Methods of treating cancer

Abstract

The present invention provides a method for predicting whether a cancer patient has a good therapeutic response to arginine deprivation therapy. The method comprises the following steps: determining the concentration of arginine in plasma of a subject; and according to the determined arginine concentration, carrying out arginine deprivation therapy on a subject by using an anti-cancer drug alone or in combination. According to some disclosed embodiments, the anti-cancer drug is selected from the following combination: FOLFOX, paclitaxel (docetaxel), cisplatin (cisplatin), pemetrexed (pemetrexed), pembrolizumab (pembrolizumab) or a combination of the FOLFOX, the paclitaxel (docetaxel), the cisplatin (cisplatin), the pemetrexed (pemetrexed), the pembrolizumab and the pemetrexed (pemetrexed) and the pembrolizumab. According to the method, a proper cancer treatment scheme is formulated by measuring the plasma arginine concentration of a subject, so that the treatment effect is improved, and the unnecessary treatment burden is reduced.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to the field of cancer treatment, and more particularly, the present invention relates to a method of treating cancer by arginine deprivation therapy. BACKGROUND

[0002] Cancer is a group of diseases characterized by the uncontrolled division of abnormal cells and the ability of these cells to invade and destroy normal tissues. Cancer is the second leading cause of death worldwide, causing an estimated 10 million deaths per year, with the most common causes of cancer deaths including lung cancer, colorectal cancer, liver cancer, stomach cancer, and breast cancer. The treatment methods for cancer vary depending on the type and stage of cancer, and the main treatment methods include surgery, chemotherapy, radiotherapy, immunotherapy, hormone therapy, and targeted therapy. However, these treatment methods have not been able to produce satisfactory results, and various adverse reactions have been observed in cancer patients. Therefore, there is a continuing need to find and develop alternative methods for treating cancer.

[0003] Certain cancers have a nutritional deficiency for a specific amino acid, such as arginine, and arginine deprivation can provide a potential therapeutic approach. Arginine can be degraded by various enzymes, including arginine deiminase (ADI), which is a microbial enzyme from Mycoplasma arginini, having a high affinity for arginine and converting arginine to citrulline and ammonia. Argininosuccinate synthetase 1 (ASS1) is expressed in normal cells, which can convert citrulline back to arginine. An ADI in a pegylated form (ADI-PEG 20) has been formulated and shown in clinical trials to target tumors with arginine nutritional deficiency by arginine deprivation therapy. However, resistance to ADI often develops through reactivation or upregulation of ASS1. In addition, it has been reported that long-term use of ADI therapy can lead to activation of different cellular pathways related to anti-apoptosis, which can limit the overall treatment of ADI.

[0004] Therefore, there is a need in the related art to optimize the treatment strategy of arginine deprivation therapy to improve the treatment effect of cancer patients. SUMMARY

[0005] The following presents a simplified summary of the invention in order to provide a basic understanding of the disclosure. This summary is not an extensive overview of the invention, and is not intended to identify key / critical elements or to delineate the scope of the invention. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0006] The present application is based, at least in part, on the discovery that plasma arginine levels in cancer patients are associated with their response to arginine deprivation therapy with polyethylene glycolated arginine deiminase (ADI-PEG 20). Accordingly, plasma arginine levels can be used to predict the therapeutic response of cancer patients to arginine deprivation therapy, and to guide the individualized development of appropriate treatment regimens.

[0007] Accordingly, the present application provides a method of predicting whether a cancer patient (e.g., a hepatocellular carcinoma patient) has a good response to arginine deprivation therapy. The method comprises: (a) determining the plasma arginine level of the subject; and (b) subjecting the subject to arginine deprivation therapy, alone or in combination with an anticancer drug, according to the plasma arginine level determined in step (a).

[0008] According to embodiments of the present application, the arginine deprivation therapy comprises an arginine deprivation agent selected from the group consisting of difluoromethylornithine (DFMO), recombinant arginine deiminase (rADI), recombinant arginine (rArg), recombinant arginine decarboxylase (rADC), polyethylene glycolated rADI (hereinafter "PEG-rADI"), polyethylene glycolated rArg, polyethylene glycolated rADC, and combinations thereof. According to some embodiments of the present application, the anticancer drug is selected from the group consisting of FOLFOX, docetaxel, cisplatin, pemetrexed, pembrolizumab, and combinations thereof.

[0009] According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 34 micromole per liter (>= 34 μmol / L), the subject is administered PEG-rADI and FOLFOX separately and independently. In some exemplary embodiments, the subject is administered about 36 mg / m2body surface area of PEG-rADI weekly, and FOLFOX is administered biweekly. 2 According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 34 micromole per liter (>= 34 μmol / L), the subject is administered PEG-rADI and FOLFOX separately and independently. In some exemplary embodiments, the subject is administered about 36 mg / m2body surface area of PEG-rADI weekly, and FOLFOX is administered biweekly.

[0010] According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 60.2 micromole per liter (>= 60.2 μmol / L), the subject is administered PEG-rADI and pembrolizumab separately and independently. In some exemplary embodiments, the subject is administered about 36 mg / m2body surface area of PEG-rADI weekly, and about 200 mg of pembrolizumab every three weeks. 2 According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 60.2 micromole per liter (>= 60.2 μmol / L), the subject is administered PEG-rADI and pembrolizumab separately and independently. In some exemplary embodiments, the subject is administered about 36 mg / m2body surface area of PEG-rADI weekly, and about 200 mg of pembrolizumab every three weeks.

[0011] According to certain embodiments, when the plasma arginine level in step (a) is greater than or equal to 68.2 micromole per liter (> = 68.2 μmol / L), the subject is administered pegylated rADI, pemetrexed, and cisplatin, each independently. In certain exemplary embodiments, the subject is administered about 36 mg / m 2 of body surface area of pegylated rADI every week, about 500 mg / m 2 of body surface area of pemetrexed every three weeks, and about 75 mg / m 2 of body surface area of cisplatin.

[0012] According to certain embodiments, when the plasma arginine level in step (a) is greater than or equal to 84.2 micromole per liter (> = 84.2 μmol / L), the subject is administered pegylated rADI, alone. In some preferred embodiments, the subject is administered about 18 mg / m 2 of body surface area of pegylated rADI every week.

[0013] According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 97.5 micromole per liter (> = 97.5 μmol / L), the subject is administered pegylated rADI and docetaxel, each independently. In certain exemplary embodiments, the subject is administered about 36 mg / m 2 of body surface area of pegylated rADI every week, and about 75 mg / m 2 of body surface area of docetaxel every three weeks.

