Method for assisting determination of appropriateness of cancer treatment by intraarterial administration of immune checkpoint inhibitor, and cancer therapeutic agent for intraarterial administration

Abstract

Method for assisting determination of appropriateness of cancer treatment by intraarterial administration of immune checkpoint inhibitor, and cancer therapeutic drug for intraarterial administration SOLUTION: A method for assisting determination of appropriateness of cancer treatment by intraarterial administration of an immune checkpoint inhibitor, wherein when tumor-infiltrating lymphocytes isolated from a subject after the intraarterial administration of the immune checkpoint inhibitor are measured by flow cytometry, if the number of immune checkpoint inhibitor-bound T lymphocytes is increased after the administration of the immune checkpoint inhibitor than before the administration, the method is for assisting determination that the cancer treatment is appropriate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in determining whether cancer treatment by intra-arterial administration of an immune checkpoint inhibitor is appropriate, and a cancer therapeutic drug for intra-arterial administration. More specifically, the present invention relates to a method for assisting in determining whether cancer treatment by intra-arterial administration of an immune checkpoint inhibitor is appropriate when specific results are shown in flow cytometry, and a cancer therapeutic drug to be administered to the artery of a patient determined to be appropriate for cancer treatment by intra-arterial administration. [Background technology]

[0002] Immune checkpoint inhibitors are attracting attention as new cancer treatments. Immune checkpoint inhibitors are a general term for drugs that promote T cell activation by inhibiting the binding of inhibitory immune checkpoint receptors to their ligands.

[0003] For example, a receptor molecule called PD-1 (Programmed cell death-1) is expressed on the surface of activated T cells. CTLA-4 (Cytotoxic T lymphocyte antigen-4) is also expressed on the surface of activated T cells and, like PD-1, has the function of suppressing T cell activation. On the other hand, PD-L1 (Programmed cell death-ligand-1), a ligand for PD-1, is expressed on the surface of cancer cells.

[0004] It is said that cancer cells suppress the activation of T cells and avoid their attack through the binding of PD-L1 on their surface with PD-1 on T cells. Therefore, it is believed that the use of immune checkpoint inhibitors that block the binding of inhibitory immune checkpoint receptors (e.g., PD-1) to their ligands (e.g., PD-L1) can promote the activation of T cells and eliminate cancer.

[0005] Representative immune checkpoint inhibitors that have already been approved include anti-CTLA-4 (Cytotoxic T-lymphocyte associated antigen-4) antibodies, anti-PD-1 (Programmed cell death-1) antibodies, and anti-PD-L1 (Programmed cell death-ligand-1) antibodies. Anti-PD-1 / anti-PD-L1 antibodies inhibit the binding of PD-1 / PD-L1, while anti-CTLA-4 antibodies inhibit the binding of CTLA4 / CD80 and CTLA4 / CD86, enhancing T cell activity and thereby achieving anti-tumor effects.

[0006] In Japan, the anti-PD-1 antibody nivolumab has been approved for melanoma, non-small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, and head and neck cancer; the anti-PD-1 antibody pembrolizumab has been approved for melanoma, non-small cell lung cancer, and head and neck cancer; and the anti-CTLA-4 antibody ipilimumab has been approved for melanoma. It is expected that the range of applicable cancer types will expand and new drugs will be approved in the future.

[0007] Although immune checkpoint inhibitors have attracted attention as cancer treatments, there are many unknowns about their mechanism of action. For example, the response rate of PD-1 immune checkpoint inhibitors alone is said to be only about 5-30%, and the literature (see Havel JJ et al., The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nat Rev Cancer 19(3): 133-150, 2019) states that the response rate to anti-PD-1 antibodies is approximately 30%.

[0008] Therefore, biomarkers that can predict the effects of immune checkpoint inhibitors have been explored, and a method has been reported in which cancer patients who can be expected to benefit from the treatment of anti-human PD-1 antibodies are selected by monitoring changes in the blood concentrations of immunoglobulins and other diagnostic markers before and after the administration of an anti-human PD-1 antibody (see, for example, Patent Document 1).

[0009] Concerns about immune-related adverse events and the economic burden on patients due to systemic administration of immune checkpoint inhibitors have arisen. Specifically, immune checkpoint inhibitors currently approved in Japan are administered intravenously. However, intravenous administration results in the drug being distributed throughout the body via the bloodstream, potentially resulting in only a portion of the drug reaching the target tumor. In particular, because immune checkpoint inhibitors have the ability to reverse T cell immunosuppression, there are concerns that systemic administration of the drug via intravenous administration could result in unnecessary adverse events (immune-related adverse events) due to excessive immune responses caused by T cell activation. According to the "Manual for Countermeasures against Immune-Related Adverse Events Caused by Immune Checkpoint Inhibitors" published by the Ministry of Health, Labor and Welfare in February 2022, a total of 14 immune-related adverse events have been reported, ranging from respiratory and digestive disorders to blood and skin disorders. Furthermore, the incidence of adverse events following intravenous administration of nivolumab alone has been reported to be 70% (Yen CJ, et al., Head & Neck, 2020;42(10), 2852-2862).

