CANCER TREATMENT METHODS

Abstract

Disclosed herein are methods for providing a prediction as to whether a subject with a cancer exhibits a beneficial response to arginine deprivation therapy. The method includes determining the plasma level of arginine in the subject, followed by administering to the subject an arginine deprivation therapy alone or in combination with an anti-cancer agent based on the determined plasma level of arginine. According to some embodiments of the present disclosure, the anti-cancer agent is selected from the group consisting of FOLFOX, docetaxel, cisplatin, pemetrexed, pembrolizumab, and a combination thereof.

Description

1 / 41 “CANCER TREATMENT METHODS BACKGROUND TO THE INVENTION FIELD OF THE INVENTION

[0001] This disclosure generally relates to the field of cancer treatment. More specifically, this disclosure relates to methods of treating cancers by means of arginine deprivation therapy. DESCRIPTION OF THE RELATED TECHNIQUE

[0002] Cancer is a group of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal tissues. Cancer is the second leading cause of death worldwide, responsible for nearly 10 million deaths each year. The most common causes of cancer death are lung cancer, colorectal cancer, liver cancer, gastric cancer, and breast cancer. Cancer treatment varies depending on the type and stage of the cancer. The mainstays of cancer treatment include surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, and targeted therapy. However, none of the treatments produces a satisfactory effect, and a variety of adverse responses are observed in cancer patients. Consequently, there is ongoing interest in identifying and developing alternative approaches to cancer treatment.

[0003] Certain cancers are auxotrophic for a specific amino acid (e.g., arginine), and amino acid deprivation (e.g., arginine deprivation) may provide a potential means to treat these cancers. Petition 870250077844, dated 01 / 09 / 2025, page 8 / 96 2 / 41 Arginine can be degraded by several enzymes, including arginine deiminase (ADI), a microbial mycoplasma enzyme that exhibits high affinity for arginine and catalyzes arginine into citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells expressing argininosuccinate synthetase 1 (ASS1). A pegylated form of ADI (ADI-PEG 20) has been formulated and demonstrated in clinical trials for the treatment of arginine auxotrophic tumors via arginine deprivation therapy. Resistance to ADI is frequently developed through reactivation or upregulation of ASS1. Furthermore, prolonged treatment with ADI is reported to lead to the activation of different cellular pathways associated with apoptosis resistance, possibly limiting the overall treatment window for ADI.

[0004] Consequently, there is a need in the related technique for therapeutic strategies to optimize arginine deprivation therapy, thereby improving treatment outcomes in cancer patients. SUMMARY OF THE INVENTION

[0005] The following is a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary does not constitute a comprehensive overview of the disclosure and does not identify the key / critical elements of this invention nor delineate its scope. Its sole purpose is to present some concepts disclosed here in a simplified form as a prelude to the more detailed description that will be presented later. Petition 870250077844, dated 01 / 09 / 2025, p. 9 / 96 3 / 41

[0006] This disclosure is based, at least in part, on the finding that plasma arginine levels are associated with the response of cancer patients to a pegylated form of ADI-based therapy (ADI-PEG 20). Therefore, plasma arginine levels can be used to predict a cancer patient's therapeutic response to arginine deprivation therapy and as a guide for the individual adaptation of an appropriate therapeutic regimen.

[0007] Consequently, the present disclosure provides a method for predicting whether an individual with cancer (e.g., HCC) will have a beneficial response to arginine deprivation therapy. The method comprises, (a) determining the plasma arginine level in the individual; and (b) administering to the individual arginine deprivation therapy, alone or in combination with an anticancer agent, based on the plasma arginine level determined in step (a).

[0008] According to the modalities of the present disclosure, arginine deprivation therapy comprises administering to the individual an arginine deprivation agent selected from the group consisting of difluoromethylornithine (DEMO), a recombinant arginine deiminase (rADI), a recombinant arginase (rArg), a recombinant arginine decarboxylase (rADC), a pegylated form of rADI (hereinafter referred to as pegylated rADI), a pegylated form of rArg (hereinafter referred to as pegylated rArg), a pegylated form of rADC (hereinafter referred to as pegylated rADC) and a combination thereof. Petition 870250077844, dated 01 / 09 / 2025, page 10 / 96 4 / 41 According to some embodiments of this disclosure, the anticancer agent is selected from the group consisting of FOLFOX, docetaxel, cisplatin, pemetrexed, pembrolizumab, and a combination thereof.

[0009] According to some embodiments, pegylated rADI and FOLFOX are administered independently to the individual when the plasma arginine level at step (a) is equal to or greater than 34 micromoles per liter (> 34 pmol / L). In some exemplary embodiments, pegylated rADI is administered in an amount of approximately 36 mg / m2 of body surface area weekly, and FOLFOX is administered biweekly.

[0010] According to some embodiments, pegylated rADI and pembrolizumab are administered independently to the individual when the plasma arginine level at step (a) is equal to or greater than 60.2 μmol / L (> 60.2 pmol / L). In some exemplary embodiments, pegylated rADI is administered in an amount of approximately 36 mg / m2 of body surface area weekly, and pembrolizumab is administered in an amount of approximately 200 mg every three weeks.

[0011] According to certain embodiments, pegylated rADI, pemetrexed, and cisplatin are administered independently to the individual when the plasma arginine level at step (a) is equal to or greater than 68.2 pmol / L (> 68.2 pmol / L). In certain exemplary embodiments, pegylated rADI is administered at a dose of approximately 36 mg / m2 of body surface area weekly, and pemetrexed is administered at a dose of approximately 500 mg / m2 of body surface area every three weeks. Petition 870250077844, dated 01 / 09 / 2025, page 11 / 96 5 / 41 weeks and cisplatin is administered in an amount of approximately 75 mg / m2 of body surface area every three weeks.

[0012] According to certain embodiments, pegylated rADI is administered to the individual alone when the plasma arginine level at step (a) is equal to or greater than 84.2 μmol / L (^ 84.2 μmol / L). In some preferred embodiments, pegylated rADI is administered in an amount of approximately 18 mg / m2 of body surface area weekly.

[0013] According to some embodiments, pegylated rADI and docetaxel are administered independently to the individual when the plasma arginine level at step (a) is equal to or greater than 97.5 μmol / L (^ 97.5 μmol / L). In certain exemplary embodiments, pegylated rADI is administered in an amount of about 36 mg / m2 of body surface area weekly, and docetaxel is administered in an amount of about 75 mg / m2 of body surface area every three weeks.

[0014] According to some embodiments, pegylated rADI and cisplatin are administered independently to the individual when the plasma arginine level at step (a) is equal to or greater than 122 μmol / L (^ 122 μmol / L). In some exemplary embodiments, pegylated rADI is administered at a dose of approximately 36 mg / m2 of body surface area weekly, and cisplatin is administered at a dose of approximately 30 mg / m2 of body surface area weekly for three weeks, followed by a one-week rest period.

