Pharmaceutical composition used for optical immune therapy targeting tumor microenvironment, marker for therapy effect confirmation, and inspection method

Abstract

To provide therapy giving therapy effects even to tumor, having its specific cancer antigen unknown and developing low, that no effect is given to by therapy using an antibody or conventional near-infrared-ray immune therapy (NIR-PIT).SOLUTION: NIT-PIT targeting immune checkpoint molecules developed in a tumor not only induces tumor cell to cell death, but also increases development of the immune checkpoint molecules. Consequently, the therapy is performed a plurality of times to give high effects. Further, immune reaction is induced, so effects are given not only locally to a light irradiation part, but also to the whole body, namely, a metastasis part.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to photoimmunotherapy targeting immune checkpoint molecules, particularly immune checkpoint molecules expressed in tumors, as well as biomarkers and testing methods for confirming the therapeutic effects of the therapy. [Background technology]

[0002] Near infrared photoimmunotherapy (NIR-PIT) is a treatment that specifically kills target cells such as tumor cells by irradiating them with near infrared light. It is a new treatment that can remove only the target cells from the local area without damaging the surrounding cells, and is attracting attention because it places less of a burden on the patient. Specifically, a complex is created by binding a molecule that is excited by near infrared light to a specific binding molecule such as an antibody that binds to a target molecule specifically expressed in the target cell. The complex is then bound to the target molecule, and light is irradiated to induce necrotic cell death in the target cell through a photochemical reaction.

[0003] NIR-PIT is being tested in clinical trials with RM-1929, a conjugate of IRDye® 700DX (IR700) bound to cetuximab, an antibody that targets the epidermal growth factor receptor (EGFR). RM-1929 has progressed to Phase III trials and was approved in Japan in September 2020, ahead of the rest of the world. EGFR is expressed in a variety of solid cancers, including head and neck cancer, which is the subject of clinical trials, as well as esophageal cancer, lung cancer, colon cancer, and pancreatic cancer, and can be applied to these cancers in which EGFR is expressed.

[0004] In addition, NIR-PIT can be applied to various target molecules because it is sufficient to prepare a complex of IR700 and a molecule such as an antibody that specifically binds to a target molecule expressed on the cell surface of tumor cells. Therefore, it is possible to target not only EGFR, which is currently undergoing clinical trials, but also so-called cancer antigens that are specifically expressed in tumors, such as HER2, PSMA, and CEA, and this is a treatment method that can be widely applied to various tumors (Patent Document 1, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2018-528268 [Patent Document 2] International Publication No. 2000 / 031588 [Non-patent literature]

[0006] [Non-Patent Document 1] Kobayashi, H. & Choyke, PL, 2019, Acc. Chem. Res., Vol.52, pp.2332-2339. Summary of the Invention [Problem to be solved by the invention]

[0007] However, in order to obtain the effect of NIR-PIT, it is necessary that the target molecule such as EGFR is highly expressed on the cell surface. This is because the complex does not bind to cells that do not express the target molecule or only weakly express it, or the binding is insufficient and the effect cannot be obtained. Therefore, patients to whom NIR-PIT can be applied are limited to those who have a high expression of the target molecule on the tumor surface. In addition, it is known that tumors become heterogeneous because new genomic abnormalities accumulate even within a single tumor. Therefore, NIR-PIT, which kills only cells that have a high expression of the target molecule, cannot induce cell death in all tumor cells, and recurrence occurs due to tumor cells in which cell death has not been induced because the target molecule is not expressed. In addition, conventional NIR-PIT is a local therapy for tumors irradiated with light, and has little effect on metastatic tumors. The present invention aims to create a treatment that is effective for patients who have low antigen expression levels and have not been effective in conventional NIR-PIT or antibody treatments by inducing an immune response, and a treatment that is effective not only in the local area where light is irradiated but also in metastatic tumors. [Means for solving the problem]

