Use of USP15 inhibitors in the preparation of medicaments for the treatment and / or prevention of renal cell carcinoma, and pharmaceutical compositions and kits thereof.

Abstract

This application discloses the use of USP15 inhibitors in the preparation of medicaments for the treatment and / or prevention of renal cell carcinoma, as well as pharmaceutical compositions and kits thereof.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to the use of an inhibitor of ubiquitin carboxyl-terminal hydrolase 15 (USP15) in the preparation of medicaments for the treatment and / or prevention of renal cell carcinoma, pharmaceutical compositions or kits thereof, and small interfering RNA (siRNA) targeting USP15. Background Technology

[0002] Renal cell carcinoma (RCC), commonly known as kidney cancer, is the most common malignant disease of the kidney, accounting for approximately 90% of all kidney malignancies (Ljungberg, B, Bensalah K, Canfield S, Dabestani S, Hofmann F, Hora M, Kuczyk MA, Lam T, Marconi L, Merseburger AS, Mulders P, Powles T, Staehler M, Volpe A, Bex A. EAU Guidelines on Renal Cell Carcinoma: 2014 Update). Eur Urol Renal cell carcinoma (RCC) ranks third in incidence among urinary tract tumors in China and is a common malignant tumor of the urinary system. Among them, clear cell renal cell carcinoma is the most common type of renal cell carcinoma, accounting for about 75% of renal cell carcinomas (Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, YuXQ, He J. Cancer statistics in China, 2015). CA Cancer J Clin 2016;66(2):115-32). Currently, surgery remains the main treatment for renal cell carcinoma. However, due to the insidious onset of renal cell carcinoma, approximately 17% of patients have already developed distant metastases at the time of diagnosis. These patients usually require systemic treatment after surgical resection (Capitanio U, Bensalah K, Bex A, Boorjian SA, Bray F, Coleman J, Gore JL, Sun M, Wood C, Russo P. Epidemiology of Renal Cell Carcinoma). Eur Urol2019 Jan;75(1):74-84). For advanced renal cell carcinoma, traditional radiotherapy and chemotherapy have little effect, and the systemic treatment with interferon (IFN) and interleukin (IL-2) has a low efficacy rate (7%-8%), significant toxic side effects, and a median survival time of only 12 months after treatment (Hsieh JJ, Purdue MP, Signoretti S, Swanton C, Albiges L, Schmidinger M, Heng DY, Larkin J, Ficarra V. Renal cell carcinoma). Nat Rev Dis Primers 2017;3:17009). In recent years, several drugs targeting VEGF, mTOR, PD-1, PD-L1, etc., have been approved by the U.S. Food and Drug Administration (FDA) for first-line and second-line treatment of renal cell carcinoma (Escudier B, Porta C, Schmidinger M, Rioux-Leclercq N, Bex A, Khoo V, et al. Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up). Ann Oncol . 2019;30:706-20; Atkins MB, Tannir NM. Current and emerging therapies for firstline treatment of metastatic clear cell renal cell carcinoma. CancerTreat Rev . 2018;70:127-37; Barata PC, Rini BI. Treatment of renal cell carcinoma: current status and future directions. CA Cancer J Clin (2017;67:507-24). However, the heterogeneity and adaptability during the development of renal cell carcinoma lead to limited efficacy of targeted drugs, increased drug resistance, and the development of death resistance, ultimately promoting renal cell carcinoma recurrence and metastasis. For example, sunitinib, a classic first-line targeted therapy for renal cell carcinoma, often leads to secondary resistance in most patients within 6-15 months of starting treatment, resulting in disease progression (Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy). Nat Rev Cancer. 2008;8:592-603; FicarraV, Novara G. Kidney cancer: Characterizing late recurrence of renal cellcarcinoma. Nat Rev Urol . 2013 Dec;10(12):687-9; Hutchinson, L. Targetedtherapies: Another option for metastatic RCC Nat Rev Clin Oncol 2013 Nov;10(11):607). Therefore, the study of the molecular mechanisms of renal cell carcinoma cell death resistance and immune checkpoint inhibitor resistance, and the search for more effective new therapeutic targets, has always been a major scientific issue in the field of renal cell carcinoma research.

