OTUB2 inhibitor and use thereof in tumor immunity

Abstract

An OTUB2 inhibitor and the use thereof in tumor immunity, specifically, the use and method of OTUB2 inhibitor for enhancing anti-tumor immune response, for reducing tumor immunosuppression, and / or for treating tumors. The present application further comprises a small molecule inhibitor targeting OTUB2.

Description

OTUB2 inhibitors and their applications in tumor immunity

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310740647.7 filed on June 21, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to OTUB2 inhibitors and their application in tumor immunity. Background Art

[0004] The ability to enhance anti-tumor immunity within the tumor microenvironment (TME) is crucial for tumor control. Cancers often evolve diverse mechanisms that enable them to evade destruction by the immune system. One such escape mechanism involves the interaction between anti-tumor cytotoxic T lymphocytes (CTLs) and tumor cells. Tumor cells resist or weaken CTL attack through a variety of tumor cell-intrinsic mechanisms, including upregulation of inhibitory immune checkpoint receptors, impaired tumor antigen presentation, secretion of inhibitory cytokines or ligands, and altered metabolic pathways.

[0005] Otubin-2 (OTUB2) is a deubiquitinating enzyme (DUB) that belongs to the ovarian tumor (OTU) superfamily (Nanao MH, Tcherniuk SO, Chroboczek J, Dideberg O, Dessen A, Balakirev MY. Crystal structure of human otubain 2. EMBO Rep 5, 783-788 (2004)). Although the molecular biology of OTUB2 has been studied, there are no reports that OTUB2 has a function in regulating tumor immunity.

[0006] Summary of the Invention

[0007] The field has reported that OTUB2 is expressed at high levels in human malignant tumors and at lower levels in various organs throughout the body (see, for example, https: / / www.proteinatlas.org / ENSG00000089723-OTUB2 / tissue). However, there are no reports linking OTUB2 to tumor immunity. The present invention is the first to discover that OTUB2 has immunomodulatory functions and plays a key role in the tumor microenvironment. Targeting OTUB2 can help improve the tumor microenvironment and enhance anti-tumor immunity, thereby exerting a therapeutic effect on tumors. Furthermore, it is worth noting that no specific small molecule inhibitors for OTUB2 have been developed. Based on the OTUB2 binding pocket structure, the present invention provides, for the first time, specific small molecule inhibitors targeting OTUB2. This provides the following invention.

[0008] Application of OTUB2 inhibitors in tumor immunity

[0009] In one aspect, the present invention provides a use of an OTUB2 inhibitor in the preparation of a medicament for anti-tumor immunotherapy. Also provided is a method for anti-tumor immunotherapy, comprising administering an effective amount of an OTUB2 inhibitor to a subject in need thereof.

[0010] In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and / or reducing tumor immunosuppression in a subject.

[0011] In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell (eg, CD8+ T cell)-mediated tumor killing in the subject.

[0012] In certain embodiments, the anti-tumor immunotherapy includes promoting immune cell function in a subject. The immune cell function is measured, for example, by one or both of the following parameters: (i) activation of T cells (such as CD8+T cells and / or CD4+T cells), NK cells, or any combination thereof; and (ii) propagation of T cells (such as CD8+T cells and / or CD4+T cells), NK cells, or any combination thereof. In certain embodiments, the activation is measured as an increased level of cytokines (such as IFN-γ, IL-2, TNF-α, etc.). In certain embodiments, the immune cell function is measured, for example, by activation and / or proliferation of CD8+T cells.

[0013] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor.

[0014] OTUB2 inhibitors

[0015] The OTUB2 inhibitors described herein can exert their inhibitory effects by any mechanism, for example, by inhibiting gene expression at the RNA or protein level (for example, reducing or inhibiting transcription of the gene, and / or reducing or inhibiting translation of the mRNA product of the gene); or, for example, by inhibiting or blocking the biological activity of the protein product of the gene; or, for example, by degrading the protein product of the gene or destroying its stability.

[0016] In certain embodiments, the OTUB2 inhibitor can inhibit or downregulate the expression of the OTUB2 gene, inhibit or block the activity of the OTUB2 protein, or degrade the OTUB2 protein.

[0017] In certain embodiments, the inhibitor inhibits or blocks the biological function of the OTUB2 protein. In certain embodiments, the biological function of the OTUB2 protein includes deubiquitinase activity. In certain embodiments, the biological function of the OTUB2 protein includes binding to substrate proteins and removing their ubiquitination (i.e., interacting with substrate proteins to stabilize them from ubiquitination and degradation). Thus, by inhibiting or blocking the above-mentioned functions of the OTUB2 protein, the substrate protein can be degraded, thereby inhibiting its expression.

[0018] In certain embodiments, the inhibitor comprises a small molecule compound. As used herein, the term "small molecule compound" refers to an organic, non-protein compound. In certain embodiments, the molecular weight of the small molecule compound is no greater than 1500 Da. The small molecule compound is capable of inhibiting the function of the aforementioned gene or its expression product. The small molecule compound can be obtained by screening existing small molecule compound libraries (e.g., Chem Bridge, Chem Div, Inter Bio Screen, Life Chemicals, Specs, or Vitas-m) and measuring the compound's inhibitory activity on OTUB2 expression levels, its binding activity to the OTUB2 protein, or its ability to inhibit the biological function of the OTUB2 protein.

[0019] In certain embodiments, the small molecule compound binds to or interacts with the OTUB2 protein, for example, as determined by BLI technology (e.g., an Octet instrument) or SPR technology (e.g., a Biacore instrument). In certain embodiments, the small molecule compound binds to the catalytic pocket of the OTUB2 protein. In certain embodiments, the small molecule compound binds to the OTUB2 protein, blocking the interaction between OTUB2 and a substrate protein (e.g., PD-L1), thereby causing the substrate protein (e.g., PD-L1) to be ubiquitinated and degraded.

[0020] In certain embodiments, the small molecule compound is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt, ester or stereoisomer thereof:

[0021] in:

[0022] R 1 -C(O)X, X is hydroxy or C1-C4 alkoxy; or is C3-7 monocyclic cycloalkyl, 4- to 6-membered monocyclic heterocyclyl or 5- to 6-membered monocyclic heteroaryl, wherein the 4- to 6-membered monocyclic heterocyclyl and the 5- to 6-membered monocyclic heteroaryl each have 1 to 3 ring heteroatoms independently selected from N, O and S;

[0023] n is an integer selected from 1 to 12;

[0024] R 2 for

[0025] Among them, R 2a is selected from (C1-C4)alkyl, (C2-C4)alkenyl, or -C(O)Y, where Y is hydroxy or (C1-C4)alkoxy;

[0026] Among them, R 2b 、R 2c 、R 2d Each is independently selected from halogen (eg, -F, -Cl, -Br or -I), nitro, amino, hydroxy, mercapto, (C1-C4) alkyl or (C1-C4) alkoxy; preferably, R 2b 、R 2c 、R 2d Different from each other;

[0027] Here, m is 1, 2, 3 or 4.

[0028] In certain embodiments, R 1 is -C(O)X, X is hydroxy or (C1-C4)alkoxy, and n is an integer selected from 1 to 12.

[0029] In certain embodiments, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0030] In certain embodiments, R 1 In certain embodiments, X is -C(O)X, wherein X is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, X is hydroxy, methoxy, or ethoxy. In certain embodiments, X is hydroxy.

[0031] In certain embodiments, R 1is a C3-7 monocyclic cycloalkyl group (e.g., a C3-6 cycloalkyl group, a C3-5 cycloalkyl group, a C3-4 cycloalkyl group, a C4-6 cycloalkyl group, or a C4-5 cycloalkyl group, such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group), a 4- to 6-membered monocyclic heterocyclyl group (e.g., an oxetanyl group, a tetrahydrofuranyl group, a tetrahydropyranyl group, a pyrrolidinyl group, or a piperidinyl group), or a 5- to 6-membered monocyclic heteroaryl group (an imidazolyl group, a triazolyl group, a tetrazolyl group, a pyrazolyl group, a phenylthio group, a thiazolyl group, a pyridinyl group, a pyrimidinyl group, or a pyrazinyl group), and n is 1, 2, 3, or 4. In certain embodiments, n is 1 or 2; preferably, n is 1.

[0032] In certain embodiments, R 1 is a C3-7 monocyclic cycloalkyl, a 4- to 6-membered monocyclic heterocyclyl, or a 5- to 6-membered monocyclic heteroaryl, each of which has 1 ring heteroatom selected from N, O, and S. In certain embodiments, R 1 is a C3-7 monocyclic cycloalkyl group or a 4 to 6 membered monocyclic heterocyclic group. 1 is a C3-6 monocyclic cycloalkyl group or a 4 to 6 membered monocyclic heterocyclic group. 1 is a cycloalkyl group selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, or a heterocyclic group selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl, for example tetrahydrofuranyl or pyrrolidinyl. In certain embodiments, R 1 For tetrahydrofuran.

[0033] In certain embodiments, R 2 for where R 2a is selected from methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, or ethoxy. 2a In some embodiments, m is 1, 2, or 3.

[0034] In certain embodiments, R 2 for Among them, R 2b 、R 2c 、R 2d Each is independently selected from nitro, hydroxy, (C1-C4) alkyl or (C1-C4) alkoxy. 2b 、R 2c 、R 2dEach is independently selected from nitro, hydroxy, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy. In certain embodiments, R 2b 、R 2c 、R 2d Each is independently selected from nitro, hydroxy, methyl, ethyl, methoxy, ethoxy. In certain embodiments, R 2b 、R 2c 、R 2d In certain embodiments, R 2b 、R 2c 、R 2d In certain embodiments, R 2 for R 2b 、R 2c 、R 2d As defined above.

[0035] In certain embodiments, the compound has the structure shown in Formula (Ia):

[0036] Among them, R 1 、R 2a , m and n are as defined in formula (I).

