Thymidine kinases as markers of immune checkpoint inhibitor efficacy

Abstract

The present invention provides a method for determining the prognosis of an individual having cancer and being considered for treatment with one or more immune checkpoint inhibitors or currently being treated with one or more immune checkpoint inhibitors, the method comprises or consists of the steps of: a) providing a sample obtained 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 said sample; c) providing a sample obtained from the individual 1-4 weeks after a first treatment with the one or more immune checkpoint inhibitors; and d) measuring the activity and / or concentration of thymidine kinase (TK) in said sample provided in step (c); wherein the activity and / or concentration of thymidine kinase (TK) measured in steps (b) and (d) is indicative of a prognosis of the individual if further treated with one or more immune checkpoint inhibitors.

Description

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

[0002] Immune checkpoint inhibitors (ICIs) are only effective for some cancer patients. Examples of ICIs in clinical development 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 eliminate the inhibition of T cell activity, which enables the activation and subsequent proliferation of tumor-reactive T cells, which can then enhance effective anti-tumor responses. Studies have shown that the number of T cells increases significantly after the first dose of ICI drugs. This increased level of T cells is associated with tumor response and patient outcomes

[28] . However, the measurement of activated and proliferated T cells requires sorting and separation of cells from whole blood populations, which is complex, time-consuming, and uses specialized flow cytometry instruments for this purpose. Currently, this is not used as a standard of care method for predicting patient response to ICI.

[0003] In recent years, effective ICI regimens with CTLA-4 and PD-1 blocking antibodies have emerged to treat a variety of cancers, including melanoma [1-6]. These treatments have revolutionized the field of oncology, but a significant proportion of patients do not respond to these treatments or achieve durable responses. Treatments can also produce significant toxicity and are expensive. Therefore, it is important to increase understanding of predictive factors and their efficacy in different patient populations.

[0004] Thymidine kinase 1 (TK) is a cytosolic enzyme, a phosphotransferase, that plays a key role in DNA synthesis and repair [7]. TK has a critical function in DNA synthesis and cell division as it is part of the chain of reactions that incorporate thymidine into the DNA chain [7]. Dividing cells release TK during mitotic exit and can therefore be detected in the blood. In addition, elevated TK enzyme activity (TKa) has been measured in blood samples from cancer patients and has been associated with tumor proliferation and tumor burden [8]. Circulating levels of TKa measured by the TKa assay have been shown to correlate with disease stage, prognosis, and treatment efficacy in several cancer types, including breast, lung, pancreatic, and renal cell carcinoma [9-15].

[0005] The inventors were surprised to find that before and during treatment with immune checkpoint inhibitors (ICIs), measurements of the activity and / or concentration of thymidine kinase (TK) in a patient's blood sample, optionally together with blood measurements of the activity or concentration of lactate dehydrogenase (LDH), can be used to provide a prognosis for subsequent treatment with ICIs. Therefore, this allows a determination to be made whether an ICI that has been given or is being considered for a patient does not work or will not work effectively. A key benefit of quickly understanding that ICI drugs are ineffective is that these drugs are very expensive and can be very toxic, so it is in the interest of the patient not to start taking or stop taking ineffective drugs as soon as possible, and alternative therapies may be administered instead.

[0006] A first aspect of the present invention provides a method for determining the prognosis of an individual who has cancer and is being considered for treatment with or is currently being treated with one or more immune checkpoint inhibitors, the method comprising or consisting of the following steps:

[0007] a) providing a sample obtained from the individual prior to treatment with one or more immune checkpoint inhibitors; and

[0008] b) measuring the activity and / or concentration of thymidine kinase (TK) in the sample;

[0009] Wherein the activity and / or concentration of thymidine kinase (TK) measured in step (b) is indicative of the prognosis of the individual if subsequently treated with one or more immune checkpoint inhibitors.

[0010] By "thymidine kinase (TK)" we mean thymidine kinase 1 (TK1). Thymidine kinase 1 is a key cell cycle regulatory enzyme that 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 before incorporation into DNA. The activity of TK1 is low or absent in resting cells, but increases dramatically as cells divide and replicate. Therefore, its presence 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. TKa is a highly sensitive and accurate blood-based assay used to measure TK activity (TKa) in serum or plasma samples.

[0011] Immune checkpoint inhibitors (ICIs) are a class of drugs that block proteins called checkpoints, which are produced by certain types of immune system cells (such as T cells) and some cancer cells. These checkpoints usually act as negative regulators of the immune system. After the checkpoint is activated, the immune cells will be inhibited from triggering cytotoxic reactions. These checkpoint proteins are essential for maintaining the balance between autoimmunity and self-tolerance. However, tumors can usually upregulate the expression of immune checkpoints, create an immunosuppressive environment and allow tumor cells to escape immune system-mediated destruction. A series of immune checkpoint inhibitors have been developed for the treatment of cancer. Among them, 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), lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin mucin-3 (TIM-3) inhibitor therapies. Antibody drugs targeting these immune checkpoints eliminate the inhibition of T cell activity, which achieves the activation and subsequent proliferation of tumor-reactive T cells, which can then enhance effective anti-tumor responses. Studies have shown that the number of T cells increases significantly after the first dose of ICI drugs. This increased level of T cells correlates with tumor response and patient outcomes (Kim et al. Clin Cancer Res; 25(7) 2020).

[0012] By "individuals with cancer who are being considered for treatment with one or more immune checkpoint inhibitors", we include cancer patients for whom doctors would typically consider and / or arrange treatment with one or more checkpoint inhibitors. For example, regulatory agencies such as the FDA (or equivalent regulatory agencies in different countries) have approved patients with tumors for treatment with immune checkpoint inhibitors. Twomey and Zhang (2021) The AAPS Journal 23:39 provide a review of FDA-approved immune checkpoint inhibitors, and the review is incorporated herein by reference. When a particular patient expresses immune checkpoint inhibitor receptors (checkpoint proteins) on their cancer cells, or expresses these receptors at higher levels than healthy individuals, treatment with immune checkpoint inhibitors may be considered, which can be determined by conventional methods (such as diagnostic assays that measure immune checkpoint inhibitor receptor expression levels). An example of such a cancer is melanoma, as the subject of the examples described herein, however the methods of the present invention are applicable to other cancers that are typically considered for treatment with one or more checkpoint inhibitors.

[0013] By “prognosis of the individual if subsequently treated with one or more immune checkpoint inhibitors” we include the expected effectiveness of treatment with one or more immune checkpoint inhibitors, including progression-free survival (PFS) and overall survival (OS) times for the individual if they are subsequently treated with one or more immune checkpoint inhibitors.

[0014] By treatment with one or more checkpoint inhibitors, we include treatment with a single ICI (ICI monotherapy) or treatment with different types of ICIs (ICI combination therapy).

