AST-3424 for treatment of cancer patient resistant to immune checkpoint inhibitor

Abstract

The present invention relates to AST-3424 for treatment of a cancer patient resistant to an immune checkpoint inhibitor, and relates to a treatment method for a human malignant tumor, and particularly to a method for an AKR1C3-activated anticancer prodrug compound AST-3424 to treat a cancer patient resistant to an immune checkpoint inhibitor, and a pharmaceutical use. Disclosed are a method for using an AST-3424 monotherapy or combination immunotherapy regimen to treat a cancer / tumor patient resistant / insensitive to an immunotherapy regimen, a use of the AST-3424 monotherapy or combination immunotherapy regimen in preparation of a drug for treating the cancer / tumor patient resistant / insensitive to the immunotherapy regimen, and a method for cross-administration of AST-3424 and the immunotherapy regimen to treat the cancer / tumor patient, wherein the immunotherapy regimen is preferably selected from among drug therapy regimens comprising the immune checkpoint inhibitor / an immune cell therapy.

Description

AST-3424 Treatment for Cancer Patients Resistant to Immune Checkpoint Inhibitors Technical Field

[0001] This invention relates to treatment methods for human malignant tumors, particularly to a method for treating cancer patients resistant to immune checkpoint inhibitors by AKR1C3-activated anticancer prodrug compound AST-3424, and its pharmaceutical applications, belonging to the field of tumor chemotherapy. Background Technology

[0002] The DNA alkylating agent prodrug AST-3424 (WO2016145092, WO2017087428), targeting overexpression of aldehyde-ketone reductase 1C3 (AKR1C3), with CAS number 2097713-69-2, has the following structure:

[0003] Chemical structural formula of AST-3424

[0004] AST-3424 (also known as OBI-3424, TH-3424) enters cancer cells and is activated by the AKR1C3 enzyme overexpressed by the cancer cells, releasing the metabolite AST-2660 (also known as AST-2660). AST-3424 itself has low toxicity to cancer cells. In animal models and in vitro pharmacological experiments, its pharmacological effects are correlated with AKR1C3 enzyme expression: the prodrug AST-3424 is metabolized into AST-2660 under the action of AKR1C3 enzyme and NADPH, and the enzyme expression level is positively correlated with the drug efficacy (Reference 1; Reference 2; Reference 3; Reference 4).

[0005] The chemical reaction formula for the metabolism of AST-3424 (OBI-3424) to AST-2660 (OBI-2660).

[0006] Currently, this drug has entered Phase I / II clinical trials in China and the United States (US NCT03592264, indication: liver cancer, pancreatic cancer and other solid tumors, sponsor: OBI-3424, Taiwan OBI Biopharma Inc. (4174); US NCT04315324, T-ALL / T-LBL (acute T-lymphoblastic leukemia / T-lymphoblastic lymphoma), drug name: OBI-3424; China CTR20191371, indication: various solid tumors, sponsor: Shenzhen Ascentawits Pharmaceuticals,LTD., drug name: AST-3424; CTR20201915, indication: acute T-lymphoblastic leukemia and acute B-lymphoblastic leukemia, sponsor: Shenzhen Ascentawits Pharmaceuticals,LTD., drug name: AST-3424).

[0007] These Phase I / II clinical trials conducted in China and the United States have preliminarily demonstrated that AST-3424 has therapeutic effects on advanced solid tumors and hematological malignancies. Summary of the Invention

[0008] Currently, PD-1 / PD-L1 immune checkpoint inhibitors, when used in combination with other therapies for the treatment of advanced cancer, often significantly improve treatment outcomes. However, not all patients respond to PD-1 / PD-L1 immune checkpoint inhibitor therapy, and some develop resistance after treatment. The ability of a tumor to resist or tolerate immunotherapy is related to many factors, such as patient characteristics (e.g., gender / HLA typing), tumor matrix properties, and tumor environmental factors (e.g., the patient's own microbiome). However, the most important factor is the tumor cell intrinsic factor. Tumor cell intrinsic factors refer to the tumor's resistance to immunotherapy determined by the properties of its genome, transcriptome, etc., including:

[0009] The antigenicity of tumor cells; tumor cells with weak antigenicity will be difficult for the immune system to recognize.

[0010] The activity of the IFN-γ signaling pathway in tumor cells; the interferon-γ signaling pathway plays an important role in regulating the expression of PD-L1 and other substances, and in the presentation of MHC-related antigens.

[0011] Tumors downregulate the expression of MHC proteins, which leads to a weakening of the tumor cell antigen-presenting ability.

[0012] Activity of tumor-related signaling pathways: Among them, the most important are the WNT–β-catenin signaling pathway, CDK4–CDK6 signaling pathway, MAPK signaling pathway, PTEN protein loss, and AKR1C3 expression upregulation (Reference 8), etc.

[0013] In response to the above-mentioned possible drug resistance mechanisms, the applicant conducted in-depth research on AST-3424 and found that:

[0014] AST-3424 can activate an immune response by promoting DNA damage;

[0015] AST-3424 can activate an immune response by inhibiting prostaglandin synthesis;

[0016] AST-3424 increases the number of immune cells that infiltrate into tumor cells;

[0017] AST-3424 is expected to activate immune responses by directly regulating immune cell function.

[0018] In other words, AST-3424, as a drug that specifically activates AKR1C3, is expected to improve or reverse the resistance or insensitivity of immune checkpoint inhibitors in cancer / tumor patients, thus allowing for the treatment of immune checkpoint inhibitor resistant cancer / tumor patients as a monotherapy or in combination with immune checkpoint inhibitors.

[0019] Accordingly, the applicant proposes the following treatment methods and pharmaceutical uses for cancer / tumor patients treated with AST-3424 monotherapy or in combination with immunotherapy regimens, with the immunotherapy regimen preferably derived from immune checkpoint inhibitor / immunotherapy drug regimens.

[0020] AST-3424 monotherapy or in combination with immunotherapy regimens is a method for treating cancer / tumor patients resistant to immunotherapy regimens, with the immunotherapy regimen preferably being an immune checkpoint inhibitor / immunotherapy drug regimen.

[0021] Use of AST-3424 as monotherapy or in combination with immunotherapy regimens in the preparation of medicines for treating cancer / tumor patients resistant to immunotherapy regimens, preferably from immune checkpoint inhibitor / immunotherapy regimens.

[0022] Immune checkpoint inhibitors (ICIs) are molecules that inhibit / block the inhibitory immune checkpoint system and have become an effective therapy for advanced neoplasmosis. Immunotherapy, represented by immune checkpoint inhibitors, works by activating the anti-tumor immune function of the patient's own T lymphocytes to kill tumor cells and has been widely used clinically. ICIs, as a series of antibodies developed targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) or their ligands PD-L1 / 2, have achieved good results in anti-tumor therapy.

[0023] Suppressive immune checkpoint antigens are selected from PD-1 / PD-L1 antigen, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam 1), leucocyte-associated immunoglobulin-like receptor-1 (LAIR-1), T-cell immunoglobulin and mucin domain-3 (TIM-3), and V-domain Ig suppressor of T cell activation. Activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), indoleamine-pyrrole 2,3-dioxygenase (IDO), B7-H3 (CD276), A2AR (adenosine A2A receptor) or CD47.

[0024] In some cases, immune checkpoint inhibitors are anti-immune checkpoint antibodies that inhibit / block inhibitory immune checkpoint antigens.

[0025] Preferably, the anti-immune checkpoint antibody is an anti-PD-1 / PD-L1 antibody, an anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibody, an anti-LAG-3 (lymphocyte activation gene 3) antibody, an anti-TIGIT (inhibitory motif domain based on T cell immunoglobulin and immune receptor tyrosine) antibody, an anti-Ceacam 1 (carcinoembryonic antigen-associated cell adhesion molecule 1) antibody, an anti-LAIR-1 (leukocyte-associated immunoglobulin receptor-1) antibody, an anti-TIM-3 (T cell immunoglobulin and mucin domain 3) antibody, an anti-VISTA (Ig inhibitor of T cell activation V domain), an anti-KIR (cytotoxic cell immunoglobulin receptor), 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.

[0026] More preferably,

[0027] The anti-PD-1 antibodies were selected from pembrolizumab, toripalimab, sintilimab, camrelizumab, nivolumab, tislelizumab, pucotelimab, penpulimab, zimberelimab, serplulimab, cemiplimab, and dostarlimab.

[0028] The anti-PD-1 / CTLA-4 bispecific antibody was selected from catonilimab and ipalomlimab (a combination of ipalomlimab and tuvonralimab in a fixed ratio, QL1706, developed by Qilu Pharmaceutical).

[0029] The anti-PD-L1 antibodies were selected from durvalumab, atezolizumab, envafolimab, sugemalimab, avelumab, and adebrelimab.

[0030] The anti-CTLA-4 antibody was selected from ipilimumab and tremelimumab.

[0031] The anti-LAG-3 / PD-1 bispecific antibody was selected from OPDUALAG (a combination of nivolumab and relatlimab in a fixed ratio).

[0032] Clearly, these antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies.

[0033] Cancer treatment resistance can be broadly classified into primary (intrinsic) resistance and secondary (acquired) resistance. Primary resistance is characterized by a lack of objective clinical response after treatment, i.e., insensitivity. In a narrower sense, secondary (acquired) resistance refers to a response initially to a certain drug, but which decreases or ceases after a period of treatment.

[0034] In some cases, for PD-1 or PD-L1, certain drugs require the detection of biomarkers and the test results must reach a certain value. If the patient does not reach this value, then the patient can be considered as insensitive to the drug.

[0035] Preferably, in some embodiments, the cancer / tumor is hepatocellular carcinoma, and the pathological paraffin block or pathological section of the ex vivo tumor tissue of the cancer / tumor patient is used to detect the expression level of AKR1C3 enzyme protein by immunohistochemical staining, and the detection result has an H-score greater than or equal to 200.

[0036] or

[0037] The pathological paraffin blocks or sections of the ex vivo tumor tissue from the cancer / tumor patients were tested for AKR1C3 enzyme protein expression level by immunohistochemical staining. The results showed that the sum of the percentages of medium-intensity staining and high-intensity staining was greater than or equal to 70%.

[0038] Immunohistochemical staining (IHC) was used to detect the expression level of AKR1C3 enzyme protein in pathological paraffin blocks or sections of isolated liver tumor tissue from patients. The specific IHC detection method is disclosed in patent application PCT / CN2021 / 114774, publication number WO2022048492A1, and the calculation method is explained as follows:

[0039] The percentage of cell staining in the focus area was evaluated on a semi-quantitative scale using AKR1C3 assays, which recorded the percentage of cytoplasmic and nuclear staining at four levels (0, 1+, 2+, and 3+).

[0040] (3) Tumor Sample Scoring Criteria

[0041] The H-score is used to assess the staining degree of tumor cells in the nuclear-cytoplasmic staining process (the total value from 0 to 3+ should not exceed 100), indicating the level of AKR1C3 enzyme expression.

[0042] 0 (Uncolored): Values ​​between 0 and 100

[0043] Tumor cell nucleus-cytoplasm 1+ (weak staining): values ​​between 0 and 100

[0044] Tumor cell nucleus-cytoplasm 2+ (moderate staining): values ​​between 0 and 100

[0045] Tumor cell nucleus-cytoplasm 3+ (high-grade staining, strong): values ​​between 0 and 100

[0046] Total percentage of positive staining in the nucleus and cytoplasm: values ​​between 0 and 100

[0047] The total H score will be calculated based on the proportion of tumors at each intensity. The final H score is calculated as follows: H-score = (%weak [1+] × 1) + (%medium [2+] × 2) + (%high [3+] × 3), with the final H score ranging from 0 to 300.

[0048] Obviously, the above-mentioned H-score of 200 is not theoretically equivalent to the sum of the percentages of moderate and high intensity staining being greater than or equal to 70%, as the latter does not take into account the case of weak staining. However, in the Phase II clinical trial conducted by the applicant, patients with advanced hepatocellular carcinoma whose test results showed a sum of the percentages of moderate and high intensity staining being greater than or equal to 70% all had corresponding H-scores greater than or equal to 200!

[0049] Preferably, in some embodiments, the patient is a patient who tests negative for p53 gene mutation or whose p53 protein expression is normal.

[0050] In the Phase II clinical trial in China disclosed in this application, all enrolled patients were advanced, refractory hepatocellular carcinoma patients who had undergone multiple treatments, including:

[0051] Interventional therapies such as liver resection surgery, transarterial chemoembolization (TACE) and hepatic artery infusion chemotherapy, systemic single-agent chemotherapy and multi-agent combination chemotherapy, antibody-based immunotherapy (including monoclonal or bispecific antibodies such as PD-1 / PD-L1 / CTAL-4, and multi-antibodies), kinase inhibitor-based targeted therapy (i.e., molecular targeted therapy, such as tyrosine kinase inhibitors), combination therapy of antibody-based and kinase inhibitor-based drugs (targeted immunotherapy combination), radiotherapy, cell therapy, and traditional Chinese medicine treatment.

[0052] According to statistical classification, all 30 patients enrolled in the aforementioned Phase II clinical trial had received either antibody-based immunotherapy or molecular targeted therapy, or a combination of antibody-based immunotherapy and molecular targeted therapy (targeted immunotherapy combination). The preliminary results of the clinical trial indicate that AST-3424 monotherapy is still effective for patients with advanced refractory hepatocellular carcinoma who have progressed to disease progression (PD) after undergoing the above-mentioned treatments.

[0053] Further p53 gene mutation testing of the aforementioned 30 patients revealed that those with negative p53 gene mutation results or positive p53 gene mutation results indicating a non-lethal mutation that does not affect normal p53 protein expression showed a significantly better response to AST-3424. In other words, compared to patients with positive p53 gene mutation results that affect normal p53 protein expression, these patients received AST-3424 treatment and achieved better therapeutic effects. Specifically, for patients whose AKR1C3 enzyme protein expression levels were measured by immunohistochemical staining, and whose H-score was greater than or equal to 200, and whose combined percentage of moderate and high-intensity staining was greater than or equal to 70%, if their p53 gene mutation results were negative or positive but indicated a non-lethal mutation that does not affect normal p53 protein expression, then receiving AST-3424 treatment would provide greater benefit.

[0054] Fatal mutations, also known as pathogenic mutations, are called disease-causing mutations.

[0055] The p53 gene, also known as the tumor protein p53 gene or tp53 gene, is a tumor protein gene.

[0056] In this application, p53 (protein) and p53 (gene) are not distinguished and are used interchangeably.

[0057] In this application, p53 gene mutation negative includes no detected mutation and a situation where a mutation is detected but does not reach the defined positive level, i.e., a non-lethal mutation or a mutation that does not affect the normal expression of p53 protein.

