OTUB2 inhibitor and application thereof in tumor immunity

Abstract

The invention belongs to the field of biological medicine, and relates to an OTUB2 inhibitor and application thereof in tumor immunity. In particular, the present invention provides uses and methods of an OTUB2 inhibitor for enhancing anti-tumor immune response, reducing tumor immunosuppression, and / or treating tumors. In addition, the invention also provides a small molecule inhibitor targeting OTUB2.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to OTUB2 inhibitors and applications thereof in tumor immunity. Background Art

[0002] The ability to enhance antitumor immunity in the tumor microenvironment (TME) is critical for tumor control. Cancers often evolve different mechanisms that allow them to evade destruction by the immune system. One of the escape mechanisms involves the interaction between antitumor cytotoxic T lymphocytes (CTLs) and tumor cells. Tumor cells resist or weaken CTLs 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.

[0003] Otubin-2 (OTUB2) is a deubiquitinating enzyme (DUB) belonging to the ovarian tumor (OTU) protein 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 protein has been studied, there is no report on the function of OTUB2 in regulating tumor immunity. Summary of the invention

[0004] It has been reported in the art that OTUB2 is expressed at a higher level in human malignant tumors and at a lower level in organs throughout the body, see, for example, https: / / www.proteinatlas.org / ENSG00000089723-OTUB2 / tissue, but there is no report on the correlation between OTUB2 and tumor immunity. The present invention is the first to discover that OTUB2 has the function of immune regulation and plays a key role in the tumor microenvironment. Targeting OTUB2 helps to improve the tumor microenvironment and enhance anti-tumor immunity, thereby exerting a tumor therapeutic effect. In addition, it is worth noting that no OTUB2-specific small molecule inhibitors have been developed so far. Based on the OTUB2 binding pocket structure, the present invention provides a specific small molecule inhibitor targeting OT UB2 for the first time. The following invention is thus provided.

[0005] Application of OTUB2 inhibitors in tumor immunity

[0006] In one aspect, the present invention provides a use of an OTUB2 inhibitor in the preparation of a medicament for anti-tumor immunotherapy, and also provides a method for anti-tumor immunotherapy, the method comprising: administering an effective amount of an OTUB2 inhibitor to a subject in need thereof.

[0007] In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and / or reducing tumor immunosuppression in a subject.

[0008] In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell (eg, CD8+ T cell)-mediated tumor killing in a subject.

[0009] 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) proliferation 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.

[0010] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor.

[0011] OTUB2 inhibitors

[0012] The OTUB2 inhibitors described herein may exert their inhibitory effects by any mechanism, such as by inhibiting the expression of a gene at the RNA or protein level (e.g., reducing or inhibiting the transcription of a gene, and / or reducing or inhibiting the translation of an mRNA product of the gene); or, for example, by inhibiting or blocking the biological activity of a protein product of the gene; or, for example, by degrading a protein product of the gene or destroying its stability.

[0013] In certain embodiments, the OTUB2 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.

[0014] 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 a substrate protein and removing its ubiquitination (i.e., interacting with the substrate protein to stabilize the substrate protein from being ubiquitinated and degraded). Thus, by inhibiting or blocking the above functions of the OTUB2 protein, the substrate protein can be degraded and the expression of the substrate protein can be inhibited.

[0015] In certain embodiments, the inhibitor comprises a small molecule compound. In this article, the expression "small molecule compound" refers to an organic non-protein compound. In certain embodiments, the molecular weight of the small molecule compound is no more than 1500Da. The small molecule compound can inhibit the function of the above-mentioned 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 inhibitory activity of the compound on the expression level of OTUB2, the binding activity to the OTUB2 protein, or the ability to inhibit the biological function of the OTUB2 protein.

[0016] In certain embodiments, the small molecule compound binds to the OTUB2 protein or interacts with the OTUB2 protein, for example, as determined by BLI technology (such as an Octet instrument) or SPR technology (such as 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, blocks the interaction between OTUB2 and a substrate protein (such as PD-L1), thereby causing the substrate protein (such as PD-L1) to be ubiquitinated and degraded.

[0017] In certain embodiments, the small molecule compound is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt or ester or stereoisomer thereof:

[0018]

[0019] in:

[0020] 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;

[0021] n is an integer selected from 1 to 12;

[0022] R 2 for

[0023] 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;

[0024] 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;

[0025] Wherein, m is 1, 2, 3 or 4.

[0026] 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.

[0027] 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.

[0028] In certain embodiments, R 1 In some embodiments, X is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy. In some embodiments, X is hydroxy, methoxy or ethoxy. In some embodiments, X is hydroxy.