[0015] ICI therapy and / or subsequent ICI therapy can be delivered alone or with additional non-ICI cancer therapy. In a specific embodiment, ICI therapy and / or subsequent ICI therapy is treatment with one or more ICIs alone and without additional non-ICI cancer therapy.

[0016] Prior to the methods of the present invention, a sample is obtained from an individual. It will be appreciated that the activity and / or concentration of thymidine kinase (TK) may be measured. It is expected that TK protein levels are correlated with TK activity levels. In one embodiment, TK activity (TKa) is measured. TKa may be measured by techniques well known in the art. It may be measured by ELISA according to the manufacturer's instructions, such as TKa assay (Biovica, Sweden), which has been 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 WO 2011 / 142719 and et al. (2013) Analytical Biochemistry 432; 155-164, the contents of which are incorporated herein by reference. TK concentration levels may also be measured by a variety of techniques well known in the art, for example by immunoassays such as ELISA.

[0017] In an additional or alternative embodiment, the method further comprises the following steps:

[0018] c) providing a sample obtained from the individual 1-4 weeks after first treatment with one or more immune checkpoint inhibitors; and

[0019] d) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (c);

[0020] Wherein 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.

[0021] In additional or alternative embodiments, the sample provided in step (c) may be obtained from the individual 3-4 weeks after the first treatment with one or more immune checkpoint inhibitors.

[0022] In additional or alternative embodiments, the sample provided in step (c) can be obtained from the individual 7-28 days after the first treatment with one or more immune check point inhibitors, or alternatively 14-28 days after the first treatment with one or more immune check point inhibitors, or alternatively 21-28 days after the first treatment with one or more immune check point inhibitors.

[0023] In an additional or alternative embodiment, the method of the present invention is used to stratify one or more individuals with cancer to treat or further treat with one or more immune checkpoint inhibitors. In other words, the method is used to determine whether an individual with cancer should receive treatment or further treatment with one or more immune checkpoint inhibitors.

[0024] In another aspect of the present invention, there is provided a method for stratifying patients with cancer for treatment or further treatment with one or more immune checkpoint inhibitors, the method comprising or consisting of the following steps:

[0025] i) performing the method of the first aspect; and

[0026] ii) based on the results of step (i), stratifying the individual for treatment or further treatment with one or more immune checkpoint inhibitors or alternative cancer therapies

[0027] Optionally wherein 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.

[0028] In one embodiment, patients are stratified for treatment or further treatment with one or more immune checkpoint inhibitors. Alternatively, patients can be stratified for no further ICI treatment. Alternatively, patients can be stratified for treatment with alternative non-ICI cancer therapies.

[0029] Alternative cancer treatments to immune checkpoint inhibitors will depend on the specific tumor type, however examples include surgery, chemotherapy, targeted therapy, immunotherapy (i.e., immunotherapy other than immune checkpoint inhibitors), chemoimmunotherapy, radiation therapy, and combinations thereof. For example, physicians can refer to the NCCN Cancer Treatment Guidelines to determine the most appropriate alternative cancer treatment for each individual tumor type for which ICIs are approved (e.g., melanoma, lung cancer, RCC, HNSCC, CRC, etc.), which are available athttps: / / www.nccn.org / guidelines / category_1 Found in.

[0030] The following examples are relevant to all aspects of the present invention.

[0031] In additional or alternative embodiments, when 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), the individual has a positive prognosis for further treatment with one or more checkpoint inhibitors (e.g., the individual will or is expected to have a progression-free survival of at least 6 months), and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

[0032] Preferably, the activity and / or concentration of thymidine kinase (TK) measured in step (d) is increased by at least 8%, such as 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%, compared to 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 ...6%, 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 %, 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 At least 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%.

[0033] In additional 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).

[0034] In additional 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).

[0035] 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).

[0036] 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).

[0037] 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).

[0038] 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).

[0039] By "positive" prognosis, we include individuals who have or are expected to have a progression-free survival of at least 6 months, i.e., the cancer does not progress 6 months after initial treatment with ICI. A "positive" prognosis may include individuals who have or are expected to have a progression-free survival of at least 12, 18, or 24 months, i.e., the cancer does not progress 12, 18, or 24 months after initial treatment with ICI.

[0040] In additional or alternative embodiments, a "positive" 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.

[0041] Progression-free survival (PFS) can be defined as the time from the start of treatment to the date of confirmed progression or death or the last follow-up.

[0042] In additional or alternative embodiments, a "positive" 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 can be defined as the time from the start of treatment to death or the last follow-up.

[0043] In additional or alternative embodiments, a "positive" prognosis means a complete response, a partial response, or stable disease. Preferably, a positive prognosis means a complete or partial response, most preferably a complete response. The response to treatment can be based on radiological examinations (CT, MRI, and / or positron emission tomography (PET) CT scans) assessed by a radiologist and assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria

[17] .

[0044] By "stratification for further treatment with one or more immune checkpoint inhibitors" we include categorizing patients or individuals with cancer into those who should receive treatment or further treatment with one or more immune checkpoint inhibitors. This may be the same or a different ICI, and may be alone or together with an alternative cancer therapy.

[0045] In an additional or alternative embodiment, when the activity and / or concentration of thymidine kinase (TK) measured in step (d) increases by at least 100% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b), the individual has a positive prognosis for further treatment with one or more checkpoint inhibitors (e.g., the individual will or is expected to have a progression-free survival of more 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) increases 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).

[0046] In additional or alternative embodiments, when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is below a cut-off value, the individual has a positive prognosis for treatment or further treatment with one or more immune checkpoint inhibitors, and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0047] In additional or alternative embodiments, when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than or equal to a cut-off value, the individual has a negative prognosis if treated or further treated with one or more immune check point inhibitors (e.g., the individual will or will be expected to have a progression-free survival of less than 6 months), and / or the patient is stratified for no further treatment with one or more immune check point inhibitors or for treatment with an alternative cancer therapy to one or more immune check point inhibitors.

[0048] In additional or alternative embodiments, when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is below a cut-off value, 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), the individual has a positive prognosis for treatment or further treatment with one or more immune checkpoint inhibitors (e.g., the individual will or is expected to have a progression-free survival of at least 6 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0049] 37%, 38%, 39%, 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%, 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%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 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%.

[0050] In additional 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).

[0051] In additional 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).

[0052] 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).

[0053] 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).

[0054] 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).

[0055] 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).

[0056] In additional or alternative embodiments, when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is below a critical value, and 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), the individual has a positive prognosis for treatment or further treatment with one or more immune checkpoint inhibitors (e.g., the individual will or is expected to have a progression-free survival of more than 24 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0057] It will be appreciated that the percent increase value may be optimized depending on the cancer or tumor type. One skilled in the art will be able to modify the percent increase value as a routine matter based on the information described herein for melanoma.