[0014] According to some embodiments, when the plasma arginine level in step (a) is greater than or equal to 122 micromole per liter (> = 122 μmol / L), the subject is administered pegylated rADI and cisplatin, each independently. In certain exemplary embodiments, the subject is administered about 36 mg / m 2 of body surface area of pegylated rADI every week, and about 30 mg / m 2 of body surface area of cisplatin for three consecutive weeks followed by one week of rest.

[0015] Cancers treatable according to the present application include, but are not limited to, breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), leukemia (e.g., acute myeloid leukemia (AML)), lymphoma, lung cancer, melanoma, mesothelioma (e.g., malignant pleural mesothelioma (MPM)), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma.

[0016] A subject suitable for the methods of the present application is a mammal, preferably a human.

[0017] The accompanying features and advantages of the disclosure will be better understood and appreciated with reference to the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] The description will be better understood from a detailed description with reference to the following drawings, in which:

[0019] Figure 1 A plot showing cancer patients treated with ADI-PEG 20, analyzed for cut-off of arginine using Maximally Selected log-rank statistics.

[0020] Figure 2 A line plot depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients treated with ADI-PEG 20 alone, according to Example 1 of the disclosure.

[0021] Figure 3 A plot showing cancer patients treated with ADI-PEG 20 + docetaxel, analyzed for cut-off of arginine using Maximally log-rank statistics.

[0022] Figure 4 A line plot depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients treated with ADI-PEG 20 + docetaxel, according to Example 1 of the disclosure.

[0023] Figure 5 A plot showing cancer patients treated with ADI-PEG 20 + cisplatin, analyzed for cut-off of arginine using Maximally log-rank statistics.

[0024] Figure 6 A line plot depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients treated with ADI-PEG 20 + cisplatin, according to Example 1 of the disclosure.

[0025] Figure 7 A plot showing cancer patients treated with ADI-PEG 20 + FOLFOX, analyzed for cut-off of arginine using Maximally log-rank statistics,

[0026] Figure 8Figure 1 is a line graph depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients receiving ADI-PEG 20 + mFOLFOX6 combination therapy according to Example 1 of the disclosure.

[0027] Figure 9 Figure 2 shows a plot of cancer patients treated with ADI-PEG 20 + pemetrexed and cisplatin, with cut-off values for arginine analyzed using maximally log-rank statistics.

[0028] Figure 10 Figure 3 is a line graph depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients receiving ADI-PEG 20 + pemetrexed and cisplatin combination therapy according to Example 1 of the disclosure.

[0029] Figure 11 Figure 4 shows a plot of cancer patients treated with ADI-PEG 20 + pembrolizumab, with cut-off values for arginine analyzed using maximally log-rank statistics.

[0030] Figure 12 Figure 5 is a line graph depicting the correlation between plasma arginine levels and overall survival (OS) in cancer patients receiving ADI-PEG 20 + pembrolizumab combination therapy according to Example 1 of the disclosure. DETAILED DESCRIPTION

[0031] The detailed description set forth below by way of illustration is not intended to represent the only constructions or applications in which the embodiments can be practiced. The description sets forth functions, constructions and sequence of steps that can be practiced in the embodiments. However, different embodiments can practice the same or equivalent functions and steps in different ways.

[0032] DEFINITIONS

[0033] In the present specification, the term "hepatocellular carcinoma" (or simply "HCC") refers to a malignant tumor that originates from hepatocytes. HCC is a type of liver cancer. Due to the lack of fibrous stroma, HCC can present with hemorrhage and necrosis. According to the Barcelona Clinic Liver Cancer classification (BCLC) system, which was updated in 2022, HCC can be classified into five stages, including: (1) Stage 0 (very early stage), defined as a single nodule less than or equal to 2 centimeters with no vascular invasion or extrahepatic spread; (2) Stage A (early stage), defined as a single nodule (regardless of size) or multifocal HCC (up to 3 nodules) with no vascular invasion or extrahepatic spread; (3) Stage B (intermediate stage), defined as multifocal HCC with no vascular invasion or extrahepatic spread; (4) Stage C (advanced stage), defined as a patient with portal vein invasion and / or extrahepatic spread; and (5) Stage D (end-stage or terminal stage), defined as a patient with major cancer-related symptoms and / or impaired liver function. According to the classification of the BCLC system, the term "advanced hepatocellular carcinoma" or "advanced HCC" refers to locally advanced HCC or metastatic HCC (i.e., HCC has spread from the liver to other parts of the body). Generally, advanced HCC is unresectable (i.e., has spread to surrounding tissues and cannot be surgically removed) and is not amenable to cure by local treatment modalities such as radiation therapy.

[0034] In the present specification, the term "arginine deprivator" refers to a compound or agent used in arginine deprivation therapy, which depletes arginine supply to cancer with impaired urea cycle, thereby preventing the growth of cancer and inducing cell death.

[0035] The term "FOLFOX" refers to a chemotherapy regimen consisting of 5-fluorouracil (5-FU), leucovorin (calcium folinate), and oxaliplatin. The term "FOLFOX" used in the present specification is not limited to any specific dosage or dosing regimen of these components. Rather, "FOLFOX" used in the present specification encompasses all combinations of these components at any dosage and dosing regimen. There are several different FOLFOX regimens known in the art depending on the dosage and administration of the three drugs, including FOLFOX-4, FOLFOX-6, modified FOLFOX-6 (mFOLFOX-6), and FOLFOX-7. According to some embodiments of the present disclosure, FOLFOX is mFOLFOX-6.

[0036] In the present specification, the term "survival" refers to the act or fact of surviving. "Overall survival" (OS) refers to the prolongation of life expectancy compared to a natural or untreated individual or patient.

[0037] The term "confidence interval" (CI) as used in the present specification has its ordinary meaning known to one of ordinary skill in the art, and refers to a statistical range within which a given parameter falls with a specified probability.

[0038] In the present specification, the terms "administered" and "administering" are used interchangeably and refer to a mode of delivery, including but not limited to intravenous, intra-tumoral, intramuscular, intraperitoneal, intra-arterial, or subcutaneous administration of a treatment (e.g., arginine deprivation therapy or an anti-cancer agent).

[0039] Unless otherwise indicated, the terms "treat," "treating," and "treatment" refer to an action that occurs while a patient is suffering from a particular disease or disorder, that reduces the severity of the disease or disorder, or one or more symptoms thereof, or retards or slows the progression of the disease or disorder.

