Thymidine kinase as a marker for the efficacy of immune checkpoint inhibitors

Abstract

The present invention provides a method of determining the prognosis of an individual suffering from cancer and who is being considered for or currently undergoing treatment with one or more immune checkpoint inhibitors, the method comprising or consisting of the steps of (a) providing a sample taken from the individual prior to treatment with one or more immune checkpoint inhibitors and (b) measuring the activity and / or concentration of thymidine kinase (TK) in the sample, wherein the activity and / or concentration of thymidine kinase (TK) measured in step (b) is indicative of the individual's prognosis if subsequently treated with one or more immune checkpoint inhibitors.

Description

Technical Field

[0001] The present invention relates to methods and uses related to determining the prognosis of cancer patients, and methods for stratifying these patients for treatment.

Background Art

[0002] Immune checkpoint inhibitors (ICIs) are effective only in some cancer patients. Examples of clinically developed ICIs include those targeting programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3). Antibody drugs targeting these immune checkpoints release the brakes on T cell activity, enabling the activation and subsequent proliferation of tumor-reactive T cells, which can then initiate an effective anti-tumor response. Studies have shown that the number of T cells significantly increases after the first administration of ICI drugs. This level of increase in T cells correlates with tumor response and patient outcome

[28] .

[0003] In recent years, effective ICI regimens using CTLA-4 and PD-1 blocking antibodies have emerged for the treatment of melanoma [1-6]. These treatments have revolutionized the field of melanoma oncology, but a significant portion of melanoma patients do not respond to these treatments or do not obtain a permanent effect from them. There is also a possibility of serious toxicity occurring during treatment, and moreover, the treatment cost is high. Therefore, it is important to increase knowledge about predictive factors and their effectiveness in various patient groups.

[0004] Thymidine kinase 1 (TK) is a cytosolic enzyme and a phosphotransferase that plays a crucial role in DNA synthesis and repair [7]. TK is part of the reaction chain that introduces thymidine into the DNA strand, and therefore plays an important role in DNA synthesis and cell division [7]. Cells in division release TK at the end of mitosis, and thus TK can be detected in the blood. Furthermore, elevated TK enzyme activity (TKa) has been measured in blood samples from cancer patients, and this elevation has been associated with tumor growth and tumor burden [8]. Circulating levels of TKa, as measured by the DiviTum® TKa assay, have been shown to be associated with the stage, prognosis, and treatment response of several cancer types, including breast cancer, lung cancer, pancreatic cancer, and renal cell carcinoma [9-15].

[0005] The inventors have surprisingly discovered that measuring the activity and / or concentration of thymidine kinase (TK), optionally along with measuring the activity and / or concentration of lactate dehydrogenase (LDH), before and during treatment with immune checkpoint inhibitors (ICIs) can provide a prognosis for subsequent treatment with ICIs. This allows for determining whether an ICI administered to or being considered for administration to a patient is not functioning or is not functioning effectively. The main advantage of quickly knowing that an ICI drug is ineffective is that, because these drugs are very expensive and potentially very toxic, it is in the patient's best interest to avoid starting treatment with an ineffective drug, or to discontinue treatment with an ineffective drug as soon as possible and instead implement an alternative therapy.

[0006] A first aspect of the present invention provides a method for determining the prognosis of an individual suffering from cancer who is being considered for treatment with one or more immune checkpoint inhibitors, or who is currently receiving treatment with one or more immune checkpoint inhibitors, and this method is: (a) providing a sample taken from an individual before treatment with one or more immune checkpoint inhibitors, The procedure includes (b) measuring the activity and / or concentration of thymidine kinase (TK) in the sample, or comprises the steps of: Here, the thymidine kinase (TK) activity and / or concentration measured in step (b) indicates the prognosis of the individual after treatment with one or more immune checkpoint inhibitors.

[0007] "Thymidine kinase (TK)" refers to thymidine kinase 1 (TK1). Thymidine kinase 1 is a key cell cycle regulatory enzyme and is important for nucleotide metabolism during DNA synthesis. TK1 catalyzes the conversion of thymidine to deoxythymidine monophosphate, which is further phosphorylated to diphosphate and triphosphate, and then incorporated into DNA. TK1 activity is low or absent in resting cells, but increases significantly as cells divide and replicate. Therefore, the presence of TK1 in cells is an indicator of active cell proliferation. TK1 diffuses from proliferating cells into the bloodstream, and its activity can be measured in serum or plasma samples from the blood. DiviTum® TKa is a highly sensitive and accurate blood-based assay for measuring TK activity (TKa) in serum or plasma samples.

[0008] Immune checkpoint inhibitors (ICIs) are a type of drug that blocks proteins called checkpoints, which are produced by certain types of immune system cells (such as T cells) and some cancer cells. These checkpoints generally function as negative regulators of the immune system. When checkpoints are activated, immune cells are inhibited from inducing cytotoxic responses. These checkpoint proteins are crucial in maintaining the balance between autoimmunity and self-tolerance. However, tumors can often upregulate the expression of immune checkpoints, creating an immunosuppressive environment that allows tumor cells to escape immune-mediated destruction. Various immune checkpoint inhibitors have been developed to treat cancer. Among these, the most clinically developed are programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and lymphocyte activator gene-3 (LAG-3) inhibitor therapies. Antibody drugs targeting these immune checkpoints release the brakes on T cell activity, enabling the activation and subsequent proliferation of tumor-responsive T cells, which can then initiate an effective anti-tumor response. Studies have shown a significant increase in T cell counts after the first dose of ICI drugs. The level of this increase in T cells correlates with tumor response and patient outcomes (Kim et al. Clin Cancer Res;25(7)2020).

[0009] "Individuals with cancer for whom treatment with one or more immune checkpoint inhibitors is being considered" includes cancer patients for whom a physician would typically consider and / or plan treatment with one or more checkpoint inhibitors. Examples include patients with tumors for which regulatory bodies such as the FDA (or equivalent regulatory bodies in different countries) have approved treatment with immune checkpoint inhibitors. Twomey and Zhang (2021) The AAPS Journal 23:39 provides an overview of FDA-approved immune checkpoint inhibitors, which are incorporated herein by reference. Certain patients may be considered for treatment with immune checkpoint inhibitors if they express immune checkpoint inhibitor receptors (checkpoint proteins) on cancer cells, or express those receptors at higher levels than healthy individuals, which can be determined by conventional methods such as diagnostic assays that measure the level of immune checkpoint inhibitor receptor expression. An example of such cancer is melanoma, which is the subject of the examples described herein, but the methods of the present invention are also applicable to other cancers for which treatment with one or more checkpoint inhibitors is typically considered.

[0010] "The prognosis of individuals subsequently treated with one or more immune checkpoint inhibitors" includes the expected efficacy of treatment with one or more immune checkpoint inhibitors, such as progression-free survival (PFS) and overall survival (OS) of individuals subsequently treated with one or more immune checkpoint inhibitors.

