Screening method for molecularly targeted Anti-tumor agent against tumors of non-human mammals

Abstract

To provide a method that enables efficient screening of molecularly targeted anti-tumor agents appropriate for personalized therapy of tumors in non-human mammals.SOLUTION: An anti-tumor agent sensitivity test kit for personalized treatment of non-human mammals having a tumor, the kit comprising a set of molecularly targeted anti-tumor agents including abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib, and optionally including toceranib, olaparib, dabrafenib, and / or encorafenib, and a screening method for an anti-tumor agent for personalized treatment of non-human mammals having a tumor, using such a set of molecularly targeted anti-tumor agents.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening molecular-targeted antitumor agents for tumors in non-human mammals. [Background technology]

[0002] Molecularly targeted antitumor agents (molecular-targeted drugs) possess specific inhibitory activity against target molecules and are known to be more effective with fewer side effects than cytotoxic anticancer drugs that target all dividing cells. However, the target molecular abnormalities targeted by molecular-targeted antitumor agents often vary from tumor to tumor, and even from patient to patient, even within the same tumor type. Therefore, personalized treatment with molecular-targeted antitumor agents, which involves screening each patient for potentially effective target molecules and selecting effective therapeutic candidates based on the results, is expected to be a promising treatment option. However, screening each patient for target molecular abnormalities with a wide variety of molecular-targeted antitumor agents is labor-intensive and places a heavy burden on the patient. Personalized treatment, particularly in non-human mammals, has yet to be established.

[0003] The three-dimensional (3D) organoid culture method was developed as a method for constructing three-dimensional epithelial tissue-like structures by mixing epithelial cells derived from various organs with Matrigel and culturing them in a medium containing factors that enhance stemness, such as Wnt, Noggin, and R-spondin (Non-Patent Document 1). In recent years, techniques have been established for producing organoids with three-dimensional structures similar to human organs and organ systems from cells obtained from patient specimens (e.g., Non-Patent Documents 2 and 3). Non-Patent Document 4 reports a technique for non-invasively producing bladder cancer organoids by culturing tumor cells in the urine of animal bladder cancer patients.

[0004] Patent Document 1 discloses a method for producing 2.5-dimensional (2.5D) organoids obtained by two-dimensionally culturing cells derived from 3D organoids, and demonstrates that an anticancer drug sensitivity test was performed using the 2.5D organoids. Non-Patent Document 5 discloses a method for directly producing 2.5D organoids from tumor tissue. Patent Document 2 discloses a method for producing feline mammary tumor organoids and demonstrates that an anticancer drug sensitivity test was performed using feline mammary tumor organoids.

[0005] However, the anticancer drug sensitivity tests disclosed in Patent Documents 1 and 2 only examined the sensitivity of specific cancer-derived organoids to anticancer drugs. These documents do not describe or suggest a molecular-targeted antitumor drug panel that enables efficient selection of individualized molecular-targeted antitumor drugs for various tumors in various individual patients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-145646 [Patent Document 2] Japanese Patent Publication No. 2022-165800 [Non-patent literature]

[0007] [Non-Patent Document 1] Sato et al., Nature, 459(7244): 262-265, (2009) [Non-patent document 2] Wetering et al., Cell, 161(4): 933-945, (2015) [Non-patent document 3] Boj et al., Cell, 160(1-2): 324-338, (2015) [Non-patent document 4] Elbadawy et al., Cancer Sci., 110(9): 2806-2821, (2019) [Non-Patent Document 5] Abugomaa A. et al., Biomedicine & Pharmacotherapy, 154 (2022) 113597 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a method for efficiently screening molecular-targeted antitumor agents suitable for personalized treatment of tumors in non-human mammals. [Means for solving the problem]

[0009] As a result of extensive research to solve the above problems, the present inventors discovered a set (combination / panel) of molecularly targeted antitumor agents that are useful for screening antitumor agents suitable for personalized treatment of tumor-bearing non-human mammals, and thus completed the present invention.

[0010] That is, the present invention includes the following. [1] An antitumor drug sensitivity test kit for personalized treatment of tumor-bearing non-human mammals, comprising a set of molecular targeted antitumor drugs including abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib. [2] The kit described in [1] above, wherein the set of molecularly targeted antitumor agents further includes at least one of toceranib and olaparib. [3] The kit according to [1] or [2] above, wherein the set of molecularly targeted antitumor agents further comprises dabrafenib or encorafenib. [4] The kit according to any one of [1] to [3] above, wherein the set of molecular targeted antitumor agents includes abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib. [5] The kit according to any one of [1] to [4] above, wherein the non-human mammal is a dog or a cat. [6] The kit according to any one of [1] to [5] above, wherein the non-human mammal has a tumor selected from the group consisting of hepatocellular carcinoma, intestinal adenocarcinoma, mucinous adenocarcinoma, malignant peripheral nerve sheath tumor, angiosarcoma, lung adenocarcinoma, adrenocortical carcinoma, adenocarcinoma, renal cell carcinoma, leiomyoma, lipoma, adrenocortical adenoma, hepatocellular adenoma, sarcoma, pancreatic islet cell carcinoma, lymphoma, transitional meningioma, giant cell tumor of tendon sheath, mast cell tumor, rectal adenocarcinoma, malignant melanoma, squamous cell carcinoma, hemangiopericytoma, urothelial carcinoma, T-cell lymphoma, thyroid carcinoma, synovial sarcoma, anal gland adenocarcinoma, gastrointestinal stromal tumor, histiocytic sarcoma, fibrosarcoma, thymoma, osteosarcoma, neuroendocrine carcinoma, and epithelial tumors. [7] A method for screening antitumor agents for personalized treatment of a non-human mammal having a tumor, using a set of molecular-targeted antitumor agents, comprising: the set of molecular targeted antitumor agents includes abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib; treating cultured cells derived from a tumor carried by a non-human mammalian individual or derived from an allogeneic tumor of the same tumor type as the tumor carried by said non-human mammalian individual with each of the molecular-targeted anti-tumor agents of said set; The proliferation level of cultured cells after treatment with each molecular target antitumor agent was evaluated. A method comprising selecting a molecularly targeted antitumor agent that inhibits the proliferation of cultured cells after treatment as an antitumor agent to which the tumor in the non-human mammal individual is sensitive. [8] The method according to [7] above, wherein the cultured cells are organoids. [9] The method according to [7] or [8] above, wherein the set of molecularly targeted antitumor agents further comprises at least one of toceranib and olaparib.

