Pharmaceutical composition containing an AKR1C3 activating compound and an immune checkpoint inhibitor

Abstract

Provided are pharmaceutical compositions comprising the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate and at least one therapeutic agent, including a chemotherapeutic agent or a biological agent, and medical uses thereof.

Description

[Technical Field] 【0001】 The present invention relates to a composition comprising the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate in combination with at least one therapeutic agent comprising a chemotherapeutic agent or a biological agent, and to the medical uses thereof. [Background technology] 【0002】 Cancer is one of the leading causes of morbidity and mortality in humans. Cancer treatment is challenging because it is difficult to kill cancer cells without damaging or killing healthy cells. Damage to or death of healthy cells during cancer treatment can cause adverse side effects in patients and may limit the amount of chemotherapy drugs that can be administered to cancer patients. 【0003】 Aldo-ketereductase family 1 member C3 (AKR1C3) is an enzyme encoded by the human AKR1C3 gene. This gene encodes a member of the aldo / ketereductase superfamily, which consists of more than 40 known enzymes and proteins. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and / or NADPH as cofactors. It is also known as type 5, 17β-hydroxysteroid dehydrogenase (17β-HSD), and prostaglandin F synthase. AKR1C3 is one of 15 gene family of aldo-ketereductases (AKR). AKR1C3 was originally found in the human prostate gland. (1) and placenta (2) It was cloned from a cDNA library. AKR1C3 is a monomeric cytosolic NAD(P)(H)-dependent oxidoreductase with 323 amino acids and a molecular weight of 37 kDa. (1)。AKR1C3 shares high sequence homology with the related human AKR1C family, including AKR1C1, AKR1C2, and AKR1C4. AKR1C3 catalyzes the metabolism of androgens, estrogens, progesterone, and prostaglandins (PG), and then is involved in the regulation of nuclear receptor activity (3、4) 。AKR1C3 is expressed in normal tissues, including steroid hormone-dependent and steroid hormone-independent cells, and the average expression level is low, except in the liver, kidney, and small intestine (5) 。Multiple studies have demonstrated that AKR1C3 is abnormally overexpressed in many malignant solid tumors and hematological tumors. The data show that more than 50% of liver cancers, bladder cancers, kidney cancers, and gastric cancers were detected with high expression of AKR1C3 with immunohistochemical score (IHC score) ≥ 4 on a scale of 0 - 6 (6) 。AKR1C is highly expressed in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (7) 。 【0004】 Upregulation of AKR1C3 in cancer is reported to be associated with the metastasis of castration-resistant prostate cancer (CRPC (8) ) and colorectal cancer (CRC (9) ), and is also associated with poor prognosis and low survival rate (10、11) 。Furthermore, many types of treatment resistance are thought to be due to the overexpression of AKR1C3. Chemotherapy resistance to doxorubicin (12、13) , enzalutamide (14) , abiraterone (15) , and methotrexate (16) has been reported to be directly related to the high level of AKR1C3 expression in cells. Esophageal cancer (17) , prostate cancer (18) , NSCL cancer cells (19)Radiotherapy resistance in this region is associated with AKR1C3 overexpression. The main mechanism of action of AKR1C3 against ionizing radiation is to reduce intracellular ROS (reactive oxygen species), increase PGF2α, and subsequently activate MAP kinase and inhibit PPARγ, resulting in a significant reduction in DNA damage. (18) Immunotherapy resistance is also thought to be caused by high expression of AKR1C3. One study used whole-genome microarrays and multiplex quantitative (q)RT-PCR gene expression analysis. (20) Based on this, high expression of AKR1C3 has been shown to be associated with failure of PD-1 targeted therapy in patients with PD-L1-positive advanced renal cell carcinoma (RCC). Because AKR1C3 is tumor-specifically overexpressed, designing AKR1C3-activated prodrugs is an attractive approach for cancer-specific targeting. One such example is PR104, an AKR1C3-activated prodrug, which was originally designed as a hypoxia-activated prodrug. (22~24) It showed excellent antitumor activity in vitro and in vivo. (6、21) . 【0005】 The anticancer prodrug of formula I-1 of this application (referred to herein as OBI-3424) is a chemically synthesized potent nitrogen mustard, which is selectively cleaved by AKR1C3 in the presence of NADPH to a cytotoxic aziridine (referred to herein as OBI-2660). The active molecule OBI-2660 released by OBI-3424 is similar to the standard chemotepa and mitomycin C, causing DNA alkylation and crosslinking at the N7 (or O6) position of guanine. The prodrug OBI-3424 is currently under development in Asian countries by Ascentawits Pharmaceuticals, LTD and in non-Asian countries by OBI Pharma, Inc. (drug code OBI-OBI-3424) for the treatment of malignant tumors. The prodrug OBI-3424 is currently being studied in multiple Phase I clinical trials in the United States (NCT04315324 and NCT03592264) and China (CXHL1900137 and CXHL2000263) for the treatment of more than 14 types of human cancer, including solid tumors and hematological malignancies. Because AKR1C3 is highly expressed in tumors, the prodrug OBI-3424 is specifically activated in tumors but not in normal cells expressing low levels of AKR1C3, thus achieving tumor-specific targeting. Furthermore, the tumor-selective activation of OBI-3424 is distinct from non-selective conventional alkylating agents such as cyclophosphamide and ifosfamide, indicating the potential of OBI-3424 as a broad-spectrum, highly selective antitumor agent. The prodrug OBI-3424 has been reported to demonstrate potent efficacy against preclinical models of T-ALL in vitro and in vivo. (25、26) . 【0006】 In the presence of NADPH, the reduction of OBI-3424 is mediated by AKR1C3, releasing the cytotoxic moiety OBI-2660, an aziridine bisalkylating agent, which leads to DNA crosslinking at the N7 (or O6) position of guanine and subsequent cell death. 【0007】 [ka] 【0008】 Prodrugs, designed to target cancer cells, have emerged as an attractive strategy for cancer treatment in recent years. However, many prodrugs have failed in Phase 3 clinical trials due to a lack of effective biomarkers for patient selection. (27) Considering that AKR1C3 expression can be assessed using RT-PCR or immunohistochemistry, OBI-3424 can be developed in a clinically efficient manner by selecting patients with high AKR1C3 expression who are most likely to respond to the prodrug. AKR1C3 is chemically resistant. (13、14) , radiation resistant (19) and immune resistance (20) It has been proven that it is overexpressed during acquisition. Furthermore, cancers with homologous recombination deficiencies (HRDs), such as ovarian cancer, breast cancer, and pancreatic cancer, are known to be sensitive to DNA damage factors. (28) As a DNA alkylating agent, OBI-3424 may also be a promising candidate drug for treating HRD cancers with AKR1C3 expression. 【0009】 There remains a need for compounds suitable for treating cancer patients, namely selective AKR1C3 reductase-activating prodrugs, and novel selective and broad-spectrum anticancer agents. This invention satisfies this need. 【0010】 Programmed death-1 (PD-1) is an inhibitory receptor expressed on T cells, B cells, or monocytes. (29、30) PD-L1 and PD-L2 are ligands for PD-1, and it has been confirmed that when they bind to PD-1, they downregulate T cell activation and cytokine secretion. (31、32)The binding of PD-1 to PD-L1 or PD-L2 leads to downregulation of the immune response. Therefore, it has been proposed that blocking the PD-1 / PD-L1 pathway weakens the central and peripheral immune response to cancer. Targeting the PD1 and PD-L1 pathways has shown clinical efficacy in more than 15 types of cancer, including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and Hodgkin lymphoma. (33) However, many patients still do not respond. Some patients showed an initial response but developed tolerance over time. Therefore, identifying the mechanisms of resistance to combination therapy is urgently needed. [Overview of the Initiative] 【0011】 The present invention provides medical applications for compounds or pharmaceutically acceptable salts thereof, or solvates thereof, as disclosed in PCT Patent Application No. PCT / US2016 / 062114 (International Publication No. 2017087428), and provides compositions comprising such compounds or pharmaceutically acceptable salts thereof, isotopic variants or solvates, and anti-cancer medical applications thereof. 【0012】 In one embodiment, the present invention provides the use of a compound represented by formula I (represented herein as OBI-2870), 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, in the manufacture of a pharmaceutical for the treatment of a patient's cancer, wherein the AKR1C3 reductase level of the cancer is expressed as an AKR1C3 protein level or RNA level and is above a predetermined value. The AKR1C3 level is measured according to routine methods well known to those skilled in the art. 【0013】 [ka] 【0014】 According to a particular embodiment of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I-1 (represented herein as OBI-OBI-3424), or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I-2 (represented herein as OBI-3423). 【0015】 [ka] 【0016】 The preparation of the compounds of formula I, formula I-1, or formula I-2 is disclosed in PCT patent application number PCT / US2016 / 062114 (International Publication No. 2017087428), which is incorporated herein by reference in its entirety. Here, compound OBI-2870 is a 1:1 racemic mixture of R-enantiomer 3423 and S-enantiomer OBI-3424. 