[0015] Examples of cancers treatable with the Petition 870250077844, dated 01 / 09 / 2025, page 12 / 96 6 / 41 present method (i.e., arginine deprivation therapy alone or in combination with an anticancer agent) includes, but is not limited to, breast cancer, brain tumor, colorectal cancer, squamous cell carcinoma of the head and neck, 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] The individual treatable with the present method is a mammal; preferably, a human being.

[0017] Many of the features and advantages inherent in the present disclosure will be better understood with reference to the following detailed description, considered in connection with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present description will be better understood from the following detailed description, read in light of the attached drawings, where:

[0019] Figure 1 shows the graph illustrating the treatment of cancer patients with ADI-PEG 20, analyzed using the maximum log-rank statistic to find the arginine cutoff point.

[0020] Figure 2 is a line graph depicting the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 alone, as per Example 1 of this disclosure.

[0021] Figure 3 shows the graph that illustrates the Petition 870250077844, dated 01 / 09 / 2025, page 13 / 96 7 / 41 treatment of cancer patients with ADI-PEG 20+ Docetaxel, analyzed using the maximum logrank statistic to find the arginine cutoff point.

[0022] Figure 4 is a line graph representing the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 in combination with docetaxel, according to Example 1 of this disclosure.

[0023] Figure 5 shows the graph illustrating the treatment of cancer patients with ADI-PEG 20+ Cisplatin, analyzed using the maximum logrank statistic to find the arginine cutoff point.

[0024] Figure 6 is a line graph representing the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 in combination with cisplatin, according to Example 1 of this disclosure.

[0025] Figure 7 shows the graph illustrating the treatment of cancer patients with ADI-PEG 20 + FOFLOX, analyzed using the maximum log-rank statistic to find the arginine cutoff point.

[0026] Figure 8 is a line graph representing the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 in combination with mFOLFOX6, according to Example 1 of this disclosure.

[0027] Figure 9 shows the graph illustrating the treatment of cancer patients with ADI-PEG 20 + Pemetrexed + Cisplatin, analyzed using the maximum log-rank statistic to find the cutoff point of Petition 870250077844, dated 01 / 09 / 2025, page 14 / 96 8 / 41 arginine.

[0028] Figure 10 is a line graph representing the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 in combination with pemetrexed and cisplatin, according to Example 1 of this disclosure.

[0029] Figure 11 shows the graph illustrating the treatment of cancer patients with ADI-PEG 20 + Pembrolizumab, analyzed using the maximum log-rank statistic to find the arginine cutoff point.

[0030] Figure 12 is a line graph depicting the correlation of plasma arginine level with OS in cancer patients receiving ADI-PEG 20 in combination with pembrolizumab, as per Example 1 of this disclosure. DETAILED DESCRIPTION OF PREFERRED MODALITIES

[0031] The detailed description provided below, in connection with the accompanying drawings, is intended to describe the present examples and not to represent the only ways in which the present example may be constructed or used. The description establishes the functions of the example and the sequence of steps for its construction and operation. However, identical or equivalent functions and sequences may be performed by different examples. I. DEFINITIONS

[0032] As used herein, the term hepatocellular carcinoma (or HCC for short) refers to a malignant tumor of hepatocellular origin. HCC is a type of liver cancer. HCC can undergo hemorrhage and necrosis due to the absence of fibrous stroma. According to the Petition 870250077844, dated 01 / 09 / 2025, page 15 / 96 9 / 41 Barcelona Clinic Liver Cancer (BCLC) staging system, updated in 2022, HCC can be classified into five stages, including (1) stage 0 (very early stage), which is defined as the presence of a single nodule equal to or smaller than 2 cm, without vascular invasion or extrahepatic spread; (2) stage A (early stage), which is defined as the presence of a nodule regardless of size or as a multifocal HCC of up to 3 nodules, without vascular invasion or extrahepatic spread; (3) stage B (intermediate stage), defined as the presence of multifocal HCC without vascular invasion or extrahepatic spread; (4) stage C (advanced stage), defined as patients with portal invasion and / or extrahepatic spread; and (5) stage D (terminal stage), defined as patients with significant cancer-related symptoms and / or compromised liver function.Based on the BCLC classification system, the term "advanced hepatocellular carcinoma" or "advanced HCC" refers to locally advanced HCC or metastatic HCC (i.e., HCC that has spread from the liver to another part of the body). In general, advanced HCC is unresectable (i.e., it has spread to surrounding tissue and cannot be surgically removed) and is not amenable to cure by local treatment modalities such as radiotherapy.

[0033] The term “arginine deprivation agent” used in arginine deprivation therapy refers to compounds or agents that remove the supply of arginine to cancers with disrupted urea cycle, thereby halting cancer growth and inducing cell death. Petition 870250077844, dated 01 / 09 / 2025, p. 16 / 96 10 / 41

[0034] The term FOLFOX refers to a chemotherapy composed of 5-fluorouracil (5-FU), folinic acid (leucovorin), and oxaliplatin. The term FOLFOX, as used herein, is not intended to be limited to any specific amounts or dosage regimens for these components. Instead, as used herein, FOLFOX includes all combinations of these components in any amounts and dosage regimens. Based on the doses and forms of administration of the three drugs, there are several FOLFOX regimens known in the art, including FOLFOX-4, FOLFOX-6, modified FOL-FOX-6 (mFOLFOX-6), and FOLFOX-7. According to some embodiments of this disclosure, FOLFOX is mFOL-FOX-6.

[0035] As used in this document, the term survival refers to the act or fact of living. The expression overall survival (OS) refers to the prolongation of life expectancy compared to individuals or patients without prior treatment or without treatment.

[0036] The term confidence interval (CI), as used in this document, has a clear meaning, known to anyone with common knowledge of the art, and refers to a statistical range with a specified probability that a given parameter will be within that range.

[0037] The terms administered and administering are used interchangeably in this document to refer to a mode of administration, including, without limitation, intravenous, intratumoral, intramuscular, intraperitoneal, intra-arterial or Petition 870250077844, dated 01 / 09 / 2025, p. 17 / 96 11 / 41 subcutaneous injection of a treatment (e.g., arginine deprivation therapy or anticancer agent).

[0038] Unless otherwise indicated, the terms treat, treating and treatment encompass an action that occurs while a patient suffers from the specified disease or disorder, that reduces the severity of the disease or disorder, or one or more of its symptoms, or slows or delays the progression of the disease or disorder.

[0039] The terms cancer and tumor are used interchangeably in this disclosure and preferably refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancers, in this sense, include metastatic cancers and / or drug-resistant cancers. Examples of cancer include, but are not limited to, breast cancer, brain tumor, colorectal cancer, squamous cell carcinoma of the head and neck, 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 a combination thereof.