[0058] Currently, relevant test kits have been approved for commercial use and can be purchased and used directly for testing, such as:

[0059] Produced by Xiamen AmoyDx Biotechnology Co., Ltd., China tp53 gene six mutation detection kit

[0060] The p53 gene amplification detection kit (fluorescence in situ hybridization) manufactured by Henan Sainote Biotechnology Co., Ltd. in China is a FISH detection kit for the p53 gene.

[0061] Manufactured by Integrated DNA Technologies, Inc., USA VariantPlex TM p53kit for

[0062] p53 protein expression is considered normal, including normal expression as well as high expression (overexpression). Normal values ​​are artificially defined based on statistical analysis of clinical practice.

[0063] Normal expression of p53 protein can be determined by typical Western blotting (WB) or clinical infiltration chemoradiography (IHC). These methods directly measure the amount of p53 protein in the sample and then compare it with normal values ​​or a set threshold to determine whether it is normally or overexpressed.

[0064] Screening can also be performed through genetic testing. Generally, if a pathogenic gene mutation is detected, it can be determined that the p53 protein is neither normally expressed nor highly expressed. If no gene mutation is detected, or if a gene mutation is detected but it is not a pathogenic mutation, then it is highly likely that the p53 protein is expressed normally. Therefore, the normal / high expression of p53 protein can be determined by detecting whether a pathogenic gene mutation has occurred in the p53 gene.

[0065] The above-mentioned gene mutation or protein expression detection is generally obtained by detecting the patient's tumor or cancerous tissue, cells or other biological test samples.

[0066] Biological testing samples include peripheral blood samples, tumor tissue or suspected tumor tissue, thin-layer cytology samples, fine needle aspiration samples, bone marrow samples, lymph node samples, urine samples, ascites samples, irrigation samples, esophageal scrubbing samples, bladder or lung lavage samples, cerebrospinal fluid samples, cerebrospinal fluid samples, catheter aspiration samples, nipple discharge samples, pleural effusion samples, fresh frozen tissue samples, paraffin-embedded tissue samples, or extracts or processed samples derived from any of these samples. Venous whole blood or saliva are commonly used.

[0067] Preferably, when AST-3424 is used as monotherapy in humans, the dosing regimen is as follows:

[0068] A cycle consists of 21 days, with one dose administered on day 1 and another on day 8. The initial dose is 6.0 mg / m². 2 They will be allowed to receive up to 34 cycles of treatment.

[0069] Preferably, the AST-3424 dosing regimen is characterized by an initial dose of 6.0 mg / m². 2 If the patient does not tolerate the medication, the dosage should be reduced sequentially to 4.5, 3.0, and 1.5 mg / m². 2 .

[0070] The AST-3424 is prepared as a concentrated solution for injection, with a specification of 10 mg AST-3424 active pharmaceutical ingredient per 1 mL. Preferably, the AST-3424 is prepared as a concentrated solution for injection, with a specification of 10 mg AST-3424 active pharmaceutical ingredient per 1 mL, and is labeled as containing 0.75 mL ethanol, 0.25 mL propylene glycol, and 10 mg AST-3424 active pharmaceutical ingredient.

[0071] Before administering AST-3424, adjust the pH by adding 0.1 ml of 5% sodium bicarbonate injection to 100 ml of sterile 5% glucose injection in an intravenous infusion bag that does not contain di(2-ethylhexyl) phthalate.

[0072] Add the calculated amount of concentrated AST-3424 injection solution, accurate to 0.01 ml, to the 5% glucose injection bag after pH adjustment to prepare AST-3424 injection solution for intravenous infusion administration.

[0073] If the patient is not suitable for glucose injection, normal saline should be used instead:

[0074] Before administering the medication, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of sterile 0.9% saline for injection in an intravenous infusion bag that does not contain di(2-ethylhexyl) phthalate to adjust the pH value.

[0075] Add the calculated amount of concentrated AST-3424 injection solution, accurate to 0.01 ml, to the pH-adjusted saline bag to prepare AST-3424 injection solution for intravenous administration.

[0076] The prepared intravenous AST-3424 solution should be injected within 8 hours, preferably within 25-35 minutes.

[0077] AST-3424 refers to a drug containing AST-3424, which is a pharmaceutical preparation or drug that can be directly applied to human patients (as opposed to an active pharmaceutical ingredient, API).

[0078] The drugs mentioned in this article refer to pharmaceuticals or preparations, and the prepared pharmaceuticals contain the active ingredient AST-3424 within a specific dosage range. The prepared pharmaceuticals are administered in a specific dosage form and via a specific route of administration.

[0079] The resulting pharmaceutical products, drugs, and formulations may also contain pharmaceutically acceptable excipients or excipients. The drug may be any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small injection needles, lyophilized powder for injection, or large-volume infusions. Depending on the specific dosage form and administration method, the pharmaceutically acceptable excipients or excipients in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and colorants, etc.

[0080] The currently available dosage form is an injection, specifically the concentrated solution for AST-3424 injection disclosed in patent application PCT / CN2020 / 101870, publication number WO2021008520 (corresponding to Chinese application CN202080001484.5, publication number CN112469394A). This solution is diluted before use and administered intravenously, preferably by intravenous infusion or injection pump. Of course, other dosage forms may be developed in the future.

[0081] AST-3424 was prepared as a concentrated solution for injection, with a specification of 10 mg AST-3424 active pharmaceutical ingredient per 1 mL.

[0082] Preferably, AST-3424 is prepared as a concentrated solution of AST-3424 for injection, with a specification of 10 mg AST-3424 active pharmaceutical ingredient per 1 mL, labeled as containing 0.75 mL ethanol, 0.25 mL propylene glycol and 10 mg AST-3424 active pharmaceutical ingredient.

[0083] Preferably, the above-mentioned concentrated solution of AST-3424 for injection is provided in the following two specifications:

[0084] 0.5ml contains 5mg of AST-3424 active pharmaceutical ingredient, labeled as containing 0.375ml of ethanol, 0.125ml of propylene glycol and 5mg of AST-3424 active pharmaceutical ingredient, and is packaged in a brown vial of 1ml or 2ml size;

[0085] Each 1.0ml vial contains 10mg of AST-3424 active pharmaceutical ingredient and is labeled as containing 0.75ml of ethanol, 0.25ml of propylene glycol, and 10mg of AST-3424 active pharmaceutical ingredient. It is packaged in a brown vial of 2ml or 5ml size.

[0086] Obviously, the above-mentioned concentrated injection solution cannot be administered directly and needs to be diluted and prepared:

[0087] Before administering the medication, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of sterile 5% glucose injection in an intravenous infusion bag that does not contain di(2-ethylhexyl) phthalate to adjust the pH value.

[0088] Add the calculated amount of concentrated AST-3424 injection solution, accurate to 0.01 ml, to the 5% glucose injection bag after pH adjustment to prepare AST-3424 injection solution for intravenous infusion administration.

[0089] If the patient is not suitable for glucose injection, normal saline should be used instead:

[0090] Before administering the medication, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of sterile 0.9% saline for injection in an intravenous infusion bag that does not contain di(2-ethylhexyl) phthalate to adjust the pH value.

[0091] Add the calculated amount of concentrated AST-3424 injection solution, accurate to 0.01 ml, to the pH-adjusted saline bag to prepare AST-3424 injection solution for intravenous administration.

[0092] AST-3424 can be used as a monotherapy for hepatocellular carcinoma or in combination with other drugs.

[0093] Monotherapy, or single-drug therapy, in this document further requires that the drug contains only the active ingredient AST-3424 and no other active ingredients; that is, the drug is not a combination drug. Monotherapy refers to the use of only one anticancer drug in a single course of treatment.

[0094] Combination therapy, also known as combined drug therapy, refers to the simultaneous or sequential use of two or more anticancer drugs during a single course of treatment.

[0095] Other anticancer drugs can be used in combination with AST-3424. Existing literature has shown that combination with abiraterone, prednisolone, 5-fluorouracil, sunitinib (WO2022178821), immune checkpoint inhibitors such as PD-1 / L1 (WO2022231580), nerabine (WO2019062919), oxaliplatin / 5-fluorouracil (literature 6) has significant synergistic effects.

[0096] The above 6.0 mg / m 2 The recommended dose is based on a Phase I clinical trial of solid tumors conducted in China (CTR20191371). However, due to the different circumstances of each patient, medical professionals may adjust the dose according to the patient's condition during treatment.

[0097] AST-3424 is a targeted chemotherapy drug, and its final metabolite, AST-2660, is a cytotoxic drug. The dosage is calculated based on the patient's body surface area. This calculated dosage is not fixed and needs to be reduced according to adverse reactions during treatment. Even at the beginning of treatment, dosage adjustments may be necessary based on the patient's age and comorbidities to ensure safe and effective treatment. The efficacy of cytotoxic drugs is linearly related to dosage; therefore, provided the patient can tolerate it, a sufficient dose should be given to ensure efficacy. Dosage reduction should follow a uniform and strict protocol: if the patient cannot tolerate it, the recommended dose of 6 mg / m² should be reduced. 2 The dosage was reduced sequentially to 4.5, 3.0, and 1.5 mg / m². 2 .

[0098] Body surface area (BSA), based on the effective dosage mentioned above, for an average patient (height 175cm, weight 75kg), the corresponding equivalent body surface area (BSA) is... 2 = ([height (cm) × weight (kg)] / 3600) 1 / 2 =1.90 (calculated using the Mosteller formula), then the corresponding dose is 11.4mg! Correspondingly, using the above 1ml:10mg concentrated AST-3424 injection solution, then 1.14ml of the above concentrated injection solution should be drawn during the dilution process.

[0099] The prepared intravenous AST-3424 solution should be administered within 8 hours, as studies have shown that the above-mentioned glucose-diluted AST-3424 solution or saline-diluted AST-3424 solution is stable at room temperature (25°C) for 8 hours. Preferably, it is best to administer the solution within 25-35 minutes.

[0100] The Phase I and Phase II clinical trials of AST-3424 (OBI-3424) in China and the United States disclosed in the above invention have all been approved by the local drug regulatory authorities, and the relevant clinical trials have been completed by clinical research institutions. The relevant clinical trial protocols have all been approved by ethics agencies, and the clinical trial process complies with the relevant medical ethics requirements and legal and regulatory requirements of the clinical trial location.

[0101] Since AST-3424 has the function of improving or reversing resistance to immune checkpoint inhibitors, it can be used in combination with immune checkpoint inhibitors to treat cancer / tumors. Preferably, when using AST-3424 in combination with immune checkpoint inhibitors for treatment, AST-3424 is given first, followed by the immune checkpoint inhibitor.

[0102] Alternatively, a monotherapy crossover approach using AST-3424 and immune checkpoint inhibitors can be employed. This involves treating cancer / tumor patients, regardless of their resistance to immune checkpoint inhibitors, with one or more cycles of AST-3424 monotherapy followed by immune checkpoint inhibitor therapy, and this cycle can be repeated. Such a crossover treatment regimen is as follows:

[0103] If a patient is insensitive / unresponsive to immune checkpoint inhibitors, or if a patient initially responded to immune checkpoint inhibitors but developed resistance and became unresponsive after a period of treatment, then AST-3424 monotherapy can be administered first to increase the number of immune cells and activate the immune response, thereby restoring the patient's sensitivity to immune checkpoint inhibitors. Then, immune checkpoint inhibitors can be administered, which can achieve a better therapeutic effect.

[0104] Immune checkpoint inhibitors, like immune cell therapy drugs, are both forms of immunotherapy. Therefore, in some embodiments, immune cell therapy drugs, like immune checkpoint inhibitors, can develop immunotherapy resistance or insensitivity when treating cancer / tumors. In this regard, those skilled in the art can infer that AST-3424 can also be expected to improve or reverse immune cell therapy drug resistance. Therefore, AST-3424 alone or in combination with immune cell therapy drugs is still effective in treating cancer / tumor patients resistant to immune cell therapy drugs.

[0105] Furthermore, when used as monotherapy, AST-3424 is cross-administered with immunotherapy drugs to treat cancer / tumor patients.

[0106] In some embodiments, immune cell therapy refers to adoptive immunotherapy.

[0107] Adoptive cell transfer therapy (ACT) refers to the isolation of immune-active cells (the main components of immunotherapy drugs) from cancer patients, their expansion and functional identification in vitro, and then the reinfusion of immunotherapy drugs into the patient. This aims to directly kill tumor cells or stimulate the body's immune response to kill tumor cells. Adoptive cell therapy mainly includes several categories such as TIL (Tumor Infiltrating Lymphocytes) cell therapy, LAK (lymphokine-activated killer cell therapy) cell therapy, CIK (cytokine-induced killer) cell therapy, DC (dendritic cell) cell therapy, NK (Natural Killer Cell) cell therapy, TCR-T (T-cell receptor engineered T cells) cell therapy, and CAR-T (Chimeric Antigen Receptor T-Cell) cell therapy.

[0108] In particular, the following CAR-T cell therapy products received marketing approval: Axicabtagene Ciloleucel (CD19), brexucabtagene autoleucel (CD19), Tisagenlecleucel (CD19), lisocabtagene maraleuce (CD19), idecabtagene vicleucel (BCMA), Relmacabtagene autoleucel (CD19), and Ciltacabtagene autoleucel (BCMA). The following CIK cell therapy product received marketing approval: ImmunCe11- Green Cross Cell Corporation (BCMA) has received marketing approval for its DC cell therapy products: Sipuleucel-T and CreaVax-RCC.

[0109] The act of “administering” or “administration of” a drug to a patient (and its grammatical equivalents) refers to the direct administration (which may be administered by a medical professional to a patient or by the physician himself) and / or the indirect administration of a drug, which may include the act of prescribing the drug. For example, instructing a patient to administer the drug himself and / or providing a prescription for the drug to a patient constitutes administering the drug to a patient.

[0110] "Cancer" refers to leukemia, lymphoma, carcinoma, and other malignant tumors (including solid tumors) that can grow with unlimited potential, spreading locally through invasion and throughout the body through metastasis. Examples of cancer include (but are not limited to) cancers of the adrenal glands, bones, brain, breast, bronchi, colon and / or rectum, gallbladder, head and neck, kidneys, larynx, liver, lungs, nervous tissue, pancreas, prostate, parathyroid glands, skin, stomach, and thyroid gland. Other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysdifferentiation and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforme, pilocytic tumor, enteric ganglionoma, proliferative corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumors, malignant melanoma, malignant hypercalcemia, and marfanoid habitus. Tumor), medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, squamous cell carcinoma of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilm's tumor.

[0111] The terms "patient" and "individual" are used interchangeably and refer to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some embodiments, "patient" or "individual" may refer to a non-human mammal used to screen, characterize, and evaluate drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0112] "Solid tumors" refers to solid tumors, including (but not limited to) metastatic tumors in bone, brain, liver, lungs, lymph nodes, pancreas, prostate, skin, and soft tissue (sarcoma).