[0011] Treatment with one or more checkpoint inhibitors includes treatment with a single ICI (ICI monotherapy) or treatment with different types of ICIs (ICI combination therapy).

[0012] ICI therapy and / or subsequent ICI therapy can be administered alone or in combination with additional non-ICI cancer therapies. In one particular embodiment, ICI therapy and / or subsequent ICI therapy consists of treatment with one or more ICIs only, without additional non-ICI cancer therapies.

[0013] Prior to the method of the present invention, a sample is taken from an individual. It will be understood that the activity and / or concentration of thymidine kinase (TK) can be measured. The TK protein level is expected to correlate with the TK activity level. In one embodiment, TK activity (TKa) is measured. TKa can be measured by techniques well known in the art. This can be measured by ELISA such as the DiviTum® TKa assay (Biovica, Sweden) in accordance with the manufacturer's instructions, as previously reported in Schwartz et al. (2003) J.Nucl.Med. 44 2027-2032, Nisman et al. (2013) Clinical Chemistry and Laboratory Medicine 51(2):439-47, and Bagegni et al. (2017) Breast Cancer Res. 19(1):123 (all incorporated herein by reference), and / or by real-time assays such as those described in International Publication No. 2011 / 142719 and Stalhandske et al. (2013) Analytical Biochemistry 432;155-164 (the contents of which are incorporated herein by reference). TK concentration levels can also be measured by several techniques well known in the art, such as immunoassays including ELISA.

[0014] In additional or alternative embodiments, this method is: (c) providing a sample taken from the individual 1 to 4 weeks after initial treatment with one or more immune checkpoint inhibitors, The process further comprises step (d) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (c), Here, the thymidine kinase (TK) activity and / or concentration measured in steps (b) and (d) indicate the prognosis of the individual if further treatment with one or more immune checkpoint inhibitors.

[0015] In additional or alternative embodiments, the sample provided in step (c) may be collected from the individual 3 to 4 weeks after initial treatment with one or more immune checkpoint inhibitors.

[0016] In additional or alternative embodiments, the sample provided in step (c) may be collected from the individual 7 to 28 days after the first treatment with one or more immune checkpoint inhibitors, or 14 to 28 days after the first treatment with one or more immune checkpoint inhibitors, or 21 to 28 days after the first treatment with one or more immune checkpoint inhibitors.

[0017] In additional or alternative embodiments, the method of the present invention is for stratifying individuals or groups of individuals with cancer for treatment with one or more immune checkpoint inhibitors or for further treatment. In other words, the method is used to determine whether an individual with cancer should receive treatment with one or more immune checkpoint inhibitors or for further treatment.

[0018] In a further aspect of the present invention, a method is provided for stratifying cancer patients for treatment with one or more immune checkpoint inhibitors or for further treatment, the method being Step (i) of carrying out the method of the first embodiment, Step (i) includes, or comprises, step (ii) stratifying the individual for treatment with one or more immune checkpoint inhibitors or alternative cancer therapies or for further treatment, based on the results of step (i). Optionally, the thymidine kinase (TK) activity and / or concentration measured in step (b) and / or (d) indicates the prognosis of the individual after treatment with one or more immune checkpoint inhibitors.

[0019] In one embodiment, patients may be stratified for treatment with one or more immune checkpoint inhibitors or for further treatment. Alternatively, patients may be stratified for treatment with alternative non-ICI cancer therapies.

[0020] Alternative cancer therapies to immune checkpoint inhibitors vary depending on the specific tumor type, but examples include surgery, chemotherapy, targeted therapy, immunotherapy (i.e., immunotherapy different from immune checkpoint inhibitors), chemoimmunotherapy, radiation therapy, and combinations thereof. For example, a physician can determine the most appropriate alternative cancer therapy by referring to the NCCN Cancer Treatment Guidelines for each individual tumor type for which an ICI is approved (e.g., melanoma, lung cancer, RCC, HNSCC, CRC), which can be found at https: / / www.nccn.org / guidelines / category_1.

[0021] The following embodiments relate to all aspects of the present invention.

[0022] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased compared to the thymidine kinase (TK) activity and / or concentration measured in step (b), the individual's prognosis is good with further treatment with one or more checkpoint inhibitors (e.g., the individual's progression-free survival is at least 6 months, or is expected to be at least 6 months), and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

[0023] Preferably, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is at least 8%, for example, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, and less than the activity and / or concentration of thymidine kinase (TK) measured in step (b). At least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 41%, at least 42%, at least 43%, at least 44% , at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least It is increasing by 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400%, or at least 500%.

[0024] In an additional or alternative embodiment, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 8% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0025] In an additional or alternative embodiment, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 23% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0026] In an additional or alternative embodiment, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 25% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0027] In an additional or alternative embodiment, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 50% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0028] In an additional or alternative embodiment, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0029] In additional or alternative embodiments, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 191% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0030] A “favorable” prognosis includes, or is expected to include, a progression-free survival period of at least 6 months for the individual, i.e., no cancer progression 6 months after initial treatment with an ICI. A “favorable” prognosis may also include, or is expected to include, a progression-free survival period of at least 12, 18, or 24 months for the individual, i.e., no cancer progression 12, 18, or 24 months after initial treatment with an ICI.

[0031] In additional or alternative embodiments, “good” prognosis means that progression-free survival or expected progression-free survival after initial treatment with an ICI is at least one year, or at least two, three, four, five, or ten years.

[0032] Progression-free survival (PFS) can be defined as the period from the start of treatment to the date on which disease progression is confirmed, or the date of death, or the date of the last follow-up.

[0033] In additional or alternative embodiments, “good” prognosis means that overall survival (OS) or expected overall survival after initial treatment with ICI is at least one year, or at least two, three, four, five, or ten years. OS may be defined as the period from the start of treatment to the date of death or the date of the last follow-up.

[0034] In additional or alternative embodiments, “good” prognosis means complete response, partial response, or disease stability. Preferably, good prognosis means complete response or partial response, most preferably complete response. The response to treatment may be based on radiological examinations (CT, MRI, and / or positron emission (PET) CT tomography) evaluated by a radiologist and may be assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria

[17] .

[0035] "Stratifying for further treatment with one or more immune checkpoint inhibitors" includes classifying patients or individuals with cancer as needing treatment with one or more immune checkpoint inhibitors or further treatment. This may involve the same ICI or different ICIs, and may be performed alone or in combination with alternative cancer therapies.

[0036] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased by at least 100% compared to the thymidine kinase (TK) activity and / or concentration measured in step (b), the individual's prognosis is good with further treatment with one or more checkpoint inhibitors (e.g., the individual's progression-free survival is greater than 24 months or is expected to be greater than 24 months), and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors. Alternatively, the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased by at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0037] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is below a cutoff value, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors, and / or the patient is stratified for treatment with or further treated with one or more immune checkpoint inhibitors.

