Treatment of malignant tumor

Abstract

Provided is a method for treating malignant tumor. Provided is a method for treating malignant tumor, the method comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate of BCV or the pharmaceutically acceptable salt, and an immunotherapeutic agent to a subject. Alternatively, provided is a pharmaceutical composition for use in the treatment of malignant tumor, wherein the treatment comprises combination treatment to a subject with BCV, a pharmaceutically acceptable salt thereof, or a solvate of BCV or the pharmaceutically acceptable salt, and an immunotherapeutic agent, and wherein the pharmaceutical composition comprises BCV, a pharmaceutically acceptable salt thereof, or a solvate of BCV or the pharmaceutically acceptable salt, or an immunotherapeutic agent.

Description

Treatment of malignant tumors

[0001] The technical field of the present invention relates to the treatment of malignant tumors.

[0002] Brincidofovir (BCV) was approved by the U.S. Food and Drug Administration (FDA) in 2021 as a treatment for smallpox. Non-patent document 1 describes a benefit-risk assessment of BCV in the treatment of smallpox. Non-patent document 2 states that monkeypox outbreaks began in May 2022, and that the use of BCV is recommended for the management of confirmed cases.

[0003] "Benefit-risk assessment for brincidofovir for the treatment of smallpox: US Food and Drug Administration's Evaluation" Chan-Tack et al., Antiviral Res. 2021 Nov;195:105182. "Monkeypox: A Mini-Review on the Globally Emerging Orthopoxvirus" Evans et al., Int J Environ Res Public Health. 2022 Nov 25;19(23):15684.

[0004] Many questions remained regarding the effectiveness and methods of treating malignant tumors using BCV.

[0005] On the other hand, the inventors of this application have demonstrated that combination therapy using BCV and immunotherapy drugs can provide excellent therapeutic effects against malignant tumors.

[0006] According to one aspect of the present invention, a method for treating a malignant tumor is provided, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug to a subject. This method provides excellent therapeutic effects against malignant tumors.

[0007] According to one aspect of the present invention, a pharmaceutical composition for use in the treatment of a malignant tumor is provided, wherein the treatment comprises a combination therapy of a target with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug, and the pharmaceutical composition comprises BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. Using this pharmaceutical composition, an excellent therapeutic effect against malignant tumors can be obtained.

[0008] The following shows the results of measuring the in vitro cytotoxic activity of BCV against various solid tumor cell lines. The dose-response curves for BCV against various solid tumor cell lines are shown. The following shows the dose-response curves for BCV against various solid tumor cell lines. The following shows the dose-response curves for BCV against various solid tumor cell lines. The following shows the results of measuring tumor size after administering various doses of BCV to humanized tumor-bearing mouse models. Figure 5(a) shows the tumor growth curve, and Figure 5(b) shows the tumor growth inhibition rate. The following shows the results of measuring tumor size in humanized tumor-bearing mouse models after administration of various drugs. Figure 6(a) shows the tumor growth curve, and Figure 6(b) shows the tumor growth inhibition rate. Anti-PD1 means anti-PD-1 antibody. PD1 and PD-1 have the same meaning. The following shows the results of measuring tumor size in humanized tumor-bearing mouse models after administration of various drugs. Figure 7(a) shows the tumor growth curve, and Figure 7(b) shows the tumor growth inhibition rate. The following shows the tumor tissue images of humanized tumor-bearing mouse models after administration of various drugs. The following are results of immunohistochemical staining of tumor tissue from humanized mouse models of tumors after administration of various drugs. The following are results of measuring the infiltration of CD4-positive T cells into tumor tissue from humanized mouse models of tumors after administration of various drugs. The following are results of measuring the infiltration of CD8-positive T cells into tumor tissue from humanized mouse models of tumors after administration of various drugs. The following are results of measuring the percentage of active Caspase-3-positive cells in p63-positive tumors from humanized mouse models of tumors after administration of various drugs. The following are results of measuring the tumor size from humanized mouse models of tumors after administration of various drugs. The graph shows the tumor growth curve for each mouse. The following are results of measuring the tumor size from humanized mouse models of tumors after administration of various drugs. The graph shows the tumor growth curve for each mouse. The following are results of measuring the tumor size from humanized mouse models of tumors after administration of various drugs. The graph shows the tumor growth suppression rate for each group. The following are results of measuring the tumor size from humanized mouse models of tumors after administration of various drugs. Figure 17(a) shows the tumor growth curve, and Figure 17(b) shows the tumor growth suppression rate. The results of measuring the infiltration of CD4-positive T cells into tumor tissue of a humanized mouse model with tumor after administration of various drugs are shown. The results of measuring the infiltration of CD8-positive T cells into tumor tissue of a humanized mouse model with tumor after administration of various drugs are also shown.The following results are shown: Analysis of the percentage of active Caspase-3-positive cells in p63-positive tumors of a humanized mouse model of tumor-bearing cells after administration of various drugs. Analysis of the movement of calreticulin molecules to the cell surface after culturing FaDu cells with various drugs for 24 hours. Analysis of the movement of calreticulin molecules to the cell surface after culturing FaDu cells with various drugs for 48 hours. Analysis of the movement of calreticulin molecules to the cell surface after culturing PC-3 cells with various drugs for 24 hours. Analysis of the movement of calreticulin molecules to the cell surface after culturing PC-3 cells with various drugs for 48 hours. Analysis of the movement of calreticulin molecules to the cell surface after culturing CaSki cells with various drugs for 24 hours. Analysis of the movement of calreticulin molecules to the cell surface after culturing CaSki cells with various drugs for 48 hours. The following shows the results of analyzing the movement of calreticulin molecules to the cell surface after culturing SNU-719 cells with various drugs for 24 hours. The following shows the results of analyzing the movement of calreticulin molecules to the cell surface after culturing SNU-719 cells with various drugs for 48 hours. The results of the results of Figures 21-24 are histograms. The results of the results of Figures 25-28 are histograms. The results of measuring PD-L1 mRNA expression after culturing PC-3 or FaDu with BCV are shown. The results of measuring PD-L1 mRNA expression after culturing A549 or SNU-719 with BCV are shown. The results of measuring PD-L1 mRNA expression after culturing CaSki or SK-OV-3 with BCV are shown. The results of measuring PD-L1 protein expression in BCV after culturing PC-3, FaDu, SNU-719, or A549 with BCV are shown. The results of measuring PD-L1 protein expression after culturing CaSki or SK-OV-3 with BCV are shown. Figures 34 and 35 are histograms of the results. They show the results of measuring the survival rate of blood cancer cells after BCV treatment. They show the results of measuring the tumor size of lymphoma mouse models after administration of various drugs. Figure 38(a) shows tumor volume, and Figure 38(b) shows body weight. They show the results of measuring the tumor weight of lymphoma mouse models after administration of various drugs.The results of gene expression analysis in lymphoma mouse models after administration of various drugs are shown. The results of gene expression analysis in lymphoma mouse models after administration of various drugs are shown. The results of gene expression analysis in lymphoma mouse models after administration of various drugs are shown. The results of gene expression analysis in lymphoma mouse models after administration of various drugs are shown. The results of tumor size measurement in colorectal cancer mouse models after administration of various drugs are shown. The results of survival rate measurement in colorectal cancer mouse models after administration of various drugs are shown.

[0009] The embodiments of the present invention will be described in detail below. To avoid repetition and unnecessary complexity, similar content will be omitted from explanation as appropriate.

[0010] (1) Method According to one embodiment of the present invention, a method for treating a malignant tumor is provided, comprising administering to a subject brincidofovir (BCV), a pharmaceutically acceptable salt thereof, or a solvate thereof. This method may be carried out in combination therapy using, for example, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug. This method may also include, for example, administering an immunotherapy drug to a subject. This method may also include, for example, performing immunotherapy on a subject. The combination therapy provides an excellent therapeutic effect against the malignant tumor. The excellent therapeutic effect may include, for example, an effect of inducing the infiltration of immune cells (e.g., CD8-positive T cells) in the tumor microenvironment, an effect of inducing tumor cell damage in the tumor microenvironment, an effect of inducing tumor cell death, an effect of suppressing variability in response between individuals, or an effect of extending the patient's life. The excellent therapeutic effect may also include, for example, a synergistic therapeutic effect of the combination therapy. In the embodiments described below, the combination therapy significantly induced the infiltration of immune cells into tumor tissue. On the other hand, monotherapy with BCV or an immunotherapy drug did not show a statistically significant difference in inducing invasion compared to vehicle administration. These results were surprising, as they demonstrate that combination therapy is a superior treatment method in terms of significantly inducing tumor cell damage in the tumor microenvironment. According to this embodiment, a method for treating malignant tumors may be provided, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject who has undergone immunotherapy. According to this embodiment, a method for treating malignant tumors may be provided, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug to a subject. According to this embodiment, a method for treating malignant tumors may be provided, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug in combination to a subject.