[0113] The "therapeutic effective dose" of a drug refers to the amount of drug that, when administered to a patient with cancer, will have the expected therapeutic effect (e.g., alleviation, improvement, relief, or elimination of clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur with a single dose and may occur only after a series of doses. Therefore, the therapeutic effective dose can be administered in one or more doses.

[0114] The term "treating" (or "treatment of") a condition or patient refers to steps taken to achieve a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) the alleviation or improvement of one or more cancer symptoms; a reduction in disease severity; a delay or slowing of disease progression; an improvement, relief, or stabilization of the disease state; or other beneficial outcomes. In some cases, cancer treatment may result in a partial response or stabilization of the disease.

[0115] "Tumor cells" or "cancer cells" refer to tumor cells or cancer cells in any appropriate species (e.g., mammals, such as rats, dogs, cats, horses, or humans).

[0116] The terms "patient" and "individual" are used interchangeably and refer to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some embodiments, "patient" or "individual" may refer to a non-human mammal used to screen, characterize, and evaluate drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0117] "Treatment" or "treatment of a patient" refers to administering, using, or applying to a patient a therapeutically effective amount of the medicine relating to this invention.

[0118] "Administering" or "using" a drug to a patient refers to the direct administration or application of a drug (which may be done by a medical professional to a patient or by the physician administering or applying the drug themselves) and / or indirect administration or application, and may include the act of prescribing the drug. For example, instructing a patient to administer or apply the drug themselves and / or providing a prescription for the drug to a patient constitutes administering or applying a drug to a patient.

[0119] "Treatment" for a condition or patient refers to steps taken to achieve a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) the alleviation or improvement of one or more cancer symptoms; a reduction in disease severity; a delay or slowing of disease progression; an improvement, relief, or stabilization of the disease state; or other beneficial outcomes. In some cases, cancer treatment may result in a partial response or stabilization of the disease.

[0120] All numerical markings, such as pH, temperature, time, concentration, and weight, including their ranges, are approximate values and usually have an error of plus or minus 10%. Description of the Drawings

[0121] Figure 1 is a graph showing the change in the average body weight of animals in each group of Example 4 over time. The body weights of the vehicle group, single-treatment group, and combination-treatment group were recorded twice a week until day 35. No weight loss was observed in all treatment groups of humanized mice carrying the human hepatocellular carcinoma HepG2 tumor. Data are shown as mean ± SEM (N = 5 per group). Statistical analysis was performed by Student's t test.

[0122] Figure 2 is a graph showing the change in the average tumor weight of animals in each group of Example 4 over time. The mice were sacrificed on day 36 after inoculation with tumor cells, and the tumor weights of the mice in the vehicle group, single-treatment group, and combination-treatment group were recorded. The tumor weights of the OBI-3424 (G2) and OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combination-treatment groups were significantly inhibited compared to the vehicle group (G1) (p < 0.001). In addition, the tumor weights of the OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combination-treatment groups were significantly inhibited compared to the anti-hPD-1 / anti-hPD-L1 (G3 / G4) treatment groups (p < 0.05). Data are shown as mean ± SEM (N = 5 per group). Statistical analysis was performed by Student's t test. A P value < 0.05 was considered significant. Single asterisks indicate 0.05 < P < 0.001, and double asterisks indicate P < 0.001.

[0123] Figure 3 is a graph showing the change in the average tumor volume of animals in each group of Example 4 over time. The tumor volumes of the mice in the vehicle group, single-treatment group, and combination-treatment group were recorded twice a week until day 35. The tumor volumes of the OBI-3424 (G2) and OBI-3424 + anti-hPD-1 / anti-hPD-L1 (G5 / G6) combination-treatment groups were significantly inhibited compared to the vehicle group (G1). Data are shown as mean ± SEM (N = 5 per group). Statistical analysis was performed by Student's t test.

[0124] Figure 4 is a schematic diagram showing the percentage of CTL cells in TILs of experimental animals in 6 dose groups in Example 4.

[0125] Figure 5 is a schematic diagram showing the percentage of TH cells in TILs of experimental animals in Example 4.

[0126] Figure 6 is a schematic diagram showing the percentage of NK cells in TILs of experimental animals in Example 4.

[0127] Figure 7 shows the percentage diagram of PD-L1 cells in the TILs of experimental animals in Example 4.

[0128] Figure 8 shows the change curve of the average body weight of each group of animals in Example 5 over time. The body weights of the vehicle group, OBI-3424 low-dose and high-dose treatment groups, and the OBI-3424 plus PD-1 antibody combination treatment group were recorded twice a week until the 30th day. No weight loss was observed in the vehicle group, OBI-3424 single-dose group, or OBI-3424 plus PD-1 antibody combination treatment group of humanized mice carrying human hepatocellular carcinoma HepG2 tumors. The data are shown as mean ± SEM (N = 5 per group). Statistical analysis was performed by Student's t-test.

[0129] Figure 9 shows the change curve of the average tumor weight of each group of animals in Example 5 over time. The mice were sacrificed on the 30th day after administration of the test item, and the tumor weights of the mice in the OBI-3424 low-dose group (G2), high-dose treatment group (G3), and OBI-3424 plus PD-1 antibody combination treatment group (G5-G7) were significantly inhibited compared to the vehicle group (G1) (p < 0.05). In addition, the tumor weight of the high-dose OBI-3424 plus PD-1 antibody combination treatment group (G6) was significantly lower than that of the high-dose OBI-3424 single-treatment group (G3) (p < 0.05). The data are shown as mean ± SEM. Statistical analysis was performed by Student's t-test. A P value < 0.05 was considered significant. One star indicates 0.05 < P < 0.001, and two stars indicate P < 0.001.

[0130] Figure 10 shows the change curve of the average tumor volume of each group of animals in Example 5 over time. The tumor volumes of the mice in the vehicle group, OBI-3424 low-dose and high-dose treatment groups, and the OBI-3424 plus PD-1 antibody combination treatment group were recorded twice a week until the 30th day. The tumor volumes of the OBI-3424 high-dose treatment group (G3) and the OBI-3424 plus anti-hPD-1 antibody (G5-G7) combination treatment group were significantly inhibited compared to the vehicle group (G1). The data are shown as mean ± SEM (N = 5 per group). Statistical analysis was performed by Student's t-test.

[0131] Figure 11 shows the percentage diagram of calreticulin cells in the TILs of experimental animals in Example 5.

[0132] Figure 12 shows the percentage diagram of PD-L1+ cells in the TILs of experimental animals in Example 5.

[0133] Figure 13 shows the percentage diagram of CTL cells in the TILs of experimental animals in Example 5.

[0134] Figure 14 is a schematic diagram of the percentage of TH cells in the experimental animals TILs in Example 5.

[0135] Figure 15 is a schematic diagram of the percentage of NK cells in the experimental animals TILs in Example 5.

[0136] Figure 16 is a schematic diagram of the percentage of dendritic cells in the experimental animals TILs in Example 5.

[0137] Figure 17 shows the weight change curves of different groups of animals in Example 7. The weight changes of tumor-bearing mice in the MH-22AhAKR1C3#20 cell subcutaneous xenograft model after administration of AST-3424, anti-PD-1Ab single drug and AST-3424 and anti-PD-1Ab combination drug are shown. The data points represent the average weight within the group, and the error bar represents the standard error (SEM).

[0138] Figure 18 shows the relative body weight change (%) curves for different groups of animals in Example 7, specifically the body weight change (%) of tumor-bearing mice in the MH-22AhAKR1C3#20 xenograft model after administration of AST-3424, anti-PD-1Ab monotherapy, and AST-3424 and anti-PD-1Ab combination therapy. Relative body weight change was calculated based on the animal's body weight at the start of administration. Data points represent the percentage change in average body weight within the group, and error bars represent standard errors (SEM).

[0139] Figure 19 shows the tumor growth curves of different groups of animals in Example 7, specifically the tumor growth curves of MH-22AhAKR1C3#20 cell subcutaneous xenograft tumor model mice after administration of AST-3424, anti-PD-1Ab single drugs, and AST-3424 and anti-PD-1Ab combination drugs. Data points represent the mean tumor volume within the group, and error bars represent standard errors (SEM).

[0140] Figure 20 shows the relative luminescence intensity (RLU) of extracellular ATP measured in the culture supernatant after treating H460 cells with OBI-3424 100 nM in Example 8. ***p<0.001, statistical analysis: Student test.

[0141] Figure 21 shows the ELISA test results of HMGB1 in the culture supernatant of H460 treated with different concentrations of OBI-3424 in Example 8. Example

[0142] Unless otherwise specified, the English abbreviations appearing in the embodiments shall be subject to the interpretations and explanations in medical textbooks or relevant medical associations.

[0143] Example 1: Phase I clinical trial of AST-3424 conducted in China

[0144] The clinical trial registration number is CTR20191371.

[0145] The trial was approved by the ethics committees of the medical institutions participating in the clinical trial, conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all participants.

[0146] Inclusion criteria

[0147] 1. Male or female, aged 18-70.

[0148] 2. Pathological histology and / or cytology confirms a malignant solid tumor (including but not limited to hepatocellular carcinoma, intrahepatic bile duct carcinoma, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer, and castration-resistant prostate cancer), and it is a metastatic or unresectable advanced case, and standard treatment has failed, or there is no standard treatment, or standard treatment is not suitable at this stage.

[0149] 3. Once the MTD is confirmed, subjects enrolled in the dose extension group (MTD group) must have at least one measurable lesion meeting RECIST 1.1 criteria. Lesions previously treated with radiotherapy are not considered measurable lesions unless they have shown clear radiographic progression after radiotherapy.

[0150] 4. The Eastern Cooperative Oncology Group (ECOG) performance status score is 0 or 1.

[0151] 5. Life expectancy ≥ 12 weeks.

[0152] 6. Before starting the investigational drug, all toxicities of previous anticancer treatments (excluding hair loss, fatigue, or peripheral neuropathy) must have been reduced to Grade 1 or baseline levels (NCI CTCAE 5th edition).

[0153] 7. The QTcF interval in the heart is ≤450 milliseconds for men and ≤470 milliseconds for women.

[0154] 8. Laboratory tests must meet the following criteria. Within 14 days prior to the screening period, the indicators cannot be corrected by blood transfusions or hematopoietic stimulating factors to meet the inclusion criteria: a. Hemoglobin ≥ 90 g / L; b. Platelet count ≥ 100 × 10⁻⁶. 9 c. Absolute neutrophil count (ANC) ≥ 1.5 × 10⁹ / L; 9 / L; d. Total bilirubin ≤1.5×ULN; e. ALT and AST ≤3.0×ULN; ≤5.0×ULN in the presence of liver tumors; f. Creatinine clearance >50 mL / min as determined by the Cockcroft-Gault equation.

[0155] 9. No history of alcoholism, drug abuse, or substance abuse within the past year.

[0156] 10. Female patients of childbearing age should have a negative pregnancy test result within 5 days prior to the start of treatment and should not be breastfeeding (a positive urine pregnancy test result needs to be confirmed by a serum pregnancy test).

[0157] 11. Female and male participants of childbearing age must agree to use effective contraception with their partners from the start of the study (e.g., surgical sterilization or condom or diaphragm contraception combined with spermicide gel or intrauterine device [IUD], etc.) until 6 months after the last dose.

[0158] 12. Participants must voluntarily participate in this study, fully understand the relevant risks, demonstrate good compliance, and sign an informed consent form.

[0159] Exclusion criteria:

[0160] 1. Untreated active central nervous system (CNS) metastases or leptomeningeal disease. Subjects with adequately treated CNS metastases are eligible to participate in the study if their CNS metastases are confirmed to be stable for at least 4 weeks by clinical examination and brain imaging (MRI or CT) during the screening period.

[0161] 2. Underwent major surgery (excluding diagnostic surgery) within 4 weeks prior to the first dose.

[0162] 3. Patients who have received radiotherapy, surgery, chemotherapy, immunotherapy, cancer-specific biotherapy, targeted therapy, or hormone therapy within 4 weeks prior to the first dose (nitrosourea or mitomycin C treatment requires a 6-week washout period; oral fluorouracil drugs require a 2-week washout period; small molecule targeted therapy requires a 2-week washout period)

[0163] 4. Participated in a study of the investigational drug (diagnostic or therapeutic) or device within 4 weeks prior to the first dose.

[0164] 5. During the study, a potent CYP3A4 inhibitor or inducer must be used in combination.

[0165] 6. Uncontrolled active bacterial, viral, or fungal infections requiring systemic treatment.

[0166] 7. Known infection with human immunodeficiency virus (HIV) or positive for syphilis.

[0167] 8. Women who are pregnant, breastfeeding, or planning to become pregnant.

[0168] 9. Accompanying diseases or symptoms that may interfere with the study, or physical abnormalities that the researchers believe pose an excessive risk to the patient, including but not limited to active peptic ulcers or gastritis, changes in mental state, or mental abnormalities that may interfere with the patient's understanding of the informed consent form.

[0169] 10. Previous allergy to ethanol or propylene glycol.

[0170] 11. Subjects who are unwilling or unable to comply with the study protocol for any reason.

[0171] Test drug:

[0172] AST-3424 Injection Concentrated Solution: Manufactured by a pharmaceutical company commissioned by Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd., specification 1mL: 10mg; contains 0.75ml ethanol, 0.25ml propylene glycol and 10mg AST-3424.

[0173] Dosage regimen:

[0174] A cycle of 21 days is followed by dosing on days 1 and 8, at doses of 1.0, 2.0, 4.0, 6.0, and 8.0 mg / m² sequentially. 2 Five dose levels are used in a dose-escalation manner, and the drug is administered continuously until disease progression or intolerable toxicity occurs, up to a maximum of two years.

[0175] Specific procedures for drug administration:

[0176] Before administration, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 5% glucose water for injection (D5W) in an intravenous infusion bag free of DEHP (di(2-ethylhexyl) phthalate). Add the calculated required volume (accurate to 0.01 ml) of concentrated AST-3424 injection solution to the pH-adjusted D5W bag to prepare the AST-3424 injection solution for intravenous administration.

[0177] The solute in this intravenous aqueous solution consists of AST-3424 active pharmaceutical ingredient, glucose, ethanol, propylene glycol, and sodium bicarbonate as a pH adjuster. The concentration of AST-3424 active pharmaceutical ingredient is 0.004-0.94 mg / ml, the pH is 7.4, the glucose content is 4.5-5.0% by mass, and it is an isotonic solution.

[0178] If the patient is not suitable for glucose injection, normal saline should be used instead:

[0179] Before administration, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 0.9% saline solution for injection in an intravenous infusion bag free of DEHP (di(2-ethylhexyl) phthalate). Add the calculated required volume (accurate to 0.01 ml) of concentrated AST-3424 injection solution to the pH-adjusted saline bag to prepare the AST-3424 injection solution for intravenous administration.