【0017】 Here, the salt may be a basic salt, and examples include salts of compounds having an inorganic base (alkali metal hydroxide, alkaline earth metal hydroxide, etc.) or an organic base (monoethanolamine, diethanolamine, triethanolamine, etc.). Alternatively, the salt may be an acidic salt, and examples include salts of compounds having an inorganic acid (hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, phosphoric acid, etc.) or an organic acid (methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.). Selecting and preparing acceptable salts, solvates, etc. of compounds is a well-known technique in the art. 【0018】 According to a specific embodiment of the present invention, the compound of formula I-1 or formula I-2 has an enantiomeric excess of 80% or more. Preferably, the compound has an enantiomeric excess of 90% or more, more preferably 95% or more. 【0019】 According to certain embodiments of the present invention, the compounds of formula I-1 or formula I-2 are substantially pure. According to certain embodiments of the present invention, cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer. 【0020】 According to a particular embodiment of the present invention, the cancer is liver cancer. The dosage of a drug used to treat cancer, or the dosage of a compound or salt contained in that drug, its isotopic variant or solvate, or other chemotherapeutic agents, usually depends on the specific compound being applied, the patient, the specific disease or condition and its severity, the route and frequency of administration, etc., and must be determined by the attending physician on a case-by-case basis. For the purposes of this disclosure, a typical daily dose may be in any of the following ranges, depending on the factors mentioned above: approximately 0.1 μg / kg to 1 μg / kg, up to 10 μg / kg, up to 100 μg / kg, up to 1 mg / kg, up to 10 mg / kg, up to 100 mg / kg, or more. In the case of repeated administration over several days or more, treatment is continued, depending on the condition, until the desired suppression of symptoms occurs, or until a sufficient level of treatment is achieved to alleviate the cancer or its symptoms. Exemplary dosing regimens include administering an initial dose of approximately 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, or higher, followed by weekly maintenance doses. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the physician wishes to achieve. For example, administration 1 to 4 times per week is possible. In specific embodiments, the frequency of administration may be once weekly, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once monthly, every 2 months, or every 3 months, or more. The progress of this treatment can be easily monitored using conventional techniques and assays. 【0021】 The above-mentioned pharmaceuticals may be in any dosage form for clinical administration, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated preparations, granules, dried powders, oral solutions, thin needles for injection, lyophilized powders for injection, or intravenous solutions. 【0022】 According to a particular embodiment of the present invention, the method further comprises the step of measuring the AKR1C3 reductase content of a patient's cancer cells using an AKR1C3 antibody, and when the AKR1C3 reductase content is measured to be above a predetermined value, the compound is administered to the patient. 【0023】 In another embodiment, the present invention provides a method for inhibiting cell proliferation, comprising the step of contacting cells with an effective amount of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, wherein the AKR1C3 reductase level of the cells is greater than or equal to a predetermined value, expressed as an AKR1C3 protein level or RNA level. 【0024】 According to a particular embodiment of the present invention, the method further comprises the step of measuring the AKR1C3 reductase content of cells using an AKR1C3 antibody, and once it is determined that the AKR1C3 reductase content is above a predetermined value, the compound is brought into contact with the cells. 【0025】 In another aspect, the present invention provides the use of a compound represented by formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, in the manufacture of a pharmacopoeia for inhibiting cell proliferation, wherein the AKR1C3 reductase level of the cells is greater than or equal to a predetermined value, expressed as an AKR1C3 protein level or RNA level. 【0026】 According to a particular embodiment of the present invention, the cells are cancer cells. In another embodiment, the present invention provides a composition comprising: (1) Compound represented by formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, or pharmaceutically acceptable salts, isotope variants or solvates thereof; and (2) At least one therapeutic agent comprising a chemotherapeutic agent or a biological agent. 【0027】 According to a particular embodiment of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I-1, or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I-2. 【0028】 According to a particular embodiment of the present invention, the anti-PD-1 / PD-L1 antibody is Bavencio® (avelumab), Opdivo® (nivolumab), Keytruda® (pembrolizumab), Imfinzi® (durvalumab), and / or Tecentriq® (atezolizumab). 【0029】 According to a particular embodiment of the present invention, when the cancer is liver cancer, the anti-PD-1 antibody is Keytruda® (pembrolizumab), and the anti-PD-1 antibody is Bavencio® (avelumab). 【0030】 According to certain embodiments of the present invention, the composition further comprises pharmaceutically acceptable excipients. Preferably, the excipients are selected from inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and oils. 【0031】 In another aspect, the present invention provides a method for treating cancer in a patient in need, comprising the step of administering an effective amount of the composition according to the present invention to the patient. According to a particular embodiment of the present invention, the method further comprises the step of measuring the AKR1C3 reductase content of a patient's cancer cells using an AKR1C3 antibody, and when the AKR1C3 reductase content is measured to be above a predetermined value, the composition is administered to the patient. 【0032】 Accordingly, embodiments of the present invention relate to combinations comprising a compound of formula I (OBI-2870), formula I-1 (OBI-3424), or formula I-2 (OBI-3423) and at least one inhibitor of an inhibitory immune checkpoint antigen. In certain embodiments, the immune checkpoint inhibitor is an anti-immune checkpoint antibody that inhibits / blocks the inhibitory immune checkpoint antigen. 【0033】 In one embodiment, the inhibitory immune checkpoint antigen is selected from the group consisting of PD-1 / PD-L1 antigen, CTLA-4 (cytotoxic T lymphocyte-associated protein 4), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif domain), Ceacam1 (carcinoembryonic antigen-associated cell adhesion molecule 1), LAIR-1 (leukocyte-associated immunoglobulin-like receptor 1), TIM-3 (T cell immunoglobulin and mucin domain 3), VISTA (T cell activation V domain Ig suppressor), KIR (killer cell immunoglobulin-like receptor), IDO (indoleamine-pyrrole 2,3-dioxygenase), B7-H3 (CD276), A2AR (adenosine A2A receptor), or CD47. 【0034】 In one embodiment, the anti-immune checkpoint antibody is an anti-PD-1 / PD-LI antibody, an anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibody, an anti-LAG-3 (lymphocyte-activating gene 3) antibody, an anti-TIGIT (T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif domain) antibody, an anti-Ceacam1 (carcinoembryonic antigen-associated cell adhesion molecule 1) antibody, an anti-LAIR-1 (leukocyte-associated immunoglobulin-like receptor-1) antibody, an anti-TIM-3 (T cell immunoglobulin and mucin domain-3) antibody, an anti-VISTA (T cell activation V domain Ig suppressor) antibody, an anti-KIR (killer cell immunoglobulin-like receptor) antibody, an anti-IDO (indoleamine-pyrrole 2,3-dioxygenase) antibody, an anti-B7-H3 (anti-CD276) antibody, an anti-A2AR (adenosine A2A receptor) antibody, or an anti-CD47 antibody. 【0035】 In one embodiment, the anti-PD-1 / PD-L1 antibody is Bavencio® (avelumab), Opdivo® (nivolumab), Keytruda® (pembrolizumab), Imfinzi® (durvalumab), and / or Tecentriq® (atezolizumab). [Brief explanation of the drawing] 【0036】 [Figure 1] Mean body weight for each group in Example 1. Body weight for the vehicle group, monotherapy group, and combination therapy group was recorded twice a week until day 35. No weight loss was observed in any of the treatment groups of humanized mice carrying human hepatocellular carcinoma (HepG2). Data are shown as mean ± SEM (N=5 for each group). Statistical analysis was performed by Student's t-test. [Figure 2]Average tumor weight of each group in Example 1. Mice were sacrificed on the 36th day after tumor cell inoculation, and the tumor weights of mice in the vehicle, single-treatment, and combined-treatment groups were recorded. The tumor weights in the OBI-3424 (G2) and OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combined-treatment groups were significantly suppressed compared with those in the vehicle (G1) group (p < 0.001). Furthermore, the tumor weights in the OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combined-treatment group were significantly suppressed compared with those in the anti-hPD-1 / anti-hPD-L1 (G3 / G4) treatment group (p < 0.05). The data were shown as mean ± SEM (N = 5 for each group). Statistical analysis was performed by Student's t-test. A P value < 0.05 was considered significant. One asterisk represents 0.05 < P < 0.001, and two asterisks represent P < 0.001. [Figure 