Cancer treatment agent

Abstract

The purpose of the present invention is to treat cancer while suppressing recurrence of cancer.　The present invention provides a cancer treatment agent containing a substance that binds to ANTXR2, wherein the average fluorescence intensity indicating bonding, to ANTXR1, of the substance that binds to ANTXR2 when said bonding is measured using the average fluorescence intensity is equal to or less than 0.65 times the average fluorescence intensity indicating bonding, to ANTXR2, of the substance that binds to ANTXR2 when said bonding is measured using the average fluorescence intensity.

Description

Cancer treatment

[0001] The present invention relates to a cancer therapeutic agent, and more specifically to a cancer therapeutic agent that can suppress the recurrence of cancer.

[0002] Pancreatic cancer has one of the poorest prognoses of all cancers. In Japan, the annual death toll is 37,677 (2020), with a five-year survival rate of only 8.5%. Pancreatic cancer is difficult to detect early because it rarely shows symptoms. At the time of detection, surgery is often not feasible, and chemotherapy remains the mainstay of treatment. However, the effectiveness of current standard treatments (e.g., gemcitabine, TS-1, FOLFIRINOX) is extremely limited, making the development of innovative, curative new therapeutic drugs highly desirable. Furthermore, methods to prevent cancer recurrence are also needed.

[0003] Regarding the relationship between ANTXR2 and pancreatic cancer, for example, Non-Patent Document 1 describes that the expression of ANTXR2 increases in pancreatic ductal adenocarcinoma.

[0004] However, Non-Patent Document 1 does not describe or suggest any experimental results using a substance that binds to only one of ANTXR1 and ANTXR2, nor does it describe or suggest anything about the relationship with cancer recurrence.

[0005] Patent Document 1 describes that ANTXR1 and ANTXR2 are hardly expressed in normal tissues but are specifically expressed only in pancreatic cancer tissues, and describes the identification of a PA63 ligand that specifically targets ANTXR1 and ANTXR2 (e.g., paragraph

[0006] of Patent Document 1). Patent Document 1 also describes the development of a CAR (chimeric antigen receptor) specific to ANTXR1 or ANTXR2 (paragraph

[0032] of Patent Document 1). Patent Document 1 also describes that the effect of CAR-T cells containing the PA63 ligand was confirmed to be highly toxic to cancer cells expressing ANTXR1 or ANTXR2, and therefore, it is expected that they will have an anticancer effect in both carcinomas expressing ANTXR1 and ANTXR2 (paragraph

[0135] of Patent Document 1). Patent Document 1 also describes that the efficacy of CAR-T cells was confirmed in an animal model using solid carcinomas expressing ANTXR1 or ANTXR2 (paragraph

[0138] of Patent Document 1).

[0006] However, in the examples of Patent Document 1, only anthrax toxin (protective factor PA63) is used to specifically target ANTXR1 and ANTXR2, and PA63 binds to both ANTXR1 and ANTXR2 (paragraph

[0104] of Patent Document 1), and no experimental results were shown using a substance that binds to only one of ANTXR1 and ANTXR2.

[0007] Patent Document 1 describes that solid cancers can be treated using the anti-ANTXR chimeric antigen receptor (CAR)-T cells of the invention of Patent Document 1, and that by administering chimeric antigen receptor (CAR)-T cells to solid cancer patients, particularly pancreatic cancer patients, for whom administration of anticancer drugs is ineffective, it is possible to limit drug administration and enable efficient, safe, and customized prevention or treatment of solid cancers.

[0008] However, Patent Document 1 does not state or suggest anything about the relationship with cancer recurrence.

[0009] Special Publication No. 2022-511885

[0010] British Journal of Surgery, Volume 108, Issue Supplement_5, July 2021, znab282.006Nat Med. 2011 Nov 6; 17(12):1685-91.

[0011] Therefore, an object of the present invention is to treat cancer while suppressing the recurrence of cancer.

[0012] We generated malignant pancreatic cancer organoids by introducing four genetic mutations frequently observed in human pancreatic cancer (K-Ras mutation, p16 deletion, p53 deletion, and Smad4 deletion) into mouse pancreatic ductal epithelial cells. These pancreatic cancer organoids were orthotopically transplanted into normal mice, and the tumors formed were analyzed by single-cell RNA sequencing after anticancer drug administration. We unbiasedly identified anticancer drug-resistant cancer stem cells using CytoTRACE analysis (a stem cell prediction algorithm). We then extracted genes whose expression patterns correlated with stemness scores and found that anthrax toxin receptor type II (ANTXR2) was most specifically expressed in pancreatic cancer stem cells. ANTXR2 is a transmembrane protein (type I) that binds to type VI collagen and plays an essential role in its endocytic degradation. Pancreatic cancer is known to be highly fibrotic, and type VI collagen fibers are particularly prominent within tumors. The inventors hypothesized that pancreatic cancer stem cells (PCSCs) might secure the space necessary for growth and metastasis by degrading surrounding type VI collagen fibrils via ANTXR2 (highly expressed by the PCSCs themselves). Indeed, epidemiological studies have shown that pancreatic cancer patients with high ANTXR2 expression have a significantly poorer prognosis. Therefore, we transfected pancreatic cancer organoids with the diphtheria toxin receptor gene downstream of the ANTXR2 gene and transplanted them into mice. After tumor formation was complete, we administered diphtheria toxin to ablate ANTXR2-expressing cells, resulting in rapid disappearance of the pancreatic cancer. In other words, ANTXR2 is a factor that has been validated in vivo as a therapeutic target for cancer control.

[0013] While ablating only ANTXR2-expressing cells may, in principle, cure pancreatic cancer, the question remains: how to put this into clinical practice? Because ANTXR2 is a cell membrane protein, antibody-based therapy is considered the first choice. However, ablating ANTXR2+ cells throughout the body is theoretically likely to have a biological effect similar to that of anthrax toxin and may potentially cause fatal side effects. To avoid this, a method that meets both the following requirements is needed: (1) ablation of specific cell populations using antibodies against cell surface antigens, and (2) a localized effect. One such method is photoimmunotherapy, which utilizes a conjugate of a cancer antigen-specific antibody and the phototoxic substance IR700 (see, for example, Non-Patent Document 2). In this method, cancer cells are specifically labeled by administering an anti-cancer cell antibody-IR700 conjugate, and then irradiated with near-infrared light to induce IR700 aggregate formation, thereby physicochemically destroying the cancer cell membrane. The advantage of this method is that it can ablate only antigen-expressing cells in the area irradiated with near-infrared light, without spreading cytotoxic effects to areas not irradiated with near-infrared light. Photoimmunotherapy has already been shown to be effective in clinical trials for some cancer types, such as head and neck cancer, and is expected to be a minimally invasive, highly effective next-generation cancer treatment. However, little progress has been made in identifying useful cancer-specific antigens for targeting many cancer types, including pancreatic cancer, which has been a major obstacle to expanding the application of photoimmunotherapy. The inventors have discovered that ANTXR2 is significantly more highly expressed in pancreatic cancer than in normal pancreas, suggesting its usefulness as a target antigen for photoimmunotherapy.

[0014] Specifically, the inventors inserted the diphtheria toxin (DT) receptor gene into the ANTXR2 gene and administered DT to ablate ANTXR2-expressing cells. This ablation almost completely eliminated pancreatic cancer in mice, and recurrence did not occur even after DT was discontinued. Furthermore, it was revealed that the expression levels of ANTXR2 and type VI collagen correlated with prognosis even in severely fibrotic scirrhous gastric cancer, and methods such as photoimmunotherapy using anti-ANTXR2 antibodies were found to be effective against scirrhous gastric cancer.