[0029] In certain embodiments, R 1 is a C3-7 monocyclic cycloalkyl (e.g., a C3-6 cycloalkyl, a C3-5 cycloalkyl, a C3-4 cycloalkyl, a C4-6 cycloalkyl, or a C4-5 cycloalkyl, such as a cyclopropyl, a cyclobutyl, a cyclopentyl, or a cyclohexyl), a 4- to 6-membered monocyclic heterocyclyl (e.g., an oxetanyl, a tetrahydrofuranyl, a tetrahydropyranyl, a pyrrolidinyl, or a piperidinyl), or a 5- to 6-membered monocyclic heteroaryl (imidazolyl, a triazolyl, a tetrazolyl, a pyrazolyl, a phenylthio, a thiazolyl, a pyridinyl, a pyrimidinyl, or a pyrazinyl), and n is 1, 2, 3, or 4. In certain embodiments, n is 1 or 2; preferably, n is 1.

[0030] In certain embodiments, R 1is 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 heterocyclyl group selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl, for example tetrahydrofuranyl or pyrrolidinyl. In certain embodiments, R 1 It is tetrahydrofuran.

[0031] 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, ethenyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, or ethoxy. 2a is selected from methyl, ethyl, ethenyl, propenyl, or -C(O)OH. In certain embodiments, m is 1, 2, or 3.

[0032] 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 2d Each 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 2for R 2b , R 2c , R 2d As defined above.

[0033] In certain embodiments, the compound has the structure shown in Formula (Ia):

[0034]

[0035] Among them, R 1 , R 2a , m and n are as defined in formula (I).

[0036] 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.

[0037] 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 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.

[0038] 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.

[0039] In certain embodiments, in Formula (Ia), m is 1, 2, or 3.

[0040] In certain embodiments, in Formula (Ia):

[0041] 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;

[0042] R 2ais 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.

[0043] In certain embodiments, the compound is selected from:

[0044] 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);

[0045] 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 (e.g., available from OTAVA chemicals, catalog number 1663785);

[0046] 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);

[0047] 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);

[0048] 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, catalog number 4349908, for example).

[0049] In certain embodiments, the small molecule compounds described herein can be chemically synthesized or purchased from commercial sources.

[0050] In certain embodiments, the inhibitor inhibits the biological function of the OTUB2 gene by inhibiting the expression level of the OTUB2 gene. In such embodiments, the expression level can be determined at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR, or real-time RT-PCR. Methods for determining 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.

[0051] 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 micro RNA (miRNA)) or an antisense oligonucleotide, a gene editing system (e.g., a CRISPR-Cas system), or a small molecule compound.

[0052] 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.

[0053] 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.

[0054] In this article, the expression "RNA interference agent" refers to any agent that suppresses the expression of a target gene by an RNA interference (RNAi) mechanism." RNA interference (RNAi) "is a kind of evolutionarily conservative process, in which the expression or introduction of RNA of a sequence identical or highly similar to a target gene causes sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of a messenger RNA (mRNA) transcribed from the target gene, thereby suppressing the expression of the target gene. Therefore, it is known to those skilled in the art that a small interfering RNA (siRNA) or a small RNA (microRNA, miRNA) molecule can be selected according to the sequence of the mRNA generated by a specific gene or its transcription to design a target sequence. The siRNA or miRNA molecule can interfere with the transcription, translation or transcription, post-translational modification of a gene, thereby affecting the expression of a protein.

[0055] In addition, in order to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed and cloned into a vector after annealing. The transcription product of the recombinant vector is short hairpin RNA (shRNA), which can fold and pair into a stem-loop structure with a stem length of 19-21 bases, wherein the 19-21 bases are derived from a specific sequence of the target gene mRNA. The precursor of this stem-loop structure is quickly cleaved in the cell to form a functional siRNA. The shRNA expressed by this vector has the characteristics of stable expression and long duration, thus causing long-term inhibition of target gene expression.

[0056] In this article, the shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polⅢ promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ. The siRNA sequence is cloned into a plasmid vector, and "small interfering RNA" (siRNA) can be delivered in vivo. When delivered into an animal, the hairpin sequence is expressed to form a stem-loop structure, which is cut into a functional siRNA to exert a gene silencing effect.

[0057] 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 RNAaseⅢ family that is specific to double-stranded RNA). siRNA is a major member of siRISC and stimulates the silencing of complementary target mRNA.

[0058] In this article, microRNA (miRNA) refers to a naturally occurring non-coding RNA molecule with a length of about 21-25 nucleotides. Based on sequence complementarity with the target mRNA, it can cause target mRNA degradation or inhibit its translation through base-specific complementary pairing with the target mRNA, thereby regulating gene expression after transcription. miRNA can refer to the miRBase database.