[0011] (2) Method According to one embodiment of the present invention, a method for treating a malignant tumor is provided, comprising administering an immunotherapy drug to a subject. This method may be carried out in combination therapy using, for example, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug. This method may, for example, involve administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject. Excellent therapeutic effects against malignant tumors can be obtained by combination therapy. According to this embodiment, a method for treating a malignant tumor is provided, comprising administering an immunotherapy drug to a subject who has been administered BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0012] (3) Method According to one embodiment of the present invention, a method for treating a malignant tumor is provided, comprising administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and combination therapy with an immunotherapy drug. This method may include, for example, administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject, or administering an immunotherapy drug to a subject. Excellent therapeutic effects against malignant tumors can be obtained by combination therapy.

[0013] (4) Method According to one embodiment of the present invention, a therapeutic method is provided for inducing tumor cell injury, comprising administering to a subject BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. This method may be carried out in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, for example, an excellent tumor cell injury induction effect can be obtained.

[0014] (5) Method According to one embodiment of the present invention, there is a method for inducing the infiltration of CD8-positive T cells, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug to a subject. This method may be carried out in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, for example, an excellent effect in inducing the infiltration of CD8-positive T cells can be obtained.

[0015] (6) Method According to one embodiment of the present invention, there is a method for inducing the expression of an immune checkpoint molecule, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug to a subject. This method may be carried out in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, for example, an excellent effect in inducing the expression of an immune checkpoint molecule can be obtained.

[0016] (7) Method According to one embodiment of the present invention, a method for treating a neuroendocrine tumor is provided, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject. This method can treat a patient suffering from a neuroendocrine tumor. This method can significantly suppress the growth of the neuroendocrine tumor.

[0017] (8) Methods One embodiment of the present invention (for example, (1) to (7) above) may include, for example, the steps of (i) testing for the presence or absence of a target malignant tumor, (ii) identifying a target having a malignant tumor, (iii) identifying a target requiring treatment for a malignant tumor, or (iv) identifying a target requiring prevention of a malignant tumor. Another embodiment of the present invention (for example, (1) to (7) above) may also include, for example, the steps of testing for the expression of a target immune checkpoint molecule or its ligand (e.g., PD-L1), detecting tumor cells positive for the target immune checkpoint molecule or its ligand, or identifying a target in which the expression of an immune checkpoint molecule or its ligand has been confirmed as a target for treatment. Furthermore, a method according to one embodiment of the present invention (for example, (1) to (7) above) may or may not include, for example, the following, if specified: a step of identifying a subject having an adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), EB virus (EBV), or baryola virus (VaV) infection, or a method for the prevention or treatment of a disease associated with AdV, CMV, BKV, EBV, or VaV infection.

[0018] (9) According to one embodiment of the composition, a pharmaceutical composition for use in the treatment of a malignant tumor is provided, the pharmaceutical composition comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. This treatment may be, for example, a combination therapy of a subject with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug. The combination therapy provides an excellent therapeutic effect against the malignant tumor. The excellent therapeutic effect may include, for example, an effect of inducing the infiltration of CD8-positive T cells in the tumor microenvironment, an effect of inducing tumor cell damage in the tumor microenvironment, an effect of inducing tumor cell death, an effect of suppressing the variability of response between individuals, or an effect of extending the patient's life. The excellent therapeutic effect may also include, for example, a synergistic therapeutic effect of the combination therapy.

[0019] (10) According to one embodiment of the composition, a pharmaceutical composition for use in the treatment of a malignant tumor is provided, wherein the pharmaceutical composition comprises an immunotherapy drug. This treatment may be, for example, a combination therapy using BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug to a subject. The combination therapy provides an excellent therapeutic effect against malignant tumors.

[0020] (11) According to one embodiment of the composition, a pharmaceutical composition for use in the treatment of malignant tumors is provided, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug. This pharmaceutical composition provides an excellent therapeutic effect against malignant tumors.

[0021] (12) According to one embodiment of the composition, a pharmaceutical composition is provided for use in the treatment of a malignant tumor, wherein the treatment comprises a combination therapy to a target using BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug. The pharmaceutical composition may, for example, contain BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. The combination therapy provides an excellent therapeutic effect against the malignant tumor. According to this embodiment, a pharmaceutical composition is provided for use in the treatment of a malignant tumor, wherein the treatment comprises a combination therapy to a target using BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug, and the pharmaceutical composition comprises BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug.

[0022] (13) According to one embodiment of the composition, a pharmaceutical composition for use in inducing tumor cell injury is provided, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. This pharmaceutical composition may be used in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, an excellent tumor cell injury induction effect can be obtained.

[0023] (14) According to one embodiment of the composition, a pharmaceutical composition is provided for use in inducing the infiltration of CD8-positive T cells, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. This pharmaceutical composition may be used in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, an excellent effect in inducing the infiltration of CD8-positive T cells can be obtained.

[0024] (15) According to one embodiment of the composition, a pharmaceutical composition is provided for use in inducing the expression of an immune checkpoint molecule, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug. This pharmaceutical composition may be used in a manner in which BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug are used in combination. Depending on the manner in which they are used in combination, an excellent immune checkpoint molecule expression induction effect can be obtained.

[0025] (16) According to one embodiment of the composition, a pharmaceutical composition is provided comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, for use in the method described in (1) to (8) above. According to another embodiment, a pharmaceutical composition is provided comprising an immunotherapy drug for use in the method described in (1) to (8) above.

[0026] (17) Kit According to one embodiment of the present invention, a kit is provided comprising the pharmaceutical compositions described in (9) to (16) above. The kit may include, for example, instructions for use, a buffer, a container, or packaging. According to another embodiment, a kit is provided for use in the treatment of malignant tumors, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug.

[0027] According to one embodiment of the present invention, there is provided the use of BCV, its pharmaceutically acceptable salt or their solvates for the manufacture of the pharmaceutical composition described in (9) to (16) above. According to another embodiment, there is provided the use of an immunotherapeutic agent for the manufacture of the pharmaceutical composition described in (9) to (16) above.

[0028] In one embodiment of the present invention (for example, (1) to (18) above), brincidofovir (BCV) includes a compound having a structure represented by the following formula. BCV can also be represented by the IUPAC name of [(2S)-1-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxypropan-2-yl]oxymethyl-(3-hexadecoxypropoxy)phosphinic acid. BCV includes a compound represented by the CAS registration number 444805-28-1. In the present specification, BCV is described by abbreviating brincidofovir, and they have the same meaning.

[0029] In one embodiment of the present invention (for example, (1) to (18) above), combination use includes, for example, when there are two or more treatments (for example, administrations), performing another treatment before, simultaneously, or after one treatment for one treatment. The previous or subsequent treatments may be performed continuously. Continuous implementation includes, for example, in one treatment plan, performing one treatment for a certain period and then performing another treatment for a certain period. Simultaneous treatment includes, for example, administering one formulation having two or more active ingredients to a subject. Combination therapy includes, for example, performing two or more treatment methods. Two or more treatment methods may be implemented in one treatment plan. The combination therapy using BCV, its pharmaceutically acceptable salt, or their solvates, and an immunotherapeutic agent includes a mode in which the administration of the former is performed before, simultaneously, or after the administration of the latter.

[0030] In one embodiment of the present invention (for example, (1) to (18) above), the administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof may be performed before, concurrently with, or after the administration of an immunotherapy drug. For example, the administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug to a subject includes the configuration in which the administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof is performed before, concurrently with, or after the administration of an immunotherapy drug. The administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and the administration of an immunotherapy drug may be carried out within a single treatment plan.

[0031] In one embodiment of the present invention (for example, (1) to (18) above), the step of identifying a target may include a step of identifying a target as a target for treatment, or a step of identifying a target as a target for administration of a pharmaceutical composition. The step of identifying a target may include a step of selecting a target or a step of identifying a target.

[0032] In one embodiment of the present invention (for example, (1) to (18) above), the treatment includes exerting an effect that brings about a beneficial outcome for the patient with respect to the patient's disease (e.g., malignant tumor) or one or more symptoms associated with the disease (e.g., improvement, reduction, relief, cure, remission, or suppression (e.g., prevention of onset (e.g., prevention), suppression of progression, or suppression of recurrence)). The treatment or method may be carried out by administering an effective amount (e.g., a therapeutically effective amount) of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to the subject. In one embodiment of the present invention (for example, (1) to (18) above), the pharmaceutical composition may be manufactured, for example, by mixing an active ingredient with one or more pharmaceutically acceptable carriers by any method known in the art of pharmaceuticals. The pharmaceutical composition is not limited in form of use as long as it is used for therapeutic purposes, and may be an active ingredient alone or a mixture of the active ingredient with any other ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (e.g., a buffer). The amount of the carrier may, for example, be a pharmacochemically effective amount. The pharmacochemically effective amount may, for example, be a sufficient amount for the pharmacochemical stability or delivery of the active ingredient. The dosage may, for example, be 0.01 to 200 mg / kg body weight per dose. The pharmaceutical composition may contain stabilizers, buffers, or pH adjusters. The dosage, administration interval, method of administration, and route of administration are not particularly limited and can be appropriately selected depending on the patient's age, weight, symptoms, target organ, etc. It is also preferable that the pharmaceutical composition contains a therapeutically effective amount or an effective amount of the active ingredient that exerts the desired effect. In one embodiment of the present invention, the effective amount includes the amount necessary for clinically observed improvement of symptoms in the patient. In one embodiment of the present invention, pharmaceutically acceptable includes a state that is suitable for use in proportion to a reasonable benefit / risk ratio, within the range of reasonable medical judgment. There are no particular restrictions on components other than BCV in the pharmaceutical composition as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose.