[0010] Additionally, immune checkpoint inhibitors are known to be extremely expensive. For example, the anti-PD-1 antibody nivolumab, when administered intravenously to treat a single adult, can cost more than 10 million yen per year. Even if patients only pay 30% of their medical expenses, they would still have to pay more than 3 million yen per year. This represents a significant burden for both the government and patients. Therefore, to reduce side effects and the economic burden on patients, microvascular blood flow reducers have been developed that selectively and significantly reduce blood flow in microvessels by injecting immune checkpoint inhibitors into nutrient arteries (see, for example, Patent Document 2).

[0011] It has also been reported that when a small amount of nivolumab was administered directly to the tumor in a model mouse, it exhibited the same tumor-suppressing effect as when administered intraperitoneally at a normal dose (see, for example, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2010 / 001617 [Patent Document 2] International Publication No. 2018 / 037595 [Non-patent literature]

[0013] [Non-Patent Document 1] Kaname Sakuma et al., “An In Vivo Study of Local Administration of Low-dose Anti-PD-1 Antibody Using an Oral Cancer Cell Line”, Anticancer Research September 2022, 42 (9) 4293-4303 Summary of the Invention [Problem to be solved by the invention]

[0014] In order to reduce side effects and the economic burden on patients, Patent Document 2 also considers local low-dose administration of immune checkpoint inhibitors, but since there are many unknowns about the mechanism of action of immune checkpoint inhibitors, it is unclear whether local low-dose administration actually results in the immune checkpoint inhibitor binding to immune checkpoint molecules or their ligands. Furthermore, Non-Patent Document 1 describes local low-dose administration of anti-PD-1 antibodies in mice with oral squamous cell carcinoma, but since the details of the mechanism of action of immune checkpoint inhibitors are unknown, it is not certain that similar results will be achieved in humans.

[0015] Patent Document 1 describes a method for predicting the effects of immune checkpoint inhibitors, but because the drug was administered intraperitoneally (i.e., intravenously) to mice transplanted with colon cancer cells, a method for predicting the effects of immune checkpoint inhibitors when administered intra-arterially has not yet been established.

[0016] The objective of the present invention is to propose a method for assisting in determining the appropriateness of cancer treatment by intra-arterial administration of immune checkpoint inhibitors, thereby increasing the success rate by delivering appropriate medical treatment to appropriate patients. [Means for solving the problem]

[0017] The present invention has been made to solve the above-mentioned problems, and embodiments of the present invention may include the following configurations. (1) A method for assisting in determining whether cancer treatment by intra-arterial administration of an immune checkpoint inhibitor is appropriate, comprising: When tumor-infiltrating lymphocytes isolated from a subject after intra-arterial administration of the immune checkpoint inhibitor were measured by flow cytometry, If the number of immune checkpoint inhibitor-binding T lymphocytes is increased after administration compared to before administration of the immune checkpoint inhibitor, A method for assisting in determining whether said cancer treatment is appropriate. (2) The flow cytometry Step 1) acquiring side scattered light, forward scattered light, and fluorescence information generated by irradiating the tumor-infiltrating lymphocytes stained with an antibody labeled with a fluorescent dye with excitation light of the fluorescent dye; Step 2) identifying a lymphocyte fraction based on the side scattered light and the forward scattered light; Step 3) identifying a T lymphocyte marker-positive fraction from the lymphocyte fraction based on the side scattered light and the fluorescence information; Step 4) counting the number of immune checkpoint inhibitor-binding T lymphocytes contained in the T lymphocyte marker-positive fraction based on the fluorescence information; The method according to (1), comprising: (3) The method according to (1), wherein the cancer is at least one selected from the group consisting of head and neck cancer, lung cancer, breast cancer, ovarian cancer, skin cancer, colon cancer, bladder cancer, liver cancer, gastric cancer, prostate cancer, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, and sarcoma. (4) The method according to (3), wherein the head and neck cancer is at least one type selected from the group consisting of cancers or tumors of the oral cavity, lip, pharynx (including the nasopharynx, oropharynx, and hypopharynx), larynx, paranasal sinuses, nasal cavity, throat, salivary glands, and thyroid gland. (5) The method according to (1) or (2), wherein the immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an anti-PD-L1 antibody. (6) A cancer therapeutic drug for intra-arterial administration, comprising an immune checkpoint inhibitor, which is administered to the cancer-feeding artery of a patient who has been determined to be suitable for cancer treatment by the method described in (1) or (2). [Effects of the Invention]