[10] The method according to any one of [7] to [9] above, wherein the set of molecular-targeted antitumor agents further comprises dabrafenib or encorafenib.

[11] The method according to any one of the above [7] to

[10] , wherein the set of molecular targeted antitumor agents comprises abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib.

[12] The method according to any one of [7] to

[11] above, wherein the non-human mammal is a dog or a cat.

[13] The method according to any one of [7] to

[12] above, wherein the non-human mammal has a tumor selected from the group consisting of hepatocellular carcinoma, intestinal adenocarcinoma, mucinous adenocarcinoma, malignant peripheral nerve sheath tumor, angiosarcoma, lung adenocarcinoma, adrenocortical carcinoma, adenocarcinoma, renal cell carcinoma, leiomyoma, lipoma, adrenocortical adenoma, hepatocellular adenoma, sarcoma, pancreatic islet cell carcinoma, lymphoma, transitional meningioma, giant cell tumor of tendon sheath, mast cell tumor, rectal adenocarcinoma, malignant melanoma, squamous cell carcinoma, hemangiopericytoma, urothelial carcinoma, T-cell lymphoma, thyroid carcinoma, synovial sarcoma, anal gland adenocarcinoma, gastrointestinal stromal tumor, histiocytic sarcoma, fibrosarcoma, thymoma, osteosarcoma, neuroendocrine carcinoma, and epithelial tumors.

[14] A method according to any one of [7] to

[13] above, in which the number of surviving cells in cultured cells after treatment with each molecular-targeted antitumor agent is determined, and the proliferation level is evaluated using the ratio of the number of surviving cells in cultured cells after treatment compared to the number of surviving cells in untreated cultured cells as an index.

[15] The method described in

[14] above, wherein a molecular targeted antitumor agent that has been shown to result in a ratio of surviving cell counts of cultured cells after treatment compared to the surviving cell counts of untreated cultured cells is selected as an antitumor agent to which the tumor of the non-human mammalian individual is sensitive. [Effects of the Invention]

[0011] According to the present invention, it is possible to efficiently screen for antitumor agents suitable for personalized treatment of tumor-bearing non-human mammals. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 1 (dog). [Figure 2] FIG. 2 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from intestinal adenocarcinoma (mucinous adenocarcinoma) in Case 2 (cat). [Figure 3]FIG. 3 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from malignant peripheral nerve sheath tumor in Case 3 (dog). [Figure 4] FIG. 4 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiosarcoma in Case 4 (dog). [Figure 5] FIG. 5 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from lung adenocarcinoma in Case 5 (dog). [Figure 6] FIG. 6 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from adrenocortical carcinoma in Case 6 (dog). [Figure 7] FIG. 7 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiosarcoma in Case 7 (dog). [Figure 8] FIG. 8 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from adenocarcinoma in Case 8 (dog). [Figure 9] FIG. 9 is a graph showing the cell growth inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from renal cell carcinoma in Case 9 (cat). [Figure 10] FIG. 10 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from leiomyoma in Case 10 (dog). [Figure 11] FIG. 11 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from a mixed tumor (adrenal cortical adenoma and lipoma) in Case 11 (dog). [Figure 12] FIG. 12 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular adenoma in Case 12 (dog). [Figure 13] FIG. 13 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from early urothelial carcinoma of Case 13 (dog). [Figure 14] FIG. 14 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from adrenocortical adenoma in Case 14 (dog). [Figure 15] FIG. 15 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from sarcoma NOS of Case 15 (cat). [Figure 16] FIG. 16 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from sarcoma NOS of Case 16 (dog). [Figure 17] Figure 17 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 17 (dog). The hepatocellular carcinoma in Figure 17 (Case 17-1) and the pancreatic islet cell carcinoma in Figure 18 (Case 17-2) were collected from the same dog. [Figure 18] Figure 18 is a graph showing the cell growth inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from pancreatic islet cell carcinoma (insulinoma) in Case 17 (dog). The hepatocellular carcinoma in Figure 17 (Case 17-1) and the pancreatic islet cell carcinoma in Figure 18 (Case 17-2) were collected from the same dog. [Figure 19] FIG. 19 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from lymphoma in Case 18 (dog). [Figure 20] FIG. 20 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from transitional meningioma of Case 19 (dog). [Figure 21] FIG. 21 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 20 (dog). [Figure 22] FIG. 22 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from giant cell tumor of tendon sheath in Case 21 (cat). [Figure 23] FIG. 23 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiosarcoma in Case 22 (dog). [Figure 24] FIG. 24 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from mast cell tumor of Case 23 (dog). [Figure 25]FIG. 25 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from rectal adenocarcinoma (recurrence) in Case 24 (dog). [Figure 26] FIG. 26 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from malignant melanoma in Case 25 (dog). [Figure 27] FIG. 27 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from squamous cell carcinoma in Case 26 (dog). [Figure 28] FIG. 28 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from lung adenocarcinoma in Case 27 (dog). [Figure 29] FIG. 29 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiopericytoma in Case 28 (dog). [Figure 30] FIG. 30 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiopericytoma in Case 29 (dog). [Figure 31] FIG. 31 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from renal cell carcinoma in Case 30 (cat). [Figure 32] FIG. 32 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 31 (dog). [Figure 33] FIG. 33 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from urothelial carcinoma in Case 32 (dog). [Figure 34] FIG. 34 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from adenocarcinoma in Case 33 (cat). [Figure 35] FIG. 35 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular adenoma in Case 34 (dog). [Figure 36] FIG. 36 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 35 (dog). [Figure 37]FIG. 37 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from T-cell lymphoma in Case 36 (cat). [Figure 38] FIG. 38 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from thyroid cancer in Case 37 (dog). [Figure 39] FIG. 39 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from malignant melanoma in Case 38 (dog). [Figure 40] FIG. 40 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from synovial sarcoma in Case 39 (dog). [Figure 41] FIG. 41 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from renal cell carcinoma in Case 40 (dog). [Figure 42] FIG. 42 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from anal gland adenocarcinoma in Case 41 (dog). [Figure 43] FIG. 43 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 42 (dog). [Figure 44] FIG. 44 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from lung adenocarcinoma in Case 43 (dog). [Figure 45] FIG. 45 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from synovial sarcoma in Case 44 (cat). [Figure 46] FIG. 46 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from gastrointestinal stromal tumor of Case 45 (dog). [Figure 47] FIG. 47 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 46 (dog). [Figure 48]Figure 48 is a graph showing the cell proliferation inhibitory effect of a molecular targeted antitumor agent on cultured cells derived from anal gland adenocarcinoma (dog) in Case 47. The anal gland adenocarcinoma (Case 47-1) in Figure 48 and the histiocytic sarcoma (Case 47-2) in Figure 49 were collected from the same dog. [Figure 49] Figure 49 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from histiocytic sarcoma (dog) in Case 47. Anal gland adenocarcinoma (Case 47-1) in Figure 48 and histiocytic sarcoma (Case 47-2) in Figure 49 were collected from the same dog. [Figure 50] FIG. 50 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from squamous cell carcinoma in Case 48 (dog). [Figure 51] FIG. 51 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from urothelial carcinoma of Case 49 (dog). [Figure 52] FIG. 52 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from squamous cell carcinoma in Case 50 (dog). [Figure 53] FIG. 53 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hemangiosarcoma in Case 51 (dog). [Figure 54] FIG. 54 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from fibrosarcoma in Case 52 (cat). [Figure 55] Figure 55 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from thymoma in Case 53 (cat). [Figure 56] FIG. 56 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from hepatocellular carcinoma in Case 54 (cat). [Figure 57] FIG. 57 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from osteosarcoma in Case 55 (dog). [Figure 58] FIG. 58 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from neuroendocrine carcinoma in Case 56 (dog). [Figure 59] FIG. 59 is a graph showing the cell proliferation inhibitory effect of a molecular-targeted antitumor agent on cultured cells derived from epithelial tumor (carcinoma) in Case 57 (cat). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. The present invention relates to a method for screening antitumor agents and an antitumor agent sensitivity test kit using a set (combination / panel) of molecular-targeted antitumor agents, which are useful for screening antitumor agents suitable for personalized treatment of tumor-bearing non-human mammals.

