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

Abstract

The present invention provides a method for determining the prognosis of an individual suffering from cancer and 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; (b) measuring the activity and / or concentration of thymidine kinase (TK) in the sample; (c) providing a sample taken from the individual 1 to 4 weeks after initial treatment with one or more immune checkpoint inhibitors; and (d) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (c), wherein the activity and / or concentration of thymidine kinase (TK) measured in steps (b) and (d) is indicative of the individual's prognosis if further treated with one or more immune checkpoint inhibitors.

Description

[Technical field]

[0001] The present invention relates to methods and uses relating to determining the prognosis of cancer patients, as well as methods of stratifying these patients for treatment. [Background technology]

[0002] Immune checkpoint inhibitors (ICIs) are effective in only a portion of 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, allowing the activation and subsequent proliferation of tumor-reactive T cells, which can then mount an effective antitumor response. Studies have shown that T cell numbers increase significantly after the first dose of ICI drugs. The level of this increase in T cells correlates with tumor response and patient outcome

[28] . However, measuring activated and proliferating T cells requires sorting and isolation of cells from a whole blood population, which is complex and time-consuming, using specialized flow cytometry instruments for this purpose. This is not currently used as a standard of care method to predict patient response to ICIs.

[0003] In recent years, effective ICI regimens using CTLA-4 and PD-1 blocking antibodies have emerged for the treatment of several cancers, including melanoma [1-6]. Although these therapies have revolutionized the field of oncology, a significant portion of patients do not respond to or achieve durable benefits from these therapies. Treatments can also cause significant toxicity, and are expensive. Therefore, it is important to increase knowledge about predictive factors and their efficacy in various patient populations.

[0004] Thymidine kinase 1 (TK) is a cytosolic enzyme and phosphotransferase that plays a pivotal role in DNA synthesis and repair [7]. TK plays a key function in DNA synthesis and cell division, as it is part of the reaction chain that introduces thymidine into the DNA strand [7]. Dividing cells release TK at the end of mitosis, and therefore 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 is associated with tumor growth and tumor burden [8]. Circulating levels of TKa, measured with the DiviTum® TKa assay, have been shown to be associated with disease stage, prognosis, and treatment response in several cancer types, including breast, lung, pancreatic, and renal cell carcinoma [9-15].

[0005] The inventors have surprisingly discovered that measuring the activity and / or concentration of thymidine kinase (TK) in a patient's blood sample before and during treatment with an immune checkpoint inhibitor (ICI) can be used, optionally together with blood measurements of lactate dehydrogenase (LDH) activity and / or concentration, to provide a prognosis for subsequent treatment with the ICI. This therefore allows one to determine whether an ICI administered to a patient, or being considered for administration, is not working or will not work effectively. The main advantage of knowing quickly that an ICI drug is ineffective is that it is to the patient's benefit not to start taking an ineffective drug, or to stop taking the ineffective drug as soon as possible, and potentially implement an alternative therapy instead, since these drugs are very expensive and potentially very toxic.

[0006] A first aspect of the present invention provides a method of determining the prognosis of an individual suffering from cancer and who is being considered for treatment with one or more immune checkpoint inhibitors or who is currently undergoing treatment with one or more immune checkpoint inhibitors, the method comprising: (a) providing a sample taken from an individual prior to treatment with one or more immune checkpoint inhibitors; (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 prognosis of the individual upon subsequent 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, which are then incorporated into DNA. TK1 activity is low or absent in resting cells, but increases significantly as cells divide and replicate. Thus, 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 blood. DiviTum® TKa is a sensitive and accurate blood-based assay for measuring TK activity (TKa) in serum or plasma samples.

[0008] Immune checkpoint inhibitors (ICIs) are a class of drugs that block proteins called checkpoints that 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 activated, checkpoints inhibit immune cells from eliciting 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 and allowing tumor cells to escape immune system-mediated destruction. A variety of immune checkpoint inhibitors have been developed to treat cancer. The most clinically developed of these are programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin mucin-3 (TIM-3) inhibitor therapy. Antibody drugs that target these immune checkpoints release the brakes on T cell activity, allowing the activation and subsequent proliferation of tumor-reactive T cells, which can then mount an effective anti-tumor response. Studies have shown that T cell numbers increase significantly 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] An "individual suffering from cancer for whom treatment with one or more immune checkpoint inhibitors is being considered" includes a cancer patient for whom a physician would normally consider and / or plan to treat with one or more checkpoint inhibitors. For example, a patient suffering from a tumor for which a regulatory body such as the FDA (or an equivalent regulatory body in a different country) has approved treatment with an immune checkpoint inhibitor. Twomey and Zhang (2021) The AAPS Journal 23:39 provides a review of FDA-approved immune checkpoint inhibitors, which is incorporated herein by reference. A particular patient may be considered for treatment with an immune checkpoint inhibitor if they express immune checkpoint inhibitor receptors (checkpoint proteins) on their cancer cells or express those receptors at higher levels than healthy individuals, which may be determined by routine methods such as diagnostic assays that measure the level of immune checkpoint inhibitor receptor expression. One example of such a 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 normally considered.

[0010] "Prognosis of the individual upon subsequent treatment with one or more immune checkpoint inhibitors" includes the predicted efficacy of treatment with one or more immune checkpoint inhibitors, such as progression-free survival (PFS) and overall survival (OS) of the individual upon subsequent treatment 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] The ICI treatment and / or subsequent ICI treatment can be performed alone or with additional non-ICI cancer therapy. In one particular embodiment, the ICI treatment and / or subsequent ICI treatment is treatment with one or more ICIs only, without additional non-ICI cancer therapy.

[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. It is expected that TK protein level correlates with 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) according to 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 of which are incorporated herein by reference, and / or by real-time assays as described in WO 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 a number of techniques well known in the art, for example by immunoassays such as ELISA.

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

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

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

[0017] In further or alternative embodiments, the methods of the present invention are for stratifying an individual or individuals suffering from cancer for treatment or further treatment with one or more immune checkpoint inhibitors, in other words, the methods are used to determine whether an individual suffering from cancer should receive treatment or further treatment with one or more immune checkpoint inhibitors.