3] Average tumor volume of each group in Example 1. The tumor volumes of mice in the vehicle, single-treatment, and combined-treatment groups were recorded twice a week until the 35th day. The tumor volumes in the OBI-3424 (G2) and OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combined-treatment groups were significantly suppressed compared with those in the vehicle (G1) group. The data were shown as mean ± SEM (N = 5 for each group). Statistical analysis was performed by Student's t-test. [Figure 4] Average body weight of each group in Example 2. The body weights of mice in the vehicle, low-dose and high-dose OBI-3424 treatment groups, and OBI-3424 + PD-1 antibody combined-treatment group were recorded twice a week until the 30th day. In humanized mice bearing human hepatocellular carcinoma HepG2 tumors, no weight loss was observed in the vehicle group, the OBI-3424 single-treatment group, or the combined-treatment group of OBI-3424 + PD-1 antibody. The data were shown as mean ± SEM (N = 5 for each group). Statistical analysis was performed by Student's t-test. [Figure 5]Average tumor weight of each group in Example 2. Mice were sacrificed on the 30th day after administration of the test article. Tumor weights of mice in the OBI-3424 low-dose (G2), high-dose treatment (G3), and combination treatment of OBI-3424 + PD-1 antibody (G5 - G7) were significantly suppressed compared to the vehicle (G1) group (p < 0.05). Furthermore, the tumor weight in the combination treatment of high-dose OBI-3424 + PD-1 antibody (G6) was significantly decreased compared to the high-dose OBI-3424 single treatment (G3) (p < 0.05). Data were shown as mean ± SEM. Statistical analysis was performed by Student's t-test. A P-value < 0.05 was considered significant. One star represents 0.05 < P < 0.001, and two stars represent P < 0.001. [Figure 6] Average tumor volume of each group in Example 2. Tumor volumes of mice in the vehicle group, OBI-3424 low-dose and high-dose treatment groups, and combination treatment group of OBI-3424 + PD-1 antibody were recorded twice a week until the 30th day. Tumor volumes in the OBI-3 high-dose treatment group (G3) and combination treatment groups of OBI-3424 + anti-hPD-1 (G5 - G7) were significantly suppressed compared to the vehicle (G1) group. Data were shown as mean ± SEM (N = 5 for each group). Statistical analysis was performed by Student's t-test. 【Mode for Carrying Out the Invention】 【0037】 Detailed Description of the Invention Hereinafter, the present invention will be described based on specific examples. Those skilled in the art will understand that these examples are only used to explain the present invention and are by no means intended to limit its scope. 【0038】 Definition The following definitions are provided for the reader's assistance. Unless otherwise defined, all technical terms, notations, and other scientific or medical or specialized terms used herein are intended to have meanings that are generally understood by those skilled in the art of chemistry and medicine. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for immediate reference. The inclusion of such definitions herein should not be construed as representing a substantial difference from the definitions of terms that are generally understood in the art. 【0039】 All numerical specifications, such as pH, temperature, time, concentration, and weight (including their respective ranges), are approximations that can typically be changed to (+) or (-) in increments of 0.1, 1.0, or 10.0 as needed. All numerical specifications should be understood to be preceded by the term "approximately." The reagents described herein are illustrative, and their equivalents may be known in the art. 【0040】 The terms “one,” “that,” and “the aforementioned” refer to multiple objects unless the context clearly indicates otherwise. Therefore, for example, a reference to one compound refers to one or more compounds, or at least one compound. Thus, the terms “one,” “one or more,” and “at least one” are used interchangeably herein. 【0041】 The terms “approximately” or “about” mean a tolerance for a particular value as determined by those skilled in the art, and which depends in part on how that value is measured or determined. In certain embodiments, the terms “approximately” or “about” mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms “approximately” or “about” mean within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. 【0042】 Where used herein, the terms “includes” or “contains” are intended to mean that a composition and method includes the enumerated elements but does not exclude other elements. “Essentially consisting of” as used to define a composition and method is intended to mean excluding other elements that are essentially important to that composition or method. “Consists of” is intended to mean, with respect to the claimed composition and substantial method steps, excluding anything beyond trace elements of other components. Embodiments defined by each of these transitional clauses are within the scope of the invention. Therefore, methods and compositions are intended to include additional steps and components (including / containing), or to include non-essential steps and compositions (essentially consisting of), or to intend only the described method steps or compositions (consisting of). 【0043】 The terms “administering” a drug to a patient or “administering” a drug to a patient (and their grammatical equivalents) refer to direct administration (which may be administration by a healthcare professional to a patient or self-administration) and / or indirect administration (which may be the act of prescribing a drug). For example, a physician who instructs a patient to self-administer a drug, and / or a physician who provides a patient with a prescription for a drug, is administering a drug to the patient. 【0044】 The term “antibody” is further intended to encompass antibodies, digestive fragments, specific parts thereof and variants, including antibody mimics or parts of antibodies that mimic the structure and / or function of an anti-cancer antibody or a specific fragment or part thereof, including single-chain antibodies and fragments thereof, each comprising at least one CDR derived from the anti-cancer antibody of the present invention. 【0045】 The term "biological agonists" includes peptides, proteins, antibodies, hormones, cytokines, chemokines, and any combination thereof. The term "cancer" refers to leukemia, lymphoma, carcinoma, and other malignant tumors, including solid tumors, that can grow uncontrollably, locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon and / or rectal cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nerve tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, and thyroid cancer. Other specific examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermal carcinoma, giant cell tumor, glioblastoma multiforme, hairy cell tumor, enteric ganglion neuroma, hyperplastic corneal neuroma, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, and marphanoid. Examples include habitus tumors, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumors, pheochromocytoma, polycythemia vera, primary brain tumors, small cell lung tumors, squamous cell carcinoma (both ulcerative and papillary types), hyperplasia, seminomas, soft tissue sarcomas, retinoblastoma, rhabdomyosarcoma, renal cell tumors, focal skin lesions, reticular sarcoma, and Wilms' tumor. 【0046】 As used herein, the term “chemotherapeutic agent” refers to compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), meltansine (DM1), anthracyclines, pyrrolobenzodiazepines, α-amanitin, tubulisin, benzodiazepines, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK222584, oxaliplatin, leucovorin, rapamycin, lapatinib, ronafarnib (SARASAR®, SCH66336), sorafenib, and gefi Tinib, AG1478, AG1571, alkylating agent, alkyl sulfonate, aziridine, ethyleneimine, methylamelamine, acetogenin, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dorastatin, duocalmycin, eleuterobin, pancratistatin, sarcodictin, spongstatin, chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide Salts, melphalan, nobembichin, fenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, dynemycin, clodronate, esperamycin, neocartinostatin chromophore, acrasinomycin, actinomycin, autramycin, azaserine, bleomycin, kakutinomycin, carabicin, camino Caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rodorubicin, streptonigrin,Streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, methotrexate, 5-fluorouracil (5-FU), denopterin, pteropterin, trimethrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone propionate, epithiostanol, mepitiostane, test Lactone, aminoglutethimide, mitotane, trilostane, floric acid, acegraton, aldofamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabucil, bisanthren, edatraxate, defofamine, demecorsin, diaziquan, elformithine, eriptinium acetate, epotilon, etogluside, gallium nitrate, hydroxyurea, lentinan, lonidainine, May Tansine, anthamitocin, mitogwazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, fenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, trichothecene, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mi Tractor, Pipobroman, Gacytosine, Arabinoside, Cyclophosphamide, Thiotepa, Taxoid, Paclitaxel, Docetaxel, Chloranbucil, Gemcitabine, 6-Thiogunine, Mercaptopurine, Methotrexate, Cisplatin, Carboplatin, Vinblastine, Platinum, Etoposide, Ifosfamide, Mitoxantrone, Vincristine, Vinorelbine, Novantrone, Teniposide, Edatrexate, Daunomycin, Aminopterin, Xeroda, Ibandronate,Examples include topoisomerase inhibitors, difluoromethylornithine (DMFO), retinoids, and capecitabine. 