[0014] The set of molecularly targeted antitumor agents of the present invention includes abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib. The set of molecularly targeted antitumor agents of the present invention may further include at least one of toceranib and olaparib (toceranib or olaparib, or both). The set of molecularly targeted antitumor agents of the present invention may further include dabrafenib or encorafenib, or may further include both dabrafenib and encorafenib. The set of molecularly targeted antitumor agents of the present invention may include i) abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib, and ii) at least one selected from the group consisting of dabrafenib, encorafenib, toceranib, and olaparib. The set of molecularly targeted antitumor agents of the present invention may further include abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib, at least one of toceranib and olaparib, and dabrafenib or encorafenib. In one embodiment, the set of molecularly targeted antitumor agents of the present invention includes or consists of abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib. In another embodiment, the set of molecularly targeted antitumor agents of the present invention includes or consists of abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and dabrafenib. In one embodiment, the set of molecularly targeted antitumor agents according to the present invention may include other molecularly targeted antitumor agents in addition to the above-mentioned molecularly targeted antitumor agents. In another embodiment, the set of molecularly targeted antitumor agents according to the present invention may consist solely of the above-mentioned molecularly targeted antitumor agents.

[0015] Abemaciclib is a CDK4 / 6 inhibitor that exhibits inhibitory activity against cyclin-dependent kinase (CDK) 4 and CDK6, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Verzenio).

[0016] Alectinib is an ALK inhibitor that exhibits inhibitory activity against ALK, which is involved in cell proliferation, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Alecensa). When the ALK gene fuses with parts of other genes to form an ALK fusion gene, ALK is constantly activated in the ALK fusion protein produced from the ALK fusion gene, and this signal is known to promote carcinogenesis and cancer cell proliferation. ALK inhibitors can suppress tumor proliferation by inhibiting ALK activation in the ALK fusion protein produced from the ALK fusion gene.

[0017] Dabrafenib is a BRAF inhibitor that inhibits mutant BRAF kinase activity and is commercially available as an orally administered molecular-targeted antitumor agent (trade name: Tafinlar).

[0018] Encorafenib is a BRAF inhibitor that inhibits mutant BRAF kinase activity, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: BRAFTOVI).

[0019] Everolimus is an mTOR (Mammalian Target of Rapamycin) inhibitor that exhibits inhibitory activity against the mTOR protein, a regulatory factor involved in cancer cell proliferation and angiogenesis, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Afinitor).

[0020] Gefitinib is an epidermal growth factor receptor (EGFR) inhibitor that inhibits EGFR tyrosine kinase activity, and is commercially available as an orally administered molecular targeted drug (trade name: Iressa).

[0021] Lapatinib is an EGFR and HER2 inhibitor that exhibits inhibitory activity against EGFR and HER2 (human epidermal growth factor receptor 2), and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Tykerb).

[0022] Olaparib is a PARP inhibitor that exhibits inhibitory activity against polyadenosine 5'-diphosphate ribose polymerase (PARP), an enzyme that repairs damaged DNA, and is commercially available as an orally administered molecular-targeted antitumor agent (trade name: Lynparza).

[0023] Pazopanib is a multikinase inhibitor that exhibits inhibitory activity against multiple receptor tyrosine kinases (multikinases), mainly vascular endothelial growth factor receptor (VEGFR)-1, -2, and -3, platelet-derived growth factor receptor (PDGFR)-α and -β, and stem cell factor receptor c-Kit. It is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Votrient).

[0024] Toceranib is a multikinase inhibitor that exhibits inhibitory activity against multiple receptor tyrosine kinases (multikinases), namely VEGFR, PDGFR, and KIT, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Palladia).