[0008] The present invention relates to the following pharmaceutical composition, testing method, and marker. (1) A test method for confirming the effect of a pharmaceutical composition that enhances tumor immunity in a subject, the pharmaceutical composition being a pharmaceutical composition for use in near-infrared photoimmunotherapy, the test method comprising measuring one or more markers, among B cells, T cells, CD4+ T cells, CD8+ T cells, MDSCs (myeloid-derived suppressor cells), monocytes / macrophages, dendritic cells, G-CSF (granulocyte colony-stimulating factor), IL-6, KC (keratinocyte-derived chemokine), and MIP-2 (macrophage inflammatory protein 2), in a sample obtained from the subject before and after near-infrared light irradiation, and confirming the therapeutic effect based on the change in the marker before and after near-infrared light irradiation. (2) The method for testing described in (1), wherein the pharmaceutical composition specifically binds to an immune checkpoint molecule expressed in tumor cells. (3) The testing method described in (2), characterized in that the immune checkpoint molecule is PD-L1. (4) The method according to any one of (1) to (3), wherein the sample is peripheral blood. (5) Markers to confirm the effect of near-infrared photoimmunotherapy in enhancing tumor immunity, evaluating the changes in at least one of B cells, T cells, CD4+ T cells, CD8+ T cells, MDSC, monocytes / macrophages, dendritic cells, G-CSF, IL-6, KC, and MIP-2 in samples before and after near-infrared photoimmunotherapy. (6) A marker for confirming the effect of near-infrared photoimmunotherapy according to (5), characterized in that the near-infrared photoimmunotherapy is carried out using a pharmaceutical composition that targets PD-L1. (7) A pharmaceutical composition for metastatic cancer and / or preventing recurrence, comprising as an active ingredient a complex in which a near-infrared light sensitive substance is bound to a specific binding molecule, and the specific binding molecule is a molecule that binds to an immune checkpoint molecule expressed in tumor cells. (8) The pharmaceutical composition for metastatic cancer and / or prevention of recurrence according to (7), wherein the immune checkpoint molecule is PD-L1. (9) The pharmaceutical composition for metastatic cancer and / or prevention of recurrence according to (7) or (8), wherein the specific binding molecule is an antibody or a functional antibody fragment. (10) The pharmaceutical composition for metastatic cancer and / or preventing recurrence according to any one of (7) to (9), wherein the near-infrared light sensitive substance is a phthalocyanine dye. (11) The pharmaceutical composition for metastatic cancer and / or prevention of recurrence according to (10), wherein the phthalocyanine dye is IR700. (12) The pharmaceutical composition for metastasis of cancer and / or prevention of recurrence according to any one of (7) to (11), which is a pharmaceutical composition for performing multiple photoirradiations. (13) A pharmaceutical composition for metastatic cancer and / or prevention of recurrence according to any one of (7) to (12), characterized in that it is used in combination with a pharmaceutical composition having as an active ingredient a complex in which a near-infrared light sensitive substance is bound to a specific binding molecule that binds to a target molecule other than an immune checkpoint molecule. (14) The pharmaceutical composition for metastatic cancer and / or prevention of recurrence according to any one of (7) to (13), which is used in combination with a chemotherapeutic agent. (15) A pharmaceutical composition for enhancing tumor immunity in a subject, comprising as an active ingredient a complex in which a specific binding molecule is bound to a near-infrared light sensitive substance, wherein the specific binding molecule is an immune checkpoint molecule expressed in tumor cells. (16) The pharmaceutical composition according to (15), wherein the immune checkpoint molecule is PD-L1. (17) The pharmaceutical composition according to (15) or (16), wherein the specific binding molecule is an antibody or a functional antibody fragment. (18) The pharmaceutical composition according to any one of (15) to (17), wherein the near-infrared light sensitive substance is a phthalocyanine dye. (19) The pharmaceutical composition according to (18), wherein the phthalocyanine dye is IR700. (20) The pharmaceutical composition according to any one of (15) to (19), which is a pharmaceutical composition for performing multiple light irradiations. (21) The pharmaceutical composition according to any one of (15) to (20), which is used in combination with a pharmaceutical composition having as an active ingredient a complex in which a near-infrared light sensitive substance is bound to a specific binding molecule that binds to a target molecule other than an immune checkpoint molecule. (22) The pharmaceutical composition according to any one of (15) to (21), which is used in combination with a chemotherapeutic agent. (23) A near-infrared photoimmunotherapy targeting a tumor microenvironment, comprising administering to a subject a pharmaceutical composition according to any one of (7) to (22), obtaining a sample from the subject before and after light irradiation, measuring one or more markers in the sample, including B cells, T cells, CD4+ T cells, CD8+ T cells, MDSC, monocytes / macrophages, dendritic cells, G-CSF, IL-6, KC, and MIP-2, confirming the therapeutic effect based on the change in the marker before and after light irradiation, and performing multiple light irradiations when the treatment is determined to be effective. (24) Near-infrared photoimmunotherapy, comprising administering to a subject a pharmaceutical composition targeting a tumor microenvironment according to any one of (7) to (12), and (15) to (20), and a pharmaceutical composition comprising, as active ingredients, a complex in which a near-infrared photosensitive substance is bound to a specific binding molecule that binds to a target molecule other than an immune checkpoint molecule, and then irradiating the subject with light. (25) A combination therapy of near-infrared photoimmunotherapy targeting a tumor microenvironment and chemotherapy, comprising administering to a subject a pharmaceutical composition according to any one of (7) to (13), or (15) to (21), and treating a tumor and ameliorating bone marrow suppression by near-infrared photoimmunotherapy. (26) A near-infrared photoimmunotherapy according to (24) or (25), comprising obtaining a sample from a subject before and after light irradiation, measuring one or more markers in the sample, including B cells, T cells, CD4+ T cells, CD8+ T cells, MDSC, monocytes / macrophages, dendritic cells, G-CSF, IL-6, KC, and MIP-2, confirming the therapeutic effect of the near-infrared photoimmunotherapy targeting immune checkpoint molecules based on the change in the marker before and after light irradiation, and performing multiple light irradiations if the treatment is determined to be effective. [Brief description of the drawings]

[0009] [Figure 1] Preparation of PD-L1-F(ab')2-IR700: (A) PD-L1-F(ab')2 was purified from an anti-mouse PD-L1 antibody, and (B) the results of testing the conjugates prepared by binding IR700 to the purified PD-L1-F(ab')2 and control IgG F(ab')2. [Diagram 2] Figure 2 shows the results of confirming PD-L1 expression in various mouse cancer cells by binding of PD-L1-F(ab')2-IR700. [Diagram 3] Figure 1 shows the effect of NIR-PIT with PD-L1-F(ab')2-IR700 in various mouse cancer cells. Microscopic images showing changes in cells after light exposure. [Figure 4]PD-L1-F(ab')2-IR700 was bound to various mouse cancer cells, and the effect of NIR-PIT was analyzed based on the percentage of dead cells after exposure to near-infrared light. [Diagram 5] Figure 1 shows the effect of NIR-PIT analyzed by luciferase assay. [Figure 6] The effect of NIR-PIT on innate immune activation was analyzed using an ATP assay. [Figure 7] Figure showing the results of investigating the effect of NIR-PIT using a mouse in vivo tumor model. Investigation results using the colon cancer cell line MC38. IVIS images are shown. [Figure 8] Figure showing the effect of NIR-PIT in a mouse in vivo tumor model, showing the results of analysis based on (A) luciferase activity and (B) tumor volume. [Figure 9] FIG. 1 shows the effect of NIR-PIT on survival in a mouse in vivo tumor model. [Figure 10] Figure showing the results of investigating the effect of NIR-PIT using a mouse in vivo tumor model. Results of investigation using the lung cancer cell line LL / 2. (A) IVIS image, (B) luciferase activity, and (C) tumor volume analysis results are shown. [Figure 11] Figure showing the results of investigating the effect of NIR-PIT using a mouse in vivo tumor model. Results of investigation using the prostate cancer cell line Tramp-C2. (A) IVIS image, (B) luciferase activity, and (C) tumor volume analysis results are shown. [Figure 12] Figure showing that NIR-PIT targeting PD-L1 is effective against metastatic sites. (A) IVIS images, (B) luciferase activity, (C) tumor volume, and (D) survival curves are shown. [Figure 13] Figure 1 shows the results of analyzing the growth inhibitory effect on tumor cells re-inoculated on the opposite side after NIR-PIT. (A) A schematic diagram of the experimental schedule. (B) A diagram showing the tumor volume of tumor cells inoculated on the opposite side after NIR-PIT. [Figure 14]Analysis of PD-L1 expression after NIR-PIT. (A) In vitro and (B) in vivo experimental results. [Figure 15] A diagram showing activation of T cells and NK cells within tumors by NIR-PIT targeting PD-L1. [Figure 16] A diagram showing the activation of T cells and NK cells in the blood by NIR-PIT targeting PD-L1. [Figure 17] A diagram showing activation of splenic T cells and NK cells by NIR-PIT targeting PD-L1. [Figure 18] FIG. 1 shows the results of an analysis of blood cell fluctuations using NIR-PIT. [Figure 19] FIG. 1 shows the results of an analysis of cytokines that change with NIR-PIT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As described above, conventional NIR-PIT is a treatment method that targets molecules that are highly expressed on the surface of tumor cells. Patent Document 1 and Non-Patent Document 1 also describe many molecules that are specifically expressed in cancer cells, including EGFR, HER2, PSMA, and CEA. In addition, they also describe targeting molecules involved in immune checkpoints, such as CD25, PD-1, PD-L1, PD-L2, and CTLA-4.