[0018] In a further aspect of the invention, there is provided a method of stratifying a cancer patient for treatment or further treatment with one or more immune checkpoint inhibitors, the method comprising: Step (i) of carrying out the method of the first aspect; and (ii) stratifying the individual for treatment or further treatment with one or more immune checkpoint inhibitors or an alternative cancer therapy based on the results of step (i); Optionally, the activity and / or concentration of thymidine kinase (TK) measured in steps (b) and / or (d) is indicative of the prognosis of the individual if subsequently treated with one or more immune checkpoint inhibitors.

[0019] In one embodiment, the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors. Alternatively, the patient may be stratified to not receive further ICI treatment. Alternatively, the patient may be stratified for treatment with alternative non-ICI cancer therapy.

[0020] Alternative cancer therapies to immune checkpoint inhibitors vary by 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, physicians can refer 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, etc.) to determine the most appropriate alternative cancer therapy, which can be found at https: / / www.nccn.org / guidelines / category_1.

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

[0022] In further or alternative embodiments, 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), then the individual's prognosis with further treatment with one or more checkpoint inhibitors is favorable (e.g., the individual's progression free survival will be or is predicted 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%, e.g., 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%, 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 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, 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 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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 An increase of 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 further or alternative embodiments, 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 further or alternative embodiments, 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 further 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).

[0027] In further 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).

[0028] In further 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).

[0029] In further 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 "good" prognosis includes an individual having or expected to have a progression-free survival of at least 6 months, i.e., no cancer progression 6 months after initial treatment with an ICI. A "good" prognosis may also include an individual having or expected to have a progression-free survival of at least 12 months, 18 months, or 24 months, i.e., no cancer progression 12 months, 18 months, or 24 months after initial treatment with an ICI.

[0031] In further or alternative embodiments, "good" prognosis means a progression-free survival or expected progression-free survival of at least 1 year, or at least 2, 3, 4, 5, or 10 years, after initial treatment with an ICI.

[0032] Progression-free survival (PFS) may be defined as the time from the start of treatment to the date of confirmed progression, or the date of death, or the date of last follow-up.

[0033] In further or alternative embodiments, "good" prognosis means an overall survival (OS) or expected overall survival of at least 1 year, or at least 2, 3, 4, 5, or 10 years after initial treatment with an ICI. OS may be defined as the time from start of treatment to date of death or last follow-up.

[0034] In further or alternative embodiments, a "good" prognosis means a complete response, a partial response, or stable disease. Preferably, a good prognosis means a complete or partial response, most preferably a complete response. Response to treatment may be based on radiological studies (CT, MRI, and / or positron emission tomography (PET) computed tomography) assessed by a radiologist and may be evaluated 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 a patient or individual with cancer as being eligible for treatment or further treatment with one or more immune checkpoint inhibitors, which may be the same or different ICIs, either alone or in combination with alternative cancer therapies.

[0036] In further or alternative embodiments, 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), the prognosis of the individual upon further treatment with one or more checkpoint inhibitors is favorable (e.g., the progression-free survival of the individual is greater than 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 activity and / or concentration of thymidine kinase (TK) 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 activity and / or concentration of thymidine kinase (TK) measured in step (b).

[0037] In further or alternative embodiments, if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than the cut-off value, the individual's prognosis when treated or further treated with one or more immune checkpoint inhibitors is good and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

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

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

[0040] Preferably, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is at least 8%, e.g. 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% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b). , 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%, 500% increase.

[0041] In further or alternative embodiments, 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).

[0042] In further or alternative embodiments, 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).

[0043] In further 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 further 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 further 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 further 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 further or alternative embodiments, if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than the cutoff value, the individual's prognosis when treated or further treated with one or more immune checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than or is expected to be greater than 24 months) and / or the patient is stratified for treatment or further treatment 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).

[0048] It will be appreciated that the increase rate values ​​may be optimized depending on the type of cancer or tumor, and one of skill in the art would be able to routinely modify the increase rate values ​​based on the information provided herein for melanoma.

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

[0050] In further or alternative embodiments of the methods of the invention, the activity of thymidine kinase (TKa) is measured and the cut-off value is selected from 549 to 1021 Du / L, such as 628 to 942 Du / L, such as 707 to 864 Du / L.

[0051] In further or alternative embodiments of the method of the invention, the activity of thymidine kinase (TKa) is measured, and the cut-off value is selected from 385 to 715 DuA, such as 440 to 660 DuA, for example 495 to 605 DuA. The relationship between Du / L and Du / A is explained below.

[0052] In further or alternative embodiments of the methods of the invention, the cut-off value is 785 Du / L (+ / - 20%). In further or alternative embodiments of the methods of the invention, the cut-off value is 785 Du / L (+ / - 10%). The 10% reflects normal assay variation.

[0053] In further or alternative embodiments of the methods of the invention, the cut-off value is 550 DuA (+ / - 20%). In further or alternative embodiments of the methods of the invention, the cut-off value is 550 DuA (+ / - 10%). 10% reflects normal assay variation.

[0054] In further or alternative embodiments of the method of the invention, the activity of thymidine kinase (TKa) is measured and the cut-off value is selected from 40-75 Du / L, such as 42-72 Du / L, for example 49-72 Du / L, such as 42-60 Du / L, for example 49-60 Du / L, for example 58-60 Du / L, or preferably 60 Du / L. In one embodiment the cut-off is 60 Du / L. In an alternative embodiment the cut-off is 58 Du / L. 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.

[0055] It will be understood that the pre-treatment cut-off value may be optimized depending on the type of cancer or tumor and the number of previous treatments the patient has already undergone. A person skilled in the art will be able to routinely modify the cut-off value based on the information provided herein.

[0056] Du / L refers to DiviTum® units per liter according to the DiviTum® TKa assay (Biovica, Sweden) and manufacturer's instructions 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, which are incorporated herein by reference.

[0057] TKa can also be measured in DuA units using the DiviTum® TKa assay. For Du / L units, recombinant thymidine kinase protein is used to assign Du / L values ​​to a panel of "gold standard sera" and calibrators. A measurement of 1 Du / L is the catalytic activity obtained from a sample with a concentration of 1 pg of recombinant thymidine kinase (TK) per ml. An optimized DiviTum® TKa assay has also been developed with a new calibration concept that better fits the assay principle of 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

[0058] In a further or alternative embodiment, the method further comprises: (e) providing one or more control samples; and (f) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (e), Here, the prognosis of the individual when 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).

[0059] One or more control samples can then be obtained from the individual with cancer prior to successful treatment with one or more immune checkpoint inhibitors.