[0025] Trametinib is a mitogen-activated protein kinase (MEK) inhibitor that exhibits inhibitory activity against mitogen-activated extracellular signal-related kinases (MEK) 1 and 2 in the MAPK pathway, and is commercially available as an orally administered molecular-targeted antitumor agent (drug name: Mekinist).

[0026] In the present invention, abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib itself may be used as part of the set of molecular-targeted antitumor agents according to the present invention. Alternatively, abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib may be included in the set of molecular-targeted antitumor agents according to the present invention and used in the form of a pharmaceutically acceptable salt, acid addition salt (e.g., hydrochloride, sulfate, nitrate, tosylate, etc.), or a hydrate or solvate thereof (e.g., DMSO adduct, ethanol adduct, etc.) of abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib, respectively. In the present invention, a set of molecularly targeted antitumor agents comprising abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib in the latter form also falls under the set of molecularly targeted antitumor agents according to the present invention comprising abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib. For example, a set of molecularly targeted antitumor agents including Verzenio, Alecensa, Tafinlar, Birafutovi, Afinitor, Iressa, Tykerb, Lynparza, Votrient, Palladia, or Mekinist corresponds to the set of molecularly targeted antitumor agents according to the present invention, which also includes abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib.

[0027] By using the set of molecularly targeted antitumor agents of the present invention, in vitro screening of antitumor agents suitable for personalized treatment of tumor-bearing non-human mammals can provide one or more antitumor agents that are expected to have a therapeutic effect against the tumor with a high probability.

[0028] The present invention provides an in vitro screening method for antitumor agents to be used in non-human mammals, using the set of molecular-targeted antitumor agents of the present invention. More specifically, the present invention provides an in vitro screening method for antitumor agents for personalized treatment of tumor-bearing non-human mammals, using the set of molecular-targeted antitumor agents of the present invention. "Personalized treatment" in the present invention means providing treatment appropriate for the tumor of each individual tumor-bearing non-human mammal. In other words, the antitumor agent selected by the screening method of the present invention is an antitumor agent appropriate for a specific tumor-bearing non-human mammal.

[0029] The method for screening antitumor agents according to the present invention may be carried out using the kit according to the present invention, which will be described later and which includes the set of molecular-targeted antitumor agents according to the present invention.

[0030] In the method for screening antitumor agents according to the present invention, cultured cells derived from tumors in non-human mammals are treated (drug treatment) with each of the molecular-targeted antitumor agents included in the set of molecular-targeted antitumor agents according to the present invention. The concentration of abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib added to the cultured cells (final concentration in the medium) is not particularly limited, but can typically be appropriately determined in the range of 0.001 μM to 1 mM, and can be determined, for example, within the range of the trough to maximum blood concentration of the corresponding drug in humans. The concentration of each molecular targeted antitumor agent added is not limited to the following, but for example, the concentration of abemaciclib added may be 0.05 μM to 5 μM, the concentration of alectinib added may be 0.1 μM to 10 μM, the concentration of dabrafenib added may be 0.1 μM to 10 μM, the concentration of encorafenib added may be 0.1 μM to 10 μM, and the concentration of everolimus added may be 0.01 μM to 1 μM. The concentration of gefitinib may be 0.05 μM to 5 μM, the concentration of lapatinib may be 0.1 μM to 10 μM, the concentration of olaparib may be 0.1 μM to 10 μM, the concentration of pazopanib may be 2 μM to 200 μM, the concentration of toceranib may be 0.01 μM to 1 μM, and the concentration of trametinib may be 0.003 μM to 0.3 μM. In one embodiment, the addition concentration of abemaciclib may be 0.4 μM to 0.6 μM, the addition concentration of alectinib may be 0.8 μM to 1.2 μM, the addition concentration of dabrafenib may be 0.8 μM to 1.2 μM, the addition concentration of encorafenib may be 0.8 μM to 1.2 μM, the addition concentration of everolimus may be 0.08 μM to 0.12 μM, and the addition concentration of gefitinib may be 0.8 μM to 1.2 μM. The added concentration may be 0.4 μM to 0.6 μM, the added concentration of lapatinib may be 0.8 μM to 1.2 μM, the added concentration of olaparib may be 0.8 μM to 1.2 μM, the added concentration of pazopanib may be 16 μM to 24 μM, the added concentration of toceranib may be 0.08 μM to 0.12 μM, and the added concentration of trametinib may be 0.024 μM to 0.036 μM.In one embodiment, the added concentrations of abemaciclib, alectinib, dabrafenib, encorafenib, everolimus, gefitinib, lapatinib, olaparib, pazopanib, toceranib, or trametinib may be 0.5 μM, 1 μM, 1 μM, 1 μM, 0.1 μM, 0.5 μM, 1 μM, 1 μM, 20 μM, 0.1 μM, and 0.03 μM, respectively.

[0031] The cultured cells to be drug-treated may be cultured cells derived from a tumor (e.g., a tumor harvested from the non-human mammalian individual) present in a non-human mammalian individual to be subjected to personalized treatment with an anti-tumor agent (i.e., the subject of personalized treatment). Alternatively, the cultured cells to be drug-treated may be cultured cells derived from a tumor (typically, an allogeneic tumor) harvested from another non-human mammalian individual of the same tumor type as the tumor present in the non-human mammalian individual to be subjected to personalized treatment with an anti-tumor agent. Such an allogeneic tumor may be a tumor harvested from another individual of the same or different breed, belonging to the same biological species as the non-human mammalian individual to be subjected to personalized treatment with an anti-tumor agent. The cultured cells to be drug-treated are cultured tumor cells.