[0040] In the present specification, the terms "cancer" and "tumor" are used interchangeably and preferably refer to a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. In this context, cancer includes metastatic cancer and / or drug-resistant cancer. Examples of cancer include, but are not limited to, breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), leukemia, acute myeloid leukemia (AML), lymphoma, lung cancer, melanoma, mesothelioma, malignant pleural mesothelioma (MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, and combinations thereof.

[0041] Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably in the present specification and refer to an animal, including a human, that can be treated by the methods of the present application. The term "patient" or "subject" is meant to refer to both males and females unless one gender is specifically indicated.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, the term "about" as used herein when used in conjunction with a given value or range is intended to mean that the amount is within 10%, 5%, 1%, or 0.5% of the given value or range. Alternatively, the term "about" is intended to mean that the amount is within an acceptable standard error of the measurement when considered in light of the experimental design and the data generated. All numerical ranges disclosed herein are understood to include all numerical values and sub-ranges within the range. All references to ranges of values are intended to include the values themselves. For example, a disclosure of a range of 1% to 50% is intended to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 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%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, and any and all sub-ranges thereof (e.g., 1 to 20%, 10 to 50%, 20 to 30%, etc.). All references to ranges of values, including ranges of material amounts, time durations, temperatures, operating conditions, ratios of amounts, etc., are understood to include the values themselves. Unless otherwise indicated, all measurements are understood to be made at standard conditions of 25°C and 1 atmosphere pressure.

[0043] The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0044] II. SUMMARY

[0045] (1) Predicting a cancer patient's response to arginine deprivation therapy

[0046] The diversity of cancer treatment response has long been recognized, primarily due to the inherent heterogeneity of cancer biology, changes in physiological function, and differences in patient genetic characteristics. It is therefore an object of the present application to provide a molecular marker that is associated with a cancer patient's response to arginine deprivation therapy. According to embodiments of the present application, arginine levels in plasma are associated with a patient's tumor (e.g., HCC) treatment response to a therapy based on a pegylated ADI (ADI-PEG 20, an arginine deiminase enzyme conjugated to a polyethylene glycol having a molecular weight of 20,000).

[0047] Accordingly, a first aspect of the present application provides a method of predicting whether a cancer (e.g., HCC) patient will respond well to arginine deprivation therapy. The method comprises:

[0048] (a) determining the subject's plasma arginine level; and

[0049] (b) making the prediction based on the plasma arginine level determined in step (a), wherein a plasma arginine level equal to or greater than 84.2 μmol / L indicates that the subject will respond well to arginine deprivation therapy.

[0050] In step (a), plasma arginine levels are determined. Assay methods suitable for determining plasma arginine levels include, but are not limited to, spectrophotometry, capillary electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), mass spectrometry (MS), Sakaguchi test, biosensors, and combinations thereof.

[0051] Then, in step (b), a skilled technician or clinician can make a prediction based on plasma arginine levels. According to some embodiments of the present invention, the plasma arginine level is equal to or greater than 84.2 μmol / L (>=84.2 μmol / L; for example, 84.2, 84.3, 84.4, 84.5, 84.6, 84.7, 84.8, 84.9, 85, 85.1, 85.2, 85.3, 85.4, 85.5, 85.6, 85.7, 85.8, 85.9, 86, 86.1, 86.2, 86.3, 86.4, 86.5, 86.6, 86.7, 86.8, 86.9, 87, 87.1, 87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8). 87.9,88, 88.1, 88.2, 88.3, ​​88.4, 88.5, 88.6, 88.7, 88.8, 88.9, 89, 89.1, 89.2,89.3, 89.4, 89.5, 89.6, 89.7, 89.8, 89.9, 90, 90.1, 90.2, 90.3, 90.4, 90.5,90.6, 90.7, 90.8, 90.9, 91, 91.1, 91.2, 91.3, 91.4, 91.5, 91.6, 91.7, 91.8,91.9, 92, 92.1, 92.2, 92.3, 92.4, 92.5, 92.6, 92.7, 92.8, 92.9, 93, 93.1, 93.2, 93.3, 93.4, 93.5, 93.6, 93.7, 93.8, 93.9, 94, 94.1, 94.2, 94.3, 94.4, 94.5, 94.6, 94.7, 94.8, 94.9, 95, 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100 pmol / L or higher) indicates that the subject has a good response to the arginine deprivation therapy, i.e. a positive response to the arginine deprivation therapy. According to certain practical examples, a good response is associated with overall survival, with subjects having a plasma arginine level >= 84.2 pmol / L having a longer overall survival after treatment compared to subjects having a plasma arginine level < 84.2 pmol / L.

[0052] According to some embodiments of the application, the arginine deprivation therapy comprises administering to the subject (e.g. a HCC patient) an agent selected from the group consisting of DFMO, rADI, rArg, rADC, pegylated rADI, pegylated rArg, pegylated rADC, and combinations thereof. In some exemplary embodiments, about 18 mg / m 2 pegylated rADI (ADI-PEG 20) per square meter of body surface area, wherein subjects having a plasma arginine level >= 84.2 pmol / L have a median overall survival of about 8.6 months (95% confidence interval (CI) ranging from 7.3 months to 10.5 months) after treatment, and subjects having a plasma arginine level < 84.2 pmol / L have a median overall survival of about 5.7 months (95% CI ranging from 4.9 months to 7.2 months) after treatment.

[0053] Examples of cancer include, but are not limited to, breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, HCC, leukemia, AML, lymphoma, lung cancer, melanoma, mesothelioma, MPM, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, and combinations thereof.

[0054] According to some embodiments of the application, the cancer is HCC. According to a specific example, the cancer is advanced HCC.

[0055] (2) Predicting a cancer patient's response to a combination therapy

[0056] According to some embodiments of the application, the arginine deprivation therapy is combined with one or more additional therapies to improve its therapeutic effect. Accordingly, a second aspect of the application relates to a method of predicting whether a subject having a cancer (e.g. HCC) has a beneficial response to a combination therapy (i.e. arginine deprivation therapy plus additional therapy). The method comprises the steps of: (a) determining the subject's plasma arginine level; and (b) making a prediction based on the plasma arginine level determined in step (a).

[0057] According to some embodiments of the application, the arginine deprivator is combined with FOLFOX therapy. In these embodiments, plasma arginine levels equal to or higher than 34 μιηοΙ / L (>= 34 μιηοΙ / L; e.g., 34, 35, 36, 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, or 100 μιηοΙ / L or more) indicate that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivator plus FOLFOX therapy. According to certain working examples, this beneficial response is associated with overall survival, with subjects having plasma arginine levels >= 34 μιηοΙ / L having a longer overall survival after treatment than subjects having plasma arginine levels < 34 μιηοΙ / L.