[0060] Successful treatment includes cessation of tumor growth, non-progression, and / or regression. This can be assessed by imaging techniques, e.g., gold standard evaluation tools. Successful treatment can be assessed by RECIST criteria (see Reference 17). Response Evaluation Criteria In Solid Tumors or (RECIST) refers to a published set of rules used to assess tumor burden to objectively evaluate response to treatment. Stable disease, partial response, complete response, progression-free survival, and overall survival are endpoints at which treatment is considered successful. Successful treatment may be defined in the same way as good prognosis, as defined above. For example, successful treatment can be considered as a progression-free survival of at least 24 months, i.e., no cancer progression 24 months after initial treatment with ICI.

[0061] In further or alternative embodiments, if the activity and / or concentration of thymidine kinase (TK) measured in step (b) corresponds to or is lower than the activity and / or concentration of thymidine kinase (TK) measured in step (f), then the individual's prognosis when treated with one or more immune checkpoint inhibitors is good and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0062] "Corresponding" includes that the thymidine kinase (TK) activity and / or concentration is the same as that of a positive control sample, or is closer to the activity and / or concentration of one or more positive control samples than to that of one or more negative control samples (or closer to a predefined reference value representing the same).

[0063] Alternatively or additionally, "corresponding" includes that the activity and / or concentration correlates in a statistically significant manner with the amount in a control sample. "Statistically significant correlation with the amount in a control sample" means or includes that the presence or amount in a test sample correlates with the presence or amount in a control sample with 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.

[0064] In further or alternative embodiments, one or more control samples may be obtained from an individual with cancer prior to subsequent unsuccessful treatment with one or more immune checkpoint inhibitors.

[0065] In further or alternative embodiments, if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than the activity and / or concentration of thymidine kinase (TK) measured in step (f), then the individual has a poor prognosis when treated with one or more immune checkpoint inhibitors and / or the patient is stratified for either no further treatment with one or more immune checkpoint inhibitors or for treatment with an alternative cancer therapy to the one or more immune checkpoint inhibitors.

[0066] In further or alternative embodiments of the method of the present invention, the method further comprises: The method further comprises the step (g) of measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a), wherein the LDH activity and / or concentration measured in step (g) is further indicative of the prognosis of the individual upon subsequent treatment with one or more immune checkpoint inhibitors.

[0067] In further or alternative embodiments, if the LDH activity and / or concentration measured in step (g) is considered to be normal, i.e., not elevated above normal levels, then the individual's prognosis if treated or further treated with one or more immune checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than 24 months) and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0068] In further or alternative embodiments, if the LDH activity and / or concentration measured in step (g) is lower than the cutoff value, the individual's prognosis when treated or further treated with one or more immune checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than 24 months) and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0069] In further or alternative embodiments, the activity of LDH is measured and the cut-off value is selected from 2 to 6 microcuts / L, for example 3 to 5 microcuts / L, or in this example 4 microcuts / L.

[0070] It will be appreciated that the cut-off value may be optimized depending on, for example, the type of cancer or tumor of an individual, which can be routinely accomplished by one of skill in the art based on the information provided herein.

[0071] In one embodiment of the method of the invention, an additional step (h) of treating the individual with one or more immune checkpoint inhibitors or an alternative cancer therapy is performed.

[0072] In additional or alternative embodiments, the cancer is a cancer approved for treatment with an immune checkpoint inhibitor. For example, patients with cancers for which a regulatory agency, such as the FDA, has approved treatment with an immune checkpoint inhibitor are included. Twomey and Zhang (2021) The AAPS Journal 23:39 provides a review of FDA-approved immune checkpoint inhibitors, which is incorporated herein by reference.

[0073] In one embodiment of any of the methods of the invention, the cancer may be one or more of melanoma, MSI-H / dMMR colorectal cancer, pleural mesothelioma, triple negative breast cancer, cutaneous squamous cell carcinoma, colorectal cancer, Bacillus Calmette-Guerin, bladder cancer, endometrial cancer, esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, primary mediastinal large B-cell lymphoma, cervical cancer, gastric cancer, urothelial carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and non-small cell lung cancer.

[0074] In one embodiment of any of the methods of the invention, the cancer is selected from the group comprising melanoma, breast cancer, and / or ovarian cancer. The cancer may be metastatic.

[0075] In one embodiment of any of the methods of the invention, the cancer is melanoma and / or breast cancer.

[0076] In one embodiment of any of the methods of the invention, the cancer is melanoma. The melanoma may be metastatic melanoma.

[0077] In one embodiment of any of the methods of the invention, the cancer is breast cancer. The breast cancer may be metastatic breast cancer. The breast cancer may be hormone receptor positive.

[0078] In one embodiment of any of the methods of the invention, the cancer is ovarian cancer. The ovarian cancer may be metastatic ovarian cancer.

[0079] In one embodiment of any of the methods of the invention, the one or more immune checkpoint inhibitors are selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, and a LAG-3 inhibitor.

[0080] In further or alternative embodiments, the one or more checkpoint inhibitors are anti-CTLA-4 and / or anti-PD-1.

[0081] In further or alternative embodiments, the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, and / or spartalizumab.

[0082] In one embodiment of any of the methods of the invention, the individual or patient is a human.

[0083] In further embodiments of any of the methods of the invention, the method is performed in vitro or ex vivo.

[0084] In a further embodiment of any of the methods of the invention, the sample is a blood, serum or plasma sample. Preferably, the sample is a serum or plasma sample.

[0085] In a further aspect of the invention there is provided a method of treating cancer in an individual comprising: (a) stratifying an individual for treatment with one or more immune checkpoint inhibitors or an alternative cancer therapy using the method of any of the previous aspects of the invention; and (b) providing the individual with a cancer therapy comprising administering one or more immune checkpoint inhibitors or an alternative cancer therapy.

[0086] Treating an individual with one or more immune checkpoint inhibitors includes administering or continuing to administer one or more immune checkpoint inhibitors to an individual according to specified treatment guidelines.

[0087] Treating an individual with an alternative cancer therapy includes administering or continuing to administer the alternative cancer therapy to the individual in accordance with specified treatment guidelines.

[0088] In a further aspect of the invention, there is provided a method of treating cancer by administering one or more immune checkpoint inhibitors to an individual selected for treatment based on TK activity and / or concentration measured prior to treatment with the one or more immune checkpoint inhibitors, and optionally, 1-4 weeks after initial treatment with the one or more immune checkpoint inhibitors.