[0038] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is above a cutoff value, the prognosis for the individual is poor if treated with or further treated with one or more immune checkpoint inhibitors (e.g., the individual's progression-free survival is less than 6 months or is expected to be less than 6 months), and / or the patient is stratified to receive no further treatment with one or more immune checkpoint inhibitors or to receive treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors.

[0039] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is below a cutoff value, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., the individual's progression-free survival is at least 6 months or is expected to be at least 6 months), and / or the patient is stratified for treatment with or further treated with one or more immune checkpoint inhibitors, where the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0040] Preferably, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is at least 8%, for example, at least 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%. It is increasing by 44%, 45%, 41%, 42%, 43%, 44%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, and 500%.

[0041] In additional or alternative embodiments, the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased by at least 8% compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0042] In additional or alternative embodiments, the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased by at least 23% compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0043] In additional or alternative embodiments, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 25% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0044] In additional or alternative embodiments, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 50% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0045] In additional or alternative embodiments, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0046] In additional or alternative embodiments, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 191% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0047] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is lower than a cutoff value, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., the individual's progression-free survival is greater than 24 months or is expected to be greater than 24 months), and / or the patient is stratified for treatment with or further treated with one or more immune checkpoint inhibitors, where the thymidine kinase (TK) activity and / or concentration measured in step (d) is increased by at least 100% compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0048] It will be understood that the growth rate value can be optimized depending on the type of cancer or tumor. Those skilled in the art will be able to routinely adjust the growth rate value based on the information provided herein with respect to melanoma.

[0049] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is above a cutoff value, the prognosis of the individual is poor if treated with or further treated with one or more immune checkpoint inhibitors (e.g., the individual's progression-free survival is less than 6 months or is expected to be less than 6 months), and / or the patient is stratified to receive no further treatment with one or more immune checkpoint inhibitors or treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors, wherein the thymidine kinase (TK) activity and / or concentration measured in step (d) is not increased compared to the thymidine kinase (TK) activity and / or concentration measured in step (b).

[0050] In additional or alternative embodiments of the method of the present invention, the activity (TKa) of thymidine kinase is measured, and the cutoff value is selected from 40-75 Du / L, e.g., 42-72 Du / L, e.g., 49-72 Du / L, e.g., 42-60 Du / L, e.g., 49-60 Du / L, e.g., 58-60 Du / L, or preferably 60 Du / L. In one embodiment, the cutoff is 60 Du / L. In an alternative embodiment, the cutoff is 58 Du / L.

[0051] In alternative or additional embodiments, the cutoff value may be 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 Du / L.

[0052] It will be understood that the cutoff value may be optimized depending on the type of cancer or tumor. Those skilled in the art will be able to routinely adjust the cutoff value based on the information provided herein.

[0053] Du / L means DiviTum® units per liter according to the manufacturer's instructions, as previously reported in the DiviTum® TKa assay (Biovica, Sweden) and in Schwartz et al. (2003) J. Nucl. Med. 44 2027-2032, Nisman et al. (2013) Clinical Chemistry and Laboratory Medicine 51(2):439-47, and Bagegni et al. (2017) Breast Cancer Res. 19(1):123 (incorporated herein by reference).

[0054] TKa can also be measured in DuA units using the DiviTum® TKa assay. In Du / L units, recombinant thymidine kinase proteins are used to assign Du / L values ​​to the "Gold Standard Serum" and calibrator panels. A 1 Du / L measurement represents the catalytic activity obtained from a sample with a recombinant thymidine kinase (TK) concentration of 1 pg per ml. An optimized DiviTum® TKa assay has also been developed with a new calibration concept that better fits the assay principle for measuring TK activity, and with a new unit (DuA) that better reflects TK activity. The conversion formula between Du / L and DuA is as follows: DuA = 134 + 0.53 (Du / L) Coefficient of determination (R 2 ) = 0.93

[0055] In additional or alternative embodiments, this method is: Step (e) provides one or more control samples, The method further comprises step (f) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (e), Here, the prognosis of an individual treated with one or more immune checkpoint inhibitors is determined by comparing the measurement in step (f) with the measurement in step (b) and / or step (d).

[0056] One or more control samples can be obtained from individuals who subsequently develop cancer before successful treatment with one or more immune checkpoint inhibitors.

[0057] Successful treatment includes cessation of tumor growth, non-progression, and / or regression. This can be assessed by imaging techniques, such as the Gold Standard Assessment Tool. Successful treatment can be assessed by RECIST criteria (see Reference 17). Response Evaluation Criteria In Solid Tumors (RECIST) refers to a published set of rules used to assess tumor burden in order to objectively evaluate the response to treatment. Disease stabilization, partial response, complete response, progression-free survival, and overall survival are endpoints in which treatment is considered successful. Successful treatment may be defined in the same way as good prognosis as defined above. For example, successful treatment may be considered to be a progression-free survival of at least 24 months, i.e., no cancer progression 24 months after initial treatment with ICI.

[0058] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) corresponds to or is lower than the thymidine kinase (TK) activity and / or concentration measured in step (f), the prognosis of the individual treated with one or more immune checkpoint inhibitors is good, and / or the patient is stratified for treatment with one or more immune checkpoint inhibitors or further treatment.

[0059] "Corresponding" includes the thymidine kinase (TK) activity and / or concentration being identical to that of a positive control sample, or being closer to (or closer to a predefined reference value representing the same) the activity and / or concentration of one or more positive control samples than one or more negative control samples.

[0060] Alternatively or additionally, “corresponding” includes the activity and / or concentration being statistically significant in correlation with the amount in the control sample. “Statistically significant correlation with the amount in the control sample” means, or includes, that the presence or amount in the test sample is correlated with the presence or amount in the control sample by a p-value of ≤0.05, e.g., ≤0.04, ≤0.03, ≤0.02, ≤0.01, ≤0.005, ≤0.004, ≤0.003, ≤0.002, ≤0.001, ≤0.0005, or ≤0.0001.

[0061] In additional or alternative embodiments, one or more control samples may be obtained from individuals who subsequently develop cancer before treatment with one or more immune checkpoint inhibitors proves unsuccessful.

[0062] In additional or alternative embodiments, if the thymidine kinase (TK) activity and / or concentration measured in step (b) is higher than the thymidine kinase (TK) activity and / or concentration measured in step (f), the prognosis for the individual treated with one or more immune checkpoint inhibitors is poor, and / or the patient is stratified to receive treatment with no further treatment with one or more immune checkpoint inhibitors or with alternative cancer therapies to one or more immune checkpoint inhibitors.

[0063] In additional or alternative embodiments of the method of the present invention, the method is: The step further comprises step (g) measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a), Here, the LDH activity and / or concentration measured in step (g) further indicates the prognosis of the individual after treatment with one or more immune checkpoint inhibitors.

[0064] In additional or alternative embodiments, if the LDH activity and / or concentration measured in step (g) is considered normal, i.e., not elevated above normal levels, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., progression-free survival of the individual is greater than 24 months), and / or the patient is stratified for treatment with or further treated with one or more immune checkpoint inhibitors.