[0037] In certain embodiments, in Formula (Ia), R 1 is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0038] In certain embodiments, in Formula (Ia), R 1 is a C3-7 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclyl group having one ring heteroatom selected from N, O and S (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl); preferably, R 1 is tetrahydrofuranyl or pyrrolidinyl; preferably, n is 1 or 2, for example, 1.

[0039] In certain embodiments, in Formula (Ia), R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; preferably, R 2a Selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

[0040] In certain embodiments, in Formula (Ia), m is 1, 2, or 3.

[0041] In certain embodiments, in Formula (Ia):

[0042] R 1 -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0043] R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; preferably, R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH; m is 1, 2, 3 or 4, for example 1, 2 or 3.

[0044] In certain embodiments, the compound is selected from:

[0045] 6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid (CAS No.: 300558-22-9);

[0046] 2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid (available from OTAVA Chemicals, Catalog No. 1663785, for example);

[0047] 2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid (CAS No.:313646-95-6);

[0048] 11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}unddecanoic acid (CAS No.: 374543-72-3);

[0049] 11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid (available from OTAVA Chemicals, for example, under Catalog No. 4349908).

[0050] In certain embodiments, the small molecule compounds described herein can be chemically synthesized or purchased from commercial sources.

[0051] In certain embodiments, the inhibitor inhibits the biological function of OTUB2 by suppressing the expression level of the gene. In such embodiments, the expression level can be measured at the nucleic acid level or the protein level. Methods for measuring expression at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR, or real-time RT-PCR. Methods for measuring expression at the protein level include, but are not limited to, Western blotting or polyacrylamide gel electrophoresis combined with protein staining techniques such as Coomassie Brilliant Blue or silver staining, mass spectrometry, ELISA, and the like.

[0052] In certain embodiments, the inhibitor is selected from an antibody, an RNA interfering agent (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), or microRNA (miRNA)) or an antisense oligonucleotide, a gene editing system (e.g., a CRISPR-Cas system), or a small molecule compound.

[0053] In some embodiments, the inhibitor is an antibody. In certain embodiments, the antibody is a blocking antibody or a neutralizing antibody. In certain embodiments, the antibody is capable of binding to the OTUB2 protein and directly interfering with its biological function.

[0054] In other embodiments, the inhibitor is a nucleic acid inhibitor, including an RNA interfering agent or an antisense oligonucleotide. The RNA interfering agent inhibits the expression of the gene. The antisense oligonucleotide specifically binds to the gene or its mRNA product to inhibit expression.

[0055] In this article, the expression "RNA interference agent" refers to any reagent that suppresses the expression of a target gene by RNA interference (RNAi) mechanism." RNA interference (RNAi) " is a kind of evolutionarily conservative process, wherein the expression of RNA of a sequence identical or highly similar to the target gene or the introduction of sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of the messenger RNA (mRNA) transcribed from the target gene leads to inhibition of the expression of the target gene. Therefore, it is known to those skilled in the art that the sequence of the mRNA generated by the transcription of a specific gene or its gene can be selected to design a small interfering RNA (siRNA) or a small RNA (microRNA, miRNA) molecule of the target sequence. The siRNA or miRNA molecule can interfere with the transcription, translation or transcription, post-translational modification of the gene, thereby affecting the expression of the protein.

[0056] Furthermore, to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed, annealed, and cloned into a vector. The transcription product of this recombinant vector, a short hairpin RNA (shRNA), folds and pairs into a stem-loop structure with a stem length of 19-21 bases. These 19-21 bases are derived from a specific sequence in the target gene mRNA. This stem-loop precursor is quickly cleaved within the cell to form a functional siRNA. The shRNA expressed by this vector, after cleavage, produces a stable expression level and a long duration of activity, thereby causing long-term inhibition of target gene expression.

[0057] In this article, shRNA refers to short hairpin RNA (shRNA). shRNA consists of two short inverted repeats separated by a stem-loop sequence, forming a hairpin structure controlled by the Pol III promoter. Five to six Ts are then attached to serve as transcriptional terminators for RNA polymerase III. Cloning the siRNA sequence into a plasmid vector allows for in vivo delivery of small interfering RNA (siRNA). When delivered into an animal, the hairpin sequence is expressed, forming a stem-loop structure that is cleaved into functional siRNA, exerting a gene silencing effect.

[0058] In this article, the siRNA refers to Small interfering RNA, which is a small RNA molecule consisting of approximately 21-25 nucleotides and is processed by Dicer (an enzyme in the RNAase III family that is specific for double-stranded RNA). siRNA is the main member of siRISC and stimulates the silencing of the target mRNA that is complementary to it.

[0059] As used herein, microRNA (miRNA) refers to a naturally occurring non-coding RNA molecule of approximately 21-25 nucleotides in length. Based on sequence complementarity with target mRNAs, they can cause target mRNA degradation or inhibit its translation through specific base pairing, thereby regulating gene expression post-transcriptionally. For more information on miRNAs, refer to the miRBase database.

[0060] In certain embodiments, the RNA interfering agent is selected from small interfering RNA (siRNA), small hairpin RNA (shRNA), or micro RNA (miRNA).

[0061] As used herein, the term "antisense oligonucleotide" refers to a molecule that is complementary to a sense nucleic acid, for example, complementary to the coding strand of the OTUB2 gene or complementary to the mRNA sequence of the OTUB2 gene. Therefore, the antisense oligonucleotide can form hydrogen bonds with (i.e., anneal to) the sense nucleic acid.

[0062] Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides, and the modified nucleotides are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as nucleotides substituted with thiophosphate derivatives and acridines. Alternatively, antisense nucleic acids can be produced biologically using an expression vector comprising a target nucleic acid subcloned therein, and the RNA transcribed from the inserted nucleic acid will have an antisense direction to the target nucleic acid.

[0063] In certain embodiments, the antisense oligonucleotide is antisense RNA or antisense DNA.

[0064] After obtaining the RNA interfering agent or antisense oligonucleotide, the inhibitory activity of the RNA interfering agent or antisense oligonucleotide on the expression level of OTUB2 can be further determined using the method described above.

[0065] In certain exemplary embodiments, the inhibitor is a shRNA, for example comprising the sequence shown in SEQ ID NO: 3 or 4.

[0066] In other embodiments, the inhibitor is a gene editing system. Exemplary gene editing systems include ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats) / Cas and other site-specific nuclease technologies. It is well known to those skilled in the art to use gene editing systems to knock down or knock out target genes (such as OTUB2). In certain embodiments, the inhibitor is CRISPR / Cas, such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a or CRISPR / Cas14a.

[0067] In other embodiments, the inhibitor is a PROTAC (protein degradation targeting chimera). PROTAC is a technology that selectively degrades target proteins by the ubiquitin-proteasome system. Typically, as a heterobifunctional molecule or bifunctional compound, PROTAC comprises an E3 ubiquitin ligase binding portion (i.e., a ligand of an E3 ubiquitin ligase) and a target protein binding portion (i.e., a protein / polypeptide targeting ligand), such that the target protein / polypeptide is placed near the ubiquitin ligase to achieve degradation (and inhibition) of the protein. The bifunctional compound may also include a chemical linker connecting the two functional groups.

[0068] In certain embodiments, the PROTAC is a bifunctional compound comprising an E3 ubiquitin ligase binding portion and an OTUB2 protein binding portion.

[0069] In certain embodiments, the E3 ubiquitin ligase binding moiety is a small molecule. In certain embodiments, the E3 ubiquitin ligase binding moiety targets an E3 ubiquitin ligase selected from Hippel-Lindau (VLM), human cerebellum (CLM), mouse double minute homolog 2 (MLM), and IAP (ILM).

[0070] In certain embodiments, the OTUB2 protein binding moiety is a small molecule. In certain embodiments, the OTUB2 protein binding moiety is selected from the small molecule compounds described herein.

[0071] Administration of OTUB2 inhibitors

[0072] The OTUB2 inhibitors described herein can be formulated into dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral administration, for example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or glucose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Preparations for parenteral administration can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0073] The dosage form suitable for injection includes sterile aqueous solution (wherein water soluble) or dispersion and the sterile powder for preparing sterile injection solution or dispersion at any time. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil ELTM or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that reaches the degree of easy injection. It must be stable under manufacturing and storage conditions and must prevent microorganisms such as bacteria and fungi from contaminating and preserving. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Preventing the effect of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, in the composition, it is preferably included isotonic agents, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0074] The OTUB2 inhibitors described herein can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. A preferred dosage form is an injection.

[0075] The OTUB2 inhibitors described herein can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or mode of administration will vary depending on the intended purpose.

[0076] The OTUB2 inhibitors described herein can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0077] The subjects described herein include any individual that can produce an immune response. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0078] Indications

[0079] The goal of anti-tumor immunotherapy is to enhance the immune system's ability to recognize and kill tumors and overcome the immunosuppressive tumor microenvironment. The term "tumor" includes any form of cancer, including solid tumors or hematological tumors.

[0080] In certain embodiments, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

[0081] In certain embodiments, the tumor expresses OTUB2 or is OTUB2 positive. Herein, OTUB2 positivity can be measured at the nucleic acid level or the protein level. Methods for measuring expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for measuring expression at the protein level include, but are not limited to, immunological detection.

[0082] OTUB2 small molecule inhibitors

[0083] Currently, no OTUB2-specific small molecule inhibitors have been developed. Based on the OTUB2 binding pocket structure, the present invention provides, for the first time, a specific small molecule inhibitor targeting OTUB2, wherein the small molecule inhibitor is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt, ester, or stereoisomer thereof:

[0084] in:

[0085] R 1 -C(O)X, X is hydroxy or C1-C4 alkoxy; or is C3-7 monocyclic cycloalkyl, 4- to 6-membered monocyclic heterocyclyl or 5- to 6-membered monocyclic heteroaryl, wherein the 4- to 6-membered monocyclic heterocyclyl and the 5- to 6-membered monocyclic heteroaryl each have 1 to 3 ring heteroatoms independently selected from N, O and S;

[0086] n is an integer selected from 1 to 12;

[0087] R 2 for

[0088] Among them, R 2a is selected from (C1-C4)alkyl, (C2-C4)alkenyl, or -C(O)Y, where Y is hydroxy or (C1-C4)alkoxy;

[0089] Among them, R 2b 、R 2c 、R 2d Each is independently selected from halogen (eg, -F, -Cl, -Br or -I), nitro, amino, hydroxy, mercapto, (C1-C4) alkyl or (C1-C4) alkoxy; preferably, R 2b 、R 2c 、R 2d Different from each other;

[0090] Here, m is 1, 2, 3 or 4.