[0089] In a further aspect of the invention, there is provided one or more immune checkpoint inhibitors for use in the treatment of cancer in an individual, where the individual is selected for treatment based on TK activity and / or concentration prior to treatment with the one or more immune checkpoint inhibitors, and optionally, TK activity and / or concentration measured 1 to 4 weeks after initial treatment with the one or more immune checkpoint inhibitors.

[0090] In one embodiment of the aforementioned aspect, the individual is selected for ICI treatment because the TK activity and / or concentration before ICI treatment is lower than a cut-off value, optionally the cut-off value being a particular cut-off value selected from the values ​​described in the aforementioned aspect of the invention.

[0091] In further embodiments of the above aspects, the individual is selected for ICI treatment because TK activity and / or concentration after 1 to 4 weeks, optionally 3 to 4 weeks, of treatment with an ICI inhibitor is increased compared to TK activity and / or concentration before ICI treatment, optionally the increase being at least a certain percentage increase selected from the values ​​described in the above aspects of the invention, for example at least a 100% increase.

[0092] In further embodiments of the above aspects, the individual is selected for ICI treatment because (i) the TK activity and / or concentration before ICI treatment is lower than a cut-off value (optionally the cut-off value is a particular cut-off value selected from the values ​​described in the above aspects of the invention), and (ii) the TK activity and / or concentration after 1 to 4 weeks, optionally 3 to 4 weeks, of treatment with an ICI inhibitor is increased compared to the TK activity and / or concentration before ICI treatment (optionally the increase is at least a particular percentage increase selected from the values ​​described in the above aspects of the invention, e.g. at least a 100% increase).

[0093] In a further embodiment of the aforementioned aspect, the individual is further selected for ICI treatment because their LDH activity and / or concentration before ICI treatment is lower than a cut-off value or is considered to be normal, optionally the cut-off value being a particular cut-off value selected from the values ​​described in the aforementioned aspect of the invention.

[0094] In further embodiments of the foregoing aspects, TK and / or LDH activity and / or concentration are measured in blood (eg, unfractionated blood), plasma, serum, tissue fluid, and / or urine samples.

[0095] In further embodiments of the above aspects, the activity and / or concentration of TK and / or LDH is measured in a serum or plasma sample.

[0096] In a further aspect of the invention, there is provided the use of thymidine kinase as a prognostic biomarker for individuals with cancer who are being considered for or are currently undergoing treatment with an immune checkpoint inhibitor.

[0097] In a further aspect of the invention, there is provided the use of thymidine kinase to stratify individuals suffering from cancer for treatment or further treatment with an immune checkpoint inhibitor.

[0098] Preferences and choices with respect to a given aspect, feature or parameter of the invention should be considered as disclosed in combination with all preferences and choices with respect to all other aspects, features and parameters of the invention, unless the context dictates otherwise. For example, preferences, choices and embodiments described in connection with the method of determining prognosis equally apply to the method of stratifying patients, the methods of treatment, and the uses described thereafter.

[0099] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0100] Reference will now be made to the following figures and examples to describe preferred, non-limiting examples embodying certain aspects of the present invention. [Brief description of the drawings]

[0101] [Figure 1] The cut-off of thymidine kinase 1 activity (TKa) is determined in the plasma of metastatic melanoma patients treated with immune checkpoint inhibitors. The rank of TKa in DiviTum® units per liter (Du / L) is shown on the x-axis, and the TKa value is shown on the y-axis. When determining the cut-off value of Tka, the most optimal cut-off value was considered to be the value closest to the median that ensured the differentiation of TKa values ​​in the situation where there were sufficient observations in each group. The median TKa at baseline was 42 Du / L. From this median (solid line), a cut-off value was obtained to separate patients with very similar TKa values ​​into different groups. A cut-off of 60 Du / L (dotted line) ensured further differentiation of TKa and sufficient observations in both groups. [Diagram 2]ROC curve analysis using optimal TKa cutoffs 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 cutoffs for each analysis are shown for each analysis (numbers above the trend line) and the TKa of 60.0, selected as the cutoff for the comparative analysis of this study, is shown below the trend line. [Diagram 3] Kaplan-Meier curves for survival of melanoma patients with high (>60 Du / L) or low plasma thymidine kinase 1 activity (TKa) before starting immune checkpoint inhibitor treatment. (A) Median progression-free survival 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). (B) Median overall survival was not reached (>60 months, 95% CI, 38.0 to not reached) in patients with low TKa and 18.5 in patients with high TKa (p=0.005). [Figure 4] Bivariate regression with pairwise analysis of TKa and one other variable for A. progression-free survival (PFS) and B. overall survival (OS) in metastatic melanoma patients treated with immune checkpoint inhibitors. [Diagram 5]Pie charts showing the number of patients with longer (>24 months, (A-C)) or shorter (<24 months, (D-F)) progression-free survival (PFS). Each pie chart shows whether 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, long PFS was predicted by both low TKa and normal LDH at baseline (correctly predicted by both variables, diamond symbols), in 3 patients, long PFS was predicted only by normal LDH because TKa was elevated (correctly predicted only by LDH, circle symbols), in 7 patients, long PFS was predicted only by low TKa because LDH levels were elevated (correctly predicted only by TKa, square symbols), and in 4 patients, long PFS was not predicted by either variable because both LDH and TKa levels were high (correctly predicted by neither, triangle symbols). [Figure 6] Plasma TKa in melanoma patients receiving immunotherapy. Plots show median TKa values ​​in Du / L at three separate time points (pretreatment, 3–4 weeks after treatment, and end of treatment or 24 months if patient was still on treatment). Patients were stratified according to progression-free survival (PFS<6 months, PFS>6 months, or still in response at 24 months). Patients with the shortest PFS <6 months had the highest pretreatment TKa levels and little or no increase in TKa during treatment. Patients with PFS 6–24 months had lower pretreatment TKa levels but less than a two-fold increase in TKa during treatment. Patients whose response to treatment was sustained beyond 24 months had lower pretreatment TKa levels and more than a two-fold increase in TKa during treatment. [Figure 7]Correlation of TK activity levels with outcome in HR+ breast and ovarian cancer patients treated with ribociclib and the PD-1 inhibitor spartalizumab. Individual patient ID numbers are shown on the horizontal axis. The height of each bar represents the fold increase in TKa levels from cycle 1 day 1 (pre-treatment) to cycle 2 day 1 (28 days after first treatment) for the indicated patient. The number above each bar is the raw baseline (pre-treatment) TKa value for that patient. Bar shading indicates best overall response as assessed by RECIST 1.1 criteria. Black / PR=partial response. Dark grey / SD>6M=stable disease for ≥6 months. Light grey / SD<6M=stable disease for <6 months. White / PD=progressive disease. NE=not evaluable. Best overall response is also shown on the horizontal axis along with progression-free survival (PFS) (in days) and tumor type for each patient. B=HR+Her2- metastatic breast cancer O=metastatic epithelial ovarian cancer