[0033] In one embodiment of the present invention (for example, (1) to (18) above), the subject (including patients) may be, for example, a subject who requires treatment for a malignant tumor. Subjects requiring treatment for a malignant tumor may include, for example, a subject diagnosed with a malignant tumor or a subject suspected of having a malignant tumor. Subjects may be, for example, a subject in which a malignant tumor marker is detected in a sample, or a subject in which the malignant tumor marker in a sample is elevated compared to a control subject (for example, a healthy person). Subjects may be, for example, a subject who has experienced a malignant tumor. Subjects may be, for example, a subject undergoing treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a subject undergoing treatment with an immunotherapy drug. Subjects may be, for example, a subject who has been administered BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a subject who has been administered an immunotherapy drug. Subjects may be, for example, a subject who has experience being administered BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a subject who has experience being administered an immunotherapy drug. The subjects may include, for example, those for whom combination therapy with cisplatin and immunotherapy is unsuitable for implementation or continuation. The subjects may include those with renal impairment. The subjects may include those who have observed, for example, leukopenia, anemia, or thrombocytopenia. The subjects may include, for example, those with decreased white blood cells, anemia, or decreased platelets. The subjects may include, for example, those in whom the expression of an immune checkpoint molecule or its ligand (e.g., PD-L1) has been confirmed, those who are positive for an immune checkpoint molecule or its ligand, those who have tumor cells expressing an immune checkpoint molecule or its ligand, or those who have tumor cells positive for an immune checkpoint molecule or its ligand. Positivity may include, for example, a TPS (Tumor Proportion Score) of ≥1%, ≥50%, or ≥1-50%. Positivity may also include, for example, a CPS (Combined Positive Score) of ≥1, ≥10, ≥20, or ≥1-20%. The subjects may, for example, be those with malignant tumors that are highly sensitive to BCV, its pharmaceutically acceptable salts, or their solvates.The subject includes a human or a mammal other than a human (e.g., one or more of mouse, guinea pig, hamster, rat, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee, etc.).

[0034] In embodiments of the present invention (for example, (1) to (18) above), malignant tumors include, for example, tumors arising from a mutation in the genes of normal cells. Malignant tumors can arise from any organ or tissue in the body. Malignant tumors may include, for example, solid tumors or hematological cancers. Solid tumors include, for example, one or more selected from the group consisting of lung cancer, sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, uterine cancer, pancreatic cancer, giant cell tumor of bone, ovarian cancer, esophageal cancer, adrenal cancer, biliary tract cancer, small intestine cancer, ovarian cancer, ureteral cancer, renal pelvis cancer, penile cancer, testicular cancer, brain tumors (e.g., glioma), central nervous system cancers, peripheral nervous system cancers, thyroid cancer, salivary gland cancers, and carcinomas. Sarcomas include, for example, uterine sarcoma or giant cell tumor of bone. Skin cancers include, for example, malignant melanoma. Kidney cancers include, for example, renal cell carcinoma. Uterine cancer includes, for example, cervical cancer. Head and neck cancer includes, for example, head and neck squamous cell carcinoma. Colorectal cancer includes, for example, colon cancer or rectal cancer. Stomach cancer includes, for example, EBV-associated stomach cancer. Liver cancer includes, for example, hepatoblastoma or hepatocellular carcinoma. Lung cancer includes, for example, non-small cell lung cancer or small cell lung cancer. Non-small cell lung cancer includes, for example, large cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. Solid tumors may include, for example, lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, uterine cancer, pancreatic cancer, giant cell tumor of bone, or ovarian cancer. Solid tumors may include, for example, lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, giant cell tumor of bone, hepatoblastoma, or uterine cancer. Solid tumors may include, for example, large cell lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, small cell lung cancer, breast cancer, skin cancer, or bladder cancer. Solid tumors may include, for example, large cell lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, small cell lung cancer, or breast cancer. Solid tumors may include, for example, large cell carcinoma, uterine sarcoma, prostate cancer, or head and neck cancer. Solid tumors may include, for example, prostate cancer or head and neck cancer. Solid tumors may include, for example, head and neck cancer. Hematological cancers include, for example, lymphoma, leukemia, or multiple myeloma.Malignant tumors include, for example, EBV-positive malignant tumors. Malignant tumors include, for example, neuroendocrine tumors. Neuroendocrine tumors include, for example, tumors originating from neuroendocrine cells. Tumors originating from neuroendocrine cells include, for example, tumors occurring in the lungs, gastrointestinal tract, or prostate. Lung cancer includes, for example, non-small cell lung cancer or small cell lung cancer. The gastrointestinal tract includes, for example, the stomach, duodenum, small intestine, appendix, or large intestine. From the viewpoint of high therapeutic efficacy, small cell lung cancer is preferred as a target neuroendocrine tumor for treatment. When specifically designated, one or more types of neuroendocrine tumors to be treated may be excluded from the group consisting of, for example, lung cancer, prostate cancer, head and neck cancer, colorectal cancer, gastric cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, uterine cancer, pancreatic cancer, ovarian cancer, esophageal cancer, adrenal cancer, biliary tract cancer, small intestine cancer, ovarian cancer, ureteral cancer, renal pelvis cancer, penile cancer, testicular cancer, brain tumors, central nervous system cancers, peripheral nervous system cancers, thyroid cancer, salivary gland cancers, and carcinomas. Malignant tumors may be, for example, malignant tumors evaluated as having a BCV IC50 value of 3.5 μM or less. From the viewpoint that combination therapy with BCV and immunotherapy drugs significantly induces tumor cell damage in the tumor microenvironment, BCV-sensitive cancers are preferred as malignant tumors. BCV-sensitive cancers may include, for example, large cell lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, gastric cancer, small cell lung cancer, breast cancer, skin cancer, bladder cancer, or lymphoma. BCV-sensitive cancers may also be cancers in which the BCV IC50 value is evaluated as 3.5 μM or less. The therapeutic effect on malignant tumors may be evaluated, for example, by observing the change in malignant tumor cell proliferation over time after drug administration. The therapeutic effect on malignant tumors may also be evaluated, for example, by measuring the induction of tumor cell damage in the tumor microenvironment. In this case, if tumor cell damage is significantly induced compared to before drug administration or after administration of a negative control, it may be judged that there is a therapeutic effect. The induction of tumor cell damage may be evaluated, for example, using the induction of CD8-positive T cell infiltration into tumor tissue as an indicator. The therapeutic effect on malignant tumors may also be evaluated, for example, by observing the reduction in tumor size or tumor volume after drug administration.In this case, a therapeutic effect may be judged if the tumor size or tumor volume is significantly reduced compared to before drug administration or during negative control administration. The therapeutic effect against malignant tumors may be measured, for example, using the amount of malignant tumor markers in the patient or patient-derived samples as an indicator. In this case, a therapeutic effect may be judged if the amount of markers is significantly reduced compared to before drug administration or during negative control administration. The induction of tumor cell damage after drug administration may increase by 5.5, 6, 6.5, 7, 8, or 9 times or more, or within the range of any two of these values, compared to before administration or during negative control administration. The tumor volume or marker amount after drug administration may decrease to 0.9, 0.7, 0.5, 0.3, or 0.1 times or less compared to before administration or during negative control administration.

[0035] In embodiments of the present invention (for example, (1) to (18) above), if the malignant tumor is a hematological cancer, the hematological cancer may include, for example, lymphoma, leukemia, or multiple myeloma. Leukemia may include, for example, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia. If the malignant tumor is a lymphoma, the lymphoma includes malignant lymphoma. Malignant lymphoma includes, for example, diseases resulting from the cancerous transformation of lymphocytes. Malignant lymphoma includes, for example, non-Hodgkin lymphoma or Hodgkin lymphoma. Non-Hodgkin lymphoma includes, for example, B-cell lymphoma, NK / T-cell lymphoma, or T-cell lymphoma. B-cell lymphoma includes, for example, B-cell lymphomas classified as one or more of the following: follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, primary exudative lymphoma, or chronic lymphocytic leukemia / small lymphocytic lymphoma. NK / T-cell lymphoma includes, for example, NK / T-cell lymphomas classified as one or more of the following: peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (nasal type), and cutaneous lymphoma (e.g., mycosis fungoides). T-cell lymphoma includes T-cell lymphomas classified as one or more of the following: peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, anaplastic large cell lymphoma, intestinal disease-associated T-cell lymphoma, or lymphoblastic lymphoma. Non-Hodgkin lymphoma includes, for example, peripheral T-cell lymphoma. Peripheral T-cell lymphoma includes, for example, peripheral T-cell lymphomas classified as one or more of the following: nodal peripheral T-cell lymphoma or extranodal peripheral T-cell lymphoma. Nodal peripheral T-cell lymphoma includes, for example, nodal peripheral T-cell lymphomas classified as one or more of the following: unspecified type PTCL (PTCL-NOS), PTCL with follicular helper T-cell phenotype, or ALK-positive anaplastic large cell lymphoma. Extranodal peripheral T-cell lymphoma includes, for example, extranodal peripheral T-cell lymphoma classified as extranodal NK / T-cell lymphoma, nasal type. Hodgkin lymphoma includes, for example, classical Hodgkin lymphoma or nodular lymphocyte-predominant Hodgkin lymphoma.For further details regarding lymphoma or its diagnostic methods, see, for example, Nirmal, J Oral Maxillofac Pathol. 2020 May-Aug;24(2):195-199, Singh et al., J Family Med Prim Care. 2020 Apr; 9(4): 1834-1840, Voltin et al., Cancers (Basel). 2020 Mar 5;12(3):601, or Pu et al., Front Immunol. 2022; 13: 1008695. From the viewpoint of improving treatment efficacy or increasing the probability of treatment efficacy, it is preferable that the hematological malignancy is lymphoma. From the viewpoint of improving treatment efficacy or increasing the probability of treatment efficacy, it is preferable that the lymphoma is T-cell lymphoma. The therapeutic effect or onset of lymphoma may be diagnosed by examination of tumor weight or by histological examination of lymph nodes or masses.