[0180] The intravenous aqueous solution consists of AST-3424 active pharmaceutical ingredient, sodium chloride, ethanol, propylene glycol, and sodium bicarbonate as a pH adjuster. The concentration of AST-3424 active pharmaceutical ingredient is 0.004-0.94 mg / ml, the pH is 7.4, the sodium chloride content is 0.81-0.90% by mass, and it is an isotonic solution.

[0181] The precise calculation method for the required volume of concentrated AST-3424 injection solution is as follows:

[0182] For a patient who is 175cm tall and weighs 75kg, the corresponding equivalent body surface area (BSA) is... 2 = ([height (cm) × weight (kg)] / 3600) 1 / 2 =1.90, then the corresponding dose is 1.90 × 6.0 = 11.40 mg, then the amount of concentrated AST-3424 injection solution of the above specification to be drawn is 11.40 ÷ 10 × 1 = 1.14 ml.

[0183] The prepared intravenous AST-3424 solution should be administered within 8 hours. In practice, it is usually injected intravenously using an infusion pump, and the injection is completed within 25-35 minutes.

[0184] Test results

[0185] The trial enrolled 21 participants. Each cycle lasted 21 days, with dosing on days 1 and 8, at doses of 1.0, 2.0, 4.0, 6.0, and 8.0 mg / m². 2 A total of 5 dose escalations were implemented. Following discussion by the Safety Review Committee (SRC), a 21-day treatment cycle was confirmed, with the MTD and RP2D for administration on days 1 and 8 being 6.0 mg / m². 2 (The total dosage for one cycle is 12 mg / m²) 2 ).

[0186] The efficacy analysis showed that among the 15 subjects who completed the evaluation, the best efficacy was stable disease (SD) in 10 cases, including 1 case of submandibular gland cancer, 1 case of prostate cancer, 1 case of breast cancer, 1 case of pancreatic cancer, 3 cases of hepatocellular carcinoma, and 3 cases of colorectal cancer.

[0187] Specifically, the enrolled hepatocellular carcinoma patients had a 100% SD (stable disease) rate, with doses distributed at 4.0, 6.0, and 8.0 mg / m². 2 The efficacy of treatment in the dosage group for patients with hepatocellular carcinoma enrolled in stage I is shown in the table below (one patient was not evaluated):

[0188] Safety data showed that 4 cases of DLT were observed among the 21 subjects. Among them, 6.0 mg / m²... 2One of the six subjects in the dosage group reported DLT, a grade 4 decrease in platelet count; 8.0 mg / m² 2 Of the nine subjects in the dosage group, three reported drug-related adverse events (DLT), including two cases of grade 4 decreased platelet count and one case of grade 2 elevated γ-GT (gamma-glutamyl transferase). The reported drug-related adverse events were mainly anemia, decreased platelet count, and fatigue, as well as gastrointestinal disorders (such as vomiting and nausea), most of which were grade 1-2 in severity. During the study, the laboratory indicators, vital signs, physical examination, electrocardiogram, and weight of the vast majority of subjects remained stable relative to baseline.

[0189] Specifically:

[0190] During the trial, a total of 147 adverse events (AEs) occurred in 21 subjects (N=21), with an AE incidence rate of 100.0%; among them

[0191] 1.0 mg / m 2 In group N=1, one subject experienced one adverse event (AE).

[0192] 2.0 mg / m 2 In group N=1, one subject experienced 12 adverse events (AEs).

[0193] 4.0 mg / m 2 In group N=4, 4 subjects experienced 27 adverse events;

[0194] 6.0 mg / m 2 In group N=6, 6 subjects experienced 37 adverse events;

[0195] 8.0 mg / m 2 In group N=9, 9 subjects experienced 70 adverse events.

[0196] During the trial, a total of 145 TEAEs occurred in 21 subjects (N=21), with a TEAE incidence rate of 100.0%; among them

[0197] 1.0 mg / m 2 In group N=1, one subject experienced one TEAE;

[0198] 2.0 mg / m 2 In group N=1, one subject experienced 12 TEAEs;

[0199] 4.0 mg / m 2 In group N=4, 4 subjects experienced 27 TEAEs;

[0200] 6.0 mg / m 2 In group N=6, 37 TEAEs occurred in 6 subjects;

[0201] 8.0 mg / m 2 In group N=9, 68 TEAEs occurred in 9 subjects.

[0202] During the trial, a total of 10 subjects (N=21) experienced 20 grade 3-5 TEAEs, with a grade 3-5 TEAE incidence rate of 47.6%; among them

[0203] 4.0 mg / m 2 In group N=4, one subject experienced two grade 3-5 TEAEs, with an incidence rate of 25.0%.

[0204] 6.0 mg / m 2 In group N=6, one subject experienced four grade 3-5 TEAEs, with an incidence rate of 16.7%.

[0205] 8.0 mg / m 2 In group N=9, 8 subjects experienced 14 grade 3-5 TEAEs, with an incidence rate of 88.9%.

[0206] 1.0 mg / m 2 Group (N=1) and 2.0 mg / m 2 No grade 3-5 TEAEs occurred in any group (N=1).

[0207] During the trial, a total of 4 subjects (N=21) experienced 4 episodes of DLT, with a DLT incidence rate of 19.0%; among them

[0208] 6.0 mg / m 2 In the group (N=6), one subject experienced one episode of DLT, with an incidence rate of 16.7%, which was grade 4 platelet count decrease;

[0209] 8.0 mg / m 2 In group N=9, 3 subjects experienced 3 episodes of DLT, with an incidence rate of 33.3%. These included 2 cases of grade 4 thrombocytopenia and 1 case of grade 2 γ-GT (gamma-glutamyl transferase) elevation (leading to a delay in medication exceeding 14 days, thus determining DLT), at 1.0 mg / m². 2 Group (N=1), 2.0 mg / m 2 Group (N=1) and 4.0 mg / m 2 No DLT occurred in any of the groups (N=4).

[0210] No adverse reactions leading to death occurred in any of the five groups during the trial.

[0211] During the study, the laboratory indicators, vital signs, physical examination, electrocardiogram, weight and other indicators of most subjects remained stable relative to baseline.

[0212] Example 2: Phase II clinical trial of AST-3424 conducted in China

[0213] The clinical trial registration number is CTR20191399.

[0214] The trial was approved by the ethics committees of the medical institutions participating in the clinical trial, conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all participants.

[0215] Inclusion criteria

[0216] 1. Male or female, aged 18 or older.

[0217] 2. Advanced HCC diagnosed by pathological histology and that cannot be controlled by surgical resection or local treatment.

[0218] 3. Patients who have previously received standard systemic therapy, including but not limited to sorafenib and / or oxaliplatin-based systemic chemotherapy, lenvatinib, regorafenib and / or nivolumab, and have experienced disease progression, intolerance to toxicity, or refusal to continue receiving these treatments.

[0219] 4. There is at least one measurable lesion that meets RECIST 1.1 criteria. Lesions that have previously undergone radiotherapy are not considered measurable lesions unless they have shown clear radiographic progression after radiotherapy.

[0220] 5. Able to provide pathological paraffin blocks or sections (including archived pathological paraffin blocks and sections) for AKR1C3 expression analysis, and confirm that the liver tumor tissue is strongly positive for AKR1C3 expression (the proportion of tumor cells with AKR1C3 staining intensity of 2+ and / or 3+ confirmed by the immunohistochemical results of the central laboratory is ≥70%).

[0221] 6. The Eastern Cooperative Oncology Group (ECOG) performance status score is 0 or 1.

[0222] 7. Life expectancy ≥ 12 weeks.

[0223] 8. With or without HBV or HCV infection. a. Subjects with HBV infection must have HBV-DNA levels less than 2,000 IU / ml and receive antiviral therapy with entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide fumarate according to the National Guidelines for the Prevention and Treatment of Chronic Hepatitis B. Maintenance therapy is required during the study and should continue for 6 months after the last dose. b. Subjects with HCV infection (with detectable HCV-RNA or anti-HCV antibodies) may be treated according to medical practice.

[0224] 9. Child-Pugh score ≤ 6.

[0225] 10. No history of hepatic encephalopathy.

[0226] 11. Before starting the investigational drug, all toxicities of previous anticancer treatments (excluding hair loss, fatigue, or peripheral neuropathy) must have been reduced to Grade 1 or baseline levels (NCI CTCAE 5th edition).

[0227] 12. Laboratory tests must meet the following criteria. Within 14 days prior to the screening period, indicators cannot be corrected to meet the inclusion criteria through blood transfusions, hematopoietic stimulating factors, or albumin infusions. a. Hemoglobin ≥ 90 g / L; b. Platelet count ≥ 80 × 10⁻⁶. 9 c. Absolute neutrophil count (ANC) ≥ 1.5 × 10⁹ / L; 9 / L; d. Serum total bilirubin ≤3mg / dL; e. ALT and AST ≤5.0×ULN; f. International normalized ratio (INR) ≤2.3 or prothrombin time prolonged ≤6 seconds; g. Albumin ≥29g / L; h. Creatinine clearance >50mL / min as determined by the Cockcroft-Gault equation.

[0228] 13. No history of alcohol abuse, drug use, or substance abuse within the past year.

[0229] 14. Female patients of childbearing age should have a negative pregnancy test result within 5 days prior to the start of treatment and should not be breastfeeding (a positive urine pregnancy test result needs to be confirmed by a serum pregnancy test).

[0230] 15. Female and male participants of childbearing age must agree to use effective contraception (e.g., surgical sterilization or condoms or diaphragm contraception combined with spermicide gel or intrauterine device [IUD], etc.) with their partners from the start of the study until 6 months after the last dose.

[0231] 16. Participants must voluntarily participate in this study, fully understand the associated risks, demonstrate good compliance, and sign an informed consent form. Participants may also sign a Future Biomedical Research (FBR) consent form. However, participants who do not participate in the FBR may still participate in the main trial.

[0232] Exclusion criteria:

[0233] 1. Untreated active central nervous system (CNS) metastases or leptomeningeal disease. Subjects with adequately treated CNS metastases are eligible to participate in the study if their CNS metastases are confirmed to be stable for at least 4 weeks by clinical examination and brain imaging (MRI or CT) during the screening period.

[0234] 2.2 years of history of other malignant tumors, excluding adequately treated basal cell carcinoma, carcinoma in situ at other sites, or natural disease history, and other tumors whose treatment would not interfere with the safety or efficacy assessment of the current study.

[0235] 3. Underwent major surgery (excluding diagnostic surgery) within 4 weeks prior to the first dose.

[0236] 4. Patients who have received radiotherapy, surgery, chemotherapy, immunotherapy, cancer-specific biotherapy, targeted therapy, or hormone therapy within 4 weeks prior to the first dose (nitrosourea or mitomycin C treatment requires a 6-week washout period; oral fluorouracil drugs require a 2-week washout period; small molecule targeted therapy requires a 2-week washout period).

[0237] 5. Participated in a study of the investigational drug (diagnostic or therapeutic) or device within 4 weeks prior to the first dose.

[0238] 6. During the study, a potent CYP3A4 inhibitor or inducer must be used in combination.

[0239] 7. Uncontrolled active bacterial, viral, or fungal infections requiring systemic treatment.

[0240] 8. Known infection with human immunodeficiency virus (HIV) or positive for syphilis.

[0241] 9. Clinically significant ascites is defined as ascites that is detected by physical examination and requires control by paracentesis or additional pharmacological intervention to maintain symptoms (patients whose ascites can only be detected by imaging examination are eligible).

[0242] 10. Women who are pregnant, breastfeeding, or planning to become pregnant.

[0243] 11. Accompanying diseases or symptoms that may interfere with the study, or physical abnormalities that the investigator believes pose an excessively high risk to the patient. These include, but are not limited to, a history of gastrointestinal bleeding within the past three months or a high risk of bleeding, active peptic ulcers or gastritis, changes in mental status, or mental abnormalities that may interfere with the patient's understanding of the informed consent form.

[0244] 12. History of allergy to ethanol and propylene glycol.

[0245] 13. Subjects who are unwilling or unable to comply with the study protocol for any reason.

[0246] Test drug:

[0247] AST-3424 Injection Concentrated Solution: Manufactured by a pharmaceutical company commissioned by Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd., specification 1mL: 10mg; contains 0.75ml ethanol, 0.25ml propylene glycol and 10mg AST-3424.

[0248] Dosage regimen:

[0249] A cycle lasts 21 days, with one dose administered on day 1 and day 8, at a dose of 6 mg / m² each time. 2They will be allowed to receive up to 34 cycles of treatment.

[0250] The specific procedure for administering the medication is the same as in Example 1.

[0251] Clinical evaluation

[0252] Effectiveness evaluation includes clinical efficacy assessment.

[0253] Clinical efficacy was assessed using RECIST 1.1, the efficacy evaluation criteria for solid tumors. MRI / CT was used to assess lesions, and the same assessment method was used for the same lesion throughout the study. Subjects must have measurable tumor lesions at baseline.

[0254] Efficacy evaluation indicators include complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).

[0255] Complete remission (CR): All target lesions disappear and the short diameter of all pathological lymph nodes (including target nodules and non-target nodules) must be reduced to <10 mm.

[0256] Partial remission (PR): The sum of the diameters of the target lesions is reduced by at least 30% compared to the baseline level.

[0257] Disease progression (PD): The minimum sum of the diameters of all target lesions measured throughout the entire experimental study is used as a reference, with a relative increase of at least 20% in the sum of diameters (or the baseline value if the baseline measurement is the minimum); in addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered disease progression).

[0258] Disease stability (SD): The degree of reduction in target lesions does not reach the PR level, nor does the degree of increase reach the PD level; it falls between the two. The minimum value of the sum of diameters can be used as a reference in studies.

[0259] Study endpoints

[0260] Based on the objective response rate (ORR), disease control rate (DCR), duration of response (DOR), and progression-free survival (PFS) of the subjects, the efficacy of AST-3424 monotherapy for HCC and other malignant tumors was initially evaluated.

[0261] Objective response rate (ORR): The percentage of cases that achieve complete remission (CR) or partial remission (PR) after treatment out of the total evaluable cases.

[0262] The Disease Control Rate (DCR) is the percentage of patients with confirmed complete remission (CR), partial remission (PR), and stable disease (SD) among those with evaluable response rates.

[0263] Test results

[0264] As of March 11, 2024, 30 subjects had been enrolled: 5 were still receiving treatment, 25 had been discharged (1 withdrew informed consent, 10 had died, and 14 were still under follow-up), with the longest current PFS > 11.5 months and the longest OS > 17.6 months. Eleven subjects were enrolled before the end of January 2023, and six of them had an OS greater than 12 months.

[0265] Researcher assessment:

[0266] Of the 26 patients who completed the investigator's initial tumor assessment, 2 achieved partial remission (PR), 15 had stable disease (SD, including 1 patient who underwent unplanned tumor assessment after C2D1), and 9 had progressive disease (PD). The disease control rate (DCR) was 60.7% (17 / 28), and the objective response rate (ORR) was 7.1% (2 / 28) (2 patients who had not yet reached the tumor assessment date were not included).