[0015] Accordingly, the present invention provides the following. [Aspect 1] A cancer therapeutic agent comprising a substance that binds to ANTXR2, wherein, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating the binding is 0.65 times or less relative to the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2. [Aspect 2] The cancer therapeutic agent according to Aspect 1, wherein, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating the binding is 0.5 times or less relative to the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2. [Aspect 3] The cancer therapeutic agent according to Aspect 1, wherein, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.1 times or less relative to the mean fluorescence intensity indicating said binding when the binding of the substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity. [Aspect 4] The cancer therapeutic agent according to Aspect 1, wherein, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.05 times or less relative to the mean fluorescence intensity indicating said binding when the binding of the substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity. [Aspect 5] The cancer therapeutic agent according to Aspect 1, wherein the substance that binds to ANTXR2 does not substantially bind to ANTXR1. [Aspect 6] The cancer therapeutic agent according to Aspect 1, wherein the cancer is pancreatic cancer, colorectal cancer, low-grade glioma, gastric cancer, head and neck cancer, renal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or germ cell tumor. [Aspect 7] The cancer therapeutic agent according to Aspect 1, wherein the substance that binds to ANTXR2 comprises an antibody, an antibody-like molecule, or a cyclic peptide. [Aspect 8] The cancer therapeutic agent according to Aspect 7, wherein the antibody-like molecule is an scFv or a nanobody. [Aspect 9] The cancer therapeutic agent according to Aspect 1, wherein the substance that binds to ANTXR2 comprises a substance that damages cells.[Aspect 10] The cancer therapeutic agent according to Aspect 9, wherein the substance for damaging cells is a substance for photoimmunotherapy. [Aspect 11] The cancer therapeutic agent according to Aspect 10, wherein the substance for photoimmunotherapy is IR700. [Aspect 12] The cancer therapeutic agent according to Aspect 9, wherein the substance for damaging cells is a drug in an antibody-drug conjugate. [Aspect 13] The cancer therapeutic agent according to Aspect 9, wherein the substance for damaging cells is a substance for antibody-dependent cellular cytotoxicity. [Aspect 14] The cancer therapeutic agent according to Aspect 9, wherein the substance for damaging cells is a substance for complement-dependent cytotoxicity. [Aspect 15] The cancer therapeutic agent according to Aspect 9, wherein the substance for damaging cells is a neutralizing antibody. [Aspect 16] The cancer therapeutic agent according to Aspect 15, wherein the neutralizing antibody is a neutralizing antibody that inhibits binding to type 6 collagen. [Aspect 17] The cancer therapeutic agent according to Aspect 1, which is for damaging cancer stem cells. [Aspect 18] The cancer therapeutic agent according to Aspect 17, wherein the cancer stem cells are pancreatic cancer stem cells. [Aspect 19] The cancer therapeutic agent according to Aspect 17, wherein cancer stem cells express ANTXR2. [Aspect 20] The cancer therapeutic agent according to Aspect 19, wherein the expression level of ANTXR2 RNA in cancer stem cells is 1.5-fold or more relative to non-cancer stem cells. [Aspect 21] A method for treating cancer, comprising the step of administering to a subject a substance that binds to ANTXR2, wherein the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR1 is 0.65-fold or less the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2. [Aspect 22] The method according to Aspect 21, wherein the substance that binds to ANTXR2 comprises a substance that damages cells, and the substance that damages cells is a substance for photoimmunotherapy, and wherein the method comprises the step of irradiating a cancer-affected area in a subject that has been administered the substance that damages ANTXR2 with near-infrared light. [Aspect 23] A pharmaceutical composition for use in treating cancer, comprising a substance that binds to ANTXR2, wherein the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR1 is 0.65 times or less the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2.[Aspect 24] Use of a substance that binds to ANTXR2 in the manufacture of a pharmaceutical composition for use in the treatment of cancer, wherein the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR1 is 0.65 times or less the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2.

[0016] According to the present invention, cancer can be treated while suppressing the recurrence of cancer.

[0017] Figure 1 shows the change in pancreatic cancer tumor burden over time when the control group received phosphate-buffered saline (PBS) and the treatment group received gemcitabine. Figure 2 illustrates the method for recovering pancreatic cancer cells from tumors in the model mice created in Figure 1. Figure 3 shows the results of single-cell RNA-seq analysis of pancreatic cancer cells recovered from mouse pancreatic tumors. Figure 4 shows the results of applying the stem cell prediction algorithm CytoTRACE to the results in Figure 3. Figure 5 shows the results of differential gene expression analysis between pancreatic cancer stem cells and other pancreatic cancer cells. Figure 6 shows the results of a correlation analysis between the expression levels of 37 genes highly expressed in pancreatic cancer stem cells and overall survival in human pancreatic cancer patients. Figure 7 shows the overall survival of human pancreatic cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the pancreatic cancer dataset from The Cancer Genome Atlas (TCGA). Figure 8 shows the plot of Antxr2 expression levels in individual cells on single-cell RNA-seq data from mouse pancreatic cancer cells. Figure 9 illustrates the method for generating pancreatic cancer organoids expressing the diphtheria toxin receptor (DTR) and tdTomato fluorescent protein in an Antxr2 gene expression-dependent manner. Figure 10 shows the flow cytometric analysis of Antxr2DTR-tdTomato organoids cultured in vitro, quantifying the proportion of Antxr2-positive cells. Figure 11A shows brightfield images comparing the organoid formation efficiency of Antxr2-positive cells (tdTomato-positive) and Antxr2-negative cells (tdTomato-negative) shown in Figure 10. Figure 11B shows the quantification results of the brightfield images shown in Figure 11A. Figure 12A shows brightfield images of Antxr2-positive cells in in vitro cultured pancreatic cancer organoids after ablation. Figure 12B shows the quantification results of the brightfield images shown in Figure 12A. Figure 13 shows the results of flow cytometry analysis of pancreatic cancer cells in a mouse pancreatic cancer orthotopic transplant model, and quantification of the proportion of Antxr2-positive cells.Figure 14 shows fluorescent images of tumor tissue sections harvested from the mouse pancreatic cancer orthotopic transplant model shown in Figure 13. Figure 15A shows bright-field images comparing the organoid formation efficiency of Antxr2-positive pancreatic cancer cells (tdTomato-positive) and Antxr2-negative pancreatic cancer cells (tdTomato-negative) shown in Figure 13. Figure 15B shows the quantification results of the bright-field images shown in Figure 15A. Figure 16A shows tumor-bearing mouse models. Figure 16A shows the time course of pancreatic cancer tumor burden after administration of phosphate-buffered saline (PBS) to the control group and diphtheria toxin to the ablation group. Figure 16B is a graph showing the time course of tumor size shown in Figure 16A. Figure 17 shows the survival curves for the mice in Figures 16A and B. Figure 18A shows tumor-bearing mouse models. Figure 18A shows the change in pancreatic cancer tumor volume over time when phosphate-buffered saline (PBS) was administered to the control group and diphtheria toxin was administered to the ablation group. Figure 18B is a graph showing the change in tumor size over time in Figure 18A. Figure 19A shows a mouse model bearing a tumor. Figure 19A shows the change in pancreatic cancer tumor volume over time when phosphate-buffered saline (PBS) was administered to the control group and diphtheria toxin was administered to the ablation group. Figure 19B is a graph showing the change in tumor size over time in Figure 19A. Figure 20 shows the overall survival of human colorectal cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the colorectal cancer (colon cancer and rectal cancer) dataset from The Cancer Genome Atlas (TCGA). Figure 21 shows the overall survival of human low-grade glioma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the low-grade glioma dataset from TCGA (The Cancer Genome Atlas).Figure 22 shows the overall survival of human gastric cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the gastric cancer dataset from The Cancer Genome Atlas (TCGA). Figure 23 shows the overall survival of human head and neck cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the head and neck cancer dataset from The Cancer Genome Atlas (TCGA). Figure 24 shows the overall survival of human papillary renal cell carcinoma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the papillary renal cell carcinoma dataset from The Cancer Genome Atlas (TCGA). Figure 25 shows the overall survival of human lung adenocarcinoma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the lung adenocarcinoma dataset from The Cancer Genome Atlas (TCGA). Figure 26 shows the overall survival of human squamous cell lung cancer patients, divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2), in the lung squamous cell carcinoma dataset from The Cancer Genome Atlas (TCGA). Figure 27 shows the overall survival of human testicular germ cell tumor patients, divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2), in the testicular germ cell tumor dataset from The Cancer Genome Atlas (TCGA). Figure 28 shows the gating conditions used in flow cytometry analysis of HEK293T cells. Figure 29 shows HEK293T cells and hANTXR1. Venus HEK293T cells, hANTXR2 VenusFigure 28 shows the results of flow cytometry analysis of HEK293T cells. Figure 30 shows brightfield images of in vitro cultured human pancreatic cancer cell line (MIA PaCa-2) subjected to photoimmunotherapy targeting ANTXR2. Figure 31 shows the quantification results of the brightfield images shown in Figure 30. Figure 32 shows that photoimmunotherapy is specific to ANTXR2. Figure 33 shows that photoimmunotherapy is effective on patient-derived organoids (PDO). Figure 34 shows that ANTXR2 deficiency strongly inhibits tumor growth.

[0018] The present invention provides a cancer therapeutic agent comprising a substance that binds to ANTXR2, wherein when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating the binding is 0.65 times or less compared to the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2, when the binding of the substance that binds to ANTXR2 is measured by mean fluorescence intensity.

[0019] ANTXR2, for example, is the anthrax toxin receptor type II. For example, ANTXR2 is a 53 kDa transmembrane protein (type I) that binds to type VI collagen and is required for its endocytic degradation. Genetic deficiency of ANTXR2 causes hyaline fibrillary storage disease (type VI collagen storage disease). It is known that ANTXR2 binds to anthrax toxin and is required for the toxin to enter cells. An example of a specific nucleic acid sequence for ANTXR2 has been published by NCBI (NCBI Reference Sequence: NM_058172.5).

