Combination drugs for treating kidney cancer and enhancers of the therapeutic effect of tyrosine kinase inhibitors

Abstract

A combination drug for treating kidney caner, said combination drug comprising a tyrosine kinase inhibitor combined with a dipeptidyl peptidase 4 inhibitor; and a therapeutic effect enhancer for a tyrosine kinase inhibitor, said therapeutic effect enhancer comprising a dipeptidyl peptidase 4 inhibitor and enhancing the therapeutic effect of the tyrosine kinase inhibitor on kidney cancer.

Description

Technical Field

[0001] The present invention relates to a combined medicine for treating renal cancer and a therapeutic effect enhancer of a tyrosine kinase inhibitor for enhancing the therapeutic effect of a tyrosine kinase inhibitor against renal cancer.

Background Art

[0002] Renal cell carcinoma (RCC) is the most common tumor among adult renal cancers, and its incidence currently exceeds 400,000 cases worldwide (see Non-Patent Documents 1 and 2). The 5-year survival rate of RCC patients who can be radically resected is usually 90% or more, while the 5-year survival rate of metastatic RCC patients is 10-20% (see Non-Patent Document 3). In recent years, molecular target drugs including immune checkpoint inhibitors and tyrosine kinase inhibitors (TKIs) have come to be used, but further improvement in the therapeutic effect is required to extend the average survival period of RCC patients.

[0003] With the recent progress of cancer research, it has become clear that cancer stem-like cells (CSCs) have infinite cell division and self-renewal ability (see Non-Patent Document 4). CSCs express cancer stem cell-related genes such as CD44, CD133, OCT3 / 4, aldehyde dehydrogenase 1 (ALDH1), and CXC-chemokine receptor 4 (CXCR4), and these are often associated with tumor progression and cancer treatment resistance. Interleukin-6 (IL6) is a pleiotropic cytokine, and it is known that the treatment with a tyrosine kinase inhibitor (TKI) induces the secretion of IL6 and the activation of the AKT-mammalian target of rapamycin (mTOR) pathway, and its overexpression may be a pathological cause of TKI resistance in RCC (see Non-Patent Document 5).

[0004] Dipeptidyl peptidase IV (DPP4) / CD26 is an endogenous membrane glycoprotein and serine exopeptidase that has recently been shown to be involved in the transformation of solid tumors, including multiple myeloma, into cancer stem cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Siegel, RL, Miller, KD & Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 68, 7-30 (2018) [Non-Patent Document 2] Bray, F. et. al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394-424 (2018) [Non-Patent Document 3] Ghatalia, P, Zibelman, M, Geynisman, DM, Plimack, ER Checkpoint Inhibitors for the Treatment of Renal Cell Carcinoma. Current treatment options in oncology 18, 7 (2017) [Non-Patent Document 4] Clevers, H. The cancer stem cell: premises, promises and challenges. Nat. Med. 17, 313-319 (2011) [Non-Patent Document 5] Ishibashi, K. et al. Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade. Oncotarget 8, 55230-55245 (2017) [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to solve the aforementioned conventional problems and achieve the following objectives. Specifically, the present invention aims to provide a combination drug for treating renal cancer that has excellent therapeutic effects against renal cancer, and a tyrosine kinase inhibitor therapeutic effect enhancer that can enhance the therapeutic effect of tyrosine kinase inhibitors against renal cancer. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objective, the present inventors have found that combining a dipeptidyl peptidase 4 inhibitor with a tyrosine kinase inhibitor can enhance the therapeutic effect of tyrosine kinase inhibitors on renal cancer, including tyrosine kinase inhibitor-resistant renal cancer.

[0008] The means to solve the aforementioned problem are as follows: <1> This is a combination drug for treating kidney cancer, characterized by the use of a tyrosine kinase inhibitor and a dipeptidyl peptidase 4 inhibitor in combination. <2> In the individual, the above <1> This is a method for treating kidney cancer characterized by administering the combination drugs described above. <3> This is a tyrosine kinase inhibitor therapeutic enhancer that contains a dipeptidyl peptidase 4 inhibitor and is characterized by enhancing the therapeutic effect of tyrosine kinase inhibitors against renal cancer. <4> In the individual, the above <3> This method enhances the therapeutic effect of tyrosine kinase inhibitors for renal cancer, characterized by administering a therapeutic effect enhancer for tyrosine kinase inhibitors described above. [Effects of the Invention]

[0009] According to the present invention, the aforementioned problems of the conventional approach can be solved, and a combination drug for treating renal cancer having excellent therapeutic effects against renal cancer can be provided, as well as a therapeutic effect enhancer for tyrosine kinase inhibitors that can enhance the therapeutic effect of tyrosine kinase inhibitors against renal cancer. [Brief explanation of the drawing]