[0091] In certain embodiments, R 1 is -C(O)X, X is hydroxy or (C1-C4)alkoxy, and n is an integer selected from 1 to 12.

[0092] In certain embodiments, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0093] In certain embodiments, R 1 In certain embodiments, X is -C(O)X, wherein X is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, X is hydroxy, methoxy, or ethoxy. In certain embodiments, X is hydroxy.

[0094] In certain embodiments, R 1is a C3-7 monocyclic cycloalkyl group (e.g., a C3-6 cycloalkyl group, a C3-5 cycloalkyl group, a C3-4 cycloalkyl group, a C4-6 cycloalkyl group, or a C4-5 cycloalkyl group, such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group), a 4- to 6-membered monocyclic heterocyclyl group (e.g., an oxetanyl group, a tetrahydrofuranyl group, a tetrahydropyranyl group, a pyrrolidinyl group, or a piperidinyl group), or a 5- to 6-membered monocyclic heteroaryl group (an imidazolyl group, a triazolyl group, a tetrazolyl group, a pyrazolyl group, a phenylthio group, a thiazolyl group, a pyridinyl group, a pyrimidinyl group, or a pyrazinyl group), and n is 1, 2, 3, or 4. In certain embodiments, n is 1 or 2; preferably, n is 1.

[0095] In certain embodiments, R 1 is a C3-7 monocyclic cycloalkyl, a 4- to 6-membered monocyclic heterocyclyl, or a 5- to 6-membered monocyclic heteroaryl, each of which has 1 ring heteroatom selected from N, O, and S. In certain embodiments, R 1 is a C3-7 monocyclic cycloalkyl group or a 4 to 6 membered monocyclic heterocyclic group. 1 is a C3-6 monocyclic cycloalkyl group or a 4 to 6 membered monocyclic heterocyclic group. 1 is a cycloalkyl group selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, or a heterocyclic group selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl, for example tetrahydrofuranyl or pyrrolidinyl. In certain embodiments, R 1 For tetrahydrofuran.

[0096] In certain embodiments, R 2 for where R 2a is selected from methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, or ethoxy. 2a In some embodiments, m is 1, 2, or 3.

[0097] In certain embodiments, R 2 for Among them, R 2b 、R 2c 、R 2d Each is independently selected from nitro, hydroxy, (C1-C4) alkyl or (C1-C4) alkoxy. 2b 、R 2c 、R 2dEach is independently selected from nitro, hydroxy, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy. In certain embodiments, R 2b 、R 2c 、R 2d Each is independently selected from nitro, hydroxy, methyl, ethyl, methoxy, ethoxy. In certain embodiments, R 2b 、R 2c 、R 2d In certain embodiments, R 2b 、R 2c 、R 2d In certain embodiments, R 2 for R 2b 、R 2c 、R 2d As defined above.

[0098] In certain embodiments, the compound has the structure shown in Formula (Ia):

[0099] Among them, R 1 、R 2a , m and n are as defined in formula (I).

[0100] In certain embodiments, in Formula (Ia), R 1 is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0101] In certain embodiments, in Formula (Ia), R 1 is a C3-7 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclyl group having one ring heteroatom selected from N, O and S (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl); preferably, R 1 is tetrahydrofuranyl or pyrrolidinyl; preferably, n is 1 or 2, for example, 1.

[0102] In certain embodiments, in Formula (Ia), R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; preferably, R 2a Selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

[0103] In certain embodiments, in Formula (Ia), m is 1, 2, or 3.

[0104] In certain embodiments, in Formula (Ia):

[0105] R 1 -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0106] R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; preferably, R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH; m is 1, 2, 3 or 4, for example 1, 2 or 3.

[0107] In certain embodiments, the compound is selected from:

[0108] 6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid (CAS No.: 300558-22-9);

[0109] 2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid (available from OTAVA Chemicals, for example, under the product number 1663785);

[0110] 2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid (CAS No.:313646-95-6);

[0111] 11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}unddecanoic acid (CAS No.: 374543-72-3);

[0112] 11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid (available from OTAVA Chemicals, for example, under Catalog No. 4349908).

[0113] In one aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt, ester or stereoisomer thereof. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0114] In one aspect, the present invention provides the use of the aforementioned compounds, or pharmaceutically acceptable salts or esters, or stereoisomers, or pharmaceutical compositions thereof, as OTUB2 inhibitors. In certain embodiments, the OTUB2 inhibitors are used to inhibit or block the activity of the OTUB2 protein in vitro. In certain embodiments, the OTUB2 inhibitors are used for non-therapeutic purposes. The present invention also provides methods for inhibiting OTUB2 activity in cells in vitro, comprising contacting the cells with the aforementioned compounds, or pharmaceutically acceptable salts or esters, or stereoisomers, or pharmaceutical compositions thereof. The present invention also provides methods for inhibiting OTUB2 activity in a subject, comprising administering an effective amount of the aforementioned compounds, or pharmaceutically acceptable salts or esters, or stereoisomers, or pharmaceutical compositions thereof, to a subject in need thereof. In certain embodiments, the subject has a tumor. In certain embodiments, the subject is a mammal, such as a human. In certain embodiments, the tumor comprises a solid tumor or a hematological tumor. In certain embodiments, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, and colorectal cancer. In certain embodiments, the tumor expresses or is positive for OTUB2.

[0115] In one aspect, the present invention provides the use of the aforementioned compound, or a pharmaceutically acceptable salt, ester, stereoisomer, or pharmaceutical composition thereof, in the preparation of a medicament for preventing and / or treating tumors. Also provided is a method for preventing and / or treating tumors, comprising administering an effective amount of the aforementioned compound, or a pharmaceutically acceptable salt, ester, stereoisomer, or pharmaceutical composition thereof, to a subject in need thereof.

[0116] In certain embodiments, the above-mentioned compound or its pharmaceutically acceptable salt, ester, stereoisomer or pharmaceutical composition is used for anti-tumor immunotherapy.

[0117] In certain embodiments, the anti-tumor immunotherapy comprises enhancing anti-tumor immune responses and / or reducing tumor immunosuppression.

[0118] In certain embodiments, the anti-tumor immunotherapy includes promoting T cell (e.g., CD8+T cell)-mediated tumor killing in a subject. In certain embodiments, the anti-tumor immunotherapy includes promoting immune cell function in a subject. The immune cell function is measured, for example, by one or both of the following parameters: (i) activation of T cells (e.g., CD8+T cells and / or CD4+T cells), NK cells, or any combination thereof; and (ii) proliferation of T cells (e.g., CD8+T cells and / or CD4+T cells), NK cells, or any combination thereof. In certain embodiments, the activation is measured as an increased level of cytokines (e.g., IFN-γ, IL-2, TNF-α, etc.). In certain embodiments, the immune cell function is measured, for example, by activation and / or proliferation of CD8+T cells.

[0119] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor.

[0120] In certain embodiments, the tumor comprises a solid tumor or a hematological tumor.

[0121] In certain embodiments, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

[0122] In certain embodiments, the tumor expresses OTUB2 or is OTUB2 positive. Herein, OTUB2 positivity can be measured at the nucleic acid level or the protein level. Methods for measuring expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for measuring expression at the protein level include, but are not limited to, immunological detection.

[0123] The above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer may be in any form known in the medical field, for example, in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections and lyophilized powders), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use.

[0124] The above-mentioned compound or its pharmaceutically acceptable salt, ester or stereoisomer may be present in a pharmaceutical composition in the form of a unit dosage for easy administration.

[0125] The above-mentioned compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof can be administered by any suitable method known in the art, including but not limited to oral, rectal, parenteral or topical administration.

[0126] An exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, etc. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl acetate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, liquid dosage forms for oral administration may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and aromatics, etc. Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In addition to the active ingredient, the solid dosage form may contain a pharmaceutically acceptable inert excipient or carrier, for example, fillers (such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, cross-linked polyvinylpyrrolidone and calcium sulfate); binders (such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia); wetting agents (such as cetyl alcohol and glyceryl monostearate); disintegrants (such as agar, calcium carbonate, starch, alginic acid, sodium carboxymethyl cellulose, sodium carboxymethyl starch); lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate); and mixtures thereof.

[0127] The above-mentioned compound or its pharmaceutically acceptable salt, ester or stereoisomer can also be administered via a non-oral route.

[0128] Therefore, another exemplary route of administration is parenteral administration, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection and infusion. The dosage form for parenteral administration can be an injection preparation, including an injection solution, a sterile powder for injection or a concentrated solution for injection. In addition to the active ingredient, the injection formulation can contain a pharmaceutically acceptable carrier such as sterile water, Ringer's solution and isotonic sodium chloride solution, and suitable additives such as antioxidants, buffers and antibacterial agents can also be added according to the properties of the drug.

[0129] Another exemplary route of administration is topical, for example, transdermal (eg, by a transdermal patch or iontophoresis device), intraocular, or intranasal or inhaled administration.

[0130] Another exemplary route of administration is rectal administration. The dosage form for rectal administration can be a suppository.

[0131] In addition, other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions comprising the above-mentioned compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof may be prepared by any known pharmaceutical process, such as effective formulations and methods of administration. The above considerations regarding effective formulations and methods of administration are well known in the art and are described in standard textbooks.

[0132] Drug screening

[0133] In one aspect, the present invention provides a method for screening drugs for anti-tumor immunotherapy, comprising the step of screening OTUB2 inhibitors.