[0065] In additional or alternative embodiments, if the LDH activity and / or concentration measured in step (g) is below a cutoff value, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., progression-free survival of the individual is greater than 24 months), and / or the patient is stratified for treatment with or further treated with one or more immune checkpoint inhibitors.

[0066] In additional or alternative embodiments, the activity of LDH is measured, and the cutoff value is selected from 2 to 6 microcuts (mikrokat) / L, for example, 3 to 5 microcuts / L, or in this example, 4 microcuts / L.

[0067] It will be understood that the cutoff value can be optimized, for example, depending on the type of cancer or tumor in the individual. This can be routinely achieved by those skilled in the art based on the information provided herein.

[0068] In one embodiment of the method of the present invention, an additional step (h) is performed in which the individual is treated with one or more immune checkpoint inhibitors or alternative cancer therapies.

[0069] In additional or alternative embodiments, cancer is cancer for which treatment with immune checkpoint inhibitors has been approved. This includes, for example, patients with cancer for which a regulatory agency such as the FDA has approved treatment with immune checkpoint inhibitors. Twomey and Zhang (2021) The AAPS Journal 23:39 provides an overview of FDA-approved immune checkpoint inhibitors, which is incorporated herein by reference.

[0070] In one embodiment of any method of the present invention, the cancer may be one or more of the following: melanoma, MSI-H / dMMR colorectal cancer, pleural mesothelioma, triple-negative breast cancer, cutaneous squamous cell carcinoma, colorectal cancer, Calmette-Guérin cancer, bladder cancer, endometrial cancer, esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, mediastinal large B-cell lymphoma, cervical cancer, gastric cancer, urothelial carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, and non-small cell lung cancer.

[0071] In one embodiment of any method of the present invention, the cancer is melanoma. The melanoma may be metastatic melanoma.

[0072] In one embodiment of any method of the present invention, one or more immune checkpoint inhibitors are selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and LAG-3 inhibitors.

[0073] In additional or alternative embodiments, one or more checkpoint inhibitors are anti-CTLA-4 and / or anti-PD-1.

[0074] In additional or alternative embodiments, one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, and / or ipilimumab.

[0075] In one embodiment of any method of the present invention, the individual or patient is human.

[0076] In further embodiments of any method of the present invention, the method is carried out in vitro or ex vivo.

[0077] In further embodiments of any method of the present invention, the sample is a sample of blood, serum, or plasma. Preferably, the sample is a sample of serum or plasma.

[0078] In a further aspect of the present invention, a method for treating cancer in an individual is provided, the method being: (a) a step of stratifying individuals for treatment with one or more immune checkpoint inhibitors or alternative cancer therapies using a method according to any of the above embodiments of the present invention, The procedure includes (b) providing an individual with a cancer therapy comprising administering one or more immune checkpoint inhibitors or alternative cancer therapies.

[0079] Treating an individual with one or more immune checkpoint inhibitors includes administering or continuing to administer one or more immune checkpoint inhibitors to the individual in accordance with designated treatment guidelines.

[0080] Treating an individual with alternative cancer therapy includes administering or continuing to administer alternative cancer therapy to the individual in accordance with designated treatment guidelines.

[0081] A further aspect of the present invention provides a method for treating cancer by administering one or more immune checkpoint inhibitors to individuals selected for treatment based on their TK activity and / or concentration before treatment with one or more immune checkpoint inhibitors, and optionally, their TK activity and / or concentration measured 1 to 4 weeks after the first treatment with one or more immune checkpoint inhibitors.

[0082] In a further aspect of the present invention, one or more immune checkpoint inhibitors are provided for use in the treatment of cancer in an individual, wherein the individual is selected for treatment based on the TK activity and / or concentration prior to treatment with one or more immune checkpoint inhibitors, and optionally, the TK activity and / or concentration measured 1 to 4 weeks after the first treatment with one or more immune checkpoint inhibitors.

[0083] In one embodiment of the aforementioned aspects, an individual is selected for ICI treatment because its TK activity and / or concentration prior to ICI treatment is below a cutoff value, and optionally, the cutoff value is a specific cutoff value selected from the values ​​described in the aforementioned aspects of the present invention.

[0084] In additional embodiments of the above-described aspects, individuals are selected for ICI treatment because, optionally, their TK activity and / or concentration 1 to 4 weeks after treatment with an ICI inhibitor, and optionally, 3 to 4 weeks after treatment, is increased compared to the TK activity and / or concentration before ICI treatment, and optionally, the increase is at least a specific percentage increase selected from the values ​​described in the above-described aspects of the present invention, e.g., at least a 100% increase.

[0085] In additional embodiments of the above-described aspects, an individual is selected for ICI treatment because (i) its TK activity and / or concentration before ICI treatment is below a cutoff value (optionally, the cutoff value is a specific cutoff value selected from the values ​​described in the above-described aspects of the present invention), and (ii) its TK activity and / or concentration 1 to 4 weeks after treatment with an ICI inhibitor, optionally 3 to 4 weeks later, is increased compared to the TK activity and / or concentration before ICI treatment (optionally, the increase is an increase of at least a specific percentage selected from the values ​​described in the above-described aspects of the present invention, e.g., an increase of at least 100%).

[0086] In further embodiments of the above-described aspects, individuals are further selected for ICI treatment because their LDH activity and / or concentration prior to ICI treatment is considered to be below or normal, and optionally, the cutoff value is a specific cutoff value selected from the values ​​described in the above-described aspects of the present invention.

[0087] In further embodiments of the above-described aspects, the activity and / or concentration of TK and / or LDH are measured in samples of blood (e.g., unfractionated blood), plasma, serum, tissue fluid, and / or urine.

[0088] In further embodiments of the above-described model, the activity and / or concentration of TK and / or LDH are measured in a serum or plasma sample.

[0089] In a further aspect of the present invention, the use of thymidine kinase as a prognostic biomarker for cancer patients who are being considered for treatment with immune checkpoint inhibitors or who are currently being treated with immune checkpoint inhibitors is provided.

[0090] A further aspect of the present invention provides the use of thymidine kinase for stratifying individuals with cancer for treatment with immune checkpoint inhibitors or for further treatment.

[0091] Any preferences and choices relating to a given aspect, feature, or parameter of the present invention should be considered, unless the context indicates otherwise, to be disclosed in conjunction with all other preferences and choices relating to all other aspects, features, and parameters of the present invention. For example, the preferences, choices, and embodiments described in relation to a method for determining prognosis also apply to methods for stratifying patients, treatment methods, and the uses described thereafter.

[0092] Any listing or discussion of previously published literature in this specification should not necessarily be construed as an endorsement that such literature is part of cutting-edge technology or common general knowledge.