[0058] In additional or alternative embodiments, when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than or equal to a cut-off value, and 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), the individual has a negative prognosis if treated or further treated with one or more immune check point inhibitors (e.g., the individual will have or will be expected to have a progression-free survival of less than 6 months), and / or the patient is stratified for no further treatment with one or more immune check point inhibitors or for treatment with an alternative cancer therapy to one or more immune check point inhibitors.

[0059] In an additional or alternative embodiment of the method of the invention, the activity of thymidine kinase (TKa) is measured and the cut-off value is selected between 549 and 1021 Du / L, such as between 628 and 942 Du / L, such as between 707 and 864 Du / L.

[0060] In an additional or alternative embodiment of the method of the invention, the activity of thymidine kinase (TKa) is measured, and the cutoff value is selected between 385 and 715 DuA, such as between 440 and 660 DuA, such as between 495 and 605 DuA. The relationship between Du / L and Du / A is as follows.

[0061] In an additional or alternative embodiment of the method of the present invention, the cut-off value is 785 Du / L (+ / - 20%). In an additional or alternative embodiment of the method of the present invention, the cut-off value is 785 Du / L (+ / - 10%). The 10% reflects normal assay variance.

[0062] In additional or alternative embodiments of the method of the present invention, the cut-off value is 550 DuA (+ / - 20%). In additional or alternative embodiments of the method of the present invention, the cut-off value is 550 DuA (+ / - 10%). The 10% reflects normal assay variance.

[0063] In an additional or alternative embodiment of the method of the present invention, the activity of thymidine kinase (TKa) is measured, and the cutoff value is selected from between 40 and 75 Du / L, such as between 42 and 72 Du / L, such as between 49 and 72 Du / L, such as between 42 and 60 Du / L, such as between 49 and 60 Du / L, such as between 58 and 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. In alternative or additional embodiments, the critical value can 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.

[0064] It should be understood that the pre-treatment cutoff value can be optimized according to the cancer or tumor type and the number of previous therapies the patient has received. Those skilled in the art will be able to modify the cutoff value as a routine matter based on the information described herein.

[0065] By Du / L we mean TKa assay (Biovica, Sweden) and manufacturer's instructions per liter Unit, which has been 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 (incorporated herein by reference).

[0066] TKa can also be passed The TKa assay is measured in DuA. For the unit Du / L, recombinant thymidine kinase protein is used to assign Du / L values ​​to a set of "gold standard serum" 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 TKa assay, which has a new calibration concept to better match 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:

[0067] DuA=134+0.53(Du / L). Determination coefficient (R 2 )=0.93

[0068] In an additional or alternative embodiment, the method further comprises the following steps:

[0069] (e) providing one or more control samples; and

[0070] (f) measuring the activity and / or concentration of thymidine kinase (TK) in the sample provided in step (e);

[0071] The prognosis of the individual if treated with one or more immune checkpoint inhibitors is determined by comparing the measurement results in step (f) with the measurement results in step (b) and / or step (d).

[0072] One or more control samples can be obtained from an individual with cancer prior to subsequently undergoing successful treatment with one or more immune checkpoint inhibitors.

[0073] By successful treatment, we include cessation, non-progression and / or regression of tumor growth. This can be assessed by imaging techniques, such as gold standard assessment tools. Successful treatment can be assessed by RECIST criteria (referring to reference 17). The response evaluation criteria for solid tumors or (RECIST) refer to a set of published rules for assessing tumor burden, to provide an objective assessment of therapeutic response. Stable disease, partial response, complete response, progression-free survival and overall survival are considered to be the endpoints of successful treatment. Successful treatment can be defined in the same manner as the positive prognosis defined above. For example, successful treatment can be considered to be at least 24 months of progression-free survival, i.e., cancer will not progress in 24 months after initial treatment with ICI.

[0074] In additional or alternative embodiments, when 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), the individual has a positive prognosis if treated with one or more immune checkpoint inhibitors, and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0075] By "corresponding to", we include the activity and / or concentration of thymidine kinase (TK) being the same as the activity and / or concentration of the positive control sample; or being closer to the activity and / or concentration of one or more positive control samples (or a predefined reference value representing the same) than to one or more negative control samples.

[0076] Alternatively or additionally, by "corresponding to", we include that the activity and / or concentration correlates with the amount in the control sample in a statistically significant manner. By "correlates with the amount in the control sample in a statistically significant manner", we mean or include that the presence or amount in the test sample correlates with the presence or amount in the control sample with a p-value of ≤0.05, such as ≤0.04, ≤0.03, ≤0.02, ≤0.01, ≤0.005, ≤0.004, ≤0.003, ≤0.002, ≤0.001, ≤0.0005, or ≤0.0001.

[0077] In additional or alternative embodiments, one or more control samples may be obtained from an individual having cancer prior to subsequently undergoing unsuccessful treatment with one or more immune checkpoint inhibitors.

[0078] In additional or alternative embodiments, when 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), the individual has a negative prognosis if treated with one or more immune check point inhibitors, and / or the patient is stratified for no further treatment with one or more immune check point inhibitors or for treatment with an alternative cancer therapy to one or more immune check point inhibitors.

[0079] In an additional or alternative embodiment of the method of the present invention, the method further comprises the following steps:

[0080] g) measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a);

[0081] Wherein the activity and / or concentration of LDH measured in step (g) is further indicative of the prognosis of the individual if subsequently treated with one or more immune checkpoint inhibitors.

[0082] In additional or alternative embodiments, when the activity and / or concentration of LDH measured in step (g) is considered normal, i.e., not elevated above normal levels, the prognosis of the individual if treated or further treated with one or more immune checkpoint inhibitors is positive (e.g., the individual will have a progression-free survival of more than 24 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0083] In additional or alternative embodiments, when the activity and / or concentration of LDH measured in step (g) is below a cut-off value, the individual has a positive prognosis for treatment or further treatment with one or more immune checkpoint inhibitors (e.g., the individual will have a progression-free survival of more than 24 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

[0084] In additional or alternative embodiments, the activity of LDH is measured and the cut-off value is selected from between 2 and 6 mikrokat / L, such as between 3 and 5 mikrokat / L, or for this example 4 mikrokat / L.

[0085] It will be appreciated that the cutoff value may be optimized, for example depending on the individual's cancer or tumor type. This can be accomplished as a routine matter by one of skill in the art based on the information described herein.

[0086] In one embodiment of the methods of the invention, an additional step (h) of treating the individual with one or more immune checkpoint inhibitors or alternative cancer therapies is performed.