[0036] In embodiments of the present invention (for example, (1) to (18) above), immunotherapy includes a therapeutic method that utilizes the immune response of a subject to attack a malignant tumor. For example, it is possible to treat a malignant tumor by inhibiting the function of an immunosuppressive immune checkpoint molecule, thereby releasing the immunosuppressive effect. Immunotherapy includes, for example, administering an immunotherapy drug to a subject. In embodiments of the present invention (for example, (1) to (18) above), the immunotherapy drug includes, for example, an immune checkpoint inhibitor. The immune checkpoint molecule includes, for example, PD-1CTLA-4 or LAG-3. The UniProt primary accession numbers for these molecules can be referenced as Q15116, P16410, and P18627, respectively. PD-1 can also be written as Programmed Cell Death 1. CTLA-4 can also be written as Cytotoxic T Lymphocyte Antigen-4. LAG-3 can also be written as Lymphocyte Activation Gene-3. Ligands for immune checkpoint molecules include, for example, PD-L1. PD-L1 can also be denoted as Programmed Cell Death Ligand 1. The UniProt primary accession number for this molecule can be referenced as Q9NZQ7. Immune checkpoint inhibitors may be, for example, antibodies that specifically bind to immune checkpoint molecules or their ligands. The antibody may be, for example, an antibody that inhibits the function of the immune checkpoint molecule or its ligand. The antibody may be, for example, a monoclonal antibody. Antibodies that specifically bind to immune checkpoint molecules or their ligands include, for example, anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, semiprimab, spartalizumab, tislerizumab, dostallimub, retifanlimub, camrelizumab, or ivonessimab), anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, or avelumab), anti-CTLA-4 antibodies (e.g., ipilimumab or tremelimumab), or anti-LAG-3 antibodies (e.g., relatrimab).Immunotherapy drugs include, for example, one or more drugs selected from the group consisting of pembrolizumab, nivolumab, semiprimab, spartalizumab, tisrelizumab, dostallimab, retifanlimab, camrelizumab, ivonessimab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab, and relatrimab. The CAS registry numbers for these molecules can be found as follows: 1374853-91-4, 946414-94-4, 1801342-60-8, 1935694-88-4, 1858168-59-8, 2022215-59-2, 2079108-44-2, 1798286-48-2, 2428381-53-5, 1380723-44-3, 1428935-60-7, 1537032-82-8, 477202-00-9, 745013-59-6, and 1673516-98-7, respectively. The PubChem SIDs for these molecules can be referenced as follows: 254741536, 163312346, 376219032, 405226472, 384585463, 384585344, 405226692, 472423441, 497621221, 312642102, 319075068, 319902617, 47206447, 47208308, and 384585328, respectively. The general names or development codes for products containing these molecules as active ingredients may include, for example, Keytruda, Opdivo, Ributayo, PDR001, TEVIMBRA, Jemperli, ZYNYZ, SHR-1210, AK112, Tecentriq, Imfinzi, Bavencio, Yervoy, Ijud, and BMS-986016. Immunotherapy drugs may also include antibodies having the amino acid sequences of these molecules (e.g., heavy chain CDR1-3 and light chain CDR1-3). The amino acid sequence may be, for example, the heavy chain, light chain, heavy chain variable region, light chain variable region, heavy chain CDR1-3, or the amino acid sequence of light chain CDR1-3. Immune checkpoint inhibitors include, for example, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, or LAG-3 inhibitors. The above immune checkpoint molecules or antibodies may be of human origin.

[0037] In one embodiment of the present invention (for example, (1) to (18) above), induction of tumor cell injury includes, for example, induction of tumor cell injury in the tumor microenvironment. In one embodiment of the present invention (for example, (1) to (18) above), induction of invasion includes, for example, induction of invasion into tumor tissue. In one embodiment of the present invention (for example, (1) to (18) above), the treatment, combination treatment, or pharmaceutical composition may involve, for example, induction of tumor cell injury in the tumor microenvironment, induction of invasion of CD8-positive T cells into tumor tissue, or induction of the expression of immune checkpoint molecules.

[0038] In one embodiment of the present invention (for example, (1) to (18) above), the salt is not particularly limited and includes, for example, inorganic salts or organic salts (see, for example, Bharate et al., Drug Discov Today. 2021 Feb;26(2):384-398. or Berge et al., J Pharm Sci. 1977 Jan;66(1):1-19.). Salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. Metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. Salts include pharmaceutically acceptable salts. In one embodiment of the present invention, pharmaceutically acceptable includes forms that have reasonable benefit for pharmaceutical use. In one embodiment of the present invention, one form of a compound or a salt thereof includes the form of its solvate. In one embodiment of the present invention (for example, (1) to (18) above), the solvate includes the form of a compound formed by a solute and a solvent (see, for example, Healy et al., Adv Drug Deliv Rev. 2017 Aug 1;117:25-46).The solvates are not particularly limited, but include, for example, hydrates (e.g., monohydrates, dihydrates, trihydrates, etc.) or organic solvent hydrates (e.g., solvates with alcohols (methanol, ethanol, propanol, etc.), acetone, dimethylformamide, or ethyl acetate, etc.). The solvent includes solvents capable of substantially maintaining the physiological activity of the solute after the formation of the solvate. The solvates include pharmaceutically acceptable solvates.

[0039] In one embodiment of the present invention (for example, (1) to (18) above), "significantly" may mean, for example, a state in which statistical significance is assessed using Student's t-test (one-sided or two-sided) and p < 0.05 or p < 0.01. Alternatively, it may mean a state in which a substantial difference exists.

[0040] All publications and substance identification numbers cited herein are incorporated herein by reference in their entirety, as well as their associated structure and information. In this specification, “or” is used when “at least one” of the items listed in the text may be adopted. The same applies to “or.” In this specification, the term “at least one of A, B, and C” means that any of (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C) may be adopted. In this specification, when “within the range of two values” is specified, that range includes the two values ​​themselves. In this specification, “A to B” includes A and B and the values ​​that fall between A and B. In this specification, “a,” “an,” and “the” may be intended to indicate that there may be one or more elements or processes unless it is clearly otherwise in the context. In this specification, “having” includes having a disease when relating to a disease. In this specification, “(1) to (18) above” includes references to one or more of (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), or (18).

[0041] Although embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various other configurations can be adopted. Furthermore, the present invention can be adopted by combining or independently the configurations or features described in the above embodiments.

[0042] The present invention will be further explained below with reference to examples, but is not limited to these.

[0043] Example 1: In vitro cytotoxic activity test against various human solid cancer cell lines. The in vitro cytotoxic activity of brincidofovir (BCV) against various solid cancer cell lines shown in Figure 1 was tested using CellTiter-Glo. TM The cells were evaluated using the CTG assay (Promega, Wisconsin, USA). The CTG assay measures the number of viable cells in culture by quantifying intracellular adenosine triphosphate (ATP), an indicator of metabolic activity, using bioluminescence. Specifically, solid cancer cells were evaluated in their respective culture media at a rate of 2 × 10⁶ 3 BCV was added to 96-well plates seeded with cells at various concentrations, and after incubation at 37°C and 5% CO2 for 5 days, the cells were treated with CTG reagent to thaw them. Then, Envision TM The fluorescence signal of the culture medium was measured using a plate reader (PerkinElmer, Connecticut, USA). Cytotoxic activity was evaluated as a percentage of the control fluorescence signal. Using GraphPad Prism (GraphPad Software, Massachusetts, USA), the percentage of cytotoxic activity against various drug concentrations was plotted to create dose-response curves (Figures 2-4). Furthermore, the 50% cytotoxic concentration (IC50 value) of BCV against various solid cancer cell lines was determined (Figure 1). All assays were performed using duplicates.