[0059] In certain embodiments, the RNA interfering agent is selected from small interfering RNA (siRNA), small hairpin RNA (shRNA) or micro RNA (miRNA).

[0060] Herein, the expression "antisense oligonucleotide" refers to a molecule 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 a hydrogen bond with the sense nucleic acid (i.e., anneal with it).

[0061] 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.

[0062] In certain embodiments, the antisense oligonucleotide is antisense RNA or antisense DNA.

[0063] After obtaining the RNA interfering agent or antisense oligonucleotide, the RNA interfering agent or antisense oligonucleotide can be further tested for its inhibitory activity on the expression level of OTUB2 using the method described above.

[0064] In certain exemplary embodiments, the inhibitor is a shRNA, for example comprising the sequence shown in SEQ ID NO: 3 or 4.

[0065] 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 (e.g., OTUB2). In certain embodiments, the inhibitor is CRISPR / Cas, such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a, or CRISPR / Cas14a.

[0066] In other embodiments, the inhibitor is 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), so 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 above two functional groups.

[0067] In certain embodiments, the PROTAC is a bifunctional compound comprising an E3 ubiquitin ligase binding portion and an OTUB2 protein binding portion.

[0068] 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 VLM, CLM, MLM and IAP (ILM).

[0069] 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.

[0070] Administration of OTUB2 inhibitors

[0071] The OTUB2 inhibitors described herein can be formulated into dosage forms compatible with their intended routes 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 applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or glucose. 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.

[0072] The dosage form suitable for injection includes sterile aqueous solution (wherein water soluble) or dispersion and 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, isotonic agents will preferably be included in the composition, 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.

[0073] The OTUB2 inhibitors described herein can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. A preferred dosage form is an injection.

[0074] 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, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). The skilled person will appreciate that the route and / or mode of administration will vary depending on the intended purpose.

[0075] The OTUB2 inhibitors described herein can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable 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.

[0076] The subject described herein includes any individual that can produce an immune response. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0077] Indications

[0078] The goal of anti-tumor immunotherapy is to enhance the ability of the immune system to recognize and kill tumors and to overcome the immunosuppressive tumor microenvironment. The term "tumor" includes any form of cancer, including solid tumors or hematological tumors.

[0079] 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.

[0080] In certain embodiments, the tumor expresses OTUB2 or is OTUB2 positive. Herein, OTUB2 positivity can be determined at the nucleic acid level or protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for determining expression at the protein level include, but are not limited to, immunological detection.

[0081] OTUB2 small molecule inhibitors

[0082] 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 a compound represented by formula (I) or a pharmaceutically acceptable salt or ester or stereoisomer thereof:

[0083]

[0084] in:

[0085] 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;

[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 (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;

[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 some embodiments, X is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy. In some embodiments, X is hydroxy, methoxy or ethoxy. In some embodiments, X is hydroxy.

[0094] In certain embodiments, R 1is a C3-7 monocyclic cycloalkyl (e.g., a C3-6 cycloalkyl, a C3-5 cycloalkyl, a C3-4 cycloalkyl, a C4-6 cycloalkyl, or a C4-5 cycloalkyl, such as a cyclopropyl, a cyclobutyl, a cyclopentyl, or a cyclohexyl), a 4- to 6-membered monocyclic heterocyclyl (e.g., an oxetanyl, a tetrahydrofuranyl, a tetrahydropyranyl, a pyrrolidinyl, or a piperidinyl), or a 5- to 6-membered monocyclic heteroaryl (imidazolyl, a triazolyl, a tetrazolyl, a pyrazolyl, a phenylthio, a thiazolyl, a pyridinyl, a pyrimidinyl, or a pyrazinyl), 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 heterocyclyl group selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl, for example tetrahydrofuranyl or pyrrolidinyl. In certain embodiments, R 1 It is 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, ethenyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy, or ethoxy. 2a is selected from methyl, ethyl, ethenyl, propenyl, or -C(O)OH. In certain 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]

[0100] Among them, R 1 , R 2a , m and n are as defined in formula (I).

[0101] 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.

[0102] 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 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.

[0103] 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.

[0104] In certain embodiments, in Formula (Ia), m is 1, 2, or 3.

[0105] In certain embodiments, in Formula (Ia):

[0106] 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;

[0107] 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.

[0108] In certain embodiments, the compound is selected from:

[0109] 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);

[0110] 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 (e.g., available from OTAVA chemicals, catalog number 1663785);

[0111] 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);

[0112] 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);

[0113] 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, catalog number 4349908, for example).