[0020] Examples of ANTXR2 include animal ANTXR2. Examples of animals include vertebrates as well as invertebrates. Examples of vertebrates include fish, amphibians, reptiles, birds, and mammals. Examples of mammals include humans, mice, rats, cows, pigs, monkeys, dogs, and cats. Examples of invertebrates include arthropods such as insects, crustaceans, arachnids, and myriapods, as well as mollusks. Preferred animals are mammals, and preferred mammals are humans. That is, ANTXR2 is preferably mammalian ANTXR2, and more preferably human ANTXR2.

[0021] Examples of ANTXR2 include the ANTXR2 gene and expression products of the ANTXR2 gene. Examples of expression products of the ANTXR2 gene include mRNA of the ANTXR2 gene and ANTXR2 protein. ANTXR2 is preferably the ANTXR2 protein.

[0022] The substance that binds to ANTXR2 is preferably a substance that specifically binds to ANTXR2. The substance that binds to ANTXR2 includes, for example, an antibody, an antibody-like molecule, a cyclic peptide, a ligand, etc. The substance that binds to ANTXR2, including an antibody, an antibody-like molecule, a cyclic peptide, a ligand, etc., may consist of an antibody, an antibody-like molecule, a cyclic peptide, a ligand, etc.

[0023] Examples of antibodies and antibody-like molecules include polyclonal antibodies, monoclonal antibodies, multivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, single-chain antibodies, nanobodies, heavy-chain antibodies, Fv, Fab, F(ab'), F(ab')2, Fc, Fc fusion proteins, bispecific antibodies, heavy chains (H chains), light chains (L chains), single-chain Fv (scFv), sc(Fv)2, tandem scFv, disulfide-linked Fv (sdFv), diabodies, single-chain diabodies (scDb), tribodies, triabodies, antibody-like molecule targeting peptides (microantibodies), single-domain antibodies (sdAb), etc. Antibodies and antibody-like molecules may be, for example, Fc-containing proteins such as Fc fusion proteins having an Fc region, or minibodies.

[0024] Examples of cyclic peptides include natural cyclic peptides and non-natural cyclic peptides. Natural cyclic peptides include naturally occurring cyclic peptides and cyclic peptides composed of natural amino acids, while non-natural cyclic peptides include cyclic peptides artificially synthesized using non-natural amino acids. Examples of cyclic peptides include cyclic peptides selected by mRNA display methods such as the RaPID method and the TRAP method, or phage display methods.

[0025] Examples of the ligand include anthrax toxin.

[0026] The cancer therapeutic agent provided by the present invention includes a substance that binds to ANTXR2, but may consist solely of a substance that binds to ANTXR2. That is, the cancer therapeutic agent provided by the present invention may include a substance other than the substance that binds to ANTXR2, but may not necessarily include a substance other than the substance that binds to ANTXR2.

[0027] Examples of cancer include pancreatic cancer, colorectal cancer (colon cancer and rectal cancer), low-grade glioma, gastric cancer, head and neck cancer, renal cancer, lung adenocarcinoma, lung squamous cell carcinoma, germ cell tumor, etc. Cancer also includes cancer accompanied by tissue fibrosis.

[0028] Examples of cancer therapeutic agents include drugs for treating cancer as well as drugs for preventing cancer. Examples of drugs for treating cancer include drugs for completely curing cancer, drugs for remission of cancer, and drugs for improving cancer prognosis. Examples of drugs for improving cancer prognosis include drugs for suppressing cancer recurrence.

[0029] In the cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating the binding is, for example, 0.65 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0.1 times or less, 0.09 times or less, 0.08 times or less, 0.07 times or less, 0.06 times or less, 0.05 times or less, 0.04 times or less, 0.03 times or less, 0.02 times or less, or 0.01 times or less relative to the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2. Note that this does not necessarily mean that the substance that binds to ANTXR2 must contain a fluorescent substance. For example, even if the substance that binds to ANTXR2 does not contain a fluorescent substance, mean fluorescence intensity can be measured to measure the binding of the substance that binds to ANTXR2 to ANTXR1 and to measure the binding of the substance that binds to ANTXR2 to ANTXR2. In this case, for example, the binding of a substance obtained by binding a new fluorescent substance to the substance that binds to ANTXR2 to ANTXR1 can be measured using mean fluorescence intensity, and the obtained mean fluorescence intensity can be used as "the mean fluorescence intensity indicating the binding when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured using mean fluorescence intensity." Similarly, for example, the binding of a substance obtained by binding a new fluorescent substance to the substance that binds to ANTXR2 can be measured using mean fluorescence intensity, and the obtained mean fluorescence intensity can be used as "the mean fluorescence intensity indicating the binding when the binding of the substance that binds to ANTXR2 to ANTXR2 is measured using mean fluorescence intensity."Furthermore, in the cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, the dissociation constant of the binding of the substance that binds to ANTXR2 to ANTXR1 is, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800x or more, 900x or more, 1,000x or more, 2,000x or more, 3,000x or more, 4,000x or more, 5,000x or more, 6,000x or more, 7,000x or more, 8,000x or more, 9,000x or more, 10,000x or more, 20,000x or more, 30,000x or more, 40,000x or more, 50,000x or more, 60,000x or more, 70,000x or more, 80,000x or more, 90,000x or more, 100,000,000x or more, 1,000,000x or more, 10,000,000x or more, 100,000,000x or more, or 1,000,000,000x or more.

[0030] Here, when the binding of a substance that binds to ANTXR2 to ANTXR1 is measured by the mean fluorescence intensity, the mean fluorescence intensity indicating the binding, and when the binding of a substance that binds to ANTXR2 to ANTXR2 is measured by the mean fluorescence intensity, the mean fluorescence intensity indicating the binding are measured by the same method as in the method described in <5. Method for evaluating ANTXR2 specificity> in the Examples below. The dissociation constant of the binding refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. The dissociation constant of the binding is Koff / Kon, where Koff is the dissociation rate and Kon is the binding rate (for details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0031] In the cancer therapeutic agent provided by the present invention, which comprises a substance that binds to ANTXR2, preferably, the substance that binds to ANTXR2 does not substantially bind to ANTXR1, more preferably, the binding of the substance that binds to ANTXR2 to ANTXR1 is not observed, and even more preferably, the substance that binds to ANTXR2 does not bind to ANTXR1.

[0032] ANTXR1, for example, is an extracellular matrix protein, also known as TEM8 (tumor endothelial marker 8). In one example, ANTXR1 is a type I transmembrane protein expressed in the endothelium of various tumors. It has already been shown that its expression increases during tumor angiogenesis (St. Croix et al., (2000) Sciences 289: 1197). It is also known to be involved in cell adhesion or migration mediated by extracellular matrix proteins in tumor vascular endothelial cells (Bradely et al., (2001) Nature 414: 228-229; Nanda et al., (2004) Curr Opin Oncol 16: 44-49). In one example, ANTXR1 acts as a receptor for extracellular ligands and is therefore a target for the treatment of angiogenesis (Carson-Walter, EB et al., (2001) Cancer Res 61: 6649). An example of a specific nucleic acid sequence of ANTXR1 has been published by NCBI (NCBI Reference Sequence: NM_032208.2).

[0033] Examples of ANTXR1 include animal ANTXR1. Examples of animals include vertebrates as well as invertebrates. Examples of vertebrates include fish, amphibians, reptiles, birds, and mammals. Examples of mammals include humans, mice, rats, cows, pigs, monkeys, dogs, and cats. Examples of invertebrates include arthropods such as insects, crustaceans, arachnids, and myriapods, as well as mollusks. Preferred animals are mammals, and preferred mammals are humans. That is, ANTXR1 is preferably mammalian ANTXR1, and more preferably human ANTXR1.

[0034] Examples of ANTXR1 include the ANTXR1 gene and expression products of the ANTXR1 gene. Examples of expression products of the ANTXR1 gene include mRNA of the ANTXR1 gene and the ANTXR1 protein. ANTXR1 is preferably the ANTXR1 protein.

[0035] In the cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, when comparing "the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR1 when the binding is measured by the mean fluorescence intensity" with "the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2 when the binding is measured by the mean fluorescence intensity," preferably, the ANTXR1 whose binding is examined in "the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR1 when the binding is measured by the mean fluorescence intensity" and the ANTXR2 whose binding is examined in "the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2 when the binding is measured by the mean fluorescence intensity" are derived from the same organism. That is, for example, if the ANTXR1 whose binding is examined in terms of "the mean fluorescence intensity indicating the binding when the binding of a substance that binds to the ANTXR2 to ANTXR1 is measured by the mean fluorescence intensity," is human ANTXR1, then the ANTXR2 whose binding is examined in terms of "the mean fluorescence intensity indicating the binding when the binding of a substance that binds to the ANTXR2 to ANTXR2 is measured by the mean fluorescence intensity," is also human ANTXR2.