[0040] Unless otherwise indicated, the terms patient and individual are used interchangeably in this disclosure and refer to an animal, including the human species, that is treatable by the method of the present invention. The term patient or individual is intended to refer to both the male and female genders, unless a gender is specifically indicated. Petition 870250077844, dated 01 / 09 / 2025, page 18 / 96 12 / 41

[0041] Although the ranges and numerical parameters that define the broad scope of the invention are approximations, the numerical values ​​presented in the specific examples are reported to the greatest possible accuracy. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective test measurements. Furthermore, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean, as considered by a professional with common knowledge in the field.Except in examples of operation / work, or unless expressly specified otherwise, all numerical ranges, quantities, values, and percentages, such as those for quantities of materials, durations of time, temperatures, operating conditions, quantity ratios, and the like disclosed herein, shall be understood as modified in all cases by the term “approximately.” Consequently, unless indicated otherwise, the numerical parameters set forth in this disclosure and the accompanying claims are approximations that may vary as desired. At a minimum, each numerical parameter shall be interpreted in light of the reported number of significant digits and by applying common rounding techniques.

[0042] The singular forms “a”, “and” and “the” are used here to include plural referents, unless the context clearly indicates otherwise. Petition 870250077844, dated 01 / 09 / 2025, page 19 / 96 13 / 41 II. DESCRIPTION OF THE INVENTION (1) Prediction of cancer patients' responses to arginine deprivation therapy

[0043] The diversity of responses to cancer treatment has long been recognized, largely due to underlying heterogeneities in cancer biology, variations in physiological function, and distinctions in patients' genetic profiles. Therefore, an objective of this disclosure is to provide a molecular marker associated with cancer patients' responses to arginine deprivation therapy. According to the modalities of this disclosure, plasma arginine levels are associated with the therapeutic response of tumors (e.g., HCC) in patients to a pegylated form of ADI (ADI-PEG 20, a polyethylene glycol-conjugated arginine deiminase with a molecular weight of 20,000).

[0044] Consequently, the first aspect of this disclosure provides a method for making a prognosis as to whether an individual with cancer (e.g., HCC) will have a beneficial response to arginine deprivation therapy. The method comprises, (a) determining the plasma arginine level in the individual; and (b) making the prognosis based on the plasma arginine level determined in step (a), wherein a plasma arginine level equal to or greater than 84.2 indicates that the individual will have a beneficial response to arginine deprivation therapy.

[0045] In step (a), the plasma arginine level is measured. Suitable assays used for Petition 870250077844, dated 01 / 09 / 2025, p. 20 / 96 14 / 41 Determining plasma arginine levels includes, but is not limited to, spectrophotometric methods, capillary electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), mass spectrometry (MS), Sakaguchi test, biosensors, and a combination thereof.

[0046] Next, in step (b), a qualified technician or clinician can make the prognosis based on the plasma arginine level. According to some modalities of the present disclosure, plasma level of the arginine being 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, 100 μmol / L indicates 99, 8 99.9 or that the or higher) individual shows a beneficial response to arginine deprivation therapy, that is, responds to therapy of Petition 870250077844, dated 01 / 09 / 2025, p. 21 / 96 15 / 41 arginine deprivation in a positive way. According to certain practical examples, the beneficial response is associated with overall survival, in which, compared to an individual with a plasma arginine level < 84.2 pmol / L, an individual with a plasma arginine level > 84.2 pmol / L has a longer post-treatment overall survival.

[0047] According to some embodiments of the present disclosure, arginine deprivation therapy comprises administering to the individual (e.g., patient with HCC) an agent selected from the group consisting of DEMO, rADI, rArg, rADC, pegylated rADI, pegylated rArg, pegylated rADC and a combination thereof. In some example modalities, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 18 mg / m2 of body surface area weekly, wherein the median overall survival of subjects with plasma arginine levels > 84.2 pmol / L is approximately 8.6 months (with a 95% confidence interval (CI) ranging from 7.3 months to 10.5 months), and the median overall survival of subjects with plasma arginine levels < 84.2 pmol / L is approximately 5.7 months (with a 95% CI ranging from 4.9 months to 7.2 months) after treatment.

[0048] Examples of cancer include, but are not limited to, breast cancer, brain tumor, colorectal cancer, squamous cell carcinoma of the head and neck, HCC, leukemia, AML, lymphoma, lung cancer, melanoma, mesothelioma, MPM, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, and a combination thereof.

[0049] In some forms of the present Petition 870250077844, dated 01 / 09 / 2025, page 22 / 96 16 / 41 disclosure, the cancer is HCC. According to a specific example, the cancer is advanced HCC. (2) Predicting cancer patients' responses to combination treatment

[0050] According to some embodiments of this disclosure, arginine deprivation therapy is combined with one or more additional treatments to enhance its therapeutic effect. Consequently, the second aspect of this disclosure relates to a method for making a prognosis as to whether an individual with cancer (e.g., HCC) will have a beneficial response to combination treatment (i.e., arginine deprivation therapy plus additional treatment(s)). The method comprises the steps of: (a) determining the plasma arginine level in the individual; and (b) making the prognosis based on the plasma arginine level determined in step (a).

[0051] According to some embodiments of the present disclosure, the arginine deprivation agent is combined with treatment with FOLFOX. In these modalities, the plasma arginine level being equal to or greater than 34 pmol / L (> 34 pmol / L; for example, 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 pmol / L, or higher) indicates that the individual shows a beneficial response to the combined treatment, that is, responds positively to the arginine deprivation agent plus treatment with FOLFOX. According to certain Petition 870250077844, dated 01 / 09 / 2025, p. 23 / 96 17 / 41 practical examples, the beneficial response is associated with overall survival, in which, compared to an individual with a plasma arginine level < 34 μmol / L, an individual with a plasma arginine level > 34 μmol / L has a longer overall survival after treatment.

[0052] In some exemplary modalities, pegylated rADI (ADI-PEG 20) is administered in an amount of approximately 36 mg / m2 of body surface area weekly, and treatment with FOLFOX is administered biweekly, where the median overall survival of individuals with plasma arginine levels > The median overall survival of individuals with plasma arginine levels < μmol / L is approximately 9.5 months (with a 95% CI ranging from 7.5 months to 15.1 months), and the median overall survival of individuals with plasma arginine levels < The μmol / L level is approximately 4.3 months (with a 95% CI ranging from 4.0 months to 4.6 months) after treatment.

[0053] According to some embodiments of this disclosure, the arginine-depriving agent is combined with pembrolizumab. In these embodiments, the plasma arginine level being 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, A plasma arginine level of 94, 95, 96, 97, 98, 99, or 100 μηΩT / E, or higher, indicates that the individual exhibits a beneficial response to the combined treatment, i.e., responding positively to arginine deprivation therapy plus treatment with pembrolizumab. According to certain examples, the beneficial response is associated with overall survival, in which, compared to an individual with a plasma arginine level < 60.2 μηΩT / E, the... Petition 870250077844, dated 01 / 09 / 2025, p. 24 / 96 18 / 41 individuals with a plasma arginine level of 60.2 μmol / L exhibit longer overall post-treatment survival.

[0054] In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m2 of body surface area weekly, and pembrolizumab is administered at a dose of approximately 200 mg every three weeks.