[0011] However, immune checkpoint molecules such as PD-1 / PD-L1 described in the above literature are merely considered as one of the tumor antigens expressed on the surface of cancer cells, similar to molecules such as EGFR. In other words, it is considered as a local therapy targeting cancers in which immune checkpoint molecules such as PD-1 / PD-L1 are highly expressed as target molecules. The present inventor has found that NIR-PIT targeting PD-L1 not only has the conventional effect of killing tumor cells at the site of light irradiation, but also enhances the expression of PD-L1 in tumor cells that have not undergone cell death, induces an immune response, and modifies the cancer microenvironment, thereby synergistically activating anti-tumor immunity. In other words, in addition to the effect of conventional NIR-PIT targeting cancer antigens, it has been found that it can be a treatment method that enhances the therapeutic effect of the anti-PD-L1 antibody itself and produces a therapeutic effect by performing NIR-PIT multiple times, even when PD-L1 expression is low. It has also been found that the induction of an immune response has an effect on tumors at sites where light irradiation has not been performed, that is, it has an effect on metastatic sites. Furthermore, they found that the effect extends to tumors that did not exist at the time of light irradiation, i.e., due to immune memory, and that it is expected to have an effect of preventing recurrence. These effects are completely different in quality from the effects of conventional NIR-PIT, which has a local effect on tumors where the target molecule is highly expressed, and also induce an immune response.

[0012] Considering the mechanism of action of NIR-PIT targeting PD-L1, it is believed that similar effects can be obtained by NIR-PIT targeting molecules involved in immune checkpoints expressed in tumor cells. In addition to PD-L1, such molecules include PD-L2, CD78, B7 (CD80 / CD86), Galectin-9, HVEM, CD137L, B7-H4, B7-H3, CD112, CD155, MHC II, CD200R, VISTA, VSIG3, LSECtin, CEACAM1, A2aR, B7RP1, FGL1, and CD48, which transmit inhibitory signals, and CD70, GITRL, ICODSL, OX40L, 4-1BBL, and CD40L, which transmit activating signals. It goes without saying that this method can also be applied to molecules that will be identified as immune checkpoint molecules in the future.

[0013] Examples of specific binding molecules that bind to immune checkpoint molecules expressed in tumors, including PD-L1, include antibodies and aptamers. Any specific binding molecule may be used as long as it specifically binds to a target molecule, but antibodies are particularly preferred. The antibody is not limited to an intact antibody, and may be any antibody that contains a region that specifically binds to an antigen, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain Fv, a disulfide-stabilized V region fragment (dsFv), a rIgG fragment, a minibody, a diabody, or a peptide containing a CDR. However, in order to suppress side effects due to ADCC (antibody dependent cellular cytotoxicity) activity, it is preferable to use an antibody that does not have an Fc region, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain Fv, or a disulfide-stabilized V region fragment (dsFv). Hereinafter, antibodies that do not have an Fc region, such as a Fab fragment, a Fab' fragment, or a F(ab')2 fragment, are referred to as functional antibody fragments.

[0014] In the following examples, an anti-mouse PD-L1 antibody is used, but as mentioned above, any molecule involved in immune checkpoints whose expression is recognized in tumor cells can be used as a target, not limited to anti-PD-L1 antibodies. Antibodies against human PD-L1 include durvalumab, atezolizumab, and avelumab, which have already been approved and used in treatment in various countries. These antibodies are used in the treatment of malignant melanoma, non-small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, etc., but any tumor with even a small amount of PD-L1 expression, not limited to these cancer types, can be subject to NIR-PIT targeting PD-L1. Furthermore, anti-PD-L1 antibodies to be developed in the future and antibodies against immune checkpoint molecules whose expression is recognized in tumor cells can also be applied to NIR-PIT.

[0015] The complex used in NIR-PIT is made by binding a photosensitizer to the specific binding molecule described above. A photosensitizer is a substance that induces cell death in target cells through a photochemical reaction. To efficiently induce a photochemical reaction, it is necessary to use high-energy photons, i.e., light with a short wavelength. However, on the other hand, it is necessary to use a longer wavelength than ultraviolet light in order to prevent nonspecific damage to the DNA of normal cells surrounding the tumor cells. For this reason, it is preferable to select a near-infrared light-sensitive substance. In addition, in order to be used by linking it to a specific binding molecule such as an antibody, it is necessary to be a small molecule. One compound that meets this condition is a phthalocyanine-based photosensitizer.

[0016] As the phthalocyanine dye, those having an absorption peak at 600 nm to 950 nm, preferably 660 nm to 740 nm, more preferably 680 nm to 720 nm can be suitably used. Particularly preferred phthalocyanine dyes include IR700 (IRDye700DX, LI-COR Biosciences), which is also used in RM-1929 (Patent Document 2). Using the NHS ester of IR700, it can be conjugated to a specific binding molecule by a covalent bond.

[0017] The complex can be administered locally or systemically by any method. Specifically, it can be administered intramuscularly, subcutaneously, intradermally, intravenously, or intraperitoneally by injection into the affected area, or parenterally by inhalation, ointment, patch, application, nasal drops, or eye drops, but it can also be administered orally. Although it can be appropriately selected depending on the target tumor, it is usually preferable to administer the complex systemically by intravenous administration or the like.

[0018] Parenteral formulations typically comprise injectable fluids that include pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, or glycerol as a vehicle, and may contain small amounts of nontoxic auxiliary substances, such as preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0019] The therapeutically effective dose of the complex depends on the target disease, but can be appropriately set according to the disease and symptoms. For example, it can be appropriately set between 0.1 mg and 1000 mg per 60 kg body weight. The dose may vary depending on the administration method, such as intravenous administration, local administration, or intraperitoneal administration, so it may be appropriately set. In addition, the administration may be a single administration or multiple administrations, which may be selected according to the disease, symptoms, and therapeutic effect.