[0044] The in vitro cytotoxic activity of BCV against various solid tumor cell lines was measured according to concentration, and the resulting cytotoxicity curves are shown in Figures 2-4. Next, the 50% cytotoxic concentration (IC50 value) obtained from this analysis is shown in Figure 1. Here, according to past or ongoing clinical trials, the Cmax of the clinical dose for BCV IV administration is approximately 3.5 μM. From this, cell lines with an IC50 value of 3.5 μM or less (NCI-H460 to SW780 in Figure 1) can be evaluated as BCV-highly sensitive cell lines, cell lines with an IC50 value greater than 3.5 μM and less than or equal to 100 μM (GCT to CaSki in Figure 1) as moderately sensitive cell lines, and cell lines with an IC50 value greater than 100 μM (PANC1 to SK-OV-3 in Figure 1) as BCV-resistant cell lines.

[0045] Example 2-1: Method for creating a humanized mouse model of tumor bearing. 8-week-old female NOG mice with immunodeficiency and a body weight of 18-22 g (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were given 5x10 6 We created mice with human immune cells by transplanting peripheral blood mononuclear cells (PBMCs) derived from individual human donors. The following day, we transplanted 2x10 FaDu cells (ATCC, Manassas, VA, USA) derived from human head and neck cancer. 6 Individual grafts were transplanted to create a tumor-bearing mouse model. In this process, donors were pre-selected whose HLA of the PBMC partially matched that of the FaDu and who were less likely to develop graft-versus-host disease (GvHD).

[0046] Example 2-2: Method for measuring tumor size after administration of various doses of BCV in a humanized tumor-bearing mouse model. The BCV dose escalation study consisted of four groups of tumor-bearing model mice, each group consisting of four mice. Group 1 (control group) received only the solvent, Group 2 received 2.5 mg / kg of BCV, Group 3 received 5 mg / kg of BCV, and Group 4 received 10 mg / kg of BCV. BCV and the solvent were administered intraperitoneally twice a week starting on day 7 after transplantation of human head and neck cancer FaDu cells. During the administration period, tumor size was measured three times a week to evaluate the inhibitory effect on tumor growth. In this evaluation, in addition to the tumor growth curve (Figure 5(a)), the tumor growth inhibition rate (TGI rate) (Figure 5(b)) was calculated using the following formula. TGI (%) = [1 - (Ti - T1) / (Vi - V1)] × 100 Ti: Mean tumor volume (size) in the group on the evaluation day T1: Mean tumor volume in the group on the treatment initiation day Vi: Mean tumor volume in the control group on the same day as the Ti evaluation day V1: Mean tumor volume in the control group on the treatment initiation day This dose escalation study is conducted when the tumor volume of any tumor-bearing mouse reaches 2,000 mm on the evaluation day. 3 The test was terminated for ethical reasons once the limit was exceeded.

[0047] Results: Tumor growth curves were plotted to analyze the antitumor activity of four BCV doses, namely 0 (vehicle), 2.5, 5, and 10 mg / kg. As shown in Figure 5(a), in the BCV 0 mg / kg dose group, tumors formed with FaDu cells grew rapidly, and the tumor volume reached 2,000 mm² on day 20 of treatment initiation. 3 This trial was terminated because it exceeded [a certain threshold]. In the groups receiving BCV doses of 2.5 and 5 mg / kg, the tumor volume at day 20 of treatment was approximately 1,600 mm². 3The growth was suppressed to a certain extent. In the group administered with 10 mg / kg of BCV, the tumor suppression effect was further enhanced. TGI (%) is the calculated value of the percentage of tumor growth suppression effect shown by BCV compared to the control group (vehicle) (Figure 5(b)). It was found that the groups administered with 2.5 and 5 mg / kg of BCV showed a 20 - 30% tumor growth suppression effect, and the group administered with 10 mg / kg showed a 50 - 60% tumor growth suppression effect. The weight loss of each mouse in each group during the treatment period was less than 20%. Based on the results of these dose escalation tests of BCV, in the future, the dosage and administration method of BCV used for treatment was set as 10 mg / kg, intraperitoneal administration twice a week.

[0048] Example 2 - 3: Measurement method of tumor size during various treatments in a humanized mouse model bearing cancer - Experiment 1. The test to observe the antitumor effects of various treatments consisted of 6 groups of 8 cancer - bearing model mice in each group. In the first group (control group), the solvent was intraperitoneally administered twice a week. In the second group, 10 mg / kg of BCV was intraperitoneally administered twice a week. In the third group, 2 mg / kg of cisplatin (CDDP) was intraperitoneally administered once a week. In the fourth group, 10 mg / kg of anti - PD - 1 antibody (pembrolizumab: an immune checkpoint inhibitor. The same in Examples 2 - 3 to 5 - 3) was intraperitoneally administered twice a week. In the fifth group, BCV and anti - PD - 1 antibody were administered in combination with the same dosage and administration method as above. And in the sixth group, CDDP and anti - PD - 1 antibody were administered in combination with the same dosage and administration method as above. These various treatments were started on the 7th day after transplantation of human head and neck cancer FaDu cells. During the administration period, the tumor size was measured three times a week, and the tumor growth suppression effect was evaluated in the same manner as in Example 2 - 2. That is, it was evaluated using the tumor growth curve (Figure 6(a)) and the tumor growth inhibition rate (TGI rate) (Figure 6(b)). Also in this test, when the tumor volume of any cancer - bearing mouse exceeded 2,000 mm 3 ³ at the evaluation day, the test was terminated from an ethical perspective. The tumor growth suppression effect and tumor growth inhibition rate of various treatments were compared and examined using the t - test.

[0049] Experiment 2. The dosage of BCV in the second and fifth groups of Experiment 1 was changed to 5 mg / kg, and the same test as in Experiment 1 was conducted. And it was evaluated using the tumor growth curve (Figure 7(a)) and the tumor growth inhibition rate (TGI rate) (Figure 7(b)).

[0050] The results of Experiment 1 are shown in Figures 6(a) and (b). In Group 1 (vehicle), the tumor consisting of FaDu cells proliferated rapidly, and on day 20 of treatment, the tumor volume reached 2,000 mm³. 3 The trial was terminated because the threshold was exceeded. Combination therapy with anti-PD-1 antibody and CDDP showed a high inhibitory effect on tumor growth (Figure 6(a)), with a TGI of approximately 60% (Figure 6(a)). BCV monotherapy showed a high inhibitory effect on tumor growth comparable to that of combination therapy with anti-PD-1 antibody and CDDP, and there was no statistically significant difference between the two (Figure 6). Furthermore, combination therapy with anti-PD-1 antibody and BCV showed the highest tumor growth inhibitory effect and was statistically significantly more effective than BCV monotherapy.

[0051] The results of Experiment 2 are shown in Figures 7(a) and (b). In Group 1 (vehicle), the tumor consisting of FaDu cells proliferated rapidly, and on day 20 of treatment, the tumor volume reached 2,000 mm³. 3 The trial was terminated because the threshold was exceeded. Combination therapy with anti-PD-1 antibody and CDDP showed a high inhibitory effect on tumor growth (Figure 7(a)), with a TGI of approximately 60% (Figure 7(a)). BCV monotherapy showed a moderate inhibitory effect on tumor growth (Figure 7). Combination therapy with anti-PD-1 antibody and BCV showed the highest tumor growth inhibitory effect and was statistically significant and more effective than BCV monotherapy.

[0052] Discussion: Combination therapy with anti-PD-1 antibodies and CDDP is the standard treatment for head and neck cancer. However, many head and neck cancer patients are elderly, and a significant number have impaired renal function. In such patients, nephrotoxic CDDP cannot be used. Based on the results of this animal experiment, it is considered beneficial to use combination therapy with anti-PD-1 antibodies and BCV instead of combination therapy with anti-PD-1 antibodies and CDDP in patients with renal impairment. Furthermore, CDDP is not only nephrotoxic but can also cause myelosuppression. Since BCV does not cause myelosuppression, it is considered beneficial to use combination therapy with anti-PD-1 antibodies and BCV in patients with leukopenia, anemia, or thrombocytopenia.

[0053] Example 2-4: Tumor tissue images during various treatments in a humanized mouse model of tumor-bearing mice At the end of Experiment 1 in Example 2-3, all mice were euthanized. Three mice were randomly selected from four of the six groups: Group 1 (vehicle treatment group), Group 2 (BCV monotherapy group), Group 4 (anti-PD-1 antibody monotherapy group), and Group 5 (BCV and anti-PD-1 antibody combination therapy group), and tumors were excised. The tumor tissue was fixed with 10% formalin, embedded in paraffin, and sectioned. The tissue sections were deparaffinized, washed with ethanol, and hydrated. The tissue sections were stained with hematoxylin and eosin. The stained tissue sections were scanned at 200x magnification using a ZEISS Axioscan scanner (Carl Zeiss Microscopy GmbH, Jena, Germany).

[0054] As shown in Figure 8, in each group, tumor tissue contained areas of clusters of living cells (darkly stained areas), as well as areas of necrosis (lightly stained areas). Necrotic areas tended to be particularly clearly observed in mice treated with anti-PD-1 antibody monotherapy and in mice treated with combination therapy of BCV and anti-PD-1 antibody.

[0055] In Examples 2-2 and 2-3, the size of the tumor was measured, and the tumor volume was calculated using a formula. However, as can be seen from the results of Example 2-4, this tumor volume includes necrotic tissue, and therefore deviates considerably from the actual tumor volume. In other words, because tumor tissue includes necrotic parts, measuring the size alone is not sufficient to adequately evaluate the effect of drug administration. To resolve this problem and to examine in more detail what changes were actually brought about by each treatment, Example 2-5 was conducted. In Example 2-5, the effect of inducing tumor cell damage (cell death) in the tumor microenvironment was measured by histological examination.