[0055] According to certain embodiments of this disclosure, the arginine-depriving agent is administered in combination with pemetrexed and cisplatin. In these modalities, a plasma arginine level equal to or greater than 68.2 μmol / L (up to 68.2 pmol / 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 pmol / L, or higher) indicates that the individual exhibits a beneficial response to the combined treatment, that is, a positive response. to treatments with arginine withdrawal agents plus pemetrexed and cisplatin. According to certain examples, the beneficial response is associated with overall survival, in which, compared to an individual with a plasma arginine level < 68.2 pmol / L, an individual with a plasma arginine level ≥ 68.2 pmol / L has a longer post-treatment overall survival.

[0056] In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m2 of body surface area weekly, pemetrexed is administered at a dose of approximately 500 mg / m2 of body surface area every three weeks, and cisplatin is administered at a dose of approximately 36 mg / m2 of body surface area weekly. Petition 870250077844, dated 01 / 09 / 2025, page 25 / 96 19 / 41 amount of approximately 75 mg / m2 of body surface area every three weeks. According to these modalities, the median overall survival of individuals with plasma arginine levels ≥ 68.2 μmol / L is approximately 12.5 months (with a 95% CI ranging from 9.8 months to 14.2 months), and the median overall survival of individuals with plasma arginine levels < 68.2 μmol / L is approximately 6.5 months (with a 95% CI ranging from 3.8 months to 8.8 months) after treatment.

[0057] According to certain embodiments of this disclosure, the arginine deprivation agent is administered in combination with docetaxel. In these embodiments, the plasma arginine level being equal to or greater than 97.5 μmol / L (≥ 97.5 μmol / L; for example, 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 A plasma arginine level (99, 6, 99, 7, 99, 8, 99, 9, or 100 μmol / Σ, or higher) indicates that the individual exhibits a beneficial response to the combined treatment, i.e., responding positively to arginine deprivation therapy plus docetaxel treatment. According to certain practical examples, the beneficial response is associated with overall survival, in which, compared to an individual with a plasma arginine level < 97.5 μmol / L, an individual with a plasma arginine level ≥ 97.5 μmol / L exhibits a longer post-treatment overall survival.

[0058] In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m2 of body surface area weekly, and docetaxel is administered at a dose of approximately 36 mg / m2 of body surface area weekly. Petition 870250077844, dated 01 / 09 / 2025, page 26 / 96 20 / 41 amount of approximately 75 mg / m2 of body surface area every three weeks. According to these modalities, the median overall survival of individuals with plasma arginine levels ≥ 97.5 μmol / L is approximately 40.7 months (with a 95% CI ranging from 7.2 months to 40.7 months), and the median overall survival of individuals with plasma arginine levels < 97.5 μmol / L is approximately 14.6 months (with a 95% CI ranging from 5.7 months to 16.0 months) after treatment.

[0059] According to some embodiments of this disclosure, the arginine deprivation agent is administered in combination with cisplatin. In these embodiments, the plasma arginine level being equal to or greater than 122 μmol / L (≥ 122 μmol / L; for example, 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, A plasma arginine level of 196, 197, 198, 199, or 200 μmol / L, or higher, indicates that the individual has a beneficial response to the combined treatment, i.e., responding positively to the arginine-depriving agent plus cisplatin treatment. According to some examples, the beneficial response is associated with overall survival, where, compared to an individual with a plasma arginine level < 122 μmol / L, an individual with a plasma arginine level ≥ 122 μmol / L has a longer post-treatment overall survival.

[0060] In some exemplary forms, Petition 870250077844, dated 01 / 09 / 2025, p. 27 / 96 21 / 41 Pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m² of body surface area weekly, and cisplatin is administered at a dose of approximately 30 mg / m² of body surface area weekly for three weeks (week 1 to week 3), followed by a one-week (week 4) rest period in a treatment cycle. According to these modalities, the median overall survival of individuals with plasma arginine levels > 122 pmol / L is approximately 15.7 months (with a 95% CI ranging from 8.9 months to 28.5 months), and the median overall survival of individuals with plasma arginine levels < 122 pmol / L is approximately 6.4 months (with a 95% CI ranging from 3.4 months to 8.2 months) after treatment.

[0061] As described above, cancer can be 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, or a combination of these. According to some practical examples, the cancer is HCC. In one specific example, the cancer is advanced HCC. (3) Cancer treatment method

[0062] Another aspect of the present disclosure relates to a method of treating cancers by means of an arginine-depriving agent alone (i.e., the administration of DEMO, rADI, rArg, rADC, pegylated rADI, pegylated rArg, pegylated rADC or a combination thereof) for arginine deprivation therapy or in combination with Petition 870250077844, dated 01 / 09 / 2025, page 28 / 96 22 / 41 one or more additional treatments. According to some embodiments of this disclosure, the method comprises the steps of: (a) determining the plasma arginine level in the individual; and (b) administering to the individual an appropriate treatment based on the plasma arginine level determined in step (a).

[0063] According to some embodiments of this disclosure, in cases where the plasma arginine level is equal to or greater than 34 μΣ / Σ, pegylated rADI (ADI-PEG 20) and FOLFOX treatment are administered independently to the individual. In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m² of body surface area weekly, and FOLFOX treatment is administered biweekly, in order to alleviate or relieve symptoms associated with cancers. As can be seen, a qualified professional or clinician may adjust the treatment regimen (including dosages, schedule, and duration of ADI-PEG 20 and FOLFOX treatments) according to practical requirements.

[0064] Alternatively, if the plasma arginine level is below 34 μmol / L, an alternative anticancer treatment is administered to the individual for therapeutic purposes, the alternative anticancer treatment preferably being selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or clinician may choose an appropriate treatment for the cancer patient according to clinical factors, such as Petition 870250077844, dated 01 / 09 / 2025, page 29 / 96 23 / 41 age, sex and physical condition of the patient, and the type and stage of cancer.

[0065] Agents commonly used in chemotherapy include, but are not limited to, doxorubicin, adriamycin, bleomycin, actinomycin, dactinomycin, mutamycin, daunorubicin, epirubicin, idarubicin, mitoxantrone, mitomycin, epipodophyllotoxins, etoposide, teniposide, antimicrotubule agent, vinblastine, vincristine, vindesine, vinorelbine, taxane, paclitaxel (taxol), nitrogen mustard, chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, aziridines, thiotepa, alkyl sulfonate, busulfan, nitrosoureas, carmustine, lomustine, streptozocin, platinum complex, carboplatin, alkylating agent, altretamine, dacarbazine, procarbazine, temozolamide, methotrexate, fludarabine, mercaptopurine, thiogaunine, cladribine, pentostatin, capecitabine, cytarabine, floxuridine, fluorouracil, gemcitabine, hydroxyurea, camptothecin, irinotecan, busufan, epothilone, azathioprine, halofuginone, sirolimus, everolimus, mitomycin and topotecan.