[0114] In one aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0115] In one aspect, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition as an OTUB2 inhibitor. In certain embodiments, the OTUB2 inhibitor is used to inhibit or block the activity of OTUB2 protein in vitro. In certain embodiments, the OTUB2 inhibitor is used for non-therapeutic purposes. The present invention also provides a method for inhibiting OTUB2 activity in a cell in vitro, comprising contacting the cell with the above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer.

[0116] In one aspect, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors. Also provided is a method for preventing and / or treating tumors, comprising: administering an effective amount of the above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition to a subject in need thereof.

[0117] In certain embodiments, the above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition is used for anti-tumor immunotherapy.

[0118] In certain embodiments, the anti-tumor immunotherapy comprises enhancing anti-tumor immune responses and / or reducing tumor immunosuppression.

[0119] 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.

[0120] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor.

[0121] In certain embodiments, the tumor comprises a solid tumor or a hematological tumor.

[0122] 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.

[0123] In certain embodiments, the tumor expresses OTUB2 or is OTUB2 positive. Herein, OTUB2 positivity can be determined at the nucleic acid level or protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for determining expression at the protein level include, but are not limited to, immunological detection.

[0124] The above compounds or their pharmaceutically acceptable salts or esters or stereoisomers may be in any form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, lyophilized powders), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0125] The above-mentioned compound or its pharmaceutically acceptable salt or ester or stereoisomer may be present in a pharmaceutical composition in the form of a unit dosage for easy administration.

[0126] The above compounds or their pharmaceutically acceptable salts or esters or stereoisomers may be administered by any suitable method known in the art, including but not limited to oral, rectal, parenteral or topical administration.

[0127] 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, isopropanol, 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, flavoring agents and aromatics, etc. Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In addition to the active ingredients, solid dosage forms may contain pharmaceutically acceptable inert excipients or carriers, for example fillers (such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, cross-linked polyvinylpyrrolidone and calcium sulfate); binders (such as carboxymethyl cellulose, alginate, 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.

[0128] The above-mentioned compound or its pharmaceutically acceptable salt, ester or stereoisomer may also be administered via a non-oral route.

[0129] 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 injection solution, sterile powder for injection or concentrated solution for injection. In addition to the active ingredient, the injection dosage form 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.

[0130] Another exemplary route of administration is topical, for example, transdermal (eg, by a transdermal patch or iontophoresis device), intraocular, or intranasal or by inhalation.

[0131] Another exemplary route of administration is rectal administration. The dosage form for rectal administration can be a suppository.

[0132] In addition, other carrier materials and modes of administration known in the pharmaceutical field may also be used. Pharmaceutical compositions comprising the above 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.

[0133] Drug Screening

[0134] In one aspect, the present invention provides a method for screening drugs for anti-tumor immunotherapy, which comprises the step of screening OTUB2 inhibitors.

[0135] 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.

[0136] In certain embodiments, the screening step is performed in vitro.

[0137] In certain embodiments, the screening step comprises:

[0138] (1) measuring the expression level or biological activity of OTUB2 in the presence of a test agent;

[0139] (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;

[0140] 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.

[0141] 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 being degraded by ubiquitination). In certain embodiments, the substrate protein is PD-L1. It is understood by those skilled in the art that there are multiple substrate proteins for OTUB2, and PD-L1 is only used as an exemplary substrate protein to detect the inhibitory ability of OTUB2 inhibitors on 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.

[0142] In certain embodiments, the screening step comprises:

[0143] (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);

[0144] (ii) detecting the deubiquitinase activity of the OTUB2 protein on the substrate protein in the presence of the test agent obtained in step (i);

[0145] (iii) comparing the result of step (ii) with the activity measured in the absence of the test agent;

[0146] 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.

[0147] In certain embodiments, the detecting the deubiquitinase activity of the OTUB2 protein on the substrate protein comprises:

[0148] (1) contacting the test agent with OTUB2 protein and ubiquitinated substrate protein;

[0149] (2) measuring the levels of ubiquitinated substrate proteins;

[0150] (3) comparing the above measurement results with the level measured in the absence of the test agent;

[0151] (4) Selecting a test agent that can increase the level of ubiquitinated substrate protein.

[0152] In certain embodiments, the detecting the deubiquitinase activity of the OTUB2 protein on the substrate protein comprises:

[0153] (1) contacting a test agent with cells (e.g., mammalian cells, such as LL / 2 or B16-F10) capable of expressing a substrate protein;

[0154] (2) determining the protein level of the substrate protein;

[0155] (3) comparing the result of step (2) with the level measured in the absence of the test agent;

[0156] (4) Select a test agent that can inhibit the level of substrate protein.

[0157] Definition of terms

[0158] 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 the relevant terms are provided below.

[0159] As used herein, the term "Otubin-2 (OTUB2)" may be of human origin or may be a homologous gene from other 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.