[0020] As shown in the following examples, NIR-PIT using an antibody that binds to PD-L1 enhances PD-L1 expression in tumor cells as a result of treatment. That is, the first treatment can induce PD-L1 expression in tumor cells. Therefore, by performing NIR-PIT multiple times, even if PD-L1 expression is low in the first treatment and a significant effect cannot be obtained, a therapeutic effect can be expected from subsequent light irradiation. Furthermore, since natural immunity is also induced, there is a high possibility that a significant effect can be obtained by multiple treatments. In general, it is said that an effective amount of antibody drugs is maintained in the body for 2 to 3 weeks. Therefore, after one administration, it is possible to obtain a strong effect by performing light irradiation multiple times within 2 to 3 weeks.

[0021] In the case of multiple light irradiations, the interval between irradiations is not particularly limited, but it is preferable to wait for the expression of PD-L1 in tumor cells to be enhanced and for natural immunity to be induced before irradiation. Depending on the tumor, various irradiation schedules can be set, such as daily irradiation, every other day irradiation, or irradiation every few days to one week.

[0022] For near-infrared light irradiation, an appropriate therapeutic dose of the appropriate wavelength may be irradiated using an LED, an LED laser, or light that has passed through a filter. The therapeutic dose is 1 to 1000 J / cm. 2 The dose and irradiation time may be appropriately determined within a range of about 5 seconds to 1 hour. Although irradiation may be performed directly from outside the body, a light guide catheter, an endoscope light guide fiber, a puncture irradiation fiber, a vascular light guide catheter, a drain-indwelling light guide device, or a light irradiation device such as an implantable type, a patch type, or a bracelet type may be used as a device for introducing the light into the diseased area. When the complex is administered systemically by intravenous injection as a pharmaceutical composition, the time required for the complex to accumulate in the lesion may be taken into consideration, and the complex may be irradiated with near-infrared light after accumulation in the lesion. The time required for the complex to accumulate in the lesion is, for example, about 5 minutes to 48 hours. In the case of local administration, light irradiation can be performed in a shorter time after administration than in the case of systemic administration. EXAMPLES

[0023] The present invention will be described in detail below with reference to examples. [Preparation of complex using anti-mouse PD-L1-F(ab')2 and IR700] We used anti-mouse PD-L1 antibody as the specific binding molecule and IR700 as the near-infrared light-sensitive substance to prepare a complex and confirm the effect of NIR-PIT. Since PD-L1 is also expressed in lymphocytes, we purified F(ab')2 and prepared a complex with IR700 to suppress side effects caused by ADCC (Figure 1).

[0024] Anti-mouse PD-L1 antibody (In vivo Mab anti-mouse PD-L1 (B7-H1), BIO XCELL) was cleaved with pepsin, and F(ab')2 was purified (Figure 1(A)). IR700 was bound to the F(ab')2 purified from the anti-mouse PD-L1 antibody and to the F(ab')2 purified from a control IgG (Rat IgG2b Isotype control (anti KLH), BIO XCELL) to prepare a complex. Fluorescence of the IR700-bound complex F(ab')2 was observed when irradiated with near-infrared light (Figure 1(B)). Hereinafter, the complex in which IR700 was bound to the anti-mouse PD-L1 antibody F(ab')2 is referred to as PD-L1-F(ab')2-IR700, and the complex in which IR700 was bound to the control IgG antibody F(ab')2 is referred to as control IgG-F(ab')2-IR700.

[0025] [Effects of NIR-PIT using anti-PD-L1-F(ab')2-IR700 in various cancer cells] 1. Microscopic observation The effect of NIR-PIT was confirmed in various mouse cancer cells using the prepared complex. First, it was confirmed whether PD-L1 was expressed in the mouse cancer cells used. Cells were established by introducing the luciferase gene into mouse colon cancer cells MC38 (obtained from ATCC), mouse lung cancer cells LL / 2 (obtained from ATCC), mouse prostate cancer cells Tramp-C2 (obtained from ATCC), and mouse melanoma cells B16F0 (obtained from ATCC). These cells are called MC38 luc, LL / 2 luc, Tramp-C2 luc, and B16F0 luc, respectively. The PD-L1 expression of each cell was confirmed by binding PD-L1-F(ab')2-IR700 and flow cytometry (Figure 2). Binding of PD-L1-F(ab')2-IR700 was observed in all cancer cell lines of different origins, confirming the expression of PD-L1. Since PD-L1 is expressed across organs, this suggests that NIR-PIT targeting PD-L1 may be broadly applicable to various cancers.

[0026] The effect of NIR-PIT was examined using these cell lines. MC38, LL / 2, and Tramp-C2 cell lines were cultured in 12-well plates at 1 × 10 5 Cells were seeded at 100 cells / well and cultured for 24 hours, after which PD-L1-F(ab')2-IR700 was added at 10μg / ml. After 6 hours, an LED with an emission wavelength of 690nm was used to irradiate 0-128J / cm 2 The images are shown in Fig. 3. The images were taken with a differential interference microscope before and after irradiation with near-infrared light, stained with IR700, propidium iodide (PI), and Hoechst, and the images were overlaid with each stain. 2 In both cell lines, the nuclei were stained with PI after light irradiation, and the Hoechst staining disappeared, indicating that cell death was induced by light irradiation.

[0027] MC38 luc, LL / 2 luc, Tramp-C2 luc, and B16F0 luc cell lines were treated with PD-L1-F(ab')2-IR700 at a concentration of 10 μg / ml and light intensity was varied from 0 to 128 J / cm. 2 The cells were irradiated with light at different intensities up to 10 J / cm, and the proportion of dead cells was analyzed by PI staining (Figure 4). In all cell lines, light irradiation did not induce cell death when PD-L1-F(ab')2-IR700 (APC: Antibody Photosensitizer Conjugate in the figure) was not added. In contrast, when PD-L1-F(ab')2-IR700 was added and light irradiation was performed, the number of PI staining positive cells increased depending on the light intensity, indicating that cell death was induced. However, in the case of PIT against highly expressed tumor antigens such as EGFR, the dose of 16 J / cm was not sufficient. 2 In contrast to the 80% to 100% cell death induction achieved by PD-L1 targeting, NIR-PIT was able to induce cell death, but the effect was limited.