[0003] Programmed cell death (PCD) is a core research area in the search for mechanisms of cell death resistance. It is a cell death pathway regulated by a series of signal transduction pathways and is one of the important biological processes by which the body eliminates unnecessary cells to maintain homeostasis. Tumor cells can also promote their own growth by inhibiting the activation of programmed cell death-related signaling pathways, thereby generating death resistance (Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation). Cell . 2011 Mar 4;144(5):646-74; Kolb JP, Oguin TH3rd, Oberst A, Martinez J. Programmed Cell Death and Inflammation: Winter IsComing. Trends Immunol2017 Oct;38(10):705-18). Based on different regulatory signals, programmed cell death is mainly divided into apoptosis mediated by Caspase3 / 7 cleavage, necroptosis mediated by activation of RIPK1 / RIPK3 / MLKL signaling, pyroptosis mediated by GSDMD / GSDME cleavage, ferroptosis mediated by iron ion-catalyzed accumulation of lipid peroxides, and autophagy mediated by LC3 cleavage (Kolb JP, Oguin TH 3rd, Oberst A, Martinez J. Programmed Cell Death and Inflammation: Winter Is Coming). Trends Immunol . 2017 Oct;38(10):705-18; TangD, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. Cell Res 2019 May;29(5):347-64). Among these, apoptosis is the most common type of programmed cell death and has been a focus of previous tumor research. However, more and more tumor cells have been found to be resistant to apoptosis (Ye J, Zhang R, Wu F, Zhai L, Wang K, Xiao M, Xie T, Sui X. Non-apoptotic cell death in malignant tumor cells and natural compounds). Cancer Lett (2018 Apr 28;420:210-27). Studies have found that when the function of caspase 8, which mediates apoptosis, is inhibited, it promotes the formation of the RIPK1 / RIPK3 complex, promotes the phosphorylation and translocation of downstream MLKL protein to the cell membrane, and forms pores in the cell membrane, significantly enhancing cell membrane permeability and ultimately leading to necroptosis. Therefore, necroptosis is also known as the "second line of defense" after the failure of the apoptosis mechanism (Legrand AJ, Konstantinou M, GoodeEF, Meier P. The Diversification of Cell Death and Immunity: Memento Mori). Mol Cell(2019 Oct 17;76(2):232-42). It is evident that, in addition to exploring mechanisms of tumor cell apoptosis resistance, in-depth exploration of other non-apoptotic programmed cell death pathways is also of significant value in identifying new targets for tumor therapy. The pro-inflammatory cell death resulting from novel cell death also holds important value for investigation regarding the sensitizing effect of immune checkpoint inhibitors.

[0004] Programmed cell death is strictly regulated by cellular signal transduction, a process inseparable from the precise regulation of post-translational modifications of proteins such as phosphorylation and ubiquitination. Ubiquitination is one of the most important post-translational modifications of proteins in cells, and its study has always been a hot topic in oncology research. Different types of polyubiquitination modifications (such as K6, K11, K27, K29, K33, K48, and K63) can play a wide range of functions in cellular signal transduction by regulating the degradation, translocation, activation, or complex formation of substrate proteins, and play an important role in tumorigenesis, development, metastasis, and cell death (Gallo LH, Ko J, Donoghue DJ. The importance of regulatoryubiquitination in cancer and metastasis). Cell Cycle . 2017 Apr 3;16(7):634-48).

[0005] Existing research indicates that USP15 plays a crucial regulatory role in tumors, but its regulatory mechanisms differ across different tumor types. USP15 can stabilize the expression of several proto-oncogene proteins, such as MDM2 (melanoma and colon cancer cells) and type I TGF-β receptor (malignant glioma cells), through its deubiquitinase activity, thereby promoting the progression of these tumors. Conversely, USP15 can also stabilize the expression of some tumor-suppressive proteins, such as APC (cervical cancer cells), through its deubiquitinase activity (Chou CK, Chang YT, Korinek M, Chen YT, Yang YT, Leu S, Lin IL, Tang CJ, Chiu CC. The Regulations of Deubiquitinase USP15 and Its Pathophysiological Mechanisms in Diseases). Int J Mol Sci2017;18(3). pii:E483). On the other hand, USP15 can also promote paclitaxel-induced apoptosis in HeLa cells by stabilizing caspase-3 expression (Xu M, Takanashi M, Oikawa K, Tanaka M, Nishi H, Isaka K, Kudo M, Kuroda M. USP15 plays an essential role for caspase-3 activation during paclitaxel-induced apoptosis). Biochem Biophys Res Commun 2009;388(2):366-71). The above studies show that the function and mechanism of action of USP15 differ in different tumors. Considering the current research status of USP15, its function in programmed cell death in renal cell carcinoma has not yet been reported.

[0006] In summary, there is an urgent need in this field to develop an active substance that can effectively inhibit the growth of clear cell renal cell carcinoma and enhance its sensitivity to immune checkpoint inhibitors. Summary of the Invention

[0007] In order to solve the above-mentioned technical problems, in one aspect, this application provides the use of an inhibitor of USP15 in the preparation of a medicament for the treatment and / or prevention of renal cancer in subjects.