[0036] In a cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, when comparing "the mean fluorescence intensity indicating the binding of a substance that binds to ANTXR2 to ANTXR1 when the binding is measured by the mean fluorescence intensity" with "the mean fluorescence intensity indicating the binding of a substance that binds to ANTXR2 to ANTXR2 when the binding is measured by the mean fluorescence intensity," it is preferable that the ANTXR1 whose binding is examined in "the mean fluorescence intensity indicating the binding of a substance that binds to ANTXR2 to ANTXR1 when the binding is measured by the mean fluorescence intensity" and the ANTXR2 whose binding is examined in "the mean fluorescence intensity indicating the binding of a substance that binds to ANTXR2 to ANTXR2 when the binding is measured by the mean fluorescence intensity" are the same type. That is, for example, if ANTXR1, whose binding is examined based on "the mean fluorescence intensity indicating the binding when the binding of a substance that binds to ANTXR2 to ANTXR1 is measured by the mean fluorescence intensity," is a protein, then ANTXR2, whose binding is examined based on "the mean fluorescence intensity indicating the binding when the binding of a substance that binds to ANTXR2 to ANTXR2 is measured by the mean fluorescence intensity," is also a protein.

[0037] In the cancer therapeutic agent provided by the present invention, which comprises a substance that binds to ANTXR2, the substance that binds to ANTXR2 is preferably a substance to be used in combination with a substance that damages cells, more preferably a substance to be used in combination with a substance that damages cells based on the binding of the substance that binds to ANTXR2 to ANTXR2, and even more preferably a substance to be used in combination with a substance that damages cells in the vicinity of the binding of the substance that binds to ANTXR2 to ANTXR2 using the binding as an indicator. Examples of cases in which the substance that binds to ANTXR2 is a substance to be used in combination with a substance that damages cells include cases in which the substance that binds to ANTXR2 includes a substance that damages cells. Examples of cases in which the substance that binds to ANTXR2 includes a substance that damages cells include cases in which the substance that binds to ANTXR2 is a substance that damages cells, and cases in which the substance that binds to ANTXR2 consists of a substance that damages cells. When the substance that binds to ANTXR2 includes a substance that damages cells, the substance that binds to ANTXR2 may, for example, form a single molecule through intramolecular bonding with the substance that damages cells, or may form a substance containing multiple molecules through intermolecular bonding with the substance that damages cells. However, even if the substance that binds to ANTXR2 is a substance that is intended to be used in combination with a substance that damages cells, it is not essential that it contains a substance that damages cells. Therefore, even if the substance that binds to ANTXR2 is a substance that is intended to be used in combination with a substance that damages cells, it may not, for example, form a single molecule through intramolecular bonding with the substance that damages cells, or may not form a substance containing multiple molecules through intermolecular bonding with the substance that damages cells, and may be a substance that is treated separately from the substance that damages cells.

[0038] Substances for damaging cells include, for example, substances for photoimmunotherapy (NIR-PIT). Photoimmunotherapy can kill specific cell groups using antibodies against cell surface antigens, potentially resulting in localized effects. Photoimmunotherapy utilizes, for example, a photosensitizer, which is a complex of a specific antibody and a phototoxic substance. An example of a phototoxic substance is IR700. That is, an example of a substance for photoimmunotherapy is IR700. Phototoxic substances such as IR700 kill cells by, for example, absorbing near-infrared light and changing their chemical structure or generating heat. Photosensitizers that can be used in photodynamic therapy, for example, can be used. Among these, photosensitizers that selectively accumulate at lesions such as cancerous lesions are preferred. Photosensitizers that selectively accumulate at lesion sites include those in which a near-infrared fluorescent dye such as phthalocyanine is conjugated to an antibody that specifically binds to the lesion site (see WO 2013 / 9475 ) and nanoparticles with particle sizes of 20–200 nm. Nanoparticles have the property of easily accumulating at vascular lesion sites such as tumors (enhanced permeability and retention; EPR effect). These photosensitizers contribute to both the diagnosis of lesion sites using molecular imaging such as fluorescence imaging and photoacoustic imaging, and treatment by irradiating the accumulation site in the body with light. Nanoparticles that can be used as photosensitizers include porphyrin nanoparticles (WO 2011 / 44671 ) and nanoparticles encapsulating near-infrared-absorbing organic molecules (WO 2013 / 51732 , WO 2012 / 128326 ). Among these, nanoparticles containing near-infrared-absorbing organic molecules with near-infrared-absorbing functional groups that absorb near-infrared light in the wavelength range of 700–1000 nm are preferred. Near-infrared-absorbing organic molecules, for example, contain functional groups that absorb near-infrared light (wavelengths of 700 to 1000 nm). Visible light (400 to 700 nm) is highly absorbed by hemoglobin and other biological components, while wavelengths longer than 1000 nm are highly absorbed by water, preventing light from traveling inside the body. In contrast, the near-infrared wavelength range easily penetrates living organisms. For this reason, near-infrared light with wavelengths of 700 to 1000 nm is used for optical measurement of biological tissues.Molecular imaging using near-infrared fluorescence can diagnose deep tumors, metastatic lymph nodes, and localized microtumors. Furthermore, photosensitizers containing near-infrared-absorbing organic molecules can also function as contrast agents in photoacoustic imaging, enabling the diagnosis of lesions using photoacoustic imaging. By administering a photosensitizer containing near-infrared-absorbing organic molecules that absorb near-infrared light and emit fluorescence or thermal energy to a living body and performing molecular imaging, accurate information on lesions where the photosensitizer has accumulated can be obtained. Examples of near-infrared-absorbing organic molecules contained in photosensitizers include those in which near-infrared-absorbing groups are bonded to polymers (near-infrared-absorbing polymers) and near-infrared-absorbing organic molecules themselves (near-infrared absorbers). Near-infrared absorbers are those that form long conjugated π-electron systems by bonding multiple aromatic rings via a conjugated system. Specific examples include cyanine dyes, phthalocyanine dyes, naphthoquinone dyes, diimmonium dyes, and azo dyes. The near-infrared absorbing organic molecule may be encapsulated in a photosensitizer as a near-infrared absorber, or the photosensitizer may contain a near-infrared absorbing organic molecule having an infrared absorbing group bound to another molecule. For example, the photosensitizer may contain the near-infrared absorbing organic molecule in nanoparticles composed of molecular assemblies containing an amphiphilic block polymer. Examples of forms in which the near-infrared absorbing organic molecule is contained in a molecular assembly include a molecular assembly formed by an amphiphilic block polymer and a near-infrared absorbing polymer; a molecular assembly formed by an amphiphilic block polymer to which a near-infrared absorbing group is bound; a molecular assembly in which a near-infrared absorbing agent is encapsulated in a molecular assembly of an amphiphilic block polymer; and a molecular assembly in which a near-infrared absorbing agent is encapsulated in a molecular assembly of an amphiphilic block polymer and a hydrophobic polymer.

[0039] Examples of substances used to kill cells include drugs in antibody-drug conjugates (ADCs). In one example, antibody-drug conjugates (ADCs) are a type of biopharmaceutical designed as targeted therapeutics for cancer treatment. Unlike chemotherapy, ADCs aim to target and kill tumor cells while sparing healthy cells. ADCs are conjugated molecules consisting of, for example, a biologically active cytotoxic (anticancer) agent or drug conjugated to an antibody. ADCs combine the targeting capabilities of, for example, monoclonal antibodies with the cancer-killing capabilities of cytotoxic agents. In ADCs, for example, an anticancer drug is covalently linked to an antibody that specifically targets a specific tumor antigen (e.g., a protein ideally present only in or on tumor cells). For example, when the antibody binds to an antigen on the surface of a cancer cell, a biochemical reaction between the antibody and the target protein (antigen) triggers a signal within the tumor cell, which then absorbs or internalizes the cytotoxin along with the antibody. For example, after the ADC is internalized, the cytotoxin is cleaved from the antibody, exerting its toxicity and killing the cancer. This prevents the drug from being distributed to cells other than the target cells, alleviating side effects and providing a wider therapeutic window than other chemotherapeutic agents. Therefore, the drug in an antibody-drug conjugate (ADC) is, for example, a cytotoxic drug, and the cytotoxic drug is, for example, an anticancer drug, and the anticancer drug is, for example, an anticancer drug for pancreatic cancer, gastric scirrhous cancer, etc., or for cancer accompanied by tissue fibrosis.

[0040] Substances for cell destruction include, for example, substances for antibody-dependent cellular cytotoxicity (ADCC). Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell-mediated cytotoxic reaction mediated by effector cells of the immune system against target cells sensitized by antibodies specific to the target cell surface antigen. In one example, ADCC is an immune defense mechanism in which effector cells of the immune system recognize and actively lyse target cells bound to membrane surface antigens using specific antibodies. In another example, ADCC is a mechanism by which antibodies function as part of the humoral immune response to limit and suppress pathogen infection. Specifically, antibody-dependent cellular cytotoxicity (ADCC) is the antibody-mediated destruction of antibody-bound cells or pathogens by immune cells such as macrophages and NK cells. In one example, ADCC is independent of the complement system, which lyses targets, but does not require any other cells. In one example, ADCC is classically mediated by effector cells known as natural killer (NK) cells, which typically interact with immunoglobulin G (IgG) antibodies. In another example, macrophages, neutrophils, and eosinophils also mediate ADCC. Mouse studies of the efficacy of monoclonal antibodies such as trastuzumab and rituximab against solid tumors have demonstrated that ADCC is an important mechanism of therapeutic efficacy. Clinically, the FcgRIII 158V / F polymorphism inhibits the ability to elicit ADCC responses in vitro during trastuzumab treatment. Multiple myeloma can be treated with the monoclonal antibody daratumumab. In vitro and patient studies have demonstrated that ADCC, along with complement-dependent cytotoxicity (CDC), are important mechanisms of ADCC. Therefore, substances for antibody-dependent cellular cytotoxicity (ADCC) include, for example, antibodies and antibody-like molecules, including, for example, antibodies belonging to the IgG class.