[0028] 2. Luciferase assay We analyzed whether cell death was induced by luciferase activity using the cell lines MC38 luc and LL / 2 luc transfected with the luciferase gene (Figure 5). Cells were seeded under the same conditions as those used for microscopy, and PD-L1-F(ab')2-IR700 was added and exposed to light. After washing with PBS, 200 μl of D-luciferin (150 μg / ml) was added and luciferase activity was measured. In both cell lines, when PD-L1-F(ab')2-IR700 was added and exposed to light, a decrease in luciferase activity was observed depending on the light energy intensity. This indicates that cell death is induced by NIR-PIT. Both analysis methods, PI staining and luciferase assay, showed that cell death was induced by binding PD-L1-F(ab')2-IR700 to cells and irradiating them with near-infrared light. However, to induce the same degree of cell death as PIT against antigens highly expressed in tumors, such as EGFR, stronger light energy is required. Similar results have been obtained in other cell lines, although not shown here.

[0029] 3. Examination of induction of innate immunity by ATP assay MC38 cells were seeded as above, and PD-L1-F(ab')2-IR700 was added. 128 J / cm 2 The cells were irradiated with light at 4°C, and the supernatant was collected and the ATP concentration was measured (Figure 6). In the control, light irradiation only (light only), and addition of PD-L1-F(ab')2-IR700 only (APC only), no intracellular ATP release was observed, but when PD-L1-F(ab')2-IR700 was added and light irradiation was performed (PIT), a significant ATP release was observed. Since it is known that the release of ATP activates innate immunity, the increase in ATP concentration in the culture supernatant is thought to contribute to the induction of innate immunity. Therefore, it is suggested that photoimmunotherapy targeting PD-L1 not only induces cell death but also activates innate immunity.

[0030] [Effects of NIR-PIT in an in vivo model] 1. Study using mouse colon cancer MC38 cells We analyzed the effect of photoimmunotherapy using PD-L1-F(ab')2-IR700 using a mouse in vivo model. The effect of photoimmunotherapy was examined in four groups: a control group in which tumor cells were subcutaneously implanted, a group in which control IgG-F(ab')2-IR700 was injected into the tail vein and then exposed to light (Cont-F(ab')2-IR700 iv + NIR-light), a group in which PD-L1-F(ab')2-IR700 was injected into the tail vein alone (PD-L1-F(ab')2-IR700 iv), and a group in which PD-L1-F(ab')2-IR700 was injected into the tail vein followed by light irradiation (PIT).

[0031] 2 × 10 subcutaneous injections were made into the dorsal rump of 9-week-old mice (C57BL / 6). 6 MC38 luc cells were inoculated into each mouse. Three days later, 100 μg of PD-L1-F(ab')2-IR700 or control IgG-F(ab')2-IR700 was administered via tail vein to each mouse in the antibody-IR700 complex administration group, and 75 J / cm 2 was administered to each mouse one day after antibody administration to the light irradiation group. 2 The day of light irradiation was designated as day 0. The experiment was conducted with 7 to 9 mice in each group.

[0032] The therapeutic effect was evaluated by measuring the estimated tumor volume and luciferase activity of the tumor. The estimated tumor volume was calculated by measuring the long and short diameters of the tumor and multiplying the long diameter by the short diameter. 2 Luciferase activity was measured by intraperitoneally administering D-luciferin (7.5 mg / ml, 200 μl) using an IVIS (registered trademark) imaging system. The luminescence unit measured was radiance, and analysis was performed using Living Image Software (registered trademark).

[0033] Images taken by IVIS are shown in Figure 7. In the control group in which tumor cells were implanted subcutaneously, the group administered control IgG-F(ab')2-IR700 and exposed to light, and the group in which PD-L1-F(ab')2-IR700 was simply injected into the tail vein, there was no attenuation of luciferase activity, and strong luminescence was observed from day 1 to day 3. In contrast, a significant decrease in luciferase activity was observed in the group in which PD-L1-F(ab')2-IR700 was injected into the tail vein and exposed to near-infrared light (PIT).

[0034] Figure 8 shows the time course of the radiance of luciferase activity and the tumor volume. The values ​​before near-infrared light irradiation on Day 0 are set to 1, and the relative changes are shown. In the group that underwent photoimmunotherapy (PIT) in which PD-L1-F(ab')2-IR700 was injected into the tail vein and irradiated with near-infrared light, luciferase activity remained significantly low until the third day of light irradiation, whereas in the control, the group that received control IgG-F(ab')2-IR700 and was irradiated with light, and the group that received only PD-L1-F(ab')2-IR700, no decrease in luciferase activity was observed, and an increase in activity over time, i.e., tumor cell proliferation, was observed (Figure 8(A)). As for tumor volume, in the photoimmunotherapy group, a significant decrease in tumor volume was observed on the first day of light irradiation, and only a gradual increase was observed until the 10th day, and the tumor volume remained almost the same as that on Day 0 even on the 10th day of light irradiation. On the other hand, in the other groups, a 15- to 20-fold increase in tumor volume was observed compared to Day 0 (FIG. 8(B)).

[0035] The results of in vitro experimental systems, microscopic observation using PI staining and luciferase assay, showed that the radiation dose was 75 J / cm 2 This light energy is not sufficient to strongly induce cell death (see Figures 4 and 5). However, in the in vivo experimental system, a significant effect was observed in both luciferase activity and tumor volume. The fact that a stronger effect was observed in the in vivo experimental system than in the in vitro experimental system is thought to be due to the unique effect of targeting immune checkpoint molecules, in addition to the effect of conventional photoimmunotherapy.

[0036] The survival curves for each group are shown (Figure 9). In the photoimmunotherapy group (PIT), a significant extension of survival was observed, with some individuals still surviving after 2 months. These individuals did not experience tumor recurrence over the observation period and survived for several more months. On the other hand, in the control and control IgG-F(ab')2-IR700 groups administered with light irradiation, all individuals died around 20 days after administration. In the group administered with PD-L1-F(ab')2-IR700 and not light irradiation, that is, the group administered with anti-PD-L1 antibody immunotherapy, some individuals survived for about 40 days, although there was no significant difference compared to the control group. Although the administration of anti-PD-L1 antibody is thought to have some effect, no individuals survived for more than 60 days, as seen in the photoimmunotherapy group.