[0018] According to the present invention, it is possible to assist in determining whether cancer treatment by intra-arterial administration of an immune checkpoint inhibitor is appropriate. This allows appropriate medical care to be provided to appropriate patients. Because the present invention performs measurements using cells collected from patients, it can assist in determination with high accuracy, unlike cases where results from model animals are used. The use of flow cytometry is also advantageous in that it can assist in determination quickly and easily. Furthermore, when the determination results of the present invention are used to administer the inhibitor to a patient, local low-dose administration can be performed, thereby reducing immune-related adverse events caused by immune checkpoint inhibitors and the economic burden on the patient. This is preferable because it allows cancer, particularly head and neck cancer, to be treated without relying on surgical procedures, and does not significantly impair the patient's quality of life or activities of daily living (ADL). [Brief explanation of the drawings]

[0019] [Figure 1]Comparison of flow cytometry analysis results of TILs before and after administration of immune checkpoint inhibitors. (A) Analysis diagram expanded two-dimensionally using side scattered light intensity and forward scattered light intensity. The area within the black frame corresponds to the lymphocyte fraction. (B) Analysis diagram expanded two-dimensionally using side scattered light intensity and fluorescence intensity. The area within the black frame corresponds to the CD3-positive fraction in the lymphocyte fraction. (C) Analysis diagram expanded two-dimensionally using fluorescence intensity. Pre indicates the analysis diagram before administration of nivolumab, and Post indicates the analysis diagram after administration of nivolumab. It can be seen that at Post, the population has shifted more toward the human IgG4-positive fraction than at Pre. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described in detail below, but the present invention is not limited to the specific embodiments given below.

[0021] <Immune checkpoint inhibitors> Immune checkpoint inhibitors bind to immune checkpoint molecules or their ligands that suppress T cell activation, thereby reversing this suppression. In other words, immune checkpoint inhibitors can be considered immune-boosting drugs. Anticancer drugs, radiation therapy, and surgery have traditionally aimed to eliminate or remove cancer cells, but these treatments place a strain on the body and may weaken the immune system, making it difficult to eliminate all cancer cells. For elderly cancer patients in particular, traditional immune-suppressing treatments have not been shown to cure cancer completely and may even shorten life expectancy. In contrast, immune checkpoint inhibitors inhibit cancer growth by suppressing the immune evasion mechanisms of tumor cells. Therefore, they can be administered to patients who are refractory to conventional treatments due to their tumor location or size, as well as to patients with poor general health due to advanced age or other factors.

[0022] Immune checkpoint inhibitors target inhibitory immune checkpoint molecules and their ligands that are presented on the surface of T cells and antigen-presenting cells. Currently identified immune checkpoint molecules include PD-1 (Programmed Death Receptor-1), CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), LAG-3 (Lymphocyte-Activation Gene 3), TIM-3 (T-cell Immunoglobulin Mucin-3), BTLA, and KIR (Killer Cell Immunoglobulin-Like Receptor). Ligands include PD-L1 (PD-1 ligand), PD-L2 (PD-1 ligand), GAL9 (TIM-3 ligand), and HVEM (BTLA ligand).

[0023] The immune checkpoint inhibitor may be any substance capable of inhibiting the functions of the immune checkpoint molecules and their ligands exemplified above, and examples include PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, BTLA inhibitors, KIR inhibitors, CCR4 inhibitors, etc. Specific examples of PD-1 inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies, and specific examples of CTLA-4 inhibitors include anti-CTLA-4 antibodies.

[0024] Examples of known immune checkpoint inhibitor pharmaceuticals include PD-1 immune checkpoint inhibitors, such as anti-PD-1 antibodies nivolumab (Opdivo (registered trademark)) and pembrolizumab (Keytruda (registered trademark)), anti-PD-L1 antibodies atezolizumab (Tecentriq (registered trademark)), durvalumab (Imfinzi (registered trademark)), and avelumab (Bavencio (registered trademark)), and anti-CTLA-4 antibodies such as ipilimumab (Yervoy (registered trademark)) and tremelimumab (Ijudo (registered trademark)).

[0025] The immune checkpoint inhibitor of the present invention is not limited to these, and any substance that inhibits an inhibitory immune checkpoint molecule or its ligand can be used as the immune checkpoint inhibitor of the present invention, but it is preferably at least one antibody selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an anti-PD-L1 antibody, and more preferably an anti-PD-1 antibody. As a pharmaceutical name, nivolumab is preferred as the anti-PD-1 antibody.

[0026] <Cancers targeted by the method of the present invention> The method of the present invention can be applied to at least one cancer selected from the group consisting of head and neck cancer, lung cancer, breast cancer, ovarian cancer, skin cancer, colon cancer, bladder cancer, liver cancer, stomach cancer, prostate cancer, renal cell cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, and sarcoma, with head and neck cancer being particularly preferred.