[0032] In the present invention, a tumor present in a non-human mammalian individual to be subjected to personalized therapy and to be treated may be a malignant tumor (cancer) or a benign tumor, and may also be a precancerous lesion, primary cancer, recurrent cancer, advanced cancer, or metastatic cancer. A tumor present in a non-human mammalian individual to be treated may be an adenocarcinoma, such as a mucinous adenocarcinoma. Examples of adenocarcinomas include, but are not limited to, intestinal adenocarcinoma, lung adenocarcinoma, adrenocortical adenoma, hepatocellular adenoma, rectal adenocarcinoma, thyroid cancer, anal gland adenocarcinoma, and thymoma, as well as other adenocarcinomas and adenocarcinomas with unspecified primary site, histological type, and other classification information. A tumor present in a non-human mammalian individual to be treated may be a sarcoma. Examples of sarcomas include, but are not limited to, angiosarcoma, synovial sarcoma, histiocytic sarcoma, and fibrosarcoma, as well as other sarcomas and sarcomas with unspecified primary site, histological type, and other classification information. In one embodiment, examples of tumors to be treated that are present in a non-human mammal include, but are not limited to, hepatocellular carcinoma, intestinal adenocarcinoma, mucinous adenocarcinoma, malignant peripheral nerve sheath tumor, angiosarcoma, lung adenocarcinoma, adrenocortical carcinoma, adenocarcinoma, renal cell carcinoma, leiomyoma, lipoma, adrenocortical adenoma, hepatocellular adenoma, sarcoma, pancreatic islet cell carcinoma, lymphoma, transitional meningioma, giant cell tumor of tendon sheath, mast cell tumor, rectal adenocarcinoma, malignant melanoma, squamous cell carcinoma, hemangiopericytoma, urothelial carcinoma, T-cell lymphoma, thyroid carcinoma, synovial sarcoma, anal gland adenocarcinoma, gastrointestinal stromal tumor (GIST), histiocytic sarcoma, fibrosarcoma, thymoma, osteosarcoma, neuroendocrine carcinoma, and epithelial tumors.

[0033] In the present invention, the non-human mammal is not particularly limited, but examples include non-human primates such as gorillas, chimpanzees, and orangutans; domestic mammals such as horses, cows, camels, donkeys, sheep, and pigs; rodents such as mice, hamsters, rats, guinea pigs, rabbits, and ferrets; dogs, cats, etc. Non-human mammals to be subjected to personalized therapy with antitumor agents are preferably rodents such as mice, hamsters, rats, guinea pigs, rabbits, and ferrets, or small animals such as dogs and cats, and are particularly preferably dogs or cats. Non-human mammals to be subjected to personalized therapy with antitumor agents have a tumor.

[0034] The cultured cells to be treated with the above-mentioned drugs are preferably organoids. Organoids are cell cultures derived from living tissues, and retain characteristics similar to those of the original living tissues (for example, their structure, function, drug sensitivity, gene expression profile, and / or tumorigenicity). The organoids used in the present invention can be prepared from tumor cells or tumor tissues. The organoids used in the present invention may be 3-dimensional organoids or 2.5-dimensional organoids, but are not limited thereto. 3-dimensional organoids are 3-dimensional cell cultures derived from living tissues that retain characteristics similar to those of the original living tissues. 2.5-dimensional organoids are 2-dimensional cell cultures derived from living tissues that retain characteristics similar to those of the original living tissues.

[0035] Organoids can be prepared according to known methods for preparing organoids. For example, 2.5-dimensional organoids can be prepared according to the methods described in the Examples below. Specifically, for example, tumor tissue is treated with collagenase, and then cells are separated using a cell strainer or the like. The obtained cells are cultured in an organoid culture medium (for example, at 36 to 38°C, typically 37°C, for 5 to 14 days), thereby preparing 2.5-dimensional organoids. In a preferred embodiment, the liquid medium used for organoid culture can include epidermal growth factor (EGF) (final concentration 50 ng / ml), N-acetyl-L-cysteine ​​(final concentration 1 mM), nicotinamide (final concentration 10 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (final concentration 10 mM), GlutaMax (final concentration 1%), A83-01 (final concentration 0.5 μM), penicillin-streptomycin (final concentration 1%), fetal bovine serum (FBS) (final concentration 5%), and Advanced Dulbecco's Modified Eagle's Medium (DMEM) / F12. Alternatively, organoids can be produced by culturing tumor cells in body fluids (including, but not limited to, for example, the urine of bladder cancer patients, the ascites of cancer patients, etc.). Organoids may be produced with reference to the organoid production methods described in, for example, Patent Documents 1-2 and Non-Patent Documents 1-5. In the present invention, 2.5-dimensional organoids may be cultures of tumor cells in a liquid medium (organoid culture medium) containing epidermal growth factor (EGF) (final concentration 50 ng / ml), N-acetyl-L-cysteine ​​(final concentration 1 mM), nicotinamide (final concentration 10 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (final concentration 10 mM), GlutaMax (final concentration 1%), A83-01 (final concentration 0.5 μM), penicillin-streptomycin (final concentration 1%), fetal bovine serum (FBS) (final concentration 5%), and Advanced Dulbecco's Modified Eagle's Medium (DMEM) / F12.

[0036] In the antitumor agent screening method of the present invention, it is preferable to evaluate the proliferation level of cultured cells (e.g., organoids) after drug treatment with each molecular-targeted antitumor agent. In a preferred embodiment, the proliferation level of cultured cells is evaluated by determining the number of surviving cells of cultured cells (drug-treated group) after treatment with each molecular-targeted antitumor agent (after incubation for a certain drug treatment time, such as 72 hours), and determining the number of surviving cells of untreated cultured cells (cultured cells cultured in the same medium, such as organoid culture medium, as described above, except that no molecular-targeted antitumor agent is added) as a control. The ratio of the number of surviving cells of cultured cells after treatment compared to the number of surviving cells of untreated cultured cells (control group) (cell viability) can be used as an index. A cell viability of less than 100% indicates that the proliferation of cultured cells is inhibited, and the lower the cell viability, the greater the inhibition of the proliferation of cultured cells. If inhibition of the proliferation of cultured cells after treatment is demonstrated, the molecular-targeted antitumor agent used can be selected as an antitumor agent to which the tumor of the non-human mammalian individual to be subjected to personalized therapy is sensitive. In one embodiment, in the method for screening antitumor agents according to the present invention, if the cell viability is 75% or less, the molecularly targeted antitumor agent used can be selected as an antitumor agent to which the tumor in the non-human mammalian individual to be subjected to personalized therapy is sensitive, i.e., an antitumor agent (promising drug) that is expected to have a therapeutic effect against the tumor. In a more preferred embodiment, in the method for screening antitumor agents according to the present invention, if the cell viability is 50% or less, the molecularly targeted antitumor agent used can be selected as an antitumor agent to which the tumor in the non-human mammalian individual to be subjected to personalized therapy is particularly sensitive, i.e., an antitumor agent (effective drug) that is expected to have a high therapeutic effect against the tumor.