[0102] Example 1 Overview and Introduction Immune checkpoint inhibitors (ICIs) are effective in a subset of patients with disseminated melanoma. 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 the activation and subsequent proliferation of tumor-reactive T cells, which can then mount an effective antitumor response. Studies have shown that T-cell numbers increase significantly after the first dose of ICI drugs. The level of this increase in T cells correlates with tumor response and patient outcome

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

[0103] In recent years, effective ICI regimens using CTLA-4 and PD-1 blocking antibodies have emerged for the treatment of melanoma [1-6]. Although these therapies have revolutionized the field of melanoma oncology, a significant portion of melanoma patients do not respond to or achieve durable benefits from these therapies. Treatments can also cause significant toxicity, and are expensive. Therefore, it is important to increase knowledge about predictive factors and their efficacy in various patient populations.

[0104] Thymidine kinase 1 (TK) is a cytosolic enzyme and phosphotransferase that plays a pivotal role in DNA synthesis and repair [7]. TK is part of the reaction chain that introduces thymidine into the DNA strand, and therefore plays a key function in DNA synthesis and cell division [7]. Dividing cells release TK at the end of mitosis, and therefore TK can be detected in the blood. Furthermore, elevated TK enzyme activity has been measured in blood samples from cancer patients, and this elevation is associated with tumor growth and tumor burden [8]. Circulating levels of TKa, measured by the DiviTum® assay, have been shown to be associated with disease stage, prognosis, and treatment response in several cancer types, including breast, lung, pancreatic, and renal cell carcinoma [9-15]. Pre-treatment TKa levels can reflect both the rate of tumor cell proliferation and the total disease burden, with high levels of TKa indicating actively growing tumor(s) and / or a large disease burden (both size and number of tumors) and low levels of TKa indicating slower growing, less progressive tumors and less disease burden (smaller and fewer tumors overall).

[0105] We analyzed for the first time the plasma activity of TK, an enzyme involved in DNA synthesis and repair, as a biomarker in melanoma patients. TK activity (TKa) levels were measured before the initiation of ICI treatment in metastatic melanoma patients and correlated with baseline clinical characteristics, treatment response, and survival.

[0106] 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. Thus, TKa was identified by the inventors as a novel prognostic and predictive marker in cancer.

[0107] Materials and Methods Patients and plasma samples: Plasma samples were collected from patients with unresectable metastatic cutaneous melanoma treated with ICIs (anti-CTLA-4 and / or anti-PD-1) at the Department of Oncology, Karolinska University Hospital, Stockholm, Sweden, between 2012 and 2019. Treatment was performed according to standard ICI regimens and doses approved for the treatment of metastatic melanoma. Blood samples were collected from patients within 5 days before treatment initiation. Blood samples were collected in EDTA tubes and centrifuged at 1500x g for 10 min, and separated plasma was centrifuged at 2400x g for 15 min and frozen at -70 °C within 1 h after processing. Baseline clinical data included age at treatment initiation, 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) level, previous lines of treatment, and ICI regimens received after TKa sample collection. The study was conducted in accordance with Good Clinical Practice, with informed consent from all patients, and approved by the Stockholm Regional Ethics Committee.

[0108] TK activity level analysis: Plasma TKa levels were measured using the DiviTum® TKa assay (Biovica, Sweden) according to the manufacturer's instructions as previously reported [7]. DiviTum® TKa is an improved ELISA-based test that reflects cell proliferation rates by measuring TKa in serum, plasma, or cells. In brief, plasma was mixed with a reaction mixture in a 96-well ELISA plate, and the TK reaction generated bromodeoxyuridine (BrdU) monophosphate, which was phosphorylated to BrdU triphosphate for incorporation 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 standards with known TKa values ​​(measurement range 20-4000 Du / L). The lower detection limit of the assay was set at 20 Du / L, and all values ​​below the threshold were reported as <20 Du / L. All plasma TKa analyses were performed at Biovica laboratories (Uppsala, Sweden), where all personnel were blinded to patient and tumor data. Samples were measured in duplicate and met the coefficient of variation (CV) criteria of the DiviTum® TKa assay (CV<20%). The optimized DiviTum® TKa assay for measuring TKa is a CE-IVD labeled assay and has been submitted to the FDA in a 510(k) application pending approval. As mentioned above, the optimized DiviTum® TKa assay has a new calibration concept that better fits the assay principle of 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

[0109] Follow-up: Regular follow-up after initiation of ICI treatment included monthly clinical evaluations and radiological evaluations every 3 months. Patients were followed up for a minimum of 24 months. Patients were grouped based on baseline plasma TKa levels (low or high) and followed for treatment response, progression-free survival (PFS), and overall survival (OS). Best response to treatment was based on radiological examinations (CT, MRI, and / or positron emission tomography (PET) computed tomography) assessed by a radiologist and was evaluated according to the Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 criteria

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

[0110] Statistical methods: Receiver operating characteristic (ROC) analysis was performed to determine the TKa cutoff with optimal sensitivity and specificity for predicting tumor stage, performance stage, response, and survival. Baseline characteristics and treatment response were compared with 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 outcomes for PFS and OS were analyzed using Kaplan-Meier curves and Cox proportional hazards regression. Median PFS and OS with 95% confidence intervals (CIs) were evaluated. Univariate, bivariate, and multivariate models of Cox regression were used to evaluate the association of each predictor with PFS and OS. Hazard ratios (HRs) and corresponding two-sided 95% CIs were estimated. Statistical analysis was performed using R version 4.1.1. Concordance is a measure of the predictive accuracy of the model and is measured as the proportion of all evaluable subject pairs in which the model correctly predicts a higher risk for the individual in the pair with the worst outcome.