[0008] In another aspect, this application provides the use of inhibitors of USP15 and immune checkpoint inhibitors in the preparation of medicaments for the treatment and / or prevention of renal cell carcinoma in subjects.

[0009] In another aspect, this application provides a pharmaceutical composition comprising an inhibitor of USP15.

[0010] In another aspect, this application provides a kit comprising an inhibitor of USP15.

[0011] In another aspect, this application provides siRNA targeting USP15, said siRNA comprising a pair of RNA molecules selected from the group consisting of or a combination thereof: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; (c) SEQ ID NO: 5 and SEQ ID NO: 6; (d) A pair of RNA molecules that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of (a)-(c) and that inhibit the activity of USP15; and A pair of RNA molecules that have been substituted, deleted and / or added to one or more bases in any of (e) (a)-(d) and have the function of inhibiting USP15. Attached Figure Description

[0012] The present invention will be further described below with reference to the accompanying drawings, which are shown only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

[0013] Figure 1 Interfering with USP15 in renal cell carcinoma cells using small interfering RNA significantly promoted cell death. Specifically: Figure 1 A is a flow cytometry diagram of cell death; Figure 1 B is a statistical analysis graph of cell death in two groups; P < 0.001. Ctrl siRNA represents the control group, and USP15 siRNA represents the USP15 interference group.

[0014] Figure 2 USP15 can inhibit RIPK1 ubiquitination levels and suppress the activation of related cell death signaling pathways. Specifically: Figure 2 A represents the activation of cell death signaling pathways by Western blot analysis after shRNA inhibits USP15 expression; where Ctrl shRNA represents the control group and USP15 shRNA represents the USP15 interference group. Figure 2 B involves immunoprecipitation and Western blot analysis using USP15 antibody and renal cell carcinoma lysate to detect the binding of USP15 to key cell death proteins. Here, IgG and USP15 serve as the control group.

[0015] Figure 2 C represents small interfering RNA (siRNA) interfering with USP15 in renal cell carcinoma cells. Immunoprecipitation and Western blotting analysis were performed using USP15 antibody and renal cell carcinoma cell lysates to detect RIPK1 ubiquitination levels. Ctrl siRNA represents the control group, and USP15 siRNA represents the USP15 interference group. IgG and RIPK1 serve as the control groups.

[0016] Figure 3USP15 from exosomes derived from renal cell carcinomas can inhibit T cell activation. Specifically: Figure 3 A represents the expression level of USP15 in exosomes secreted by HK-2 and 786-O cells after siRNA treatment. Figure 3 B is Figure 3 A. Detection of intracellular USP15 expression in Jurket T cells after treatment with exosomes; Figure 3 C is Figure 3 The production of secreted IL-2 was measured after T cells in B cells were stimulated with CD3 / CD28.

[0017] P<0.05; P < 0.01. Ctrl siRNA represents the control group, and USP15 siRNA represents the USP15 interference group.

[0018] Figure 4 Intratumoral injection of USP15 siRNA can inhibit the growth of renal cell carcinoma-bearing tissue and promote the sensitivity of renal cell carcinoma to immune checkpoint inhibitors (anti-PD1). Specifically: Figure 4 A is a comparison diagram of the size of the four groups of tumor-bearing tissues; Figure 4 B represents the growth curves of four groups of renal cell carcinoma-bearing tissues; P < 0.0001. Ctrl siRNA represents the control group, USP15 siRNA represents the USP15 interference group, and the black arrows represent the time of intratumoral injection of siRNA and intraperitoneal injection of anti-PD1 antibody after tumor formation. Detailed Implementation