[0093] Herein, preferred non-limiting embodiments that embody a particular aspect of the present invention will be described with reference to the following drawings and examples. [Brief explanation of the drawing]

[0094] [Figure 1] This study determines the cutoff value for thymidine kinase 1 activity (TKa) in the plasma of metastatic melanoma patients treated with immune checkpoint inhibitors. The x-axis shows the TKa rank in DiviTum® units / liter (Du / L), and the y-axis shows the TKa value. When determining the TKa cutoff value, the most optimal cutoff value was considered to be the value closest to the median that ensures differentiation of TKa values ​​with a sufficient number of observations in each group. The baseline median TKa was 42 Du / L. From this median (solid line), a cutoff value was obtained to differentiate patients with very similar TKa values ​​into different groups. A cutoff of 60 Du / L (dotted line) ensured further differentiation of TKa values ​​and sufficient observations in both groups. [Figure 2] ROC curve analysis using the optimal TKa cutoff for sensitivity and specificity in predicting A. performance stage (ECOG ≥ 1 vs. ECOG 0), B. tumor stage (M1c-d vs. M1a-b), C. response (SD and PD vs. CR and PR), D. progression-free survival (shorter or longer than 24 months), and E. overall survival (shorter or longer than 24 months). The optimal TKa cutoff for each analysis is shown for each analysis (above the trend line), and TKa 60.0, selected as the cutoff for the comparative analysis of this study, is shown below the trend line. [Figure 3]Kaplan-Meier curves for survival in melanoma patients with high (>60 Du / L) or low plasma thymidine kinase 1 activity (TKa) before initiation of immune checkpoint inhibitor therapy. (A) Median progression-free survival was 19.9 months (95% CI, 11.0–not reached) in patients with low TKa and 12.6 months (95% CI, 3.6–28.3) in patients with high TKa (p=0.021). (B) Median overall survival was not reached (>60 months, 95% CI, 38.0–not reached) in patients with low TKa and 18.5 in patients with high TKa (p=0.005). [Figure 4] Bivariate regression analysis of TKa and one other variable for A. progression-free survival (PFS) and B. overall survival (OS) in patients with metastatic melanoma treated with immune checkpoint inhibitors. [Figure 5] Pie charts showing the number of patients with long (>24 months, (A-C)) or short (<24 months, (D-F)) progression-free survival (PFS). Each pie chart indicates whether the longer or shorter PFS was correctly predicted by TKa level and one other baseline variable (ECOG, LDH, or M stage). For example, (B) shows that in 17 patients, a long PFS was predicted by both low baseline TKa and normal LDH (correctly predicted by both variables, diamond symbol); in 3 patients, a long PFS was predicted only by normal LDH because TKa was elevated (correctly predicted only by LDH, circle symbol); in 7 patients, a long PFS was predicted only by low TKa because LDH levels were elevated (correctly predicted only by TKa, square symbol); and in 4 patients, a long PFS was not predicted by either variable because both LDH and TKa levels were high (not correctly predicted by either, triangle symbol). [Figure 6]Plasma TKa in melanoma patients receiving immunotherapy. The plot shows the median TKa value in Du / L at three separate time points (pre-treatment, 3-4 weeks post-treatment, and at the end of treatment or 24 months if the patient was still receiving treatment). Patients were stratified according to progression-free survival (PFS < 6 months, PFS > 6 months, or still responding at 24 months). Patients with the shortest PFS (less than 6 months) had the highest pre-treatment TKa levels and little to no increase in TKa during treatment. Patients with a PFS of 6-24 months had low pre-treatment TKa levels but less than a twofold increase in TKa during treatment. Patients whose response to treatment lasted beyond 24 months had low pre-treatment TKa levels and more than a twofold increase in TKa during treatment. [Examples]

[0095] Overview and Introduction Immune checkpoint inhibitors (ICIs) are effective in some patients with disseminated melanoma. The most clinically developed ICIs target programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3). Antibody drugs targeting these immune checkpoints release the brakes on T cell activity, allowing for the activation and subsequent proliferation of tumor-responsive T cells, which can then initiate an effective antitumor response. Studies have shown a significant increase in T cell counts after the first dose of ICI drugs. The level of this increase in T cells correlates with tumor response and patient outcomes

[28] . The DiviTum® TKa assay described herein can detect this T cell proliferation burst in patients within the first month of ICI treatment.

[0096] In recent years, effective ICI regimens using CTLA-4 and PD-1 blocking antibodies have emerged for the treatment of melanoma [1-6]. While these treatments have revolutionized the field of melanoma oncology, a significant portion of melanoma patients do not respond to these treatments or do not achieve lasting effects from them. These treatments also carry the potential for serious toxicity and are expensive. Therefore, it is crucial to increase our knowledge of predictors and their efficacy in various patient populations.

[0097] Thymidine kinase 1 (TK) is a cytosolic enzyme and a phosphotransferase that plays a crucial role in DNA synthesis and repair [7]. TK is part of the reaction chain that introduces thymidine into the DNA strand, and therefore plays an important role in DNA synthesis and cell division [7]. Cells during division release TK at the end of mitosis, and thus TK can be detected in the blood. Furthermore, elevated TK enzyme activity has been measured in blood samples from cancer patients, and this elevation has been associated with tumor growth and tumor burden [8]. Circulating levels of TKa, as measured by the DiviTum® assay, have been shown to be associated with the stage, prognosis, and treatment response of several cancer types, including breast cancer, lung cancer, pancreatic cancer, and renal cell carcinoma [9-15]. Pre-treatment TKa levels can reflect both the rate of tumor cell growth and the total disease burden. High TKa levels indicate that the tumor(s) are actively growing and / or have a high disease burden (both tumor size and number), while low TKa levels indicate that the tumor is growing slowly, progressing slowly, and has a low disease burden (the overall tumor is smaller and less numerous).

[0098] The inventors have for the first time analyzed the plasma activity of TK, an enzyme involved in DNA synthesis and repair, as a biomarker in melanoma patients. TK activity (TKa) levels in metastatic melanoma patients were measured before the initiation of ICI treatment and correlated with baseline clinical characteristics, treatment response, and survival rates.

[0099] The inventors found that high TKa levels in melanoma patients were associated with poor baseline factors such as poor performance status, high plasma lactate dehydrogenase levels, and advanced tumor stage. High TKa levels were also associated with reduced efficacy of immune checkpoint inhibitors. Therefore, TKa was identified by the inventors as a novel prognostic and predictive marker in cancer.

[0100] Materials and methods Patients and plasma samples: Plasma samples were collected from patients with unresectable metastatic cutaneous melanoma treated with ICI (anti-CTLA-4 and / or anti-PD-1) at the Department of Oncology, Karolinska University Hospital, Stockholm, Sweden, between 2012 and 2019. Treatment was carried out according to standard ICI regimens and dosages approved for the treatment of metastatic melanoma. Blood samples were collected from patients within 5 days prior to the start of treatment. Blood samples were collected in EDTA tubes, centrifuged at 1500 x g for 10 minutes, and the separated plasma was centrifuged at 2400 x g for 15 minutes and frozen at -70°C within 1 hour after processing. Baseline clinical data included age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, baseline tumor stage according to the American Joint Committee (AJCC) on Cancer, Eighth Edition

[16] , number of affected organs, baseline lactate dehydrogenase (LDH) levels, previous treatment lines, and ICI regimens received after TKa sample collection. This study was conducted in accordance with Good Clinical Practice, with informed consent obtained from all patients, and was approved by the Stockholm Regional Ethics Committee.