[0041] Substances for damaging cells include, for example, substances for complement-dependent cytotoxicity (CDC). In one example, complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When antibodies bind to surface antigens on target cells (e.g., cells infected with bacteria or viruses), the C1q protein bound to these antibodies activates the classical pathway of the complement system, forming the membrane attack complex (MAC) and lysing the target cell. The complement system is said to be efficiently activated by IgG1, IgG3, and IgM antibodies, but weakly by IgG2 antibodies and not activated by IgG4 antibodies. In one example, CDC is one of the mechanisms by which therapeutic antibodies and antibody fragments exert their antitumor effects. Therefore, substances for complement-dependent cytotoxicity include, for example, antibodies and antibody-like molecules. Examples of antibodies and antibody-like molecules include antibodies belonging to the IgG or IgM class, and examples of antibodies belonging to the IgG class include IgG1, IgG3, and IgG2 antibodies.

[0042] Substances that damage cells include, for example, neutralizing antibodies, such as those that inhibit binding to type 6 collagen.

[0043] The cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, is preferably a cancer therapeutic agent for damaging cancer stem cells. Examples of cancer stem cells include pancreatic cancer stem cells. Examples of cancer stem cells that express ANTXR2 include cancer stem cells whose ANTXR2 RNA expression level is 1.5-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 4.5-fold or more, 5.0-fold or more, 5.5-fold or more, 6.0-fold or more, 6.5-fold or more, 7.0-fold or more, 7.5-fold or more, 8.0-fold or more, 8.5-fold or more, 9.0-fold or more, 9.5-fold or more, or 10.0-fold or more compared to non-cancer stem cells. Here, the expression level of ANTXR2 RNA can be measured, for example, by RT-PCR.

[0044] When the cancer therapeutic agent provided by the present invention contains a substance other than a substance that binds to ANTXR2, the cancer therapeutic agent provided by the present invention may also contain, for example, a pharmaceutically acceptable carrier and additive. The pharmaceutically acceptable carrier or additive is not particularly limited and can be selected appropriately depending on the dosage form, etc., and may include any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener. The dosage form of the cancer therapeutic agent provided by the present invention is not particularly limited and can be selected appropriately depending on the desired administration method, and examples include injections (solutions, suspensions, solids for dissolution before use, etc.), solids (tablets, capsules, suppositories, powders, etc.), etc. Injectables can be prepared, for example, by adding a pH adjuster, buffer, stabilizer, isotonicity agent, local anesthetic, etc. to the composition and using conventional methods to prepare injections for subcutaneous, intraarticular, intramuscular, intravenous, etc. Examples of pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of isotonicity agents include sodium chloride and glucose. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Solid preparations may be enteric-coated. That is, the cancer therapeutic agent provided by the present invention may be prepared as a liquid or solid preparation and may be formulated into various dosage forms, such as injections, jellies, sprays, tablets, and granules. When prepared as a liquid preparation, sterile water, physiological saline, aqueous glucose solution, and the like may be used as a carrier, and, if desired, a bactericide, an isotonicity agent, a stabilizer, and the like may be further blended. When prepared as a solid preparation, additives such as starch, lactose, mannitol, and inorganic salts may be added, and, if desired, binders, disintegrants, lubricants, and the like may be further blended.

[0045] Administering the cancer therapeutic agent of the present invention to a subject contributes to treating cancer in the subject. The subject may be, for example, any vertebrate, but is preferably a mammal such as a mouse, rat, rabbit, pig, cow, monkey, or human, and more preferably a human. The subject may be of any age, including infants, young children, adolescents, adults, and elderly subjects.

[0046] The cancer therapeutic agents provided by the present invention may be administered by any administration method appropriate for the dosage form, for example, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, intra-articular administration, nasal administration, intraperitoneal administration, direct injection into target tissue, inhalation administration, enteral administration, enema administration, tube feeding, etc. The dosage of the cancer therapeutic agents provided by the present invention may vary depending on the age, weight, health condition, etc. of the subject. The cancer therapeutic agents provided by the present invention may be administered once per day or multiple times per day, and the administration frequency can be, for example, 1 to 100 times per month.

[0047] In the cancer therapeutic agent provided by the present invention, which contains a substance that binds to ANTXR2, if the substance that binds to ANTXR2 is a substance to be used in combination with a substance that damages cells, when administering the cancer therapeutic agent provided by the present invention to a subject to treat cancer in the subject, a treatment step may be performed depending on the substance that damages cells to be used in combination. For example, if the substance that damages cells is a substance for photoimmunotherapy (NIR-PIT), after administering the cancer therapeutic agent provided by the present invention to the subject, near-infrared rays may be irradiated onto the cancer-affected area of ​​the subject that has been administered the cancer therapeutic agent provided by the present invention. Examples of the near-infrared rays to be irradiated include near-infrared rays with a wavelength of 660 to 740 nm, near-infrared rays with a wavelength of 670 to 710 nm, and near-infrared rays with a wavelength of 680 to 700 nm. The near-infrared rays to be irradiated may have a wavelength of, for example, 0.01 mW / cm. 2 More than 0.1mW / cm 2 More than 1mW / cm 2 More than 10mW / cm 2 More than 20mW / cm 2 More than 30mW / cm2 More than 40mW / cm 2 More than 50mW / cm 2 More than 60mW / cm 2 More than 70mW / cm 2 More than 80mW / cm 2 More than 90mW / cm 2 More than 100mW / cm 2 More than 110mW / cm 2 More than 120mW / cm 2 More than 130mW / cm 2 or more than 140mW / cm 2 and above 100,000 mW / cm 2 Below 10,000 mW / cm 2 Below 1,000 mW / cm 2 Below, 900 mW / cm 2 Below 800 mW / cm 2 Below 700 mW / cm 2 Below 600 mW / cm 2 Below 500 mW / cm 2 Below 400 mW / cm 2 Below 300 mW / cm 2 Below 200 mW / cm 2 Below 190 mW / cm 2 Below 180 mW / cm 2 Below 170 mW / cm 2 or less than 160 mW / cm 2 The near-infrared radiation to be irradiated is, for example, 0.005 J / cm 2 More than 0.05 J / cm 2 More than 0.5 J / cm 2 More than 1 J / cm 2 more than 10 J / cm 2 more than 20 J / cm 2 More than 30 J / cm 2 or more than 40 J / cm 2 and above 500,000 J / cm 2 Below 50,000 J / cm 2 Below, 5,000 J / cm 2 Below, 500 J / cm 2 Below, 400 J / cm 2Below, 300 J / cm 2 Below, 200 J / cm 2 Below, 100 J / cm 2 Below, 90 J / cm 2 Below, 80 J / cm 2 Below, 70 J / cm 2 or less than 60 J / cm 2 The following is the result.

[0048] The present invention provides a method for treating cancer, comprising the step of administering to a subject a substance that binds to ANTXR2, wherein when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.65 times or less compared to the mean fluorescence intensity indicating said binding when the binding of the substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity.

[0049] The present invention provides a pharmaceutical composition for use in the treatment of cancer, which comprises a substance that binds to ANTXR2, and in which, when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.65 times or less compared to the mean fluorescence intensity indicating said binding of the substance that binds to ANTXR2 to ANTXR2, when the binding of the substance that binds to ANTXR2 is measured by mean fluorescence intensity.

[0050] The present invention provides the use of a substance that binds to ANTXR2 in the manufacture of a pharmaceutical composition for use in the treatment of cancer, wherein when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating the binding is 0.65 times or less compared to the mean fluorescence intensity indicating the binding of the substance that binds to ANTXR2 to ANTXR2, when the binding of the substance that binds to ANTXR2 is measured by mean fluorescence intensity.

[0051] 1. Animal Experiments

[0052] Figure 1 shows a mouse model with tumors. The mouse model was generated as follows: mouse pancreatic cancer organoids (Kras) were implanted into the pancreatic tail of 8-week-old wild-type C57BL / 6 mice. G12D, Cdkn2a KO, p53 KO, Smad4 KO) 3 × 10 5 A mouse pancreatic cancer orthotopic transplantation model was obtained by transplanting individual cells. After a 10-day follow-up observation,

[0053] In Figure 1, 1 mM AkaLumine-HCl was administered intraperitoneally to model mice every 12 days. The chemiluminescence from the transplanted pancreatic cancer cells was measured using an IVIS imaging system, and the amount of pancreatic cancer tumors in the model mice was confirmed over time.