[0019] This application relates to the use of an inhibitor of USP15 in the preparation of a medicament for treating and / or preventing renal cell carcinoma in a subject. In one embodiment, the renal cell carcinoma includes clear cell renal carcinoma. In one embodiment, the renal cell carcinoma includes clear cell renal adenocarcinoma. As used herein, the term "USP15" has its broad meaning, including... USP15The gene, USP15 mRNA, cDNA, USP15 precursor, USP15 protein, its modified or cleaved products, or active fragments. In one embodiment, treatment and / or prevention of renal cell carcinoma includes treating and / or preventing one or more symptoms of renal cell carcinoma. In one embodiment, the object includes mammals, such as humans, non-human primates (e.g., chimpanzees, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits), preferably humans. As used herein, the term "USP15 inhibitor" or "inhibitor" has its broad meaning and refers to a substance capable of reducing, weakening, or eliminating the level or function of USP15. USP15 inhibitors may include, but are not limited to: substances that reduce the transcription, translation, or expression levels of USP15 (e.g., substances that knock down or eliminate USP15 expression), substances that inhibit USP15 function, etc. In some embodiments, inhibitors of USP15 include, but are not limited to: substances that inhibit USP15 (such as compounds, e.g., small molecule compounds), antibodies against upstream metabolic enzymes that generate USP15 or its precursors (e.g., antibodies against IRG1 or OGDH), siRNA, miRNA, antisense oligonucleotides, CRISPR / Cas9 gene editing products, exosomes, zinc finger proteins, compounds that inhibit the function and / or activity of USP15 or its precursors, promoter elements and / or expression vectors that downregulate the gene, mRNA, or protein expression levels of USP15. In one embodiment, inhibitors of USP15 include inhibitors selected from the group consisting of: siRNA, shRNA, miRNA, antisense oligonucleotides, exosomes, zinc finger proteins, CRISPR / Cas9 gene editing products, antibodies, chemical inhibitors, or combinations thereof. In one embodiment, inhibitors of USP15 include inhibitors selected from the group consisting of: naturally purified substances, modified naturally purified substances, semi-synthetic substances, chemically synthesized substances, or combinations thereof. In one embodiment, the inhibitor of USP15 is derived from mammals. In one embodiment, the inhibitor of USP15 is derived from: humans, non-human primates (e.g., chimpanzees, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits). In a preferred embodiment, the inhibitor of USP15 comprises siRNA targeting USP15.In one embodiment, the siRNA comprises a pair of RNA molecules selected from the group consisting of: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; (c) SEQ ID NO: 5 and SEQ ID NO: 6; (d) a pair of RNA molecules having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of (a)-(c) and having the function of inhibiting USP15; and (e) a pair of RNA molecules having one or more bases substituted, deleted, and / or added in any one of (a)-(d) and having the function of inhibiting USP15. As used herein, the term “inhibits USP15” has its broad meaning and refers to the ability to reduce, weaken, or eliminate the level or function of USP15. In a preferred embodiment, the siRNA comprises a pair of RNA molecules selected from the group consisting of: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; and (c) SEQ ID NO: 5 and SEQ ID NO: 6. In a preferred embodiment, the siRNA comprises a pair of RNA molecules selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 4; a pair of RNA molecules having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and having the function of inhibiting USP15; and a pair of RNA molecules having one or more bases substituted, deleted, and / or added to either or both of SEQ ID NO: 3 and SEQ ID NO: 4, and having the function of inhibiting USP15. In a preferred embodiment, the siRNA comprises SEQ ID NO: 3 and SEQ ID NO: 4. In this application, the terms "homology," "identity," and "identity" are used interchangeably.

[0020] This application also relates to the use of inhibitors of USP15 and immune checkpoint inhibitors in the preparation of medicaments for treating and / or preventing renal cell carcinoma in subjects. In one embodiment, this use enhances the sensitivity of renal cell carcinoma to immune checkpoint inhibitor drugs. In one embodiment, the immune checkpoint inhibitor comprises inhibitors selected from the group consisting of PD1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, or combinations thereof. In a preferred embodiment, the immune checkpoint inhibitor comprises a PD1 inhibitor. In a more preferred embodiment, the immune checkpoint inhibitor comprises an anti-PD1 antibody. The inhibitor and subject of USP15 may include the inhibitor and subject of USP15 as described in any of the foregoing embodiments.

[0021] This application also relates to a pharmaceutical composition comprising an inhibitor of USP15. In one embodiment, the pharmaceutical composition further comprises an immune checkpoint inhibitor. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically or immunologically acceptable carrier or excipient. As used herein, the term "pharmaceutically or immunologically acceptable" means a substance suitable for human and / or animal use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically or immunologically acceptable carrier or excipient" refers to a carrier or excipient used for therapeutic administration, including various diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers or excipients are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). In one embodiment, an acceptable carrier or excipient may include liquids such as water, saline, glycerol, and ethanol. In another embodiment, an acceptable carrier or excipient may also include auxiliary substances such as fillers, disintegrants, lubricants, flow aids, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, and more preferably about 7. In one embodiment, the pharmaceutical composition is suitable for administration via a method selected from the group consisting of: oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA, or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by replication-deficient bacteria, target DNA carried by replication-deficient adenovirus, or protein encoded by a target gene, electroporation, nasal administration, pulmonary administration, oral administration, and transdermal administration. The inhibitor and immune checkpoint inhibitor of USP15 may include the inhibitor and immune checkpoint inhibitor of USP15 as described in any of the foregoing embodiments. In one implementation, the inhibitor and / or immune checkpoint inhibitor of USP15 are administered by injection.