[0058] In some exemplary embodiments, the arginine deprivator is administered at about 36 mg / m2of body surface area of pegylated rADI (ADI-PEG 20) and the FOLFOX therapy is administered once every two weeks, wherein the median overall survival of subjects having plasma arginine levels >= 34 μιηοΙ / L is about 9.5 months (95% confidence interval (CI) ranging from 7.5 months to 15.1 months) and the median overall survival of subjects having plasma arginine levels < 34 μιηοΙ / L is about 4.3 months (95% CI ranging from 4.0 months to 4.6 months) after treatment. 2 In some exemplary embodiments, the arginine deprivator is administered at about 36 mg / m2of body surface area of pegylated rADI (ADI-PEG 20) and the FOLFOX therapy is administered once every two weeks, wherein the median overall survival of subjects having plasma arginine levels >= 34 μιηοΙ / L is about 9.5 months (95% confidence interval (CI) ranging from 7.5 months to 15.1 months) and the median overall survival of subjects having plasma arginine levels < 34 μιηοΙ / L is about 4.3 months (95% CI ranging from 4.0 months to 4.6 months) after treatment.

[0059] According to some embodiments of the invention, an arginine deprivation agent is used in combination with pembrolizumab. In these embodiments, a plasma arginine level equal to or greater than 60.2 μmol / L (>= 60.2 μmol / L; for example, 60.2, 60.3, 60.4, 60.5, 60.6, 60.7, 60.8, 60.9, 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, or 100 μmol / L or higher) indicates a good response to the combination therapy, i.e., a positive response to arginine deprivation therapy plus pembrolizumab. According to some examples, this beneficial response is associated with overall survival, with subjects with plasma arginine levels >= 60.2 μmol / L having longer overall survival after treatment compared to subjects with plasma arginine levels < 60.2 μmol / L.

[0060] In some exemplary embodiments, approximately 36 mg / m² is administered weekly. 2 The body surface area is PEGylated rADI (ADI-PEG 20), and approximately 200 mg of pembrolizumab is administered every three weeks. According to certain embodiments of the invention, the arginine depriver is used in combination with pemetrexed and cisplatin. In these embodiments, a plasma arginine level equal to or greater than 68.2 μmol / L (>= 68.2 μmol / L; for example, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 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, or 100 μmol / L or higher) indicates a good response to combination therapy, i.e., a positive response to arginine deprivation agent plus pemetrexed and cisplatin. According to some examples, this beneficial response is associated with overall survival, with subjects with plasma arginine levels >= 68.2 μmol / L having longer overall survival after treatment compared to subjects with plasma arginine levels < 68.2 μmol / L.

[0061] In some exemplary embodiments, approximately 36 mg / m² is administered weekly. 2 PEGylated rADI (ADI-PEG 20) per body surface area, approximately 500 mg / m² every three weeks. 2 Pemetrexed per body surface area, and approximately 75 mg / m² every three weeks. 2Platinum. According to these embodiments, a plasma arginine level equal to or higher than 68.2 μιηοΙ / L (>= 68.2 μιηοΙ / L; e.g., 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 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, or 100 μιηοΙ / L or higher) indicates that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivation therapy plus pemetrexed and cisplatin treatment. According to certain examples, this beneficial response is associated with overall survival, with subjects having a plasma arginine level >= 68.2 μιηοΙ / L having a longer overall survival after treatment than subjects having a plasma arginine level < 68.2 μιηοΙ / L.

[0062] According to certain embodiments of the application, the arginine deprivation agent is used in combination with docetaxel. In these embodiments, a plasma arginine level equal to or higher than 97.5 μιηοΙ / L (>= 97.5 μιηοΙ / L; e.g., 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 μιηοΙ / L or higher) indicates that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivation therapy plus docetaxel treatment. According to certain working examples, this beneficial response is associated with overall survival, with subjects having a plasma arginine level >= 97.5 μιηοΙ / L having a longer overall survival after treatment than subjects having a plasma arginine level < 97.5 μιηοΙ / L.

[0063] In certain exemplary embodiments, about 36 mg / m^2 body surface area of pegylated rADI (ADI-PEG 20) is administered weekly, about 75 mg / m^2 body surface area of docetaxel is administered every three weeks, and about 500 mg / m^2 body surface area of pemetrexed is administered every three weeks. 2European paclitaxel (docetaxel). According to these embodiments, a plasma arginine level equal to or higher than 97.5 pmol / L (> = 97.5 pmol / L; e.g., 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 pmol / L or more) indicates that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivation therapy plus European paclitaxel treatment. According to certain working examples, this beneficial response is associated with overall survival, with subjects having a plasma arginine level > = 97.5 pmol / L having a longer overall survival after treatment than subjects having a plasma arginine level < 97.5 pmol / L.

[0064] According to certain embodiments of the application, the arginine deprivation agent is used in combination with cisplatin. In these embodiments, a plasma arginine level equal to or higher than 122 pmol / L (> = 122 pmol / L; e.g., 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 pmol / L or more) indicates that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivation therapy plus cisplatin treatment. According to certain examples, this beneficial response is associated with overall survival, with subjects having a plasma arginine level > = 122 pmol / L having a longer overall survival after treatment than subjects having a plasma arginine level < 122 pmol / L.

[0065] In certain exemplary embodiments, about 36 mg / m 2 PEGylated rADI (ADI-PEG 20) at about 30 mg / m 2cisplatin, with treatment cycles of three weeks (Week 1 to Week 3) followed by one week of rest (Week 4). According to these working examples, a plasma arginine level equal to or higher than 122 pmol / L (> 122 pmol / L; e.g., 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 pmol / L or higher) indicates that the subject has a good response to the combination therapy, i.e., a positive response to the arginine deprivation therapy plus cisplatin treatment. According to certain working examples, this beneficial response is associated with overall survival, with subjects having a plasma arginine level >= 122 pmol / L having a longer overall survival after treatment compared to subjects having a plasma arginine level < 122 pmol / L.

[0066] As mentioned above, the cancer can be breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), leukemia, acute myeloid leukemia (AML), lymphoma, lung cancer, melanoma, mesothelioma, mesothelioma peritoneal (MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, or a combination thereof. According to some working examples, the cancer can be hepatocellular carcinoma (HCC). In one specific example, the cancer is advanced HCC.