[0101] TK activity level analysis: Plasma TKa levels were measured using the DiviTum® TKa assay (Biovica, Sweden) according to previously reported manufacturer instructions [7]. DiviTum® TKa is an improved ELISA-based test that reflects cell proliferation rate by measuring TKa in serum, plasma, or cells. In summary, plasma was mixed with the reaction mixture in a 96-well ELISA plate, and the TK reaction produced bromodeoxyuridine (BrdU) monophosphate, which was phosphorylated to BrdU triphosphate and incorporated into a synthetic DNA strand. BrdU incorporation was detected using an anti-BrdU monoclonal antibody conjugated to the enzyme alkaline phosphatase and a chromogenic substrate. Absorbance readings were converted using a standard with a known TKa value (measurement range 20–4000 Du / L). The assay's detection limit was set to 20 Du / L, and all values ​​below the threshold were reported as <20 Du / L. All plasma TKa analyses were performed at the Biovica Laboratory (Uppsala, Sweden), where all staff were not informed about patient and tumor data. Samples were measured in two sequences, meeting the coefficient of variation (CV) criterion (CV < 20%) for the DiviTum® TKa assay. The optimized DiviTum® TKa assay for measuring TKa is a CE-IVD labeled assay and has been submitted to the FDA as a 510(k) application and is awaiting approval. As mentioned above, the optimized DiviTum® TKa assay has a new calibration concept that better fits the assay principle for measuring TK activity and a new unit (DuA) that better reflects TK activity. The conversion formula between Du / L and DuA is as follows: DuA = 134 + 0.53 (Du / L) Coefficient of determination (R 2 ) = 0.93

[0102] Follow-up: Regular follow-up after initiation of ICI treatment included monthly clinical evaluations and quarterly radiological evaluations. Patients received a minimum of 24 months of follow-up. Patients were grouped based on baseline plasma TKa levels (low or high) and tracked for treatment response, progression-free survival (PFS), and overall survival (OS). The best response to treatment was based on radiological examinations (CT, MRI, and / or positron emission (PET) CT tomography) assessed by a radiologist and evaluated according to the Response Evaluation Criteria in Solid Time (RECIST) 1.1 criteria

[17] . The response rate (RR) was defined as the frequency of patients showing a partial response (PR) or complete response (CR) as the best response. The disease control rate (DCR) was defined as the frequency of patients showing a PR, CR, or stable disease (SD) as the best response after at least 3 months of treatment. PFS was defined as the period from the initiation of treatment to the day progression was confirmed, or the day of death, or the day of the last follow-up. OS was defined as the period from the start of treatment to the date of death or the date of the last follow-up.

[0103] Statistical methods: Receiver operating characteristic (ROC) analysis was performed to determine the optimal TKa cutoff with the best sensitivity and specificity for predicting tumor stage, performance stage, response, and survival. Baseline characteristics and treatment response were compared using the chi-square test for categorical variables and Student's t-test for continuous variables. A p-value < 0.05 was considered statistically significant. Time to event outcome for PFS and OS was analyzed using Kaplan-Meier curves and Cox proportional hazards regression. Median PFS and OS with 95% confidence intervals (CIs) were assessed. The association between each predictor and PFS and OS was evaluated using univariate, bivariate, and multivariate models of Cox regression. Hazard ratios (HRs) and corresponding two-sided 95% CIs were estimated. Statistical analysis was performed using R version 4.1.1. The agreement rate is a measure of the predictive accuracy of the model, measured as the proportion of all evaluable target pairs in which the model correctly predicts a higher risk for individuals in pairs with the worst outcome.

[0104] result Baseline characteristics: A total of 90 patients with metastatic melanoma were included in this study. The median plasma TKa level before treatment was 42 Du / L (range <20 to 1787 Du / L). There were no significant differences in TKa levels related to patient age or sex (Table 1). However, plasma TKa levels were significantly higher in patients with an ECOG performance status of 1 or higher compared to those with a status of 0 to 1 (p=0.003), in patients with M1c-M1d disease compared to those with M1a-M1b disease (p=0.015), or in patients with elevated LDH levels compared to those without (p<0.001). Patients who had previously received treatment or who had more than three affected organs had higher TKa levels, but the difference in TKa levels was not significant in this case. In patients with M1b-d disease, TKa levels were compared according to the presence or absence of metastasis to specific organs. This analysis was conducted separately from the analysis of M1a patients to examine whether TKa levels are affected by metastases to specific organs, rather than to assess tumor burden (as M1a patients generally have significantly lower tumor burden). In conclusion, no significant differences were observed regarding which organs were affected.

[0105] TKa cutoff determination: Since TKa had not been previously studied in melanoma, a key objective was to determine an appropriate cutoff value and compare patients with high and low plasma TKa levels. A cutoff was obtained from the median baseline TKa (42 Du / L) to differentiate patients with very similar TKa levels into different groups (Figure 1). In this sense, 60 Du / L was considered a more appropriate cutoff value, as it ensured further differentiation of TKa levels and provided a sufficient number of patients with both high and low TKa levels. In the next step, ROC analysis was performed to determine the TKa cutoff with the optimal sensitivity and specificity for predicting baseline characteristics and outcomes (Figure 2). ROC analysis demonstrated that the highest sensitivity and specificity for predicting tumor stage, performance stage, ICI response, and PFS and OS at 24 months were achieved when the TKa cutoff was between 49 and 72 (the median of these cutoffs was 58). Therefore, ROC analysis further supported the use of 60 Du / L as a reasonable cutoff.

[0106] Characteristics and outcomes of patients with high or low TKa: There were no significant differences in age, sex, or tumor BRAF mutation status between melanoma patients with high (≥60 Du / L) or low plasma TKa levels (Table 2). However, high TKa levels were significantly associated with ECOG performance status ≥1 (p<0.001), M1c or M1d disease (p=0.002), three or more affected organs (p=0.031), elevated LDH (p<0.001), and higher median LDH (p<0.001). There were no significant differences between patients with high and low TKa levels regarding prior treatment lines or the ICI regimen chosen for the patient. The majority of patients were treated with PD-1 inhibitor monotherapy (nivolumab or pembrolizumab) as first-line treatment. A small number of patients received CTLA-4 inhibitor monotherapy (ipilimumab) or a combination of CTLA-4 inhibitor and PD-1 inhibitor (ipilimumab and nivolumab). Although the difference was not statistically significant, more patients in the low TKa group (n=6) received combination immunotherapy compared to the high TKa group (n=0). A plausible explanation is that low TKa patients had better performance status, were slightly younger, were evaluated more frequently, and were found to be able to tolerate more toxic combination therapies.