[0027] The subject of the method of the present invention is humans and non-human mammals for which cancer treatment is desired or required. Non-human mammals include, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters, including pet animals, livestock, and laboratory animals. A preferred subject for administration is humans. Hereinafter, one embodiment of the present invention will be described using humans as an example.

[0028] The cancer therapeutic drug for intra-arterial administration of the present invention is administered to patients who have been determined to be appropriate for cancer treatment by the method described below, but can be administered regardless of the phase of the cancer as long as it is determined to be appropriate. For example, it may be administered to patients who have cancer, those suspected of having cancer, or patients who have had tumor tissue removed. Because the cancer therapeutic drug for intra-arterial administration of the present invention is administered locally to the target tissue, it also has the effect of minimizing drug distribution to normal surrounding tissue other than the target tissue. Therefore, the patient's current or past medication history is not a factor.

[0029] <Head and neck cancer> The head and neck region refers to the area of ​​the face and neck below the base of the skull and above the collarbone. Head and neck cancer is the general term for cancers that develop in this area, including the nose, oral cavity, salivary glands, and thyroid gland. Head and neck cancer develops in an area that contains organs that perform important daily functions, such as eating, speaking, and breathing. Depending on the treatment method, it can have a significant impact on the patient's quality of life and activities of daily living (ADL). While achieving a cure is generally considered the most important goal in cancer treatment, as the head and neck region is directly linked to QOL and ADL, treatment must maintain a high level of both cure and functional preservation, making treatment selection difficult.

[0030] Furthermore, the current standard of care for advanced head and neck cancer, especially recurrent or metastatic head and neck cancer, is intravenously administered 200 mg every three weeks with pembrolizumab (anti-PD-1 antibody). Even with this treatment, the response rate is reported to be around 10-15%, despite the physical and financial burden it places on patients (Harrington KL, et al., Oral Oncology. 2023; 147: 106587). Therefore, administering this drug to patients with a high response rate will also help reduce the physical and financial burden on patients.

[0031] The head and neck cancer in the present invention is preferably at least one type selected from the group consisting of cancers or tumors of the oral cavity, lip, pharynx (including the nasopharynx, oropharynx, and hypopharynx), larynx, paranasal sinuses, nasal cavity, throat, salivary glands, and thyroid gland. Alternatively, the present invention is preferably applied to head and neck malignant melanoma. Head and neck malignant melanoma often metastasizes lymphatic and hematogenously at an early stage, making it a disease with an extremely poor prognosis.

[0032] <Method for assisting in determining the suitability of cancer treatment by intra-arterial administration of immune checkpoint inhibitors> The present invention is a method for confirming binding between T lymphocytes and the drug even at low doses by intra-arterial injection of an immune checkpoint inhibitor, and using the results to assist in determining the appropriateness of cancer treatment, i.e., whether or not to continue the cancer treatment drug. Therefore, an administration method and dosage that mimic actual treatment are preferred.

[0033] Therefore, in the present invention, attention is focused on lymphocytes infiltrating tumor tissue (tumor-infiltrating lymphocytes, hereinafter also referred to as "tumor-infiltrating lymphocytes" or "TILs"), and the degree of binding between TILs and immune checkpoint inhibitors is confirmed by flow cytometry. The use of flow cytometry in the present invention allows for simple and rapid measurement. The obtained flow cytometry results serve as auxiliary data for determining whether or not intra-arterial administration of an immune checkpoint inhibitor to a subject (patient) is appropriate for cancer treatment.

[0034] <Sample processing process> First, tumor tissue is collected from the subject and pre-administration TILs are prepared. Next, the immune checkpoint inhibitor is injected into the subject's artery. More specifically, it is administered into the tumor-feeding artery. The tumor-feeding artery is an artery that carries blood containing nutrients and oxygen necessary for tumor growth. Specific examples of arterial administration methods are described below, but general methods can be used. The subject's tumor-feeding artery is identified by angiography, and the inhibitor can be administered using a catheter. Immune checkpoint inhibitors are administered at a dose that is less than one-tenth of the standard intravenous dose per dose.

[0035] After injecting an immune checkpoint inhibitor into the subject's artery, tumor tissue is collected and the administered TILs are prepared. A specific method for preparing TILs is to collect tumor tissue from the subject's local tumor or tumor tissue with visible metastasis during biopsy or surgery. The collected tissue is then shredded with scissors and placed in culture medium. Lymphocytes that have leaked from the tumor tissue into the culture medium are filtered through a filter to produce TILs, which are then used for subsequent evaluation. 104 ~10 7 It is preferable to prepare 10 cells / 100 μL. 5 ~10 6 It is more preferable to prepare the solution at cells / 100 μL.

[0036] The prepared TILs are stained by adding a fluorescent dye-labeled monoclonal antibody, and then detected by flow cytometry. The monoclonal antibodies used are preferably monoclonal antibodies that serve as markers for T lymphocytes, immune checkpoint inhibitors, or receptor molecules recognized by the immune checkpoint inhibitors.