[0087] In additional or alternative embodiments, the cancer is a cancer that is approved for treatment with an immune checkpoint inhibitor. For example, we include patients with cancer for whom a regulatory agency such as the FDA has approved treatment with an immune checkpoint inhibitor. Twomey and Zhang (2021) The AAPS Journal 23:39 provide a review of FDA-approved immune checkpoint inhibitors, and the review is incorporated herein by reference.

[0088] In one embodiment of any method of the present invention, the cancer can 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, BCG cancer, 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, head and neck squamous cell carcinoma, non-small cell lung cancer.

[0089] In one embodiment of any of the methods of the invention, the cancer is selected from the group consisting of: melanoma; breast cancer; and / or ovarian cancer. The cancer may be metastatic.

[0090] In one embodiment of any of the methods of the invention, the cancer is melanoma and / or breast cancer.

[0091] In one embodiment of any of the methods of the invention, the cancer is melanoma. The melanoma may be metastatic melanoma.

[0092] 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.

[0093] In one embodiment of any of the methods of the invention, the cancer is ovarian cancer. The ovarian cancer may be metastatic ovarian cancer.

[0094] 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, a LAG-3 inhibitor.

[0095] In additional or alternative embodiments, the one or more checkpoint inhibitors are anti-CTLA-4 and / or anti-PD-1.

[0096] In additional or alternative embodiments, the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab and / or spartalizumab.

[0097] In one embodiment of any of the methods of the invention, the individual or patient is a human.

[0098] In another embodiment of any of the methods of the invention, the method is performed in vitro or ex vivo.

[0099] In another 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.

[0100] In another aspect of the present invention, there is provided a method of treating cancer in an individual, the method comprising the steps of:

[0101] a) using the method of any of the previous aspects of the invention to stratify individuals for treatment with one or more immune checkpoint inhibitors or alternative cancer therapies; and

[0102] b) providing the individual with cancer therapy comprising administering one or more immune checkpoint inhibitors or alternative cancer therapy.

[0103] By treating an individual with one or more immune checkpoint inhibitors, we include administering or continuing to administer one or more immune checkpoint inhibitors to the individual in accordance with specified treatment guidelines.

[0104] By treating an individual with an alternative cancer therapy we include administering or continuing to administer the alternative cancer therapy to the individual in accordance with designated treatment guidelines.

[0105] In another aspect of the present invention, a method of treating cancer is provided by administering one or more immune check point inhibitors to an individual selected for treatment based on their TK activity and / or concentration prior to treatment with the one or more immune check point inhibitors and optionally also based on their TK activity and / or concentration measured 1-4 weeks after the first treatment with the one or more immune check point inhibitors.

[0106] In another aspect of the invention, one or more immune checkpoint inhibitors are provided for use in treating cancer in an individual, wherein the individual is selected for treatment based on their TK activity and / or concentration prior to treatment with the one or more immune checkpoint inhibitors and optionally also based on their TK activity and / or concentration measured 1-4 weeks after the first treatment with the one or more immune checkpoint inhibitors.

[0107] In embodiments of the aforementioned aspects, the individual is selected for ICI treatment because prior to ICI treatment, the individual has a TK activity and / or concentration below a cut-off value, optionally wherein the cut-off value is a specific cut-off value selected from those described in the previous aspects of the invention.

[0108] In further embodiments of the aforementioned aspects, the individual is selected for ICI treatment because the individual has an increased TK activity and / or concentration 1-4 weeks, optionally 3-4 weeks after treatment with the ICI inhibitor compared to their TK activity and / or concentration before ICI treatment, optionally wherein the increase is at least a specific percentage increase selected from those values ​​described in the previous aspects of the invention, such as at least a 100% increase.

[0109] In further embodiments of the aforementioned aspects, the individual is selected for ICI treatment due to: (i) having a TK activity and / or concentration below a cut-off value prior to ICI treatment, optionally wherein the cut-off value is a specific cut-off value selected from those described in the previous aspects of the invention; and (ii) having an increased TK activity and / or concentration 1-4 weeks, optionally 3-4 weeks after treatment with the ICI inhibitor compared to the TK activity and / or concentration prior to ICI treatment, optionally wherein the increase is at least a specific percentage increase selected from those described in the previous aspects of the invention, such as at least a 100% increase.

[0110] In another embodiment of the aforementioned aspects, the individual is additionally selected for ICI treatment because prior to ICI treatment, the individual had an LDH activity and / or concentration that was below a cutoff value or considered normal, optionally wherein the cutoff value is a specific cutoff value selected from those described in the previous aspects of the invention.

[0111] In another embodiment of the aforementioned aspects, TK and / or LDH activity and / or concentration is measured in a blood (eg, unfractionated blood), plasma, serum, interstitial fluid, and / or urine sample.

[0112] In another embodiment of the aforementioned aspects, TK and / or LDH activity and / or concentration is measured in a serum or plasma sample.

[0113] In another 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 treatment with, or are currently being treated with, an immune checkpoint inhibitor.

[0114] In another aspect of the present invention, there is provided the use of thymidine kinase for stratifying individuals with cancer for treatment or further treatment with immune checkpoint inhibitors.

[0115] Unless the context indicates otherwise, preferences and options for a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences and options for all other aspects, features and parameters of the invention. For example, preferences, options and embodiments described with respect to methods for determining prognosis also apply to methods, treatment methods and uses for stratifying patients described later.

[0116] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an admission that the document is prior art or part of the common general knowledge.

[0117] Preferred non-limiting examples embodying certain aspects of the invention will now be described with reference to the following figures and examples:

[0118] Figure 1 : To determine the cut-off value of plasma thymidine kinase 1 activity (TKa) in patients with metastatic melanoma treated with immune checkpoint inhibitors. The TKa grade (order) expressed in units (Du / L) is shown on the x-axis, and the TKa value is shown on the y-axis. When determining the cutoff value of TKa, the optimal cutoff is considered to be the value closest to the median to ensure the difference in TKa values ​​when there is a sufficient number of observations in each group. The median TKa at baseline is 42Du / L. This median (solid line) gives a cutoff that divides patients with very similar TKa values ​​into different groups. A cutoff of 60Du / L (dashed line) ensures more differentiation of TKa and sufficient observations in both groups.

[0119] Figure 2 : ROC curve analysis of the best TKa cutoff for predicting sensitivity and specificity: A. Physical staging (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 best TKa cutoff for each analysis is shown in each analysis (numbers above the trend line), and the TKa of 60.0, which was selected as the cutoff for comparison analysis in the study, is shown below the trend line.

[0120] Figure 3: Kaplan-Meier curves of survival of melanoma patients with high (>60Du / L) or low thymidine kinase 1 activity (TKa) in plasma before starting treatment with immune checkpoint inhibitors. (A). The median progression-free survival of patients with low TKa was 19.9 months (95% CI, 11.0 to not reached), while the median progression-free survival of patients with high TKa was 12.6 months (95% CI, 3.6 to 28.3) (p=0.021). (B). Patients with low TKa did not reach the median overall survival (>60 months, 95% CI, 38.0 to not reached), while the median overall survival of patients with high TKa was 18.5 (p=0.005).