[0160] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relieving 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 relieving 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", it includes, but is not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in the various physiological symptoms associated with the cancer condition.

[0161] 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 beneficial effect exceeds any toxic or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following result, 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 complications 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 objects suffering from the disease, reducing the dosage of other drugs required for the treatment of 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 to some extent) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing down and desirably stopping to some extent) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment.

[0162] As used herein, the terms "cancer" and "tumor" are used interchangeably and 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 benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies such as lymphomas, leukemias, myelomas or lymphatic malignancies, as well as spleen cancer and lymph node tumors.

[0163] As used herein, "alkyl" refers to a straight (i.e., non-branched) or branched, substituted or unsubstituted fully saturated hydrocarbon chain. As used herein, an alkyl group 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, 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 contemplated; 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 obtained by removing a hydrogen atom from a straight or branched chain alkane containing 1 to 4 carbon atoms, 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.

[0164] 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 vinyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0165] 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.

[0166] As used herein, "aryl" refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or comprising multiple rings (e.g., bicyclic or tricyclic) of 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 with 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.

[0167] As used herein, "cycloalkyl" refers to a saturated or partially saturated cyclic alkyl group with a single ring or multiple rings comprising condensed, bridged and spirocyclic 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.

[0168] As used herein, "bridged" refers to ring fusions in which non-adjacent atoms on the ring are linked 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.

[0169] As used herein, "fused" refers to a ring that is bound to an adjacent ring.

[0170] As used herein, "spiro" refers to a ring substituent connected 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.

[0171] As used herein, "halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

[0172] 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 cover 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 whose ring structure contains heteroatoms selected from N, O, S and P. Specific examples include but are not limited to furanyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and the like.

[0173] 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., a C2-20 heterocyclyl group), 2 to 12 carbon ring atoms (i.e., a C2-12 heterocyclyl group), 2 to 10 carbon ring atoms (i.e., a C2-10 heterocyclyl group), 2 to 8 carbon ring atoms (i.e., a C2-8 heterocyclyl group), 3 to 12 carbon ring atoms (i.e., a C3-12 heterocyclyl group), 3 to 8 carbon ring atoms (i.e., a C3-8 heterocyclyl group), or 3 to 6 carbon ring atoms (i.e., a C3-6 heterocyclyl group); 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.

[0174] As used herein, "oxo" refers to the group (=O) or (O).

[0175] 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 by the present invention and 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), and including but not limited to, hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, 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.

[0176] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed by -COOH present in the compound provided by the present invention and a suitable alcohol, or an ester formed by -OH present in the compound provided by the present invention 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 reactions to generate corresponding acids or alcohols in the presence of acids or bases.

[0177] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers mainly include cis-trans isomers and optical isomers. The compounds described herein may exist in the form of stereoisomers, and therefore all possible stereoisomer forms, and any combination or any mixture thereof are encompassed. For example, a single enantiomer, a single diastereomer or a mixture of the above.

[0178] 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 a subject and an 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, flavoring agents, preservatives, taste masking agents, etc. For example, non-limiting examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, cross-linked 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. Reagents that maintain osmotic pressure include but are not limited to sugar, NaCl and the like. Reagents 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.

[0179] Beneficial Effects

[0180] The present invention provides for the first time a tumor immunotherapy targeting OTUB2, which helps improve the tumor microenvironment and enhance anti-tumor immunity by inhibiting OTUB2, thereby exerting a tumor therapeutic effect. In addition, based on the OTUB2 binding pocket structure, the present invention provides for the first time a specific small molecule inhibitor targeting OTUB2, which can inhibit tumor growth by enhancing anti-tumor immune response and reducing immunosuppression.

[0181] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present 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

[0182] Figure 1 :Effects of knocking down, knocking out or overexpressing OTUB2 on the growth and proliferation of tumor cells in vivo and in vitro. Among them:

[0183] Figure 1 A shows the in vitro growth and proliferation curves of MC38 cells with OTUB2 knockdown and control MC38 cells;

[0184] Figure 1 B shows the tumor growth curves of OTUB2 knockdown MC38 cells and control MC38 cells in C57BL / 6J immune-competent mice;

[0185] Figure 1 C shows the survival curves of mice transplanted with OTUB2 knockdown MC38 tumors and control MC38 tumors;

[0186] Figure 1 D shows that blocking CD8+ T cells in vivo reversed the growth inhibition of MC38 tumors with OTUB2 knockdown in vivo;

[0187] Figure 1 E shows the tumor growth curves of OTUB2-knockout MC38 cells and control MC38 cells in C57BL / 6J immune-competent mice;

[0188] Figure 1 F shows the tumor growth curves of OTUB2-knockout MC38 cells and control MC38 cells in NOD-SCID immunodeficient mice;

[0189] Figure 1 G shows the tumor growth curves of OTUB2 knockout LL / 2 cells and control LL / 2 cells in C57BL / 6J immune-competent mice;

[0190] Figure 1 H shows the tumor growth curves of OTUB2 knockout B16-F10 cells and control B16-F10 cells in C57BL / 6J immune-competent mice;

[0191] Figure 1 I shows the tumor growth curves of B16-F10 cells overexpressing OTUB2 and control B16-F10 cells in C57BL / 6J immunocompetent mice.