[0037] Monoclonal antibodies that are T lymphocyte markers include monoclonal antibodies that can recognize commonly used T lymphocyte markers (CD3, CD2, CD5, or CD7). In particular, since all T lymphocytes express CD3, it is preferable to use an anti-human CD3 monoclonal antibody.

[0038] It is preferable to use a monoclonal antibody that matches the isotype of the immune checkpoint inhibitor used as a marker for the immune checkpoint inhibitor. For example, when using anti-PD-1 antibodies such as nivolumab or pembrolizumab, an anti-human IgG4 antibody is preferred, and when using anti-CTLA-4 antibody ipilimumab, an anti-human IgG1 antibody is preferred.

[0039] The monoclonal antibody used as a marker for the receptor molecule recognized by the immune checkpoint inhibitor is selected depending on the immune checkpoint inhibitor used. For example, when using nivolumab or pembrolizumab, an anti-human PD-1 monoclonal antibody is preferred, and when using ipilimumab, an anti-human CTLA-4 antibody is preferred.

[0040] Any antibody is labeled with a substance that can be detected or measured. Examples include radioactive labels, luminescent labels, and fluorescent labels. From the perspective of ease of operation, fluorescent labeling is preferred. For example, as fluorescent labels, Cy3, Cy5, FITC, Hilyte Fluor TM 647, phycoerythrin, allophycocyanin, etc. can be mentioned. Alternatively, BD Horizon TM Brillian's violet dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786), ultraviolet dyes (e.g., BUV395, BUV496, BUV737, BUV805), blue dyes (e.g., BB515), etc. may also be used.

[0041] Add a fluorescently labeled antibody to the prepared TIL (TIL before and after administration of an immune checkpoint inhibitor), react, wash thoroughly, and then observe using flow cytometry. The excitation light depends on the fluorescent dye used.

[0042] <Step 1: Obtaining side scatter light, forward scatter light, and fluorescence information generated by irradiating the lymphocytes stained with an antibody labeled with a fluorescent dye with the excitation light of the fluorescent dye> Flow cytometry is a device that measures the optical properties of cells from a homogeneous suspension of cells. Cells ride on a fluid flow and pass through the focus of a laser beam. At this time, the optical properties of side scatter light, forward scatter light, and fluorescence of one or more different wavelengths are simultaneously measured for each individual cell, and the cell size, internal structure, presence or absence of binding between cells and drugs, etc. can be measured accurately and quickly. In the present invention, three types of fluorescently labeled antibodies are used, so the obtained optical properties are also three types. The obtained side scatter light, forward scatter light, and fluorescence information (optical properties) are converted into electrical signals and analyzed by a computer. It is represented by a dot plot, etc.

[0043] In the present invention, since the purpose is to confirm that the immune checkpoint inhibitor is bound to T lymphocytes even at a low-dose administration, the binding between the immune checkpoint inhibitor and lymphocytes is confirmed by applying a gate as follows.

[0044] <Step 2: Step of identifying a lymphocyte fraction based on the side scatter light and the forward scatter light> The results of flow cytometry are two-dimensionally developed as a dot plot with side scatter (Side scatter / SSC) on the X-axis and forward scatter (Forward scatter / FSC) on the Y-axis. Lymphocytes, which are relatively small in size and have a simple internal structure, form a population on the left side, so a gate is applied to that part (which may also be referred to as the "lymphocyte fraction").

[0045] <Step 3: Step of identifying a T lymphocyte marker-positive fraction from the lymphocyte fraction based on the side scatter light and the fluorescence information> The lymphocyte fraction is further two-dimensionally developed based on the side scatter light and the fluorescence information of a monoclonal antibody, which is a marker for T lymphocytes. A gate is applied to the T lymphocyte marker-positive fraction within the lymphocyte fraction. Thereby, T lymphocytes can be sorted.

[0046] <Step 4: Step of measuring the number of immune checkpoint inhibitor-bound T lymphocytes contained in the T lymphocyte marker-positive fraction based on the fluorescence information> The T lymphocyte marker-positive fraction is further two-dimensionally developed based on the fluorescence information of a monoclonal antibody, which is a marker for the immune checkpoint inhibitor, and the fluorescence information of a monoclonal antibody, which is a marker for the receptor molecule recognized by the immune checkpoint inhibitor.

[0047] The fraction positive for the monoclonal antibody, which is a marker for immune checkpoint inhibitors, is considered to contain immune checkpoint inhibitor-binding T lymphocytes, and their number is counted. Taking Figure 1(C) as an example, these correspond to the upper left and upper right quadrants of Figure 1(C), and the cell populations counted here are considered to be immune checkpoint inhibitor-binding T lymphocytes.

