antigen-binding fragment thereof, is selected from the group consisting of: a monoclonal antibody, or an antigen-binding fragment thereof, having specificity for GEAVAARKLF (SEQ ID NO: 1) of human TK1 ;a monoclonal antibody, or an antigen-binding fragment thereof, having specificity 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 an antigen-binding fragment thereof, having specificity for a conformation dependent epitope of human TK1 .

15. The method according to claim 14, wherein the monoclonal antibody, or the antigen-binding fragment thereof, has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence SEQ ID NO: 5; a VH domain CDR2 having amino acid sequence SEQ ID NO: 6; a VH domain CDR3 having amino acid sequence SEQ ID NO: 7; a variable light (VL) domain CDR1 having amino acid sequence SEQ ID NO: 8; a VL domain CDR2 having amino acid sequence SEQ ID NO: 9; and a VL domain CDR3 having amino acid sequence SEQ ID NO: 10.

16. The method according to claim 14, wherein the monoclonal antibody, or the antigen-binding fragment thereof, has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence SEQ ID NO: 5; a VH domain CDR2 having amino acid sequence SEQ ID NO: 11 ; a VH domain CDR3 having amino acid sequence SEQ ID NO: 12; a variable light (VL) domain CDR1 having amino acid sequence SEQ ID NO: 13; a VL domain CDR2 having amino acid sequence SEQ ID NO: 9; and a VL domain CDR3 having amino acid sequence SEQ ID NO: 10.

17. The method according to claim 14, wherein the monoclonal antibody, or the antigen-binding fragment thereof, has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence SEQ ID NO: 14; a VH domain CDR2 having amino acid sequence SEQ ID NO: 15; a VH domain CDR3 having amino acid sequence SEQ ID NO: 16; a variable light (VL) domain CDR1 having amino acid sequence SEQ ID NO: 17; a VL domain CDR2 having amino acid sequence SEQ ID NO: 18; anda VL domain CDR3 having amino acid sequence SEQ ID NO: 19.

18. The method according to any of the claims 14 to 17, wherein determining the level of STK1 material comprises determining the level of STK1 material in the body fluid sample using a kit for determining a level of STK1 material in a body fluid sample comprising: a first monoclonal antibody, or a first antigen-binding fragment thereof, having specificity for an epitope selected from the group consisting of:GEAVAARKLF (SEQ ID NO: 1) of human TK1 ; 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 conformation dependent epitope of human TK1 ; and a second monoclonal antibody, or a second antigen-binding fragment thereof, having specificity for an epitope selected from the group consisting of:GEAVAARKLF (SEQ ID NO: 1) of human TK1 ; 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 conformation dependent epitope of human TK1 .

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