[0037] Molecularly targeted antitumor agents selected by the antitumor agent screening method of the present invention are highly useful as antitumor agents (candidate therapeutic agents) to be used for the treatment of tumors in non-human mammalian individuals (individualized treatment). Generally, even therapeutic agents that are considered effective vary in effectiveness for each individual. Therefore, for personalized treatment, it is preferable to narrow down the candidate therapeutic agents to those expected to be therapeutically effective for each individual, select a therapeutic agent from among them, and administer it to the individual. The antitumor agent screening method of the present invention makes it possible to select with a high probability one or more drugs expected to be therapeutically effective for each individual, thereby efficiently providing antitumor agents that serve as candidate therapeutic agents for personalized treatment of tumor-bearing non-human mammalian individuals.

[0038] The present invention also provides a method for treating tumors in non-human mammals (personalized treatment), by selecting a molecularly targeted anti-tumor agent as an anti-tumor agent to which a tumor in a non-human mammal is sensitive using the anti-tumor agent screening method of the present invention, and administering the selected molecularly targeted anti-tumor agent to the non-human mammal. In one embodiment, in the treatment method of the present invention, a molecularly targeted anti-tumor agent that shows a cell viability rate of 75% or less is selected as an anti-tumor agent to which a tumor in a non-human mammal to be subjected to personalized treatment is sensitive, and administered to the non-human mammal. In a more preferred embodiment, in the treatment method of the present invention, a molecularly targeted anti-tumor agent that shows a cell viability rate of 50% or less is selected as an anti-tumor agent to which a tumor in a non-human mammal to be subjected to personalized treatment is sensitive, and administered to the non-human mammal.

[0039] The dosage of the molecularly targeted antitumor agent is not particularly limited, and can be appropriately determined depending on the body weight, condition, etc. of the non-human mammal, preferably with reference to the dosage of a human or veterinary pharmaceutical containing the molecularly targeted antitumor agent as an active ingredient. The route of administration to the non-human mammal is not particularly limited, but is preferably oral (oral administration). The non-human mammal and its tumor to be subjected to personalized treatment in the treatment method of the present invention are as described above.

[0040] It is further preferable to evaluate the actual efficacy (antitumor effect) of the administered therapeutic agent in the non-human mammalian individual after administration. The actual efficacy of the therapeutic agent can be evaluated, for example, based on the change in tumor volume over time. When evaluating the actual efficacy of the therapeutic agent, it is also preferable to evaluate side effects, etc. at the same time. Based on such evaluation, a therapeutic agent that is particularly suitable for a non-human mammalian individual bearing a tumor can be selected.

[0041] The present invention also provides a kit comprising the set of molecularly targeted antitumor agents according to the present invention, which can be used for screening antitumor agents for tumor-bearing non-human mammals. In one embodiment, the present invention relates to an antitumor agent sensitivity test kit for personalized treatment of tumor-bearing non-human mammals, which comprises the set of molecularly targeted antitumor agents according to the present invention. In one embodiment, the present invention relates to a kit for screening antitumor agents for personalized treatment of tumor-bearing non-human mammals, which comprises the set of molecularly targeted antitumor agents according to the present invention.

[0042] The kit of the present invention includes a set of molecularly targeted antitumor agents of the present invention, specifically a set of molecularly targeted antitumor agents including abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib. In one embodiment, the set of molecularly targeted antitumor agents included in the kit of the present invention may further include at least one of toceranib and olaparib (toceranib or olaparib, or both). The set of molecularly targeted antitumor agents included in the kit of the present invention may further include dabrafenib or encorafenib, or may further include both dabrafenib and encorafenib. In one embodiment, the kit of the present invention may comprise a set of molecularly targeted antitumor agents comprising i) abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib, and ii) at least one selected from the group consisting of dabrafenib, encorafenib, toceranib, and olaparib.The kit of the present invention may comprise a set of molecularly targeted antitumor agents comprising abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib, at least one of toceranib and olaparib, and dabrafenib or encorafenib. In one embodiment, the kit of the present invention comprises a set of molecularly targeted antitumor agents comprising or consisting of abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib. In one embodiment, the kit of the present invention comprises a set of molecularly targeted antitumor agents comprising or consisting of abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and dabrafenib. In one embodiment, the set of molecularly targeted antitumor agents included in the kit of the present invention may include other molecularly targeted antitumor agents in addition to the above-mentioned molecularly targeted antitumor agents. In another embodiment, the set of molecularly targeted antitumor agents included in the kit of the present invention may consist solely of the above-mentioned molecularly targeted antitumor agents.

[0043] The kit of the present invention may further comprise instructions for using the kit in screening for antitumor agents in tumor-bearing non-human mammals, or in testing for susceptibility to antitumor agents for personalized treatment of tumor-bearing non-human mammals.

[0044] The kit of the present invention can be used in the method of screening for antitumor agents of the present invention, and based on the results, typically one or more molecularly targeted antitumor agents can be selected as antitumor agents to which the tumor in the non-human mammal is sensitive. The non-human mammal and its tumor to be subjected to personalized therapy in relation to the kit of the present invention are described above. The kit of the present invention can be used to test the sensitivity of a tumor-bearing non-human mammal to an antitumor agent for personalized therapy. Therefore, the present invention also provides a method of testing the sensitivity of a tumor-bearing non-human mammal to an antitumor agent for personalized therapy using the kit of the present invention. [Example]

[0045] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0046] [Example 1] Screening using 2D cultured cells For spontaneous tumors in dogs and cats, only a limited number of molecularly targeted drugs have been used for one or two tumor types, and molecularly targeted drug therapy has not yet been established. Furthermore, there is little information on the molecular abnormalities of each tumor type in dogs and cats, or on effective antitumor agents. Therefore, we first narrowed down the target molecules expected to be effective by performing a cell growth inhibition assay on two-dimensional (2D) cultured cells using five strains of two culturable canine tumor cell lines and a library of 364 inhibitors.