[0048] If the number of immune checkpoint inhibitor-binding T lymphocytes measured in Step 4 increases before and after administration of the immune checkpoint inhibitor, this indicates a high efficacy of the immune checkpoint inhibitor, and helps determine whether the cancer treatment is appropriate. Specifically, the number of immune checkpoint inhibitor-binding T lymphocytes after administration is preferably at least two-fold higher than that before administration, more preferably at least three-fold higher, and even more preferably at least four-fold higher.

[0049] <Cancer treatment drugs for intra-arterial administration including immune checkpoint inhibitors> When it is determined according to the present invention that administration to a subject (patient) is appropriate, the cancer therapeutic drug for intra-arterial administration comprising the immune checkpoint inhibitor of the present invention is administered to the subject (patient).

[0050] The immune checkpoint inhibitor contained in the cancer therapeutic agent for intra-arterial administration of the present invention includes those mentioned above, but is preferably at least one selected from the group consisting of anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-PD-L1 antibody, and more preferably an anti-PD-1 antibody. As the pharmaceutical name of the anti-PD-1 antibody, nivolumab is preferred.

[0051] <Administration method> The cancer therapeutic agent for intra-arterial administration of the present invention is administered intra-arterially. This is also called selective intra-arterial infusion, and more specifically, the cancer therapeutic agent is administered into a tumor-feeding artery. The tumor-feeding artery is an artery that carries blood and oxygen containing nutrients necessary for tumor growth. By administering the agent into this artery, the agent can be delivered to tumor tissue at a high concentration and efficiently, thereby enhancing the therapeutic effect and reducing the amount of agent circulating throughout the body, thereby reducing side effects.

[0052] Head and neck cancer, a preferred embodiment of the present invention, will now be described in detail. To ensure reliable delivery of the drug to head and neck cancer tumors, the intra-arterial cancer therapeutic agent of the present invention is preferably administered to each tumor's nutrient artery, primarily the external carotid artery. The external carotid artery, which supplies nutrients to head and neck cancer, originates from a large cervical artery called the common carotid artery. The internal carotid artery, which connects to the brain, also originates from this common carotid artery. Thus, head and neck cancers are primarily nourished by the external carotid artery, another branch of the carotid artery, and each is governed by its own artery. The preferred method of administering cancer therapeutic drugs to these tumor-feeding arteries is head and neck IVR. IVR stands for "Interventional Radiology," and refers to a method of inserting a catheter while viewing the inside of the body using diagnostic imaging devices such as X-ray fluoroscopy, CT, and ultrasound. For example, using X-rays allows doctors to view the transmitted images in real time, confirm the location of the blood vessels, and insert the catheter. Below, we will explain in detail the currently common method of IVR via the femoral artery in the thigh.

[0053] First, imaging diagnostics such as X-rays, CT scans, and MRI scans are used to identify the location of the tumor and the terminal arteries from the external carotid artery that nourishes the tumor. While viewing real-time X-ray images, the doctor inserts a catheter through a femoral artery puncture under local anesthesia in the subject's body. A contrast agent is then injected into the catheter, and while checking the position of the catheter and blood vessels in real time using X-rays, the catheter is inserted into the target blood vessel and the drug is injected for local administration.

[0054] More specifically, while compressing the central side of the femoral artery, a few millimeters (1–3 mm) of arterial wall are incised distally. A double-layered needle is then inserted through the incision. The outer tube (outer tube) remains inside the vessel. The needle is removed, leaving the outer tube. Instead, a thin wire (guidewire) is threaded through the outer tube and inserted into the vessel. A thick tube (sheath) is then inserted along the guidewire, creating an opening for the catheter. Passing the catheter through the sheath allows for free insertion and removal of the catheter. The catheter is then placed over the guidewire within the sheath, and inserted into the vessel along with the guidewire. The catheter is then advanced to the neck, and the target tumor-feeding artery is identified. If there are branch vessels proximal to the catheter tip that nourish the tumor, it is preferable to position the catheter slightly in front of the tumor to provide broad coverage of the entire tumor, including the surrounding tissue.

[0055] When the cancer therapeutic agent of the present invention is administered using IVR, i) most of the agent can be delivered to the superior thyroid artery, thereby enabling uniform delivery of the agent to tumors nourished by the superior thyroid artery and the superior laryngeal artery branching from it. This makes it effective for treating, for example, laryngeal cancer and pharyngeal cancer, for which the superior thyroid artery and the superior laryngeal artery branching from it are the main nutrient arteries. ii) It can also be delivered to the lingual artery, thereby enabling uniform delivery of the agent to tumors nourished by the lingual artery. This makes it effective for treating most oral cancers, such as tongue cancer, for which the lingual artery is the main nutrient artery. iii) It can also be delivered to the facial artery, thereby enabling uniform delivery of the agent to tumors nourished by the facial artery. This makes it effective for oral cancers, such as buccal mucosa cancer and gingival cancer, for which the facial artery is the main nutrient artery. Furthermore, administration to the ascending pharyngeal artery, etc., allows administration to nasopharyngeal cancer. iv) It can also be delivered to the maxillary artery, thereby enabling uniform delivery of the agent to tumors nourished by the maxillary artery. For example, in addition to tongue cancer, which is primarily nourished by the facial artery, this drug is also effective for oral cancer, which is mostly nourished by the maxillary artery, and maxillary sinus cancer, which is nourished by the maxillary artery. And v) because it can be diverted to the stylomastoid artery, etc., it is possible to administer drugs evenly to tumors nourished by the stylomastoid artery, etc. For example, this drug is also effective for external auditory canal cancer and middle ear cancer, which are primarily nourished by the stylomastoid artery, etc. As mentioned above, this drug can be administered to all types of head and neck cancer.