[0054] Figure 1 shows the time course of pancreatic cancer tumor burden in a control group receiving phosphate-buffered saline (PBS) and a treatment group receiving gemcitabine. Gemcitabine was administered intraperitoneally (ip) at 120 mg / kg every three days. Mice in the control group received PBS intraperitoneally every three days.

[0055] The results of the observations in Figure 1 are as follows: In a mouse pancreatic cancer orthotopic transplant model, tumors grew over time in mice administered phosphate-buffered saline (PBS). In mice administered gemcitabine, tumor growth was suppressed up to 36 days after the start of administration, but tumor recurrence was confirmed from day 48 onwards.

[0056] Figure 2 shows the method for recovering pancreatic cancer cells from the tumor in the model mouse created in Figure 1. The method for recovering pancreatic cancer cells is as follows. First, the tumor removed from the mouse pancreas was enzymatically digested to recover cells from within the tumor. Next, pancreatic cancer cells that had been previously labeled with Venus fluorescent protein were recovered using flow cytometry.

[0057] Figure 2 shows that pancreatic cancer cells (Venus-positive) collected from mouse pancreatic tumors were used for single-cell RNA-seq analysis.

[0058] 2. Identification of anthrax toxin receptor type II expressed on the surface of pancreatic cancer stem cells

[0059] Figure 3 shows the results of single-cell RNA-seq analysis of pancreatic cancer cells recovered from tumors in mice treated with phosphate-buffered saline (PBS) for 12 days (1 mouse), gemcitabine for 12 days (2 mice), and gemcitabine for 63 days (1 mouse).

[0060] Figure 4 shows the results of applying CytoTRACE, a stem cell prediction algorithm, to the results of Figure 3. Cells with high scores predicted by CytoTRACE are highly likely to be pancreatic cancer stem cells. In this example, cells with a CytoTRACE score of 0.75 or higher are referred to as pancreatic cancer stem cells.

[0061] Figure 5 shows the results of differential gene expression analysis between pancreatic cancer stem cells and other pancreatic cancer cells. Compared to other pancreatic cancer cells, pancreatic cancer stem cells contained genes that were highly expressed (red) and genes that were less expressed (blue).

[0062] The data shown in Figure 5 were tested as follows. Pancreatic cancer cells were divided into two groups: pancreatic cancer stem cells (cells with a CytoTRACE score of 0.75 or higher) and other pancreatic cancer cells (cells with a CytoTRACE score of less than 0.75). Differences in gene expression between the two groups were tested using the Wilcoxon rank-sum test, and adjusted p-values ​​based on the Bonferroni correction were calculated.

[0063] In Figure 5, genes with a fold change (FC) of 1.5 times or more and an adjusted p-value of less than 0.05 were determined to be highly expressed in pancreatic cancer stem cells compared to other pancreatic cancer cells. There were only 37 genes that were highly expressed in pancreatic cancer stem cells.

[0064] Figure 6 shows the results of a correlation analysis between the expression levels of 37 genes highly expressed in pancreatic cancer stem cells and overall survival in human pancreatic cancer patients. Of the 37 genes highly expressed in pancreatic cancer stem cells, three (Tubb5, Ctla2a, and A100a10) have no human homologues and were therefore excluded from the correlation analysis. Correlation analysis between expression levels and overall survival for the remaining 34 genes was performed using the pancreatic cancer dataset from The Cancer Genome Atlas (TCGA). The expression levels of 13 genes (Top2a, Tpm4, Racgap1, Cdk1, Ccna2, Rrm2, Cks1b, Antxr2, Anxa1, Ppia, Banf1, Tagln2, and Ywhah) were significantly correlated with overall survival (p-value less than 0.05).

[0065] The data shown in Figure 6 was tested for each gene as follows. The pancreatic cancer dataset from TCGA (The Cancer Genome Atlas) was divided into two groups, high expression and low expression, with the median gene expression level as the boundary. The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated.

[0066] Figure 6 shows that among the 13 genes highly expressed in pancreatic cancer stem cells and significantly correlated with the overall survival of human pancreatic cancer patients, anthrax toxin receptor type II (Antxr2) was the only gene localized on the cell membrane surface (shown in red).

[0067] Figure 7 shows the overall survival of human pancreatic cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the pancreatic cancer dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 177 pancreatic cancer patients were divided into two groups, a high expression group (89 patients) and a low expression group (88 patients), with the median ANTXR2 expression level as the dividing line. The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0068] Figure 8 shows the results of plotting the Antxr2 expression levels of individual cells on single-cell RNA-seq data of mouse pancreatic cancer cells. Compared to the results in Figure 4, Antxr2 was highly expressed in pancreatic cancer stem cells (cells with a CytoTRACE score of 0.75 or higher).

[0069] <3. Verification of the effect of eliminating Antxr2-positive pancreatic cancer stem cells>

[0070] Figure 9 shows the method for generating pancreatic cancer organoids that express the diphtheria toxin receptor (DTR) and tdTomato fluorescent protein in an Antxr2 gene expression-dependent manner. The method for generating these pancreatic cancer organoids is as follows. Using the CRISPR-Cas9 system, a gene cassette consisting of T2A peptide-DTR-T2A peptide-tdTomato fluorescent protein was inserted into the pancreatic cancer organoids by homologous recombination immediately before the stop codon of the Antxr2 gene. These pancreatic cancer organoids are called Antxr2DTR-tdTomato organoids. In Antxr2DTR-tdTomato organoids, DTR and tdTomato fluorescent protein are expressed specifically in Antxr2-positive cells.

[0071] Figure 10 shows the results of flow cytometry analysis of in vitro cultured Antxr2DTR-tdTomato organoids to quantify the percentage of Antxr2-positive cells. Flow cytometry was performed as follows. The percentage of Antxr2-positive cells was assessed by quantifying tdTomato fluorescent protein-positive cells within live cells (7AAD-negative). Pancreatic cancer organoids that do not express tdTomato fluorescent protein were used as a control.

[0072] Figure 10 shows that approximately 9.6% of Antxr2-positive cells were present in Antxr2DTR-tdTomato organoids cultured in vitro.

[0073] Figure 11A shows bright-field images comparing the organoid formation efficiency of Antxr2-positive cells (tdTomato-positive) and Antxr2-negative cells (tdTomato-negative) shown in Figure 10. Observations were performed as follows. Equal numbers of tdTomato-positive and tdTomato-negative cells were collected from in vitro cultured Antxr2DTR-tdTomato organoids by flow cytometry and cultured for 6 days.

[0074] Figure 11B shows the quantification results of the bright-field observation image in Figure 11A. Data were analyzed using Welch's t-test.

[0075] The results of the observations in Figure 11A and B are as follows: Antxr2-positive cells showed significantly higher organoid formation efficiency in in vitro cultured pancreatic cancer organoids. In other words, Antxr2-positive cells exhibited high stemness.

[0076] Figure 12A shows brightfield images of Antxr2-positive cells in in vitro cultured pancreatic cancer organoids after ablation. Observations were performed as follows: Phosphate-buffered saline (PBS) or diphtheria toxin was added to the culture medium of in vitro cultured Antxr2DTR-tdTomato organoids, and the organoids were cultured for 3 days. Pancreatic cancer organoids that do not express the anthrax toxin receptor (DTR) were used as a control.

[0077] Figure 12B shows the quantification results of the brightfield images in Figure 12A. Organoid formation was quantified by chemiluminescence. Data were analyzed by Welch's t-test.

[0078] The results of the observations in Figures 12A and 12B are as follows: ablation of Antxr2-positive cells in in vitro cultured pancreatic cancer organoids significantly reduced organoid formation. This indicates that Antxr2-positive cells are essential for organoid formation and have high stemness.

[0079] Figure 13 shows the results of flow cytometry analysis of pancreatic cancer cells in an orthotopic mouse pancreatic cancer transplant model, quantifying the proportion of Antxr2-positive cells. Flow cytometry was performed as follows. First, Antxr2DTR-tdTomato organoids pre-labeled with Venus fluorescent protein were transplanted into the pancreas of mice to form tumors. Cells were then harvested from the tumor and evaluated by quantifying tdTomato fluorescent protein-positive cells within live cells (7AAD-negative), non-immune cells (CD45-negative), and pancreatic cancer cells (Venus-positive). Pancreatic cancer organoids that did not express tdTomato fluorescent protein were used as a control.

[0080] Figure 13 shows that Antxr2-positive cells account for approximately 10% in a mouse pancreatic cancer orthotopic transplant model.

[0081] Figure 14 shows a fluorescent image of a tumor tissue section taken from the mouse pancreatic cancer orthotopic transplant model shown in Figure 13. The location of the cells was confirmed by DAPI (4',6-diamidino-2-phenylindole) signals. This shows that among the pancreatic cancer cells (Venus positive), some cells are Antxr2 positive (tdTomato positive).