[0010] [Figure 1a] Figure 1a shows the results of a correlation analysis (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids) of the expression levels of DPP4 and cancer stem cell-related genes (CD44). [Figure 1b] Figure 1b shows the results of a correlation analysis (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids) of the expression levels of DPP4 and cancer stem cell-related genes (CD133) in RCC patient-derived spheroids. [Figure 1c] Figure 1c shows the results of a correlation analysis (co-expression of DPP4 and cancer stem cell-related genes (OCT3 / 4)) performed on spheroids derived from RCC patients. [Figure 1d] Figure 1d shows the results of a correlation analysis (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids) of the expression levels of DPP4 and cancer stem cell-related genes (CXCR4) in RCC patient-derived spheroids. [Figure 1e]Figure 1e shows the results of a correlation analysis of the expression levels of DPP4 and the cancer stem cell-related gene (ALDH1A1) in spheroids derived from RCC patients (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids). [Figure 1f] Figure 1f shows the results of a correlation analysis of the expression levels of DPP4 and the cancer stem cell-related gene (ALDH1A2) in spheroids derived from RCC patients (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids). [Figure 1g] Figure 1g shows the results of a correlation analysis of the expression levels of DPP4 and the cancer stem cell-related gene (ALDH1A3) in spheroids derived from RCC patients (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids). [Figure 1h] Figure 1h shows the results of a correlation analysis of the expression levels of DPP4 and the cancer stem cell-related gene (IL6) in spheroids derived from RCC patients (co-expression of DPP4 and cancer stem cell-related genes in patient-derived RCC spheroids). [Figure 2a] Figure 2a shows the results of representative morphological features, hematoxylin and eosin (H / E) staining, and DPP4 immunohistochemical staining in primary RCC tumors and their spheroids in RCC-A and RCC-B patients with high DPP4 expression. All panels are at the same magnification, and the scale bar is 50 μm. [Figure 2b] Figure 2b shows the results of examining the combined effect of SUN and the DPP4 inhibitor SITA on the cell viability of RCC-A spheroids. [Figure 2c] Figure 2c shows the results of examining the combined effect of SUN and the DPP4 inhibitor SITA on the cell viability of RCC-B spheroids. [Figure 2d] Figure 2d shows the results of examining the DPP4 expression levels in RCC-A spheroids treated with control siRNA (siControl) or DPP4-specific siRNA (siDPP4 #1 or #2). [Figure 2e]Figure 2e shows the results of examining the DPP4 expression levels in RCC-B spheroids treated with control siRNA (siControl) or DPP4-specific siRNA (siDPP4 #1 or #2). [Figure 2f] Figure 2f shows the results of examining the effect of DPP4-specific siRNA on the cell viability of RCC-A spheroids treated with SUN. [Figure 2g] Figure 2g shows the results of examining the effect of DPP4-specific siRNA on the cell viability of RCC-B spheroids treated with SUN. [Figure 3a] Figure 3a shows the results of comparing the mRNA expression levels of DPP4 in ACHN-R cells and its parental strain (ACHN cells). [Figure 3b] Figure 3b shows the results of comparing the mRNA expression levels of the CSC-related gene (CD44) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3c] Figure 3c shows the results of comparing the mRNA expression levels of the CSC-related gene (CD133) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3d] Figure 3d shows the results of comparing the mRNA expression levels of the CSC-related gene (OCT3 / 4) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3e] Figure 3e shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A1) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3f] Figure 3f shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A2) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3g] Figure 3g shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A3) in ACHN-R cells and its parental strain (ACHN cells). [Figure 3h]Figure 3h shows the results of comparing the mRNA expression levels of the CSC-related gene (IL6) in ACHN-R cells and their parent cell line (ACHN cells). [Figure 3i] Figure 3i shows the results of comparing the mRNA expression levels of DPP4 in 769-PR cells and their parent cell line (769-P cells). [Figure 3j] Figure 3j shows the results of comparing the mRNA expression levels of the CSC-related gene (CD44) in 769-PR cells and their parent cell line (769-P cells). [Figure 3k] Figure 3k shows the results of comparing the mRNA expression levels of the CSC-related gene (CD133) in 769-PR cells and their parent cell line (769-P cells). [Figure 3l] Figure 3l shows the results of comparing mRNA expression levels of CSC-related genes (OCT3 / 4) in 769-PR cells and their parent cell line (769-P cells). [Figure 3m] Figure 3m shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A1) in 769-PR cells and their parent cell line (769-P cells). [Figure 3n] Figure 3n shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A2) in 769-PR cells and their parent cell line (769-P cells). [Figure 3o] Figure 3o shows the results of comparing the mRNA expression levels of the CSC-related gene (ALDH1A3) in 769-PR cells and their parent cell line (769-P cells). [Figure 3p] Figure 3p shows the results of comparing mRNA expression levels of the CSC-related gene (IL6) in 769-PR cells and their parental strain (769-P cells). [Figure 4a] Figure 4a shows the results of investigating the dose-response effect of sitagliptin (SITA) on three-dimensional spheroid proliferation in ACHN-R cells and their parental cell line. [Figure 4b]Figure 4b shows the results of investigating the dose-response effect of sitagliptin (SITA) on three-dimensional spheroid proliferation in 769-PR cells and their parental cell line. [Figure 4c] Figure 4c shows the results of an investigation into the combined effects of SITA and SUN in three-dimensional culture of ACHN cells. [Figure 4d] Figure 4d shows the results of investigating the combined effects of SITA and SUN in three-dimensional culture of ACHN-R cells. [Figure 4e] Figure 4e shows the results of investigating the combined effects of SITA and SUN in three-dimensional culture of 769-P cells. [Figure 4f] Figure 4f shows the results of investigating the combined effects of SITA and SUN in three-dimensional culture of 769-PR cells. [Figure 4g] Figure 4g shows the results of an investigation into the effect of siRNA-mediated DPP4 knockdown on the therapeutic effect of SUN in 3D culture of ACHN cells. [Figure 4h] Figure 4h shows the results of investigating the effect of siRNA-mediated DPP4 knockdown on the therapeutic effect of SUN in a three-dimensional culture of ACHN-R cells. [Figure 4i] Figure 4i shows the results of investigating the effect of siRNA-mediated DPP4 knockdown on the therapeutic effect of SUN in 3D culture of 769-P cells. [Figure 4j] Figure 4j shows the results of an investigation into the effect of siRNA-mediated DPP4 knockdown on the therapeutic effect of SUN in 3D culture of 769-PR cells. [Figure 4k] Figure 4k shows the results of investigating the effect of SITA treatment on IL6 mRNA levels in 3D culture of ACHN-R cells. [Figure 4l] Figure 4l shows the results of investigating the effect of SITA treatment on IL6 mRNA levels in 3D culture of 769-PR cells. [Figure 4m]Figure 4m shows the results of an investigation into the effect of DPP4 overexpression on the cell viability of RCC spheroids (ACHN) induced by SUN. [Figure 4n] Figure 4n shows the results of an investigation into the effect of DPP4 overexpression on the cell viability of RCC spheroids (769-P) mediated by SUN. [Figure 5a] Figure 5a shows the results of an investigation into the effect of RA replacement in ACHN-R cells on the suppression of DPP4 by the ALDH1 inhibitor disulfiram. [Figure 5b] Figure 5b shows the results of an investigation into the effect of RA replacement in 769-PR cells on the suppression of DPP4 by the ALDH1 inhibitor disulfiram. [Figure 5c] Figure 5c is a schematic diagram illustrating the location of the functional retinoic acid-responsive element (RARE) and reference region near the DPP4 locus on chromosome 2q24. [Figure 5d] Figure 5d shows the results of confirming the recruitment of retinoic acid receptor α (RARα) to RARE in the DPP4 promoter of ACHN-R cells. [Figure 5e] Figure 5e shows the results of confirming the recruitment of retinoic acid receptor α (RARα) to RARE in the DPP4 promoter of 769-PR cells. [Figure 5f] Figure 5f shows the results of confirming the recruitment of retinoid X receptor α (RXRα) to RARE in the DPP4 promoter of ACHN-R cells. [Figure 5g] Figure 5g shows the results of confirming the recruitment of retinoid X receptor α (RXRα) to RARE in the DPP4 promoter of 769-PR cells. [Figure 5h] Figure 5h shows the results of investigating the effect of RA treatment on RARE sequences in 293T cells. [Figure 6a]Figure 6a shows the tumor growth curves for the ACHN / SUN group, the ACHN-R / SUN group, and the ACHN-R / SUN+SITA group. [Figure 6b] Figure 6b shows the results of measuring tumor weight in the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group. [Figure 6c] Figure 6c shows the results of measuring body weight in the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group. [Figure 6d] Figure 6d shows a representative photograph of xenograft nude mice in the ACHN / SUN group on day 13. [Figure 6e] Figure 6e shows a representative photograph of xenograft nude mice in the ACHN-R / SUN group on day 13. [Figure 6f] Figure 6f shows a representative photograph of xenograft nude mice in the ACHN-R / SUN+SITA group on day 13. [Figure 6g] Figure 6g shows the results of Ki67 immunohistochemical staining of resected tumors in the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group, and the percentage of Ki67-positive cells (Ki67 index) to be determined. [Figure 6h] Figure 6h shows the results of examining DPP4 mRNA levels in the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group. [Figure 6i] Figure 6i shows the results of examining IL6 mRNA levels in the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group. [Figure 7a] Figure 7a shows the results of examining the Kaplan-Meier 10-year overall survival (OS) from the initial visit for 73 patients who received TKI treatment, stratified by the presence or absence of type 2 diabetes mellitus (T2DM) and the administration of DPP4 inhibitors (DPP4i). [Figure 7b]Figure 7b shows the results of a waterfall plot analysis of the maximum tumor change rate in 73 RCC patients who received TKI treatment, stratified according to the presence or absence of T2DM and the presence or absence of DPP4i administration. [Figure 7c] Figure 7c shows the results of a box plot analysis of the maximum tumor change rate in 73 RCC patients treated with TKI therapy, stratified by the presence or absence of T2DM and the presence or absence of DPP4i administration. [Figure 7d] Figure 7d shows the results of a Kaplan-Meier analysis of 10-year overall survival regarding the association between DPP4 expression and prognosis. [Figure 7e] Figure 7e shows the results of a Kaplan-Meier analysis conducted on 10-year overall survival regarding the association between DPP4 expression and prognosis, focusing on 31 non-T2DM cases. [Figure 7f] Figure 7f shows the results of a Kaplan-Meier analysis conducted on 10-year overall survival regarding the association between DPP4 expression and prognosis, focusing on 18 T2DM cases. [Figure 7g] Figure 7g is a schematic diagram of the ALDH1 / retinoic acid / DPP4 system (axis) in DPP4-highly expressing renal cancer stem cell-like cells. [Modes for carrying out the invention]

[0011] (Concomitant medication) The combination drug of the present invention is a combination drug for treating renal cancer, comprising a tyrosine kinase inhibitor and a dipeptidyl peptidase 4 inhibitor in combination, and optionally containing other components. In the present invention, the term "treatment" refers to curing, alleviating, or preventing the progression of cancer symptoms.

[0012] <Kidney cancer> The aforementioned renal cancer is not particularly limited and can be appropriately selected depending on the purpose, and examples include renal cell carcinoma and renal pelvis cancer. The aforementioned renal cancer may be metastatic or non-metastatic. Furthermore, the aforementioned renal cancer may be resistant to tyrosine kinase inhibitors (hereinafter sometimes referred to as "resistant") or not resistant to tyrosine kinase inhibitors, but the combination drug of the present invention can be suitably used for renal cancer resistant to tyrosine kinase inhibitors.

[0013] <Tyrosine kinase inhibitors> The tyrosine kinase inhibitors mentioned above are not particularly limited as long as they can inhibit the enzymatic activity of tyrosine kinase or suppress the expression of tyrosine kinase, and can be appropriately selected according to the purpose. Examples include sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib. These may be used individually or in combination of two or more. The tyrosine kinase inhibitor may be a commercially available product or a synthesized product.

[0014] <Dipeptidyl peptidase-4 inhibitors> The dipeptidyl peptidase 4 inhibitors mentioned above are not particularly limited as long as they can inhibit the enzymatic activity of dipeptidyl peptidase 4 or suppress the expression of dipeptidyl peptidase 4, and can be appropriately selected according to the purpose. Examples include sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omaligliptin, gemigliptin, evogliptin, gosogliptin, and dipeptidyl peptidase 4 expression inhibitors. These may be used individually or in combination of two or more. The aforementioned dipeptidyl peptidase 4 inhibitor may be a commercially available product or a synthesized product.

[0015] <<Dipeptidyl peptidase 4 expression inhibitor>> The dipeptidyl peptidase 4 expression inhibitor is not particularly limited as long as it can suppress the expression of dipeptidyl peptidase 4, and can be appropriately selected depending on the purpose. Examples include double-stranded nucleic acid molecules for suppressing the expression of the dipeptidyl peptidase 4 gene (hereinafter sometimes referred to as "double-stranded nucleic acid molecules"), DNA containing the base sequence encoding the double-stranded nucleic acid molecule (hereinafter sometimes referred to as "DNA"), and vectors containing the DNA (hereinafter sometimes referred to as "vectors"). These may be used individually or in combination of two or more.

[0016] -Double-stranded nucleic acid molecule- The double-stranded nucleic acid molecule is not particularly limited as long as it can suppress the expression of the dipeptidyl peptidase 4 gene, and can be appropriately selected depending on the purpose. Examples include a double-stranded nucleic acid molecule comprising (a) a sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 1 or SEQ ID NO: 4, and (b) an antisense strand containing a base sequence complementary to the sense strand that forms a double helix with the sense strand in (a). These may be used individually or in combination of two or more. In this invention, "double-stranded nucleic acid molecule" refers to a double-stranded nucleic acid molecule formed by the hybridization of a sense strand and an antisense strand.