[0066] Exemplary agents for targeted therapy include, but are not limited to, trastuzumab or pertuzumab (an antibody specific for the HER-2 / neu tumor antigen); bevacizumab (an antibody specific for vascular endothelial growth factor (VEGF)); ramucirumab (an antibody specific for the VEGF receptor); nivolumab or cemiplimab (an antibody specific for programmed cell death protein 1 (PD-1)); atezolizumab, avelumab, or durvalumab (an antibody Petition 870250077844, dated 01 / 09 / 2025, page 30 / 96 24 / 41 specific for programmed cell death protein ligand 1 (PD-L1), ipilimumab (an antibody specific for cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)), rituximab (an antibody specific for CD20 on B cells), etc.

[0067] Non-limiting examples of immunomodulatory agents for immunotherapy include thalidomide, lenolidomide, pomalidomide, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, interleukin (IL)-2, IL-6, IL-12, interferon-alpha (IFN-α), IFN-β, IFN-γ, granulocyte-macrophage colony-stimulating factor (GMCSF), granulocyte colony-stimulating factor (G-CSF), and cancer vaccine (e.g., human papillomavirus (HPV) vaccine and hepatitis B vaccine).

[0068] According to some embodiments of this disclosure, in cases where the plasma arginine level is equal to or greater than 60.2 mIU / mg, pegylated rADI (ADI-PEG 20) and pembrolizumab are administered independently to the individual. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m² of body surface area weekly, and pembrolizumab is administered at a dose of approximately 200 mg every three weeks, in order to alleviate or relieve symptoms associated with cancers. As can be seen, a qualified professional or clinician may adjust the treatment regimen (including dosages, schedule, and duration of treatments with ADI-PEG 20 and pembrolizumab) according to practical requirements.

[0069] Alternatively, in the case where the level Petition 870250077844, dated 01 / 09 / 2025, p. 31 / 96 If plasma arginine levels are less than 60.2 mIU / L, an alternative anticancer treatment is administered to the individual for therapeutic purposes. As described above, the alternative anticancer treatment is preferably selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or clinician can choose an appropriate treatment for the cancer patient according to the patient's clinical factors.

[0070] According to some embodiments of this disclosure, in cases where the plasma arginine level is equal to or greater than 68.2 μmol / L, pegylated rADI (ADI-PEG 20), pemetrexed, and cisplatin are administered independently to the individual. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m² of body surface area weekly, pemetrexed is administered at a dose of approximately 500 mg / m² of body surface area every three weeks, and cisplatin is administered at a dose of approximately 75 mg / m² of body surface area every three weeks, in order to improve or alleviate symptoms associated with cancers. A qualified professional or clinician may adjust the treatment regimen (including dosages, schedule, and duration of treatments with ADI-PEG 20, pemetrexed, and cisplatin) according to practical requirements.

[0071] Alternatively, if the plasma arginine level is less than 68.2 μmol / L, one Petition 870250077844, dated 01 / 09 / 2025, page 32 / 96 26 / 41 Alternative cancer treatment is administered to the individual for therapeutic purposes. Alternative cancer treatment is preferably selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or clinician can choose an appropriate treatment for the cancer patient according to the patient's clinical factors.

[0072] According to certain embodiments of this disclosure, in cases where the plasma arginine level is equal to or greater than 84.2 μmol / L, pegylated rADI (ADI-PEG 20) is administered to the individual alone, without combination with any additional treatments. In certain exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered in an amount of approximately 18 mg / m2 of body surface area weekly, in order to improve or alleviate symptoms associated with cancer. A qualified professional or clinician may adjust the treatment regimen (including dosage, schedule, and duration of treatment with ADI-PEG 20) according to practical requirements.

[0073] Conversely, if the plasma arginine level is below 84.2 μmol / L, an alternative anticancer treatment is administered to the individual for therapeutic purposes. The alternative anticancer treatment is preferably selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or Petition 870250077844, dated 01 / 09 / 2025, page 33 / 96 27 / 41 A clinician can choose an appropriate treatment for a cancer patient based on the patient's clinical factors.

[0074] According to certain embodiments of this disclosure, in cases where the plasma arginine level is equal to or greater than 97.5 μN / L, pegylated rADI (ADI-PEG 20) and docetaxel are administered independently to the individual. In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m² of body surface area weekly, and docetaxel is administered at a dose of approximately 75 mg / m² of body surface area every three weeks, in order to alleviate or relieve symptoms associated with cancers. A qualified professional or clinician may adjust the treatment regimen (including dosages, schedule, and duration of treatments with ADI-PEG 20 and docetaxel) according to practical requirements.

[0075] Alternatively, if the plasma arginine level is below 97.5 μK / L, an alternative anticancer treatment is administered to the individual for therapeutic purposes. The alternative anticancer treatment is preferably selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or clinician can choose an appropriate treatment for the cancer patient according to the patient's clinical factors.

[0076] According to certain modalities of Petition 870250077844, dated 01 / 09 / 2025, page 34 / 96 28 / 41 present disclosure, in the case where the plasma arginine level is equal to or greater than 122 μmol / L, pegylated rADI (ADI-PEG 20) and cisplatin are administered independently to the individual. In some exemplary embodiments, pegylated rADI (ADI-PEG 20) is administered at a dose of approximately 36 mg / m2 of body surface area weekly, and cisplatin is administered at a dose of approximately 30 mg / m2 of body surface area weekly for three weeks (week 1 to week 3), followed by a one-week (week 4) rest period in a treatment cycle, in order to improve or alleviate symptoms associated with cancers. A qualified professional or clinician may adjust the treatment regimen (including dosages, schedule and duration of treatments with ADI-PEG 20 and cisplatin) according to practical requirements.

[0077] Conversely, if the plasma arginine level is below 122 μmol / L, an alternative anticancer treatment is administered to the individual for therapeutic purposes. The alternative anticancer treatment is preferably selected from the group consisting of surgery, chemotherapy, targeted therapy, radiotherapy, hormone therapy, immunotherapy, and a combination thereof. A qualified professional or clinician can choose an appropriate treatment for the cancer patient according to the patient's clinical factors.

[0078] Cancers treatable with the present method include, but are not limited to, breast cancer, brain tumor, colorectal cancer, squamous cell carcinoma. Petition 870250077844, dated 01 / 09 / 2025, page 35 / 96 29 / 41 head and neck cancer, HCC, leukemia, AML, lymphoma, lung cancer, melanoma, mesothelioma, MPM, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, and sarcoma. According to some realizations, the cancer is HCC. In one specific example, the cancer is advanced HCC.

[0079] The individual is a mammal, such as a human, a mouse, a rat, a guinea pig, a monkey, a sheep, a goat, a cat, a dog, a horse, or a chimpanzee. Preferably, the individual is a human.

[0080] The present invention will now be described more specifically with reference to the following embodiments, which are provided for demonstration purposes and not for limitation. Although these are typically the embodiments that can be used, other procedures, methodologies or techniques known to those skilled in the art may alternatively be used. EXAMPLES Materials and methods Patient registration

[0081] Plasma samples from patients on arginine deprivation therapy were used in this study. Patients (including cohort 1 to cohort 6) included in the study were diagnosed with advanced-stage HCC and participated in the ADI-PEG 20 clinical trial in Taiwan. Unbound samples and clinicopathological parameters were used for post-hoc analysis.