[0037] 2. Study using mouse lung cancer LL / 2 cells As in the experiment using MC38 luc cells, 2 × 10 6 LL / 2 luc cells were inoculated subcutaneously into the dorsal buttocks of mice, and 3 days later PD-L1-F(ab')2-IR700 was injected into the tail vein, followed by light irradiation one day later (photoimmunotherapy group, PIT). As above, the control group was an untreated group in which LL / 2 luc cells were administered subcutaneously. The IVIS images (Figure 10(A)), luciferase activity (Figure 10(B)), and tumor volume (Figure 10(C)) of the photoimmunotherapy group and control are shown over time. The day of light irradiation is designated as day 0.

[0038] In a system using mouse lung cancer cells LL / 2 luc, luciferase activity was barely observed in the PIT-treated group until the third day of light irradiation, and a significant difference was observed compared to the control. In addition, the increase in tumor volume was suppressed, and a significant difference was observed compared to the control until the eighth day of photoimmunotherapy.

[0039] 3. Study using mouse prostate cancer Tramp-C2 cells As above, 2×10 6Tramp-C2 luc cells were inoculated subcutaneously into the dorsal buttocks of mice, and 3 days later, PD-L1-F(ab')2-IR700 was injected into the tail vein, followed by light irradiation one day later (photoimmunotherapy group, PIT). The IVIS images (Figure 11(A)), luciferase activity (Figure 11(B)), and tumor volume (Figure 11(C)) of the photoimmunotherapy group and the control untreated group are shown over time. The day of light irradiation is designated as day 0.

[0040] In a system using mouse prostate cancer cells, Tramp-C2 luc, luciferase activity was barely observed in the PIT-treated group until the third day of light irradiation. In addition, the increase in tumor volume was also suppressed, and a significant difference from the control group was observed up to the sixth day of photoimmunotherapy.

[0041] From the above results, it is recognized that photoimmunotherapy targeting the immune checkpoint molecule PD-L1 expressed in cancer is effective regardless of the type of cancer. Conventional photoimmunotherapy targets cancer markers that are highly expressed on the surface of cancer cells, and cannot be applied to tumors in which the target molecule is not expressed or is expressed at a low level. However, when targeting immune checkpoint molecules, as long as the immune checkpoint molecule is expressed, it is possible to treat any tumor, regardless of the organ it originates from, and it is thought that it can be applied to a larger number of patients. This is a crucial difference from conventional near-infrared photoimmunotherapy.

[0042] [Effects on metastases] We analyzed whether photoimmunotherapy targeting immune checkpoint molecules expressed in cancer would act not only locally at the site of light irradiation, but also at other sites, i.e., metastatic sites. We injected 2 × 10 6 Three days later, 100 μg of PD-L1-F(ab')2-IR700 or control IgG-F(ab')2-IR700 was injected intravenously into the tail, and 1 day later, 75 J / cm was administered to the right side only. 2The day of light irradiation is designated as day 0. IVIS images (Figure 12(A)), luciferase activity (Figure 12(B)), tumor volume up to day 13 (Figure 12(C)), and survival curve (Figure 12(D)) are shown before light irradiation on the day of light irradiation and from day 1 to day 3 after light irradiation. The experiment was conducted with 3 mice in each group.

[0043] In the PD-L1-F(ab')2-IR700-administered group (PD-L1-F(ab')2-IR700), the left side (L) that was not irradiated with light showed a tendency to show a decreased luciferase activity up to the third day compared to the control IgG-F(ab')2-IR700-administered group (Cont-F(ab')2-IR700), but no significant difference was observed (Figure 12(B)). However, in the PD-L1-F(ab')2-IR700-administered group, the left side (L) that was not irradiated with light showed a significant difference in tumor volume on the first, third, and sixth days compared to the control IgG-F(ab')2-IR700-administered group. The fact that NIR-PIT was effective in reducing tumor volume even in tumors that were not irradiated with light indicates that a therapeutic effect also occurs in metastatic sites. In radiation therapy, the abscopal effect is known to have an effect not only on the site irradiated with radiation, but also on distant metastatic sites. Near-infrared photoimmunotherapy targeting molecules involved in immune checkpoints produced an effect that could be called a photoabscopal effect, which was also effective on metastatic lesions. The photoabscopal effect is an effect unique to near-infrared photoimmunotherapy targeting molecules involved in immune checkpoints, and does not occur in conventional near-infrared photoimmunotherapy for highly expressed targets such as EGFR. The therapeutic effect also improved prognosis, and while all individuals in the control IgG-F(ab')2-IR700 group died on day 15, some individuals in the PD-L1-F(ab')2-IR700 group survived until day 25, demonstrating a significant improvement in prognosis.

[0044] [Inhibitory effect on tumor cell proliferation inoculated on the opposite side after NIR-PIT] To further confirm that the tumor regression effect of NIR-PIT is mediated by the immune system, cancer cells were inoculated on the opposite side after NIR-PIT and the effect on proliferation was analyzed. As shown in Fig. 13(A), 2 × 10 6 MC38 luc cells were inoculated, and 3 days later, 100 μg of PD-L1-F(ab')2-IR700 or control IgG-F(ab')2-IR700 was administered via tail vein (day -1). One day later, 75 J / cm 2 The next day, the cells were irradiated with 2 × 10 6 MC38 luc cells were inoculated subcutaneously into the left dorsal buttock (day 1), and the tumor volume on the left dorsal buttock was measured over time (Figure 13(B)). Compared to the group in which the control IgG-F(ab')2-IR700 was injected into the tail vein, the group in which NIR-PIT was performed showed suppression of the proliferation of tumor cells inoculated after NIR-PIT in all individuals. Since there was suppression of proliferation of tumor cells inoculated after NIR-PIT, it is suggested that NIR-PIT targeting PD-L1 is mediated by an immune memory response. Therefore, it can be expected to prevent tumor recurrence.

[0045] [Induction of immune response and expression of targeted molecules] Since photoimmunotherapy targeting PD-L1 was effective even against tumors that had not been irradiated with light, it was suggested that the activation of antitumor immunity may be induced. Therefore, we analyzed the change in PD-L1 expression by photoimmunotherapy targeting PD-L1 using both in vitro and in vivo systems.

[0046] Mouse colon cancer cells MC38 luc were cultured in a 12-well plate at 1 × 10 5 / well, and 24 hours later, PD-L1-F(ab')2-IR700 was added at 10μg / mL, and 6 hours later, 32J / cm 2After light irradiation, PD-L1 expression was evaluated by flow cytometry 5 days later. PD-L1 was detected using an anti-PD-L1 antibody. As shown in Figure 14(A), no change in mean fluorescence intensity was observed in the control (no PD-L1-F(ab')2-IR700 added, no light irradiation), light irradiation only (light), and PD-L1-F(ab')2-IR700 added only (APC only). However, the PD-L1-F(ab')2-IR700 added, light irradiation group, i.e., the group that underwent photoimmunotherapy targeting PD-L1 (PIT), showed a significant decrease in mean fluorescence intensity compared to the control. This indicates that in an in vitro system, cells with high PD-L1 expression were killed by photoimmunotherapy, and only cells with low expression selectively survived.