[0017] In this invention, the dipeptidyl peptidase 4 gene's mRNA is targeted by the double-stranded nucleic acid molecule, and its expression is suppressed by the double-stranded nucleic acid molecule. Therefore, in this specification, the dipeptidyl peptidase 4 gene may be referred to as the "target gene" of the double-stranded nucleic acid molecule.

[0018] --Sense chain, antisense chain-- The double-stranded nucleic acid molecule preferably includes (a) a sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 1 or SEQ ID NO: 4, and (b) an antisense strand containing a base sequence complementary to the sense strand that forms a double helix with the sense strand in (a). Here, the sense strand and the antisense strand may be RNA strands or RNA-DNA chimeric strands. The sense strand and the antisense strand can hybridize with each other to form the double-stranded nucleic acid molecule.

[0019] The sense strand in the double-stranded nucleic acid molecule may contain a base sequence corresponding to the target sequence, may contain other base sequences, or may consist solely of the base sequence corresponding to the target sequence. Furthermore, the antisense strand in the double-stranded nucleic acid molecule only needs to contain a base sequence that is complementary to the sense strand to the extent that it can hybridize with the sense strand. It may also contain other base sequences, but it is preferable that it contains 70% or more of the base sequence complementary to the sense strand, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0020] There are no particular restrictions on the type of double-stranded nucleic acid molecule, and it can be appropriately selected depending on the purpose. Examples include double-stranded RNA (dsRNA) and double-stranded RNA-DNA chimeras. Here, "double-stranded RNA" refers to a double-stranded nucleic acid molecule in which both the sense strand and the antisense strand are composed of RNA sequences, and "double-stranded RNA-DNA chimera" refers to a double-stranded nucleic acid molecule in which both the sense strand and the antisense strand are composed of chimeric sequences of RNA and DNA.

[0021] The double-stranded RNA and the double-stranded RNA-DNA chimera are preferably siRNA (small interfering RNA) or chimeric siRNA, and more preferably siRNA.

[0022] Here, siRNA is a small double-stranded RNA molecule with a length of 18 to 29 nucleotides, and it has the function of cleaving target RNA having a sequence complementary to the antisense strand (guide strand) of the siRNA, thereby suppressing the expression of the target RNA. The siRNA has a sense strand and an antisense strand as described above, and is capable of suppressing the expression of the target RNA. There are no particular restrictions on its terminal structure, and it can be appropriately selected depending on the purpose. For example, the siRNA may have a blunt end or an overhang. In particular, the siRNA preferably has a structure in which 2 to 6 nucleotides protrude from the 3' end of each strand, and more preferably has a structure in which 2 nucleotides protrude from the 3' end of each strand.

[0023] Furthermore, chimeric siRNA refers to a small double-stranded RNA-DNA chimera with a length of 18 to 29 base pairs, in which a portion of the RNA sequence of siRNA is converted to DNA. In particular, it is preferable to have a small double-stranded RNA-DNA chimera with a length of 21 to 23 base pairs, in which the bases within the 8 bases on the 3' end of the sense strand and the bases within the 6 bases on the 5' end of the antisense strand of siRNA are converted to DNA. The chimeric siRNA has the function of suppressing the expression of target genes, similar to the siRNA described above. It should be noted that the chimeric siRNA also includes a form in which a portion of the sequence converted to DNA is converted back to RNA. The terminal structure of the chimeric siRNA is not particularly limited, just like that of the siRNA, and can be appropriately selected depending on the purpose. For example, it may have a blunt end or an overhanging end.

[0024] Specific examples of the aforementioned siRNA include the following:

[0025] An example of an siRNA in which the target sequence is the base sequence represented by Sequence ID No. 1 is an siRNA consisting of a sense strand of Sequence ID No. 2 and an antisense strand of Sequence ID No. 3. Sense chain 5'-GGAGGGUACGUAACCUCAAUG-3'(Sequence ID: 2) • Antisense chain 5'-UUGAGGUUACGUACCCUCCAU-3'(Sequence ID: 3)

[0026] Furthermore, an example of an siRNA in which the target sequence is the base sequence represented by Sequence ID No. 4 is an siRNA consisting of the sense strand of Sequence ID No. 5 and the antisense strand of Sequence ID No. 6 shown below. Sense chain 5'-CAGUCGCAAAACUUACACUCU-3'(Sequence ID: 5) • Antisense chain 5'-AGUGUAAGUUUUGCGACUGUC-3'(Sequence ID: 6)

[0027] Furthermore, the double-stranded RNA may also be shRNA (short hairpin RNA). Here, shRNA is a single-stranded RNA containing a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases. When expressed in vivo, shRNA forms base pairs to become a hairpin-shaped double-stranded RNA. Subsequently, shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to suppress the expression of target RNA. The terminal structure of the shRNA is not particularly limited, just like that of the siRNA and double-stranded RNA-DNA chimera, and can be appropriately selected depending on the purpose. For example, it may have a blunt end or an overhanging end.

[0028] Furthermore, the double-stranded nucleic acid molecule may be modified as appropriate depending on the purpose. For example, to confer resistance to nucleases and improve stability in culture medium or in vivo, the double-stranded nucleic acid molecule can be subjected to 2'O-methylation, phosphorothioate (S-modification), LNA (Locked Nucleic Acid) modification, etc. Also, for example, to improve the efficiency of introduction into cells, the 5' or 3' end of the sense strand of the double-stranded nucleic acid molecule can be modified with nanoparticles, cholesterol, cell membrane-penetrating peptides, etc. There are no particular restrictions on the method of performing such modifications on the double-stranded nucleic acid molecule, and conventionally known methods can be used as appropriate.

[0029] There are no particular restrictions on how the double-stranded nucleic acid molecules can be obtained, and each can be prepared using conventionally known methods. For example, the siRNA can be produced by chemically synthesizing single-stranded RNA molecules of 18 to 29 nucleotide lengths, corresponding to the desired sense strand and antisense strand, using existing automated DNA / RNA synthesis equipment, and then annealing them. Alternatively, commercially available annealed double-stranded siRNA can be obtained, or it can be obtained by commissioning an siRNA synthesis company to perform the synthesis. Furthermore, siRNA can also be produced by constructing a desired siRNA expression vector, such as the vector of the present invention described later, and introducing the expression vector into cells, thereby utilizing intracellular reactions. Furthermore, the chimeric siRNA can be produced, for example, by chemically synthesizing a sense strand and an antisense strand, which are chimeric nucleic acid molecules, and then annealing them.

[0030] -DNA- The DNA in question is not particularly limited as long as it contains a base sequence encoding the double-stranded nucleic acid molecule, and can be appropriately selected depending on the purpose. However, it is preferable that a promoter sequence for controlling the transcription of the double-stranded nucleic acid molecule is linked upstream (5' side) of the base sequence encoding the double-stranded nucleic acid molecule. The promoter sequence is not particularly limited and can be appropriately selected depending on the purpose. Examples include pol II promoters such as the CMV promoter, and pol III promoters such as the H1 promoter and U6 promoter. Furthermore, it is even more preferable that a terminator sequence for terminating the transcription of the double-stranded nucleic acid molecule is linked downstream (3' side) of the base sequence encoding the double-stranded nucleic acid molecule. There are no particular restrictions on the terminator sequence, and it can be appropriately selected depending on the purpose. The transcription unit comprising the promoter sequence, the base sequence encoding the double-stranded nucleic acid molecule, and the terminator sequence is a preferred embodiment in the DNA. The transcription unit can be constructed using conventionally known methods.

[0031] -vector- The vector is not particularly limited as long as it contains the DNA, and can be appropriately selected depending on the purpose. Examples include plasmid vectors and viral vectors. Preferably, the vector is an expression vector capable of expressing the double-stranded nucleic acid molecule. There are no particular restrictions on the expression mode of the double-stranded nucleic acid molecule, and it can be appropriately selected depending on the purpose. For example, methods for expressing siRNA as a double-stranded nucleic acid molecule include expressing two short single-stranded RNAs (tandem type) and expressing single-stranded RNA as shRNA (hairpin type). The tandem siRNA expression vector includes a DNA sequence encoding the sense strand of the siRNA and a DNA sequence encoding the antisense strand, wherein a promoter sequence is ligated upstream (5' side) of the DNA sequence encoding each strand, and a terminator sequence is ligated downstream (3' side) of the DNA sequence encoding each strand. Furthermore, the hairpin-type siRNA expression vector contains DNA in which a DNA sequence encoding the sense strand and a DNA sequence encoding the antisense strand constituting the siRNA are arranged in opposite directions, the sense strand DNA sequence and the antisense strand DNA sequence are connected via a loop sequence, and a promoter sequence is ligated upstream (5' side) and a terminator sequence is ligated downstream (3' side). Each of the aforementioned vectors can be constructed using conventionally known methods, for example, by ligating the DNA to the cleavage sites of a vector that has been previously cleaved with restriction enzymes.

[0032] By introducing (transfecting) the DNA or vector into cells, the promoter is activated, and the double-stranded nucleic acid molecule can be generated. For example, in the case of the tandem vector, the DNA is transcribed in the cell to generate a sense strand and an antisense strand, and these hybridize to produce siRNA. In the case of the hairpin vector, the DNA is transcribed in the cell to first generate hairpin RNA (shRNA), and then siRNA is produced by processing with a dicer.

[0033] <Other ingredients> Other components in the aforementioned combination drug are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected from pharmacologically acceptable carriers depending on the purpose, such as additives, auxiliaries, and water. These may be used individually or in combination of two or more.