[0134] In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and / or reducing tumor immunosuppression. In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell-mediated tumor killing in a subject.

[0135] In certain embodiments, the screening step is performed in vitro.

[0136] In certain embodiments, the screening step comprises:

[0137] (1) Determine the expression level or biological activity of OTUB2 in the presence of a test agent;

[0138] (2) comparing the measurement result of step (1) with the expression level or biological activity of OTUB2 measured in the absence of the test agent;

[0139] Wherein, if the determination result of step (1) is lower than the determination result in the absence of the test agent, it indicates that the test agent is an inhibitor of OTUB2 and is a candidate drug for anti-tumor immunotherapy.

[0140] In certain embodiments, the biological activity of OTUB2 includes: deubiquitinase activity. In certain embodiments, the biological activity of OTUB2 includes: binding to substrate proteins and removing their ubiquitination (i.e., interacting with substrate proteins to stabilize substrate proteins from degradation by ubiquitination). In certain embodiments, the substrate protein is PD-L1. Those skilled in the art will appreciate that there are multiple substrate proteins for OTUB2, and PD-L1 is merely an exemplary substrate protein used to detect the ability of OTUB2 inhibitors to inhibit the biological activity of OTUB2. However, regardless of whether PD-L1 expression is high or low, the growth of OTUB2-knockout tumor cells is inhibited, indicating that the regulation of tumor immunity by OTUB2 inhibitors is not limited to the PD-L1 pathway, that is, the use of OTUB2 inhibitors is not limited to the expression of PD-L1.

[0141] In certain embodiments, the screening step comprises:

[0142] (i) screening agents that interact with the OTUB2 protein as test agents, for example, by measuring using BLI technology (such as an Octet instrument) or SPR technology (such as a Biacore instrument);

[0143] (ii) detecting the deubiquitinating activity of the OTUB2 protein on the substrate protein in the presence of the test agent obtained in step (i);

[0144] (iii) comparing the result of the determination in step (ii) with the activity determined in the absence of the test agent;

[0145] Wherein, if the determination result of step (ii) is lower than the determination result in the absence of the test agent, it indicates that the test agent is an inhibitor of OTUB2 and is a candidate drug for anti-tumor immunotherapy.

[0146] In certain embodiments, detecting the deubiquitinating enzyme activity of the OTUB2 protein on the substrate protein comprises:

[0147] (1) contacting the test agent with OTUB2 protein and ubiquitinated substrate protein;

[0148] (2) measuring the levels of ubiquitinated substrate proteins;

[0149] (3) comparing the above measurement results with the levels measured in the absence of the test agent;

[0150] (4) Selecting a test agent that can increase the level of ubiquitinated substrate protein.

[0151] In certain embodiments, detecting the deubiquitinating enzyme activity of the OTUB2 protein on the substrate protein comprises:

[0152] (1) contacting the test agent with cells capable of expressing the substrate protein (e.g., mammalian cells, such as LL / 2 or B16-F10);

[0153] (2) measuring the protein level of the substrate protein;

[0154] (3) comparing the result of step (2) with the level determined in the absence of the test agent;

[0155] (4) Select a test agent that can inhibit the level of substrate protein.

[0156] Definition of terms

[0157] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0158] As used herein, the term "Otubin-2 (OTUB2)" may be of human origin or a homologous gene from another species (e.g., non-human mammals, fish, reptiles, or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, zebrafish, etc.). An exemplary amino acid sequence of human OTUB2 can be found in SEQ ID NO: 7.

[0159] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period. For "anti-tumor effect", including but not limited to, for example, tumor volume reduction, cancer cell number reduction, metastatic lesion number reduction, life expectancy increase, cancer cell proliferation reduction, cancer cell survival reduction, or improvement of the various physiological symptoms associated with cancer conditions.

[0160] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount is also the amount at which the therapeutic benefit exceeds any toxic or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following results, such as eliminating or reducing risk, alleviating severity, or delaying the onset of the disease, including the biochemistry of the disease, histology and / or behavioral symptoms, the intermediate pathological phenotype presented during its complication and disease formation. For therapeutic use, the beneficial or desired result includes clinical results, such as reducing one or more symptoms from the disease, improving the quality of life of those subjects suffering from the disease, reducing the dosage of other drugs needed for treating the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug can have an effect in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing down or desirably stopping) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing down and desirably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the condition. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve, directly or indirectly, prophylactic or therapeutic treatment.

[0161] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies, such as lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as spleen cancer and lymph node tumors.

[0162] As used herein, " alkyl " refers to a straight chain (i.e., non-branched) or branched, substituted or unsubstituted fully saturated hydrocarbon chain. As used herein, alkyl has 1 to 20 carbon atoms (i.e., C1-20 alkyl), 1 to 12 carbon atoms (i.e., C1-12 alkyl), 1 to 8 carbon atoms (i.e., C1-8 alkyl), 1 to 6 carbon atoms (i.e., C1-6 alkyl), 1 to 4 carbon atoms (i.e., C1-4 alkyl), 1 to 3 carbon atoms (i.e., C1-3 alkyl) or 1 to 2 carbon atoms (i.e., C1-2 alkyl). The example of an alkyl group includes methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, the tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. When an alkyl residue having a particular number of carbon atoms is designated by a chemical name or identified by a molecular formula, all positional isomers having that number of carbon atoms are encompassed; thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3); and "propyl" includes n-propyl (i.e., (CH2)2CH3) and isopropyl (i.e., -CH(CH3)2). The term "C1-C4 alkyl" refers to a radical derived from a straight or branched chain alkane containing 1 to 4 carbon atoms with one hydrogen atom removed, including, for example, "C 1- C2 alkyl", "C 1- C3 alkyl", "C 2- C3 alkyl", "C 2- C4 alkyl", "C 3- C4 alkyl" etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0163] As used herein, "alkenyl" refers to a monovalent straight or branched hydrocarbon group containing one or more carbon-carbon double bonds, for example, an aliphatic group containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms (i.e., C2-20 alkenyl), 2 to 8 carbon atoms (i.e., C2-8 alkenyl), 2 to 6 carbon atoms (i.e., C2-6 alkenyl), or 2 to 4 carbon atoms (i.e., C2-4 alkenyl). Examples of alkenyl groups include ethenyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0164] As used herein, "alkoxy" refers to the group "alkyl-O-." Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.

[0165] As used herein, " aryl " refers to an aromatic carbocyclic group with a single ring (such as a monocycle) or a plurality of rings (such as a bicyclic or tricyclic) comprising a fused system. As used herein, aryl has 6 to 20 ring carbon atoms (i.e., C6-20 aryl), 6 to 12 carbon ring atoms (i.e., C6-12 aryl) or 6 to 10 carbon ring atoms (i.e., C6-10 aryl). The example of aryl includes phenyl, naphthyl, fluorenyl and anthracenyl. However, aryl does not encompass heteroaryl defined below or overlaps with it in any way. If one or more aryl groups are fused to a heteroaryl ring, the resulting ring system is a heteroaryl. As used herein, the term "C5-C6 aryl" refers to an aromatic group containing 5-6 ring members, and specific examples include but are not limited to phenyl etc.

[0166] As used herein, " cycloalkyl " refers to the saturation or partially saturated cyclic alkyl group with a single ring or a plurality of rings comprising condensed, bridged and spirocyclic ring systems.Term " cycloalkyl " includes cycloalkenyl groups (i.e., cyclic groups with at least one double bond).As used herein, cycloalkyl has 3 to 20 ring carbon atoms (i.e., C3-20 cycloalkyl), 3 to 12 ring carbon atoms (i.e., C3-12 cycloalkyl), 3 to 10 ring carbon atoms (i.e., C3-10 cycloalkyl), 3 to 8 ring carbon atoms (i.e., C3-8 cycloalkyl) or 3 to 6 ring carbon atoms (i.e., C3-6 cycloalkyl).The example of cycloalkyl group includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0167] As used herein, "bridged" refers to ring fusions in which non-adjacent atoms on the ring are connected by a divalent substituent (such as an alkylene group, an alkylene group containing one or two heteroatoms) or a single heteroatom. Quinuclidinyl and adamantyl are examples of bridged ring systems.

[0168] As used herein, "fused" refers to a ring that is bound to an adjacent ring.

[0169] As used herein, "spiro" refers to a ring substituent joined by two bonds at the same carbon atom. Examples of spiro groups include 1,1-diethylcyclopentane, dimethyl-dioxolane, and 4-benzyl-4-methylpiperidine, wherein cyclopentane and piperidine are spiro substituents, respectively.

[0170] As used herein, "halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

[0171] As used herein, " heteroaryl " refers to an aromatic group with a single ring, multiple rings or multiple fused rings, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl includes 1 to 20 carbon ring atoms (i.e., C1-20 heteroaryl), 3 to 12 carbon ring atoms (i.e., C3-12 heteroaryl) or 3 to 8 carbon ring atoms (i.e., C3-8 heteroaryl);And 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms or 1 ring heteroatom independently selected from nitrogen, oxygen and sulfur. The example of heteroaryl groups includes pyrimidyl, purinyl, pyridyl, pyridazinyl, benzothiazolyl and pyrazolyl. Heteroaryl does not encompass aryl as defined above or overlaps with it. As used herein, the term "C5-C6 heteroaryl" refers to an aromatic group containing 5-6 ring members, and its ring structure contains a heteroatom selected from N, O, S and P. Specific examples include but are not limited to furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and the like.