[0056] Example 2-5: Inducement of tumor cell damage by combination therapy of BCV and anti-PD-1 antibody (histological examination) Method At the end of Experiment 1 in Example 2-3, all mice were euthanized. Three mice were randomly selected from four of the six groups, namely Group 1 (vehicle therapy group), Group 2 (BCV monotherapy group), Group 4 (anti-PD-1 antibody monotherapy group), and Group 5 (combination therapy group of BCV and anti-PD-1 antibody), and tumors were excised. The tumor tissue was fixed with 10% formalin, embedded in paraffin, and sectioned. The tissue sections were deparaffinized, washed with ethanol, and hydrated. The tissue sections were then stained with multicolor immunofluorescence using the LEICA BOND RX (Leica Biosystems, Nussloch, Germany), an automated staining system from LEICA. For this immunohistochemistry, rabbit anti-CD4 monoclonal antibody (product number 133616, Abcam, Cambridge, UK), rabbit anti-CD8 monoclonal antibody (product number 178089, Abcam), rabbit anti-active Caspase-3 monoclonal antibody (product number 9661S, Cell Signaling, Danvers, MA, USA), and rabbit anti-p63 monoclonal antibody (product number 32353, Abcam) were used as primary antibodies. These primary antibodies were labeled with various fluorescent dyes from Alpha TSA (Alpha X Bio, Beijing, China). Cell nuclei were fluorescently stained using DAPI. Immunostained tissue sections were scanned at 200x magnification using a ZEISS Axioscan scanner (Carl Zeiss Microscopy GmbH, Jena, Germany). All images were analyzed using HALO AI software version 3.3.2541 (Indica Labs, Inc., Albuquerque, NM, USA).

[0057] The results were histologically examined to determine the inducing effect of each of the above treatments on tumor cell damage in the tumor microenvironment. As shown in Figures 9-12, infiltration of CD4 and CD8-positive T cells was only limited in the tumors of mice in the Vehicle treatment group. No statistically significant differences were observed in the BCV monotherapy group or the anti-PD-1 antibody monotherapy group compared to the Vehicle treatment group. On the other hand, infiltration of CD8-positive T cells was significantly observed in the tumors of mice in the BCV and anti-PD-1 antibody combination therapy group. Compared to the Vehicle treatment group, a statistically significant increase in the number of infiltrating CD8-positive T cells was observed in this two-drug combination group (Figure 11).

[0058] Furthermore, in addition to the infiltration of immune cells into tumor tissue, the extent to which tumor cells were undergoing cell death due to attack by CD8-positive cytotoxic T cells was analyzed by counting cells that were positive for p63, a marker for squamous cell carcinoma, and also positive for activated Caspase-3. As a result, there was no statistically significant difference between the BCV monotherapy group and the anti-PD-1 antibody monotherapy group compared to the vehicle group. On the other hand, a large statistically significant difference was observed in the combination therapy group of BCV and anti-PD-1 antibody (Figure 12). In other words, it was found that adding an anti-PD-1 antibody to BCV induces significant tumor cell death.

[0059] In the studies using examples 2-5, it was found that administering BCV in combination with an anti-PD-1 antibody significantly induced the invasion of CD8-positive cytotoxic T cells into tumor tissue. On the other hand, regarding the induction of invasion, no statistically significant difference was observed in the monotherapy group compared to the vehicle-administered group. Furthermore, the induction rate was significantly higher with combination therapy. Therefore, the effect of combination therapy on infiltration is considered to be a synergistic effect of the two drugs. CD8-positive cytotoxic T cells damage cancer cells in tumor tissue, leading to cell death. Combination therapy with BCV and an anti-PD-1 antibody has been shown to be an excellent treatment method from the perspective of significantly inducing tumor cell damage in the tumor microenvironment.

[0060] Example 2-6: Method for measuring tumor size during various treatments in a humanized mouse model of tumor-bearing cancer. The test to examine the antitumor effect of these various treatments used tumors formed from EBV-associated human gastric cancer SNU-719 cells. Except for this, this test was conducted in exactly the same manner as Experiment 1 of Example 2-3. The inhibitory effect on tumor growth was also evaluated in the same manner as Experiment 1 of Example 2-3. That is, it was evaluated using tumor growth curves (Figures 13-15) and tumor growth inhibition rate (TGI rate) (Figure 16).

[0061] Figures 13 and 14 show the tumor growth curves for each mouse belonging to the treatment group (8 mice per group). In the mice belonging to Group 1 (control), the tumors consisting of SNU-719 cells grew rapidly, and by day 21 after the start of treatment, the tumor volume in all mice was 600 mm². 3 It exceeded (shown in the gray area). In the second group (BCV monotherapy group), the tumor volume was 600 mm in half of the mice (4 mice). 3 It exceeded [a certain value]. In the third group (anti-PD-1 antibody monotherapy group), the tumor volume was 600 mm in 2 out of 8 animals. 3 It exceeded [a certain value]. However, the fourth group (combination therapy group with anti-PD-1 antibody and BCV) showed the highest tumor growth inhibitory effect, and the tumor volume in all mice was 600 mm. 3 The following was suppressed:

[0062] Figure 15 shows the tumor growth curves based on the mean and standard error for each treatment group. In Group 1 (control), tumors grew rapidly, but in Group 2 (BCV monotherapy group), tumor growth was moderately suppressed. In Group 3 (anti-PD-1 antibody monotherapy group), tumor growth was highly suppressed, but in Group 4 (combination therapy with anti-PD-1 antibody and BCV group), an even higher tumor growth suppression effect was observed.

[0063] Figure 16 shows the tumor growth inhibition rate (TGI rate) curves for each treatment group. The TGI rates for Group 2 (BCV monotherapy group), Group 3 (anti-PD-1 antibody monotherapy group), and Group 4 (combination therapy with anti-PD-1 antibody and BCV group) were 46%, 77%, and 84%, respectively.

[0064] Discussion: Unlike BCV monotherapy or anti-PD-1 antibody monotherapy, when these are used in combination, the tumor volume in all mice was 600 mm. 3 It was suppressed to the following extent: This 600 mm 3 Regarding tumor load, all mice in the control group exceeded this threshold, indicating that it represents an effective threshold. The fact that only the combination therapy suppressed growth below this threshold is highly significant. Regarding the TGI rate, while BCV monotherapy resulted in a TGI rate of 46%, combining it with anti-PD-1 antibody therapy increased it to 84%, suggesting that BCV therapy has affinity with anti-PD-1 antibody therapy.

[0065] Furthermore, the variability in tumor volume was significantly reduced in the combination therapy group compared to the monotherapy group. Specifically, as shown in Figure 15, the standard error for the BCV monotherapy group was 114.36, and the standard error for the anti-PD-1 antibody monotherapy group was 100.56, while the standard error for the combination therapy group was 61.93, a reduction of approximately 40% in variability. Variability in treatment effectiveness indicates differences in responsiveness between individuals and is a factor that increases treatment uncertainty in clinical practice. In addition, when multiple drugs are used in combination, it is generally expected that the variability in responsiveness between individuals will be large because each drug has a different mechanism of action. On the other hand, in this example, the unexpected result was shown that the combination of BCV and anti-PD-1 antibody significantly suppressed the variability in responsiveness between individuals. In other words, it was shown that combination therapy with BCV and anti-PD-1 antibody provides a dual effect of reducing tumor volume and improving treatment certainty.

[0066] Example 2-7: Method for measuring tumor size during various treatments in a humanized mouse model of tumor-bearing cancer. The test to examine the antitumor effect of these various treatments used tumors formed from human cervical cancer CaSki cells. Except for this, this test was conducted in exactly the same manner as Experiment 1 of Example 2-3. The inhibitory effect on tumor growth was also evaluated in the same manner as Experiment 1 of Example 2-3. That is, it was evaluated using the tumor growth curve (Figure 17(a)) and the tumor growth inhibition rate (TGI rate) (Figure 17(b)).

[0067] Results: In Group 1 (vehicle), the tumors composed of CaSki cells gradually proliferated, and by day 18 of treatment, the tumor volume was approximately 600 mm². 3 The results were as follows: The growth inhibitory effects of the anti-PD-1 antibody and CDDP monotherapy groups were similar (Figure 17(a)), and their TGI was approximately 20-30% (Figure 17(b)), which was the same value as in Examples 2-3. Regarding BCV monotherapy, BCV and anti-PD-1 antibody combination therapy, and anti-PD-1 antibody and CDDP combination therapy, there was no significant difference in the growth inhibitory effects of the three (Figure 17(b)), and their TGI was approximately 40% (Figure 17(b)).

[0068] Discussion: In Examples 2-3, the TGI of BCV monotherapy for FaDu cells was approximately 60%. On the other hand, in Example 2-7, the TGI of BCV monotherapy for CaSki cells was approximately 40%. These in vivo results are consistent with the evaluation of FaDu cells as a BCV-highly sensitive cell line and CaSki as a BCV-moderately sensitive cell line in Example 1.