[0034] There are no particular limitations on the additives or auxiliary agents, and they can be appropriately selected according to the purpose. Examples include disinfectants, preservatives, binders, thickeners, adhesives, binders, colorants, stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, defoamers, property improvers, and preservatives.

[0035] There are no particular restrictions on the amount of other components in the aforementioned combination drug, and they can be appropriately selected depending on the purpose.

[0036] <Use> The aforementioned combination drug may be used solely as a combination of the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor, or it may be used in combination with a drug containing other active ingredients. Furthermore, the aforementioned combination drug may be used in a state in which it is incorporated into a drug containing another active ingredient.

[0037] The aforementioned combination drug may use the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor as separate formulations, or both may be used as a single formulation (combination drug).

[0038] <Dosage form> There are no particular restrictions on the dosage form of the aforementioned concomitant medication, and it can be appropriately selected according to the desired method of administration. Examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral liquids, syrups, elixirs, etc.), injectable preparations (solutions, suspensions, solid preparations for immediate dissolution, etc.), ointments, patches, gels, creams, topical powders, sprays, and inhaled powders. When the aforementioned combination drug uses the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor as separate formulations, there are no particular restrictions on the dosage forms, and they can be appropriately selected according to the purpose. The two may be the same dosage form or different dosage forms.

[0039] The oral solid preparation can be manufactured, for example, by adding an excipient, and optionally other additives such as a binder, disintegrant, lubricant, colorant, or flavoring / odorizing agent, to the active ingredient using a conventional method. Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of disintegrants include dried starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of lubricants include refined talc, stearate, borax, and polyethylene glycol. Examples of colorants include titanium dioxide and iron oxide. Examples of flavoring and deodorizing agents include sucrose, orange peel, citric acid, and tartaric acid.

[0040] The oral liquid preparation can be manufactured, for example, by adding additives such as flavoring agents, odor-masking agents, buffering agents, and stabilizers to the active ingredient using a conventional method. Examples of flavoring and odor-modifying agents include sucrose, orange peel, citric acid, and tartaric acid. Examples of buffering agents include sodium citrate. Examples of stabilizers include tragacanth, gum arabic, and gelatin.

[0041] For example, the injectable preparation can be manufactured by adding a pH adjuster, buffer, stabilizer, isotonic agent, local anesthetic, etc., to the active ingredient, and using a conventional method to produce an injectable preparation for subcutaneous, intramuscular, or intravenous use. Examples of the pH adjusting agent and buffering agent include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

[0042] For example, the ointment can be manufactured by mixing the active ingredient with a known base, stabilizer, humectant, preservative, etc., using a conventional method. Examples of the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.

[0043] The aforementioned adhesive patch can be manufactured, for example, by applying a cream, gel, paste, or the like, as an ointment, to a known support using a conventional method. Examples of the support include woven fabrics and nonwoven fabrics made of cotton, rayon, and chemical fibers, films and foam sheets made of soft polyvinyl chloride, polyethylene, and polyurethane.

[0044] <Administration> There are no particular restrictions on the method of administration, dosage, timing of administration, or target recipients of the aforementioned concomitant medications; they can be appropriately selected according to the purpose.

[0045] There are no particular restrictions on the method of administering the concomitant drug. For example, depending on the dosage form of the concomitant drug, the patient's condition, etc., either local administration or systemic administration can be selected. For example, in local administration, the active ingredient of the concomitant drug can be administered by directly injecting it into the desired site (e.g., the tumor site). Conventional known methods such as injection can be used for the injection. In systemic administration (e.g., oral administration, intraperitoneal administration, administration into the bloodstream, etc.), it is preferable to appropriately apply conventionally known drug delivery techniques so that the active ingredient of the concomitant drug is delivered stably and efficiently to the desired site (e.g., the tumor site). When the aforementioned combination drug uses the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor as separate formulations, the method of administration may be the same for both, or they may be different.

[0046] There are no particular restrictions on the dosage, and it can be appropriately selected considering various factors such as the age, weight, constitution, symptoms, and whether or not the individual receiving the treatment is taking other medicines or drugs containing other active ingredients. There are no particular restrictions on the ratio of the dosages of the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor in the aforementioned combination drug, and they can be appropriately selected depending on the purpose. Furthermore, there are no particular restrictions on the number of administrations; the number can be appropriately selected considering various factors such as the age, weight, constitution, symptoms of the individual being administered the drug, and whether or not they are receiving other medications or drugs containing other active ingredients.

[0047] There are no particular restrictions on the timing of administration, and it can be selected as appropriate depending on the purpose. The aforementioned combination drug may be administered simultaneously with the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor, or at different times. When the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor are administered at different times, there are no particular restrictions on the order of administration of the two, and they can be appropriately selected according to the purpose. Furthermore, there are no particular restrictions on the administration interval when the tyrosine kinase inhibitor and the dipeptidyl peptidase 4 inhibitor are administered at different times; the interval can be appropriately selected depending on the purpose.

[0048] There are no particular restrictions on the target population for administration, and they can be appropriately selected according to the purpose. Examples include humans, monkeys, pigs, cattle, sheep, goats, dogs, cats, mice, rats, and birds, but among these, it can be suitably used in humans.

[0049] (Treatment methods for kidney cancer) As shown in the examples section below, the aforementioned combination drug, when used in combination with a dipeptidyl peptidase 4 inhibitor, can enhance the tumor growth inhibitory effect of the tyrosine kinase inhibitor (enhance the therapeutic effect). Therefore, the present invention also relates to a method for treating renal cancer, characterized by administering the combination drug of the present invention to an individual. There are no particular restrictions on the aforementioned renal cancers; the same types as those listed in the section on renal cancers in combination therapy above can be included. The aforementioned method for treating kidney cancer may also involve the action of other kidney cancer drugs.

[0050] (An agent that enhances the therapeutic effect of tyrosine kinase inhibitors) The therapeutic effect enhancer for tyrosine kinase inhibitors of the present invention comprises at least a dipeptidyl peptidase 4 inhibitor and optionally other components. The aforementioned tyrosine kinase inhibitor therapeutic effect enhancer can enhance the therapeutic effect of the tyrosine kinase inhibitor on renal cancer. In the present invention, "enhancing the therapeutic effect" includes enhancing the tumor growth inhibitory effect and restoring the therapeutic effect of tyrosine kinase inhibitors (overcoming resistance) to tumors that have developed resistance to tyrosine kinase inhibitors.

[0051] <Kidney cancer> Examples of the aforementioned renal cancers include those listed in the section on renal cancers under the combination therapy mentioned above.

[0052] <Dipeptidyl peptidase-4 inhibitors> Examples of the aforementioned dipeptidyl peptidase-4 inhibitors include those listed in the section on dipeptidyl peptidase-4 inhibitors in the concomitant medications section above. There are no particular restrictions on the amount of the dipeptidyl peptidase 4 inhibitor in the therapeutic effect enhancer for the tyrosine kinase inhibitor, and it can be appropriately selected depending on the purpose. The therapeutic effect enhancer for the tyrosine kinase inhibitor may consist only of the dipeptidyl peptidase 4 inhibitor.

[0053] <Other ingredients> Other components in the therapeutic effect enhancer for the tyrosine kinase inhibitor are not particularly limited and can be appropriately selected from pharmacologically acceptable carriers depending on the purpose. Examples include those similar to those listed in the section on other components of the concomitant drug above. These may be used individually or in combination of two or more. There are no particular restrictions on the content of other components in the therapeutic effect enhancer for the tyrosine kinase inhibitor, and they can be appropriately selected depending on the purpose.

[0054] <Tyrosine kinase inhibitors> Examples of tyrosine kinase inhibitors targeted by the aforementioned tyrosine kinase inhibitor therapeutic effect enhancers include those listed above under the section on tyrosine kinase inhibitors in combination therapy.

[0055] <Use> The tyrosine kinase inhibitor therapeutic effect enhancer may be used in combination with the tyrosine kinase inhibitor alone, or in combination with the tyrosine kinase inhibitor and a pharmaceutical product containing another active ingredient. Furthermore, the tyrosine kinase inhibitor therapeutic effect enhancer may be used in combination with the tyrosine kinase inhibitor or a pharmaceutical product containing another active ingredient.

[0056] <Dosage form> There are no particular restrictions on the dosage form of the therapeutic effect enhancer for the tyrosine kinase inhibitor, and it can be appropriately selected depending on the purpose. For example, the same forms as those listed in the section on dosage forms of concomitant medications above can be used.

[0057] <Administration> There are no particular restrictions on the method of administration, dosage, timing of administration, or target recipients of the tyrosine kinase inhibitor therapeutic effect enhancer; these can be appropriately selected according to the purpose. For example, the same methods as those described in the section on the administration of concomitant medications above can be cited.

[0058] (Methods to enhance the therapeutic effect of tyrosine kinase inhibitors on kidney cancer) As shown in the examples below, the tyrosine kinase inhibitor therapeutic effect enhancer can enhance the tumor growth inhibitory effect of the tyrosine kinase inhibitor. Therefore, the present invention also relates to a method for enhancing the therapeutic effect of a tyrosine kinase inhibitor against renal cancer, characterized by administering the tyrosine kinase inhibitor therapeutic effect enhancer of the present invention to an individual. There are no particular restrictions on the aforementioned renal cancers, and they can be appropriately selected according to the purpose. For example, the same ones listed in the section on renal cancers in combination drugs above can be cited. There are no particular restrictions on the tyrosine kinase inhibitor to be administered in combination with the therapeutic effect enhancer of the aforementioned tyrosine kinase inhibitor. It can be appropriately selected according to the purpose, for example, the same as those listed in the section on tyrosine kinase inhibitors in combination drugs above. The method for enhancing the therapeutic effect of tyrosine kinase inhibitors on renal cancer may involve further action of other renal cancer treatment agents. [Examples]

[0059] The following describes some test examples of the present invention, but the present invention is not limited in any way to these test examples.