[0047] Next, the effect of photoimmunotherapy on PD-L1 expression was analyzed in an in vivo system. 6 MC38 luc cells were inoculated, and 3 days later, 100 μg of PD-L1-F(ab')2-IR700 was administered via tail vein. One day later, 32 J / cm 2 The irradiation energy was 32 J / cm. 2 is the light intensity of 75 J / cm at which tumor regression was observed in the above example. 2 The light energy was about half that of the normal light energy. This light energy is thought to be the light energy that weakly induces cell death and causes inflammation in the tumor area. Seven days after light irradiation, the mice were euthanized, tumor cells were extracted, and PD-L1 expression was analyzed using flow cytometry as in the in vitro system (Figure 14(B)). Compared to the control group that did not receive photoimmunotherapy, the photoimmunotherapy group showed a significant increase in PD-L1 expression in tumor cells. It is thought that NIR-PIT using PD-L1-F(ab')2-IR700 induced strong inflammation at the tumor site, inducing PD-L1 expression in the remaining tumor. This can be said to be the immunoediting function of this treatment.

[0048] NIR-PIT targeting PD-L1 induces the expression of PD-L1 in tumors. In other words, it acts negatively as a tumor immunity mechanism. However, it has been shown that by inducing the expression of PD-L1, even in tumors where PD-L1 expression is not expressed or is low, NIR-PIT targeting PD-L1 enhances PD-L1 expression. If photoimmunotherapy using PD-L1-F(ab')2-IR700 is performed again after the expression enhancement, it becomes possible to further induce cell death of tumor cells that could not be killed by the initial treatment. In addition, it is thought that the synergistic effect of the immune checkpoint inhibitory action of PD-L1-F(ab')2-IR700 itself can be expected by enhancing the expression. The enhancement of cancer immunity by these positive feedback mechanisms can be said to be a unique and noteworthy mechanism of this treatment.

[0049] The effect of enhancing cancer immunity by NIR-PIT targeting immune checkpoint molecules is expected to be more effective when combined with other NIR-PIT. For example, by combining NIR-PIT targeting a target highly expressed in tumors such as EGFR with NIR-PIT targeting PD-L1, tumor cells highly expressing EGFR in tumors irradiated with near-infrared light can be killed first. Furthermore, tumor cells that do not express EGFR or express it at a low level, as well as tumor cells in metastatic sites, can be killed by NIR-PIT targeting PD-L1. Furthermore, since PD-L1 expression is induced by multiple treatments, tumor cells that do not express EGFR due to mutations can also be completely removed. As a result, remission may be achieved even for tumors that may recur with conventional NIR-PIT.

[0050] To further analyze the effect of photoimmunotherapy targeting PD-L1 on tumor immunity, we measured activation markers of CD8(+) T cells and NK cells after photoirradiation. 6 MC38 luc cells were inoculated into the mice. Three days later, 100 μg of PD-L1-F(ab')2-IR700 was administered via tail vein, and 1 day later, 75 J / cm 2The mice were euthanized 1.5 hours later, and the expression of IFNγ, IL-2, CD69, CD25, and CD107a in CD8(+) T cells (CD3+CD8+) and NK cells (CD3-NK1.1+) in the tumor (Figure 15), blood (Figure 16), and spleen (Figure 17) was analyzed by flow cytometry.

[0051] The antibodies used in the analysis are as follows. CD4:anti-mouse CD4 cloneRM4-5, CD8:anti-mouse CD8a clone53.67, CD3:anti-mouse CD3e clone145-2c11, NK:anti-mouse NK1.1 clonePK136, IFNγ:anti-mouseIFNγ cloneXMG1.2, IL-2:anti-mouse IL-2 cloneJES6-5H4, CD69:anti-mouse CD69 cloneH1.2F3, CD25:anti-mouse CD25 clone eBio3C7, CD107a:anti-mouse CD107a clone 1D4B.

[0052] Within the tumor, the expression of IFNγ was significantly increased in NK cells, and the expression of CD69 and CD25 was significantly increased in CD8(+) T cells and NK cells by photoimmunotherapy (Figure 15). In addition, in the blood, the expression of IFNγ and IL-2 was significantly increased in NK cells, and the expression of CD69 was significantly increased in CD8(+) T cells and NK cells (Figure 16). Furthermore, in the spleen, the expression of IFNγ was significantly increased in NK cells, and the expression of CD69 was significantly increased in CD8(+) T cells and NK cells. Although there was no significant difference in the expression of other activation markers, an increase in expression was observed after photoimmunotherapy, making it clear that natural immunity was activated. In particular, the activation of CD8(+) T cells and NK cells in the blood is in line with the regression of tumor cells in metastatic sites and the suppression of tumor growth inoculated on the opposite side after NIR-PIT, as shown above.

[0053] [Marker Consideration] Early determination of whether a certain treatment is effective for a patient is important not only in providing the most appropriate treatment to the patient, but also from the perspective of medical economics. Therefore, we investigated markers that can determine whether a patient will respond to photoimmunotherapy targeting PD-L1. We performed NIR-PIT targeting PD-L1 and comprehensively analyzed the markers that change after photoirradiation by CyTOF analysis of peripheral blood. 2 × 10 6 MC38 luc cells were inoculated, and 3 days later, 100 μg of PD-L1-F(ab')2-IR700 was administered via tail vein, and 1 day later, 75 J / cm 2 Blood samples were collected before, 6 hours after, and 24 hours after light irradiation and analyzed by CyTOF (Figure 18).

[0054] B cells, T cells, CD4+ T cells, and CD8+ T cells were significantly decreased both 6 and 24 hours after light irradiation compared to before irradiation (naive). It is thought that these cells migrated to the light-irradiated tumor area, which is why their numbers in the blood decreased. On the other hand, MDSCs (myeloid-derived suppressor cells) were significantly increased 6 hours after light irradiation, and monocytes / macrophages (Mono / MΦ) and dendritic cells (DCs) were significantly increased 24 hours after light irradiation compared to before light irradiation.