[0056] If a tumor is present in an area controlled by multiple blood vessels, it is preferable to inject the human head and neck cancer therapeutic drug into each of the blood vessels that control it. If the blood vessel being considered for injection is significantly narrowed and close to occlusion, or if many small arterial branches are involved as tumor blood vessels, it is preferable to administer a small amount of MRI contrast agent through a port, check on MRI images which arterial branches will return the drug to the entire tumor, and then use those arterial branches to inject the human head and neck cancer therapeutic drug from a location closer to the tumor.

[0057] This administration method is preferably applied to head and neck cancers associated with tumor-feeding vessels supplied by the external carotid artery, such as the lingual artery, facial artery, and carotid artery.

[0058] <Dosage and administration interval> The cancer therapeutic drug for intracranial administration of the present invention can be administered locally and is effective at a much lower dose than in the case of systemic administration. The dose of the cancer therapeutic drug for intracranial administration of the present invention varies depending on the type of immune checkpoint inhibitor contained, the age of the patient, the type of disease, the severity of the disease, etc., but is preferably 1 / 10 or less of the standard dose for intravenous administration per dose.

[0059] For patients for whom cancer treatment is deemed appropriate by the method of the present invention, it is expected that high efficacy will be achieved even at a dose of, for example, 1 / 10 or less of the standard dose. Furthermore, low-dose administration can reduce the amount of drug circulating throughout the body, thereby reducing immune-related adverse events and the cost burden on patients.

[0060] The standard dosage refers to the dosage of a therapeutic drug in standard cancer treatment. For example, the dosage and administration described in the guidelines for promoting optimal use prepared by the Ministry of Health, Labor and Welfare are considered to be standard dosages. In addition, the recommended dosage of a therapeutic drug described in a product insert by a therapeutic drug manufacturer is also considered to be standard dosages.

[0061] The standard dosage varies depending on the drug and the type of cancer being treated. Table 1 shows the standard dosages for the anti-PD-1 antibodies nivolumab and pembrolizumab for head and neck cancer, as outlined in the Optimal Use Promotion Guidelines prepared by the Ministry of Health, Labour and Welfare. [Table 1]

[0062] The dosing interval also refers to the dosing interval of the standard treatment. As with the standard dosage mentioned above, the dosing interval described in the guidelines for promoting optimal use prepared by the Ministry of Health, Labor and Welfare or the dosing interval recommended by the therapeutic drug manufacturer can be adopted.

[0063] The cancer therapeutic drug for intra-arterial administration of the present invention is effective at 1 / 10 or less of the standard dose. Therefore, for example, when administering nivolumab as a therapeutic drug for head and neck cancer, referring to the guidelines for promoting optimal use prepared by the Ministry of Health, Labour and Welfare shown in Table 1, it is recommended to administer nivolumab arterially at a dose of 24 mg or less every two weeks, or at a dose of 48 mg or less every four weeks.

[0064] The immune checkpoint inhibitor of the present invention may be used in combination with other drugs, as long as the object of the present invention is not impaired. For example, it may be used in combination with anticancer drugs, anti-inflammatory drugs, etc. Alternatively, it may be used in combination with other cancer immunotherapies. Cancer immunotherapies include cancer vaccine therapy, immune cell infusion therapy, and methods for inducing the removal of regulatory T cells.

[0065] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to the examples shown below. [Example]

[0066] <Administration of immune checkpoint inhibitors to subjects and cell collection> To prepare TIL cells before administration, tumor tissue from the patient's head and neck cancer (right tonsil) was collected during biopsy, minced with scissors, and plated in culture medium. Lymphocytes exuded from the tumor tissue into the medium were then filtered through a 40 μM filter to prepare TIL cells before administration. The patient's tumor-feeding artery was identified using an IVR system, and 20 mg of nivolumab was administered intra-arterially via a catheter. One week later, 20 mg of nivolumab was administered intra-arterially again. Two weeks later, cells were again collected from the patient's head and neck cancer (right tonsil), and post-administration TILs were prepared using the method described above. The standard dose of nivolumab for intravenous administration is 240 mg, so less than one-tenth of that amount was administered.