【0047】 The term "combination therapy" refers to combination therapy, where the amounts of the OBI-3423 / OBI-3424 compound and / or other biological or chemical agents, when administered sequentially or simultaneously (as co-administration and / or co-formulation) on the same or different days during the treatment cycle, are therapeutically effective and produce a therapeutically additive or greater synergistic effect. 【0048】 The terms “contact” or “in contact” refer to bringing a therapeutic agent together with cells or tissues so that physiological and / or chemical effects result from such contact. Contact can be performed in vitro, ex vivo, or in vivo. In one embodiment, a therapeutic agent is brought into contact with cells in a cell culture (in vitro) to measure the effect of the therapeutic agent on the cells. In another embodiment, contact between a therapeutic agent and cells or tissues includes administering the therapeutic agent to a subject having the cells or tissues to be contacted. 【0049】 The term "optically active" refers to a collection of molecules having an enantiomeric excess of approximately 10% or more, approximately 20% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, approximately 99.5% or more, approximately 99.8% or more, or approximately 99.9% or more. In certain embodiments, the enantiomeric excess of an optically active compound is approximately 90% or more, approximately 95% or more, approximately 98% or more, or approximately 99% or more. The enantiomeric excess of a compound can be determined by any standard method used by those skilled in the art, including, but not limited to, chiroptical chromatography (gas chromatography, high-performance liquid chromatography, and thin-layer chromatography) using an optically active stationary phase, isotopic dilution, electrophoresis, calorimetry, polarimetric analysis, NMR spectroscopy by chiral derivatization, and NMR spectroscopy using chiral solvating agents or chiral shift reagents. 【0050】 When describing optically active compounds, the prefixes R and S are used to indicate the absolute configuration of the molecule around its chiral center. The term “substantially pure” means that a substance is sufficiently homogeneous to appear to be free of readily detectable impurities when determined by standard analytical methods used by those skilled in the art, such as, but not limited to, thin-layer chromatography (TLC), gel electrophoresis, high-performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectrometry (MS), or is sufficiently pure to the extent that further purification does not alter the physical, chemical, biological, and / or pharmacological properties of the substance (such as enzyme activity or biological activity) to a detectable degree. In certain embodiments, “substantially pure” refers to a collection of molecules in which, when measured by standard analytical methods, at least about 50% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 98% by weight, at least about 99% by weight, or at least about 99.5% by weight of the molecule is a single stereoisomer of the compound. 【0051】 The terms "patient" and "subject" are used interchangeably to refer to mammals requiring cancer treatment. Typically, a patient is a human. Typically, a patient is a human being diagnosed with cancer. In certain embodiments, "patient" or "subject" may refer to non-human mammals, such as non-human primates, dogs, cats, rabbits, pigs, mice, or rats, used for screening, characterizing, and evaluating drugs and treatments. 【0052】 The term "prodrug" refers to a compound that, after administration, is metabolized or otherwise converted into a biologically active compound (or drug) that is more active in at least one property. Prodrugs are chemically modified in such a way that their activity is reduced or inactive compared to the drug, but the chemical modification is such that the corresponding drug is produced by metabolism or other biological processes after the prodrug is administered. Compared to the active drug, prodrugs may have altered metabolic stability or transport properties, fewer side effects or lower toxicity, or improved flavor (see, for example, Nogrady, 1985, Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pp. 388-392, incorporated herein by reference). Prodrugs may be synthesized using reactants other than the corresponding drug. 【0053】 The term "solid tumor" refers to solid tumors that include, but are not limited to, metastatic tumors in the bone, brain, liver, lungs, lymph nodes, pancreas, prostate, skin, and soft tissues (sarcomas). The term "therapeutic dose" of a drug refers to the amount of drug that, when administered to a cancer patient, would produce the intended therapeutic effect, such as reduction, improvement, mitigation, or elimination of one or more symptoms of cancer in that patient. The therapeutic effect does not necessarily occur with a single dose; it may only occur after a series of doses. Therefore, a therapeutic dose may be administered in more than one dose. 【0054】 The term “treatment” of a condition or patient refers to taking measures to obtain beneficial or desirable outcomes, including clinical outcomes. For the purposes of this invention, beneficial or desirable clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms of cancer; reduction of the severity of the disease; delay or slowing of the progression of the disease; relief, reduction, or stabilization of the disease state; or other beneficial outcomes. In some cases, treatment of cancer may result in a partial response or stable disease. 【0055】 The term "tumor cell" refers to tumor cells of any appropriate species of mammal, such as rodents, canids, felines, equids, or humans. The term "isotope variant" refers to a compound that contains an unnatural proportion of isotopes in one or more atoms that make up such a compound. In certain embodiments, the "isotope variant" of a compound is hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12( 12 C), carbon-13( 13 C), carbon-14( 14 C), nitrogen 13( 13 N), Nitrogen 14( 14 N), Nitrogen 15( 15 N), oxygen 14( 14 O), oxygen 15 ( 15 O), oxygen 16( 16 O), oxygen 17( 17 O), oxygen 18( 18 O), fluorine 17( 17 F), Fluorine 18( 18 F), Lin31( 31 P), Lin32( 32 P), Lin33( 33 P), sulfur 32( 32 S), sulfur 33( 33 S), Sulfur 34 ( 34 S), sulfur 35( 35 S), sulfur 36( 36 S), Chlorine 35 ( 35 Cl), Chlorine 36( 36 Cl), Chlorine 37( 37 Cl), bromine 79( 79 Br), bromine 81 ( 81 Br), Iodine-123 ( 123 I) Iodine-125 ( 125 I) Iodine-127 ( 127 I) Iodine-129 ( 129 I), and iodine-131 ( 131contains one or more isotopes including, but not limited to, these in unnatural proportions. In certain embodiments, an "isotope variant" of a compound is in a stable form, i.e., non-radioactive. In certain embodiments, an "isotope variant" of a compound contains one or more isotopes including, but not limited to, hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-37 ( 37 Cl), bromine-79 ( 79 [[ID=3)]]Br), bromine-81 ( 81 Br), and iodine-127 ( 127 I) in unnatural proportions. In certain embodiments, an "isotope variant" of a compound is in an unstable form, i.e., radioactive. In certain embodiments, an "isotope variant" of a compound contains one or more isotopes including, but not limited to, tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-35 ( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I), iodine-125 ( 125 I), iodine-129 ( 129 I), and iodine-131 ( 131The compounds provided herein contain, but are not limited to, one or more isotopes in unnatural proportions. Where possible in the judgment of those skilled in the art, any hydrogen may be, for example 2 It could be H, or any carbon as an example 13 It could be C, or any nitrogen as an example. 15 It can be N, and any oxygen is 18 It will be understood that it can be O. In certain embodiments, the "isotope variant" of the compound contains an unnatural proportion of deuterium. 【0056】 The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute (e.g., compounds provided herein) and one or more molecules of a solvent present in stoichiometric or non-stoichiometric amounts. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in crystalline form. In another embodiment, the complex or aggregate is in amorphous form. When the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate. 【0057】 The term “pharmaceutically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense that it is compatible with other components of a pharmaceutical formulation and is suitable for use in contact with human and animal tissues or organs without causing excessive toxicity, irritation, or allergic reactions. 【0058】 The term "immune checkpoint inhibitor" refers to molecules that inhibit / block the suppressive immune checkpoint system and has emerged as an effective treatment for advanced tumors. These include therapeutic antibodies that block cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1), which are used for several types of tumors. (34) PD-1 (programmed cell death protein, CD279), a member of the B7 / CD28 family of receptors, is a monomeric molecule expressed on the cell surface of activated leukocytes such as T cells, B cells, NK cells, and bone marrow-derived suppressor cells. Its expression is finely regulated by interactions between genetic and epigenetic mechanisms. Known ligands for PD-1 are PD-L1 and PD-L2. (35) . 【0059】 PD-L1 (Programmed Cell Death Protein Ligand 1, B7H1, CD274) is expressed at low levels and is upregulated by cell activation in hematopoietic cells, including T cells, B cells, bone marrow cells, and dendritic cells, as well as non-hematopoietic cells (e.g., lung, heart, endothelial, pancreatic islet cells, keratinocytes, etc.), especially cancer cells. PD-L2 (Programmed Cell Death Protein Ligand 2, B7-DC, CD273) is upregulated in macrophages, dendritic cells (DCs), and activated CD4 + and CD8 +PD-L1 and PD-L2 are expressed in lymphocytes and some solid tumors (ovarian cancer, small cell lung cancer, esophageal cancer). PD-L1 and PD-L2 expression has also been detected in normal cells and cancer-associated fibroblasts. Both PD-L1 and PD-L2 interact with additional receptors. PD-L1 interacts with the CD28 ligand CD80, while PD-L2 interacts with repulsion-inducing molecule (RGM)b, which is expressed in macrophages and other cell types. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). In T lymphocytes, the interaction of PD-1 and its ligand leads to phosphorylation of two tyrosines in the intracellular tail of PD-1. When SH2 domain-containing protein tyrosine phosphatases (SHP-1 and / or SHP-2) are recruited to the ITSM cytoplasmic region of PD-1, downstream signaling of the T cell receptor is inhibited, thereby inhibiting T cell proliferation and cytokine production. PD-1 also exerts other effects on T cells. For example, by inhibiting the Akt and Ras pathways, PD-1 induction suppresses the transcription of the ubiquitin ligase component SKP2. This impairs the degradation of p27(kipl), a cyclin-dependent kinase inhibitor mediated by SKP2, thereby blocking cell cycle progression. Furthermore, PD-1 can promote apoptosis through multiple mechanisms. In addition to directly inhibiting T cell activation, PD-L1-mediated induction of PD-1 can induce the development of regulatory T cells (Tregs), which are important mediators of peripheral tolerance that actively suppress effector T cells. PD-1-induced Treg induction is mediated by the regulation of key signaling molecules such as phospho-Akt, whose levels are kept low by PD-1-induced PTEN activity. Several types of cancer cells express PD-L1. Furthermore, non-tumor cells (endothelial cells, leukocytes, fibroblasts) within the tumor microenvironment can also express PD-L1. This is because they are tumor-infiltrating PD-1 +This suggests resistance to T lymphocytes (TILs) and / or the ability to induce the development of Tregs. In fact, there is growing evidence that treating patients with certain types of cancer (such as melanoma, renal cancer, and non-small cell lung cancer) with anti-PD-1 / PD-L1 monoclonal antibodies (mAbs) can reduce tumor growth. 【0060】 Immune checkpoint inhibitors are known to produce some degree of antitumor activity in humans, although this partial antitumor activity is observed only in a subset of treated subjects. Checkpoint inhibitors may or may not include proteins, polypeptides containing amino acid residues, and monoclonal or polyclonal antibodies. The compositions described herein may contain or be administered together with two or more checkpoint inhibitors. In some embodiments, the checkpoint inhibitors bind to ligands or proteins found in any of the T cell regulatory factor families, including CD28 / CTLA-4. Targets of checkpoint inhibitors may include, but may not include, receptors or co-receptors expressed on immune system effector cells or regulatory cells (e.g., T cells) (e.g., CTLA-4, CD8); proteins expressed on the surface of antigen-presenting cells (i.e., expressed on the surface of activated T cells, and may or may not include PD-1, PD-2, PD-L1, and PD-L2); metabolic enzymes, or metabolic enzymes expressed by both tumor and tumor-infiltrating cells (e.g., indoleamine-pyrrole 2,3-dioxygenase (IDO), including isoforms such as IDO1 and IDO2); proteins belonging to the immunoglobulin superfamily (e.g., lymphocyte-activating gene 3, also known as LAG3); and proteins belonging to the B7 superfamily (e.g., B7-H3 / CD276 or its homologs). B7 proteins are found in both activated antigen-presenting cells and T cells. In some embodiments, two or more checkpoint inhibitors may be combined or paired together. For example, B7 family checkpoint inhibitors present on antigen-presenting cells can be paired with inhibitors of CD28 or CTLA-4 expressed on the surface of T cells to generate a co-inhibitory signal that reduces the activity of these two types of cells. Co-receptors refer to two different receptors present on the same cell that, after binding to an external ligand, can regulate intracellular processes. Co-receptors can be stimulant or inhibitory. Co-receptors are sometimes called accessory receptors or co-signaling receptors.As used herein, the term “co-inhibition” refers to the result of multiple molecules binding to their respective receptors on the cell surface, thereby slowing down or interfering with the development of intracellular processes. 【0061】 In certain embodiments, immune checkpoint inhibitors may include antagonists of inhibitory receptors that inhibit the PD-1 or CTLA-4 pathway, such as anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies or inhibitors. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as lambrolizumab (anti-PD-1Ab, trade name Keytruda®) or pidilizumab (anti-PD-1Ab), Bavencio® (anti-PD-L1Ab, avelumab), Imfinzi® (anti-PD-L1Ab, durvalumab), Tecentriq® (anti-PD-L1Ab, atezolizumab), and fully human antibodies, such as nivolumab (anti-PD-1Ab, trade name Opdivo®). Other PD-1 inhibitors may include, but are not limited to, the presentation of soluble PD-1 ligands, including PD-L2Fc fusion proteins also known as B7-DC-Ig or AMP-244, and other PD-1 inhibitors currently under investigation and / or development for therapeutic use. Furthermore, immune checkpoint inhibitors may include, but are not limited to, humanized or fully human antibodies that block PD-L1, such as durvalumab and MIH1, as well as other PD-L1 inhibitors currently under investigation. In some embodiments, the immune checkpoint inhibitor is a CTLA-4, PD-L1, or PD-1 antibody. In some embodiments, PD-1 or CTLA-4 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as lambrolizumab (anti-PD-1Ab, trade name Keytruda®) or pidilizumab (anti-PD-1Ab), nivolumab (anti-PD-1Ab, trade name Opdivo®), tisilimucob (anti-CTLA-4Ab), ipilimumab (anti-CTLA-4Ab), MPDL3280A, BMS-936559, AMP-224, IMP321 (ImmuFact), MGA271, indoximod, and INCB024360. 【0062】 Examples Example 1. Evaluation of the efficacy of OBI-3424 + anti-PD-1 (pembrolizumab) or anti-PD-L1 antibody (avelumab) in a humanized mouse model carrying HepG2 tumors. The objective of this study is to evaluate the efficacy of OBI-3424 as monotherapy or in combination with anti-hPD-1 or anti-hPD-L1 antibodies in a humanized mouse model carrying HepG2 tumors. 【0063】 material 1. OBI-3424-DP Lot number: FLC-INJ-1711-01 Number of test samples: 2 vials / 1 mL per vial Ingredients: DNA alkylating agent Concentration: 10mg / mL Appearance: Transparent liquid Storage conditions: -20℃ 2. Anti-human PD-1, pembrolizumab, Merck & Co., Inc. Lot number: 7302614A13 Number of test samples: 1 vial / 1.2 mL per vial Component: Antibody Concentration: 25mg / mL Appearance: Transparent liquid Storage conditions: 2~8℃ 3. Anti-human PD-L1, avelumab, Merck & Co., Inc. Lot number: AU024788 Number of test samples: 1 tube / 1.5 mL per tube Component: Antibody Concentration: 20mg / mL Appearance: Transparent liquid Storage conditions: 2~8℃ 4. Sterile physiological saline (Taiwan Biotech Co., LTD) Lot number: 1PD2A054 Number of test samples: 6 x 20mL tubes Concentration: 0.9% sodium chloride Appearance: Transparent liquid Solubility: No information available Storage conditions: room temperature 5. Matrigel (Corning catalog number: 354248, lot number: 8228001) 6. Human PBMC (Lot: PBMC102219D, Zenbio, USA) 7. Collagenase (Sigma Aldrich, C5138), DNase I (Sigma Aldrich, D5025), Hyaluronidase (Sigma Aldrich, H6254) 8. RBC erythrocyte lysis buffer (Biolegend, 420302) 9. Cell staining buffer (Biolegend, 420201) 10. Human TruStain FcX (trademark) (Fc receptor blocking solution) (Biolegend, 422302) 11. Antibodies: Anti-human CD45 antibody (Beckman, IM0782U), anti-human CD8 antibody (Beckman, IM0452U and Biolegend, 300911), anti-human CD4 antibody (Beckman, B16491), anti-human CD56 antibody (Beckman, IM2474U), anti-human CD16 antibody (Beckman, IM1238U), anti-human CD25 antibody (Beckman, IM0479U). 【0064】 mouse 1. Species: House mouse Strand: Progressive immunodeficiency mouse. 【0065】 (NOD.Cg-Prkdc scid Il2rg) Supplier: Trineo Biotechnology Co.,LTD. Sex: Female Age at the start of the study: 6-8 weeks old Body weight range at the start of the study: 17-28g 2. Numbering and Identification: Each mouse was numbered using an ear tag. The housing cages were identified by cage cards containing information including the study number, cage number, animal number, sex, and dose level. 【0066】 Animal grouping: Mice were divided into six groups: G1 (vehicle), G2 (OBI-3424), G3 (anti-hPD-1), G4 (anti-hPD-L1), G5 (OBI-3424 + anti-hPD-1), and G6 (OBI-3424 + anti-hPD-L1). Each group contained 5 mice. Human hepatocellular carcinoma cell line HepG2 was subcutaneously inoculated into progressively immunodeficient mice. A total of 30 mice were included in this study. 【0067】 3. Reasons for Animal Selection: According to the TFDA's guidelines for nonclinical studies of anticancer drugs in nonclinical safety studies of drug proposals, animal xenograft tumor models can be used to evaluate the efficacy of new drugs or new anticancer agents. While BALB / c and C57BL / 6 are commonly used mouse strains, BALB / c nude, Nu / Nu, and NOD / SCID mice are typically selected to evaluate the antitumor effects of the drug of interest. These strains are managed globally based on well-known genetic and breed backgrounds and can provide valuable insights into the functional importance of appropriate responses in the human body. 【0068】 4. Adaptation Period: Mice were adapted for at least 3 days prior to randomization. Clinical observations and weight measurements were performed during the adaptation period. No signs of illness or behavioral changes were observed in the animals during this period. 