[0082] Cohort 1, containing 422 patients, received monotherapy with ADI-PEG 20, in which ADI-PEG 20 was administered intramuscularly to each patient in Petition 870250077844, dated 01 / 09 / 2025, page 36 / 96 30 / 41 dose of 18 mg / m2 weekly, until disease progression or unacceptable adverse event occurred or other withdrawal criteria were met.

[0083] Cohort 2, containing 31 patients, received ADI-PEG 20 in combination with docetaxel, in which ADI-PEG 20 was administered intramuscularly to each patient at a dose of 36 mg / m2 weekly, and docetaxel was administered intravenously at a dose of 75 mg / m2 every three weeks, until disease progression or an unacceptable adverse event occurred or other withdrawal criteria were met.

[0084] Cohort 3, containing 78 patients, received ADI-PEG 20 in combination with cisplatin, in which ADI-PEG 20 was administered intramuscularly to each patient at a dose of 36 mg / m2 weekly, and cisplatin was administered intravenously to each patient at a dose of 30 mg / m2 weekly for three weeks (week 1 to week 3), followed by a one-week (week 4) rest period in a treatment cycle, until disease progression or an unacceptable adverse event occurred or other withdrawal criteria were met.

[0085] Cohort 4, containing 39 patients, received ADI-PEG 20 in combination with the modified FOLFOX6 regimen (mFOLFOX6 regimen, consisting of 85 mg / m2 oxaliplatin, 400 mg / m2 bolus of 5-FU, and 400 mg / m2 leucovorin on day one, followed by 2,400 mg / m2 of 5-FU in continuous infusion over 2 days), in which ADI-PEG 20 was administered intramuscularly to each patient at a dose of 36 mg / m2 weekly, and the mFOLFOX6 regimen was administered intravenously to each patient every Petition 870250077844, dated 01 / 09 / 2025, page 37 / 96 31 / 41 two weeks, until disease progression or an unacceptable adverse event occurred, or other abstinence criteria were met.

[0086] Cohort 5, containing 111 patients, received ADI-PEG 20 in combination with pemetrexed and cisplatin, in which ADI-PEG 20 was administered intramuscularly to each patient at a dose of 36 mg / m2 weekly, pemetrexed was administered intravenously to each patient at a dose of 500 mg / m2 every three weeks, and cisplatin was administered intravenously to each patient at a dose of 75 mg / m2 every three weeks, until disease progression or an unacceptable adverse event occurred or other withdrawal criteria were met.

[0087] Cohort-6, containing 27 patients, received ADI-PEG 20 in combination with pembrolizumab, in which ADI-PEG 20 was administered intramuscularly to each patient at a dose of 36 mg / m2 weekly, and pembrolizumab was administered intravenously to each patient at a dose of 200 mg every three weeks, until disease progression or an unacceptable adverse event occurred or other withdrawal criteria were met. Evaluation of therapeutic results

[0088] Overall survival (OS) duration for patients in cohorts 1 and 5 was calculated from the date patients were included for randomization until the date of death, regardless of any causes, or the date of loss to follow-up. OS duration for patients in cohorts 2, 3, 4, and 6 was calculated as the Petition 870250077844, dated 01 / 09 / 2025, page 38 / 96 32 / 41 time from the first dose of study treatment until death in any case; in the case of an individual being alive or lost to follow-up, they were censored at the last contact date. Statistical analysis

[0089] The ideal plasma arginine level cutoff point for survival outcomes was determined using maximum selected log-rank statistics. The Kaplan-Meier method was employed to estimate the median overall survival (OS) of the patients. A p-value < 0.05 determined by the log-rank test was considered statistically significant. Statistical analysis was conducted using software.

[0090] Example 1. Correlation of plasma arginine level with OS in patients with advanced HCC receiving arginine deprivation therapy alone or in combination with additional treatment.

[0091] To investigate whether plasma arginine levels were correlated with overall survival (OS) in cancer patients, a maximum selected log-rank statistic was performed. The analytical results are presented in Figures 1 to 12 and summarized in Tables 1 to 3, respectively. Table 1. Correlation of plasma arginine levels with overall survival (OS) in cancer patients receiving specific treatments. Cohort Treatment Indication Sample Size cohort·11 Arginine (μηοΙ / L) # of Event # of Censored SG Media121 (95% Cl)131 p-value[4] cohort1 ADI-PEG 20 (18 mg / m2) HCC 184 84.2 >= 84.2 143 41 8.6 (7.3, 10.5) 0.0057 Petition 870250077844, dated 01 / 09 / 2025, page 39 / 96 33 / 41 238 <84.2 202 36 5.7(4.9, 7.2) cohort5 ADI-PEG 20 (36 mg / m2) + Pemetrexed (500 mg / m2) + Cisplatin (75 mg / m2) Malignant Pleural Mesothelium (MPM) 72 8.2 >= 68.2 58 14 12.5 (9.8, 14.2) 0.0011 39 < 68.2 36 3 6.5 (3.8, 8.8) [1] Log-rank statistics selected to the maximum, [2] Me ses, [3] Kaplan-Meier product boundary estimates, [4] p-value comparing treatment groups is based on the log-rank test. Table 2. Correlation of plasma arginine levels with overall survival in cancer patients receiving specific treatments.

[0092] Protocol No. Treatment Indication Sample Size Cutoff [1] Arginine (μηοΙ / L) Event # Censored # SG Media·21 (95% Cl)131 p-value[4] cohort-2 ADI-PEG 20 (36 mg / m2) + Docetaxel (75 mg / m2) Advanced solid tumors with emphasis on castration-resistant prostate cancer (CRPC) and advanced non-small cell lung cancer (NSCLC) 12 97.5 >= 97.5 5 7 40.7 (7.2, 40.7) 0.0117 19 <97.5 14 5 14.6 (5.7, 16.0) cohort-3 ADI-PEG 20 (36 mg / m2) + Cisplatin (30 mg / m2) HCC HCC with coexisting biliary carcinoma (BTC) or just BTC Cutaneous melanoma Carcinoma 23 122 >= 122 16 7 15.7 (8.9, 28.5) 0.0006 55 < 122 47 8 6.4 (3.4, 8.2) Petition 870250077844, dated 01 / 09 / 2025, page 40 / 96 34 / 41 Ovarian melanoma Uveal cohort-4 ADI-PEG 20 (36 mg / m2) + FOLFOX Gastroesophageal cancer Colorectal carcinoma Hepatocellular carcinoma 37 34 >=34 30 7 9.5 (7.5, 15.1) 0.0083 2 <34 2 0 4.3 (4.0, 4.6) cohort-6 ADI-PEG 20 36 mg / m2 + Pembrolizumab 200 mg Advanced Solid Cancers 10 60.2 >= 60.2 2 8 , (2.5 ,,) 0.0119 17 < 60.2 13 4 6.6 (1.8, 7.8) [1] Maximum selected log-rank statistics, [2] Mes, [3] Kaplan-Meier product limit estimates, [4] p-value comparing treatment groups is based on the log-rank test Example 2. The procedure for determining the arginine (e.g., HCC) cutoff point.