[0172] As used herein, "heterocyclyl" or "heterocycle" refers to a non-aromatic cyclic alkyl group, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen and sulfur. As used herein, unless otherwise indicated, "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated ring, for example, in some embodiments, "heterocyclyl" or "heterocycle" refers to a partially saturated ring under specified circumstances. The term "heterocyclyl" or "heterocycle" includes heterocycloalkenyl groups (i.e., heterocyclyl groups with at least one double bond). Heterocyclyl can be a single ring or multiple rings, wherein the multiple rings can be fused, bridged or spiral. As used herein, a heterocyclyl group has 2 to 20 carbon ring atoms (i.e., C2-20 heterocyclyl), 2 to 12 carbon ring atoms (i.e., C2-12 heterocyclyl), 2 to 10 carbon ring atoms (i.e., C2-10 heterocyclyl), 2 to 8 carbon ring atoms (i.e., C2-8 heterocyclyl), 3 to 12 carbon ring atoms (i.e., C3-12 heterocyclyl), 3 to 8 carbon ring atoms (i.e., C3-8 heterocyclyl), or 3 to 6 carbon ring atoms (i.e., C3-6 heterocyclyl); has 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, sulfur, or oxygen. Examples of heterocyclic rings include, but are not limited to, the following monocyclic rings: 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1- Piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, oxetanyl, dioxolanyl, azetidinyl.

[0173] As used herein, "oxo" refers to the group (=O) or (O).

[0174] As used herein, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided herein with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups (e.g., -NH2) present in the compounds provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0175] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed between the -COOH group present in the compounds provided herein and a suitable alcohol, or an ester formed between the -OH group present in the compounds provided herein and a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, ethylsuccinates, stearic acid esters, or palmitates. Esters can undergo hydrolysis in the presence of an acid or base to produce the corresponding acid or alcohol.

[0176] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers primarily include cis-trans isomers and optical isomers. The compounds described herein may exist in the form of stereoisomers and therefore encompass all possible stereoisomeric forms, and any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer, or a mixture thereof.

[0177] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, disintegrants, binders, surfactants, glidants, lubricants, pH adjusters, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, antioxidants, colorants, flavorings, preservatives, taste-masking agents, etc. For example, non-limiting examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate. Non-limiting examples of binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Non-limiting examples of diluents include lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, glucose, sucrose, sorbitol, microcrystalline cellulose, starch and calcium hydrogen phosphate dihydrate. Non-limiting examples of surfactants include sodium lauryl sulfate and polysorbate 80. Non-limiting examples of glidants include silicon dioxide and talc. Non-limiting examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate and a mixture of magnesium stearate and sodium lauryl sulfate. Non-limiting examples of pH regulators include, but are not limited to, phosphate buffers. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Beneficial effects

[0178] This invention provides the first tumor immunotherapy targeting OTUB2. By inhibiting OTUB2, it helps improve the tumor microenvironment and enhance anti-tumor immunity, thereby exerting a tumor therapeutic effect. Furthermore, based on the OTUB2 binding pocket structure, this invention provides the first specific small molecule inhibitor targeting OTUB2, which can inhibit tumor growth by enhancing anti-tumor immune responses and reducing immunosuppression.

[0179] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1: Effects of knocking down, knocking out, or overexpressing OTUB2 on the growth and proliferation of tumor cells in vivo and in vitro.

[0181] Figure 1A shows the in vitro growth and proliferation curves of MC38 cells with OTUB2 knockdown and control MC38 cells;

[0182] Figure 1B shows the tumor growth curves of OTUB2-knockdown MC38 cells and control MC38 cells in C57BL / 6J immunocompetent mice;

[0183] Figure 1C shows the survival curves of mice transplanted with OTUB2-knockdown MC38 tumors and control MC38 tumors;

[0184] Figure 1D shows that blocking CD8+ T cells in vivo reversed the growth inhibition of MC38 tumors with OTUB2 knockdown in vivo;

[0185] Figure 1E shows the tumor growth curves of OTUB2-knockout MC38 cells and control MC38 cells in C57BL / 6J immunocompetent mice;

[0186] Figure 1F shows the tumor growth curves of OTUB2-knockout MC38 cells and control MC38 cells in NOD-SCID immunodeficient mice;

[0187] Figure 1G shows the tumor growth curves of OTUB2-knockout LL / 2 cells and control LL / 2 cells in C57BL / 6J immunocompetent mice;

[0188] Figure 1H shows the tumor growth curves of OTUB2-knockout B16-F10 cells and control B16-F10 cells in C57BL / 6J immunocompetent mice;

[0189] Figure 1I shows the tumor growth curves of OTUB2-overexpressing B16-F10 cells and control B16-F10 cells in C57BL / 6J immunocompetent mice.

[0190] Figure 2: Effect of targeting OTUB2 on tumor cell killing by tumor-specific T cells.

[0191] Figure 2A shows that compared with control LL / 2 cells, the cytotoxic activity of OVA-specific mouse CD8+ T cells against OTUB2-knockout LL / 2 tumor cells was significantly enhanced;

[0192] Figure 2B shows that compared with control B16-F10 cells, the cytotoxic activity of OVA-specific mouse CD8+ T cells against OTUB2-knockout B16-F10 tumor cells was significantly enhanced;

[0193] Figure 2C shows that the cytotoxic activity of human PBMCs against OTUB2-knockout NCI-H358 tumor cells was significantly enhanced compared with control NCI-H358 cells.

[0194] Figure 3: Identification of OTUB2 substrate proteins.

[0195] Figure 3A shows the interaction between OTUB2 and substrate proteins in cells;

[0196] Figure 3B shows that knockout of OTUB2 in B16-F10 cells reduces the expression level of substrate proteins;

[0197] Figure 3C shows that knockdown of OTUB2 in NCI-H358 cells reduces the expression levels of substrate proteins;

[0198] Figure 3D shows that knockdown of OTUB2 in LoVo cells reduces the expression levels of substrate proteins.

[0199] Figure 4: Anti-tumor immune effects of OTUB2-IN-1, a novel inhibitor targeting OTUB2.

[0200] Figure 4A schematically shows the chemical name and formula of OTUB2-IN-1;

[0201] Figure 4B schematically shows the action mode of OTUB2 and OTUB2-IN-1;

[0202] Figure 4C shows the affinity between OTUB2-IN-1 and recombinant OTUB2 protein;

[0203] Figure 4D shows Western Blot identification of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0204] Figure 4E shows the response curve of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0205] Figure 4F shows the reduction of substrate protein levels by OTUB2-IN-1 in LL / 2 cells;

[0206] Figure 4G shows the reduction of substrate protein levels by OTUB2-IN-1 in B16F10 cells;

[0207] Figure 4H shows the therapeutic effect of OTUB2-IN-1 in a subcutaneous lung cancer (LL / 2) mouse model;

[0208] Figure 4I shows the safety evaluation of OTUB2-IN-1 in a murine lung cancer (LL / 2) subcutaneous tumor mouse model.

[0209] Figure 5: Anti-tumor immune effects of OTUB2-IN-2, a novel inhibitor targeting OTUB2.

[0210] Figure 5A schematically shows the chemical name and formula of OTUB2-IN-2;

[0211] Figure 5B shows the affinity between OTUB2-IN-2 and recombinant OTUB2 protein;

[0212] Figure 5C shows Western Blot identification of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0213] Figure 5D shows the response curve of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0214] Figure 5E shows the reduction of substrate protein levels by OTUB2-IN-2 in B16F10 cells.

[0215] Figure 6: Anti-tumor immune effects of OTUB2-IN-3, a novel inhibitor targeting OTUB2.

[0216] Figure 6A schematically shows the chemical name and formula of OTUB2-IN-3;

[0217] Figure 6B shows the affinity between OTUB2-IN-3 and recombinant OTUB2 protein;

[0218] Figure 6C shows Western Blot identification of OTUB2-IN-3 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0219] FIG6D shows the response curve of OTUB2-IN-3 in inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0220] Figure 7: Anti-tumor immune effects of OTUB2-IN-4, a novel inhibitor targeting OTUB2.

[0221] Figure 7A schematically shows the chemical name and formula of OTUB2-IN-4;

[0222] Figure 7B shows the affinity between OTUB2-IN-4 and recombinant OTUB2 protein;

[0223] Figure 7C shows Western Blot identification of OTUB2-IN-4 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0224] FIG7D shows the response curve of OTUB2-IN-4 in inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0225] Figure 8: Anti-tumor immune effects of OTUB2-IN-5, a novel inhibitor targeting OTUB2.

[0226] Figure 8A schematically shows the chemical name and formula of OTUB2-IN-5;

[0227] Figure 8B shows the affinity between OTUB2-IN-5 and recombinant OTUB2 protein;

[0228] Figure 8C shows Western Blot identification of OTUB2-IN-5 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0229] FIG8D shows the response curve of OTUB2-IN-5 in inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0230] Notes on sequence information

[0231] The information of the sequences involved in the present invention is provided in Table 1.

[0232] Table 1: Sequence information Example

[0233] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0234] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0235] Example 1. Knockdown or knockout of OTUB2 inhibits tumor cell growth

[0236] In this example, lentiviral technology was used to prepare tumor cell lines with knockdown, knockout, or overexpression of OTUB2, and the growth and proliferation abilities of these cell lines were evaluated in vivo and in vitro.

[0237] 1.1 Construction of plasmids and cell lines

[0238] Knockdown: A shRNA nucleotide fragment targeting mouse OTUB2 (NCBI Reference Sequence Number: NM_001177841.2, Gene ID: 68149) and a shRNA nucleotide fragment targeting an unrelated sequence (SEQ ID NO: 1 and SEQ ID NO: 2; synthesized by Sangon) were respectively ligated into the pLKO.1 vector (Addgene, Catalog No. 10878) to construct shRNA plasmids capable of knocking down mouse OTUB2, named pLKO.1-sh-OTUB2 and pLKO.1-sh-Scramble. The pLKO.1-sh-OTUB2 or pLKO.1-sh-Scramble plasmids and the packaging plasmids psPAX2 (Addgene, Catalog No. 12260) and pMD2.G (Addgene, Catalog No. 12259) were co-transfected into 293T cells (purchased from ATCC, Catalog No. CRL-3216) using lipofectamine 2000 transfection reagent (Invitrogen, Catalog No. 11668019). After 72 hours, MC38 cells were infected with the recombinant viruses to express the corresponding shRNAs. The cells were then pressurized with medium containing 1 μg / mL puromycin (InvivoGen, Catalog No. ant-pr-5) to generate MC38 cell lines with stable knockdown of mouse OTUB2 (sh-OTUB2) and a control cell line (Scramble).