[0082] Figure 15A shows the results of bright-field observation comparing the organoid formation efficiency of Antxr2-positive pancreatic cancer cells (tdTomato positive) and Antxr2-negative pancreatic cancer cells (tdTomato negative) shown in Figure 13. The observation was performed as follows. Antxr2-positive pancreatic cancer cells (7AAD - CD45 - Venus+ tdTomato + ) and Antxr2-negative pancreatic cancer cells (7AAD - CD45 - Venus + tdTomato - ) were collected in equal numbers by flow cytometry and cultured for 6 days.

[0083] Figure 15B shows the results of quantifying the bright-field observation image in Figure 15A. Data were analyzed using Welch's t-test.

[0084] The results of the observations in Figures 15A and 15B are as follows: Antxr2-positive pancreatic cancer cells showed significantly higher organoid formation efficiency in an orthotopic mouse pancreatic cancer transplant model. In other words, Antxr2-positive pancreatic cancer cells exhibited high stemness.

[0085] Figure 16A shows a tumor-bearing mouse model. The mouse model was generated as follows: 3 × 10 Antxr2DTR-tdTomato organoids were inoculated into the pancreatic tail of 8-week-old wild-type C57BL / 6 mice. 5 A mouse pancreatic cancer orthotopic transplantation model was obtained by transplanting individual cells. A follow-up observation period of 34 days was performed.

[0086] Figure 16A shows the time course of changes in pancreatic cancer tumor burden when phosphate-buffered saline (PBS) was administered to the control group and diphtheria toxin was administered to the ablation group. Diphtheria toxin was administered intraperitoneally at 10 ng / g for five consecutive days starting on day 34 after transplantation. The same amount of PBS was administered intraperitoneally to the control group for five consecutive days starting on day 34 after transplantation.

[0087] Figure 16B is a graph showing the time course of the tumor size shown in Figure 16A. The cases of phosphate-buffered saline (PBS) administration and diphtheria toxin administration are shown. In both cases, the tumor size on day 34 after implantation is expressed as a relative value, with the value set as 100%. Data were analyzed using the Wilcoxon rank-sum test.

[0088] The results of the observations in Figures 16A and 16B are as follows: In a mouse pancreatic cancer orthotopic transplant model, tumors grew over time in mice administered phosphate-buffered saline (PBS), whereas tumor growth was significantly reduced in mice administered diphtheria toxin.

[0089] Figure 17 shows the survival curves for the mice in each case shown in Figures 16A and 16B. Data were analyzed using the log-rank test. The diphtheria toxin-administered group showed a significantly longer survival period than the phosphate-buffered saline (PBS)-administered group.

[0090] Figure 18A shows a tumor-bearing mouse model. The mouse model was generated as follows: 3 × 10 Antxr2DTR-tdTomato organoids were placed under the capsule of the left liver lobe of 8-week-old wild-type C57BL / 6 mice. 5 A mouse model of pancreatic cancer liver metastasis was obtained by transplantation of 100 individual cells. After a 34-day follow-up observation,

[0091] Figure 18A shows the time course of changes in pancreatic cancer tumor burden when phosphate-buffered saline (PBS) was administered to the control group and diphtheria toxin was administered to the ablation group. Diphtheria toxin was administered intraperitoneally at 10 ng / g for five consecutive days starting on day 34 after transplantation. The same amount of PBS was administered intraperitoneally to the control group for five consecutive days starting on day 34 after transplantation.

[0092] Figure 18B is a graph showing the time course of the tumor size shown in Figure 18A. The cases of phosphate-buffered saline (PBS) administration and diphtheria toxin administration are shown. In both cases, the tumor size on day 34 after implantation is expressed as a relative value, with the value set as 100%. Data were analyzed using the Wilcoxon rank-sum test.

[0093] The results of the observations in Figures 18A and 18B are as follows: In a mouse pancreatic cancer liver metastasis model, tumors increased over time in mice administered phosphate-buffered saline (PBS), whereas tumor growth was significantly reduced in mice administered diphtheria toxin.

[0094] Figure 19A shows a tumor-bearing mouse model. The mouse model was generated as follows: 3 × 10 Antxr2DTR-tdTomato organoids were injected intraperitoneally into 8-week-old wild-type C57BL / 6 mice. 5 A mouse model of peritoneal dissemination of pancreatic cancer was obtained by transplantation of 100 individual cells. After a 34-day follow-up observation,

[0095] Figure 19A shows the time course of changes in pancreatic cancer tumor burden when phosphate-buffered saline (PBS) was administered to the control group and diphtheria toxin was administered to the ablation group. Diphtheria toxin was administered intraperitoneally at 10 ng / g for five consecutive days starting on day 34 after transplantation. The same amount of PBS was administered intraperitoneally to the control group for five consecutive days starting on day 34 after transplantation.

[0096] Figure 19B is a graph showing the time course of the tumor size shown in Figure 19A. The cases of phosphate-buffered saline (PBS) administration and diphtheria toxin administration are shown. In both cases, the tumor size on day 34 after implantation is expressed as a relative value, with the value set as 100%. Data were analyzed using the Wilcoxon rank-sum test.

[0097] The results of the observations in Figures 19A and 19B are as follows: In a mouse model of pancreatic cancer peritoneal dissemination, tumors increased over time in mice administered phosphate-buffered saline (PBS), whereas tumor growth was significantly reduced in mice administered diphtheria toxin.

[0098] <4. Verification of effects on other cancers> Figure 20 shows the overall survival of human colorectal cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the colorectal cancer (colon cancer and rectal cancer) dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 588 colorectal cancer patients were divided into two groups: high ANTXR2 expression group (294 patients) and low ANTXR2 expression group (294 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0099] Figure 21 shows the overall survival of human low-grade glioma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the low-grade glioma dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 513 low-grade glioma patients were divided into two groups: high ANTXR2 expression group (257 patients) and low ANTXR2 expression group (256 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0100] Figure 22 shows the overall survival of human gastric cancer patients in two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the gastric cancer dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 407 gastric cancer patients were divided into two groups: high ANTXR2 expression group (103 patients) and low ANTXR2 expression group (304 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0101] Figure 23 shows the overall survival of human head and neck cancer patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the head and neck cancer dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 514 head and neck cancer patients were divided into two groups: high ANTXR2 expression group (74 patients) and low ANTXR2 expression group (440 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0102] Figure 24 shows the overall survival of human papillary renal cell carcinoma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the papillary renal cell carcinoma dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows: Data from 282 papillary renal cell carcinoma patients were divided into two groups: high ANTXR2 expression group (52 patients) and low ANTXR2 expression group (230 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0103] Figure 25 shows the overall survival of human lung adenocarcinoma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the lung adenocarcinoma dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 501 lung adenocarcinoma patients were divided into two groups: high ANTXR2 expression group (126 patients) and low ANTXR2 expression group (375 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0104] Figure 26 shows the overall survival of human lung squamous cell carcinoma patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the lung squamous cell carcinoma dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 478 lung squamous cell carcinoma patients were divided into two groups: high ANTXR2 expression group (241 patients) and low ANTXR2 expression group (237 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0105] Figure 27 shows the overall survival of human testicular germ cell tumor patients divided into two groups based on the expression level of anthrax toxin receptor type II (ANTXR2) in the testicular germ cell tumor dataset from The Cancer Genome Atlas (TCGA). The analysis was performed as follows. Data from 133 testicular germ cell tumor patients were divided into two groups: high ANTXR2 expression group (61 patients) and low ANTXR2 expression group (72 patients). The overall survival between these two groups was tested using the log-rank test, and the p-value was calculated. The high ANTXR2 expression group had a significantly shorter overall survival.

[0106] 5. Evaluation of ANTXR2 specificity Expression vectors were prepared by cloning human ANTXR1-Venus fusion protein and human ANTXR2-Venus fusion protein into the pcDNA3 vector. The Venus fluorescent protein is fused to the C-terminus of the human ANTXR1 protein or human ANTXR2 protein. In other words, the expression levels of both human ANTXR1-Venus fusion protein and human ANTXR2-Venus fusion protein can be monitored by observing Venus fluorescence. These vectors were transfected into human embryonic kidney cells (HEK293T) and selected with puromycin to establish stable expression lines. These stable expression lines were then used to identify hANTXR1 and hANTXR2 specificity. Venus HEK293T cells, hANTXR2 Venus These cells are called HEK293T cells.

[0107] Figure 28 shows the gating conditions used when HEK293T cells were analyzed by flow cytometry. Samples used for flow cytometry were prepared as follows: HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 Venus Harvest 1 x 10 HEK293T cells 5 The cells were stained with anti-human ANTXR2 antibody (final concentration: 500 ng / ml) diluted in 4% FCS / PBS for 30 minutes at 4°C. The anti-human ANTXR2 antibody was pre-labeled with the fluorescent dye PE. The stained cells were washed with 4% FCS / PBS (500 μl) and stained with 7-AAD (4°C, 10 minutes). After 7-AAD staining, the cells were analyzed using a BD FACSymphony.