[0067] (3) Methods of treating cancer

[0068] Another aspect of the present disclosure relates to treating cancer by arginine deprivation therapy using an arginine depleting agent alone (i.e., administering DFMO, rADI, rArg, rADC, PEGylated rADI, PEGylated rArg, PEGylated rADC, or a combination thereof), or in combination with one or more additional therapies. According to certain embodiments of the present disclosure, the method comprises the steps of: (a) determining the plasma arginine level of the patient; and (b) administering to the patient an appropriate therapy according to the plasma arginine level determined in step (a).

[0069] According to certain embodiments of the present disclosure, in the case where the plasma arginine level is equal to or greater than 34 μιηοΙ / L, then PEGylated rADI (ADI-PEG 20) and FOLFOX therapy are independently administered to the patient. In some example embodiments, PEGylated rADI (ADI-PEG 20) is administered at a dose of about 36 mg / m2body surface area per week, and FOLFOX therapy is administered every two weeks to ameliorate or alleviate the symptoms associated with the cancer. The treatment regimen, including the dosage, schedule, and duration of ADI-PEG 20 and FOLFOX therapy, can be adjusted according to actual needs, as understood by the skilled artisan or clinician.

[0070] Alternatively, in the case where the plasma arginine level is less than 34 μιηοΙ / L, then an alternative anti-cancer therapy is administered to the patient to achieve the therapeutic purpose, wherein the alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, or a combination thereof. The skilled artisan or clinician can select the appropriate treatment modality according to the clinical factors such as the patient's age, gender, and physical condition, as well as the type and stage of the cancer.

[0071] Here is a list of common chemotherapy drugs, including but not limited to: Adriamycin (doxorubicin), Adrucil, Bleomycin, Actinomycin, Dactinomycin, Idarubicin, Mitoxantrone, Mitomycin, Etoposide, Fluorouracil, Flurouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, Fluorouracil, FluTeniposide), Antimicrotubule Agents, Vinblastine, Vincristine, Vindesine, Vinorelbine, Taxanes, Paclitaxel / Taxol, Nitrogen Mustards, Chlorambucil, Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Melphalan, Aziridines, Thiotepa, Alkyl Sulfonates, Busulfan, Nitrosoureas, Carmustine, Lomustine, Platinum Complexes, Carboplatin, Alkylators, Altretamine, Dacarbazine, Procarbazine, Temozolomide, Methotrexate, Fludarabine, Mercaptopurine, Thioguanine, Cladribine, Pentostatin, Capecitabine, Cytarabine, Floxuridine, Fluorouracil, Gemcitabine, Hydroxyurea, Camptothecin, Irinotecan, Busulfan, Epothilones, Azathioprine, Halofuginone, Sirolimus, Everolimus, Mitomycin, Topotecan.

[0072] Exemplary agents for targeted therapy include, but are not limited to, trastuzumab or pertuzumab (antibodies against the tumor antigen HER-2 / neu); bevacizumab (an antibody against vascular endothelial growth factor (VEGF)); Ramucirumab (an antibody against VEGF receptor); Nivolumab or cemiplimab (antibodies against programmed death protein 1 (PD-1)); atezolizumab, avelumab, or durvalumab (antibodies against the ligand of programmed death protein 1 (PD-L1)), Ipilimumab (an antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)), Rituximab (an antibody against CD20 on B cells), and the like.

[0073] Non-limiting examples of immunomodulators for immunotherapy include thalidomide, lenalidomide, pomalidomide, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, interleukin (IL)-2, IL-6, IL-12, interferon-alpha (IFN-alpha), IFN-beta, IFN-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and cancer vaccines (e.g., human papillomavirus (HPV) vaccine and hepatitis B vaccine).

[0074] According to some embodiments of the present disclosure, when the plasma arginine level is equal to or higher than 60.2 μmol / L, then pegylated rADI (ADI-PEG 20) and pembrolizumab are independently administered to the subject. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of about 36 mg per square meter of body surface area per week, while pembrolizumab is administered at a dose of 200 mg every three weeks, to alleviate or reduce symptoms associated with cancer. As can be appreciated, the skilled artisan or clinician can adjust the treatment regimen (including the dosage, schedule, and duration of ADI-PEG 20 and pembrolizumab therapy) according to actual needs.

[0075] Alternatively, when the plasma arginine level is lower than 60.2 μmol / L, then the subject is administered an alternative anti-cancer therapy to achieve the therapeutic purpose. As described above, the alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and combinations thereof. The skilled artisan or clinician can select the appropriate treatment method according to the clinical factors of the patient.

[0076] According to some embodiments of the present disclosure, when the plasma arginine level is equal to or higher than 68.2 μmol / L, then pegylated rADI (ADI-PEG 20), pemetrexed and cisplatin are administered to the subject independently. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of about 36 mg per square meter of body surface area per week, pemetrexed is administered at a dose of about 500 mg per square meter of body surface area every three weeks, and cisplatin is administered at a dose of about 75 mg per square meter of body surface area every three weeks, to alleviate or reduce the symptoms associated with the cancer. The treatment regimen, including the dosage, schedule and duration of ADI-PEG 20, pemetrexed and cisplatin treatment, can be adjusted by the skilled person or clinician according to the actual needs.

[0077] Alternatively, when the plasma arginine level is lower than 68.2 μmol / L, then an alternative anti-cancer therapy is administered to the subject to achieve the therapeutic purpose. The alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy and combinations thereof. The skilled person or clinician can choose the appropriate treatment method according to the clinical factors of the patient.

[0078] According to some embodiments of the present disclosure, when the plasma arginine level is equal to or higher than 84.2 μmol / L, then pegylated rADI (ADI-PEG 20) is administered to the subject alone without being combined with any other therapy. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of about 18 mg per square meter of body surface area per week, to alleviate or reduce the symptoms associated with the cancer. The treatment regimen, including the dosage, schedule and duration of ADI-PEG 20 treatment, can be adjusted by the skilled person or clinician according to the actual needs.

[0079] Conversely, when the plasma arginine level is lower than 84.2 μmol / L, then an alternative anti-cancer therapy is administered to the subject to achieve the therapeutic purpose. The alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy and combinations thereof. The skilled person or clinician can choose the appropriate treatment method according to the clinical factors of the patient.