[0192] Figure 2 :The effect of targeting OTUB2 on tumor-specific T cells killing tumor cells. Among them:

[0193] Figure 2 A shows that compared with control LL / 2 cells, the killing activity of OVA-specific mouse CD8+T cells against OTUB2-knockout LL / 2 tumor cells was significantly increased;

[0194] Figure 2 B shows that compared with control B16-F10 cells, the killing activity of OVA-specific mouse CD8+T cells against OTUB2-knockout B16-F10 tumor cells was significantly increased;

[0195] Figure 2 C shows that the cytotoxic activity of human PBMCs against OTUB2 knockout NCI-H358 tumor cells was significantly enhanced compared with the control NCI-H358 cells.

[0196] Figure 3 :Identification of OTUB2 substrate proteins. Among them:

[0197] Figure 3 A shows the interaction between OTUB2 and substrate proteins in cells;

[0198] Figure 3 B shows that knocking out OTUB2 in B16-F10 cells reduces the expression level of substrate proteins;

[0199] Figure 3 C shows that knockdown of OTUB2 in NCI-H358 cells reduces the expression level of substrate proteins;

[0200] Figure 3 D shows that knockdown of OTUB2 in LoVo cells reduces the expression levels of substrate proteins.

[0201] Figure 4 :Anti-tumor immune effects of OTUB2-IN-1, a novel inhibitor targeting OTUB2. Among them:

[0202] Figure 4 A schematically shows the chemical name and formula of OTUB2-IN-1;

[0203] Figure 4 B schematically shows the action mode of OTUB2 and OTUB2-IN-1;

[0204] Figure 4 C shows the affinity between OTUB2-IN-1 and recombinant OTUB2 protein;

[0205] Figure 4 D shows Western Blot identification of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0206] Figure 4 E shows the response curve of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0207] Figure 4 F shows the reduction of substrate protein levels by OTUB2-IN-1 in LL / 2 cells;

[0208] Figure 4 G shows the reduction of substrate protein levels by OTUB2-IN-1 in B16F10 cells;

[0209] Figure 4 H shows the therapeutic effect of OTUB2-IN-1 in a mouse model of subcutaneous lung cancer (LL / 2);

[0210] Figure 4 I shows the safety evaluation of OTUB2-IN-1 in a subcutaneous tumor mouse model of murine lung cancer (LL / 2).

[0211] Figure 5 :The anti-tumor immune effect of OTUB2-IN-2, a novel inhibitor targeting OTUB2. Among them:

[0212] Figure 5 A schematically shows the chemical name and formula of OTUB2-IN-2;

[0213] Figure 5 B shows the affinity between OTUB2-IN-2 and recombinant OTUB2 protein;

[0214] Figure 5 C shows Western Blot identification of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0215] Figure 5 D shows the response curve of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0216] Figure 5 E shows the reduction of substrate protein levels by OTUB2-IN-2 in B16F10 cells.

[0217] Figure 6 :Anti-tumor immune effects of OTUB2-IN-3, a novel inhibitor targeting OTUB2. Among them:

[0218] Figure 6 A schematically shows the chemical name and formula of OTUB2-IN-3;

[0219] Figure 6 B shows the affinity between OTUB2-IN-3 and recombinant OTUB2 protein;

[0220] Figure 6 C shows Western Blot identification of OTUB2-IN-3 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0221] Figure 6 D shows the response curve of OTUB2-IN-3 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0222] Figure 7 :Anti-tumor immune effects of OTUB2-IN-4, a novel inhibitor targeting OTUB2. Among them:

[0223] Figure 7 A schematically shows the chemical name and formula of OTUB2-IN-4;

[0224] Figure 7 B shows the affinity between OTUB2-IN-4 and recombinant OTUB2 protein;

[0225] Figure 7 C shows Western Blot identification of OTUB2-IN-4 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0226] Figure 7 D shows the response curve of OTUB2-IN-4 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0227] Figure 8 :Anti-tumor immune effects of OTUB2-IN-5, a novel inhibitor targeting OTUB2. Among them:

[0228] Figure 8 A schematically shows the chemical name and formula of OTUB2-IN-5;

[0229] Figure 8 B shows the affinity between OTUB2-IN-5 and recombinant OTUB2 protein;

[0230] Figure 8 C shows the Western Blot identification of OTUB2-IN-5 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins;

[0231] Figure 8 D shows the response curve of OTUB2-IN-5 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate proteins.