[0022] This application also relates to a kit comprising an inhibitor of USP15. In one embodiment, the kit further comprises an immune checkpoint inhibitor. In another embodiment, the kit further comprises a pharmaceutically or immunologically acceptable carrier or excipient. The USP15 inhibitor, immune checkpoint inhibitor, and pharmaceutically or immunologically acceptable carrier or excipient may comprise the USP15 inhibitor, immune checkpoint inhibitor, and pharmaceutically or immunologically acceptable carrier or excipient as described in any of the foregoing embodiments.

[0023] This application also relates to siRNA targeting USP15, said siRNA comprising a pair of RNA molecules selected from the group consisting of: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; (c) SEQ ID NO: 5 and SEQ ID NO: 6; (d) a pair of RNA molecules having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of (a)-(c) and having inhibitory activity against USP15; and (e) a pair of RNA molecules having undergone substitution, deletion, and / or addition of one or more bases in any one of (a)-(d) and having inhibitory activity against USP15. The siRNA for USP15 may include the siRNA for USP15 described in any of the foregoing embodiments.

[0024] Optionally, this application also relates to a method for treating and / or preventing renal cell carcinoma in a subject, the method comprising administering to the subject an effective amount of an inhibitor of USP15, a pharmaceutical composition, or a kit. The USP15 inhibitor, pharmaceutical composition, or kit may include the USP15 inhibitor, pharmaceutical composition, or kit described in any of the foregoing embodiments. As used herein, the term "effective amount" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0025] Compared with the prior art, the present invention has the following beneficial effects: Through extensive research and animal model experiments, the applicant has discovered that interfering with the expression of USP15 in renal cell carcinoma can effectively promote the death of renal cell carcinoma cells, inhibit the growth of renal cell carcinoma tissue, and enhance the sensitivity of renal cell carcinoma tissue to immune checkpoint inhibitors.

[0026] The applicant's research revealed that USP15 knockdown significantly promotes renal cell carcinoma cell death. Furthermore, USP15 in exosomes derived from renal cell carcinoma cells can inhibit T cell activation, suggesting that interfering with USP15 knockdown may have potential applications in the treatment of renal cell carcinoma. Therefore, this application provides a method and strategy for applying small interfering RNA of USP15 in the treatment of renal cell carcinoma.

[0027] This application investigates the effects of a novel USP15 small interfering RNA on the growth and death of renal cell carcinoma cells and verifies the therapeutic effect of this small interfering RNA on renal cell carcinoma. Experiments demonstrate that: 1) USP15 knockdown in renal cell carcinoma cells promotes cell death; 2) USP15 knockdown in renal cell carcinoma cells promotes the activation of death-related signaling pathways; 3) USP15 in exosomes derived from renal cell carcinoma cells inhibits T cell activation; 4) Intratumoral injection of USP15 knockdown inhibits the growth of renal cell carcinoma-bearing tissue and enhances the sensitivity of renal cell carcinoma-bearing tissue to the immune checkpoint inhibitor anti-PD1 antibody.

[0028] This application demonstrates that inhibitors of USP15 can promote renal cell carcinoma death and enhance the sensitivity of renal cell carcinoma tissue to the immune checkpoint inhibitor anti-PD1 antibody. These findings confirm that inhibitors of USP15 (e.g., small interfering RNA) hold promise as an effective treatment for renal cell carcinoma.

[0029] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0030] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0031] Example

[0032] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present application, and such modifications and variations are all within the scope of the present application.

[0033] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0034] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in the methods of this application. The preferred methods and materials described herein are for illustrative purposes only.

[0035] Unless otherwise specified, all reagents and equipment are commonly used in the field or are commercially available.

[0036] Small interfering RNA

[0037] The sequence of the small interfering RNA used in the examples is shown below:

[0038] The knockdown experiment was conducted according to the procedure described in Example 1 below.

[0039] Example 1: Interference with USP15 knockdown promotes renal cell carcinoma cell death

[0040] Materials and Methods

[0041] 786-O cells (purchased from ATCC, CRL-1932) were passaged in DMEM (10% FBS). Human USP15 small interfering RNA (H-siRNA2) was synthesized by Shanghai Gemma Gene Technology Co., Ltd., and control siRNAs (5'-UUCUCCGAACGUGUCACGUTT-3', 5'-ACGUGACACGUUCGGAGAATT-3') were purchased from Shanghai Gemma Gene Technology Co., Ltd. After co-incubation with Lipofectamine RNAiMAX liposomes (purchased from Life Technology Co., Ltd.) according to the manufacturer's instructions, RNA knockdown experiments were performed.

[0042] Cell death detection experiments were performed according to the instructions for the Annexin V / PI flow cytometry kit (purchased from Invitrogen).