[0121] Figure 4 : Bivariate regression of paired analysis of TKa with another variable A. progression-free survival (PFS) and B. overall survival (OS) in patients with metastatic melanoma treated with immune checkpoint inhibitors.

[0122] Figure 5 : Pie charts showing the number of patients with longer (>24 months, (AC)) or shorter (<24 months, (DF)) progression-free survival (PFS). Each pie chart shows whether TKa level and other baseline variables (ECOG, LDH or M stage) correctly predicted longer or shorter PFS. For example (B) shows that in 17 patients, long PFS was predicted by both low TKa and normal LDH at baseline (both variables predicted correctly, 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, neither variable predicted long PFS because both LDH and TKa levels were high (no correct prediction, triangle symbols).

[0123] Figure 6: Plasma TKa in melanoma patients receiving immunotherapy. The figure shows the median TKa value in Du / L at 3 different time points: before treatment, 3-4 weeks after treatment, at the end of treatment, or 24 months if the patient is still receiving treatment. Patients were stratified according to their progression-free survival time (PFS <6 months, PFS>6 months, or response still ongoing at 24 months). Patients with the shortest PFS time of less than 6 months had the highest pre-treatment TKa levels and almost no increase in TKa during treatment. Patients with PFS times between 6-24 months had lower pre-treatment TKa, but the TKa increased less than 2-fold during treatment. Patients whose response to treatment lasted more than 24 months had lower pre-treatment TKa levels and their TKa increased by 2-fold or more during treatment.

[0124] Figure 7 : Correlation between TK activity levels and outcomes in HR+ breast and ovarian cancer patients treated with ribociclib and the PD-1 inhibitor spartalizumab. The ID numbers of individual patients are listed on the horizontal axis. The height of each bar represents the fold increase in the TKa level of the specified patient from Day 1 of Cycle 1 (before treatment) to Day 1 of Cycle 2 (28 days after first treatment). The number above each bar is the patient's original baseline (before treatment) TKa value. The bar shading indicates the best overall response assessed according to the RECIST 1.1 criteria, where:

[0125] Black / PR = partial response;

[0126] Dark grey / SD>6M=stable disease greater than or equal to 6 months;

[0127] Light grey / SD<6M=stable condition for less than 6 months;

[0128] White / PD=progressive disease;

[0129] NE = Not evaluable.

[0130] Also shown on the horizontal axis are the best overall response and progression-free survival (PFS) times (in days) for each patient and tumor type, where:

[0131] B=HR+Her2-metastatic breast cancer

[0132] O = metastatic epithelial ovarian cancer

[0133] Example 1

[0134] Summary and Introduction

[0135] Immune checkpoint inhibitors (ICIs) are effective in a subset of patients with disseminated melanoma. The ICIs most in clinical development are those that 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 eliminate the inhibition of T cell activity, which enables the activation and subsequent proliferation of tumor-reactive T cells, which are then able to enhance effective anti-tumor responses. Studies have shown that the number of T cells increases significantly after the first dose of an ICI drug. This increased level of T cells correlates with tumor response and patient outcomes

[28] . The TKa assay is able to detect this proliferative burst of T cells in patients within the first month of ICI treatment.

[0136] In recent years, effective ICI regimens with 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 proportion of melanoma patients do not respond to these treatments or achieve durable responses. Treatments can also produce significant toxicity and are expensive. Therefore, it is important to increase understanding of predictive factors and their efficacy in different patient populations.

[0137] Thymidine kinase 1 (TK) is a cytosolic enzyme, a phosphotransferase, that plays a key role in DNA synthesis and repair [7]. TK has a critical function in DNA synthesis and cell division as it is part of the reaction chain that incorporates thymidine into the DNA chain [7]. Dividing cells release TK during mitotic exit and can therefore be detected in the blood. In addition, elevated TK enzyme activity has been measured in blood samples from cancer patients and has been associated with tumor proliferation and tumor burden [8]. Circulating levels of TKa measured by the assay have been shown to correlate with disease stage, prognosis, and treatment efficacy in several cancer types, including breast, lung, pancreatic, and renal cell carcinomas [9-15]. Pre-treatment levels of TKa can reflect both the rate of tumor cell proliferation and the overall disease burden, with high levels of TKa indicating actively proliferating tumors and / or greater disease burden (in both tumor size and number), and low levels of TKa indicating slower-growing, indolent tumors and lower disease burden (smaller and fewer overall tumors).

[0138] The inventors first analyzed 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 in metastatic melanoma patients before starting ICI treatment and correlated with baseline clinical characteristics, treatment response and survival.

[0139] 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 are also associated with poor efficacy of immune checkpoint inhibitors. Therefore, the inventors have identified TKa as a new prognostic and predictive marker in cancer.

[0140] Materials and Methods

[0141] Patients and plasma samples:

[0142] 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, from 2012 to 2019. Treatment was administered according to standard ICI regimens and doses approved for the treatment of metastatic melanoma. Blood samples were collected from patients within 5 days before the start of treatment. Blood samples were collected in EDTA tubes and centrifuged at 1500× g for 10 min, and the separated plasma was centrifuged at 2400× g for 15 min and frozen at −70°C within 1 hour of processing. Baseline clinical data included age at the start of treatment, sex, Eastern Cooperative Oncology Group (ECOG) performance status, baseline tumor stage according to the eighth edition of the American Joint Committee on Cancer (AJCC)

[16] , number of affected organs, baseline lactate dehydrogenase (LDH) level, previous lines of treatment, and ICI regimen received after TKa sampling. The study was conducted in accordance with good clinical practice, with informed consent from all patients and approved by the Stockholm Regional Ethics Committee.