[0232] Notes on sequence information

[0233] The information of the sequences involved in the present invention is provided in Table 1.

[0234] Table 1: Sequence information

[0235]

[0236] Example

[0237] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0238] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0239] Example 1. Knockdown or knockout of OTUB2 inhibits the growth of tumor cells

[0240] In this example, tumor cell lines with knockdown, knockout or overexpression of OTUB2 were prepared by lentiviral technology, and the growth and proliferation abilities of these cell lines in vivo and in vitro were evaluated.

[0241] 1.1 Construction of plasmids and cell lines

[0242] Knockdown: The shRNA nucleotide fragment targeting mouse OTUB2 (NCBI reference sequence number: NM_001177841.2, Gene ID: 68149) and the shRNA nucleotide fragment targeting an unrelated sequence (SEQ ID NO: 1 and SEQ ID NO: 2; synthesized by Sangon) were respectively connected into the pLKO.1 vector (Addgene, catalog number 10878) to construct shRNA plasmids that can knockdown mouse OTUB2, named pLKO.1-sh-OTUB2 and pLKO.1-sh-Scramble. The pLKO.1-sh-OTUB2 or pLKO.1-sh-Scramble plasmid and packaging plasmid psPAX2 (Addgene, Catalog No. 12260) and pMD2.G (Addgene, Catalog No. 12259) were co-transfected into 293T cells (purchased from ATCC, USA, Catalog No. CRL-3216) using lipofectamine 2000 transfection reagent (Invitrogen, Catalog No. 11668019). After 72 hours, the recombinant virus was used to infect MC38 cells to express the corresponding shRNA, and the MC38 cell line (sh-OTUB2) with stable knockdown of mouse OTUB2 and the control cell line (Scramble) were obtained by pressurization with a culture medium containing 1 μg / mL puromycin (InvivoGen, Catalog No. ant-pr-5).

[0243] The shRNA nucleotide fragments (SEQ ID NO: 3 and SEQ ID NO: 4; synthesized by Sangon) targeting human OTUB2 (NCBI reference sequence number: NM_023112.4, Gene ID: 78990) were connected to the pLKO.1 vector to construct shRNA plasmids that can knock down mouse OTUB2, named pLKO.1-shOTUB2#1 and pLKO.1-shOTUB2#2. The pLKO.1-shOTUB2#1 and pLKO.1-shOTUB2#2 plasmids and packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells using lipofectamine2000 transfection reagent. After 72 hours, the recombinant virus was used to infect NCI-H358 cells to express the corresponding shRNA, and the NCI-H358 cell lines (shOTUB2#1 and shOTUB2#2) with stable knockdown of human OTUB2 were obtained by pressurization with a medium containing 1 μg / mL puromycin.

[0244] Knockout: The sgRNA nucleotide fragment targeting mouse OTUB2 and the sgRNA nucleotide fragment targeting an unrelated sequence (SEQ ID NO: 5 and SEQ ID NO: 6; synthesized by Sangon) were respectively connected to the lentiCRISPR v2 vector (Addgene, Cat. No. 52961) to construct CRISPR plasmids capable of knocking out mouse OTUB2, named lentiCRISPR-sg-mOTUB2 and lentiCRISPR-sg-Scramble. The lentiCRISPR-sg-mOTUB2 or lentiCRISPR-sg-Scramble plasmids and packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells using lipofectamine 2000 transfection reagent. After 72 hours, the recombinant viruses were used to infect MC38, LL / 2 and B16-F10 cells to express the corresponding sgRNA, and the stable integrated tumor cell lines were obtained by pressurization with a medium containing 1 μg / mL puromycin. Single clones were obtained by limiting dilution. Finally, anti-OTUB2 antibody (Sangon Biotechnology Co., Ltd., catalog number D199590) was used to screen monoclonal tumor cell lines with complete knockout of OTUB2 for subsequent experiments. Finally, MC38, LL / 2 and B16-F10 cell lines with complete knockout of mouse OTUB2 (OTUB2 KO) and control cell lines (Control) were obtained.