[0060] (Test Example 1) <Materials and Methods> <<Collection of clinical data and patient selection>> This retrospective analysis examined 73 patients with renal cell carcinoma (RCC) who received tyrosine kinase inhibitor (TKI) therapy at Saitama Medical University Saitama Medical Center between 2008 and 2019. Overall survival from initial presentation and maximum tumor reduction (MAX) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) (Eisenhauer, EA et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur. J. Cancer 45, 228-247 (2009)) were evaluated. For MAX tumor reduction, only the earliest available TKI treatment was evaluated. Patients who did not consent to this study, cases for which objective images before and after TKI treatment were unavailable, and patients who received only immunotherapy or mTOR inhibitors were excluded. Furthermore, patients with missing clinical factor values ​​were excluded from statistical analyses for comparing patient characteristics. This clinical analysis was approved by the Ethics Committee of Saitama Medical University Saitama Medical Center (Nos. 117 and 2308).

[0061] <<Patient-derived cell generation and RCC cell line used in experiments>> After obtaining informed consent at Saitama Medical University General Medical Center, patient-derived cells (PDCs) were generated from resected tumors of RCC patients. Tumor sample processing was performed according to the literature (Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019)). The protocol for this study has been approved by the Ethics Committee of Saitama Medical University Saitama Medical Center (No. 1363-IV). The human RCC cell lines ACHN and 769-P were obtained from the American Type Culture Collection (ATCC) and certified by BEX Corporation using short tandem repeat (STR) analysis. ACHN and 769-P cells were cultured in DMEM and RPMI medium (Nacalai Tesque) supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin, respectively, in a 5% CO2 incubator at 37°C. Exposure of ACHN and 769-P cells to sunitinib (SUN) up to 10 μM for more than 6 months generated SUN-resistant (sometimes referred to as "resistant") RCC cell lines ACHN-R and 769-PR.

[0062] <<siRNAのトランスフェクション> > Dipeptidyl peptidase 4 (DPP4)-targeted siRNAs (siDPP4 #1 and #2) and a control siRNA (siControl) were purchased from RNAi Inc. and introduced into cells using RNAiMAX reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. The sequences of each siRNA are shown below.

[0063] [siDPP4 #1] -Target sequence- 5’-GGAGGGTACGTAACCTCAATG-3’ (SEQ ID NO: 1) -Sequence of double-stranded nucleic acid molecule (siRNA)- · Sense strand 5’-GGAGGGUACGUAACCUCAAUG-3’ (SEQ ID NO: 2) · Antisense strand 5’-UUGAGGUUACGUACCCUCCAU-3’ (SEQ ID NO: 3)

[0064] [siDPP4 #2] -Target sequence- 5’-CAGTCGCAAAACTTACACTCT-3’ (SEQ ID NO: 4) -Sequence of double-stranded nucleic acid molecule (siRNA)- · Sense strand 5’-CAGUCGCAAAACUUACACUCU-3’ (SEQ ID NO: 5) · Antisense strand 5’-AGUGUAAGUUUUGCGACUGUC-3’ (SEQ ID NO: 6)

[0065] [siControl] -Sequence of double-stranded nucleic acid molecule (siRNA)- · Sense strand 5’-GUACCGCACGUCAUUCGUAUC-3’ (SEQ ID NO: 7) · Antisense strand 5’-UACGAAUGACGUGCGGUACGU-3’ (SEQ ID NO: 8)

[0066] [Cell viability assay for spheroid culture] The cell viability on the 3rd day after drug treatment and siRNA transfection was evaluated by CellTiter-Glo 3D Assay (Promega).

[0067] [cDNA synthesis and quantitative reverse transcription PCR] RNA extraction, cDNA synthesis, and quantitative reverse transcription PCR (qRT-PCR) were performed using the method described in "Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019)". The primers used for qRT-PCR are as follows:

[0068] [DPP4] • Forward 5'-CCAAACGGCACTTTTTTAGCA-3'(Sequence ID: 9) Reverse 5'-GAGTATTCAATAAGTGGGACTTCTGTGT-3'(Sequence ID: 10) [CD44] • Forward 5'-GTGATGGCACCCGCTATG-3'(Sequence ID: 11) Reverse 5'-ACTGTCTTCGTCTGGGATGG-3'(Sequence ID: 12) [CD133] • Forward 5'-CAGAGTACAAACGCCAAACCA-3'(Sequence ID: 13) Reverse 5'-AAATCACGATGAGGGTCAGC-3'(Sequence ID: 14) [OCT3 / 4] • Forward 5'-TTCAGCCAAACGACCATCTG-3'(Sequence ID: 15) Reverse 5'-CACGAGGGTTTCTGCTTTGC-3'(Sequence ID: 16) [CXCR4] • Forward 5'-GCATGACGGACAAGTACAGGCT-3'(Sequence ID: 17) Reverse 5'-AAAGTACCAGTTTGCCACGGC-3'(Sequence ID: 18) [ALDH1A1] • Forward 5'-CGCAAGACAGGCTTTTCAGAT-3'(Sequence ID: 19) Reverse 5'-CCCTCTCGGAAGCATCCA-3'(Sequence ID: 20) [ALDH1A2] • Forward 5'-AGGCCCTCACAGTGTCTTCT-3'(Sequence ID: 21) Reverse 5'-ACATCTTGAATCCCCCAAAG-3'(Sequence ID: 22) [ALDH1A3] • Forward 5'-TGGATCAACTGCTACAACGC-3'(Sequence ID: 23) Reverse 5'-CACTTCTGTGTATTCGGCCA-3'(Sequence ID: 24) [IL6] • Forward 5'-AGACAGCCACTCACCTCTTCAGA-3'(Sequence ID: 25) Reverse 5'-CAGCCATCTTTGGAAGGTTCA-3'(Sequence ID: 26) [36B4] • Forward 5'-CCACGCTGCTGAACATGCT-3'(Sequence ID: 27) Reverse 5'-GATGCTGCCATTGTCGAACA-3'(Sequence ID: 28)

[0069] <<Immunohistochemical staining>> Formalin-fixed tissue was embedded in paraffin and sectioned. For immunohistochemical staining of DPP4, the Histofine kit (Nichirei Corporation) using streptavidin-biotin amplification was employed. Ki67 (dilution 1:100; MIB1) and DPP4 (dilution 1:100; AF1180-SP) were used as primary antibodies, and secondary antibodies were purchased from Agilent Technologies and R&D Systems, respectively. Immunohistochemical staining was evaluated by a specialist pathologist.

[0070] <<Chromatin Immunoprecipitation>> Chromatin immunoprecipitation (ChIP) was performed using the method described in "Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019)". The primer sequences used in the chromatin immunoprecipitation PCR method are as follows:

[0071] [peak] • Forward 5'-CTAATGTGTTGGCCAGCTGCTACC-3'(Sequence ID: 29) Reverse 5'-GTCCGATGGGGCATTACCACATGA-3'(Sequence ID: 30) [Reference] • Forward 5'-CCTCTTCAAGGGGACTAACTACAA-3'(Sequence ID: 31) Reverse 5'-CTATCATTGCAAGATTGCTCTCTC-3'(Sequence ID: 32)

[0072] <<Luciferase assay>> The retinoic acid responsive element (RARE), identified by JASPAR (Wasserman WW & Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5, 276-287 (2004)), and its mutant oligonucleotides or direct repeats (DR5) of RARE (Bulens, F. et al. Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DR5) located at -7 kilobases. J. Biol. Chem. 270, 7167-7175 (1995)) were generated by annealing the corresponding oligonucleotides (see primer sequences below) and inserted into the pGL3 promoter (Promega). Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) with the reniral luciferase signal used as an internal standard. The primer sequences used in the luciferase assay are as follows:

[0073] · RARE_WT_U 5'-CTCTAGAAAGACTGGTGACACAGAGGTCAAGGTGGGGGAGGCATGA-3'(Sequence ID: 33) · RARE_WT_D 5'-CATGGAGATCTTTCTGACCACTGTGTCTCCAGTTCCACCCCCTCCGTACTGCGC-3'(Sequence ID: 34) · RARE_Mut_U 5'-CTCTAGAAAGACCAACATCACAGCAACATAGGTGCAACATGCATGA-3'(Sequence ID: 35) · RARE_Mut_D 5'-CGCGTCATGCATGTTGCACCTATGTTGCTGTGATGTTGGTCTTTCTAGAGGTAC-3'(Sequence ID: 36)

[0074] <<Animal Experiments>> All animal experiments were conducted with the approval of the Animal Management and Use Committee of Saitama Medical University, and in accordance with the guidelines and regulations for the management and use of laboratory animals at Saitama Medical University. Male nude mice (BALB / c-nu / nu, 6 weeks old) were purchased from CREA Japan. ACHN cells and ACHN-R cells were trypsinized, washed with PBS, and 5 × 10⁶ cells were prepared. 6 Individual cells were subcutaneously transplanted using 150 μL of Matrigel (BD Biosciences). The size of the tumor was measured every other day. The tumor volume (V) was determined by measuring the length (L) and width (W) and using the following formula. V = (L × W) 2 ) × 0.52 The transplanted tumor was 180 mm in size. 3 When the volume reached a certain level, mice with ACHN cells were orally administered SUN (20 mg / kg), and mice with ACHN-R cells were randomly selected and orally administered either SUN (20 mg / kg) or SUN (20 mg / kg) and the DPP4 inhibitor sitagliptin (SITA) (30 mg / kg). The solvents for each drug were carboxymethylcellulose sodium (0.5% wt / vol) with NaCl (1.8% wt / vol), Tween 80 (0.4% wt / vol), and benzyl alcohol (0.9% wt / vol), according to the literature (Stany, MP et al. Identification of novel therapeutic targets in microdissected clear cell ovarian cancers. PLoS One 6, e21121 (2011)). The drugs were administered in a 2-day administration, 1-day rest cycle.