[0267] Among them, patient 03003 has completed 6 investigator tumor assessments, all of which were partial responses (PR). The target lesion shrinkage was 41.5%, 44.1%, 58.4%, 62.2%, 60.3%, and 64.9% respectively, showing a trend of tumor shrinkage. The alpha-fetoprotein (AFP) also gradually decreased, with values ​​of >1210 (baseline), 436.1, 80.31, 21.2, 6.09, 3.41, and 2.46 ng / ml respectively.

[0268] Patient 06010 has completed three investigator tumor assessments. The first two assessments showed a partial response (PR), with the target lesions shrinking by 50% and 53.8% from baseline, respectively. The third assessment showed a progressive disease (PD), with the target lesions shrinking by 57.7% from baseline, but due to new lesions in the right parietal lobe, retroperitoneum, and diaphragm. The AFP levels were 2630.30 (baseline), 58.52, 174.29, and 1377.57 ng / ml, respectively.

[0269] The details are shown in Table 6 below.

[0270] Table 6: Clinical data on the relationship between AST-3424-hepatocellular carcinoma treatment efficacy and gene mutation as of March 11, 2024

[0271] Of the 26 enrolled subjects with efficacy evaluation results, 20 had p53 gene mutation detection results. Efficacy was observed by grouping subjects according to their p53 detection results.

[0272] Wild-type without mutation (WT), meaning the p53 gene mutation test result is negative (-);

[0273] The mutation significance is unknown (VUS), meaning that the p53 gene mutation test result is positive (+), but it is unclear whether it affects protein expression;

[0274] Mutations that may affect protein function (MUT), i.e., p53 gene mutations that test positive (+), may affect protein expression. They are classified into three groups for statistical analysis, and the results are shown in Tables 7, 8, and 9 below.

[0275] Table 7: Treatment data of 8 patients with P53 mutation-positive (MUT)

[0276] Statistical results showed that 2 of the 8 cases had died, and 6 were still under follow-up. The longest progression-free survival (PFS) was 1.6 months, the longest overall survival (OS) was >14.0 months, the average PFS was 1.4 months, and the average OS was >9.3 months. Among the 8 cases with positive p53 gene mutations (+), which may affect protein expression, there were 0 partial responses (PR), 2 stable disease (SD), and 7 progressive disease (PD). This subgroup had a disease control rate (DCR) of 25% (2 / 8) and an objective response rate (ORR) of 0% (0 / 5). This data was significantly different from the overall disease control rate (DCR) of 60.7% (17 / 28) and objective response rate (ORR) of 7.1% (2 / 28) for the 28 patients in the whole group. The subgroup with positive p53 gene mutations (+), which may affect protein expression, had significantly lower DCR and ORR than the whole group.

[0277] Table 8: Treatment data of 9 patients with P53 mutation-negative (WT) cases

[0278] Statistical results show that 4 of the 9 cases have died, and 5 are still under follow-up. The longest PFS is greater than 11.5 months, the longest OS is greater than 13.8 months, the average PFS is greater than 4.1 months, and the average OS is greater than 7.7 months.

[0279] Among the 9 cases with negative p53 gene mutations, 2 achieved partial response (PR), 5 had stable disease (SD), and 2 had progressive disease (PD). This means that the disease control rate (DCR) in this subgroup was 77.8% (7 / 9), and the objective response rate (ORR) was 22.2% (2 / 9). These figures are significantly different from the overall disease control rate (DCR) of 60.7% (17 / 28) and objective response rate (ORR) of 7.1% (2 / 28) for the total 28 patients. The disease control rate (DCR) and objective response rate (ORR) of the p53 gene mutation negative (-) subgroup were significantly higher than those of the overall group.

[0280] Table 9: Treatment data for 3 patients with P53 mutation-positive cases where it is unclear whether protein expression (VUS) is affected.

[0281] Therefore, based on the current clinical results, those skilled in the art have reason to believe that AST-3424 has a significantly better therapeutic effect on patients with p53 gene mutation or defect-negative (-) cancers and tumors than on patients with p53 gene mutation or defect-positive (+) cancers and tumors. For this reason, the applicant speculates that AST-3424 will have a better therapeutic effect on patients with p53 gene mutation or defect-negative (-) cancers and tumors, that is, patients with p53 gene mutation or defect-negative (-) cancers and tumors who receive AST-3424 treatment will have more significant clinical benefits.

[0282] Patient's previous treatment

[0283] Table 10: Patient's Previous Treatment History

[0284] UK, unknown.

[0285] Chinese medicine.

[0286] Based on the previous treatment history of the 28 enrolled patients and the efficacy evaluation, it can be seen that AST-3424 still has therapeutic effects on patients with progressive disease who have undergone targeted therapy and immunotherapy, or patients with advanced hepatocellular carcinoma whose disease has progressed after targeted and immunotherapy combined.

[0287] Example 3: Follow-up of patients' subsequent treatment after treatment in the Phase II clinical trial of AST-3424 conducted in China.

[0288] We followed up on the survival and treatment of some patients (on October 12, 2024), and extracted the results of patients who received immunotherapy as a follow-up treatment. The results are shown in Table 11 below.

[0289] Table 11: Follow-up on subsequent survival and treatment outcomes of some patients Note: Targeted therapy refers to the administration of various small molecule kinase inhibitors.

[0290] Traditional Chinese medicine decoction.

[0291] Example 9: Follow-up of patients after treatment in the Phase II clinical trial of AST-3424 conducted in China.

[0292] As of the last survival follow-up data on June 26, 2025, the progress of the above clinical trials is as follows.

[0293] Test results

[0294] As of June 26, 2025, 30 subjects had been enrolled; all 30 have been discharged (24 died, 6 are still under follow-up). The longest PFS is currently 14.5 months; the longest OS is >30.2 months. Details are shown in Table 36 below.

[0295] Table 36: Clinical data on the relationship between AST-3424-hepatocellular carcinoma treatment efficacy and gene mutation as of June 26, 2025

[0296] Of the 28 enrolled subjects with efficacy evaluation results, 22 had p53 gene mutation detection results. Efficacy was observed by grouping subjects according to their p53 detection results.

[0297] Wild-type without mutation (WT), meaning the p53 gene mutation test result is negative (-);

[0298] The mutation significance is unknown (VUS), meaning that the p53 gene mutation test result is positive (+), but it is unclear whether it affects protein expression;

[0299] Mutations that may affect protein function (MUT), i.e., p53 gene mutations that test positive (+), may affect protein expression. They are classified into three groups for statistical analysis, and the results are shown in Tables 37, 38, and 39 below.

[0300] Table 37: Treatment data of 9 patients with P53 mutation-positive (MUT)

[0301] Statistical results showed that 8 out of 9 cases had died, and 1 case was still under follow-up. The longest progression-free survival (PFS) was 3.2 months, the longest overall survival (OS) was >28.1 months, the average PFS was 1.6 months, and the average OS was greater than 10.8 months. Among the 9 cases with positive p53 gene mutations (+), which may affect protein expression, there were 0 partial responses (PR), 2 stable disease (SD), and 7 progressive disease (PD). This means that the disease control rate (DCR) in this subgroup was 22% (2 / 9), and the objective response rate (ORR) was 0% (0 / 9).

[0302] Table 38: Treatment data of 9 patients with P53 mutation-negative (WT) cases

[0303] Statistical results showed that 4 of the 9 cases had died, and 5 were still under follow-up. The longest progression-free survival (PFS) was 14.5 months, the longest overall survival (OS) was >26.6 months, the mean PFS was 4.9 months, and the mean OS was greater than 14.5 months. Among the 9 cases with negative p53 gene mutations, 2 had partial response (PR), 5 had stable disease (SD), and 2 had progressive disease (PD). That is, the disease control rate (DCR) in this subgroup of cases was 77.8% (7 / 9), and the objective response rate (ORR) was 22.2% (2 / 9).

[0304] Table 39: Treatment data for 4 patients with P53 mutation-positive cases where it is unclear whether protein expression (VUS) is affected.

[0305] We followed up on the survival and treatment of some patients (follow-up on June 26, 2025), and extracted the results of patients who received immunotherapy in subsequent treatment. The results are shown in Table 40 below.

[0306] Table 40: Follow-up on subsequent survival and treatment outcomes of some patients

[0307] Analysis revealed that the seven patients were insensitive to or resistant to immunotherapy (immune checkpoint inhibitor therapy or combined targeted therapy, cell immunotherapy) before receiving AST-3424. Most of them showed good results during AST-3424 treatment (only one of the seven had PD, while the rest had SD or PR). Notably, these patients responded to immunotherapy (immune checkpoint inhibitor therapy or combined targeted therapy, cell immunotherapy) again in subsequent anti-tumor treatments. In other words, AST-3424 may have the ability to improve or reverse immunotherapy insensitivity or resistance.

[0308] To verify the above hypothesis and demonstrate that AST-3424 has the ability or function to improve or reverse resistance to immune checkpoint inhibitors, the following experiments were conducted.

[0309] Example 4: Therapeutic efficacy of OBI-3424 monotherapy or in combination with anti-PD-1 antibody pembrolizumab (embrolizumab) or anti-PD-L1 antibody avelumab in a HepG2 tumor-humanized mouse CDX model.

[0310] The aim of this study was to evaluate the efficacy of different doses of the OBI-3424 assay against tumor growth and CD8 in the presence of the anti-PD-1 antibody pembrolizumab in a HepG2 humanized mouse CDX model. + The impact of T cell exhaustion on the antitumor effect of combination therapy (OBI-3424 / anti-PD-1 antibody / PD-L1)

[0311] The experimental design, experimental groups, injection dosage and volume, route of administration, and number of animals are listed in Table 12 below.

[0312] Table 12: Dosage regimen and sampling

[0313] *Starting from day 15, the intraperitoneal injection dose is changed to 20 mg / kg.

[0314] The key testing drug information is as follows.

[0315] 1. OBI-3424-DP

[0316] Batch number: FLC-INJ-1711-01

[0317] Number of test samples: 2 vials / 1 mL per vial

[0318] Ingredients: DNA alkylating agent

[0319] Concentration: 10 mg / mL

[0320] Physical appearance: Transparent liquid

[0321] Storage conditions: -20℃

[0322] 2. Anti-human PD-1 antibody, pembrolizumab, Merck & Co., Inc.

[0323] Batch number: 7302614A13

[0324] Number of samples tested: 1 bottle / 1.2 mL per bottle

[0325] Ingredients: Antibodies

[0326] Concentration: 25 mg / mL

[0327] Physical appearance: Transparent liquid

[0328] Storage conditions: 2~8℃

[0329] 3. Anti-human PD-L1 antibody, avelumab, Merck.

[0330] Batch number: AU024788

[0331] Number of test samples: 1 tube / 1.5 mL per tube

[0332] Ingredients: Antibodies

[0333] Concentration: 20 mg / mL

[0334] Physical appearance: Transparent liquid

[0335] Storage conditions: 2~8℃

[0336] Other biological and chemical reagents were purchased from commercial reagent companies and are not listed here.

[0337] 2. Establishment of xenograft mouse model

[0338] 2.1. Animal hair removal: Hair was removed only on the right side of the abdomen before injection of the human liver cancer cell line HepG2.

[0339] 2.2. Subcutaneous inoculation of tumor cells: 1×10 7 One HepG2 cell and 0.25 × 10 7 Premix 1 hPBMC (cell ratio 4:1) and then mix with matrix gel (volume ratio 1:1) (Corning, model: 354248, batch number: 8228001). Subcutaneous injection volume: 200 μL / mouse.

[0340] 3. Method of application of the test item:

[0341] 3.1. On day 8 after tumor cell inoculation, mice were administered the test product anti-hPD-1 antibody or the reference product via intraperitoneal injection. The injection was performed using an insulin injector at a dose of 10 mg / kg, with an injection volume of 10 mL / kg (the injection dose was changed to 20 mg / kg from day 15). For G3 and G5 mice, the test product anti-hPD-1 antibody was administered consecutively on days 8, 11, 15, 18, 22, 25, 29, and 32. The reference product was administered to the G1 group. This procedure followed standard sample administration guidelines.

[0342] 3.2. On day 8 after tumor cell inoculation, mice were administered the test product anti-hPD-L1 antibody or the reference product via intraperitoneal injection. The injection was performed using an insulin injector at a dose of 10 mg / kg, with an injection volume of 10 mL / kg (the injection dose was changed to 20 mg / kg from day 15). For G4 and G6 mice, the test product anti-hPD-L1 antibody was administered consecutively on days 8, 11, 15, 18, 22, 25, 29, and 32. The reference product was administered to the G1 group. This procedure followed standard sample administration guidelines.

[0343] 3.3. On day 14 after tumor cell inoculation, mice were administered the test product OBI-3424 and the reference product intravenously. Insulin was used to administer the product at a dose of 1 mg / kg and an injection volume of 5 mL / kg. For groups G2, G5, and G6, OBI-3424 was administered on days 14, 21, 28, and 35. The reference product was administered to group G1. This procedure followed standard sample administration guidelines.

[0344] 3.4. Preparation of test or reference samples:

[0345] Before administration, dilute the test sample with the reference sample. The concentration of the OBI-3424 test sample solution is 0.2 mg / mL, and the concentrations of the anti-hPD-1 antibody and anti-hPD-L1 antibody are 1 mg / mL (2 mg / mL from day 15).

[0346] 4. Weight measurement:

[0347] Measurements should begin the day after vaccination. Animal weight should be measured and recorded twice a week.

[0348] 5. Tumor diameter measurement:

[0349] Measurements were taken starting the day after vaccination. Tumor volume was measured and recorded twice a week (Monday and Thursday). Based on the records, the tumor volume (major axis × minor axis × minor axis) × (π / 6) was calculated using the equation of an ellipse.

[0350] 6. Calculation of tumor growth inhibition rate:

[0351] The tumor volume is used to calculate the tumor growth inhibition (TGI) rate 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 the load cell group at the end of the experiment (day 35), and T0 and C0 represent the mean tumor volume of the treatment group and the load cell group at the start of the experiment (day 1).

[0352] 7. Blood sampling:

[0353] Collect a submandibular blood sample at the end of the experiment. Blood samples can be collected via cardiac puncture upon animal euthanasia. Centrifuge the collected blood sample at 1500 x g for 15 minutes at 4 ± 2 °C to separate serum and sediment. Collect the supernatant serum and store it at a temperature below -70 °C. This procedure follows standard animal blood sampling procedures.

[0354] 8. Determine the endpoint of the study:

[0355] The study ended on day 36.