【0069】 5. Animal housing conditions: Mice were housed in individually ventilated cages (IVCs) equipped with sterile bedding (10054, Andersons, USA) in a controlled environment with a temperature of 22±3°C, relative humidity of 50±20%, and a 12 / 12-hour light-dark cycle. Food (LabDiet5010, PMI, USA) and water (sterile RO water) were provided ad libitum throughout the study period. 【0070】 6. Randomization: The body weight of all animals was measured and their health status was observed before the study. Animals without abnormal clinical signs were selected for the experiment. Healthy animals were randomly divided into different groups to ensure that there were no significant differences in body weight between groups. The variability in animal body weight was kept within ±20% of the mean body weight. This procedure was carried out in accordance with the standards of practice for experimental animals. 【0071】 7.IACUC approval number: IACUC-2020-SH-016 Device Cell culture incubator (Shel Lab / 3552) Safety cabinet (BAKER / SG604) Electronic balance (PRECISA / XS 225A-SCS) Pipette (Thermo / Finnpipette F1) Positive and negative pressure verified individual cage housing system (Allentown / NEXGEN) Analytical balance (PRECISA / XS3250C-SCS) Animal euthanasia device (Forward Biotech Supply) Flow cytometer (Beckman Coulter / Navios EX) Vernier (Mitutoyo / CD-6"ASX) method Experimental Design 1. Sampling: Table 1 shows the experimental design, experimental groups, injection dose and volume, route of administration, and number of animals. 【0072】 [Table 1] 【0073】 2. Establishment of xenograft mouse models 2.1. Removal of animal hair: Before injecting the human hepatocellular carcinoma cell line HepG2, only the hair on the right flank was removed by clipping. 【0074】 2.2. Subcutaneous inoculation of tumor cells: 1 × 10 7 0.25 × 10⁶ HepG2 cells 7 The hPBMCs (cell number ratio 4:1) were pre-mixed with Matrigel (volume ratio 1:1) (Corning catalog number: 354248, lot number: 8228001). The subcutaneous injection volume was 200 μL / mouse. 【0075】 3. Route and administration of the test product: 3.1. Eight days after inoculation of tumor cells, the test anti-hPD-1 antibody or the reference product was intraperitoneally injected into mice. The injection was performed using an insulin syringe at a dose of 10 mg / kg (changed to 20 mg / kg from day 15) and an injection volume of 10 mL / kg. The test anti-hPD-1 antibody was administered to G3 and G5 mice consecutively on days 8, 11, 15, 18, 22, 25, 29, and 32. The reference product was administered to G1 mice. The procedure followed the sample administration criteria. 【0076】 3.2. Eight days after inoculation of tumor cells, the test anti-hPD-L1 antibody or the reference antibody was intraperitoneally injected into mice. The injection was performed using an insulin syringe at a dose of 10 mg / kg (changed to 20 mg / kg from day 15) and an injection volume of 10 mL / kg. The test anti-hPD-L1 antibody was administered to G4 and G6 mice consecutively on days 8, 11, 15, 18, 22, 25, 29, and 32. The reference antibody was administered to G1 mouse. The procedure followed the sample administration criteria. 【0077】 3.3. Fourteen days after inoculation of tumor cells, the test product OBI-3424 and the reference product were intravenously injected into mice. Injections were performed using an insulin syringe at a dose of 1 mg / kg and an injection volume of 5 mL / kg. The test product OBI-3424 was administered to G2, G5, and G6 mice consecutively on days 14, 21, 28, and 35. The reference product was administered to G1 mouse. The procedure followed the sample administration criteria. 【0078】 3.4. Preparation of test specimens or reference specimens: Prior to administration, the test product was diluted with a reference product. The solution concentration of test product OBI-3424 was 0.2 mg / mL, while the concentrations of test product anti-hPD-1 and test product anti-hPD-L1 were 1 mg / mL (2 mg / mL from day 15). 【0079】 4. Weight measurement: Measurements were taken starting the day after vaccination. The animals' weight was measured and recorded twice a week. 5. Measurement of tumor diameter: Measurements were taken starting the day after vaccination. Tumor volume was measured and recorded twice a week (Monday and Thursday). Tumor volume was calculated using the ellipsoid equation according to the recorded values ​​((major axis × minor axis × minor axis) × (π / 6)). 【0080】 6. Calculation of tumor growth inhibition rate: The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T0) / (Ci - C0)] × 100, where Ti and Ci represent the mean tumor volume of the treatment group and vehicle group at the end of the experiment (day 35). Meanwhile, T0 and C0 represent the mean tumor volume of the treatment group and vehicle group at the start of the experiment (day 1). 【0081】 7. Blood sampling: Submandibular blood samples were collected at the endpoint. During slaughter, blood samples could be collected using cardiac puncture. The collected blood samples were centrifuged at 1500 × g for 15 minutes at 4 ± 2°C to separate the serum from the pellet. The upper serum was collected and stored at a temperature below -70°C. The procedure followed the standards for animal blood sampling. 【0082】 8. Determining the study endpoint: The study concluded on the 36th day. 9. Tumor resection: At the end of the study period, mice were euthanized with CO2, and the connective tissue surrounding the tumor was excised. Tumor samples were then removed and weighed. Half of the tissue was fixed with 10% formaldehyde and embedded in paraffin. The remaining half was prepared for the isolation of tumor-infiltrating lymphocytes (TILs). 【0083】 10. Isolation of tumor-infiltrating lymphocytes (TILs): Half of the mouse tumor was dissected into smaller fragments using a scalpel and digested with a cocktail of collagenase, DNase I, and hyaluronidase (collagenase #C5138, DNase I #D5025, hyaluronidase #H6254, Sigma Aldrich) for at least 2 hours. The tumor digests were then passed through a 70 μm mesh cell strainer (Falcon #352350) using a syringe plunger and washed with PBS. The cells were treated with RBC erythrocyte lysis buffer (Biolegend #420302) to prepare single-cell suspensions for flow cytometry. 【0084】 11. Flow cytometry analysis of the TIL population: Cells were washed with staining buffer (Biolegend#420201), resuspended in staining buffer containing Fc receptor blocking solution (Biolegend#422302), and incubated at 4°C for 15 minutes. Cells were stained with a fluorescently coupled surface antibody, incubated at 4°C for 30 minutes, and then resuspended in staining buffer for flow cytometry analysis. Flow cytometry was performed using a Navios EX flow cytometer (Beckman Coulter). Data were analyzed using Kaluza Analysis software (Beckman Coulter). 【0085】 12.Statistical analysis: Results are presented as the mean and the standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with parallel negative control data were calculated using Student's t-test (Microsoft Excel®, 2007). P ≤ 0.05 was considered statistically significant. 【0086】 result Table 2 summarizes the body weight of each group. At the start of the study, there were no statistically significant differences in average body weight among the groups. At the end of the study, the body weight of G5 (OBI-3424 + anti-hPD-1) and G6 (OBI-3424 + anti-hPD-L1) was slightly increased compared to the other groups (Figure 1 and Table 2). Tumor response was examined using various test products, and the average tumor response was recorded on days 1, 4, 7, 11, 14, 18, 21, 25, 28, 32, and 35 (Figure 3 and Table 3). 【0087】 [Table 2-1] 【0088】 [Table 2-2] 【0089】 [Table 3-1] 【0090】 [Table 3-2] 【0091】 First, the effects of all test products on tumor response were examined, starting with G1 (vehicle), G2 (OBI-3424), G3 (anti-hPD-1), and G4 (anti-hPD-L1). In the presence of OBI-3424, a significant reduction in mean tumor volume was observed on day 35 (G1 vehicle: 1538.51 ± 195.78 mm). 3 , G2 OBI-3424:590.62±164.32mm 3 (p=0.003<0.05). No statistically significant difference was observed at day 35 in anti-hPD-1 and anti-hPD-L1 treatment (G1 vehicle: 1538.51±195.78mm). 3 , G3 anti-hPD-1:1613.37±338.07mm 3 , G4 anti-hPD-L1:1148.64±193.00mm 3). 【0092】 Furthermore, in combination therapy with G5 (anti-hPD-1 + OBI-3424) and G6 (anti-hPD-L1 + OBI-3424), the results showed a significant reduction in mean tumor volume on day 35 (G1 vehicle: 1538.51 ± 195.78 mm). 3 , G5 anti-hPD-1+OBI-3424:267.43±32.10mm 3 , p=0.0001<0.001, G6 anti-hPD-L1+OBI-3424:452.75±80.72mm 3 (p=0.0005<0.001). On the other hand, the mean tumor volume on day 35 was significantly reduced between G3 and G5 (G3 anti-hPD-1: 1613.37±338.07 mm). 3 , G5 anti-hPD-1+OBI-3424:267.43±32.10mm 3 (p=0.002<0.05). Furthermore, tumor volume at day 35 between G4 and G6 also showed a significant decrease in the mean tumor volume at day 35 (G4 anti-hPD-L1: 1148.64±193.00 mm). 3 , G6 anti-hPD-L1+OBI-3424:452.75±80.72mm 3 (p=0.004<0.05) (Figure 3 and Table 3). A similar trend was observed in tumor weight (Figure 2 and Table 3). Furthermore, one G4 (anti-hPD-L1) mouse was found dead on day 34, and its tumor weight was recorded on the same day. 【0093】 Tumor-infiltrating lymphocytes (TILs) were isolated from fresh tumor tissue, and the expression levels of surface markers CD45, CD4, CD8, CD56, CD16, CD25, PD-1, and PD-L1 were measured between groups using flow cytometry. Numerical data for individual mice are shown in Tables 4-7. This shows the difference in CD8 expression levels in tumors treated with OBI-3424 (G2) compared to the vehicle. +While a significant increase in cytotoxic T cell (CTL) count was observed, this increase was not seen in tumors treated with anti-PD-1 (G3) or anti-PD-L1 (G4). However, in tumors treated with OBI-3424 + anti-PD-1 (G5) and OBI-3424 + anti-PD-L1 (G6), a significant increase in CTL count was found in both groups. This suggests that the increase in CTL count was caused by OBI-3424, rather than by anti-PD-1 or anti-PD-L1 treatment. Furthermore, CD4 + T helper cell counts were significantly increased in G5 and G6 tumors. There were no significant differences in NK cell counts between the vehicle and each treatment group. 