[0075] <<Statistical analysis>> For clinical data, the log-rank test was used for the Kaplan-Meier method. The Holm method was used as a post hoc test for multiple comparisons. The Fisher's exact probability test was used for partial efficacy rates and patient characteristics. The Mann-Whitney U test was used for the maximum tumor shrinkage rate. In the cell proliferation assay or quantitative reverse transcription PCR method, the two-sided Student's t-test was used for comparison between two groups, and the two-way ANOVA test was used for multiple comparisons. JMP 9.0.0 (SAS Institute) was used for statistical calculations.

[0076] <Results> <<DPP4 expression level correlates with the expression levels of cancer stem cell-like cell-related genes in RCC stem cell-like cells>> To analyze the correlation between DPP4 expression and the expression of cancer stem cell-like cell (CSC)-related genes in RCC stem cell-like cells, primary-derived cells (PDCs) were prepared from 15 cases of clear cell RCC using a three-dimensional spheroid culture method that applied the technique of culturing normal tissue stem cells. The expression level of DPP4 was evaluated using quantitative reverse transcription PCR. 36B4 was used as an internal standard. The Spearman rank correlation coefficient (R) and statistical significance (P) were calculated using JMP software version 9.0.0.0 of SAS Institute. The results are shown in Figs. 1a to 1h.

[0077] As shown in Figs. 1a to 1h, the mRNA expression levels of DPP4 were significantly correlated with the mRNA expression levels of CD133, aldehyde dehydrogenase 1 (ALDH1) A2, ALDH1A3, and IL6, respectively.

[0078] Next, hematoxylin and eosin (HE) staining and DPP4 immunohistochemical staining were performed on the original tumor morphology of RCC-A and RCC-B, which are representative PDCs. The results are shown in Fig. 2a.

[0079] As shown in Fig. 2a, the PDC maintained the morphology of tumor cells similar to the original tumor, and DPP4 staining was also positive. These RCC-A and RCC-B were used in the following further experiments.

[0080] <<DPP4 inhibition enhances the tumor growth inhibitory effect of sunitinib in RCC cells in RCC patient-derived spheroid culture>> To evaluate whether DPP4 is associated with cancer stemness and contributes to cancer cell survival, an experiment was conducted using sunitinib (SUN), a multi-target receptor tyrosine kinase inhibitor, which is a RCC therapeutic agent. To examine the effect of DPP4 inhibition on the therapeutic effect of SUN, SITA, a DPP4 inhibitor already used as a therapeutic agent for type 2 diabetes mellitus (T2DM), or siRNAs targeting DPP4 (siDPP4 #1 and #2), were combined with SUN, and the effect on spheroid growth was evaluated.

[0081] The results of treating RCC-A and RCC-B spheroids with SITA (100 μM) and SUN are shown in Figs. 2b to 2c. The data are shown as the mean ± SD of the relative luciferase activity in each spheroid analyzed by an ATP-based luciferase assay. A two-sided Student's t-test was performed with a significance level of P < 0.05 compared with the vehicle and SITA. In the two bar graphs of each item on the horizontal axis in Figs. 2b to 2c, the results of treatment with the vehicle and SITA are shown in order from the left. Also, in Figs. 2b to 2c, "*" represents P < 0.05. As shown in Figs. 2b to 2c, when the ATP production of spheroids on the third day in RCC-A cells or RCC-B cells treated with SUN alone or SUN combined with SITA was measured by luciferase activity using CellTiter-Glo, the growth inhibitory effect of SUN was enhanced when combined with SITA.

[0082] Figures 2d (RCC-A) to 2e (RCC-B) show the results of examining DPP4 expression levels in RCC-A or RCC-B spheroids treated with either control siRNA (siControl) or DPP4-specific siRNA (siDPP4 #1 or #2). The data are presented as the mean ± SD percentage of the relative DPP4 level in each siRNA-treated spheroid. A two-sided Student's t-test was performed to compare siControl and DPP4-specific siRNA (siDPP4) under each condition, with P<0.05 as the significance level. The horizontal axis in Figures 2d to 2e shows, from left to right, the results for treatment with siControl, siDPP4 #1, and siDPP4 #2. In Figures 2d to 2e, "*" indicates P<0.05. As shown in Figures 2d-2e, it was confirmed that siDPP4 suppresses DPP4 expression.

[0083] Figures 2f (RCC-A) to 2g (RCC-B) show the results of investigating the effect of DPP4-specific siRNA on the cell viability of RCC-A or RCC-B spheroids treated with SUN. The data are shown as the mean ± SD of relative luciferase activity in each spheroid culture analyzed by an ATP-based luciferase assay. A two-sided Student's t-test was performed to compare siControl and siDPP4 under each condition, with P<0.05 as the significance level. In Figures 2f to 2g, the three bar graphs for each item on the horizontal axis show, from left to right, the results for treatment with siControl, siDPP4 #1, and siDPP4 #2. In Figures 2f to 2g, "*" indicates P<0.05. As shown in Figures 2f-2g, when DPP4 expression was suppressed by siDPP4, the growth inhibitory effect of SUN was similarly enhanced.

[0084] Next, using the RCC cell lines ACHN and 769-P, we experimentally generated SUN-resistant RCC cell lines, ACHN-R and 769-PR, and compared the mRNA expression levels of DPP4 and CSC-related genes in ACHN-R and 769-PR cells with those of their respective parental cells. Figures 3a-3h show the results of qRT-PCR evaluation of DPP4 and CSC-related gene expression levels in 3D cultures of ACHN and ACHN-R cells, and Figures 3i-3p show the results of qRT-PCR evaluation of DPP4 and CSC-related gene expression levels in 3D cultures of 769-P and 769-PR cells. Data are shown as mean ± SD, n=3. A two-tailed Student's t-test was performed with a significance level of P<0.05. In Figures 3a-3p, the horizontal axis shows the results for the parental strain and the resistant strain, from left to right. In Figures 3a-3j and 3l-3p, "*" indicates P<0.05. As shown in Figures 3a-3p, mRNA expression levels of DPP4, OCT3 / 4, ALDH1A1, ALDH1A3, and IL6 were all increased in resistant strains.

[0085] Furthermore, the effects of SITA and siDPP4 in combination with SUN were evaluated using ACHN-R cells and 769-PR cells on spheroid proliferation.

[0086] First, we investigated the dose-response effect of sitagliptin (SITA) on three-dimensional spheroid proliferation in ACHN-R, 769-PR, and their parent strains when SITA was administered alone. The results for ACHN-R and its parent strain are shown in Figure 4a, and the results for 769-PR and its parent strain are shown in Figure 4b. Data are presented as mean ± SD, n=4, and a two-tailed Student's t-test was performed with a significance level of P<0.05. In Figures 4a and 4b, the two bar graphs for each item on the horizontal axis show the results for the parent strain and the resistant strain, respectively, from left to right. In Figures 4a and 4b, "ns" indicates "no significant difference". As shown in Figures 4a-4b, SITA monotherapy did not affect spheroid proliferation of ACHN cells, ACHN-R cells, 769-P cells, and 769-PR cells.

[0087] Figures 4c-4f show the results of a study investigating the combined effects of SITA and SUN in three-dimensional culture of ACHN cells (Figure 4c), ACHN-R cells (Figure 4d), 769-P cells (Figure 4e), and 769-PR cells (Figure 4f). The SITA concentration used was 100 μM. In Figures 4c-4f, the two bar graphs for each item on the horizontal axis show the results for treatment with the vehicle and treatment with SITA, respectively, from left to right. In Figures 4c, 4d, and 4f, "*" indicates P<0.05. As shown in Figures 4c-4f, combined administration of SUN and SITA suppressed spheroid proliferation of ACHN-R cells and 769-PR cells.

[0088] Figures 4g-4j show the results of an investigation into the effect of siRNA-mediated DPP4 knockdown on the therapeutic effect of SUN in three-dimensional cultures of ACHN cells (Figure 4g), ACHN-R cells (Figure 4h), 769-P cells (Figure 4i), and 769-PR cells (Figure 4j). The data are shown as the mean ± SD of relative luciferase activity in each spheroid, with n=3. The three bar graphs for each item on the horizontal axis in Figures 4g-4j show, from left to right, the results when treated with siControl, when treated with siDPP4 #1, and when treated with siDPP4 #2. In Figures 4h and 4j, "*" indicates P<0.05. As shown in Figures 4g-4j, it was confirmed that suppression of DPP4 expression by siDPP4 enhances the inhibitory effect on SUN proliferation in ACHN-R cells and 769-PR cells, thereby improving the therapeutic effect of SUN.