[0043] Specific experimental procedures

[0044] 786-O cells were cultured, and small interfering RNA (USP15 H-siRNA2 and control siRNA) was mixed with Lipofectamine RNAiMAX liposomes to a final concentration of 20 nM. After mixing for 10 minutes, the mixture was added to the cell culture supernatant, and the interference time was 72 hours. Subsequently, 786-O cells were digested and collected: 1. IP lysis buffer (Thermo Fisher) containing PMSF and protease inhibitor (Abcam) was added, and the cells were incubated on ice for 15 minutes for lysis. The cell lysate was thoroughly pipetted and transferred to a new 1.5 mL EP tube. Centrifuge at 12,000 rpm, 4℃ for 15 minutes, collect the supernatant and transfer it to a new EP tube. Add protein sample preparation buffer (Sangon Biotech) to prepare protein samples. Perform Western blotting experiments according to standard laboratory procedures. USP15 antibody was purchased from Proteintech (1. Electrophoresis: Prepare an appropriate concentration of polyacrylamide gel according to the molecular weight of the target protein. Clamp the gel and place it in the electrophoresis tank, add electrophoresis buffer and remove the comb. Add the marker and sample one by one into the wells of the gel, close the electrophoresis tank lid, and then perform electrophoresis. The electrophoresis conditions are set to 80V for 30 minutes, 120V for 30 minutes. 1 hour. Electrophoresis ends when bromophenol blue reaches the bottom of the gel. 2. Transfer: Cut the NC membrane (0.45μm) and filter paper to the gel area and immerse them in transfer buffer for at least 1 minute to activate. Remove the gel and clamp it in the transfer tank in the following order: negative electrode - filter paper - gel - NC membrane - filter paper - positive electrode, and place it in the transfer tank. Transfer the transfer cassette to an ice-water bath and fill it with transfer buffer. Cover the tank and then perform the transfer. The transfer conditions are a constant current of 250mA. 2 hours. 3. Blocking: Prepare 5% skim milk (w / v) by diluting skim milk powder with TBST. Remove the NC membrane and immerse it in the skim milk with the protein side facing up. Incubate at room temperature for 1 hour at 80 rpm. 4. Primary antibody incubation (USP15 protein primary antibody purchased from Proteintech, other primary antibodies purchased from CST): Cut the band containing the target molecule according to the molecular weight of the target protein and the marker indication. Dilute the antibody with antibody diluent according to the proportions in the antibody instructions. Add the solution to the NC membrane strip and incubate overnight at 4°C. After incubation, transfer the strip to TBST washing buffer and wash the membrane for 10 minutes on a shaker at 80 rpm. Repeat the washing process three times. 5. Secondary antibody incubation: Dilute the antibody with antibody diluent according to the ratio specified in the antibody instructions. Add the secondary antibody solution (purchased from CST) to the NC membrane strip and incubate for 1 hour on a shaker at 50 rpm. After incubation, transfer the strip to TBST washing buffer and wash the membrane for 10 minutes on a shaker at 80 rpm. Repeat the washing process three times.6. Development: Develop and photograph the target band with ECL developing solution; 2. Label and perform flow cytometry detection according to the Annexin V / PI flow cytometry kit instructions to observe cell death.

[0045] Results and Discussion

[0046] like Figure 1 As shown in Figure A, USP15 siRNA effectively knocked down the protein expression level of USP15 in 786-O cells. The cell death profile of 786-O cells is shown in Figure A. Figure 1 As shown in B and 1C. The results showed that after USP15 knockdown, the proportion of PI-positive 786-O cells increased significantly, and 786-O cells underwent more cell membrane rupture-related cell death.

[0047] The results of this embodiment indicate that the small interfering RNA of USP15 can promote a non-apoptotic predominant form of cell death in renal cancer cells.

[0048] Example 2: USP15 inhibits RIPK1 ubiquitination and phosphorylation, thereby inhibiting the activation of death-related signaling pathways.

[0049] Materials and Methods

[0050] The 786-O cell culture, USP15 knockdown with small interfering RNA (H-siRNA2), and Western blotting methods were the same as in Example 1. Immunoprecipitation experiments following knockdown were performed according to standard laboratory procedures.