[0080] According to certain embodiments of the present disclosure, when the plasma arginine level is equal to or higher than 97.5 μmol / L, then pegylated rADI (ADI-PEG 20) and docetaxel are independently administered to the subject. In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of about 36 mg per square meter of body surface area per week, while docetaxel is administered at a dose of about 75 mg per square meter of body surface area every three weeks, to alleviate or reduce the symptoms associated with cancer. The treatment regimen, including the dosage, schedule, and duration of ADI-PEG 20 and docetaxel treatment, can be adjusted by the skilled artisan or clinician according to the actual needs.

[0081] Conversely, when the plasma arginine level is lower than 97.5 μmol / L, then an alternative anti-cancer therapy is administered to the subject to achieve the therapeutic purpose. The alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and combinations thereof. The skilled artisan or clinician can choose the appropriate treatment method according to the clinical factors of the patient.

[0082] According to certain embodiments of the present disclosure, when the plasma arginine level is equal to or higher than 122 μmol / L, then pegylated rADI (ADI-PEG 20) and cisplatin are independently administered to the subject. In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of about 36 mg per square meter of body surface area per week, while cisplatin is administered at a dose of about 30 mg per square meter of body surface area per week for three consecutive weeks (week 1 to week 3), followed by a one-week rest (week 4), to complete one treatment cycle, to alleviate or reduce the symptoms associated with cancer. The treatment regimen, including the dosage, schedule, and duration of ADI-PEG 20 and cisplatin treatment, can be adjusted by the skilled artisan or clinician according to the actual needs.

[0083] Conversely, when the plasma arginine level is lower than 122 μmol / L, then an alternative anti-cancer therapy is administered to the subject to achieve the therapeutic purpose. The alternative anti-cancer therapy is preferably selected from surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and combinations thereof. The skilled artisan or clinician can choose the appropriate treatment method according to the clinical factors of the patient.

[0084] Cancers treatable by the present method include, but are not limited to, breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), leukemia, acute myeloid leukemia (AML), lymphoma, lung cancer, melanoma, mesothelioma, malignant pleural mesothelioma (MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma. According to some exemplary embodiments, the cancer is HCC. In one specific example, the cancer is advanced HCC.

[0085] The subject is a mammal, such as a human, mouse, rat, guinea pig, monkey, sheep, goat, cat, dog, horse, or chimpanzee. Preferably, the subject is a human.

[0086] The present application will now be more particularly described with reference to the following examples, which are provided for the purpose of illustration and not by way of limitation. Although these examples generally relate to those that can be used, the skilled artisan can substitute other known procedures, methods, or techniques.

[0087] Materials and Methods

[0088] Patient Enrollment

[0089] Plasma samples from patients who received arginine deprivation therapy were used in the present study. Patients enrolled in the study (cohort-1 through cohort-6) were diagnosed with advanced HCC and had participated in ADI-PEG 20 clinical trials in Taiwan. These de-identified samples and clinicopathological parameters were used for post-hoc analysis.

[0090] Cohort-1 included 422 patients who received ADI-PEG 20 monotherapy, with each patient receiving intramuscular injection of 18 mg / m2 of ADI-PEG 20 per week until disease progression, occurrence of unacceptable adverse events, or other withdrawal criteria were met.

[0091] Cohort-2 included 31 patients who received ADI-PEG 20 in combination with EU therapy, with each patient receiving a weekly intramuscular injection of 36 mg / m2 of ADI-PEG 20 and a weekly intravenous injection of 75 mg / m2 of EU for three weeks, followed by one week off (week 4), for a treatment cycle, until disease progression, occurrence of an unacceptable adverse event, or other withdrawal criteria were met.

[0092] Cohort-3 included 78 patients who received ADI-PEG 20 in combination with cisplatin therapy, with each patient receiving a weekly intramuscular injection of 36 mg / m2 of ADI-PEG 20 and a weekly intravenous injection of 30 mg / m2 of cisplatin for three consecutive weeks (weeks 1-3), followed by one week off (week 4), for a treatment cycle, until disease progression, occurrence of an unacceptable adverse event, or other withdrawal criteria were met.

[0093] Cohort-4 included 39 patients who received ADI-PEG 20 in combination with the modified FOLFOX6 regimen (mFOLFOX6 regimen, consisting of 85 mg / m2 oxaliplatin, 400 mg / m2 bolus 5-FU, and 400 mg / m2 leucovorin on day 1, followed by continuous infusion of 2,400 mg / m2 5-FU over 2 days), with each patient receiving a weekly intramuscular injection of 36 mg / m2 of ADI-PEG 20 and a biweekly intravenous injection of the mFOLFOX6 regimen, until disease progression, occurrence of an unacceptable adverse event, or other withdrawal criteria were met.

[0094] Cohort-5 included 111 patients who received ADI-PEG 20 in combination with pemetrexed and cisplatin therapy, with each patient receiving a weekly intramuscular injection of 36 mg / m2 of ADI-PEG 20 and a triweekly intravenous injection of 500 mg / m2 of pemetrexed and 75 mg / m2 of cisplatin, until disease progression, occurrence of an unacceptable adverse event, or other withdrawal criteria were met.

[0095] Cohort-6 included 27 patients who received ADI-PEG 20 in combination with pembrolizumab therapy, with each patient receiving a weekly intramuscular injection of 36 mg / m2 of ADI-PEG 20 and a triweekly intravenous injection of 200 mg of pembrolizumab, until disease progression, occurrence of an unacceptable adverse event, or other withdrawal criteria were met.

[0096] Treatment outcome assessment

[0097] Overall survival (OS) for patients in Cohort-1 and Cohort-5 was calculated from the date of patient randomization to the date of death from any cause or the date of loss of follow-up. Overall survival (OS) for patients in Cohort-2, Cohort-3, Cohort-4, and Cohort-6 was calculated from the date of first dose of study treatment to death from any cause; if the subject was still alive or lost to follow-up, data were censored at the date of last contact.

[0098] Statistical Analysis

[0099] Maximally selected log-rank statistics was used to determine the optimal cutpoint value of plasma arginine levels on survival outcome. The median overall survival of patients was estimated using the Kaplan-Meier method. A p-value < 0.05 from the log-rank test was considered statistically significant. Statistical analyses were performed using associated software.

[0100] Example 1. Correlation of plasma arginine levels with overall survival (OS) in patients with advanced hepatocellular carcinoma (HCC) receiving arginine-deprivation therapy alone or in combination with other treatments.

[0101] To investigate whether plasma arginine levels are correlated with overall survival (OS) in cancer patients, a Maximally Selected log-rank statistics analysis was performed. The results of the analysis are shown in Figures 1 to 12 and summarized in Tables 1-3.