【0094】 [Table 4-1] 【0095】 [Table 4-2] 【0096】 [Table 5-1] 【0097】 [Table 5-2] 【0098】 [Table 6-1] 【0099】 [Table 6-2] 【0100】 [Table 7-1] 【0101】 [Table 7-2] 【0102】 The results showed that administration of anti-hPD-1 or anti-hPD-L1 had no effect on tumor growth. In contrast, administration of OBI-3424, combination therapy with OBI-3424 and anti-hPD-1, or combination therapy with OBI-3424 and anti-hPD-L1 demonstrated efficient antitumor effects against tumor growth in the HepG2 humanized mouse model. 【0103】 Example 2. Evaluation of the efficacy of OBI-3424 + anti-PD-1 (pembrolizumab) in a humanized mouse model carrying HepG2 tumors. The purpose of this study is to evaluate the efficacy of different doses of the test product OBI-3424 against tumor growth in the presence of the anti-PD-1 antibody (pembrolizumab) in a HepG2 humanized mouse model, and to assess the antitumor effect of combination therapy (OBI-3424 / anti-PD-1) against CD8 + The objective is to evaluate the effects of T cell depletion. 【0104】 material 1. OBI-3424-DP Lot number: FLC-INJ-1711-01 Number of test samples: 2 vials / 1 mL per vial Ingredients: DNA alkylating agent Concentration: 10mg / mL Appearance: Transparent liquid Storage conditions: -20℃ 2. Anti-human PD-1, pembrolizumab, Merck & Co., Inc. Lot number: 7006846900 Number of test samples: 1 vial / 100 mg per vial Component: Antibody Concentration: 25mg / mL Appearance: Transparent liquid Storage conditions: 2~8℃ 3. Isotonic sodium chloride solution (Taiwan Biotech Co., LTD) Lot number: 1OP2A092 Concentration: 0.9% sodium chloride Appearance: Transparent liquid Solubility: No information available Storage conditions: room temperature 4. Matrigel (Corning, Catalog Number: 354248, Lot Number: 0261002) 5. Human PBMC (Lot: PBMC102219D, Zenbio, USA) 6. Collagenase (Sigma Aldrich, C5138), DNase I (Sigma Aldrich, D5025), Hyaluronidase (Sigma Aldrich, H6254) 7. RBC erythrocyte lysis buffer (Biolegend, 420302) 8. Cell staining buffer (Biolegend, 420201) 9. Human TruStain FcX (trademark) (Fc receptor blocking solution) (Biolegend, 422302) 10. Antibodies: Anti-human CD45 antibody (Biolegend, 368508), anti-human CD8 antibody (Biolegend, 344710), anti-human CD4 antibody (Biolegend, 317429), anti-human CD56 antibody (Biolegend, 318332), anti-human CD11c antibody (Biolegend, 301614), anti-human CD25 antibody (Biolegend, 302610), anti-human CD69 antibody (Biolegend, 310914), anti-human CD86 antibody (Biolegend, 305406), anti-human CD91 antibody Biolegend (46-0919-42), anti-human Foxp3 antibody (Biolegend, 320108), anti-human IFN-γ antibody (Biolegend, 506507), anti-human granzyme B antibody (Biolegend, 372204), anti-human calreticulin antibody (Abcam, ab209577), anti-human PD-1 antibody (Biolegend, 329907), anti-human PD-L1 antibody (Biolegend, 329738), and ViaKrome405 (Biolegend, 302610) mouse 1. Species: House mouse Strand: Progressive immunodeficiency mouse. 【0105】 (NOD.Cg-Prkdc scid Il2rg) Supplier: Trineo Biotechnology Co.,LTD. Sex: Female Age at the start of the study: 6-8 weeks old Body weight range at the start of the study: 17-30g 2. Numbering and Identification: Each mouse was numbered using an ear tag. The housing cages were identified by cage cards containing information including the study number, cage number, animal number, sex, and dose level. 【0106】 Animal grouping: Mice were divided into nine groups: G1 (vehicle), G2 (OBI-3424 0.3 mg / kg), G3 (OBI-3424 1 mg / kg), G4 (anti-hPD-1), G5 (OBI-3424 0.3 mg / kg + anti-hPD-1), G6 (OBI-3424 1 mg / kg + anti-hPD-1), and G7 (OBI-3424 1 mg / kg + anti-hPD-1, CD8) + (PBMCs were excluded). Each group consisted of 6 mice. Human hepatocellular carcinoma cell line HepG2 was subcutaneously inoculated into progressively immunodeficient mice. A total of 42 mice were included in this study. 【0107】 3. Reasons for animal selection: According to the TFDA's guidelines for nonclinical studies of anticancer drugs in nonclinical safety studies of drug proposals, animal xenograft tumor models can be used to evaluate the efficacy of new drugs or new anticancer agents. Commonly used mouse strains include BALB / c and C57BL / 6, but BALB / c nude, Nu / Nu, and NOD / SCID mice are typically selected to evaluate the antitumor effects of the drug of interest. These strains are managed globally based on well-known genetic and breed backgrounds and can provide valuable insights into the functional importance of appropriate responses in the human body. 【0108】 4. Adaptation Period: Mice were adapted for at least 3 days prior to randomization. Clinical observations and weight measurements were performed during the adaptation period. No signs of disease or behavioral changes were observed in the animals during this period. 【0109】 5. Animal housing conditions: Mice were housed in individually ventilated cages (IVCs) equipped with sterile bedding (10054, Andersons, USA) in a controlled environment with a temperature of 22±3°C, relative humidity of 50±20%, and a 12 / 12-hour light-dark cycle. Food (LabDiet5010, PMI, USA) and water (sterile RO water) were provided ad libitum throughout the study period. 【0110】 6. Randomization: The body weight of all animals was measured and their health status was observed before the study. Animals without abnormal clinical signs were selected for the experiment. Healthy animals were randomly divided into different groups to ensure that there were no significant differences in body weight between groups. The variability in animal body weight was kept within ±20% of the mean body weight. This procedure was carried out in accordance with the standards of practice for experimental animals. 【0111】 7.IACUC approval number: IACUC-2020-SH-024 Device Cell culture incubator (Shel Lab / 3552) Safety cabinet (BAKER / SG604) Electronic balance (PRECISA / XS 225A-SCS) Pipette (Thermo / Finnpipette F1) Positive and negative pressure verified individual cage housing system (Allentown / NEXGEN) Analytical balance (PRECISA / XS3250C-SCS) Animal euthanasia device (Forward Biotech Supply) Flow cytometer (Beckman Coulter / Navios EX) Vernier (Mitutoyo / CD-6"ASX) method Experimental Design Sampling: Table 8 shows the experimental design, experimental groups, injection dose and volume, route of administration, and number of animals. 【0112】 [Table 8] 【0113】 2. Establishment of xenograft mouse models 2.1. Removal of animal hair: Before injecting the human hepatocellular carcinoma cell line HepG2, only the hair on the right flank was removed by clipping. 【0114】 2.2. Subcutaneous inoculation of tumor cells: 1 × 10 7 0.25 × 10⁶ HepG2 cells 7 The hPBMCs (cell number ratio 4:1) were pre-mixed with Matrigel (volume ratio 1:1) (Corning, 354248, lot number: 0261002). The subcutaneous injection volume was 200 μL / mouse. 【0115】 The day of tumor cell injection was designated as the first day of the incubation period (L0). 3. Route and administration of the test product: 3.1. Test sample OBI-3424 or the reference sample was intravenously injected into mice on day 0. The injection was performed using an insulin syringe at a dose of 0.3 mg / kg or 1 mg / kg, with an injection volume of 5 mL / kg. Test sample OBI-3424 was administered consecutively to G2, G3, G5, G6, and G7 on days 7, 14, 21, and 28. The reference sample was administered to G1. The procedure followed the sample administration standard. The start date of test sample administration is indicated as the first experimental day (D0). 【0116】 3.2. The test anti-hPD-1 antibody was intraperitoneally injected into mice on day 2. The injection was performed using an insulin syringe at a dose of 20 mg / kg and an injection volume of 10 mL / kg. The test anti-hPD-1 antibody was administered to G4, G5, G6, and G7 mice consecutively on days 5, 9, 12, 16, 19, 23, and 26. The procedure followed the sample administration criteria. 【0117】 3.3. Preparation of test specimens or reference specimens: Before administration, the test product was diluted with a reference product. The solution concentrations of test product OBI-3424 were 0.06 mg / mL and 0.2 mg / mL, and the test product anti-hPD-1 was 2 mg / mL. 【0118】 4. Weight measurement: Measurements were taken starting the day after vaccination. The animals' weight was measured and recorded twice a week. 5. Measurement of tumor diameter: Measurements were taken starting the day after vaccination. Tumor volume was measured and recorded twice a week (Monday and Thursday). Tumor volume was calculated using the ellipsoid equation according to the recorded values ​​((major axis × minor axis × minor axis) × (π / 6)). 【0119】 6. Calculation of tumor growth inhibition rate: The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T0) / (Ci - C0)] × 100, where Ti and Ci represent the mean tumor volume of the treatment group and vehicle group at the end of the experiment (day 30). Meanwhile, T0 and C0 represent the mean tumor volume of the treatment group and vehicle group at the start of the experiment (day 0). 【0120】 7. Blood sampling: Submandibular blood samples were collected at the endpoint. During slaughter, blood samples could be collected using cardiac puncture. The collected blood samples were centrifuged at 1500 × g for 15 minutes at 4 ± 2°C to separate the serum from the pellet. The upper serum was collected and stored at a temperature below -70°C. The procedure followed the standards for animal blood sampling. 