[0051] Specific experimental procedures

[0052] 786-O cells were cultured, and small interfering RNA (SRNA) was mixed with Lipofectamine RNAiMAX liposomes to a final concentration of 20 nM. After mixing for 10 minutes, the mixture was added to the cell culture supernatant, and the interference time was 72 hours. Subsequently, 786-O cells were digested and collected: 1. Proteins were directly extracted for Western blotting to detect the activation of death-related signaling pathways (experimental steps were the same as in Example 1). Figure 2A); 2. After protein extraction, perform immunoprecipitation (IP) using RIPK1 antibody (CST Biotechnology). (Take 80 μL of supernatant as the input sample, add 20 μL of 5× protein loading buffer (Sangon Biotech), heat at 95℃ for 10 minutes to boil the sample; take 800 μL as the IP protein sample, take 50 μL of Protein G magnetic beads (CST Biotechnology) into a 1.5 mL EP tube, add 1 mL of pre-chilled TBST, invert and mix several times to balance the magnetic beads, place the EP tube on a magnetic rack, wait for the magnetic beads to adhere to one side of the tube wall, carefully discard the supernatant. Repeat the washing once. Add 500 μL of pre-chilled TBST to resuspend the magnetic beads, and add 4 μg of...) Antibody (IP grade) was incubated for 4-6 hours on a rotary suspending apparatus at 4°C. The magnetic beads were removed and placed on a magnetic rack. Once the beads adhered to one side, the supernatant was discarded. 1 mL of pre-chilled TBST was added, and the mixture was inverted several times to wash the beads. The EP tube was then placed on a magnetic rack, and the beads adhered to one side of the tube wall. The supernatant was carefully discarded. This washing process was repeated once. 1 mL of protein lysate supernatant was added to the antibody-magnetic bead complex, and the mixture was incubated overnight on a rotary suspending apparatus at 4°C. The EP tube was placed on a magnetic rack, and the beads adhered to one side of the tube wall. The protein supernatant was carefully discarded. 1 mL of pre-chilled TBST solution was added, and the mixture was inverted several times to wash the beads. The EP tube was then placed on a magnetic rack, and the beads adhered to one side of the tube wall. The supernatant was carefully discarded. This washing process was repeated twice. 80 μL of... Resuspend the magnetic beads in 1× protein loading buffer and boil at 95°C for 10 minutes to prepare IP samples. Input and IP samples can be directly used for subsequent immunoblotting experiments to analyze the interaction between bait and prey proteins, or temporarily stored in a -80°C freezer. Western blotting is then performed to detect the ubiquitination level of RIPK1. Figure 2 C).

[0053] 786-O cells were cultured, and proteins were directly extracted. Immunoprecipitation was performed using a USP15 antibody (purchased from Proteintech), followed by Western blotting to detect USP15-interacting proteins. Figure 2 B). Figure 2 B and Figure 2 In C, IgG (purchased from CST) represents the isotype control antibody group.

[0054] Results and Discussion

[0055] The results are as follows Figure 2 As shown in Figure A, knocking down USP15 in renal cell carcinoma cells can promote phosphorylation of the death-related signaling RIPK1 and cleavage of Caspase3.

[0056] Figure 2As shown in B and 2C, USP15 can bind to RIPK1 in renal cell carcinoma cells and inhibit its ubiquitination level.

[0057] The results of this embodiment show that knocking down USP15 can activate death-related signaling pathways by promoting RIPK1 ubiquitination and its activation.

[0058] Example 3: Knockdown of USP15 inhibits T cell activation

[0059] Materials, Methods and Experimental Procedures

[0060] 786-O cells (purchased from ATCC, CRL-1932) and HK-2 cells (purchased from ATCC, CRL-2190) were passaged in DMEM (10% FBS). The small interfering RNA (H-siRNA2) knockdown assay was performed according to Example 1. Exosome extraction reagents were purchased from Invitrogen, and exosomes were extracted from the cell culture supernatant according to the product instructions. Exosomal proteins were extracted, and USP15 expression was detected using Western blotting (reagents and procedures were the same as in Example 1).

[0061] Jurket T-cell line (purchased from ATCC, TIB-152) was passaged in RPMI 1640 (10% FBS). Exosomes extracted as described above were co-incubated with Jurket cells. After 48 hours, Jurket cell proteins were extracted, and USP15 expression was detected by Western blotting. Alternatively, after 48 hours of incubation, CD3 / CD28 antibody (purchased from Invitrogen) was added, and Jurket cells were stimulated as shown in the diagram. Cell supernatant was collected, and IL-2 secretion levels were detected using an IL-2 ELISA kit (purchased from R&D).

[0062] Results and Discussion

[0063] After USP15 was knocked down in HK-2 and 786-O cells, the level of USP15 in the secreted exosomes was as follows: Figure 3 As shown in Figure A, after co-incubation with Jurket cells using the corresponding exosomes, the intracellular USP15 level in Jurket cells was as follows: Figure 3 As shown in Figure B, the IL-2 secretion level after Jurket cell activation is as follows: Figure 3 As shown in C.

[0064] The results showed that knocking down USP15 significantly reduced the level of USP15 in exosomes secreted by HK-2 and 786-O cells, and also affected the expression level of USP15 in co-incubated Jurket cells. The expression level of USP15 in Jurket cells was negatively correlated with the expression level of IL-2 after activation of Jurket cells.