[0356] 9. Tumor resection:

[0357] At the end of the study, mice were euthanized with carbon dioxide, and the connective tissue surrounding the tumor was removed. Tumor samples were then taken and weighed. Half of the tissue was fixed in 10% formaldehyde and then embedded in paraffin; the other half was prepared for the isolation of tumor-infiltrating lymphocytes (TILs).

[0358] 10. Isolation of tumor-infiltrating lymphocytes (TILs):

[0359] Half of the mouse tumors were surgically cut into smaller fragments and then digested for at least 2 hours with a mixture of collagenase, DNA hydrolase I, and hyaluronidase (collagenase, model C5138; DNA hydrolase I, model D5025; hyaluronidase, model H6254, Sigma Aldrich). The tumor digest was then passed through a 70 μm sieve cell filter (Falcon, model 352350) using a syringe plunger and washed with PBS. Cells were treated with RBC lysis buffer (Biolegend, model 420302) to prepare single-cell suspensions for flow cytometry.

[0360] 11. Flow cytometry analysis of TIL populations:

[0361] Cells were washed with staining buffer (Biolegend, model 420201), resuspended in staining buffer containing Fc receptor blocking solution (Biolegend, model 422302), and incubated at 4°C for 15 minutes. Cells were then stained with a fluorescently conjugated surface antibody and incubated at 4°C for 30 minutes, before being resuspended in staining buffer for flow cytometry analysis. Flow cytometry analysis was performed using a Navios EX flow cytometer (Beckman Coulter). Data were analyzed using Kaluza analysis software (Beckman Coulter).

[0362] 12. Statistical Analysis:

[0363] Results are expressed as mean ± standard error of mean (Mean ± SEM). The Stulton t-test (Microsoft Excel, 2007) was used to compare all data collected for each treatment group with concurrent negative control data. P ≤ 0.05 was considered statistically significant.

[0364] The aforementioned research and the research process were commissioned to a professional CRO, complied with animal welfare regulations, and were approved by the ethics committee.

[0365] result

[0366] Table 13 summarizes the weight distribution for each group. At the start of the study, there were no statistically significant differences in mean weight among the groups. At the end of the study, compared to other groups, G5 (OBI-3424 + anti-hPD-1 antibody) and G6 (OBI-3424 + anti-hPD-L1 antibody) showed a slight increase in weight (Figure 1 and Table 13). Tumor response was examined using different assays, and mean tumor response was recorded on days 1, 4, 7, 11, 14, 18, 21, 25, 28, 32, and 35 (Figure 3 and Table 14).

[0367] Table 13: Summary of Animal Weights in Example 4

[0368] *: MI052 was found dead on day 34.

[0369] Table 14: Summary of Tumor Volume and Weight in Experimental Animals in Example 4

[0370] *MI052 was found dead on day 34, and its tumor weight was recorded. MI052 was not included in the average tumor weight calculation.

[0371] First, we examined the efficacy of all test samples from G1 (loader), G2 (OBI-3424), G3 (anti-hPD-1 antibody), and G4 (anti-hPD-L1 antibody) on tumor response. At day 35, a significant reduction in mean tumor volume was observed in the presence of OBI-3424 (G1 loader: 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 on day 35 between anti-hPD-1 antibody and anti-hPD-L1 treatment (G1 load: 1538.51 ± 195.78 mm). 3 G3 anti-hPD-1 antibody: 1613.37±338.07 mm 3 G4 anti-hPD-L1 antibody: 1148.64±193.00 mm 3 ).

[0372] Furthermore, the results of combination therapy G5 (anti-hPD-1 antibody + OBI-3424) and G6 (anti-hPD-L1 antibody + OBI-3424) showed a significant reduction in mean tumor volume on day 35 (G1 loader: 1538.51 ± 195.78 mm). 3G5 anti-hPD-1 antibody + OBI-3424: 267.43±32.10 mm 3 p = 0.0001 < 0.001; G6 anti-hPD-L1 antibody + OBI-3424: 452.75 ± 80.72 mm 3 (p = 0.0005 < 0.001), and at the same time, the mean tumor volume between G3 and G5 was significantly reduced on day 35 (G3 anti-hPD-1 antibody: 1613.37 ± 338.07 mm). 3 G5 anti-hPD-1 antibody + OBI-3424: 267.43±32.10 mm 3 (p = 0.002 < 0.05). Furthermore, the mean tumor volume between G4 and G6 was significantly reduced on day 35 (G4 anti-hPD-L1 antibody: 1148.64 ± 193.00 mm). 3 G6 anti-hPD-L1 antibody + OBI-3424: 452.75±80.72mm 3 (p = 0.004 < 0.05) (Figure 3 and Table 14). A similar trend was observed in tumor weight (Figure 2 and Table 14). In addition, one G4 (anti-hPD-L1 antibody) mouse died on day 34, and tumor weight was recorded on the same day.

[0373] 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 detected in each group by flow cytometry. Tables 15–18 provide numerical data for individual mice.

[0374] Compared to loading agents, OBI-3424 treatment (G2) showed increased CD8 levels in tumors. + The number of cytotoxic T cells (CTLs) was significantly increased, but this increase was not observed in tumors treated with anti-PD-1 antibody (G3) or anti-PD-L1 antibody (G4) (Table 15 and Figure 4). However, in tumors treated with OBI-3424 + anti-PD-1 antibody (G5) and OBI-3424 + anti-PD-L1 antibody (G6), the number of CTLs was significantly increased in both groups. This indicates that the increased CTL population was caused by OBI-3424 rather than by anti-PD-1 antibody or anti-PD-L1 antibody treatment.

[0375] Furthermore, in G5 and G6 tumors, CD4 +The number of T helper cells (TH) increased significantly (Table 16 and Figure 5). There were no significant differences in the number of natural killer cells (NK) between the loading agents and between each treatment group (Table 17 and Figure 6). There were no significant differences in the number of PD-L1 cells between the loading agents and between each treatment group (Table 18 and Figure 7).

[0376] Table 15: Summary of CTL cell percentage in experimental animal TILs in Example 4

[0377] CD45 + Gated% = Selected CD45 + cell

[0378] Cell count (×10) 4 = Total number of cells × Total number of cells % × 10

[0379] CTL selection % = selected CD45 + CD8 + cell

[0380] As shown in Figure 4, OBI-3424 monotherapy significantly increased the proportion of tumor-infiltrating CTL cells, and the proportion of CTL cells further increased when OBI-3424 was combined with PD-1 or PD-L1 antibodies, respectively.

[0381] Table 16: Summary of the percentage of TH cells in experimental animals TILs in Example 4

[0382] CD45 + Selection % = Selected CD45 + cell

[0383] Cell count (×10) 4 = Total number of cells × Total number of cells % × 10

[0384] TH selection % = selected CD45 + CD4 + cell

[0385] As shown in Figure 5, the proportion of tumor-infiltrating TH cells did not change significantly after OBI-3424 monotherapy, but the proportion of TH cells increased significantly after OBI-3424 was combined with PD-1 or PD-L1 antibodies.

[0386] Table 17: Summary of NK cell percentage in experimental animals with TILs in Example 4

[0387] CD45 + Selection % = Selected CD45 + cell

[0388] Cell count (×10) 4 = Total number of cells × Total number of cells % × 10

[0389] NK selection % = selected CD45 + CD56 + cell

[0390] As shown in Figure 6, treatment with OBI-3424 alone did not significantly change the proportion of tumor-infiltrating NK cells. However, when OBI-3424 was combined with either PD-1 or PD-L1 antibodies, the proportion of NK cells increased significantly. Furthermore, the combination of OBI-3424 and PD-L1 antibodies significantly increased the proportion of PD-1+ NK cells, further enhancing the cytotoxic ability of PD-1-expressing T cells.

[0391] Table 18: Summary of PD-L1 cell percentage in experimental animal TILs in Example 4

[0392] As shown in Figure 7, OBI-3424 monotherapy significantly increased CD45 levels. + The proportion of cells, and the CD45 after OBI-3424 was used in combination with PD-1 or PD-L1 antibodies, respectively. + The proportion of cells also increased.

[0393] The results showed that for resistance to the immune checkpoint inhibitor PD-1 / PD-L1 antibody, on the one hand, AST-3424 monotherapy can still exert a good therapeutic effect; on the other hand, the administration of AST-3424 can increase the number of related immune cells, thereby reversing the resistance to the immune checkpoint inhibitor PD-1 / PD-L1, thus enabling AST-3424 to exert a combined effect with immune checkpoint inhibitors.

[0394] Example 5: Efficacy evaluation of OBI-3424 + anti-PD-1 antibody pembrolizumab in a HepG2 tumor-humanized mouse CDX model

[0395] The aim of this study was to evaluate the efficacy of different doses of OBI-3424 assay against tumor growth in the presence of the anti-PD-1 antibody pembrolizumab in a HepG2 humanized mouse model, and the impact of CD8+ T cell depletion on the anti-tumor effect of combination therapy (OBI-3424 / anti-PD-1 antibody).

[0396] method

[0397] Experimental Design

[0398] Sampling: Experimental design, experimental groups, injection dosage and volume, route of administration and number of animals are listed in Table 19.

[0399] Table 19: Dosing regimen and sampling for experimental animals in Example 4

[0400] The rest of the process is the same as in Example 4, except for the following part which is different from Example 4, and the corresponding operation is as follows:

[0401] 3. Method of application of the test item:

[0402] 3.1. On day 0, mice were administered the test sample OBI-3424 or the reference sample intravenously. Insulin injectors were used to administer the sample at doses of 0.3 mg / kg or 1 mg / kg, with an injection volume of 5 mL / kg. For groups G2, G3, G5, G6, and G7, OBI-3424 was administered on days 7, 14, 21, and 28. The reference sample was administered to group G1. The procedure followed standard sample administration guidelines. The start date of administration of the test sample was designated as day 1 (D0).

[0403] 3.2. On day 2, the test sample anti-hPD-1 antibody was administered to mice via intraperitoneal injection. The injection was performed using an insulin injector at a dose of 20 mg / kg and an injection volume of 10 mL / kg. For G4, G5, G6, and G7 mice, the test sample anti-hPD-1 antibody was administered consecutively on days 5, 9, 12, 16, 19, 23, and 26. This procedure followed standard sample administration guidelines.

[0404] 3.3. Preparation of test or reference samples:

[0405] Before application, dilute the test sample with the reference sample. The solution concentrations of test sample OBI-3424 are 0.06 mg / mL and 0.2 mg / mL, and the concentration of test sample anti-hPD-1 antibody is 2 mg / mL.

[0406] result

[0407] Table 20 summarizes the body weight of each group. At the start of the study or at sacrifice, there were no statistically significant differences in mean body weight among groups G1–G7 (Figure 8 and Table 20).

[0408] Table 20: Summary of Animal Weights in Example 5

[0409] Tumor response was examined using different test products. The mean tumor response was recorded on days L1, L3, and L7 after tumor cell injection, and on days D0, D2, D6, D9, D13, D16, D20, D23, D27, and D30 after test product administration (Figure 10 and Table 21).

[0410] First, we examined the efficacy of the G1 (loador, i.e., blank solvent group), G2 (OBI-3424 0.3 mg / kg), G3 (OBI-3424 1 mg / kg), and G4 (anti-hPD-1 antibody, 20 mg / kg) assays on tumor response. On day 30, in the presence of OBI-3424 (0.3 mg / kg and 1 mg / kg), a dose-related reduction in mean tumor volume was observed (G1 carrier: 882.92 ± 158.14 mm). 3 ;G2 OBI-3424 0.3mg / kg: 716.44±31.12mm 3 ;G3 OBI-34241mg / kg: 216.90±22.20mm 3 (p = 0.00096 < 0.001). No statistically significant difference was observed in anti-hPD-1 antibody treatment on day 30 (G1 load: 882.92 ± 158.14 mm). 3 G4 anti-hPD-1 antibody: 983.84±266.44 mm 3 ).

[0411] Combination therapy for G5 (OBI-3424 0.3 mg / kg + anti-hPD-1 antibody 20 mg / kg), G6 (OBI-3424 1 mg / kg + anti-hPD-1 antibody 20 mg / kg), and G7 (OBI-3424 1 mg / kg + anti-hPD-1 antibody 20 mg / kg, excluding CD8) + Results from PBMC showed that, at day 30, the mean tumor volume was significantly reduced in all these groups compared to the load cell group (G1 load cell: 882.92 ± 158.14 mm). 3 G5 OBI-3424 0.3 mg / kg + anti-hPD-1 antibody: 429.41 ± 106.14 mm 3 p = 0.0193 < 0.05; G6 OBI-3424 1 mg / kg + anti-hPD-1 antibody: 197.74 ± 19.62 mm 3 p = 0.00078 < 0.001; G7 OBI-3424 1 mg / kg + anti-hPD-1 antibody, excluding CD8 + PBMC: 374.44±36.97mm 3(p = 0.0053 < 0.05).

[0412] Compared to monotherapy, at day 30, combination therapy tended to improve the inhibition of mean tumor volume between G2 and G5, and between G3 and G6, but these reductions were not significant (G2 OBI-3424 0.3 mg / kg: 716.44 ± 31.12 mm). 3 G5 OBI-3424 0.3 mg / kg + anti-hPD-1 antibody: 429.41 ± 106.14 mm 3 ;G3 OBI-3424 1mg / kg: 216.90±22.20mm 3 G6 OBI-3424 1mg / kg + anti-hPD-1 antibody: 197.74±19.62mm 3 Furthermore, the percentage of tumor growth inhibition (TGI) was calculated to quantify the treatment effect. Monotherapy with low-dose and high-dose OBI-3424 resulted in TGIs of 27.82% and 113.27%, respectively. Combination therapy with anti-hPD-1 antibody and low-dose and high-dose OBI-3424 resulted in TGIs of 77.22% and 117.66%, respectively (Figure 10 and Table 21).

[0413] However, for G6 (OBI-3424 1mg / kg + anti-hPD-1 antibody) and G7 (OBI-3424 1mg / kg + anti-hPD-1 antibody, excluding CD8) + Comparison with PBMC, CD8 + Cell exhaustion led to a significant increase in mean tumor volume on day 30 (G6 OBI-3424 1 mg / kg + anti-hPD-1 antibody: 197.74 ± 19.62 mm). 3 G7 OBI-3424 1mg / kg + anti-hPD-1 antibody, excluding CD8 + PBMC: 374.44±36.97mm 3 (p = 0.00088 < 0.001), despite using the same dose of combination therapy, the percentage of TGI decreased from 117.66% to 87.35% (Figure 10 and Table 21). A similar trend was observed in tumor weight (Figure 9 and Table 21).

[0414] Table 21: Summary of Tumor Volume and Weight in Experimental Animals in Example 5

[0415] 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 were measured by flow cytometry. Numerical data for individual mice are provided in Tables 22–27.