[0239] ShRNA nucleotide fragments (SEQ ID NO: 3 and SEQ ID NO: 4; synthesized by Sangon) targeting human OTUB2 (NCBI Reference Number: NM_023112.4, Gene ID: 78990) were ligated into the pLKO.1 vector to construct shRNA plasmids capable of knocking down mouse OTUB2, designated pLKO.1-shOTUB2#1 and pLKO.1-shOTUB2#2. 293T cells were co-transfected with the psPAX2 and pMD2.G packaging plasmids using lipofectamine 2000 transfection reagent. Seventy-two hours later, NCI-H358 cells were infected with the recombinant viruses to express the corresponding shRNAs. Stable NCI-H358 cell lines (shOTUB2#1 and shOTUB2#2) expressing human OTUB2 were generated by pressurizing with medium containing 1 μg / mL puromycin.

[0240] Knockout: A sgRNA targeting mouse OTUB2 and an sgRNA targeting an unrelated sequence (SEQ ID NO:5 and SEQ ID NO:6; synthesized by Sangon) were ligated into the lentiCRISPR v2 vector (Addgene, Catalog No. 52961) to construct CRISPR plasmids capable of knocking out mouse OTUB2. These plasmids were designated lentiCRISPR-sg-mOTUB2 and lentiCRISPR-sg-Scramble. The lentiCRISPR-sg-mOTUB2 or lentiCRISPR-sg-Scramble plasmids were co-transfected into 293T cells using lipofectamine 2000 transfection reagent along with the packaging plasmids psPAX2 and pMD2.G. 72 hours later, the recombinant viruses were used to infect MC38, LL / 2, and B16-F10 cells expressing the corresponding sgRNAs. Stably integrated tumor cell lines were generated by transfection with culture medium containing 1 μg / mL puromycin. Individual clones were isolated by limiting dilution. Finally, an anti-OTUB2 antibody (Sangon Biotechnology, Cat. No. D199590) was used to screen monoclonal tumor cell lines with complete OTUB2 knockout for subsequent experiments. Ultimately, MC38, LL / 2, and B16-F10 cell lines with complete mouse OTUB2 knockout (OTUB2 KO) and a control cell line (Control) were obtained.

[0241] Overexpression: Gene fragments expressing OTUB2 and PD-L1 (SEQ ID NO:7 and SEQ ID NO:8; synthesized by Sangon) were ligated into the lentiCRISPR v2 vector (Addgene, Catalog No. 52961) to construct plasmids stably expressing the corresponding genes, designated pLenti-OTUB2-Flag (with a Flag tag) and pLenti-PD-L1. pLenti-OTUB2 or pLenti were co-transfected with the packaging plasmids psPAX2 and pMD2.G, respectively, into 293T cells using lipofectamine 2000 transfection reagent. Seventy-two hours later, B16-F10 cells were infected with the recombinant viruses to express the corresponding genes. Stable OTUB2-expressing B16-F10 cell lines (OTUB2 OE) and control cell lines (Control) were generated by pressurizing with medium containing 1 μg / mL puromycin.

[0242] Ovalbumin (OVA) was overexpressed in control, LL / 2, and B16-F10 OTUB2 KO cells. A gene fragment expressing OVA (SEQ ID NO: 9; synthesized by Sangon) was ligated into the lentiCas9-Blast vector (Addgene, Cat. No. 52962) to construct a plasmid stably expressing the corresponding gene, named pLenti-OVA-blast. pLenti-OVA-blast or pLenti-Blast was co-transfected with the packaging plasmid into 293T cells using lipofectamine 2000 transfection reagent. 72 hours later, the recombinant virus was used to infect B16-F10 and LL / 2 cells to express the corresponding gene. The cells were then pressurized with culture medium containing 6 μg / mL (B16-F10) or 2 μg / mL (LL / 2) blasticidin (InvivoGen, catalog number ant-pr-5) to obtain OTUB2 knockout tumor cell lines (OTUB2 KO-OVA) stably expressing OVA and control cell lines (OTUB2 KO-Control).

[0243] 1.2 CCK-8 assay to detect the growth and proliferation ability of OTUB2 knockout tumor cells

[0244] MC38-Scramble and MC38-sh-OTUB2 cells were cultured at 2×10 3Plate a 96-well plate with the number of cells / well. On days 0, 1, 2, and 3 after complete cell attachment, add 10 μl of CCK-8 cell proliferation assay reagent (MCE, Cat. No. HY-K-03001) to each well. After incubation at 37°C for 4 hours, the 96-well plate was placed in a microplate reader (Thermo Fisher Scientific, Multiskan Go 1510) and read at a wavelength of 450 nm to plot cell proliferation curves.

[0245] The results in Figure 1A showed that knockdown of OTUB2 in MC38 had no effect on cell proliferation compared with the control group.

[0246] 1.3 Evaluation of tumor growth in OTUB2-knockdown MC38 cells and control MC38 cells in immunocompetent C57BL / 6J mice

[0247] Six-week-old C57BL / 6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were selected as experimental animals and subcutaneously inoculated with 100 μL of 5×10 5 MC38 cell suspension (sh-OTUB2 and Scramble) was used to monitor tumor size 8 days after tumor inoculation. The length (L) and width (W) of the tumor were measured with a vernier caliper and the formula Volume = (L × (W) 2 ) / 2 to calculate the tumor volume. The tumor volume was measured every two days and the tumor volume was continuously measured. The tumor volume was 1000 mm 3 As an ethical endpoint, the survival time of mice was recorded.

[0248] The experimental results in Figures 1B and 1C showed that compared with the control group, in the immune-competent mouse model, knockdown of OTUB2 significantly inhibited MC38 tumor growth (P < 0.05) and significantly prolonged the survival of tumor-bearing mice (P = 0.0034).

[0249] 1.4 Tumor growth inhibition of MC38 with OTUB2 knockdown in immunocompetent C57BL / 6J mice is CD8 dependent + T cells

[0250] Six-week-old C57BL / 6J mice were selected as experimental animals and treated with anti-CD8 antibody (BioXCell, Cat. No. BE0061) and control antibody 2 days before tumor inoculation, and treated every 3 days until the end of the experiment. Then, 100 μL of 5×10 5 MC38 cell suspension (sh-OTUB2 and Scramble), tumor size was monitored starting 7 days after tumor inoculation, and tumor volume was measured every two days until day 22.

[0251] The experimental results in Figure 1D show that, compared with the control group, knockdown of OTUB2 significantly inhibited MC38 tumor growth in an immune-competent mouse model (P = 0.0059). However, when CD8 cell function was blocked, the inhibitory effect of OTUB2 knockdown on MC38 tumor growth disappeared, indicating that the tumor suppression effect of targeting OTUB2 is dependent on the immune system.

[0252] 1.5 Evaluation of tumor growth in OTUB2-knockout MC38 cells and control MC38 cells in C57BL / 6J immunocompetent mice and NOD-SCID immunodeficient mice

[0253] Six-week-old C57BL / 6J mice and NOD-SCID mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were selected as experimental animals and subcutaneously inoculated with 100 μL of 5×10 5 MC38 cell suspensions (OTUB2 KO and Control) were used to monitor tumor size starting 8 days after inoculation. Tumor volume was measured every two days until day 20 or day 28.

[0254] The experimental results in Figures 1E and 1F show that compared with the control, OTUB2 knockout significantly inhibited MC38 tumor growth in immunocompetent mice (P < 0.0001), but failed to inhibit MC38 tumor growth in immunodeficient mice, indicating that the anti-tumor immunomodulatory effect of OTUB2 depends on the immune system.

[0255] 1.6 Evaluation of the effect of OTUB2 knockout on tumor growth in LL / 2 cells and control LL / 2 cells in immunocompetent C57BL / 6J mice

[0256] Six-week-old C57BL / 6J mice were selected as experimental animals and subcutaneously inoculated with 100 μL of 5×10 5 LL / 2 cell suspension (OTUB2 KO and Control) was used to monitor tumor size starting 8 days after tumor inoculation. Tumor volume was measured every two days until day 20.

[0257] The experimental results in Figure 1G showed that compared with the control, knockout of OTUB2 significantly inhibited the growth of LL / 2 tumors in immune-competent mice (P < 0.0001).

[0258] 1.7 Evaluation of tumor growth in OTUB2-knockout B16-F10 cells and control B16-F10 cells in immunocompetent C57BL / 6J mice

[0259] Six-week-old C57BL / 6J mice were selected as experimental animals and subcutaneously inoculated with 100 μL of 3×10 5B16-F10 cell suspensions (OTUB2 KO and Control) were used to monitor tumor size starting 7 days after tumor inoculation. Tumor volume was measured every other day and continued until day 17.

[0260] The experimental results in Figure 1H showed that compared with the control, knockout of OTUB2 significantly inhibited the growth of B16-F10 tumors in immunocompetent mice (P=0.0036).

[0261] 1.8 Evaluation of tumor growth in OTUB2-overexpressing and control B16-F10 cells in immunocompetent C57BL / 6J mice

[0262] Six-week-old C57BL / 6J mice were selected as experimental animals and subcutaneously inoculated with 100 μL of 3×10 5 B16-F10 cell suspensions (OTUB2 OE and Control) were used to monitor tumor size starting 7 days after tumor inoculation. Tumor volume was measured every two days and continued until day 16.

[0263] The experimental results in Figure 1I showed that compared with the control, overexpression of OTUB2 significantly promoted the growth of B16-F10 tumors in immunocompetent mice (P=0.0097).

[0264] The above results indicate that OTUB2 serves as a potential negative regulatory target for tumor immunity, promoting tumor immune escape.

[0265] Example 2. Tumor-targeted OTUB2 enhances CD8+ T cell killing of tumor cells

[0266] This example uses T cell killing experiments in vitro to verify that targeting OTUB2 in tumor cells can increase CD8 + T cell responses to tumor cells.