[0065] The results indicate that knocking down USP15 can inhibit T cell activation.

[0066] Example 4: Intratumoral injection of USP15 small interfering RNA inhibits tumor tissue growth and enhances the tumor tissue's resistance to immune detection. Sensitivity of checkpoint inhibitors

[0067] Renca cell line (purchased from ATCC, CRL-2947) was passaged in RPMI 1640 (10% FBS). Wild-type C57 mice were purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd. Each mouse was administered 1 x 10⁻⁶ cells subcutaneously. 7 Renal cell carcinomas were implanted to bear tumors, and the tumor tissue was allowed to grow to 70-100 mm. 3 Intratumoral injection of USP15 small interfering RNA (H-siRNA2) (2 μg / animal) or / and intraperitoneal injection of anti-PD1 antibody (purchased from Bio X Cell) (50 μg / animal) were performed. During the injection, 2 μg of USP15 was dissolved in 20 μl of water and injected into multiple sites within the tumor. At the same time, 50 μg of anti-PD1 antibody was dissolved in PBS water and injected intraperitoneally. The injections were performed 3 times, with an interval of 4 days between each injection. The growth curve was recorded at the same time.

[0068] Results and Discussion

[0069] Mouse tumor-bearing tissue size and growth curve as shown in the figure. Figure 4 As shown.

[0070] The results showed that intratumoral knockdown of USP15 expression significantly inhibited the growth of renal cell carcinoma tumor-bearing tissue, and intraperitoneal injection of anti-PD1 antibody also inhibited the growth of renal cell carcinoma tumor-bearing tissue. Furthermore, the combination of intratumoral knockdown of USP15 and intraperitoneal injection of anti-PD1 antibody could more significantly inhibit the growth of tumor-bearing tissue, suggesting that in addition to inhibiting the growth of tumor-bearing tissue, USP15 can also enhance the sensitivity of renal cell carcinoma to immune checkpoint inhibitors.

[0071] The above are merely specific application examples of this application and do not constitute any limitation on the scope of protection of this application. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to list and describe all embodiments here. Any technical solutions similar to these obtained through equivalent transformations or substitutions fall within the scope of protection of this application.

[0072] Sequence information

Claims

1. Use of the inhibitor of USP15 in the preparation of a medicament for the treatment and / or prevention of renal cell carcinoma in subjects.

2. Use of USP15 inhibitors and immune checkpoint inhibitors in the preparation of medicaments for the treatment and / or prevention of renal cell carcinoma in subjects.

3. The use as described in claim 1 or 2, wherein the object includes mammals, such as humans, non-human primates, rodents, pets, livestock, preferably humans.

4. A pharmaceutical composition comprising an inhibitor of USP15.

5. A kit comprising an inhibitor of USP15.

6. The pharmaceutical composition of claim 4 or the kit of claim 5, further comprising an immune checkpoint inhibitor.

7. The use as described in any one of claims 1-3, the pharmaceutical composition as described in claim 4 or 6, or the kit as described in claim 5 or 6, wherein the inhibitor of USP15 comprises inhibitors selected from the group consisting of siRNA, shRNA, miRNA, antisense oligonucleotides, exosomes, zinc finger proteins, CRISPR / Cas9 gene editing products, antibodies, chemical inhibitors, or combinations thereof targeting USP15.

8. The use, pharmaceutical composition, or kit of claim 7, wherein the inhibitor of USP15 comprises siRNA targeting USP15, preferably, the siRNA comprises a pair of RNA molecules selected from the group consisting of: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; (c) SEQ ID NO: 5 and SEQ ID NO: 6; (d) A pair of RNA molecules that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of (a)-(c) and that inhibit the activity of USP15; and A pair of RNA molecules that have been substituted, deleted and / or added to one or more bases in any of (e) (a)-(d) and have the function of inhibiting USP15.

9. The use as described in claim 2 or the pharmaceutical composition or kit as described in claim 6, wherein the immune checkpoint inhibitor comprises an inhibitor selected from the group consisting of PD1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors or combinations thereof, preferably PD1 inhibitors, more preferably anti-PD1 antibodies.

10. A siRNA targeting USP15, said siRNA comprising a pair of RNA molecules selected from the group consisting of: (a) SEQ ID NO: 1 and SEQ ID NO: 2; (b) SEQ ID NO: 3 and SEQ ID NO: 4; (c) SEQ ID NO: 5 and SEQ ID NO: 6; (d) A pair of RNA molecules that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of (a)-(c) and that inhibit the activity of USP15; and A pair of RNA molecules that have been substituted, deleted and / or added to one or more bases in any of (e) (a)-(d) and have the function of inhibiting USP15.

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