[0416] Compared to the loading group, high-dose OBI-3424 treatment, whether alone or in combination with anti-hPD-1 antibodies, resulted in a decrease in cytotoxic lymphocytes (CTLs) (CD45+). + CD8 + T cells and T helper cells (TH) (CD45) + CD4 + The number of T cells increased significantly (CTL cells: G1 carrier: 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 antibody: 38.46±2.63%, p=0.00215<0.05. TH cells: G1 carrier: 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 antibody: 30.62±2.07%, p=0.00012<0.001) (Tables 24-25, Figures 13 and 14).

[0417] Table 22: Summary of the percentage of calreticulin-containing cells in experimental animal TILs in Example 5

[0418] CD45 - Selection % = Selected CD45 - cell

[0419] Live cell selection % = selected CD45 - ViaKrome405 弱 cell

[0420] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0421] As shown in Figure 11, the proportion of calreticulocytes did not change after OBI-3424 monotherapy and OBI-3424 combined with PD-1 antibody.

[0422] Table 23: PD-L1 in experimental animals TILs in Example 5 + Summary of cell percentages

[0423] CD45 - Selection % = Selected CD45 - cell

[0424] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0425] As shown in Figure 12, the proportion of cells expressing PD-L1+ did not change significantly after OBI-3424 was used alone or in combination with PD-1 antibody, indicating that OBI-3424 does not affect the expression of PD-L1 on the surface of tumor cells.

[0426] Table 24: Summary of CTL cell percentage in experimental animal TILs in Example 5

[0427] CD45 - Selection % = Selected CD45 - cell

[0428] CTL selection % = selected CD45 + CD8 + cell

[0429] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0430] As shown in Figure 13, OBI-3424 monotherapy or in combination with PD-1 antibody significantly increased the proportion of granzyme B+ CTL cells and CD69+ CTL lymphocytes with tumor-killing function. Simultaneously, OBI-3424 monotherapy dose-dependently increased the expression of PD-1 antibody on the surface of T cells. In mice with depleted CD8+ PBMCs, due to the lack of the aforementioned tumor-killing lymphocytes, the proportions of granzyme B+ CTL cells and CD69+ CTL lymphocytes did not change significantly after drug administration.

[0431] Table 25: Summary of the percentage of TH cells in experimental animals TILs in Example 5

[0432] CD45 - Selection % = Selected CD45 - cell

[0433] TH selection percentage = selected CD45 + CD4 + cell

[0434] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0435] As shown in Figure 14, after OBI-3424 was used alone or in combination with PD-1 antibody, the proportion of TH cells increased significantly, while the proportion of Treg cells remained unchanged. This indicates that OBI-3424 can increase the promoting effect of TH cells on tumor killing, but does not enhance the inhibitory effect of Treg cells on tumor killing.

[0436] Table 26: Summary of NK cell percentage in TILs of experimental animals in Example 5

[0437] CD45 + Selection % = Selected CD45 + cell

[0438] NK selection % = selected CD45 + CD56 + cell

[0439] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0440] As shown in Figure 15, the proportion of NK cells did not increase significantly after OBI-3424 was used in combination with PD-1 antibody.

[0441] Table 27: Summary of the percentage of dendritic cells in TILs of experimental animals in Example 5

[0442] CD45 + Selection % = Selected CD45 + cell

[0443] DC selection % = Selected CD45 + CD11c + cell

[0444] Cell count (×10) 5 = Total number of cells × Total number of cells % × 10

[0445] As shown in Figure 16, the proportion of dendritic cells (DC cells) increased significantly after OBI-3424 was used alone or in combination with PD-1 antibody, indicating that OBI-3424 can promote the tumor-killing ability of DC cells.

[0446] The results showed that for resistance to the immune checkpoint inhibitor PD-1 antibody, on the one hand, AST-3424 monotherapy (requiring a sufficiently high dose) can still exert a good therapeutic effect; on the other hand, the administration of AST-3424 can increase the number of relevant immune cells, thereby reversing the resistance to the immune checkpoint inhibitor PD-1, thus enabling the combination therapy of low-dose AST-3424 and immune checkpoint inhibitors, which are ineffective in monotherapy, to exert a combined effect.

[0447] Example 6: Experiment on changes in blood prostaglandin levels in cynomolgus monkeys before and after administration of AST-3424

[0448] Three cynomolgus monkeys were used in the experiment as shown in Table 28.

[0449] Table 28: Experiments with AST-3424 administration in cynomolgus monkeys

[0450] Four male cynomolgus monkeys were purchased from Guangxi Xiongsen Primate Development and Experimentation Co., Ltd. All animals were healthy and passed physical examinations without any abnormalities. Three were used for drug administration experiments, and the remaining animals were used to prepare blank plasma.

[0451] Before administration, and at 6, 24, 48, and 72 hours after administration. Collect 1 mL of blood via the femoral vein or other suitable vein and place it in an anticoagulant-free blood collection tube. After collection, place the blood sample on ice and centrifuge for 30–60 minutes to separate the serum (centrifugation conditions: 3500 rpm, 10 minutes, 2–8°C). Store the collected serum at –80°C before analysis.

[0452] Prostaglandins E2 and F2 in serum samples were analyzed using a standard ELISA method. The results are shown in the table below.

[0453] The serum concentrations of prostaglandins E2 and F2 in cynomolgus monkeys after a single intravenous infusion are shown in Table 29 below.

[0454] Table 29: Changes in blood prostaglandin E2 and F2 levels in cynomolgus monkeys before and after administration of AST-3424

[0455] After administration of AST-3424 to cynomolgus monkeys, both prostaglandin E2 and F2 decreased, but fluctuated and increased at 24 hours. This is presumably related to the timing characteristics of prostaglandin E2 and F2 secretion in the animals themselves, indicating that administration of AST-3424 can inhibit the secretion of prostaglandin E2 and F2 in cynomolgus monkeys.

[0456] AST-3424 reduces tumor-induced immunosuppression by inhibiting the production of prostaglandin E2.

[0457] Example 7: Efficacy and safety evaluation of AST-3424 alone and in combination with mouse PD-1Ab in the MH-22A hAKR1C3 C3H syngeneic mouse model.

[0458] The purpose of this experiment is to evaluate the in vivo efficacy and safety of compound AST-3424 as a monotherapy and in combination with mouse PD-1 antibody using the MH-22A hAKR1C3 C3H syngeneic mouse model.

[0459] The experimental design, experimental groups, injection dosage and volume, route of administration, and number of animals are listed in Table 30 below.

[0460] Table 30: Grouping and Dosing Regimens of Animals in In Vivo Efficacy Experiments

[0461] Note:

[0462] N: Number of mice in each group.

[0463] Dosage volume: 10 μL / g based on mouse body weight.

[0464] The day of grouping is recorded as PG-D0, and medication begins from PG-D0.

[0465] Vehicle 2 is the solvent control, which is glucose injection solution D5W (pH=7.4).

[0466] The key testing drug information is as follows.

[0467] 1. AST-3424 Injection

[0468] Provided by: Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd.

[0469] Batch number: 219210701

[0470] Physical characteristics: Pale yellow-green clear liquid

[0471] MW: 460.43

[0472] Packaging: 1mL:10mg

[0473] Concentration: 10 mg / mL

[0474] Storage conditions: -20℃

[0475] Name: AST-001

[0476] Provided by: Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd.

[0477] Batch number: 20210101

[0478] Properties: Colorless liquid

[0479] MW: 555.40

[0480] Concentration: 10 mg / mL

[0481] Packaging: 2mL solution

[0482] Storage conditions: -20℃

[0483] 2. Positive compounds

[0484] Name: Anti-PD-1Ab

[0485] Supplier: BioXCell

[0486] Brand code: BioXCell-BP0146

[0487] Batch number: 810421S1

[0488] Packaging: 55.4mg

[0489] Concentration: 9.90 mg / mL

[0490] Storage conditions: 4℃

[0491] Other biological and chemical reagents were purchased from commercial reagent companies and are not listed here.

[0492] Cell culture

[0493] Mouse hepatocellular carcinoma cells MH-22AhAKR1C3#20 (custom-made mouse hepatocellular carcinoma cells highly expressing human AKR1C3 enzyme protein from a CRO; the specific process is detailed in the references) were cultured in vitro as a monolayer. The culture conditions were: DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 1.5 μg / mL blisterdin; cultured at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When the MH-22AhAKR1C3#20 cells reached 80%-90% saturation and the required number was achieved, the cells were harvested, counted, and seeded.

[0494] Tumor cell inoculation

[0495] 5 × 10⁵ cells were subcutaneously injected into the right nape of the neck of each mouse. 6 Ninety-six MH-22AhAKR1C3#20 cells were inoculated at a volume of 0.1 mL, with PBS as the cell suspension. In vivo efficacy experiments were conducted on day 5 post-inoculation, with the average tumor volume reaching 61 mm². 3At that time, mice were randomly assigned to groups of 6 and started receiving medication. The specific experimental grouping and administration regimens are shown in Table 1.

[0496] The preparation of the solvent and test drug is shown in Table 31 below.

[0497] Table 31: Preparation methods of solvents and test drugs Note: Before administration, bring the AST-3424 solution to room temperature and mix thoroughly.

[0498] Daily observation of laboratory animals

[0499] The experimental protocol and any modifications thereof were evaluated and approved by the International Association for the Management and Use of Laboratory Animals (IACUC). The use and welfare of laboratory animals were conducted in accordance with the regulations of the International Committee for the Evaluation and Accreditation of Laboratory Animals (AAALAC). Animal health and mortality were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on daily behavior, such as activity levels, food and water intake (visual assessment only), weight changes (measured twice weekly), physical appearance, and any other abnormalities. The number of deaths and side effects within each group were recorded based on the number of animals in each group.

[0500] Tumor measurements and experimental indicators

[0501] The experimental endpoint is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0502] The relative tumor-suppressive efficacy of the compounds was evaluated using TGI (%) or relative tumor proliferation rate T / C (%).

[0503] Tumor growth inhibition rate (TGI) (%): The calculation formula is as follows: TGI (%) = [1 - (T... i -T0) / (C i -C0)]×100%. Where T i T0 is the average tumor volume at a certain measurement after the drug administration group is grouped, and C is the average tumor volume at the time of grouping. i C0 is the average tumor volume at a certain measurement after the solvent control group is grouped, and C0 is the average tumor volume at the solvent control group group.

[0504] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTVThe relative tumor volume (RTV) was calculated based on the results of tumor measurements using the solvent control group. The formula is: RTV = V i / V0, where V0 is the average tumor volume measured at the time of grouping (i.e., PG-D0), V i T represents the average tumor volume at a given measurement. RTV With C RTV Take data from the same day.

[0505] Tumor clearance rate: The calculation formula is as follows: N TV＝0 / N×100%(N) TV＝0 Tumor volume in a certain group of mice = 0 mm 3 N: Total number of mice in a group.

[0506] Sample collection

[0507] When the tumor volume of a single mouse exceeds 3,000 mmHg 3 Alternatively, on day 28 after grouping, tumor samples are collected, weighed, and recorded. The tumors are then photographed and flash-frozen in liquid nitrogen. All samples are stored at -80°C before being sent to the customer. See section 9.4 for tumor photographs.

[0508] Statistical analysis

[0509] Statistical analysis was performed, including the mean and standard error (SEM) of tumor volume at each time point for each group (see Table 5 in Section 6.2 for specific data). Statistical analysis was conducted based on the data to assess differences between groups. Comparisons between two groups were performed using the T-test. Comparisons between three or more groups were performed using one-way ANOVA. If the variances were homogeneous (F-test p-value > 0.05), Dunnett's test was used; if the variances were unequal (F-test p-value < 0.05), Games-Howell's multiple comparison test was used. p > 0.05 was considered statistically insignificant, p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

[0510] Experimental results

[0511] The body weight of experimental animals is used as a reference indicator for indirectly determining drug toxicity. The effects of AST-3424 alone and in combination with mouse PD-1Ab on the body weight of MH-22A hAKR1C3 C3H syngeneic mouse model are shown in Figures 17 and 18.

[0512] In the MH-22A hAKR1C3#20 cell subcutaneous xenograft C3H syngeneic mouse model, the changes in tumor volume in each group after administration of AST-3424, anti-PD-1Ab monotherapy, and AST-3424 and anti-PD-1Ab combination therapy are shown in Table 32.

[0513] Table 32: Tumor volume at different time points in each group (mm) 3 ) Note: a. Mean ± SEM, n = 6. b. Number of days after grouping.

[0514] Tumor change curve

[0515] The tumor growth curves of tumor-bearing mice after treatment with the test substance are shown in Figure 19.

[0516] Starting from day 18 after grouping, some experimental animals in the MH-22AhAKR1C3#20 model were euthanized due to tumor volume exceeding 3,000 mm3. Therefore, the tumor volume on day 18 after grouping was used to evaluate the antitumor efficacy. The results of the antitumor efficacy evaluation are shown in Tables 33 and 34.

[0517] On day 28 after grouping, tumors in the MH-22AhAKR1C3#20 model were completely cleared. Therefore, the tumor clearance rate was calculated based on the tumor volume on day 28 after grouping. The results of the tumor clearance rate are shown in Table 35.

[0518] The p-value was analyzed using a T-test based on the tumor volume (compared to the solvent control group, Vehicle 1). p>0.05 was considered to be of no significant difference, p<0.05 was considered to be of significant difference, and p<0.01 was considered to be of highly significant difference.

[0519] Table 33: In vivo efficacy evaluation of AST-3424 monotherapy and AST-3424 in combination with mouse PD-1Ab in the MH-22AhAKR1C3#20 model, based on tumor volume calculation on day 18 after grouping. Note: Mean ± SEM.

[0520] Tumor growth inhibition rate (TGI) (%) = [1 - (T18 - T0) / (C18 - C0)] × 100%.

[0521] Relative tumor proliferation rate T / C% = TRTV / CRTV × 100%, RTV = V18 / V0.

[0522] The p-value was analyzed using one-way ANOVA based on tumor volume (compared to the solvent control group Vehicle 2). p>0.05 was considered as no significant difference, p<0.05 was considered as a significant difference, and p<0.01 was considered as a highly significant difference.

[0523] Table 34: In vivo efficacy evaluation of AST-3424 on the MH-22AhAKR1C3#20 model, based on tumor volume calculation on day 18 after grouping. Note: Mean ± SEM.

[0524] The p-value was analyzed using a T-test based on tumor volume (compared to Group 2). p>0.05 was considered to be of no significant difference, p<0.05 was considered to be of significant difference, and p<0.01 was considered to be of highly significant difference.

[0525] The p-value was analyzed using a T-test based on tumor volume (compared to Group 4). p>0.05 was considered to indicate no significant difference, p<0.05 was considered to indicate a significant difference, and p<0.01 was considered to indicate a highly significant difference.

[0526] Table 35: Tumor clearance rates of AST-3424 monotherapy and AST-3424 in combination with mouse PD-1Ab in the MH-22A hAKR1C3 C3Hsyngeneic model, calculated based on tumor volume on day 28 after grouping.