[0102] Table 1. Correlation of plasma arginine levels with overall survival (OS) in cancer patients receiving specific treatments

[0103]

[0104]

[0105] [1] Maximally selected log-rank statistics.

[0106] [2] Months.

[0107] [3] Kaplan-Meier survival analysis estimate.

[0108] [4] P-value based on log-rank test comparing between treatment groups.

[0109] Table 2. Correlation of plasma arginine levels with overall survival (OS) in cancer patients receiving specific treatments

[0110]

[0111]

[0112] [1] Maximally selected log-rank value ranking statistics.

[0113] [2] Month.

[0114] [3] Kaplan-Meier survival analysis estimate.

[0115] [4] P-value comparison between treatment groups based on log-rank test.

[0116] Example 2. Procedure to determine arginine cutpoint (e.g. HCC)

[0117] Step 1, Determine arginine cutpoint value. HCC patients agree to participate in the Polaris HCC clinical study, and Polaris collects patients' plasma before they receive ADI-PEG 20 treatment for the first time. Then, the pharmacodynamics is assessed by measuring arginine and citrulline levels in peripheral blood by LCMS. A total of 633 patients are enrolled in this study (422 in the ADI-PEG 20 group and 211 in the placebo group). Subsequently, once the number of deaths reaches the sample size calculation requirement at the beginning of the study design, the clinical database is locked. In addition, the statistician calculates the overall survival of each patient. The arginine cutpoint value is determined using the maximally selected log-rank statistics (the statistician inputs the arginine value and OS of each patient into the statistical model)

[0118] Step 2, Validate the efficacy of the arginine cutpoint. The 422 HCC patients who received ADI-PEG 20 treatment are divided into two groups: high arginine group and low arginine group. Then, the Kaplan-Meier survival analysis estimate is used to estimate overall survival. The treatment groups will provide point estimates (25th, 50th and 75th percentiles), as well as 95% confidence intervals. Survival estimates will also be displayed graphically for each treatment group. In addition, stratified log-rank test will be used to compare treatment effects.

[0119] In one embodiment, a method of identifying an arginine threshold in a biological sample of a cancer subject in vitro to predict whether the subject is responsive to an arginine deprivation therapy, comprising the steps of: providing a biological sample taken from the subject prior to receiving an arginine deprivation agent; determining arginine concentration, measuring the arginine concentration in the biological sample; calculating overall survival, assessing overall survival statistics after the subject receives the arginine deprivation therapy; determining an arginine cutpoint value by: (1) determining the highest value of the standardized log-rank statistics on the Y-axis of a statistical plot; (2) determining the highest point on the Y-axis and finding the corresponding arginine value on the X-axis; and (3) the arginine level corresponding to this highest point is considered the arginine cutpoint value. (For example, the arginine cutpoint value in cohort-1 is 84.2 µmol / L, see Figure 1 ); calculating an arginine threshold, based on the arginine cutpoint value being the highest arginine value, gradually decreasing the arginine value and calculating the corresponding p-value for each arginine value, continuing this process until the p-value for the nth arginine value is higher than 0.05, then the arginine value corresponding to the (n-1)th arginine value is the arginine threshold (see Table 3); wherein when the arginine concentration of the subject is greater than or equal to the arginine threshold, it means that the cancer subject is responsive to the arginine deprivation therapy.

[0120] According to the results, 422 patients in cohort-1 received ADI-PEG 20 18 mg / m 2 treatment, the estimated arginine cutpoint value for overall survival was set at 84.2 µmol / L. The maximum log-rank statistic recorded was M = 2.8792 ( Figure 1 ). The figure shows the difference in overall survival time between the two groups defined by the arginine cutpoint value of 84.2 µmol / L. The median overall survival for the high arginine group (arginine >= 84.2 µmol / L) was 8.6 months (95% CI: 7.3, 10.5), while the median overall survival for the low arginine group (arginine < 84.2 µmol / L) was 5.7 months (95% CI: 4.9, 7.2), indicating that the high arginine group experienced longer survival in cohort-1 (p-value = 0.0057) ( Figure 2 and Table 1).

[0121] Based on previous ADI-PEG 20 monotherapy studies in HCC, the cutpoint value of arginine was set at 84.2 pmol / L according to the Maximally Selected log-rank statistics. Patients with arginine level >= 84.2 pmol / L were likely to have longer survival time than patients with arginine level < 84.2 pmol / L.

[0122] To enroll more patients, prognostic variable analysis was used to determine the arginine threshold. Based on the arginine cutpoint value of 84.2 pmol / L, the highest arginine value was considered, and then the arginine value was gradually reduced, in order: 83 pmol / L, 82 pmol / L, 81 pmol / L, 80 pmol / L, and 79 pmol / L. In Table 3, the significance P value corresponding to each arginine value was less than 0.05. However, when the arginine value was 78 pmol / L, the corresponding P value was higher than 0.05 for the first time. Therefore, 78 pmol / L was defined as the nth arginine value. The previous (n-1) arginine value, i.e., 79 pmol / L, was used as the arginine threshold. Survival above the arginine threshold was longer than below the arginine threshold, reaching statistical significance. According to the statistical results, the arginine threshold was usually 5% to 10% lower than the arginine cutpoint value (Table 3).

[0123] Table 3. Cutpoint of cohort-1

[0124]

[0125] Significant if *p value is less than 0.05

[0126] According to the results, 31 patients in cohort-2 received ADI-PEG 20 36 mg / m 2 + Docetaxel 75mg / m 2 treatment, and the arginine cutpoint value of overall survival was estimated to be 97.5 pmol / L. The recorded Maximally log-rank statistics value was M=3.0599 ( Figure 3). The figure shows the difference in overall survival between the two groups defined by the arginine cutpoint value of 97.5 pmol / L. The median overall survival in the high arginine group (arginine > 97.5 pmol / L) was 40.7 months (95% CI: 7.2, 40.7) while the median overall survival in the low arginine group (arginine < 97.5 pmol / L) was 14.6 months (95% CI: 5.7, 16.0), indicating that in Group 2, the high arginine group had a longer survival (p-value = 0.0117) Figure 4 and Table 2).

[0127] According to the results, 78 patients in Group 3 received ADI-PEG 20 36 mg / m 2 + Cisplatin 30 mg / m 2 The arginine cutpoint value estimated for overall survival was 122 pmol / L. The recorded Maximally log-rank statistics value was M = 3.6943 (p-value = 0.0006) Figure 5 ). The figure shows the difference in overall survival between the two groups defined by the arginine cutpoint value of 122 pmol / L. The median overall survival in the high arginine group (arginine > 122 pmol / L) was 15.7 months (95% CI: 8.9, 28.5) while the median overall survival in the low arginine group (arginine < 122 pmol / L) was 6.4 months (95% CI: 3.4, 8.2), indicating that in Group 3, the high arginine group had a longer survival (p-value = 0.0006) Figure 6 and Table 2).