[0047] The cell lines used were as follows: - Canine bladder cancer cell lines: OMTCC (OM), Sora, K9TCC-Pu-Sh (Sh) - Canine melanoma cell lines: Pu(CMM1) strain (CMM1 strain), KMeC strain

[0048] The OM, Sora, and Sh lines are 2D culture cell lines that were established as cell lines capable of long-term culture after more than 50 passages by primary culture of tumor cells surgically removed from dogs with canine bladder cancer. The OM and Sora lines were independently created by the present inventors, while the Sh line was created and provided by Dr. Debbie Knapp of Purdue University (USA).

[0049] The CMM1 and KMeC lines are 2D culture cell lines that were independently established by the present inventors as cell lines capable of long-term culture after more than 50 passages by primary culture of tumor cells surgically removed from dogs with canine melanoma.

[0050] In this screening test, the OM, Sora, CMM1, and KMeC cell lines were cultured in RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing L-glutamine and phenol red, supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 50 μg / mL gentamicin (Sigma-Aldrich) as an antibiotic. The Sh cell line was cultured in D-MEM / Ham's F-12 medium (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 5% fetal bovine serum (FBS) (Thermo Fisher Scientific). All of the OM, Sora, Sh, CMM1, and KMeC cell lines were cultured in an incubator at 37°C under 5% CO2 conditions.

[0051] The inhibitor library used was the 364 inhibitors contained in Standard Inhibitor Kits 1–4 (provided by the Molecular Profiling Committee, Grant-in-Aid for Scientific Research on Innovative Areas “Advanced Animal Model Support (AdAMS)” from the Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI 16H06276)); these are libraries of a systematic collection of various inhibitors.

[0052] For the cell growth inhibition assay using the inhibitor library, OM, Sora, Sh, CMM1, and KMeC cells were seeded in 96-well plates at 3,000 cells / well, 11,000 cells / well, 2,000 cells / well, 3,000 cells / well, and 3,000 cells / well, respectively. All cell lines were seeded at a density that allowed logarithmic growth for 72 hours. Twenty-four hours after seeding, 364 inhibitors were added to individual wells at a concentration of 10 μM (inhibitor-treated group). Each inhibitor was dissolved in 0.001% dimethyl sulfoxide (DMSO) for a final addition concentration. As a control, the same concentration of DMSO without inhibitors was added to the cell culture medium (control group).

[0053] Forty-eight hours after the addition of inhibitors, the number of viable cells in each well was determined using sulforhodamine B (SRB) assay for canine bladder cancer cell lines (OM, Sora, and Sh) and WST-1 assay for canine melanoma cell lines (CMM1 and KMeC). To evaluate the growth inhibitory effect of each inhibitor, the ratio of viable cells in the inhibitor-treated group to the number of viable cells in the control group (cell viability) was calculated.

[0054] As a result, for each cell line, the number of inhibitors that showed cell viability less than 100% compared to the control group was as follows: - OM strain: 244 agents - Sora strain: 261 agents - Sh strain: 229 agents - CMM1 strain: 302 agents - KMeC strain: 276 agents

[0055] Among the inhibitors that resulted in less than 100% cell viability in one or more cell lines, we excluded cytotoxic anticancer drugs. Furthermore, we excluded inhibitors for which there are currently no commercially available drugs (human or non-human animal drugs) that inhibit the target molecule or target pathway of the inhibitor. We then selected target molecules or target pathways targeted by two or more of the remaining inhibitors.

[0056] Molecularly targeted drugs with inhibitory activity against the target molecules or pathways selected in this way were selected from a wide range of commercially available drugs, not limited to drugs containing inhibitors from the inhibitor library mentioned above as active ingredients. From the selected commercially available drugs, the following 11 types of commercially available drugs were selected, which have small dosage forms that can be administered in divided doses to dogs and cats (Table 1).

[0057] [Table 1]

[0058] [Example 2] Screening using organoids -1) In this example, in order to select a molecular targeted drug suitable for the tumor of each patient animal, organoids were prepared for each patient animal using tumor tissue collected from the patient animal, and 10 of the drugs (molecular targeted antitumor agents) that are active ingredients of the pharmaceuticals shown in Table 1 (including dabrafenib or encorafenib, but the other drugs are common) were added to the organoids, and a cell proliferation inhibition assay was performed.

[0059] Organoids were prepared according to the method described in Non-Patent Document 5 (Abugomaa A. et al., Biomedicine & Pharmacotherapy, 154 (2022) 113597). Specifically, tumor tissue was surgically removed from each patient animal, which was a dog or cat with a spontaneous tumor. The tumor tissue obtained from each patient was minced and then treated with Liberase containing collagenase I and II. TM Advanced TH (Roche Diagnostics) TM Collagenase treatment was performed in DMEM medium (Thermo Fisher Scientific) with shaking at 37°C. After collagenase treatment, the tumor tissue was passed through a cell strainer (70 μm) to separate individual cells, and the resulting filtrate (cell solution) was centrifuged at 600 × g for 3 minutes to recover the cells. The recovered cells were washed 3-4 times with PBS and then cultured in 2.5D organoid culture medium (Table 2) at 37°C.

[0060] [Table 2]

[0061] After culturing the cells in 2.5D organoid culture medium for 7–10 days, the resulting 2.5D organoids were seeded at 1,000 cells per well in a 96-well plate. The organoids were treated with each molecular-targeted antitumor agent by adding the agent and incubating at 37°C for 72 hours (drug-treated group). The drug concentrations (final concentrations in the medium) were set as shown in Table 3, within the range of the trough to maximum blood concentrations in humans for the drugs shown in Table 1. As a control, organoids (1,000 cells) were incubated in the same 2.5D organoid culture medium as above without the addition of the molecular-targeted antitumor agent at 37°C for 72 hours (control group).

[0062] [Table 3]

[0063] After drug treatment, the number of viable cells in each well was determined using the Alamar Blue assay. To evaluate the cell proliferation inhibitory effect of each drug, the ratio of viable cells in the drug-treated group to the number of viable cells in the control group (cell viability) was calculated. For each patient animal, drugs showing a cell viability of 75% or less were selected as promising drugs, and drugs showing a cell viability of 50% or less, indicating a stronger cell proliferation inhibitory effect, were selected as effective drugs. The results are shown in Table 4 and Figures 1 to 56.