[0093] First step - determining the arginine cutoff value. HCC patients agree to participate in the Polaris HCC clinical trial, and Polaris collects plasma from patients before the first treatment with ADI-PEG 20. Then, pharmacodynamics will be assessed by measuring arginine and citrulline levels in peripheral blood using LCMS. A total of 633 patients (422 for the ADI-PEG 20 group and 211 for the placebo group) were included in the study. Then, the clinical database is locked when the number of deaths reaches the sample size calculation requirement at the beginning of the study design. In addition, the statistician calculates the overall survival of each patient. The arginine cutoff point is determined using the maximum selected log-rank statistic (the statistician enters the arginine value and the Petition 870250077844, dated 01 / 09 / 2025, page 41 / 96 35 / 41 overall survival rate for each patient in the statistical model).

[0094] Second stage - validating the efficiency of the arginine cutoff point. 422 HCC patients treated with ADI-PEG 20 are categorized into two groups: a high-arginine group and a low-arginine group. Then, overall survival is summarized using Kaplan-Meier limit-product estimates. Point estimates (25th, 50th, and 75th percentiles), along with 95% confidence intervals, will be provided for each treatment group. Survival estimates will also be presented graphically for each treatment group. In addition, treatments will be compared using a stratified log-rank test.

[0095] In one embodiment, a method for identifying in vitro the arginine threshold in biological samples from individuals with cancer to predict whether individuals respond to arginine deprivation therapy, comprising: providing a biological sample, the biological sample collected from an individual before administration of an arginine deprivation agent; determining an arginine concentration; measuring the arginine concentration in the biological sample; calculating overall survival, overall survival statistics are evaluated after individuals undergo arginine deprivation therapy; the arginine cutoff point is determined through the following steps: (1) Identifying the highest value of the standardized log-rank statistics on the Y-axis of the statistical graph; (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 point plus Petition 870250077844, dated 01 / 09 / 2025, p. 42 / 96 36 / 41 high is considered the arginine cutoff value. (e.g., the arginine cutoff point is 84.2 μmol / L in cohort-1, and see Figure 1); Calculate an arginine threshold, based on the arginine cutoff point as the highest arginine value, gradually decrease the arginine value and calculate the corresponding p-value for each arginine value, continuing this process until the p-value for the nth arginine value is greater than 0.05. Then, the arginine value corresponding to the (n-1)th arginine value is the arginine threshold (see Table 3); where, when the arginine concentration of individuals is greater than or equal to the arginine threshold, it means that the individual with cancer responds well to arginine deprivation therapy.

[0096] According to the results, 422 patients in cohort 1 were treated with ADI-PEG 20 18 mg / m2, with the estimated arginine cutoff point for overall survival set at 84.2 μmol / L. The maximum recorded log-rank statistic was M = 2.8792 (Figure 1). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 84.2 μmol / L, is illustrated in the figure. The group with high arginine content (arginine >= 84.2 μmol / L) had a median overall survival of 8.6 months (95% CI: 7.3; 10.5), while the group with low arginine content (arginine < 84.2 μmol / L) had a median overall survival of 5.7 months (95% CI: 4.9; 7.2), indicating that the group with high arginine content had greater survival (p-value = 0.0057) in cohort 1 (Figure 2 and Table 1). Petition 870250077844, dated 01 / 09 / 2025, page 43 / 96 37 / 41

[0097] According to the previous study of ADI-PEG 20 monotherapy for HCC in the treatment phase, the arginine cutoff value was 84.2 μmol / L, based on selected maximum classification statistics. Patients with arginine levels > 84.2 μmol / L may survive longer than those with arginine levels < 84.2 μmol / L.

[0098] To include more patients, prognostic variable analysis was used to determine the arginine threshold. Based on the cutoff point of 84.2 μmol / L for arginine, the highest arginine value is considered, followed by a gradual decrease in arginine values ​​as follows: 83 μmol / L, 82 μmol / L, 81 μmol / L, 80 μmol / L, and 79 μmol / L. In Table 3, the corresponding significant *P value for each arginine is less than 0.05. However, when arginine is 78 μmol / L, and for the first time, the *P value is greater than 0.05. Therefore, 78 μmol / L is defined as the nth arginine. The anterior arginine (n-1), which is 79 μmol / L, serves as the arginine threshold. The survival of individuals above the arginine threshold is greater than that of individuals with low arginine levels, reaching statistical significance. According to the statistical results, the arginine threshold is mostly lower than the cutoff point for arginine between 5% and 10% (Table 3). Table 3. Cutoff value for cohort 1 Arginine Cutoff Point (gmol / L) Level N # of event # of censor SG Average (95% Cl) p-value Judgment of the nth value of Arginine 78 >=78 215 172 43 8.17 (6.9, 9.4) 0.0671 nth Petition 870250077844, dated 01 / 09 / 2025, p. 44 / 96 38 / 41 <78 207 173 34 6.17 (4.93, 7.63) 79 >=79 211 168 43 8.17 (6.9, 9.53) 0.0414* (n1) eth <79 211 177 34 5.9 (4.9, 7.43) 80 >=80 208 165 43 8.3 (7.13, 9.57) 0.0223* <80 214 180 34 5.7 (4.9, 7.4) 81 >=81 203 161 42 8.3 (7.13, 9.93) 0.0383* <81 219 184 35 5.7 (4.9, 7.4) 82 >=82 199 158 41 8.3 (7.13, 9.93) 0.0342* <82 223 187 36 5.7 (4.93, 7.4) 83 >= 83 196 155 41 8.47 (7.3, 10.0) 0.0211* <83 226 190 36 5.7 (4.93, 7.33) 84.2 >= 84.2 184 143 41 8.57 (7.33, 10.47) 0.0057* Arginine cutoff point < 84.2 238 202 36 5.7 (4.9, 7.3) * A p-value of less than 0.05 is considered significant.

[0099] According to the results, 31 patients from cohort 2 were treated with ADI-PEG 20 36 mg / m2 + Docetaxel 75 mg / m2, with the estimated arginine cutoff point for overall survival set at 97.5 μmol / L. The maximum recorded log-rank statistic was M = 3.0599 (Figure 3). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 97.5 μmol / L, is illustrated in the figure. The high-arginine group (arginine >= 97.5 μmol / L) had a median overall survival of 40.7 months (95% CI: 7.2, 40.7), while the low-arginine group Petition 870250077844, dated 01 / 09 / 2025, page 45 / 96 39 / 41 (arginine < 97.5 μηοI / L) had a median overall survival of 14.6 months (95% CI: 5.7, 16.0), indicating that the group with high arginine content had greater survival (p-value = 0.0117) in cohort 2 (Figure 4 and Table 2).