[0067] <Flow cytometry measurement> TIL(10 5The cells were stained by adding 10 μL each of BUV737-labeled anti-human CD3 monoclonal antibody (BD Bioscience), PE-labeled anti-human IgG4 antibody (SouthenBiotech), and BV421-labeled anti-human PD-1 monoclonal antibody (Biolegend) to the cells (100 μL each). After washing once with phosphate-buffered saline (PBS), flow cytometry analysis was performed.

[0068] The lymphocyte fraction was identified by two-dimensional analysis using FSC and SSC (Figure 1(A)). The cells were then expanded in two dimensions using SSC and CD3, and gated on the CD3-positive fraction (Figure 1(B)). The cells were then expanded in two dimensions using IgG4 and PD-1 (Figure 1(C)). The results of TIL before administration are shown as Pre in Figure 1(C), and the results after administration are shown as Post. As is clear from the Post results, cell accumulation was observed in the human IgG4-positive fraction (upper left and right of Figure 1(C)). This means that the immune checkpoint inhibitor was binding to T lymphocytes. More specifically, in Figure 1(C), PD-1-positive human IgG4-negative T lymphocytes accounted for 49.3% of the pre-stage data, whereas this cell population shifted to the human IgG4-positive fraction at post-stage data. In other words, the proportion of the human IgG4-positive fraction increased more than fivefold from pre-stage data (0.31 + 3.67 = 3.98%) to post-stage data (11.4 + 19.3 = 30.7%). This indicates that nivolumab was able to bind to T lymphocytes in TILs despite being administered at a low dose of 20 mg, less than one-tenth the standard dose of nivolumab (240 mg). (Note: The small number of cells observed in the human IgG4-positive fraction at pre-stage data is presumed to be nonspecific.) This suggests that the intra-arterial administration of the cancer therapeutic agent of the present invention will be effective even at low doses. This is also thought to be useful in reducing the physical and economic burden on patients.

[0069] It was speculated from these results that some of the cancer treatment drug also leaked into the periphery, but because the intra-arterial administration cancer treatment drug of the present invention is less than one-tenth of the conventional dose, even if it leaked into the whole body, the incidence of immune-related adverse events is thought to be low.

[0070] The method of the present invention is expected to be applicable to other molecularly targeted therapeutic agents. Molecularly targeted therapeutic agents are drugs designed to target only specific molecules and regulate their function. In addition to immune checkpoint inhibitors, examples include ADC (Antibody Drug Conjugate) preparations, CAR-T preparations, antibody-photosensitizer conjugates, and peptide receptor radionuclide therapy agents. For example, the method is expected to be useful for predicting the rate at which molecularly targeted therapeutic agents bind to tumor sites, allowing for administration to patients after confirming the rate at which ADC agents bind to tumor sites.

Claims

1. A method for assisting in determining whether cancer treatment by intra-arterial administration of an immune checkpoint inhibitor is appropriate, comprising: When tumor-infiltrating lymphocytes isolated from a subject after intra-arterial administration of the immune checkpoint inhibitor were measured by flow cytometry, If the number of immune checkpoint inhibitor-binding T lymphocytes is increased after administration compared to before administration of the immune checkpoint inhibitor, A method for assisting in determining whether said cancer treatment is appropriate.

2. The flow cytometry Step 1) acquiring side scattered light, forward scattered light, and fluorescence information generated by irradiating the tumor-infiltrating lymphocytes stained with an antibody labeled with a fluorescent dye with excitation light of the fluorescent dye; Step 2) identifying a lymphocyte fraction based on the side scattered light and the forward scattered light; Step 3) identifying a T lymphocyte marker-positive fraction from the lymphocyte fraction based on the side scattered light and the fluorescence information; Step 4) counting the number of immune checkpoint inhibitor-binding T lymphocytes contained in the T lymphocyte marker-positive fraction based on the fluorescence information; The method of claim 1 , comprising:

3. 2. The method of claim 1, wherein the cancer is at least one selected from the group consisting of head and neck cancer, lung cancer, breast cancer, ovarian cancer, skin cancer, colon cancer, bladder cancer, liver cancer, gastric cancer, prostate cancer, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, and sarcoma.

4. 4. The method of claim 3, wherein the head and neck cancer is at least one selected from the group consisting of cancers or tumors in the oral cavity, lip, pharynx (including the nasopharynx, oropharynx, and hypopharynx), larynx, paranasal sinuses, nasal cavity, throat, salivary glands, and thyroid gland.

5. The method of claim 1 or 2, wherein the immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an anti-PD-L1 antibody.

6. A cancer therapeutic agent for intra-arterial administration comprising an immune checkpoint inhibitor, which is administered to a cancer-feeding artery of a patient determined to be appropriate for cancer treatment by the method of claim 1 or 2.

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