[0108] Figure 28 shows the gating conditions used when HEK293T cells were analyzed by flow cytometry. Flow cytometry was performed as follows. First, cells were developed with FSC-A and SSC-A to select the cell fraction ((1) in the figure). Next, cells were developed with FSC-A and FSC-W to extract the single-cell fraction ((2) in the figure). Next, cells were developed with 7-AAD and SSC-A to extract live cells (7-AAD-negative) ((3) in the figure). Finally, cells were developed with Venus and PE ((4) in the figure). Venus indicates the expression level of human ANTXR1-Venus fusion protein and human ANTXR2-Venus fusion protein. PE indicates the expression level of human ANTXR2. The scales for Venus and PE are based on HEK293T cells at 1 x 10 3 The scales for Venus and PE are set as follows: Venus The brightest cells in HEK293T cells were 1 × 10 4 Greater than or equal to 1 x 10 5 It was set to the following:

[0109] Figure 29 shows HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 VenusThis shows the results of flow cytometry analysis of HEK293T cells as shown in Figure 28. Venus indicates the expression levels of human ANTXR1-Venus fusion protein and human ANTXR2-Venus fusion protein. PE indicates the expression level of human ANTXR2. The mean fluorescence intensity (MFI) of each cell is shown in the figure.

[0110] Figure 29 shows HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 Venus The results of flow cytometry analysis of HEK293T cells are shown in Figure 28. The observation results are as follows: HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 Venus The Venus MFI in HEK293T cells was 27.0, 2384, and 2536, respectively. Venus HEK293T cells were transfected with human ANTXR1-Venus fusion protein, hANTXR2 Venus HEK293T cells express the human ANTXR2-Venus fusion protein. Venus HEK293T cells, hANTXR2 Venus The difference in Venus MFI in HEK293T cells was within ±10%, indicating that the expression levels of the human ANTXR1-Venus fusion protein and the human ANTXR2-Venus fusion protein were comparable.

[0111] Figure 29 shows HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 Venus The results of flow cytometry analysis of HEK293T cells are shown in Figure 28. The observation results are as follows: HEK293T cells, hANTXR1 Venus HEK293T cells, hANTXR2 Venus The PE MFI of HEK293T cells was 105, 101, and 12329, respectively. Therefore, the anti-human ANTXR2 antibody does not recognize human ANTXR1 and is a human ANTXR2-specific antibody.

[0112] 6. Verification of the Effect of ANTXR2-Positive Cell Elimination Therapy in Human Pancreatic Cancer Figure 30 shows the results of bright-field observation of in vitro cultured human pancreatic cancer cell line (MIA PaCa-2) subjected to photoimmunotherapy targeting ANTXR2. The experiment was carried out as follows. 4 One day after seeding the cells onto a 24-well plate, the cells were exposed to the anti-human ANTXR2 antibody used in Figures 28 and 29 labeled with IR700 dye (hereafter referred to as anti-human ANTXR2 antibody-IR700) at a final concentration of 10 μg / ml and incubated at 37°C for 1 hour. Afterwards, excess antibody was washed off with PBS, and phenol red-free medium was added. The cells were then exposed to near-infrared light (690 nm, 150 mW / cm 2 , 50 J / cm 2 ) and observed after 2 hours.

[0113] Figure 30 shows brightfield images of ANTXR2-targeted photoimmunotherapy performed on a human pancreatic cancer cell line (MIA PaCa-2) cultured in vitro. The results are as follows: "Control" represents the sample that was not exposed to either anti-human ANTXR2 antibody-IR700 or near-infrared light (NIR). "Antibody" represents the sample that was exposed only to anti-human ANTXR2 antibody-IR700. "NIR" represents the sample that was exposed only to NIR light. "NIR-PIT" represents the sample that was exposed to both anti-human ANTXR2 antibody-IR700 and NIR light. The scale bar is 50 μm. This demonstrates that ANTXR2-targeted photoimmunotherapy kills pancreatic cancer cells in vitro.

[0114] Figure 31 shows the results of quantification of the bright-field observation images in Figure 30. Quantification was performed using Cell Counting Kit-8. Data were analyzed by the Tukey method. This shows that photoimmunotherapy targeting ANTXR2 significantly reduces human pancreatic cancer cells in vitro.

[0115] 7. Photoimmunotherapy is specific to ANTXR2 (specificity test) Figure 32 shows that photoimmunotherapy is specific to ANTXR2. In human pancreatic cancer cells in which the ANTXR2 gene was disrupted, no killing effect was observed by photoimmunotherapy using the ANTXR2 antibody-IR700. In other words, the fact that no killing effect was observed in ANTXR2 knockout cells suggests that this treatment is highly specific.

[0116] 8. Photoimmunotherapy is effective on patient-derived organoids (PDO) (efficacy study) Figure 33 shows that photoimmunotherapy is effective on patient-derived organoids (PDO). Photoimmunotherapy using the ANTXR2 antibody-IR700 was performed on organoids derived from human pancreatic cancer patients, and efficient killing was observed. In other words, photoimmunotherapy was applied to PDAC organoids derived from human patients, and high efficacy was confirmed.

[0117] 9. ANTXR2 Deficiency Strongly Inhibits Tumor Growth. Figure 34 shows that ANTXR2 deficiency strongly inhibits tumor growth. When human pancreatic cancer cells with a disrupted ANTXR2 gene were transplanted into mice, their tumorigenicity was significantly attenuated. Specifically, two independent ANTXR2 knockout cell lines were established from the human pancreatic cancer cell line MIA PaCa-2 and transplanted into mouse pancreases. As shown in Figure 34, wild-type cells formed large tumors, while ANTXR2 knockout cells formed significantly smaller tumors. This is likely due to the inability of ANTXR2 knockout cells to degrade collagen VI surrounding the tumor. These results support the idea that antibodies that inhibit ANTXR2 function can exert antitumor effects by antibody administration alone, without the need for photoimmunotherapy.

Claims

1. A cancer therapeutic agent comprising a substance that binds to ANTXR2, wherein when the binding of the substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.65 times or less than the mean fluorescence intensity indicating said binding when the binding of the substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity.

2. A cancer therapeutic agent as described in claim 1, wherein, when the binding of a substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.5 times or less than the mean fluorescence intensity indicating said binding when the binding of a substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity.

3. A cancer therapeutic agent as described in claim 1, wherein, when the binding of a substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.1 times or less than the mean fluorescence intensity indicating said binding when the binding of a substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity.

4. A cancer therapeutic agent as described in claim 1, wherein, when the binding of a substance that binds to ANTXR2 to ANTXR1 is measured by mean fluorescence intensity, the mean fluorescence intensity indicating said binding is 0.05 times or less than the mean fluorescence intensity indicating said binding when the binding of a substance that binds to ANTXR2 to ANTXR2 is measured by mean fluorescence intensity.

5. The cancer therapeutic agent according to claim 1, wherein the substance that binds to ANTXR2 does not substantially bind to ANTXR1.

6. The cancer therapeutic agent according to claim 1, wherein the cancer is pancreatic cancer, colon cancer, low-grade glioma, gastric cancer, head and neck cancer, renal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or germ cell tumor.

7. The cancer therapeutic agent of claim 1, wherein the substance that binds to ANTXR2 comprises an antibody, an antibody-like molecule, or a cyclic peptide.

8. The cancer therapeutic agent according to claim 7, wherein the antibody-like molecule is an scFv or a nanobody.

9. The cancer therapeutic agent according to claim 1, wherein the substance that binds to ANTXR2 includes a substance that damages cells.

10. The cancer therapeutic agent according to claim 9, wherein the substance for damaging cells is a substance for photoimmunotherapy.

11. The cancer therapeutic agent according to claim 10, wherein the substance for photoimmunotherapy is IR700.

12. The cancer therapeutic agent according to claim 9, wherein the substance for damaging cells is a drug in an antibody-drug complex.

13. The cancer therapeutic agent according to claim 9, wherein the substance for damaging cells is a substance for antibody-dependent cellular cytotoxicity.

14. The cancer therapeutic agent according to claim 9, wherein the substance for damaging cells is a substance for complement-dependent cytotoxicity.

15. The cancer therapeutic agent according to claim 9, wherein the substance for damaging cells is a neutralizing antibody.

16. The cancer therapeutic agent according to claim 15, wherein the neutralizing antibody is a neutralizing antibody for inhibiting binding to type 6 collagen.

17. The cancer therapeutic agent described in claim 1 for damaging cancer stem cells.

18. The cancer therapeutic agent according to claim 17, wherein the cancer stem cells are pancreatic cancer stem cells.

19. The cancer therapeutic agent described in claim 17, wherein cancer stem cells express ANTXR2.

20. The cancer therapeutic agent according to claim 19, wherein the expression level of ANTXR2 RNA in cancer stem cells is 1.5 times or more higher than that in non-cancer stem cells.

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