[0064] [Table 4] TIFF2025133103000005.tif90165

[0065] A cell proliferation inhibition assay in organoids using a panel of 10 drugs showed promising results in all 54 cases (100%), and 51 of the 54 cases (94.4%) showed at least one effective drug. Furthermore, all 54 cases (100%) showed at least one effective or promising drug from the following: abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib.

[0066] [Example 3] Screening using organoids - 2) In this example, using a method similar to that used in Example 2, 10 types of drugs were added to organoids prepared for each patient animal (Cases 55-57), and cell proliferation inhibition assays were performed to select molecular targeted drugs suitable for the tumors of each patient animal.

[0067] The results are shown in Figures 57 to 59. For case 55, osteosarcoma (dog), there were two promising drugs and one effective drug (Figure 57). For case 56, neuroendocrine carcinoma (dog), there were three promising drugs and one effective drug (Figure 58). For case 57, epithelial tumor (cat), there were five promising drugs and one effective drug (Figure 59).

[0068] Cell proliferation inhibition assays in organoids using a panel of 10 drugs revealed promising and effective drugs in all three cases (100%).Furthermore, all three cases (100%) showed effective or promising drugs, including one or more of the following: abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib.

[0069] Therefore, the results of Examples 2 and 3 demonstrated that by subjecting the above-mentioned combinations of 7 to 11 drugs to in vitro cell growth inhibition assays using cultured cells, including organoids, it is possible to efficiently select candidates for personalized molecular-targeted antitumor agents for tumors in individual patient animals.

Claims

1. An antitumor agent sensitivity test kit for personalized treatment of a non-human mammal having a tumor, comprising a set of molecular targeted antitumor agents including abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib.

2. The kit of claim 1, wherein the set of molecular targeted antitumor agents further comprises at least one of toceranib and olaparib.

3. The kit according to claim 1 or 2, wherein the set of molecular-targeted antitumor agents further comprises dabrafenib or encorafenib.

4. The kit of claim 1 , wherein the set of molecularly targeted antitumor agents comprises abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib.

5. The kit of claim 1 , wherein the non-human mammal is a dog or a cat.

6. The kit of claim 1, wherein the non-human mammal has any tumor selected from the group consisting of hepatocellular carcinoma, intestinal adenocarcinoma, mucinous adenocarcinoma, malignant peripheral nerve sheath tumor, angiosarcoma, lung adenocarcinoma, adrenocortical carcinoma, adenocarcinoma, renal cell carcinoma, leiomyoma, lipoma, adrenocortical adenoma, hepatocellular adenoma, sarcoma, pancreatic islet cell carcinoma, lymphoma, transitional meningioma, tendon sheath giant cell tumor, mast cell tumor, rectal adenocarcinoma, malignant melanoma, squamous cell carcinoma, hemangiopericytoma, urothelial carcinoma, T-cell lymphoma, thyroid carcinoma, synovial sarcoma, anal gland adenocarcinoma, gastrointestinal stromal tumor, histiocytic sarcoma, fibrosarcoma, thymoma, osteosarcoma, neuroendocrine carcinoma, and epithelial tumors.

7. A method for screening antitumor agents for personalized treatment of a non-human mammal having a tumor using a set of molecular-targeted antitumor agents, comprising: the set of molecular targeted antitumor agents includes abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, and trametinib; treating cultured cells derived from a tumor carried by a non-human mammalian individual or derived from an allogeneic tumor of the same tumor type as the tumor carried by said non-human mammalian individual with each of the molecular-targeted anti-tumor agents of said set; The proliferation level of cultured cells after treatment with each molecular target antitumor agent was evaluated. A method comprising selecting a molecularly targeted antitumor agent that inhibits the proliferation of cultured cells after treatment as an antitumor agent to which the tumor in the non-human mammal individual is sensitive.

8. The method of claim 7, wherein the cultured cells are organoids.

9. The method of claim 7, wherein the set of molecularly targeted antitumor agents further comprises at least one of toceranib and olaparib.

10. The method of claim 7 or 9, wherein the set of molecular targeted antitumor agents further comprises dabrafenib or encorafenib.

11. 8. The method of claim 7, wherein the set of molecular targeted antitumor agents comprises abemaciclib, alectinib, everolimus, gefitinib, lapatinib, pazopanib, trametinib, toceranib, olaparib, and encorafenib.

12. 8. The method of claim 7, wherein the non-human mammal is a dog or a cat.

13. The method of claim 7, wherein the non-human mammal has any tumor selected from the group consisting of hepatocellular carcinoma, intestinal adenocarcinoma, mucinous adenocarcinoma, malignant peripheral nerve sheath tumor, angiosarcoma, lung adenocarcinoma, adrenocortical carcinoma, adenocarcinoma, renal cell carcinoma, leiomyoma, lipoma, adrenocortical adenoma, hepatocellular adenoma, sarcoma, pancreatic islet cell carcinoma, lymphoma, transitional meningioma, giant cell tumor of tendon sheath, mast cell tumor, rectal adenocarcinoma, malignant melanoma, squamous cell carcinoma, hemangiopericytoma, urothelial carcinoma, T-cell lymphoma, thyroid carcinoma, synovial sarcoma, anal gland adenocarcinoma, gastrointestinal stromal tumor, histiocytic sarcoma, fibrosarcoma, thymoma, osteosarcoma, neuroendocrine carcinoma, and epithelial tumors.

14. The method of claim 7, wherein the number of surviving cells in culture after treatment with each molecular targeted antitumor agent is determined, and the proliferation level is evaluated using the ratio of the number of surviving cells in culture after treatment compared to the number of surviving cells in untreated cultured cells as an index.

15. The method of claim 14, wherein a molecular targeted antitumor agent that has been shown to have a ratio of cell viability of cultured cells after treatment compared to cell viability of untreated cultured cells of 50% or less is selected as an antitumor agent to which the tumor of the non-human mammalian individual is sensitive.

Images