[0128] According to the results, 39 patients in Group 4 received ADI-PEG 20 36 mg / m² + FOFLOX, the arginine cutpoint value estimated for overall survival was 34 pmol / L. The recorded Maximally log-rank statistics value was M = 1.6007 (p-value = 0.00083) Figure 7 ). The figure shows the difference in overall survival between the two groups defined by the arginine cutpoint value of 34 pmol / L. The median overall survival in the high arginine group (arginine > 34 pmol / L) was 9.5 months (95% CI: 7.5, 15.1) while the median overall survival in the low arginine group (arginine < 34 pmol / L) was 4.3 months (95% CI: 4.0, 4.6), indicating that in Group 4, the high arginine group had a longer survival (p-value = 0.00083) Figure 8 and Table 2).

[0129] Based on the results, 111 patients in Group 5 received treatment with ADI-PEG 20 36 mg / m2+ Pemetrexed 500 mg / m2+ Cisplatin 75 mg / m2, and the arginine cutpoint value for overall survival was estimated to be 68.2 pmol / L. The recorded maximally log-rank statistics value was M = 3.043 (see Figure 9 ). The difference in overall survival between the two groups defined by the arginine cutpoint value of 68.2 pmol / L is shown in the figure. The median overall survival in the high arginine group (arginine >= 68.2 pmol / L) was 12.5 months (95% CI: 9.8, 14.2), while the median overall survival in the low arginine group (arginine < 68.2 pmol / L) was 6.5 months (95% CI: 3.8, 8.8), indicating that in Group 5, the high arginine group had a longer survival (p-value = 0.0011) (see Figure 10 and Table 1).

[0130] Based on the results, 27 patients received treatment with ADI-PEG 20 36 mg / m2+ Pembrolizumab 200 mg, and the arginine cutpoint value for overall survival was estimated to be 60.2 pmol / L. The recorded maximally log-rank statistics value was M = 3.0899 (see Figure 11 ). The difference in overall survival between the two groups defined by the arginine cutpoint value of 60.2 pmol / L is shown in the figure. Since most subjects were still alive, the median overall survival could not be estimated. The median overall survival in the low arginine group (arginine < 60.2 pmol / L) was 6.6 months (95% CI: 1.8, 7.8), indicating that in Group 6, the high arginine group had a longer survival (p-value = 0.0119) (see Figure 12 and Table 2).

[0131] In summary, the level of arginine in plasma can predict the outcome (i.e., overall survival) of arginine deprivation therapy alone or in combination with different anticancer treatments, including folffox, docetaxel, cisplatin, pemetrexed, and pembrolizumab.

[0132] It is to be understood that the above- described embodiments are only exemplary and various modifications can be made by those skilled in the art without departing from the spirit or scope of the application. The above specification, examples and data provide a complete description of the structure and use of the inventive embodiments. Although the various embodiments of the application have been described above with a certain degree of particularity, those skilled in the art could make various modifications to the embodiments without departing from the spirit or scope of the application as set forth in the claims.

Claims

1. A method of identifying an arginine threshold in a biological sample of a cancer subject in vitro to predict whether the subject is responsive to an arginine deprivation therapy, comprising: providing a biological sample taken from a subject prior to administration of an arginine deprivation agent; determining an arginine concentration, measuring the arginine concentration in the biological sample; calculating overall survival, assessing the statistical data of the overall survival of the subject after receiving the arginine deprivation therapy; calculating an arginine cutpoint, inputting the arginine concentration and the overall survival of the subject into a Maximally Selected log-rank statistics method to determine an arginine cutpoint; and calculating an arginine threshold, based on the arginine cutpoint as the highest arginine value, gradually decreasing arginine values and calculating the P value corresponding to each arginine value, continuing this process until the P value of the nth arginine value is higher than 0.05, then the (n-1)th arginine value is the arginine threshold; wherein when the arginine concentration of the subject is greater than or equal to the arginine threshold, it indicates that the cancer subject is well responsive to the arginine deprivation therapy.

2. The method of claim 1 wherein, The result of the Maximally Selected log-rank statistics method further comprises generating a statistical plot, in which the arginine level is plotted on the X-axis and the standardized log-rank statistics is plotted on the Y-axis.

3. The method of claim 2, wherein, The arginine cutpoint is determined by the following steps: (1) identifying the highest value of the standardized log-rank statistics on the Y-axis in the statistical plot; (2) determining the highest point on the Y-axis and finding the corresponding arginine value on the X-axis; and (3) the arginine level corresponding to the highest point is considered as the arginine cutpoint. The arginine threshold is analyzed by log-rank value test statistics.

4. The method of claim 1 wherein, The arginine threshold is 5-10% lower than the arginine cutpoint.

5. The method of claim 1 wherein, The cancer is selected from the group consisting of breast cancer, brain tumor, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), leukemia, acute myeloid leukemia (AML), lymphoma, lung cancer, melanoma, mesothelioma, malignant pleural mesothelioma (MPM), neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, and combinations thereof.

6. The method as described in claim 1, characterized in that, The cancer is hepatocellular carcinoma (HCC), and the arginine cutpoint is 84.2 μmol / L.

7. The method of claim 6 wherein, The cancer is hepatocellular carcinoma (HCC), and the arginine threshold is 79 μmol / L.

8. The method of claim 6 wherein, The biological sample is plasma.

9. The method of claim 1 wherein, The arginine deprivation agent is selected from the group consisting of recombinant arginine deiminase (rADI), recombinant arginase (rArg), recombinant arginine decarboxylase (rADC), pegylated form of rAD, pegylated form of rArg, pegylated form of rADC, difluoromethylornithine (DFMO), and combinations thereof.

10. The method of claim 1 wherein, The arginine deprivation agent is further used in combination with an anti-cancer agent.

11. The method of claim 10 wherein, ​ 12. The method of claim 1 wherein, The anti-cancer agent is selected from the group of FOLFOX, docetaxel, cisplatin, pemetrexed, pembrolizumab, and combinations thereof.

13. The method of claim 1 wherein, The arginine depleting agent is recombinant arginine deiminase (rADI) in pegylated form, the anti-cancer agent is cisplatin, and the arginine cutpoint value is 122 pmol / L.

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