[0069] Example 2-8: Inducement effect of BCV and anti-PD-1 antibody combination therapy on tumor cell damage (histological examination) Method All mice were euthanized at the end of the experiment in Example 2-7. Tumors were removed from all mice in four of the six groups: Group 1 (vehicle therapy group), Group 2 (BCV monotherapy group), Group 4 (anti-PD-1 antibody monotherapy group), and Group 5 (BCV and anti-PD-1 antibody combination therapy group). Tissue sections were prepared from the tumor tissue and immunohistochemical analysis was performed in the same manner as in Example 2-5.

[0070] As with Example 2-5, the histological effect of each treatment on inducing tumor cell damage in the tumor microenvironment was examined in Example 2-8. As shown in Figure 18, there was no significant difference in the infiltration of CD4-positive T lymphocytes into CaSki tumors among the groups. Regarding CD8-positive T lymphocytes, the combination therapy group with BCV and anti-PD-1 antibody tended to show slightly more cell infiltration (Figure 19).

[0071] Furthermore, similar to Examples 2-5, in addition to the infiltration of immune cells into tumor tissue, the extent to which tumor cells were undergoing cell death due to attack by CD8-positive cytotoxic T cells was analyzed by counting cells that were positive for p63, a marker for squamous cell carcinoma, and also positive for activated Caspase-3 (Figure 20). As a result, a slightly higher tendency was observed in the BCV monotherapy group compared to the BCV and anti-PD-1 antibody combination therapy group.

[0072] Discussion: In Examples 2-5, when FaDu cells were used, significant infiltration of CD8-positive T cells into the tumors was observed in the combination therapy group, with a statistically significant difference. On the other hand, in Example 2-8, when CaSki cells were used, the infiltration of CD8-positive T cells into the tumors in the combination therapy group was only slightly more pronounced. These in vivo test results are consistent with the evaluation of FaDu cells as a BCV-highly sensitive cell line and CaSki as a BCV-moderately sensitive cell line in Example 1.

[0073] Example 3: Method for inducing immunogenic cell death of various cancer cells with BCV FaDu, PC-3, CaSki, and SNU-719 cells were seeded and cultured in plates, and BCV was added to final concentrations of 0, 0.1, 1, and 10 μg / mL, respectively. In addition to these wells, for FaDu, PC-3, and CaSki cells, wells containing bleomycin (BLM), which induces strong immunogenic cell death, were prepared as positive controls to final concentrations of 1 and 10 μg / mL, respectively. For SNU-719 cells, wells containing BLM were prepared as positive controls to final concentrations of 0.1, 1, and 10 μg / mL, respectively. These wells were cultured for 24 or 48 hours in the presence of 5% CO2 at 37°C in a steam-saturated environment. After that, the cells were harvested using 0.25% trypsin solution, and the culture medium was added to inactivate the trypsin, followed by washing with PBS. Subsequently, 150 μL of either rabbit anti-calreticulin antibody (D3E6, Cell Signaling, Danvers, MA, USA) or rabbit IgG isotype control (DA1E, Cell Signaling), diluted to 0.5 μg / mL in staining solution, was used to react each collected tumor cell with the solution at 4°C for 60 minutes. After the reaction, the cells were washed with 150 μL of PBS and reacted with a secondary antibody diluted to 4 μg / mL in 150 μL of staining solution at 4°C for 60 minutes. This secondary antibody was Alexa Flour TM This is a goat anti-rabbit IgG (H+L) antibody labeled with 488 (Invitrogen, Waltham, MA, USA). The labeled cells were then washed twice with 150 μL of PBS, and finally suspended in 150 μL of PBS. Flow cytometry analysis was performed using Attune NxT (Invitrogen), and Alexa Fluor TM The fluorescence intensity of 488 was quantified. The obtained results were analyzed using the flow cytometry data analysis software FlowJo. TM The analysis was performed using Ver.10 (Ashland, OR, USA).

[0074] In immunogenic cell death, it is known that calreticulin molecules migrate from inside the cell to the cell surface. In this verification experiment, different concentrations of BCV or BLM (positive control) were added to the culture medium, and the migration of calreticulin molecules to the cell surface of three types of cells was analyzed by flow cytometry. Figures 21 and 22 show the flow cytometry results for FaDu cells (derived from head and neck cancer), Figures 23 and 24 show the results for PC-3 cells (derived from prostate cancer), Figures 25 and 26 show the results for CaSki cells (derived from cervical cancer), and Figures 27 and 28 show the results for SNU-719 cells (derived from gastric cancer). These results are histograms in Figures 29 and 30. It was found that in all three types of cells, the proportion of positive cells expressing calreticulin on the cell surface increased with increasing culture time in BCV. Furthermore, in FaDu, CaSki, and SNU-719, this cell positivity increased in a concentration-dependent and culture time-dependent manner with BCV. Regarding the results for the positive control BLM, all three cell types showed migration of calreticulin molecules to the cell surface, but the degree of migration varied among cells. Interestingly, in FaDu and SNU-719, BCV showed stronger calreticulin molecule migration than BLM.

[0075] Discussion: Similar to BLM, BCV induced immunogenic cell death in four different solid tumor cells: FaDu, PC-3, CaSki, and SNU-719. In particular, it induced immunogenic cell death more strongly in FaDu and SNU-719 than BLM. These results suggest that the strong immunogenic cell death-inducing activity of BCV may be a contributing factor to the intratumoral invasion of immune cells observed in Examples 2-5.

[0076] Example 4-1: Method for inducing PD-L1 mRNA expression in various solid tumors using BCV Cells of five types of solid tumors were placed in 1 mL of culture medium, inoculated into a 12-well plate, and cultured overnight in the presence of 5% CO2 at 37°C in a steam-saturated environment. After that, the cultured cells on the plate were washed, and 1 mL of culture medium adjusted to BCV concentrations of 0, 0.1, 1, and 10 μg / mL was added to each well, and the cells were cultured for 6, 24, and 48 hours. RNA was then recovered from the cells in each well using an Ultrapure RNA kit (product number CW0581M, CoWin Biosciences, Jiangsu, China). 0.4 μg of this RNA was taken and processed using PrimeScript containing gDNA Eraser. TM The mRNA was converted to cDNA using the RT reagent kit (product number RR047A, Takara Bio, Tokyo). Next, PCR was performed using the TB Green Premix Ex Taq II (Tli RNaseH Plus) kit (Takara Bio) on a QuantStudio 7 Real-Time PCR System (Thermo Fisher Scientific). In this qPCR, mRNA expression levels were examined, with GAPDH used as the internal standard. Histograms were plotted using Prism 10.1.2 (GraphPad, Boston, USA).

[0077] The results are shown in Figures 31-33. In PC-3, FaDu, A549, and SNU-719, the expression level of PD-L1 mRNA tended to increase with longer culture times. Furthermore, after 48 hours of culture, PD-L1 mRNA expression increased in a BCV concentration-dependent manner in all cell lines. After 24 hours of culture, PD-L1 mRNA expression increased in a BCV concentration-dependent manner in the PC-3, FaDu, and SK-OV-3 cell lines. From these results, it was found that BCV is involved in the regulation of PD-L1 transcription in a time-dependent or concentration-dependent manner in some solid tumor cell lines, and increases the expression level of PD-L1 mRNA.

[0078] Example 4-2: Method for inducing PD-L1 protein expression in various solid tumors using BCV Six types of solid tumor cells (PC-3, FaDu, SNU-719, A549, CaSki, SK-OV3) were cultured in plates, and BCV was added to achieve final concentrations of 0, 0.1, 1, and 10 μg / mL, respectively. The cells were then cultured for 24 hours or 48 hours in a 5% CO2 environment at 37°C and saturated with water vapor. The cells were harvested using a 0.25% trypsin solution, and the trypsin was inactivated by adding the culture medium, after which the cells were washed with PBS. Subsequently, to fix the cells and increase cell membrane permeability, they were treated with 100 μL of BD Cytofix / Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 20 minutes. Furthermore, to enhance cell membrane permeability, the cells were washed with a saponin-containing solution and then treated with either rabbit anti-PD-L1 antibody (product number ab213524, Abcam, Cambridge, UK) diluted to 1 μg / mL in 100 μL of washing solution or rabbit IgG isotype control (DA1E, Cell Signaling) at 4°C for 1 hour. After washing the cells, they were reacted with a secondary antibody diluted to 4 μg / mL in 150 μL of solution at 4°C in the dark for 60 minutes. This secondary antibody was Alexa Flour TM The antibody used was a goat anti-rabbit IgG (H+L) antibody labeled with 488 (product number A-11008, Invitrogen). The labeled cells were then washed twice with 200 μL of solution, and finally suspended in 150 μL of PBS. Flow cytometry analysis was performed using an ACEA NovoCyte flow cytometer (Agilent, Santa Clara, CA, USA).