[0107] The response rate (RR) was significantly higher in patients with low TKa (63.2%) than in patients with high TKa (30.3%) (p=0.022) (Table 3). The complete response rate was also higher in the low TKa group (33.3%) than in the high TKa group (6.0%) (p=0.016). The disease control rate (DCR) was also higher in patients with low TKa (80.7%) than in patients with high TKa (54.3%) (p=0.022). There were no differences in the reasons for discontinuing treatment (disease progression, sufficient response, or toxicity) (Table 3).

[0108] The median PFS was 19.9 months (95% CI, 11.0 to not reached) in patients with low TKa and 12.6 months (95% CI, 3.6 to 28.3) in patients with high TKa (p=0.021) (Figure 3). The median OS was not reached (>60 months, 95% CI, 38.0 to not reached) in patients with low TKa and 18.5 months (95% CI, 11.7 to not reached) in patients with high TKa (p=0.005).

[0109] Univariate Cox regression analysis showed that PFS and OS were significantly worse in patients with baseline ECOG performance status ≥1, M1c or M1d disease, elevated LDH, and high TKa (Table 4). For TKa, the HR for PFS was 1.83 (95% CI, 1.08–3.08), p=0.024, and the HR for OS was 2.25 (95% CI, 1.25–4.05), p=0.007. In multivariate analysis, TKa was not significant for PFS or OS. High levels of multicollinearity among the analyzed variables were identified as a factor contributing to the results where HRs for many variables that were significant in the univariate model were not significant in the multivariate model.

[0110] To assess how TKa is affected by each covariate, we performed a bivariate regression analysis in which TKa was paired with one other baseline factor (Figure 4). Regarding PFS, TKa was independent only of the patient's sex.

[0111] Furthermore, in the bivariate analysis of OS, TKA was independent of age, sex, and tumor stage. Figure 5 shows the number of patients with longer (>24 months, Figures 5A-5C) or shorter (<24 months, Figures 5D-5F) PFS, and each chart shows whether the longer or shorter PFS was correctly predicted by TKa level and one other baseline variable (ECOG, LDH, or M stage). The chart shows that there was considerable overlap between TKa and the other variables (diamond symbol), but in some patients, only low or high TKa levels were associated with longer or shorter PFS, respectively (square symbol).

[0112] As part of the same study, TKa levels were also measured in 58 patients undergoing immunotherapy (3-4 weeks after the start of treatment and at the end of treatment). The results are shown in Table 5 and Figure 6. This indicates that patients with the shortest PFS (less than 6 months) had the highest pre-treatment TKa levels and little to no increase in TKa during treatment. Patients with a PFS of 6-24 months had low pre-treatment TKa levels, but their TKa increase during treatment was less than twofold. Patients whose response to treatment lasted longer than 24 months had low pre-treatment TKa levels and their TKa increase during treatment was more than twofold. This indicates a T-cell proliferation burst in patients within the first month after successful ICI treatment.

[0113] Consideration In patients with advanced cutaneous melanoma included in the study, significantly higher TKa levels were observed in patients with poor performance status, advanced tumor stage, and high LDH levels at the start of treatment. The median TKa was 42 Du / L (range <20 to 1787 Du / L), compared to <20 Du / L in 123 healthy subjects (data not shown). In the cohort of preoperative pancreatic cancer patients, the median TKa was 40 Du / L, and in the cohort of preoperative renal cell carcinoma patients, the median TKa was 38 Du / L [12,14]. Furthermore, in the cohort of non-small cell lung cancer patients, the median TKa before the start of systemic treatment was 129 Du / L, compared to 57 Du / L in patients with localized disease and 101 Du / L in patients with visceral metastases in the breast cancer cohort [15,18]. In summary, this data indicates that TKa levels in patients with metastatic melanoma are elevated compared to those in healthy individuals, as is the case with other cancer types studied, and are even higher in patients with more advanced disease.

[0114] Patients with high TKa levels had significantly poorer response to ICI treatment and significantly shorter survival (both PFS and OS). In multivariate analysis, TKa was not an independent predictor of PFS and OS. Bivariate analysis showed that the association between TKa and PFS and OS depended to varying degrees on ECOG, LDH, and tumor stage; however, in a significant portion of patients, there were no corresponding pairs of good or bad baseline variables (Figure 5). Clinical factors such as performance status, tumor stage, and tumor burden are well-known prognostic factors and also predict the efficacy of ICI in melanoma [1-4,16]. Regarding serum markers, elevated LDH levels are the most potent known prognostic factor and are routinely used when monitoring melanoma patients in clinical practice and are the only biomarker included as a marker in clinical trials

[16] . Serum LDH levels reflect the hypoxic environment commonly seen in melanoma, where oxidative phosphorylation is reduced and anaerobic glycolysis is increased, with LDH catalyzing the conversion of pyruvate to lactate when oxygen supply is low or insufficient

[19] . Since LDH is not a secreted enzyme, elevated serum levels are thought to be secondary to LDH leakage when melanoma cells proliferate beyond the blood supply. LDH is also often elevated in a variety of conditions affecting the liver, both malignant and non-malignant

[19] . TK enzymes play a crucial role in DNA synthesis and repair. These processes are highly active in proliferating cells, and dividing tumor cells release TK at the end of mitosis [7,8]. Thus, both LDH and TKa are markers of cell proliferation and tumor burden, but through different cellular processes.

[0115] The data herein also demonstrate that a positive patient response to ICI can be predicted by measuring TKa levels in cancer patients at two critical time points: pre-treatment and 1–4 weeks after the first ICI treatment administration. Low pre-treatment TKa levels indicate a level of disease burden that the immune system can effectively manage once activated. A TKa level increase of at least twofold during ICI treatment compared to baseline levels indicates a successful response to treatment, with sufficient T-cell activation and proliferation to achieve tumor killing.

[0116] Several other markers have been reported to predict the efficacy of ICI, including peripheral blood leukocyte composition, circulating tumor DNA (ctDNA) and exosomes, tumor mutational burden (TMB), high interferon-gamma-related gene expression signatures in tumors, gut microbiota diversity, and invasive tumor biopsy (now used for patient selection) measuring ICI receptor expression [20-27]. In a clinical setting, widely performing predictive assays such as TMB, ctDNA, exosomes, tumor RNA expression signatures, or microbiome analysis is difficult, for example, due to the need for complex and expensive techniques and equipment, and although many different assays are available, measuring TKa is a simpler and less expensive (ELISA-based) test for a single plasma marker, and this assay can be easily set up in a typical hospital laboratory.