[0089] Figures 4k to 4l are diagrams showing the results of examining the effect of SITA treatment on the IL6 mRNA levels in three-dimensional cultures of ACHN-R cells (Figure 4k) and 769-P-R cells (Figure 4l) that are SUN-resistant. The data were shown as mean ± SD, n = 3 using 36B4 as an internal standard. A two-sided Student's t-test was performed with P < 0.05 as the significance level. For each item on the horizontal axis of Figures 4k to 4l, the two bar graphs show the results when treated with vehicle and when treated with SITA in order from the left. Also, in Figures 4k to 4l, "*" represents P < 0.05. As shown in Figures 4k to 4l, the DPP4 inhibitor decreased the mRNA expression of IL6.

[0090] Figures 4m to 4n are diagrams showing the results of examining the effect of DPP4 overexpression on the cell viability of RCC spheroids by SUN. ACHN cells (Figure 4m) and 769-P cells (Figure 4n) stably expressing a control vector (vector #1 or #2) or DPP4 (DPP4#1 or #2) were treated with SUN and analyzed by an ATP-based luciferase assay. The data were shown as mean ± SD of the relative luciferase activity in each spheroid, n = 4. A two-sided Student's t-test was performed with P < 0.05 as the significance level. For each item on the horizontal axis of Figures 4m to 4n, the four bar graphs show the results when treated with vector #1, when treated with vector #2, when treated with DPP4#1, and when treated with DPP4#2 in order from the left. Also, in Figures 4m to 4n, "*" represents P < 0.05. As shown in Figures 4m to 4n, in ACHN cells and 769-P cells with stable overexpression of DPP4, cell viability under SUN administration was improved, and it was confirmed that DPP4 overexpression restored the cell viability of RCC spheroids by SUN.

[0091] <<DPP4 expression in SUN-resistant RCC cells is regulated by the retinoic acid signal>> Since ALDH1 and DPP4 expression levels are correlated, we hypothesized that there might be a relationship between ALDH1 function and DPP4 expression. ALDH1 is an enzyme involved in retinol metabolism, converting retinol to retinoic acid (RA) and acting as a ligand for RA receptors such as retinoic acid receptor alpha (RARα).

[0092] Figures 5a-5b show the results of an investigation into the effect of RA supplementation on the suppression of DPP4 by disulfiram (DSF), one of the FDA-approved ALDH inhibitors used to treat alcohol dependence, in sunitinib (SUN)-resistant RCC cells (Figure 5a: ACHN-R cells, Figure 5b: 769-PR cells). ACHN-R cells were treated with vehicle alone, 15 μM DSF, or 15 μM DSF and 1 μM RA. 769-PR cells were treated with vehicle alone, 25 μM DSF, or 25 μM DSF and 1 μM RA. Data are presented as mean ± SD, n=3. A two-sided Student's t-test was performed with a significance level of P<0.05. The horizontal axis in Figures 5a-5b shows the results from left to right: treatment with vehicle alone, treatment with DSF alone, and treatment with DSF and RA. Also, in Figures 5a-5b, "*" represents P < 0.05. As shown in Figures 5a-5b, we found that the addition of disulfiram (DSF) suppressed DPP4 expression in both ACHN-R and 769-PR cells, and that this effect was restored by RA administration. This indicates that RA signaling regulates DPP4 expression in RCC cells.

[0093] Next, to identify the RARα binding site in the DPP4 gene promoter region, the DPP4 promoter region retrieved from the hg19 human genome dataset was searched using the RARα matrix profile in the open-access transcription factor binding profile database JASPAR (http: / / jaspar.genereg.net / ) (Wasserman WW & Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5, 276-287 (2004)).

[0094] One RARE candidate was identified in the DPP4 promoter region located between -1619 bp and -1647 bp from the transcription start site (TSS), where the JASPAR algorithm's relative profile score threshold exceeded 85% (Figure 5c).

[0095] Figures 5d-5e show the results of confirming the recruitment of retinoic acid receptor α (RARα) to RARE via the DPP4 promoter in SUN-resistant RCC cells, and Figures 5f-5g show the results of confirming the recruitment of retinoid X receptor α (RXRα) to RARE via the DPP4 promoter in SUN-resistant RCC cells. Chromatin immunoprecipitation was performed in ACHN-R cells and 769-PR cells using anti-RARα antibody (Figures 5d-5e) or anti-RXRα antibody (Figures 5f-5g) and control IgG, and quantification was performed by PCR. Data are shown as mean ± SD, n=3. A two-sided Student's t-test was performed with a significance level of P<0.05. In Figures 5d-5g, the left side shows the results for the RARE case, and the right side shows the results for the reference region. In Figures 5d-5e, the horizontal axis shows the results using IgG and RARα antibody, respectively from left to right. In Figures 5f-5g, the horizontal axis shows the results using IgG and RXRα antibody, respectively from left to right. Also, in Figures 5d-5g, "*" indicates P<0.05. As shown in FIGS. 5d to 5g, when performing ChIP-qPCR assays using RARα antibody, RXRα antibody, and IgG, it was revealed that both ACHN-R cells and 769-P-R cells recruited RARα and RXRα to RARE.

[0096] Next, to examine whether RA modifies DPP4 promoter activity, a luciferase reporter assay was conducted. Luciferase reporters containing wild-type (WT) and mutant (Mut) RARE were used. Also, as a known RARE, a RARE having direct repeat 5 (DR5) (Bulens, F. et al. Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DR5) located at -7 kilobases. J. Biol. Chem. 270, 7167-7175 (1995)) was inserted into the luciferase reporter and used as a positive control. The results are shown in FIG. 5h. The data are shown as mean ± SD, n = 3. A two-sided Student's t-test was performed with P < 0.05 as the significance level. In each of the two bar graphs for each item on the horizontal axis of FIG. 5h, from left to right, the results without RA treatment and with RA treatment are shown. Also, in FIG. 5h, "*" represents P < 0.05. As shown in FIG. 5h, in 293T cells, the wild-type (WT) RARE showed a significant increase in transcriptional activity in response to RA treatment, whereas, in contrast, the mutant (Mut) RARE did not show activation by RA treatment. This suggests that the RA / RAR / RXR signal regulates the transcription of DPP4 via a functional RARE.

[0097] <<DPP4 inhibitor SITA inhibits the growth of SUN-resistant RCC xenograft tumors>> The combined effect of SITA on the growth of SUN-resistant RCC tumors was verified using a xenograft tumor model. ACHN or ACHN-R nude mouse xenograft tumor volume of 180 mm 3 When this condition was reached, oral administration of SUN, or SUN and SITA, was initiated.

[0098] The growth curves for ACHN xenograft tumors treated with sunitinib (SUN) (ACHN / SUN group, n=6), ACHN-R xenograft tumors treated with SUN (ACHN-R / SUN group, n=6), and ACHN-R xenograft tumors treated with SUN and SITA (ACHN-R / SUN+SITA group, n=6) are shown in Figure 6a. The tumor weight at day 13 (endpoint) for each group is shown in Figure 6b, and the tumor weight at day 13 (endpoint) for each group is shown in Figure 6c. Data are presented as mean ± SD, n=6, and a two-sided Student's t-test was performed with a significance level of P<0.05. Tumor volume was estimated by measuring the size of the tumor with calipers. The horizontal axis in Figures 6b-6c shows the results for the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group from left to right. In addition, in Figures 6a and 6b, "*" represents P < 0.05. Furthermore, representative photographs of xenografted nude mice from each group on day 13 are shown in Figures 6d-6f. The scale bar represents 10 mm. As shown in Figures 6a-6f, ACHN-R xenograft tumors showed resistance to SUN compared to ACHN xenograft tumors, but tumor growth was suppressed to a similar extent as in ACHN xenograft tumors by using SUN in combination with SITA.

[0099] Figure 6g shows the results of Ki67 immunohistochemical staining of resected tumors in each group, and the percentage of Ki67-positive cells (Ki67 index) was determined. The scale bar is 50 μm. Data are shown as mean ± SD, n=6, and a two-sided Student's t-test was performed with P<0.05 as the significance level. The horizontal axis of Figure 6g shows the results for the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN+SITA group from left to right. In Figure 6g, "*" indicates P<0.05. As shown in Fig. 6g, the combination of SITA and SUN decreased the Ki67 index, which is a marker of cell proliferation, in ACHN-R xenograft tumors.

[0100] The results of examining the mRNA levels of DPP4 and IL6 in each group are shown in Fig. 6h (DPP4) and Fig. 6i (IL6). The expression levels were evaluated by qRT-PCR. The data are shown as mean ± SD, n = 6, and a two-sided Student's t-test was performed with P < 0.05 as the significance level. In addition, the horizontal axes of Figs. 6h to 6i show the results of the ACHN / SUN group, ACHN-R / SUN group, and ACHN-R / SUN + SITA group in order from the left. Also, in Figs. 6h to 6i, "*" represents P < 0.05. As shown in Figs. 6h to 6i, the expression of DPP4 and IL6 increased in ACHN-R tumors compared with ACHN tumors, and the expression of IL6 decreased in ACHN-R xenograft tumors combined with SITA.

[0101] From the above, it was confirmed that sitagliptin (SITA) overcame sunitinib resistance in RCC xenograft tumors.