[0111] result Baseline characteristics: A total of 90 patients with metastatic melanoma were included in the study. The median plasma TKa level before treatment was 42 Du / L (range <20–1787 Du / L). There were no significant differences in TKa levels related to patient age or sex (Table 1). However, plasma TKa was found to be significantly higher in patients with ECOG performance status ≥1 vs. 0–1 (p=0.003), in patients with M1c-M1d disease vs. M1a-M1b disease (p=0.015), or in patients with elevated vs. non-elevated LDH levels (p<0.001). TKa levels were higher in previously treated patients or patients with more than three affected organs, but here the TKa differences were not significant. In patients with M1b-d disease, TKa levels were compared according to the presence or absence of metastases to specific organs. This analysis was performed separately from the analysis of M1a patients, not to assess tumor burden (as M1a patients generally have a significantly lower tumor burden) but to address whether TKa levels were influenced by metastasis to specific organs. In conclusion, no significant differences were observed regarding which organs were affected.

[0112] Determination of TKa cutoff: Since TKa has not been studied in melanoma before, an important objective was to determine an appropriate cut-off value and compare patients with high and low plasma TKa levels. The median TKa value at baseline (42 Du / L) provided a cut-off to separate patients with very similar TKa levels into different groups (Figure 1). In that sense, 60 Du / L was considered to be a more appropriate cut-off value, since it ensured further differentiation of TKa levels and provided a sufficient number of patients with high and low TKa levels. In the next step, a ROC analysis was performed to determine the TKa cut-off with the most optimal sensitivity and specificity in predicting baseline characteristics and outcomes (Figure 2). The ROC analysis demonstrated that a TKa cut-off of 49–72 achieved the highest sensitivity and specificity for predicting tumor stage, performance stage, ICI response, PFS and OS at 24 months (the median of these cut-offs was 58). Thus, the ROC analysis further supported the use of 60 Du / L as a reasonable cut-off.

[0113] Characteristics and outcomes of patients with high or low TKa: No significant differences were found in age, sex, or tumor BRAF mutation status in 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), ≥3 organs involved (p=0.031), elevated LDH (p<0.001), and higher median LDH (p<0.001). No significant differences were found in patients with high and low TKa regarding whether they had received a previous line of treatment or the ICI regimen selected for them. The majority of patients were treated with PD-1 inhibitor monotherapy (nivolumab or pembrolizumab) in the first-line setting. A minority of patients received a CTLA-4 inhibitor (ipilimumab) alone or a combination of a CTLA-4 inhibitor and a 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 a better performance status and were somewhat younger, therefore they were evaluated more frequently and were found to be able to tolerate more toxic combination therapy.

[0114] The RR was significantly higher in patients with low TKa (63.2%) than in those 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 DCR was also higher in patients with low TKa (80.7%) than in those with high TKa (54.3%) (p=0.022). No differences were observed regarding reasons for treatment discontinuation (disease progression, adequate response, or toxicity) (Table 3).

[0115] 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). 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).

[0116] 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. A high degree of multicollinearity among the analyzed variables was identified as a factor leading to the results that HRs of many variables that were significant in the univariate model were not significant in the multivariate model.

[0117] To assess how TKa was affected by each covariate, bivariate regression analyses were performed analyzing TKa pairwise with one other baseline factor (Figure 4).With regard to PFS, TKa was independent only of patient sex.

[0118] Furthermore, in bivariate analysis of OS, TKA was independent of age, sex, and tumor stage. Figure 5 shows the number of patients with longer (>24 months, Figure 5A-5C) or shorter (<24 months, Figure 5D-5F) PFS, and each chart indicates whether longer or shorter PFS was correctly predicted by TKa level and one other baseline variable (ECOG, LDH, or M stage). The charts show that although there was a significant overlap between TKa and other variables (diamond symbols), in some patients only low or high TKa levels were associated with longer or shorter PFS, respectively (square symbols).

[0119] As part of the same study, TKa levels were also measured in 58 patients during immunotherapy treatment (3-4 weeks after initiation 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 duration, less than 6 months, had the highest pretreatment TKa levels and little or no increase in TKa during treatment. Patients with PFS durations of 6-24 months had lower pretreatment TKa levels but less than a two-fold increase in TKa during treatment. Patients whose response to treatment lasted beyond 24 months had lower pretreatment TKa levels and more than a two-fold increase in TKa during treatment. This indicates a burst of T cell proliferation in patients within the first month of successful ICI treatment.

[0120] Consideration In the patients with advanced cutaneous melanoma included in the study, significantly higher TKa levels were found 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 <20–1787 Du / L), whereas, for reference, in 123 healthy subjects, the median TKa was <20 Du / L (data not shown). In a cohort of patients with preoperative pancreatic cancer, the median TKa was 40 Du / L, and in a cohort of patients with preoperative renal cell carcinoma, the median TKa was 38 Du / L [12,14]. Furthermore, in a cohort of patients with non-small cell lung cancer, the median TKa before the start of systemic treatment was 129 Du / L, whereas in a cohort of patients with breast cancer, the pretreatment TKa was 57 Du / L in patients with locoregional disease and 101 Du / L in patients with visceral metastases [15,18]. Taken together, the data show that TKa levels in metastatic melanoma patients, similar to other cancer types studied, are elevated compared to levels in healthy individuals and are even higher in patients with more advanced disease.

[0121] Patients with high TKa had significantly worse 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 of TKa with PFS and OS depended to various degrees on ECOG, LDH, and tumor stage, however, a significant portion of patients did not have a matched pair of baseline variables that were good or bad (Figure 5). Clinical factors such as performance status, tumor stage, and tumor burden are well-known prognostic factors and predictors of ICI efficacy in melanoma [1-4,16]. With regard to serum markers, elevated LDH levels are the strongest known prognostic predictor and the only biomarker routinely used in monitoring melanoma patients in the clinic and included as a marker in clinical trials

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

[19] . Because LDH is not a secreted enzyme, elevated serum levels are likely secondary to leakage of LDH when melanoma cells outgrow the blood supply. LDH is also frequently elevated in a variety of conditions affecting the liver, both malignant and nonmalignant

[19] . TK enzymes play important roles 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.

[0122] The data herein also show that measuring TKa levels in cancer patients at two key time points, pre-treatment and 1-4 weeks after the first ICI treatment dose, can predict a positive patient response to ICIs. If pre-treatment TKa levels are low, this indicates a level of disease burden that the immune system can effectively manage once it is activated. If TKa levels increase at least two-fold during ICI treatment compared to baseline levels, it indicates a successful response to treatment and increased T cell activation and proliferation sufficient to achieve tumor killing.