[0143] TK activity level analysis:

[0144] Plasma TKa levels were used according to the manufacturer's instructions. The TKa assay (Biovica, Sweden) was used for determination, which has been previously reported [7]. TKa is an elaborate ELISA-based test that reflects the cell proliferation rate by measuring TKa in serum, plasma or cells. In summary, plasma is mixed with a reaction mixture in a 96-well ELISA plate and bromodeoxyuridine (BrdU) monophosphate is generated by the TK reaction, which is phosphorylated to BrdU triphosphate and incorporated into the synthetic DNA strand. BrdU incorporation is detected using an anti-BrdU monoclonal antibody conjugated to alkaline phosphatase and a chromogenic substrate. Absorbance readings are converted using standards with known TKa values ​​(working range of 20 to 4000 Du / L). The lower limit of detection of the assay is set at 20 Du / L, and all values ​​below the threshold are reported as <20 Du / L. All plasma TKa analyses were performed at the Biovica laboratory (Uppsala, Sweden), where laboratory staff were blinded to patient and tumor data. Samples were measured twice and met Coefficient of variation (CV) criteria for TKa assay (CV < 20%). Optimization for measuring TKa The TKa assay is a CE-IVD marked assay and has been submitted to the FDA in a 510(k) application pending approval. The TKa assay has a new calibration concept to better match 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:

[0145] DuA=134+0.53(Du / L). Determination coefficient (R 2 )=0.93

[0146] Follow-up:

[0147] Routine follow-up after initiation of ICI treatment included monthly clinical assessments and three-month radiological assessments. Patients were followed up for a minimum of 24 months. Patients were grouped according to baseline plasma TKa levels (low or high) and followed up 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 assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria

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

[0148] Statistical methods:

[0149] Receiver operating characteristic (ROC) analysis was performed to investigate the TKa cutoff with the best sensitivity and specificity to predict tumor stage, performance stage, response, and survival. Baseline characteristics and treatment response were compared with chi-square tests for categorical variables and Student's t-tests for continuous variables. P values ​​< 0.05 were 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 (CI) were assessed. Univariate, bivariate, and multivariate models of Cox regression were used to assess the association of each predictor with PFS and OS. Hazard ratios (HRs) and corresponding two-sided 95% CIs were estimated. Statistical analyses were performed using R version 4.1.1. Consistency is a measure of model predictive accuracy, measured as the proportion of all evaluable pairs of subjects in which the model correctly predicted a higher risk for individuals in the pair of subjects with the worst outcome.

[0150] result

[0151] Baseline characteristics:

[0152] 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). TKa levels did not differ significantly with patient age or sex (Table 1). However, plasma TKa was significantly higher in patients with ECOG performance status ≥1 compared with 0-1 (p = 0.003), patients with M1c-M1d compared with M1a-M1b disease (p = 0.015), or patients with elevated LDH compared with patients with non-elevated LDH (p < 0.001). TKa levels were higher in patients who had previously received treatment or had more than three affected organs, but the differences in TKa were not significant here. In patients with M1b-d disease, TKa levels were compared according to whether the patient had metastatic spread to a certain organ. This analysis was performed separately from the analysis of M1a patients in an attempt to determine whether TKa levels were affected by spread to a specific organ rather than assessment of tumor burden (because M1a patients generally have significantly less tumor burden). Overall, no significant differences were observed in which organs were affected.

[0153] Determine the critical value of TKa:

[0154] Since TKa has not been studied before in melanoma, the basic goal was to determine an appropriate cutoff and then compare patients with high or low plasma TKa levels. The median TKa at baseline (42 Du / L) gave a cutoff that divided patients with very similar TKa levels into different groups ( Figure 1 ). In this sense, 60Du / L was considered a more appropriate cutoff because it ensured more differentiation of TKa levels and provided a sufficient number of patients with high or low TKa levels. In the next step, ROC analysis was performed to determine the TKa cutoff with the best sensitivity and specificity in predicting baseline characteristics and outcomes ( Figure 2 ). ROC analysis showed that a TKa cutoff between 49 and 72 achieved the highest sensitivity and specificity in predicting tumor stage, physical stage, ICI response, PFS, and OS at 24 months (the median of these cutoffs was 58). Therefore, ROC analysis further supported the use of 60Du / L as a reasonable cutoff. Characteristics and outcomes of patients with high or low TKa:

[0155] There were no significant differences in age, sex, or tumor BRAF mutation status among melanoma patients with high (≥60Du / 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 affected organs (p=0.031), elevated LDH (p<0.001), and higher median LDH (p<0.001). No significant differences were observed in patients with high or low TKa in terms of whether they had received previous lines of treatment or the ICI regimen selected for the patients. Most patients received first-line treatment with PD-1 inhibitor monotherapy (nivolumab or pembrolizumab). A smaller portion received a single CTLA-4 inhibitor (ipilimumab) or a combination of CTLA-4 and PD-1 inhibitors (ipilimumab and nivolumab). Although not statistically significant, more patients in the TKa low group (n=6) received combination immunotherapy compared to the TKa high group (n=0). A plausible explanation is the fact that the better performance status and younger age of TKa low patients led to their being assessed more frequently and found to be able to tolerate the more toxic combination therapy.

[0156] The RR of patients with low TKa (63.2%) was significantly higher than that of patients with high TKa (30.3%) (p=0.022) (Table 3). The complete response rate in the low TKa group (33.3%) was also higher than that in the high TKa group (6.0%) (p=0.016). The DCR of patients with low TKa (80.7%) was also higher than that in patients with high TKa (54.3%) (p=0.022). No differences were found in the reasons for treatment termination (progressive disease, adequate response, or toxicity) (Table 3).

[0157] The median PFS for patients with low TKa was 19.9 months (95% CI, 11.0 to not reached), while the median PFS for patients with high TKa was 12.6 months (95% CI, 3.6 to 28.3) (p = 0.021) ( Figure 3 ). The median OS was not reached for patients with low TKa (>60 months, 95% CI, 38.0 to not reached), whereas the median OS for patients with high TKa was 18.5 months (95% CI, 11.7 to not reached) (p=0.005).

[0158] Univariate Cox regression analysis showed that patients with baseline ECOG performance status ≥1, M1c or M1d disease, elevated LDH, and high TKa had significantly worse PFS and OS (Table 4). For TKa, the HR for PFS was 1.83 (95% CI, 1.08–3.08), p = 0.024), while the HR for OS was 2.25 (95% CI, 1.25–4.05), p = 0.007. In multivariate analysis, TKa was not statistically significant for PFS or OS. A high degree of multicollinearity among the variables analyzed was identified as a factor that caused the HRs of many variables that were significant in the univariate model to be nonsignificant in the multivariate model.

[0159] To assess how TKa was affected by each of the covariates, bivariate regression analyses were performed in which TKa was analyzed pairwise with another baseline factor ( Figure 4 ). As for PFS, TKa was only independent of the patient's gender.

[0160] Furthermore, in bivariate analysis of OS, TKA was not associated with age, sex, and tumor stage. Figure 5 showed a longer (>24 months, Figure 5 A-5C) or shorter (<24 months, Figure 5 D-5F) Number of patients with PFS; each graph shows whether TKa level correctly predicted longer or shorter PFS together with other baseline variables (ECOG, LDH, or M stage). The graphs show that, despite a large overlap between TKa and other variables (diamond symbols), in a small subset of patients, low or high TKa levels alone were associated with longer or shorter PFS, respectively (square symbols).