[0102] <<In RCC patients receiving TKI treatment, DPP4 inhibitor (DPP4i) contributes to a good prognosis>> To verify whether the data shown in previous in vitro and in vivo experiments are related to prognosis and drug efficacy in clinical cases, a retrospective clinical analysis was performed on 73 cases of RCC treated with TKI (sunitinib, sorafenib, axitinib, or pazopanib) at the Saitama Medical University Comprehensive Medical Center. Among the 73 cases, 47 cases had no T2DM and did not take DPP4i (sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, or vildagliptin) (T2DM- / DPP4i-), 12 cases had T2DM but did not take DPP4i (T2DM+ / DPP4i-), and 14 cases had T2DM and took DPP4i (T2DM+ / DPP4i+).

[0103] Figure 7a shows the results of a Kaplan-Meier analysis of 10-year overall survival to determine whether DPP4i use contributed to prognosis in these three groups. P-values ​​were evaluated using the log-rank test. Holm's test was used as a post-hoc test for multiple comparisons. As shown in Figure 7a, DPP4i use was a favorable prognostic factor in T2DM patients. It was confirmed that DPP4 inhibitors (DPP4i) contributed to the prognosis and maximum tumor reduction rate in RCC patients treated with TKIs.

[0104] Next, Figure 7b shows the results of a waterfall plot analysis of the maximum tumor change rate in 73 RCC patients treated with TKI, stratified according to the presence or absence of T2DM and DPP4i use. Partial response rates were compared between groups using Fisher's exact test. DPP4i use increased the partial response rate under TKI treatment (see Table 1 below).

[0105] [Table 1]

[0106] Figure 7c shows the results of a box plot analysis of the maximum tumor change rate in 73 RCC patients treated with TKI, stratified by the presence or absence of T2DM and the presence or absence of DPP4i administration. P values ​​were evaluated using the Mann-Whitney U test. As shown in Figure 7c, we observed an enhanced tumor-reducing effect with DPP4i.

[0107] Furthermore, to evaluate the relationship between the immunohistochemical staining properties of DPP4 and prognosis and TKI treatment efficacy, immunohistochemical staining analysis of DPP4 was performed on 49 of the 73 cases for which immunohistochemical staining was possible. As shown in Figure 7d, a Kaplan-Meier analysis of 10-year overall survival revealed that high DPP4 expression was a poor prognostic factor. Even when the analysis was limited to 31 non-T2DM cases, high DPP4 expression remained a poor prognostic factor (Figure 7e). However, when the analysis was limited to 18 T2DM cases, DPP4 expression was not a significant prognostic factor (Figure 7f). The p-value was evaluated using the log-rank test. Of these 49 cases, no significant difference was found in the partial response rate for the maximum tumor change rate among the 20 RCC patients with low DPP4 immunoresponsiveness (see Table 2 below). On the other hand, a significant difference in the maximum tumor change rate was observed among the 29 RCC patients with low DPP4 immunoresponsiveness with DPP4 inhibitor administration (see Table 3 below). A schematic diagram of the ALDH1 / RA / DPP4 system in DPP4-highly expressing RCC stem cell-like cells in this study is shown in Figure 7g.

[0108] [Table 2]

[0109] [Table 3]

[0110] Examples of embodiments of the present invention include the following: <1> This is a combination drug for treating kidney cancer, characterized by the use of a tyrosine kinase inhibitor and a dipeptidyl peptidase 4 inhibitor in combination. <2> The kidney cancer is a kidney cancer that is resistant to tyrosine kinase inhibitors. <1> It is a combination drug as described above. <3> The tyrosine kinase inhibitor is at least one selected from the group consisting of sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib. <1> from <2> It is a combination drug described in any of the following. <4> The dipeptidyl peptidase 4 inhibitor is at least one selected from the group consisting of sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omaligliptin, gemigliptin, evogliptin, gosogliptin, and dipeptidyl peptidase 4 expression inhibitors. <1> from <3> It is a combination drug described in any of the following. <5> The dipeptidyl peptidase 4 expression inhibitor is at least one selected from the group consisting of a double-stranded nucleic acid molecule for suppressing the expression of the dipeptidyl peptidase 4 gene, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, and a vector containing the DNA. <4> It is a combination drug as described above. <6> A double-stranded nucleic acid molecule that suppresses the expression of the dipeptidyl peptidase 4 gene, (a) A sense strand containing a nucleotide sequence corresponding to a target sequence consisting of a nucleotide sequence represented by either SEQ ID NO:1 or SEQ ID NO:4, (b) The sense strand of (a) and an antisense strand having a complementary base sequence to the sense strand that forms a double helix with the sense strand of (a) <5> It is a combination drug as described above. <7> The double-stranded nucleic acid molecule is either double-stranded RNA or a double-stranded RNA-DNA chimera. <5> from <6> It is a combination drug described in any of the following. <8> The double-stranded nucleic acid molecule is either siRNA or chimeric siRNA. <5> from <7> It is a combination drug described in any of the following. <9> The double-stranded nucleic acid molecule is siRNA. <5> from <8> It is a combination drug described in any of the following. <10> In the individual, the above <1> from <9> This is a method for treating kidney cancer characterized by administering one of the concomitant medications described in any of the above. <11> This is a tyrosine kinase inhibitor therapeutic enhancer that contains a dipeptidyl peptidase 4 inhibitor and is characterized by enhancing the therapeutic effect of tyrosine kinase inhibitors against renal cancer. <12> The kidney cancer is a kidney cancer that is resistant to tyrosine kinase inhibitors. <11> It is an enhancer of the therapeutic effect of the tyrosine kinase inhibitors described above. <13> The tyrosine kinase inhibitor is at least one selected from the group consisting of sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib. <11> from <12> It is an enhancer of the therapeutic effect of tyrosine kinase inhibitors as described in any of the above. <14> The dipeptidyl peptidase 4 inhibitor is at least one selected from the group consisting of sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omaligliptin, gemigliptin, evogliptin, gosogliptin, and dipeptidyl peptidase 4 expression inhibitors. <11> from <13> It is an enhancer of the therapeutic effect of tyrosine kinase inhibitors as described in any of the above. <15> The dipeptidyl peptidase 4 expression inhibitor is at least one selected from the group consisting of a double-stranded nucleic acid molecule for suppressing the expression of the dipeptidyl peptidase 4 gene, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, and a vector containing the DNA. <14> It is an enhancer of the therapeutic effect of the tyrosine kinase inhibitors described above. <16> A double-stranded nucleic acid molecule that suppresses the expression of the dipeptidyl peptidase 4 gene, (a) A sense strand containing a nucleotide sequence corresponding to a target sequence consisting of a nucleotide sequence represented by either SEQ ID NO:1 or SEQ ID NO:4, (b) The sense strand of (a) and an antisense strand having a complementary base sequence to the sense strand that forms a double helix with the sense strand of (a) <15> It is an enhancer of the therapeutic effect of the tyrosine kinase inhibitors described above. <17> The double-stranded nucleic acid molecule is either double-stranded RNA or a double-stranded RNA-DNA chimera. <15> from <16> It is an enhancer of the therapeutic effect of tyrosine kinase inhibitors as described in any of the above. <18> The double-stranded nucleic acid molecule is either siRNA or chimeric siRNA. <15> from <17> It is an enhancer of the therapeutic effect of tyrosine kinase inhibitors as described in any of the above. <19> The double-stranded nucleic acid molecule is siRNA. <15> from <18> It is an enhancer of the therapeutic effect of tyrosine kinase inhibitors as described in any of the above. <20> In the individual, the above <11> from <19> This method enhances the therapeutic effect of tyrosine kinase inhibitors for renal cancer, characterized by administering a therapeutic effect enhancer for tyrosine kinase inhibitors described in any of the above.

Claims

1. A combination drug for treating renal cancer resistant to tyrosine kinase inhibitors, comprising a tyrosine kinase inhibitor and a dipeptidyl peptidase 4 inhibitor, The tyrosine kinase inhibitor is at least one selected from the group consisting of sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib. The dipeptidyl peptidase 4 inhibitor is at least one selected from the group consisting of sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omaligliptin, gemigliptin, evogliptin, gosogliptin, and dipeptidyl peptidase 4 expression inhibitors. The combination drug is characterized in that the dipeptidyl peptidase 4 expression inhibitor is at least one selected from the group consisting of a double-stranded nucleic acid molecule for suppressing the expression of the dipeptidyl peptidase 4 gene that targets the dipeptidyl peptidase 4 gene, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, and a vector containing the DNA.

2. A dipeptidyl peptidase 4 inhibitor is provided to enhance the therapeutic effect of a tyrosine kinase inhibitor on renal cancer resistant to tyrosine kinase inhibitors, The tyrosine kinase inhibitor is at least one selected from the group consisting of sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib. The dipeptidyl peptidase 4 inhibitor is at least one selected from the group consisting of sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omaligliptin, gemigliptin, evogliptin, gosogliptin, and dipeptidyl peptidase 4 expression inhibitors. The therapeutic effect enhancer for tyrosine kinase inhibitors is characterized in that the dipeptidyl peptidase 4 expression inhibitor is at least one selected from the group consisting of a double-stranded nucleic acid molecule for suppressing the expression of the dipeptidyl peptidase 4 gene that targets the dipeptidyl peptidase 4 gene, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, and a vector containing the DNA.

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