[0123] Several other markers have been reported as predictive of ICI efficacy, including peripheral blood leukocyte composition, circulating tumor DNA (ctDNA) and exosomes, tumor mutation burden (TMB), high interferon-gamma-related gene expression signatures in tumors, gut microbiota diversity, and invasive tumor biopsy tests measuring ICI receptor expression (currently used for patient selection) [20-27]. In clinical settings, it is difficult to widely implement predictive assays such as TMB, ctDNA, exosomes, tumor RNA expression signatures, or microbiome analysis, for example, because they require complex and expensive techniques and equipment, and there are also many different assays that can be used. Measuring TKa is a simpler, lower-cost test (ELISA-based) for a single plasma marker, and this assay can be easily set up in a general hospital laboratory.

[0124] conclusion High pre-treatment plasma TKa levels were significantly associated with worse baseline characteristics, response and survival in ICI-treated melanoma patients.The inventors are the first to identify TKa as an interesting and previously unexplored 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]

[0125] Example 2 A phase 1b trial of the CDK4 / 6 inhibitor ribociclib in combination with the PD-1 inhibitor spartalizumab (also known as PDR001) was conducted in patients with hormone receptor-positive metastatic breast cancer (HR+MBC) and metastatic ovarian cancer (MOC).

[0126] Twenty-four patients were enrolled in this study. Ribociclib was administered orally once daily on days 1 through 21 of a 28-day cycle. Spartalizumab was administered intravenously on day 1 of the same 28-day cycle. Blood was drawn from each patient at baseline (before treatment) and on day 1 of every 28-day treatment cycle. Plasma was separated from the blood samples and then analyzed for thymidine kinase activity using the DiviTum-TKa assay as previously described. Data are shown in Figure 7 and the accompanying data table, Table 6.

[0127] As seen in the melanoma PD-1 inhibitor trial with DiviTim-TKa, patients who achieved the best clinical responses had low baseline TKa values ​​and a ≥2-fold increase in TKa during treatment.

[0128] Patient #19 was a breast cancer patient with a baseline TKa level of 590 DuA. After the first cycle of treatment, on day 28, the patient's TKa level increased 2-fold to 1173 DuA. The patient had a partial response to treatment with a 68% reduction in tumor volume and the patient was treated for 334 days, the second longest treatment duration. Patient #27 was also a breast cancer patient. The patient's baseline TKa level was 174 DuA (the lowest TKa baseline level in the study). After the first cycle of treatment, on day 28, the patient's TKa level increased 3.8-fold to 411 DuA (the highest fold increase in the study). The patient had a partial response to treatment with a 54% reduction in tumor volume and the patient was treated for 411 days, the longest treatment duration in the study. None of the other 22 patients in this study (except for numbers 19 and 27 above) met the criteria for both a low baseline TKa level and a two-fold increase in TKa during treatment. And none of them subsequently achieved meaningful clinical benefit. The only exception was patient #3, who had a low TKa BL level (296 DuA) and a 2.6-fold increase in TKa during treatment to 758 DuA, but was only on treatment for 95 days. Further investigation revealed that this patient had clinical symptoms, which were the reason for treatment discontinuation. It is quite possible that this patient would have benefited if he had been able to continue treatment. Patient #24 also had a more than two-fold increase in TKa during treatment (2.21), but because this patient had a very high baseline TKa (1316 DuA), he did not meet the criteria to predict benefit from immune checkpoint inhibitors based on a pre-treatment TKa cutoff value of 550 DuA + / - 20%, and in fact experienced rapid disease progression over the course of 35 days.

[0129] This data adds to and builds on previous data on TKa exemplified in melanoma patients treated with immune checkpoint inhibitors (ICIs). This data supports the applicability of the method of the present invention to other cancers, such as metastatic HR+ breast and ovarian cancer, as exemplified. It also supports the applicability of the method when using a different ICI (spartalizumab). [Table 7] References [1] Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al.Improved survival with ipilimumab in patients with metastatic melanoma.N Engl J Med.2010;363:711-23. [2] Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al.Nivolumab in previously untreated melanoma without BRAF mutation.N Engl J Med.2015;372:320-30. [3] Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, et al.Pembrolizumab versus Ipilimumab in Advanced Melanoma.N Engl J Med.2015;372:2521-32. [4]Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al.Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma.N Engl J Med.2015;373:23-34. [5]Robert C,Karaszewska B,Schachter J,Rutkowski P,Mackiewicz A,Stroiakovski D,et al.Improved overall survival in melanoma with combined dabrafenib and trametinib.N Engl J Med.2015;372:30-9. [6]Dummer R,Ascierto PA,Gogas HJ,Arance A,Mandala M,Liszkay G,et al.Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma(COLUMBUS):a multicentre,open-label,randomised phase 3 trial.Lancet Oncol.2018;19:603-15. [7]Schwartz JL,Tamura Y,Jordan R,Grierson JR,Krohn KA.Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.J Nucl Med.2003;44:2027-32. [8]Bitter EE,Townsend MH,Erickson R,Allen C,O’Neill KL.Thymidine kinase 1 through the ages:a comprehensive review.Cell Biosci.2020;10:138. [9]Bagegni N,Thomas S,Liu N,Luo J,Hoog J,Northfelt DW,et al.Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclin-dependent kinase 4 / 6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib.Breast Cancer Res.2017;19:123.

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Claims

1. 1. A method for 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, comprising: (a) providing a sample taken from said individual prior to treatment with one or more immune checkpoint inhibitors; (b) measuring the activity and / or concentration of thymidine kinase (TK) in said sample; (c) providing a sample taken from said individual 1 to 4 weeks after initial treatment with said one or more immune checkpoint inhibitors; and (d) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (c), wherein the activity and / or concentration of thymidine kinase (TK) measured in steps (b) and (d) indicates the prognosis of the individual when further treated with one or more immune checkpoint inhibitors.

2. 10. The method of claim 1, wherein the method is for stratifying an individual or a plurality of individuals suffering from cancer for treatment or further treatment with one or more immune checkpoint inhibitors.

3. 1. A method of stratifying a cancer patient for treatment or further treatment with one or more immune checkpoint inhibitors, comprising: Step (i) of carrying out a method according to any one of the preceding claims, and (ii) stratifying the individual for treatment or further treatment with one or more immune checkpoint inhibitors or an alternative cancer therapy based on the result of step (i).