[0117] conclusion High pre-treatment plasma TKa levels were significantly associated with worsened baseline characteristics, reduced response, and decreased survival in melanoma patients treated with ICIs. We are the first to identify TKa as an interesting, previously unstudied biomarker in cancer patients that can be used to indicate response to subsequent ICI treatment. table [Table 1] [Table 2]

Table 3

Table 4

Table 5

Table 6

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Claims

1. A method for assisting in determining the prognosis of individuals suffering from cancer who are being considered for treatment with one or more immune checkpoint inhibitors, or who are currently receiving treatment with one or more immune checkpoint inhibitors, (a) providing a sample taken from the individual before treatment with one or more immune checkpoint inhibitors, (b) a step of measuring the activity and / or concentration of thymidine kinase (TK) in the sample, or comprising the steps thereof, Herein, the activity and / or concentration of thymidine kinase (TK) measured in step (b) indicates the prognosis of the individual after treatment with one or more immune checkpoint inhibitors.

2. (c) providing a sample taken from the individual one to four weeks after the initial treatment with one or more immune checkpoint inhibitors, The step further comprises measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (c), The method according to claim 1, wherein the activity and / or concentration of thymidine kinase (TK) measured in steps (b) and (d) indicates the prognosis of the individual after further treatment with one or more immune checkpoint inhibitors.

3. The method according to claim 1 or 2, wherein the method is for stratifying individuals or groups of individuals suffering from cancer in order to assist in determining whether or not they should be treated with one or more immune checkpoint inhibitors or further treatment.

4. A method for stratifying cancer patients to assist in determining whether or not they should receive treatment with one or more immune checkpoint inhibitors or further treatment, Step (i) of carrying out the method according to claim 1 or 2, A method comprising or consisting of the steps of step (i) stratifying an individual to assist in determining whether or not to treat with one or more immune checkpoint inhibitors or alternative cancer therapies or further treatment, based on the results of step (i).

5. The method according to claim 4, wherein the patient is stratified for treatment with one or more immune checkpoint inhibitors or for further treatment.

6. The method according to claim 2, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b), it indicates that the individual will have a good prognosis if further treated with one or more checkpoint inhibitors (for example, the individual will have a progression-free survival of at least six months), and / or stratifies the patient for further treatment with one or more immune checkpoint inhibitors.

7. The method according to claim 6, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 100% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b), it indicates that the individual will have a good prognosis if further treated with one or more checkpoint inhibitors (e.g., progression-free survival of the individual is greater than 24 months), and / or stratifies the patient for further treatment with one or more immune checkpoint inhibitors.

8. The method according to claim 1 or 2, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cutoff value, the prognosis of the individual to be treated with or further treated with one or more immune checkpoint inhibitors is good, and / or the patient is stratified for treatment with one or more immune checkpoint inhibitors or further treatment.

9. The method according to claim 1 or 2, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is above a cutoff value, it indicates that the prognosis of the individual is poor if treated with one or more immune checkpoint inhibitors or further treatment (for example, the progression-free survival of the individual will be less than 6 months), and / or stratifies the patient for no further treatment or treatment with cancer therapies other than one or more immune checkpoint inhibitors.

10. The method according to claim 1 or 2, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cutoff value, it indicates that the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (for example, the progression-free survival of the individual is at least 6 months), and / or stratifies the patient for treatment with or further treated with one or more immune checkpoint inhibitors, wherein the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

11. The method according to claim 10, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cutoff value, it indicates that the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., the progression-free survival of the individual is greater than 24 months), and / or stratifies the patient for treatment with or further treated with one or more immune checkpoint inhibitors, wherein the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 100% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

12. The method according to claim 1 or 2, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than or equal to a cutoff value, it indicates that the prognosis of the individual is poor if treated with one or more immune checkpoint inhibitors or further treatment (for example, the progression-free survival of the individual will be less than 6 months), and / or stratifies the patient to receive no further treatment or treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors, wherein the activity and / or concentration of thymidine kinase (TK) measured in step (d) has not increased compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

13. The method according to claim 8, wherein the activity (TKa) of thymidine kinase is measured in step (b), and the cutoff value is selected from 40 to 75 Du / L, for example 42 to 72 Du / L, for example 49 to 72 Du / L, for example 42 to 60 Du / L, for example 49 to 60 Du / L, or preferably 60 Du / L.

14. The aforementioned method, Step (e) providing one or more control samples, The method further comprises step (f) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (e), The method according to claim 1 or 2, wherein the prognosis of the individual treated with one or more immune checkpoint inhibitors is determined by comparing the measurement value in step (f) with the measurement value in step (b) and / or step (d).

15. The method according to claim 14, wherein the one or more control samples are obtained from individuals who subsequently develop cancer before successful treatment with one or more immune checkpoint inhibitors.

16. The method according to claim 14, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) corresponds to or is lower than the activity of thymidine kinase (TKa) measured in step (f), the prognosis of the individual treated with one or more immune checkpoint inhibitors is good, and / or the patient is stratified for treatment with one or more immune checkpoint inhibitors or further treatment.

17. The method according to claim 14, wherein the one or more control samples are obtained from individuals who subsequently develop cancer before treatment with one or more immune checkpoint inhibitors is unsuccessful.

18. The method according to claim 14, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is higher than the activity and / or concentration of thymidine kinase (TK) measured in step (f), it indicates that the prognosis of the individual when treated with one or more immune checkpoint inhibitors is poor, and / or the patient is stratified to receive no further treatment or treatment with cancer therapies other than one or more immune checkpoint inhibitors.

19. The step (g) further comprises measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a), The method according to claim 1 or 2, wherein the activity and / or concentration of LDH measured in step (g) further indicates the prognosis of the individual after treatment with one or more immune checkpoint inhibitors.

20. The method according to claim 19, wherein if the activity and / or concentration of LDH measured in step (g) is lower than a cutoff value, the prognosis of the individual is good when treated with or further treated with one or more immune checkpoint inhibitors (e.g., progression-free survival of the individual is greater than 24 months), and / or stratifies the patient for treatment with or further treated with one or more immune checkpoint inhibitors.

21. The method according to claim 20, wherein the activity of LDH is measured, and the cutoff value is selected from 2 to 6 microcuts / L, for example from 3 to 5 microcuts / L, or for example from 4 microcuts / L.

22. The method according to claim 1 or 2, wherein the activity (TKa) of thymidine kinase is measured.

23. The method according to claim 1 or 2, wherein the cancer is a cancer for which treatment with an immune checkpoint inhibitor has been approved.

24. The method according to claim 1 or 2, wherein the cancer is melanoma.

25. The method according to claim 24, wherein the cancer is metastatic melanoma.

26. The method according to claim 1 or 2, wherein the one or more immune checkpoint inhibitors are selected from a list consisting of CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and LAG-3 inhibitors.

27. The method according to claim 1 or 2, wherein the individual or patient is human.

28. The method according to claim 1 or 2, wherein the method is carried out in vitro.

29. The method according to claim 1 or 2, wherein the sample is a sample of plasma or serum.

30. The use of thymidine kinase as a biomarker to aid in determining the prognosis of cancer patients who are being considered for or are currently receiving treatment with immune checkpoint inhibitors.

31. The use of thymidine kinase to stratify cancer patients in order to assist in determining whether they should receive treatment with immune checkpoint inhibitors or further treatment.

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