[0079] In Example 4-1, it was found that BCV increased the expression level of PD-L1 mRNA in a time-dependent or concentration-dependent manner depending on the solid cancer cell line. In Example 4-2, we investigated whether this led to an increase in protein expression. Figures 34 and 35 show the results of flow cytometry analysis of PD-L1 expression on the cell membrane. Figure 36 shows a histogram based on this analysis. In this figure, for each cell line, the expression level of PD-L1 protein at a BCV concentration of 0 μg / mL is defined as 1, and the histograms show the PD-L1 expression levels at other BCV concentrations. A concentration-dependent increase in PD-L1 protein expression was observed in each cell line. In particular, in FaDu, the PD-L1 protein expression level increased significantly during 24-hour and 48-hour cultures. As can be seen from the vertical axis scale, this increase in expression was more pronounced in 48-hour cultures than in 24-hour cultures.

[0080] Discussion: It was shown that BCV treatment induces PD-L1 protein expression. Tumors in which PD-L1 protein expression is induced are thought to be more susceptible to the effects of PD-1 or PD-L1 inhibitors compared to tumors in which expression is not induced. Therefore, combining BCV treatment with the administration of PD-1 or PD-L1 inhibitors is considered to be very beneficial in cancer treatment. Furthermore, PD-L1 protein expression induced by BCV treatment was particularly strong in FaDu cells. This cell line is a BCV-sensitive cell line. From this, it is suggested that combination therapy of BCV and PD-1 or PD-L1 inhibitors shows a remarkable therapeutic effect, especially in BCV-sensitive cancers. In fact, a remarkable therapeutic effect was observed in mouse models in Examples 2-5.

[0081] Example 5-1: Effect of BCV on the viability of hematological cancer cells The effect of BCV on cell viability was investigated for two mouse T-cell lymphoma cell lines (EL-4, TK-1). Cell viability was quantified using the Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol. Specifically, cells were 2 × 10⁶ cells in 100 μL of medium. 3 Cells were seeded in 96-well plates at various concentrations, and BCV was added at each concentration. After each time point, Promega CellTiter-Glo® 2.0 Cell Viability Assay reagent was added to the wells, and the cells were incubated at room temperature for 10 minutes. Absorbance at 480 nm was then measured using a Tecan M200 Infinite 96-well plate reader and iConrol software 1.6 (Tecan, Mönndorf, Switzerland). Cell viability was calculated as a percentage of the control absorbance. Growth inhibitory effects were analyzed by generating dose-response curves as plots of the percentage of viable cells against BCV concentration, and their IC50s were estimated using GraphPad Prism version 8.0.2 (GraphPad Software). All reactions were performed in triple replication.

[0082] The results are shown in Figure 37. In both cell lines (EL-4 and TK-1), the BCV IC50 value was 3.5 μg / mL or less, indicating high responsiveness.

[0083] Example 5-2: Syngeneic mouse in vivo test 0.5x10 6 Six-week-old female C57BL6 mice were inoculated with EL-4 cells, and then each drug was administered intraperitoneally. Specifically, Group 1 received BCV at a dose of 40 mg / kg twice a week. Group 2 received anti-mouse PD-1 antibody at a dose of 200 μg once a week. Group 3 received BCV and anti-mouse PD-1 antibody at the same dosage and administration as Groups 1 and 2, respectively. The control group received intraperitoneal administration of a solution containing isotype IgG. The tumor size in the control group was approximately 2000 mm². 3Tumor measurements were recorded at each time point until a certain value was reached. Mice were euthanized according to IACUC guidelines. Tumor sizes of the experimental and control groups (n=8 per group) were averaged at each time point (days 1, 3, 5, and 8) and statistically compared. Signs of toxicity, including diarrhea and weight loss, were monitored throughout the experiment. Tumor weight was measured at the end of the study.

[0084] The results are shown in Figures 38 and 39. BCV administration significantly suppressed lymphoma growth, and the animals were in good health with no diarrhea observed (Figures 38(a) and (b)). The mean tumor weight at the end of the study decreased slightly in the BCV-administered group, no decrease was observed in the anti-PD-1 antibody-administered group, and the greatest decrease was observed in the BCV and anti-PD-1 antibody combination group (Figure 39). These results indicate that combination therapy with BCV and anti-PD-1 antibodies is a particularly excellent treatment method in terms of reducing tumor weight.

[0085] Example 5-3: Gene expression analysis in syngeneic mouse in vivo studies. After removing tumors from the animals used in Example 5-2, mRNA expression analysis was performed. The analysis was performed using NanoString nCounter, Mouse Immunology Panel (NanoStrings Inc.).

[0086] The results are shown in Figures 40-43. Scores for adaptive immune response, cytokines / chemokines and receptors, cytotoxic cells, dendritic cells, NK CD56dim cells, and neutrophils were highest in the BCV and anti-PD-1 antibody combination group, and significantly higher immune cell infiltration was observed only in this group (Kruskal-Wallis test). Furthermore, the score for cytotoxic immune cells (Figure 42(c)) increased slightly in the BCV administration group, showed no increase in the anti-PD-1 antibody administration group, and increased most significantly in the BCV and anti-PD-1 antibody combination group. These results indicate that combination therapy with BCV and anti-PD-1 antibody is a particularly excellent treatment method in terms of inducing immune cell infiltration or tumor cell damage.

[0087] Example 6: Effects of BCV in an in vivo model of colorectal cancer in syngeneic mice 1x10 6After inoculating 7-week-old female C57BL6 mice with a colon cancer cell line (CT26), the average tumor size was 80 mm on days 9-10. 3 The patients were divided into groups once they reached a certain size, and intraperitoneal administration of each drug was initiated. Specifically, Group 1 received BCV at a dose of 20 mg / kg twice a week. Group 2 received anti-mouse PD-1 antibody (clone RMP1-14, Bioxcell) at a dose of 10 mg / kg once every three days for a total of four doses. Group 3 received BCV and anti-mouse PD-1 antibody at the same dosage and administration as Groups 1 and 2, respectively. The control group received intraperitoneal administration of a solution containing isotype IgG (clone 2A3, Bioxcell). The tumor size of the control group was approximately 2000 mm². 3 Tumor measurements were recorded at each time point until a certain number of mice were reached. Mice were euthanized according to IACUC guidelines. Tumor sizes of the experimental and control groups (n=5 per group) were averaged at each time point (days 3, 6, 9, 12, and 16) and statistically compared. Survival numbers in each group were also monitored up to day 44, and signs of toxicity, including diarrhea, weight loss, and activity, were monitored throughout the experiment.

[0088] The results are shown in Figures 44 and 45. In both the BCV administration group and the anti-PD-1 antibody administration group, tumor growth was significantly suppressed, the animals' health status was good, and neither weight loss nor diarrhea was clearly observed. On the other hand, the BCV and anti-PD-1 antibody combination group showed significant suppression compared to the control group from day 9, and unlike treatment with each drug alone, there was almost no tumor growth. Furthermore, on days 12 and 16, it showed significant suppression compared to the BCV administration group (Figure 45). In addition, the survival rate on day 44 was 20% and 40% for the BCV administration group and the anti-PD-1 antibody administration group, respectively, while the BCV and anti-PD-1 antibody combination group showed a survival rate of 100%. These results indicate that combination therapy using BCV and anti-PD-1 antibodies is a particularly excellent treatment method.

[0089] The present invention has been described above based on examples. These examples are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible, and that such modifications also fall within the scope of the present invention.

Claims

1. A pharmaceutical composition for use in the treatment of a malignant tumor, wherein the treatment comprises combination therapy to a subject using brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy drug, and the pharmaceutical composition comprises brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof, or an immunotherapy drug.

2. The pharmaceutical composition according to claim 1, comprising brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof.

3. The pharmaceutical composition according to claim 1, comprising brincidofovir.

4. The pharmaceutical composition according to claim 1, comprising an immunotherapy drug.

5. The pharmaceutical composition of claim 4, wherein the immunotherapy drug is an immune checkpoint inhibitor.

6. The pharmaceutical composition of claim 1, comprising brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof, and an immunotherapy agent.

7. A pharmaceutical composition according to any one of claims 1 to 6, which induces the infiltration of CD8-positive T cells into tumor tissue.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the subject is a subject for whom combination therapy with cisplatin and immunotherapy is not suitable for implementation or continuation.

9. The subject is a pharmaceutical composition according to any one of claims 1 to 8, wherein the subject has renal dysfunction.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the malignant tumor is a solid tumor.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the malignant tumor is lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, uterine cancer, pancreatic cancer, giant cell tumor of bone, or ovarian cancer.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the malignant tumor is lung cancer, uterine sarcoma, prostate cancer, head and neck cancer, colorectal cancer, stomach cancer, breast cancer, skin cancer, or bladder cancer.

13. The pharmaceutical composition according to any one of claims 1 to 10, wherein the malignant tumor is head and neck cancer or gastric cancer.

14. The pharmaceutical composition according to any one of claims 1 to 9, wherein the malignant tumor is a blood cancer.

15. The pharmaceutical composition according to any one of claims 1 to 9, wherein the malignant tumor is lymphoma.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the immunotherapy drug is an immune checkpoint inhibitor.

17. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is an antibody that specifically binds to an immune checkpoint molecule or its ligand.

18. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-LAG-3 antibody.

19. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

20. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor.

21. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

22. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, spartalizumab, tislerizumab, dostallimab, retifanlimab, camrelizumab, ivonessimab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab, or relatrimab.

23. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, spartalizumab, atezolizumab, durvalumab, or avelumab.

24. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is pembrolizumab.

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