4. 4. The method of claim 3, wherein the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

5. 13. The method of any one of the preceding claims, 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), then the individual's prognosis upon further treatment with one or more checkpoint inhibitors is good (e.g. the individual's progression free survival is at least 6 months) and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

6. 6. The method of claim 5, 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), the individual's prognosis upon further treatment with one or more checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than 24 months) and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

7. 13. The method according to any one of the preceding claims, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cut-off value, the prognosis of the individual when treated or further treated with one or more immune checkpoint inhibitors is good and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

8. 13. The method of any one of the preceding claims, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is equal to or greater than a cut-off value, then the individual's prognosis upon treatment or further treatment with one or more immune checkpoint inhibitors is poor (e.g. the individual's progression free survival will be less than 6 months) and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to the one or more immune checkpoint inhibitors.

9. 5. The method of any one of the preceding claims, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cut-off value, the individual's prognosis upon treatment or further treatment with one or more immune checkpoint inhibitors is good (e.g. the individual's progression free survival is at least 6 months) and / or the patient is stratified for treatment or further treatment 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).

10. 10. The method of claim 9, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is lower than a cut-off value, the individual's prognosis when treated or further treated with one or more immune checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than 24 months) and / or the patient is stratified for treatment or further treatment 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).

11. 10. The method of any one of the preceding claims, wherein if the activity and / or concentration of thymidine kinase (TK) measured in step (b) is equal to or greater than a cut-off value, the individual's prognosis upon treatment or further treatment with one or more immune checkpoint inhibitors is poor (e.g. the individual's progression free survival will be less than 6 months) and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to the one or more immune checkpoint inhibitors, wherein the activity and / or concentration of thymidine kinase (TK) measured in step (d) is not increased compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b).

12. The method according to any one of claims 8 to 11, wherein the activity of thymidine kinase (TKa) is measured in step (b), and said cut-off value is 550 DuA + / - 20%.

13. The method comprises: (e) providing one or more control samples; and (f) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (e), 10. The method of any one of the preceding claims, wherein the prognosis of the individual upon treatment with one or more immune checkpoint inhibitors is determined by comparing the measurements of step (f) with the measurements of step (b) and / or step (d).

14. 14. The method of claim 13, wherein the one or more control samples are obtained from an individual with cancer prior to subsequent successful treatment with one or more immune checkpoint inhibitors.

15. 15. The method of claim 13 or 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 when treated with one or more immune checkpoint inhibitors is good and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

16. 16. The method of claim 15, wherein the one or more control samples are obtained from an individual with cancer prior to subsequent unsuccessful treatment with one or more immune checkpoint inhibitors.

17. 17. The method of claim 13 or 16, 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), then the individual's prognosis when treated with one or more immune checkpoint inhibitors is poor and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to the one or more immune checkpoint inhibitors.

18. The method further comprises the step (g) of measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a), 10. The method of any one of the preceding claims, wherein the activity and / or concentration of LDH measured in step (g) is further indicative of the prognosis of the individual upon subsequent treatment with one or more immune checkpoint inhibitors.

19. 20. The method of claim 18, wherein if the activity and / or concentration of LDH measured in step (g) is lower than a cut-off value, the individual's prognosis when treated or further treated with one or more immune checkpoint inhibitors is good (e.g., the individual's progression free survival is greater than 24 months) and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

20. 20. The method of claim 19, wherein the activity of LDH is measured, said cut-off value being selected from 2 to 6 microcuts / L, such as from 3 to 5 microcuts / L, or such as being 4 microcuts / L.

21. 13. The method of any one of the preceding claims, wherein an additional step (h) of treating the individual with one or more immune checkpoint inhibitors or an alternative cancer therapy is performed.

22. 1. A method of treating cancer in an individual, comprising: (a) stratifying an individual for treatment with one or more immune checkpoint inhibitors or an alternative cancer therapy using the method of any one of claims 1 to 21; and (b) providing said individual with a cancer therapy comprising administering one or more immune checkpoint inhibitors or an alternative cancer therapy.

23. 1. A method of treating cancer in an individual, comprising: (a) selecting an individual for treatment with one or more immune checkpoint inhibitors using the method of any one of claims 1 to 21; and (b) providing said individual with a cancer therapy comprising administering one or more immune checkpoint inhibitors.

24. 21. One or more immune checkpoint inhibitors for use in the treatment of cancer in an individual, wherein said individual has been selected for treatment using the method of any one of claims 1 to 20.

25. 25. The method or use of an immune checkpoint inhibitor according to claim 23 or 24, wherein the individual is selected for treatment because (i) the serum or plasma TK activity and / or concentration before ICI treatment is lower than a cut-off value, and (ii) the serum or plasma TK activity and / or concentration after 1 to 4 weeks of treatment with the ICI inhibitor is increased compared to the serum or plasma TK activity and / or concentration before ICI treatment.

26. 2. The method or use of an immune checkpoint inhibitor according to any one of the preceding claims, wherein the activity of thymidine kinase (TKa) is measured.

27. 2. The method or use of an immune checkpoint inhibitor according to any one of the preceding claims, wherein the cancer is a cancer for which treatment with an immune checkpoint inhibitor is approved.

28. 20. The method or use of an immune checkpoint inhibitor according to any one of the preceding claims, wherein said cancer is selected from the group comprising melanoma, breast cancer, and / or ovarian cancer.

29. 13. The method or use of immune checkpoint inhibitors according to any one of the preceding claims, wherein said one or more immune checkpoint inhibitors are selected from the list consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor.

30. 2. The method or use of an immune checkpoint inhibitor according to any one of the preceding claims, wherein the individual or patient is a human.

31. 13. The method according to any one of the preceding claims, wherein the method is carried out in vitro.

32. 2. The method or use of an immune checkpoint inhibitor according to any one of the preceding claims, wherein the sample is a plasma or serum sample.

33. Use of thymidine kinase as a prognostic biomarker for individuals with cancer who are being considered for treatment with an immune checkpoint inhibitor or who are currently undergoing treatment with an immune checkpoint inhibitor.

34. Use of thymidine kinase to stratify individuals with cancer for treatment or further treatment with an immune checkpoint inhibitor.

35. 20. A method or use substantially as described herein.

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