[0161] As part of the same study, TKa levels were also measured in 58 patients during immunotherapy treatment (3-4 weeks after the start of treatment, and at the end of treatment). The results are shown in Tables 5 and Figure 6This suggests that patients with the shortest PFS time of less than 6 months had the highest pre-treatment TKa levels and little increase in TKa during treatment. Patients with PFS time between 6-24 months had lower pre-treatment TKa, but their TKa increased less than 2-fold during treatment. Patients whose response to treatment lasted more than 24 months had lower pre-treatment TKa levels and their TKa increased by 2-fold or more during treatment. This suggests a proliferative burst of T cells in patients within the first month of successful ICI treatment.

[0162] discuss

[0163] Among patients with advanced cutaneous melanoma enrolled in this study, significantly higher TKa levels were observed in patients with poorer performance status, more advanced tumor stage, and higher LDH levels at the start of treatment. The median TKa was 42 Du / L (range <20–1787 Du / L), whereas, as a reference, the median TKa value was <20 Du / L in 123 healthy subjects (data not shown). In a group of patients with preoperative pancreatic cancer, the median TKa value was 40 Du / L, whereas in a group of patients with preoperative renal cell carcinoma, the median TKa value was 38 Du / L [12,14]. In addition, in a group of patients with non-small cell lung cancer, the median TKa before the start of systemic therapy was 129 Du / L, whereas in a group 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]. Overall, these data suggest that, as with the other cancer types studied, patients with metastatic melanoma have elevated TKa levels compared with levels in healthy individuals and are higher in patients with more advanced disease.

[0164] Patients with high TKa had significantly worse responses 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 on ECOG, LDH, and tumor stage to varying degrees; however, a considerable number of patients did not have corresponding good or poor baseline variables ( Figure 5). Clinical factors such as performance status, tumor stage, and tumor burden are well-known prognostic factors and can also predict the efficacy of ICIs in melanoma [1–4,16]. As for serum markers, elevated LDH levels are the strongest known prognostic and predictive factor and are the only biomarker routinely used in the monitoring of melanoma patients in the clinic and included as a marker in clinical trials

[16] . Serum LDH levels reflect the hypoxic environment that often exists in melanoma tumors, with reduced oxidative phosphorylation and increased anaerobic glycolysis, in which LDH catalyzes the conversion of pyruvate to lactate when oxygen supply is insufficient or absent

[19] . LDH is not a secreted enzyme; therefore, elevated serum levels are thought to be secondary to LDH overflow when melanoma cells outgrow their blood supply. LDH is also frequently elevated in a variety of conditions that affect the liver, both malignant and nonmalignant

[19] . TK enzymes have important roles in DNA synthesis and repair. These processes are highly active in proliferating cells, and dividing tumor cells release TK during mitotic exit [7,8]. Thus, both LDH and TKa are markers of cell proliferation and tumor burden, but through different cellular processes.

[0165] The data in this article also show that by measuring the TKa levels of cancer patients at 2 key time points (before treatment and 1-4 weeks after the first ICI treatment dose), the patient's positive response to ICI can be predicted. If the TKa level is low before treatment, it indicates that the immune system can effectively control the level of disease burden once activated. If the TKa level during ICI treatment increases to at least 2 times compared to the baseline level, it indicates a successful response to therapy and a sufficient increase in T cell activation and proliferation to achieve tumor killing.

[0166] Several other markers have been reported to predict ICI efficacy, including the composition of peripheral blood leukocytes, circulating tumor DNA (ctDNA) and exosomes, tumor mutational burden (TMB), high gene expression signatures associated with interferon-γ in tumors, diversity of the gut microbiome, and invasive tumor biopsy tests that measure ICI receptor expression (currently used for patient selection) [20-27]. In the clinical setting, widespread implementation of predictive assays (such as TMB, ctDNA, exosomes, tumor RNA expression signatures, or microbiome analysis) is challenging, for example due to the complex and expensive techniques and equipment required, and there are many different assays that can be used. Measuring TKa is a simpler and less expensive test (based on ELISA) than a single plasma marker, and the assay can be easily established in a routine hospital laboratory.

[0167] in conclusion

[0168] In ICI-treated melanoma patients, high pre-treatment plasma TKa levels were significantly associated with poor baseline characteristics and poor response and survival. The inventors identified TKa for the first time as an interesting, previously unexplored biomarker in cancer patients that can be used to indicate response to subsequent ICI treatment.

[0169] surface

[0170] Table 1: Pre-treatment thymidine kinase 1 (TK) activity (Du / L) in plasma of patients with unresectable melanoma.

[0171]

[0172] Table 2: Characteristics of melanoma patients with low (<60 Du / L) or high thymidine kinase 1 (TK) activity (Du / L) in plasma before starting immune checkpoint inhibitor treatment.

[0173]

[0174] * Patients started immune checkpoint inhibitor regimen after baseline TKa test

[0175] Table 3: Response assessment in melanoma patients with low (<60 Du / L) or high thymidine kinase 1 (TK) activity (Du / L) in plasma before starting immune checkpoint inhibitor treatment.

[0176]

[0177] Table 4: Cox regression of survival of patients with metastatic melanoma treated with immune checkpoint inhibitors.

[0178]

[0179] Table 5. TK activity before treatment (pre-trm), during treatment (on-trm) and at the end of treatment (eot) with immunotherapy.

[0180] TKa PRE-TRM TKa ON-TRM TKM PFS < 6 months 54 58 148 PFS>6 months 36 44 147 PFS>24 months 34 99 44

[0181] Example 2

[0182] A phase 1b study 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).

[0183] The study enrolled 24 patients. Ribociclib was taken orally once daily on days 1-21 of a 28-day cycle. Spartalizumab was given 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 each 28-day treatment cycle. Plasma was separated from the blood samples and then analyzed for thymidine kinase activity using the DiviTum-TKa assay as described previously. Data are presented in Figure 7 and Table 6 of the accompanying data sheet.

[0184] As seen in the melanoma PD-1 inhibitor study using DiviTim-TKa, patients with the best clinical response had lower baseline TKa values ​​and a 2-fold or greater increase in TKa on treatment.

[0185] Patient #19 is a breast cancer patient with a baseline TKa level of 590DuA. After the first treatment cycle, on day 28, the patient's TKa level increased 2-fold to 1173DuA. The patient had a partial response to therapy, with a 68% reduction in tumor volume, and the patient continued to receive therapy for 334 days. This is the second longest duration of therapy. Patient #27 is also a breast cancer patient. The patient's baseline TKa level was 174DuA (the lowest TKa baseline level in the study). After the first treatment cycle, on day 28, the patient's TKa level increased 3.8-fold to 411DuA (the highest multiple increase in the study). The patient had a partial response to therapy, with a 54% reduction in tumor volume, and the patient continued to receive therapy for 411 days. This is the longest duration of therapy in this study. The other 22 patients in the study (except #19 and #27 as described above) did not meet the criteria of low baseline TKa levels and a two-fold increase in TKa during treatment. And none of them subsequently achieved any meaningful clinical benefit. The only exception was patient #3. This patient had a low BL level of TKa (296DuA) and a 2.6-fold increase in TKa to 758DuA during treatment, but this patient only continued treatment for 95 days. Upon further investigation, it was found that the patient developed clinical symptoms, which was the reason for the treatment interruption. It is entirely reasonable that this patient would have benefited if he had been able to continue on treatment. Patient #24 also achieved a greater than 2-fold increase in TKa during treatment (2.21), but this patient's baseline TKa was very high (1316DuA), so this patient did not meet the criteria for predicting benefit from immune checkpoint inhibitors based on a pre-treatment TKa cutoff of 550DuA + / -20%, and did experience rapid disease progression within 35 days.