[0267] 2.1 Experiment on the specific killing of tumor cells by tumor-specific T cells

[0268] OT-1 T cells isolated from the spleen of OT-1 transgenic mice (Jackson Laboratory, Cat. No. 003831) were cultured at a concentration of 2 × 10 6Cells were cultured at a density of 10 cells / ml in RPMI 1640 medium supplemented with 1 μg / ml SIINFEKL peptide (Sangon Biotechnology) and 50 U / ml mouse recombinant IL-2 protein (Sino Biological, 51061-MNAE-20). After 7 days of activation, OT-1 T cells were added to tumor cells (OTUB2 KO and control cells) pretreated with 1 ng / ml IFN-γ and co-cultured for 24 hours. The ratio of activated cells (effector cells) to tumor cells (target cells) was 1:1. Tumor cell survival was assessed by flow cytometry.

[0269] The experimental results in Figures 2A-2B show that compared with control cells, OTUB2 knockout enhances the killing ability of tumor-specific OT-1T cells against B16-F10 and LL / 2 tumor cells.

[0270] 2.2 Experiment on the specific killing of tumor cells by tumor-specific T cells

[0271] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy subjects using Ficoll-Paque PLUS (GE Healthcare, Cat. No. 17-1440-02) density gradient centrifugation. 6 cells were added to the microwells and 0.1 μg / well of Dynabeads TM Human T-Activator CD3 / CD28 (Thermo Fisher Scientific, Catalog No. 11131D) and 50 U / ml human recombinant IL-2 protein (STEMCELL, Catalog No. 78036) were used. After 3 days of stimulation, PBMCs were added to tumor cells (shOTUB2 and control scramble cells) pretreated with 1 ng / ml IFN-γ and co-cultured for 120 hours. The ratio of activated cells (effector cells) to tumor cells (target cells) was 5:1. Flow cytometry was used to assess tumor cell survival.

[0272] The experimental results in Figure 2C showed that compared with control cells, knockdown of OTUB2 in tumor cells increased the killing ability of PBMCs against tumor cells.

[0273] In summary, targeting OTUB2 enhances the ability of tumor-specific T cells to kill tumor cells, thereby inhibiting tumor growth.

[0274] Example 3. OTUB2-IN-1, a novel inhibitor targeting OTUB2, can promote anti-tumor immunity

[0275] This example develops novel, specific inhibitors against OTUB2 and evaluates the inhibitory effect of the inhibitors on tumor growth in vivo.

[0276] 3.1 Identification of substrate proteins

[0277] OTUB2 is a deubiquitinating enzyme that binds to and removes ubiquitin from substrate proteins. This interaction stabilizes the substrate proteins against ubiquitination and degradation. Therefore, the enzymatic activity of OTUB2 can be determined by examining the ubiquitination and degradation of OTUB2 substrate proteins. In this study, we identified a substrate protein of OTUB2 and used it as an example to investigate the effects of OTUB2 inhibitors on OTUB2 enzymatic activity.

[0278] 3.1.1 Detection of the interaction between OTUB2 and substrate proteins by co-immunoprecipitation

[0279] The pLenti-OTUB2-Flag or pLenti and pLenti-PD-L1 plasmids were co-transfected into 293T cells using lipofectamine 2000 transfection reagent. After 72 hours, the total protein sample was collected and the protein was expressed in 293T cells. Immunoprecipitation was performed using M2Affinity Gel (Sigma-Aldrich, Cat. No. A2220). The co-precipitated proteins and total protein samples were then analyzed by western blotting. PD-L1 and OTUB2 were detected using corresponding antibodies: Abcam, ab213480 for PD-L1; and anti-Flag (Sigma-Aldrich, F1804) for OTUB2. The experimental results in Figure 3A show that, compared to the control, PD-L1 protein was detected upon capture of OTUB2 protein, indicating a direct interaction between OTUB2 and PD-L1.

[0280] 3.1.2 Knockout of OTUB2 in tumor cells reduces substrate protein levels

[0281] OTUB2 knockout and control tumor cells in the logarithmic growth phase were cultured at 5×10 5Cells were seeded at a density of 100 μg / well in 6-well culture plates. When the cell density reached 70%, they were treated with fresh culture medium or culture medium containing 0.5 ng / ml IFN-γ (R&D Systems, Catalog No. 285-IF). 24 hours later, the cells were harvested and stained with APC-labeled anti-mouse PD-L1 antibodies (BioLegend, Catalog No. 124312) and APC-labeled anti-human PD-L1 antibodies (BioLegend, Catalog No. 329708). PD-L1 protein expression levels were then determined by flow cytometry. The experimental results in Figures 3B-3D show that compared with the control, knocking out OTUB2 in B16-F10 cells or knocking down OTUB2 in NCI-H358 and LoVo cells reduced PD-L1 expression levels in tumor cells.

[0282] The above results indicate that PD-L1 is a substrate protein of OTUB2 and can be used to detect the enzymatic activity of OTUB2.

[0283] 3.2 OTUB2-IN-1 inhibitors can target the catalytic center of OTUB2

[0284] Recombinant OTUB2 protein was coupled to a CM5 chip (GE Healthcare, Catalog No. BR100530) and tested and analyzed using Biacore 8K using 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM OTUB2-IN-1 inhibitor (Lifechemical, Catalog No. F0444-0064) as the mobile phase. The experimental data were processed using Biacore Insight Evaluation Software.

[0285] Figure 4A shows that through virtual screening, we identified an OTUB2-IN-1 inhibitor that specifically binds to the OTUB2 protein. The OTUB2-IN-1 inhibitor is CAS No. 300558-22-9, and its chemical formula is 6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid.

[0286] Figure 4B shows a schematic diagram of the docking interactions between the OTUB2-IN-1 inhibitor and the amino acid residues in the catalytic pocket of OTUB2.

[0287] Figure 4C shows that the interaction affinity between the OTUB2-IN-1 inhibitor and OTUB2 was 12 μM as determined by SPR.

[0288] 3.3 Effects of OTUB2-IN-1 inhibitors on substrate protein ubiquitination

[0289] 10 ng of recombinant OTUB2 protein was co-incubated with different gradients of OTUB2-IN-1 inhibitor (0 μM, 0.39 μM, 1.56 μM, 6.25 μM, 25 μM, 100 μM), and then 100 ng of ubiquitinated PD-L1 protein was added. After incubation for 4 hours, ubiquitinated PD-L1 protein was detected by western blotting. The experimental data were processed using Image J software (NIH).

[0290] Figures 4D and 4E show that as the concentration of OTUB2-IN-1 inhibitor increased, the level of ubiquitinated substrate proteins increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0291] Effects of OTUB2-IN-1 inhibitors on substrate protein degradation in cells

[0292] LL / 2 and B16-F10 cells in the logarithmic growth phase were selected and cultured at 5×10 5 Cells were seeded at a density of 10 cells / well in a 6-well culture plate. When the cell density reached 70%, the OTUB2-IN-1 inhibitor was serially diluted in fresh culture medium (0, 0.15, 0.6, 2.5, 10, and 40 μM) and treated with the drug. After 24 hours of drug treatment, samples were collected for western blotting. Specific antibodies were used to detect the expression of the substrate proteins PD-L1 and OTUB2.

[0293] Figures 4F and 4G show that, in both LL / 2 and B16-F10 cells, as the concentration of the OTUB2-IN-1 inhibitor increased, the content of substrate proteins decreased gradually, while the content of OTUB2 did not change significantly. This indicates that the OTUB2-IN-1 inhibitor inhibits the function of OTUB2 by binding to OTUB2 without changing its stability, thereby degrading substrate proteins.

[0294] 3.5 Antitumor effect of OTUB2-IN-1 inhibitor in tumor-bearing mice

[0295] Six-week-old C57BL / 6J mice were selected as experimental animals and 100 μL of 5×10 5 LL / 2 cell suspension, wait until the tumor grows to 50mm 3At the same time, the patients were treated with OTUB2-IN-1 inhibitor and control drug once a day for a total of 6 times until the end of the experiment. Tumor size was monitored after treatment and the tumor was measured every two days. The tumor volume was continuously measured and the tumor volume was 1000mm 3 The end of ethics.

[0296] Figures 4H and 4I show that compared with the control group, the OTUB2-IN-1 inhibitor can significantly inhibit tumor growth (P<0.001) without significantly affecting the body weight of mice, indicating that OTUB2-IN-1 has a good anti-tumor effect in vivo and shows a certain degree of safety.

[0297] Example 4. Other novel inhibitors targeting OTUB2 can inhibit the activity of OTUB2

[0298] In this example, a series of novel, specific inhibitors targeting OTUB2 were developed and their inhibitory effects on OTUB2 enzyme activity were evaluated in vitro. The experimental method was the same as that in Example 3.

[0299] 4.1 OTUB2-IN-2 can effectively inhibit the activity of OTUB2

[0300] Figure 5A shows that through virtual screening, we identified the inhibitor OTUB2-IN-2, which specifically binds to the OTUB2 protein. Its chemical formula is: 2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid (obtained from OTAVA Chemicals, Catalog No. 1663785).

[0301] Figure 5B shows that the interaction affinity between the OTUB2-IN-2 inhibitor and OTUB2 was 41 μM as determined by SPR.

[0302] Figures 5C and 5D show that as the concentration of the added OTUB2-IN-2 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of the OTUB2 enzyme was significantly inhibited.

[0303] Figure 5E shows that in B16-F10 cells, as the concentration of the OTUB2-IN-2 inhibitor increased, the substrate protein content decreased gradually, while the OTUB2 content remained unchanged. This indicates that the OTUB2-IN-2 inhibitor inhibits OTUB2 function by binding to OTUB2 without altering OTUB2 stability, thereby degrading the substrate protein.