[0055] Any blood cells that showed a significant difference can be used as a marker to confirm the therapeutic effect of photoimmunotherapy targeting PD-L1. In particular, since MDSCs are hardly found in the blood before the start of photoimmunotherapy, if they are used as an indicator of therapeutic effect, it is possible to distinguish patients who will benefit from photoimmunotherapy. In other words, if an increase in MDSCs is observed after photoimmunotherapy, it can be predicted that the patient will benefit from the treatment.

[0056] In photoimmunotherapy targeting PD-L1, it is desirable to perform multiple phototherapy sessions, since it is predicted that PD-L1 expression will increase after the first treatment, further enhancing the therapeutic effect. Since antibody drugs usually remain in the body for 2 to 3 weeks, multiple light irradiations within this period can be expected to be effective in the tumor and metastatic areas that have been irradiated with light. In humans, the usual dose is 300 J / cm2. 2 If the effect is weak, stronger light irradiation may be performed. The number of treatments, treatment intervals, and irradiation energy can be appropriately set using the changes in blood cell markers, including MDSC, after light irradiation as indicators.

[0057] Furthermore, changes in serum cytokines were analyzed by ELISA using a Cytokine Array. The amounts of G-CSF (granulocyte-colony stimulating factor), IL-6, KC (keratinocyte-derived chemokines), and MIP-2 (macrophage inflammatory protein-2) were measured (Figure 19). An increase was observed in all of these levels 6 hours after photoimmunotherapy. The increase in these cytokines may be used as an indicator of treatment.

[0058] The blood cell and cytokine markers that were shown to change as a result of PD-L1-targeting photoimmunotherapy can all be measured in peripheral blood, and the results can be confirmed as early as six hours, making this a very effective means of confirming the therapeutic effects of PD-L1-targeting photoimmunotherapy.

[0059] In addition, an increase in G-CSF was observed with NIR-PIT targeting PD-L1. Since G-CSF has the effect of stimulating the bone marrow and increasing the number of blood cells, it is expected to have the effect of suppressing the side effects of chemotherapy that frequently cause bone marrow suppression. Bone marrow suppression is a side effect in which bone marrow stem cells are damaged, and the function of producing blood cell components such as white blood cells, platelets, and red blood cells does not work normally, resulting in a decrease in blood cell components. Specific symptoms include infections, anemia, and bleeding, but they are difficult to notice, and if infections are complicated, problems such as being unable to continue treatment can occur. Bone marrow suppression can appear as a side effect of almost all anticancer drugs. Necrosis-inducing anticancer drugs, which are particularly common as a side effect of drugs classified as necrosis-inducing anticancer drugs, include taxane drugs such as paclitaxel and docetaxel, anthracycline drugs such as daunorubicin and epirubicin, vinca alkaloid drugs such as vinblastine and vincristine, and anticancer antibiotics such as mitomycin C. In addition, some molecular targeted drugs and radiation therapy may cause bone marrow suppression depending on the irradiation site. NIR-PIT, which targets PD-L1, increases G-CSF and increases blood cell counts, so by combining it with these chemotherapy drugs or radiation therapy, which may cause bone marrow suppression, it may be possible to suppress the side effect of bone marrow suppression.

[0060] Immune checkpoint molecules such as PD-L1 that are specifically expressed in tumor cells are expressed to some extent in any cancer, regardless of the type of cancer or organ. Therefore, NIR-PIT, which targets immune checkpoint molecules expressed in tumor cells, can be applied to any cancer. Furthermore, as shown in the above example, the complex of an anti-PD-L1 antibody and a photosensitizer induces PD-L1 expression after the first NIR-PIT, so that a therapeutic effect can be expected even in cases with low PD-L1 expression by multiple light irradiation. Conventional NIR-PIT is not effective unless the target molecule is highly expressed on the cell surface, and there have been many cases in which treatment has been difficult. However, NIR-PIT, which targets PD-L1, is thought to be applicable to many cases because it induces PD-L1 expression by treatment even if PD-L1 expression is low at the start of treatment. In addition, unlike conventional NIR-PIT, it not only induces cell death at the site of light irradiation, but also induces natural immunity, which gives it an excellent systemic therapeutic effect, i.e., an effect on metastases and an effect of preventing recurrence. In addition, since it is expected to enhance tumor immunity, it can also be used as a method to enhance the effects of other immune checkpoint inhibitors.

[0061] In addition, when used in combination with NIR-PIT, which targets molecules highly expressed in tumor cells such as EGFR, it is possible to induce cell death in the target molecule and also to be effective against tumor cells in which gene mutations have occurred in the target molecule, in which the target molecule is not expressed, or which have been mutated so that antibodies cannot bind to the target molecule. Even in tumor cells in which cell death could not be induced by conventional NIR-PIT targeting highly expressed molecules, it is possible to induce cell death by combining it with NIR-PIT targeting immune checkpoint molecules, which is expected to improve prognosis and prevent recurrence. Furthermore, since an increase in G-CSF is observed in NIR-PIT targeting PD-L1, it is expected to have the effect of increasing blood cell counts, and therefore it is expected to have the effect of reducing side effects by combining it with chemotherapy or radiation therapy that causes bone marrow suppression.

Claims

1. A test method for confirming the effect of a pharmaceutical composition that enhances tumor immunity in a subject, comprising: The pharmaceutical composition is a pharmaceutical composition for use in near-infrared photoimmunotherapy, Measure one or more markers of B cells, T cells, CD4+ T cells, CD8+ T cells, MDSCs (myeloid-derived suppressor cells), monocytes / macrophages, dendritic cells, G-CSF (granulocyte colony-stimulating factor), IL-6, KC (keratinocyte-derived chemokine), and MIP-2 (macrophage inflammatory protein 2) in peripheral blood obtained from the subject before and after near-infrared light irradiation; By comparing the values ​​before and after near-infrared light exposure, Decrease in B cells, T cells, CD4+ T cells, CD8+ T cells, or If any one or more of increases in MDSC, monocytes / macrophages, dendritic cells, G-CSF, IL-6, KC, and MIP-2 are observed, Determine whether the treatment is effective A testing method to confirm the effectiveness of treatment by observing changes in markers before and after near-infrared light irradiation.

2. The pharmaceutical composition comprises: The method for testing according to claim 1, which specifically binds to an immune checkpoint molecule expressed in tumor cells.

3. The method according to claim 2, wherein the immune checkpoint molecule is PD-L1.

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