[00100] According to the results, 78 patients in cohort 3 were treated with ADI-PEG 20 36 mg / m2 + Cisplatin 30 mg / m2, with the estimated cutoff point for overall survival for arginine set at 122 μmol / L. The maximum recorded log-rank statistic was M = 3.6943 (Figure 5). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 122 μmol / L, is illustrated in the figure. The group with high arginine content (arginine >= 122 μmol / L) had a median overall survival of 15.7 months (95% CI: 8.9; 28.5), while the group with low arginine content (arginine < 122 μmol / L) had a median overall survival of 6.4 months (95% CI: 3.4; 8.2), indicating that the group with high arginine content had greater survival (p-value = 0.0006) in cohort 3 (Figure 6 and Table 2).

[00101] According to the results, 39 patients from cohort 4 were treated with ADI-PEG 20 36 mg / m2 + FOFLOX, with the estimated cutoff point for overall arginine survival set at 34 μmol / L. The maximum recorded log-rank statistic was M = 1.6007 (Figure 7). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 34 μmol / L, is illustrated in the figure. The high-arginine group (arginine >= 34 μmol / L) had a median overall survival of 9.5 months (95% CI: 7.5, 15.1), while the low-arginine group (arginine < 34 μmol / L) Petition 870250077844, dated 01 / 09 / 2025, page 46 / 96 Group 40 / 41 showed a median overall survival of 4.3 months (95% CI: 4.0, 4.6), indicating that the group with high arginine content had greater survival (p-value = 0.00083) in cohort 4 (Figure 8 and Table 2).

[00102] According to the results, 111 patients in cohort 5 were treated with ADI-PEG 20 36 mg / m2 + Pemetrexed 500 mg / m2 + Cisplatin 75 mg / m2, with the estimated cutoff point for overall arginine survival set at 68.2 μmol / L. The maximum recorded logrank statistic was M = 3.043 (Figure 9). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 68.2 μmol / L, is illustrated in the figure below. The group with high arginine content (arginine >= 68.2 μmol / L) had a median overall survival of 12.5 months (95% CI: 9.8; 14.2), while the group with low arginine content (arginine < 68.2 μmol / L) had a median overall survival of 6.5 months (95% CI: 3.8; 8.8), indicating that the group with high arginine content had greater survival (p-value = 0.0011) in cohort 5 (Figure 10 and Table 1).

[00103] According to the results, 27 patients were treated with ADI-PEG 20 36 mg / m2 + Pembrolizumab 200 mg, with the estimated cutoff point for overall arginine survival set at 60.2 μmol / L. The maximum recorded log-rank statistic was M = 3.0899 (Figure 11). The difference in overall survival time between the two groups, defined by an arginine cutoff point of 60.2 μmol / L, is illustrated in the figure below. Median OS cannot be estimated due to the high censoring rate, as most individuals are still alive. The group with low Petition 870250077844, dated 01 / 09 / 2025, page 47 / 96 41 / 41 arginine content (arginine < 60.2 μηοI / L) showed a median survival of 6.6 months (95% CI: 1.8, 7.8), indicating that the group with high arginine content had greater survival (p-value = 0.0119) in cohort 6 (Figure 12 and Table 2).

[00104] Taken together, plasma arginine levels can predict the outcome (i.e., overall survival) of arginine deprivation therapy, alone or in combination with different anticancer treatments, including FOLFOX, docetaxel, cisplatin, pemetrexed, and pembrolizumab.

[00105] It should be understood that the above description of embodiments is given only as an example and that various modifications may be made by those with average skill in the art. The specification, examples, and data above provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with common skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of the present disclosure. Petition 870250077844, dated 01 / 09 / 2025, p. 48 / 96

Claims

1 / 3 CLAIMS 1. A method for identifying in vitro the arginine threshold in biological samples from individuals with cancer to predict whether individuals respond to arginine deprivation therapy, characterized in that it comprises: providing a biological sample, the biological sample collected from an individual before administration of an arginine deprivation agent; determining an arginine concentration by measuring the arginine concentration in the biological sample; calculating an overall survival, the overall survival statistics of which are evaluated after individuals undergo arginine deprivation therapy; calculating an arginine cutoff point by entering the arginine concentration and overall survival of individuals into the statistical method of maximally selected log-rank statistics to determine the arginine cutoff point;and calculate an arginine threshold, based on the arginine cutoff point as the highest arginine value, gradually decrease the arginine value and calculate the corresponding p-value for each arginine value, continuing this process until the p-value for the nth arginine value is greater than 0.05, then the (n-1)-th arginine value is the arginine threshold;wherein, when the arginine concentration of individuals is greater than or equal to the arginine threshold, it means that the individual with cancer responds well to arginine deprivation therapy.

2. Method, according to claim 1, characterized in that the results of the maximum selected log-rank statistic, further comprising a statistical graph, will be generated with arginine levels plotted on the X-axis and standardized log-rank statistics on the Y-axis.

3. Method, according to claim 2, characterized in that the arginine cutoff point is determined through the following steps: (1) identifying the highest value of the standardized log-rank statistic on the Y-axis of the statistical graph; (2) determine the highest point on the Y-axis and find the corresponding arginine value on the X-axis; and (3) the arginine level corresponding to this highest point is considered the arginine cutoff value.

4. Method according to claim 1, characterized in that the arginine limit is analyzed by log-rank test statistics.

5. Method according to claim 1, characterized in that the arginine limit is less than the arginine cutoff point between 5% and 10%. 6.A method according to claim 1, characterized in that the cancer is selected from the group consisting of breast cancer, brain tumor, colorectal cancer, squamous cell carcinoma of the head and neck, 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 a combination thereof.

7. A method according to claim 6, Petition 870250077844, dated 01 / 09 / 2025, page 50 / 96 3 / 3, characterized in that the cancer is hepatocellular carcinoma (HCC) and the arginine cutoff point is 84.2 μmol / L.

8. Method according to claim 6, characterized in that the cancer is hepatocellular carcinoma (HCC) and the arginine limit is 79 μmol / L.

9. Method according to claim 1, characterized in that the biological sample is plasma. 10.A method according to claim 1, characterized in that the arginine-depriving agent is selected from the group consisting of a recombinant arginine deiminase (rADI), a recombinant arginase (rArg), a recombinant arginine decarboxylase (rADC), a pegylated form of rADI, rArg, rADC, difluoromethylornithine (DFMO), and a combination thereof.

11. A method according to claim 1, characterized in that the arginine-depriving agent is further combined with an anticancer agent.

12. A method according to claim 11, characterized in that the anticancer agent is selected from the group consisting of FOLFOX, docetaxel, cisplatin, pemetrexed, pembrolizumab, and a combination thereof. 13.Method, according to claim 12, characterized in that the arginine-depriving agent is the pegylated form of recombinant arginine deiminase (rADI) and the anticancer agent is cisplatin and the arginine cutoff value is 122 μmol / L. Petition 870250077844, dated 01 / 09 / 2025, page 51 / 96.

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