[0527] AST-3424 1mpk QW×2 (group 4) and 1mpk QD×5, 2 weeks off, QD×5 (group 4) single drug group showed significant efficacy.

[0528] The combination of AST-3424 1mpk QW×2 and mouse PD-1 (group 6) showed better efficacy than the individual monotherapy groups (groups 4 and 5). At the end of the experiment, tumors were completely cleared in three mice, with a clearance rate of 50%.

[0529] In the AST-3424 1mpk QD×5, 2 weeks off, the tumors of 4 mice in the QD×5 group and the PD-1 combination group (group 8) were completely cleared, with a clearance rate of 66.7%; in the monotherapy group (group 7), only 2 mice had their tumors completely cleared, with a clearance rate of 33.3%.

[0530] Example 8: In vitro immunogenicity-induced death ICD experiment using OBI-3424

[0531] To demonstrate that AST-3424 can induce immunogenic death, the following experiment was conducted.

[0532] 1. Experiment on ATP changes after OBI-3424 treatment of cancer cells

[0533] The NCI-H460 cell suspension was plated.

[0534] Cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0535] Compound treatment: The test compound OBI-3424 was added at 100 nM (DMSO as solvent) for treatment.

[0536] Sample collection: Collect cell supernatant into sterile EP tubes, centrifuge at 13000 rpm and 4℃ for 10 min, and then transfer the supernatant into new EP tubes.

[0537] Prepare the standard and working solution according to the instructions (ATP Assay Kit (Bioluminescence, product number: abs580117)).

[0538] Set up blank control wells, standard wells, and sample wells: Add 50 μL of ATP Assay Buffer to the blank control wells, add 50 μL of gradient concentration ATP Standard (0.0078-1 μM) to the standard wells, and add 50 μL of sample to the sample wells.

[0539] Add 50 μL of ATP detection working solution to each well, mix well, and incubate at room temperature for 5 min.

[0540] After the reaction was complete, the relative luminescence intensity (RLU) was measured using a chemiluminescent microplate reader. A standard curve was plotted based on the standards, and the ATP concentration in each sample well was calculated from the RLU readings using the standard curve. The results are shown in Figure 20. OBI-3424 treatment significantly increased the level of extracellular ATP in the cells.

[0541] 2. Experiment on changes in human high-mobility group box 1 (HMGB-1) after OBI-3424 treatment of cancer cells

[0542] The NCI-H460 cell suspension was plated.

[0543] Cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0544] Compound treatment: The test compound OBI-3424 was treated with 25, 50, and 100 nM (DMSO as solvent).

[0545] Sample collection: Collect cell supernatant into sterile EP tubes, centrifuge at 13000 rpm and 4℃ for 10 min, and then transfer the supernatant into new EP tubes.

[0546] Prepare the standard, washing solution, biotinylated antibody working solution, and enzyme conjugate working solution according to the instructions (Human High Mobility Group Box B1 ELISA Kit, Product No.: SEKH-0409).

[0547] Set up standard wells, blank control wells (0 wells), and sample wells: Add 100 μL of standard / sample diluent to the blank control well, add 100 μL of graded concentration standard to the standard well, and add 100 μL of the sample to be tested to the sample well.

[0548] After incubating at room temperature with shaking for 120 min, add 300 μL of washing buffer to each well to wash the plate 4 times.

[0549] Add 100 μL of biotinylated antibody to each well for detection, incubate at room temperature with shaking for 60 min, then add 300 μL of washing buffer to each well to wash the plate 4 times.

[0550] Add 100 μL of enzyme conjugate working solution to each well, incubate at room temperature with shaking for 30 min, then add 300 μL of washing buffer to each well to wash the plate 5 times.

[0551] Add 100 μL of the chromogenic substrate TMB to each well and allow it to develop at room temperature in the dark for 5-30 minutes.

[0552] Add 50 μL of stop solution to each well.

[0553] Within 5 minutes, dual-wavelength detection was performed using a microplate reader to measure the OD value at the maximum absorption wavelength of 450 nm, and the concentration was finally calculated. The results are shown in Figure 21. OBI-3424 treatment significantly increased the amount of extracellular HMGB1 protein in cells.

[0554] 3. Experiment on changes in calreticulin (CRT) after OBI-3424 treatment of cancer cells.

[0555] NCI-H460 and HepG2 cell suspensions were plated.

[0556] Cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0557] Compound treatment: Add the test compound OBI-3424 100 nM (DMSO as solvent) and treat for 48 or 72 hours.

[0558] Sample collection: Centrifuge at 4°C, wash once with pre-cooled PBS, fix with 1 mL of pre-cooled 4% polymethanol at room temperature for 15 min, centrifuge at 4°C, wash once with pre-cooled PBS, resuspend in 0.5 mL of PBS, and proceed to the next step or store at 4°C.

[0559] Add 4.5 mL of pre-cooled 100% methanol for permeation, resulting in a final methanol concentration of 90%. Permeate on ice for 10 min, then stain or store at -20°C.

[0560] After centrifugation to remove methanol, wash once with pre-cooled PBS, and dilute the primary antibody (Calreticulin (D3E6)XP Rabbit mAb, #12238) 1:1000 with 0.5% BSA PBS buffer. Add 100 μL of the diluted primary antibody to each sample and incubate at room temperature for 1 h.

[0561] Centrifuge and discard the primary antibody, wash twice with PBS, then add the secondary antibody (Anti-Rabbit IgG (H+L), F(ab)2Fragment (Alexa)). Dilute 488Conjugate#4412) 1:1000, add 100μL and incubate at room temperature for 30min in the dark; after centrifugation, discard the secondary antibody, wash twice with PBS, and resuspend in 200-500μL PBS.

[0562] Flow cytometry was used to detect the cells, with an excitation wavelength of 488 nm.

[0563] Results: After treating H460 and HepG2 cells with OBI-3424 for 48 h, the proportion of Ecto-CRT positive cells increased from 2.8% and 3.7% to 15.9% and 9.3%, respectively. After treating HepG2 cells with OBI-3424 for 72 h, the proportion of Ecto-CRT positive cells increased from 3.3% to 24%.

[0564] The results of the three in vitro cell experiments above demonstrate that AST-3424 can significantly enhance the expression of multiple ICD indicators (ATP, HMGB1, Ecto-CRT) by activating immune cell death (ICD).

[0565] The above experiments proved AST-3424:

[0566] Activates immunogenic cell death;

[0567] By inhibiting the activation of immune responses by prostaglandins;

[0568] Increase the number of functional immune cells infiltrating tumor cells;

[0569] Increases the expression of PD-1 antigen on the surface of T cells infiltrating tumor cells;

[0570] This can improve or reverse the resistance or insensitivity of immunotherapy in cancer / tumor patients, thus enabling the treatment of cancer / tumor patients resistant to immune checkpoint inhibitors as monotherapy or in combination with immunotherapy; or the cross-application of AST-3424 with immunotherapy regimens (immune checkpoint inhibitors / immunotherapy drugs) as monotherapy to treat cancer / tumor patients, especially those who are insensitive to or resistant to immunotherapy regimens.

[0571] All patent applications cited in this application are incorporated in their entirety into this specification.

[0572] Non-patent literature cited in this application (academic journal articles, academic conference papers, etc.):

[0573] Document 1, Meng F, Li WF, Jung D, et al. A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. Am J Cancer Res. 2021; 11(7): 3645-3659;

[0574] Document 2, Evans K, Duan J, Pritchard T, et al. OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL.Clin Cancer Res.2019;25(14):4493-4503.doi:10.1158 / 1078-0432.CCR-19-0551;

[0575] Document 3, Wang Y, Liu Y, Zhou C, et al. An AKR1C3-specific prodrug with potent anti-tumor activities against T-ALL. Leuk Lymphoma. 2020; 61(7): 1660-1668.doi:10.1080 / 10428194.2020.1728746;

[0576] Reference 4, He P, Wang C, Wang Y, et al. A Novel AKR1C3 Specific Prodrug TH3424 With Potent Antitumor Activity in Liver Cancer[retracted in: Clin Pharmacol Ther. 2021 Jul;110(1):262]. Clin Pharmacol Ther. 2021, 110(1):229-237. doi:10.1002 / cpt.2171;

[0577] Reference 5, Tsimberidou, Apostolia & Verschraegen, Claire & Hsu, Pei & Pearce, Tillman. (2022). Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. Journal of Clinical Oncology. 40. 3030-3030. 10.1200 / JCO.2022.40.16_suppl.3030, poster can be downloaded from the official website of OBIPharma Inc, the download address is https: / / www.obipharma.com / news / news-2022 / poster-presentations-at-the-2022-asco-annual-meeting-for-adagloxad-simolenin-obi-999-and-obi-3424 / ; or Tsimberidou, A.M., Verschraegen, C.F., Wesolowski, R. et al. Phase 1 dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer 129, 266–274 (2023). https: / / doi.org / 10.1038 / s41416-023-02280-4;

[0578] Reference 6: Zhang Y, Qin S, Chao J, Luo Y, Sun Y and Duan J (2022) The In-Vitro Antitumor Effects of AST-3424 Monotherapy and Combination Therapy With Oxaliplatin or 5-Fluorouracil in Primary Liver Cancer. Front. Oncol. 12: 885139. doi: 10.3389 / fonc.2022.885139;

[0579] Reference 7: Tsimberidou, A.M., Verschraegen, C.F., Wesolowski, R. et al. Phase 1 dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer 129, 266–274 (2023). https: / / doi.org / 10.1038 / s41416-023-02280-4.

[0580] Reference 8: Ascierto ML, McMiller TL, Berger AE, Danilova L, Anders RA, Netto GJ, Xu H, Pritchard TS, Fan J, Cheadle C, Cope L, Drake CG, Pardoll DM, Taube JM, Topalian SL. The Intratumoral Balance between Metabolic and Immunologic Gene Expression Is Associated with Anti-PD-1 Response in Patients with Renal Cell Carcinoma. Cancer Immunol Res. 2016 Sep 2; 4(9): 726-33. doi: 10.1158 / 2326-6066.CIR-16-0072.

[0581] Unless otherwise specified, all English abbreviations used in this application shall be as defined in pharmacy and medical textbooks.

Claims

1. A method for treating a cancer / tumor patient who is resistant or insensitive to an immunotherapy regimen, wherein the immunotherapy regimen is preferably selected from an immune checkpoint inhibitor / immune cell therapy drug treatment regimen, by AST-3424 monotherapy or in combination with the immunotherapy regimen.

2. Use of AST-3424 monotherapy or in combination with an immunotherapy regimen in the preparation of a medicament for treating a cancer / tumor patient who is resistant or insensitive to an immunotherapy regimen, wherein said immunotherapy regimen is preferably selected from an immune checkpoint inhibitor / immune cell therapy drug treatment regimen.

3. A method for treating a cancer / tumor patient by cross administration of AST-3424 monotherapy and an immunotherapy regimen, wherein said immunotherapy regimen is preferably selected from an immune checkpoint inhibitor / immune cell therapy drug treatment regimen.

4. The use or method according to claim 1 or 2, wherein: the cancer / tumor is hepatocellular carcinoma, and the cancer / tumor patient has a pathological paraffin block or pathological section of an ex vivo tumor tissue with AKR1C3 enzyme protein expression level detected by immunohistochemical staining method, and the detection result has an H-score score greater than or equal to 200; or the cancer / tumor patient has a pathological paraffin block or pathological section of an ex vivo tumor tissues with AKR1C3 enzyme protein expression level detected by immunohistochemical staining method, wherein the detection result shows that the sum of the percentages of medium intensity staining and high intensity staining is greater than or equal to 70%.

5. The use or method according to claim 4, wherein: the patient is a patient with negative p53 gene mutation detection or normal p53 protein expression.

6. Use or method according to claim 4, characterized in that the administration regimen of AST-3424 monotherapy is: Each 21 -day cycle, on days 1 and 8, with a first dose of 6.0 mg / m 2 The longest treatment will be allowed to receive 34 cycles.

7. Use or method according to claim 6, characterized in that The dosing regimen for AST-3424 is 6.0 mg / m 2 at the first dose, and if the patient is intolerant, then the dose is reduced by 4.5, 3.0, 1.5 mg / m 2 , in that order.

8. The use or method according to claim 1 or 2, wherein the AST-3424 is prepared as an AST-3424 concentrated solution for injection, and the specification is 10 mg of AST-3424 active pharmaceutical ingredient per 1 mL.

9. The use or method according to claim 8, wherein the AST-3424 is prepared as an AST-3424 concentrated solution for injection with the specification of 10 mg of AST-3424 active pharmaceutical ingredient per 1 mL, and is labeled as containing 0.75 ml of ethanol, 0.25 ml of propylene glycol and 10 mg of AST-3424 active pharmaceutical ingredient.

10. The use or method according to claim 9, wherein: before administration, 0.1 ml of 5% sodium bicarbonate injection is added to 100 ml of sterile 5% glucose injection to adjust the pH value in a bag containing no di(2-ethylhexyl) phthalate; the calculated milliliter number of AST-3424 concentrated solution for injection is added to the 5% glucose injection bag after pH adjustment, and the preparation is accurate to 0.01 ml, and is used for intravenous infusion administration of AST-3424 injection for intravenous injection administration; if the patient is not suitable for injection of glucose, use normal saline instead: before administration, 0.1 ml of 5% sodium bicarbonate injection is added into 100 ml of sterile 0.9% injection of normal saline to adjust the pH value in a bag containing no di(2-ethylhexyl) phenthalate; To the bag of physiological saline after adjusting pH value, add the calculated required milliliter of AST-3424 injection concentrated solution, accurate to 0.01 ml, to prepare AST-3424 injection for intravenous infusion administration for intravenous injection administration.

11. Use or method according to claim 10, characterized in that, The prepared intravenous AST-3424 injection should be injected within 8 hours, preferably within 25-35 minutes.

12. Use or method according to claim 1 or 2, characterized in that, When using AST-3424 in combination with an immune checkpoint inhibitor for treatment, AST-3424 is administered first, and the immune checkpoint inhibitor is administered later.

13. Use or method according to claim 1 or 2, characterized in that, The immune checkpoint inhibitor is an anti-immune checkpoint antibody that inhibits / blocks an inhibitory immune checkpoint antigen, and the anti-immune checkpoint antibody is preferably an anti-PD-1 / PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-Ceacam 1 antibody, an anti-LAIR-1 antibody, an anti-TIM-3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-IDO antibody, an anti-CD276 antibody, an anti-A2AR antibody, or an anti-CD47 antibody.

14. The use or method of claim 13, wherein the anti-PD-1 / PD-L1 antibody is avelumab, nivolumab, pembrolizumab, durvalumab, and / or atezolizumab, and in the case of cancer being liver cancer, the anti-PD1 antibody is pembrolizumab or avelumab.

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