[0186] This data complements and builds on previous data of TKa exemplified in melanoma patients treated with immune checkpoint inhibitors (ICIs). This data confirms the applicability of the method of the present invention to other cancers, such as exemplary metastatic HR+ breast cancer and ovarian cancer. It also confirms the applicability of the method when using a different ICI (spartalizumab).

[0187] Table 6. Figure 7 Data table.

[0188]

[0189]

[0190] References

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Claims

1. A method for determining the prognosis of an individual who has cancer and is being considered for treatment with or is currently being treated with one or more immune checkpoint inhibitors, the method comprising or consisting of the following steps: a) providing a sample obtained 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 obtained from the individual 1-4 weeks after the first treatment with the one or more immune checkpoint inhibitors; as well as 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 prognosis of the individual if further treated with one or more immune checkpoint inhibitors.

2. The method of claim 1, wherein the method is used to stratify one or more individuals with cancer for treatment or further treatment with one or more immune checkpoint inhibitors.

3. A method for stratifying patients with cancer for treatment or further treatment with one or more immune checkpoint inhibitors, the method comprising or consisting of the following steps: i) carrying out a method according to any preceding claim; as well as ii) based on the results of step (i), stratifying the individual for treatment or further treatment with one or more immune checkpoint inhibitors or alternative cancer therapies.

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

5. The method according to any preceding claim, wherein when 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), the prognosis of the individual if further treated with one or more checkpoint inhibitors is positive (e.g., the individual will have a progression-free survival of at least 6 months), and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

6. The method of claim 5, wherein when the activity and / or concentration of thymidine kinase (TK) measured in step (d) increases by at least 100% compared to the activity and / or concentration of thymidine kinase (TK) measured in step (b), the prognosis of the individual if further treated with one or more checkpoint inhibitors is positive (e.g., the individual will have a progression-free survival of more than 24 months), and / or the patient is stratified for further treatment with one or more immune checkpoint inhibitors.

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

8. A method according to any preceding claim, wherein when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than or equal to a cut-off value, the prognosis of the individual if treated or further treated with one or more immune checkpoint inhibitors is negative (e.g., the individual will have a progression-free survival of less than 6 months), and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors.

9. The method according to any preceding claim, wherein when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is below a cut-off value, 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), the prognosis of the individual if treated or further treated with one or more immune checkpoint inhibitors is positive (e.g., the individual will have a progression-free survival of at least 6 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

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

11. The method according to any preceding claim, wherein when the activity and / or concentration of thymidine kinase (TK) measured in step (b) is greater than or equal to a cut-off value, and 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), the prognosis of the individual if treated or further treated with one or more immune checkpoint inhibitors is negative (e.g., the individual will have a progression-free survival of less than 6 months), and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors.

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 the cut-off value is 550 DuA + / - 20%.

13. A method according to any preceding claim, wherein the method further comprises the steps of: (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); Wherein the prognosis of the individual if treated with one or more immune checkpoint inhibitors is determined by comparing the measurement results in step (f) with the measurement results in step (b) and / or step (d).

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. The method according to claim 13 or 14, wherein when the activity and / or concentration of thymidine kinase (TK) measured in step (b) corresponds to or is lower than the activity (TKa) of thymidine kinase measured in step (f), the prognosis of the individual if treated with one or more immune checkpoint inhibitors is positive, and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

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. The method according to claim 13 or 16, wherein when 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), the prognosis of the individual if treated with one or more immune checkpoint inhibitors is negative, and / or the patient is stratified for no further treatment or for treatment with an alternative cancer therapy to one or more immune checkpoint inhibitors.

18. The method according to any preceding claim, further comprising the steps of: g) measuring the activity and / or concentration of lactate dehydrogenase (LDH) in the sample provided in step (a); wherein said activity and / or concentration of LDH measured in step (g) is further indicative of said prognosis of said individual if subsequently treated with one or more immune checkpoint inhibitors.

19. A method according to claim 18, wherein when the activity and / or concentration of LDH measured in step (g) is below a cut-off value, the prognosis of the individual if treated or further treated with one or more immune checkpoint inhibitors is positive (e.g., the individual will have a progression-free survival of more than 24 months), and / or the patient is stratified for treatment or further treatment with one or more immune checkpoint inhibitors.

20. The method according to claim 19, wherein the activity of LDH is measured and the cut-off value is selected between 2 and 6 mikrokat / L, such as between 3 and 5 mikrokat / L, or such as 4 mikrokat / L.

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

22. A method of treating cancer in an individual, the method comprising the steps of: a) using the method of any one of claims 1 to 21 to stratify individuals for treatment with one or more immune checkpoint inhibitors or alternative cancer therapies; as well as b) providing cancer therapy to the individual comprising administering one or more immune checkpoint inhibitors or alternative cancer therapy.

23. A method of treating cancer in an individual, the method comprising the steps of: 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 the individual with a cancer therapy comprising administering one or more immune checkpoint inhibitors.

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

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

26. An immune checkpoint inhibitor for use according to any preceding claim, wherein thymidine kinase activity (TKa) is measured.

27. An immune checkpoint inhibitor for use according to any preceding claim, wherein the cancer is a cancer approved for treatment with an immune checkpoint inhibitor.

28. An immune checkpoint inhibitor for use according to any preceding claim, wherein the cancer is selected from the group consisting of: melanoma; breast cancer; and / or ovarian cancer.

29. An immune checkpoint inhibitor for use according to any preceding claim, wherein the one or more immune checkpoint inhibitors are selected from the list consisting of: CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors.

30. An immune checkpoint inhibitor for use according to any preceding claim, wherein the individual or patient is a human.

31. The method according to any preceding claim, wherein the method is performed in vitro.

32. An immune checkpoint inhibitor for use according to any preceding claim, wherein the sample is a plasma or serum sample.

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

34. Use of thymidine kinase for stratifying individuals with cancer for treatment or further treatment with immune checkpoint inhibitors.

35. A method or use substantially as described herein.

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