【0121】 8. Determining the study endpoint: The study concluded on the 30th day. 9. Tumor resection: At the end of the study period, mice were euthanized with CO2, and the connective tissue surrounding the tumor was excised. The weight of the tumor sample was then measured, and if the tumor weight exceeded 400 mg, it was cut into three equal parts. The tumor tissue was prepared for the isolation of tumor-infiltrating lymphocytes (TILs). One of the remaining sections was fixed with 10% formaldehyde and embedded in paraffin. The other section was stored at a temperature below -70°C. 【0122】 10. Isolation of tumor-infiltrating lymphocytes (TILs): Tumor samples were cut into small fragments using a scalpel and digested with a cocktail of collagenase, DNase I, and hyaluronidase (collagenase #C5138, DNase I #D5025, hyaluronidase #H6254, Sigma Aldrich) for at least 2 hours. The tumor digests were then passed through a 70 μm mesh cell strainer (Falcon #352350) using a syringe plunger and washed with PBS. Cells were treated with RBC erythrocyte lysis buffer (Biolegend #420302) to prepare single-cell suspensions for flow cytometry. 【0123】 11. Flow cytometry analysis of the TIL population: Cells were washed with staining buffer (Biolegend#420201), resuspended in staining buffer containing Fc receptor blocking solution (Biolegend#422302), and incubated at 4°C for 15 minutes. Cells were stained with a fluorescently coupled surface antibody, incubated at 4°C for 30 minutes, and then resuspended in staining buffer for flow cytometry analysis. Flow cytometry was performed using a Navios EX flow cytometer (Beckman Coulter). Data were analyzed using Kaluza Analysis software (Beckman Coulter). 【0124】 12.Statistical analysis: Results are presented as the mean and the standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with parallel negative control data were calculated using Student's t-test (Microsoft Excel®, 2007). P ≤ 0.05 was considered statistically significant. 【0125】 result Table 9 summarizes the body weight of each group. No statistically significant differences in average body weight were observed among groups G1 to G7 at the start of the study or at slaughter (Figure 4 and Table 9). 【0126】 [Table 9-1] 【0127】 [Table 9-2] 【0128】 [Table 9-3] 【0129】 Tumor responses were examined using various test products, and the mean tumor response was recorded at LI, 3, and 7 after tumor cell injection, and at D0, 2, 6, 9, 13, 16, 20, 23, 27, and 30 after test product administration (Figure 6 and Table 10). 【0130】 First, the effects of the test products on tumor response were investigated using G1 (vehicle), G2 (OBI-3424 0.3 mg / kg), G3 (OBI-3424 1 mg / kg), and G4 (anti-hPD-1, 20 mg / kg). In the presence of OBI-3424 (0.3 mg / kg and 1 mg / kg), a dose-dependent decrease in mean tumor volume was observed at D30 (G1 vehicle: 882.92 ± 158.14 mm). 3 , G2 OBI-3424 0.3mg / kg:716.44±31.12mm 3 , G3 OBI-3424 1mg / kg:216.90±22.20mm 3(p=0.00096<0.001). No statistically significant difference was observed in anti-hPD-1 treatment at D30 (G1 vehicle: 882.92±158.14mm). 3 , G4 anti-hPD-1:983.84±266.44mm 3 ). 【0131】 G5 (OBI-3424 0.3 mg / kg + anti-hPD-1 20 mg / kg), G6 (OBI-3424 1 mg / kg + anti-hPD-1 20 mg / kg), and G7 (OBI-3424 1 mg / kg + anti-hPD-1 20 mg / kg, CD8) + In combination therapy (excluding PBMCs), the results showed that the mean tumor volume in all these groups was significantly reduced compared to the vehicle group at D30 (G1 vehicle: 882.92 ± 158.14 mm). 3 ), G5 OBI-3424 0.3mg / kg+anti-hPD-1:429.41±106.14mm 3 , p=0.0193<0.05, G6 OBI-3424 1mg / kg+anti-hPD-1:197.74±19.62mm 3 , p=0.00078<0.001, G7 OBI-3424 1mg / kg+anti-hPD-1, CD8 + Excluding PBMC: 374.44±36.97mm 3 (=0.0053 < 0.05). 【0132】 Compared to monotherapy, combination therapy tended to improve the inhibitory effect on mean tumor volume at D30 between G2 and G5, and between G3 and G6, but these reductions were not statistically significant (G2 OBI-3424 0.3 mg / kg: 716.44 ± 31.12 mm). 3 , G5 OBI-3424 0.3mg / kg+anti-hPD-1:429.41±106.14mm 3 ;G3 OBI-3424 1mg / kg:216.90±22.20mm 3 , G6 OBI-3424 1mg / kg+anti-hPD-1:197.74±19.62mm 3Furthermore, the treatment effect was quantified by calculating the rate of tumor growth inhibition (TGI). Monotherapy with low-dose and high-dose OBI-3424 resulted in TGIs of 27.82% and 113.27%, respectively. Combination therapy with low-dose and high-dose OBI-3424 against hPD-1 resulted in TGIs of 77.22% and 117.66%, respectively (Figure 6 and Table 10). 【0133】 Nevertheless, G6 (OBI-3424 1 mg / kg + anti-hPD-1) and G7 (OBI-3424 1 mg / kg + anti-hPD-1, CD8) + Comparing (excluding PBMC), CD8 + Cell depletion led to a significant increase in mean tumor volume at D30 (G6 OBI-3424 1 mg / kg + anti-hPD-1: 197.74 ± 19.62 mm). 3 , G7 OBI-3424 1mg / kg+anti-hPD-1, CD8 + Excluding PBMC: 374.44±36.97mm 3 Despite combination therapy at the same dose, the rate of TGI decreased from 117.66% to 87.35% (Figure 6 and Table 10). A similar trend was observed in tumor weight (Figure 5 and Table 10). 【0134】 [Table 10-1] 【0135】 [Table 10-2] 【0136】 [Table 10-3] 【0137】 Tumor-infiltrating lymphocytes (TILs) were isolated from fresh tumor tissue, and the expression levels of surface markers CD45, CD4, CD8, CD56, CD11c, CD69, CD25, CD86, CD91, granzyme B, IFN-γ, Foxp3, calreticulin, PD-1, and PD-L1 in each group were measured by flow cytometry. Numerical data for individual mice are shown in Tables 11-16. Cytotoxic lymphocytes (CTLs) (CD45 + CD8 + T cells) and helper T(TH) cells (CD45 + CD4 + The T cell population was significantly increased with high-dose OBI-3424 monotherapy or in combination with anti-hPD-1 compared to the vehicle group (CTL cells: G1 vehicle: 15.53±5.66%, G3 OBI-3424 1mg / kg: 33.50±3.38%, p=0.0107<0.05, G6 OBI-3424 1mg / kg + anti-hPD-1: 38.46±2.63%, p=0.00215<0.05; TH cells: G1 vehicle: 14.59±2.00%, G3 OBI-3424 1mg / kg: 25.05±2.08%, p=0.0023<0.05, G6 OBI-3424 1mg / kg+anti-hPD-1: 30.62±2.07%, p=0.00012<0.001) (Tables 13-14). 【0138】 [Table 11-1] 【0139】 [Table 11-2] 【0140】 [Table 11-3] 【0141】 [Table 12-1] 【0142】 Table 12-2 【0143】 Table 12-3 【0144】 Table 13-1 【0145】 Table 13-2 【0146】 Table 13-3 【0147】 Table 14-1 【0148】 Table 14-2 【0149】 Table 14-3 【0150】 Table 15-1 【0151】 Table 15-2 【0152】 [Table 15-3] 【0153】 [Table 16-1] 【0154】 [Table 16-2] 【0155】 [Table 16-3] 【0156】 The results showed that administration of OBI-3424 significantly suppressed tumor growth and exhibited a dose-dependent trend in antitumor activity. Furthermore, high-dose OBI-3424 combined with anti-hPD-1 therapy showed the most efficient antitumor effect against tumor growth in the HepG2 humanized mouse model. However, CD8+ cell depletion significantly impaired the antitumor effect of the combination therapy. 【0157】 The above description relating to embodiments of the present invention is not intended to limit the invention. Those skilled in the art can make various modifications and changes in accordance with the invention, and any modifications and changes within the scope of the invention shall be included in the claims appended to the invention. 【0158】 All references cited herein are incorporated herein by reference to the maximum extent permitted by law. The discussion of these references is solely for the purpose of summarizing the authors' arguments. No reference (or any part thereof) claims to be related prior art. The applicant reserves the right to object to the accuracy and appropriateness of the cited references. 【0159】 References 【0160】 Table 17-1 【0161】 Table 17-2 【0162】 Table 17-3 【0163】 Table 17-4

Claims

[Claim 1] (1) Compound represented by formula I: 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate 【Chemistry 1】 or its pharmaceutically acceptable salts, isotopic variants or solvates, and (2) An anti-PD-1 / PD-LI antibody, at least one of avelumab, nivolumab, pembrolizumab, durvalumab, and atezolizumab. A pharmaceutical composition containing the following: [Claim 2] The compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate represented by formula I-1, or 【Chemistry 2】 It is (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, represented by formula I-2. 【Transformation 3】 The pharmaceutical composition according to claim 1. [Claim 3] The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable excipient. [Claim 4] A pharmaceutical composition for use in the treatment of cancer in a patient requiring cancer treatment, wherein the treatment comprises administering to the patient a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 3. [Claim 5] The pharmaceutical composition according to claim 4, wherein the therapeutically effective dose is 0.1 mg / kg to 100 mg / kg. [Claim 6] The pharmaceutical composition according to claim 4, wherein the cancer is a cancer that overexpresses AKR1C3 reductase. [Claim 7] The pharmaceutical composition according to claim 4, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer. [Claim 8] A pharmaceutical composition for use in inhibiting the proliferation of cancer cells, comprising a compound represented by formula I-1 or formula I-2 in combination with an anti-PD-1 / PD-LI antibody which is at least one of avelumab, nivolumab, pembrolizumab, durvalumab, and atezolizumab. 【Chemistry 4】 [Claim 9] The pharmaceutical composition according to claim 8, wherein the combination of the compound and the anti-PD-1 / PD-LI antibody acts comprehensively or synergistically to rescue T cell inactivation and improve therapeutic effect. [Claim 10] The pharmaceutical composition according to claim 8, wherein the cancer cells are cancer cells that overexpress AKR1C3 reductase. [Claim 11] The pharmaceutical composition according to claim 10, wherein the cancer cells are cells of liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer.

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