[0304] 4.2 OTUB2-IN-3 can effectively inhibit the activity of OTUB2

[0305] Figure 6A shows that through virtual screening, we identified the inhibitor OTUB2-IN-3, which specifically binds to the OTUB2 protein. The OTUB2-IN-3 inhibitor is CAS No. 313646-95-6, and its chemical formula is: 2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid (obtained from Lifechemicals, Catalog No. F3224-0128).

[0306] Figure 6B shows that the interaction affinity between the OTUB2-IN-3 inhibitor and OTUB2 was 32.6 μM as determined by SPR.

[0307] Figures 6C and 6D show that as the concentration of the added OTUB2-IN-3 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of the OTUB2 enzyme was significantly inhibited.

[0308] 4.3 OTUB2-IN-4 inhibitor can effectively inhibit the activity of OTUB2

[0309] Figure 7A shows that through virtual screening, we identified the inhibitor OTUB2-IN-4, which specifically binds to the OTUB2 protein. The OTUB2-IN-4 inhibitor is CAS No. 374543-72-3, and its chemical formula is: 11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid (obtained from OTAVA Chemicals, Catalog No. 4349982).

[0310] Figure 7B shows that the interaction affinity between the OTUB2-IN-4 inhibitor and OTUB2 was 25.9 μM as determined by SPR.

[0311] Figures 7C and 7D show that as the concentration of the added OTUB2-IN-4 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of the OTUB2 enzyme was significantly inhibited.

[0312] 4.4 OTUB2-IN-5 inhibitor can effectively inhibit the activity of OTUB2

[0313] Figure 8A shows that through virtual screening, we identified the inhibitor OTUB2-IN-5, which specifically binds to the OTUB2 protein. Its chemical formula is: 11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid (obtained from OTAVA Chemicals, Catalog No. 4349908).

[0314] Figure 8B shows that the interaction affinity between the OTUB2-IN-5 inhibitor and OTUB2 was 3.91 μM as determined by SPR.

[0315] Figures 8C and 8D show that as the concentration of the added OTUB2-IN-4 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of the OTUB2 enzyme was significantly inhibited.

[0316] The results in Figures 5 to 8 above indicate that these small molecule compounds can act as OTUB inhibitors to regulate tumor immunity.

[0317] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of OTUB2 inhibitors in the preparation of drugs for anti-tumor immunotherapy.

2. The use according to claim 1, wherein the drug is used to enhance anti-tumor immune response and / or reduce tumor immunosuppression in a subject; Preferably, the medicament is used to promote T cell-mediated tumor killing in a subject; Preferably, the subject suffers from a tumor.

3. The use according to claim 1 or 2, wherein The inhibitor can inhibit or down-regulate the expression of the OTUB2 gene, inhibit or block the activity of the OTUB2 protein, or degrade the OTUB2 protein.

4. The use according to claim 3, wherein The inhibitor is selected from antibodies, RNA interfering agents (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA) or micro RNA (miRNA)) or antisense oligonucleotides, gene editing systems (e.g., CRISPR-Cas systems), small molecule compounds, or PROTACs (protein degradation targeting chimeras).

5. The use according to claim 3, wherein The inhibitor is an RNA interference agent, for example, small interfering RNA (siRNA), small hairpin RNA (shRNA) or micro RNA (miRNA)) or an antisense oligonucleotide; Preferably, the inhibitor is shRNA.

6. The use according to claim 3, wherein The inhibitor is selected from small molecule compounds.

7. The use according to claim 6, wherein The small molecule compound is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt, ester or stereoisomer thereof: in: R 1 -C(O)X, X is hydroxy or C1-C4 alkoxy; or is C3-7 monocyclic cycloalkyl, 4-6-membered monocyclic heterocyclyl or 5-6-membered monocyclic heteroaryl, wherein the 4-6-membered monocyclic heterocyclyl and the 5-6-membered monocyclic heteroaryl each have 1 to 3 ring heteroatoms independently selected from N, O and S; n is an integer selected from 1 to 12; R 2 for Among them, R 2a is selected from (C1-C4)alkyl, (C2-C4)alkenyl, or -C(O)Y, where Y is hydroxy or (C1-C4)alkoxy; Among them, R 2b , R 2c , R 2d Each is independently selected from halogen (e.g., -F, -Cl, -Br or -I), nitro, amino, hydroxy, mercapto, (C1-C4) alkyl or (C1-C4) alkoxy; preferably, R 2b , R 2c , R 2d Different from each other; Here, m is 1, 2, 3 or 4.

8. The use according to claim 7, wherein R 1 -C(O)X, X is hydroxy or (C1-C4)alkoxy, and n is an integer selected from 1 to 12; Preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

9. The use according to claim 8, wherein R 1 is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy; Preferably, X is hydroxy, methoxy or ethoxy. Preferably, X is hydroxy.

10. The use according to claim 7, wherein R 1 is a C3-7 monocyclic cycloalkyl, a 4- to 6-membered monocyclic heterocyclyl, or a 5- to 6-membered monocyclic heteroaryl, and n is 1, 2, 3 or 4; Preferably, n is 1 or 2; preferably, n is 1.

11. The use according to claim 10, wherein R 1 is a C3-7 monocyclic cycloalkyl, a 4- to 6-membered monocyclic heterocyclyl, or a 5- to 6-membered monocyclic heteroaryl, each of which has 1 ring heteroatom selected from N, O and S; Preferably, R 1 is a C3-7 monocyclic cycloalkyl group or a 4- to 6-membered monocyclic heterocyclic group; Preferably, R 1 is a C3-6 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclyl group (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl); Preferably, R 1 is tetrahydrofuranyl or pyrrolidinyl; Preferably, R 1 It is tetrahydrofuran.

12. The use according to any one of claims 7 to 11, wherein R 2 for Where R 2a is selected from methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy; Preferably, R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; Preferably, R 2a Selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

13. The use according to any one of claims 7 to 11, wherein R 2 for Among them, R 2b , R 2c , R 2d Each is independently selected from nitro, hydroxy, (C1-C4) alkyl or (C1-C4) alkoxy; Preferably, R 2b , R 2c , R 2d Each is independently selected from nitro, hydroxy, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy; Preferably, R 2b , R 2c , R 2d Each is independently selected from nitro, hydroxy, methyl, ethyl, methoxy, ethoxy; Preferably, R 2b , R 2c , R 2d Different from each other; Preferably, R 2b , R 2c , R 2d They are nitro, hydroxy and methoxy respectively.

14. The use according to any one of claims 7 to 13, wherein The compound has a structure shown in formula (Ia): Among them, R 1 , R 2a , m and n are as defined in formula (I).

15. The use according to claim 14, wherein R 1 Possessing characteristics selected from the following: (1)R 1 is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or (2)R 1 is a C3-7 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclyl group having 1 ring heteroatom selected from N, O and S (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl); preferably, R 1 is tetrahydrofuranyl or pyrrolidinyl; preferably, n is 1 or 2, for example, 1.

16. The use according to claim 14 or 15, wherein R 2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy; Preferably, R 2a Selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

17. The use according to any one of claims 7 to 16, wherein The compound is selected from the following compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof: 6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid; 2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid; 2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid; 11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}unddecanoic acid; 11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanic acid.

18. The use according to any one of claims 1 to 17, wherein The tumor includes a solid tumor or a blood tumor; Preferably, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

19. Use of a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof as an OTUB2 inhibitor, wherein: The compound is as defined in any one of claims 7 to 17; Preferably, the OTUB2 inhibitor is used to inhibit or block the activity of OTUB2 protein in vitro; Preferably, the OTUB2 inhibitor is used for non-therapeutic purposes.

20. Use of a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof in the preparation of a medicament for preventing and / or treating tumors, wherein: The compound is as defined in any one of claims 7 to 17; Preferably, the drug is used for anti-tumor immunotherapy; Preferably, the medicament is used to enhance anti-tumor immune response and / or reduce tumor immunosuppression in a subject; Preferably, the medicament is used to promote T cell-mediated tumor killing in a subject; Preferably, the tumor comprises a solid tumor or a blood tumor; Preferably, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

21. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof, and a pharmaceutically acceptable carrier and / or excipient; wherein: The compound is as defined in any one of claims 7-17.

22. A method for screening drugs for anti-tumor immunotherapy, comprising the step of screening OTUB2 inhibitors.

23. The method of claim 22, wherein: The method comprises: (i) screening agents that interact with the OTUB2 protein as test agents, for example, by measuring using BLI or SPR technology; (ii) in the presence of the test agent obtained in step (i), detecting the effect of OTUB2 protein on the substrate protein Deubiquitinase activity; (iii) comparing the result of step (ii) with the activity measured in the absence of the test agent.

24. A method for anti-tumor immunotherapy, comprising: administering an effective amount of an OTUB2 inhibitor to a subject in need thereof; Preferably, the OTUB2 inhibitor is as defined in any one of claims 3-17; Preferably, the anti-tumor immunotherapy comprises enhancing anti-tumor immune response and / or reducing tumor immunosuppression in the subject; Preferably, the anti-tumor immunotherapy comprises promoting T cell-mediated tumor killing in the subject; Preferably, the subject suffers from a tumor; Preferably, the tumor comprises a solid tumor or a blood tumor; Preferably, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

25. A method for preventing and / or treating tumors, comprising: Administering an effective amount of a compound as defined in any one of claims 7 to 17 or a pharmaceutically acceptable salt or ester or stereoisomer thereof, or a pharmaceutical composition as claimed in claim 21 to a subject in need thereof; Preferably, the tumor comprises a solid tumor or a blood tumor; Preferably, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

26. A method for inhibiting OTUB2 activity in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound as defined in any one of claims 7 to 17 or a pharmaceutically acceptable salt or ester or stereoisomer thereof, or a pharmaceutical composition as claimed in claim 21.

27. A method for inhibiting OTUB2 activity in a cell, the method comprising contacting the cell with a compound as defined in any one of claims 7 to 17 or a pharmaceutically acceptable salt or ester or stereoisomer thereof, or a pharmaceutical composition according to claim 21.

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