[0245] Overexpression: The gene fragments expressing OTUB2 and PD-L1 (SEQ ID NO:7 and SEQ ID NO:8; synthesized by Sangon) were connected to the lentiCRISPR v2 vector (Addgene, Cat. No. 52961) to construct plasmids that can stably express the corresponding genes, named pLenti-OTUB2-Flag (with Flag tag) and pLenti-PD-L1. Lipofectamine2000 transfection reagent was used to co-transfect pLenti-OTUB2 or pLenti with packaging plasmids psPAX2 and pMD2.G into 293T cells. After 72 hours, the recombinant virus was used to infect B16-F10 cells to express the corresponding genes, and the B16-F10 cell line (OTUB2 OE) and the control cell line (Control) that stably expressed OTUB2 were obtained by pressurization with a culture medium containing 1 μg / mL puromycin.

[0246] Ovalbumin (OVA) was overexpressed in Control, LL / 2 and B16-F10 OTUB2 KO cells: The gene fragment expressing OVA (SEQ ID NO: 9; synthesized by Sangon) was connected to the lentiCas9-Blast vector (Addgene, Cat. No. 52962) to construct a plasmid that can stably express 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. After 72 hours, the recombinant virus was used to infect B16-F10 and LL / 2 cells to express the corresponding genes, and the cells were 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) and control cell lines (OTUB2 KO-Control) that stably expressed OVA.

[0247] 1.2 CCK-8 assay to detect the growth and proliferation ability of OTUB2 knockout tumor cells

[0248] MC38-Scramble and MC38-sh-OTUB2 cells were cultured at 2×10 3 The number of cells / well was plated on a 96-well plate. On days 0, 1, 2, and 3 after the cells were fully attached, 10 μl of CCK-8 cell proliferation detection reagent (MCE, catalog number HY-K-03001) was added to each well. After incubation at 37°C for 4 hours, the 96-well plate was placed in an ELISA reader (Thermo Fisher Scientific, model Multiskan Go 1510) at a wavelength of 450 nm for data reading, and a cell proliferation curve was plotted.

[0249] Figure 1 A The results showed that compared with the control group, knockdown of OTUB2 in MC38 had no effect on cell proliferation.

[0250] 1.3 Evaluation of tumor growth in OTUB2 knockdown MC38 cells and control MC38 cells in C57BL / 6J immune-competent mice estimate

[0251] Six-week-old C57BL / 6J mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were selected as experimental animals and inoculated subcutaneously with 100 μL of 5×10 5 MC38 cell suspension (sh-OTUB2 and Scramble), tumor size was monitored 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) 2The tumor volume was calculated by 1000 mm. 3 As an ethical endpoint, the survival time of mice was recorded.

[0252] Figure 1 B and Figure 1 The experimental results of C showed that compared with the control group, in the immune-competent mouse model, knocking down OTUB2 significantly inhibited the growth of MC38 tumors (P < 0.05) and significantly prolonged the survival of tumor-bearing mice (P = 0.0034).

[0253] 1.4 Tumor growth inhibition of MC38 with OTUB2 knockdown in C57BL / 6J immune-competent mice is dependent on CD8 + T cells

[0254] 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 once 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 the tumor was measured every two days, and the tumor volume was continuously measured until the 22nd day.

[0255] Figure 1 The experimental results of D showed that compared with the control group, in the immune-competent mouse model, knocking down OTUB2 significantly inhibited the growth of MC38 tumors (P=0.0059). When the function of CD8 cells was blocked, the inhibitory effect of knocking down OTUB2 on MC38 tumor growth disappeared, indicating that the tumor suppression effect of targeting OTUB2 depends on the immune system.

[0256] 1.5 In vitro culture of OTUB2-knockout MC38 cells and control MC38 cells in C57BL / 6J immune-competent mice and NOD-SCID immune-competent mice Assessment of tumor growth in immunodeficient mice

[0257] Six-week-old C57BL / 6J mice and NOD-SCID mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were selected as experimental animals and inoculated subcutaneously with 100 μL of 5×10 5 MC38 cell suspension (OTUB2 KO and Control), tumor size was monitored starting 8 days after tumor inoculation. Tumors were measured every two days and tumor volume was continuously measured until day 20 or day 28.

[0258] Figure 1 E and Figure 1The experimental results of F showed that compared with the control, knocking out OTUB2 significantly inhibited the growth of MC38 tumors in immune-competent mice (P < 0.0001), but could not inhibit the growth of MC38 tumors in immunodeficient mice, indicating that the anti-tumor immunomodulatory effect of OTUB2 depends on the immune system.

[0259] 1.6 Evaluation of the effect of OTUB2 knockout on tumor growth of LL / 2 cells and control LL / 2 cells in C57BL / 6J immune-competent mice estimate

[0260] 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), tumor size was monitored starting 8 days after tumor inoculation. Tumors were measured every two days and tumor volume was continuously measured until day 20.

[0261] Figure 1 The experimental results of G showed that compared with the control, knockout of OTUB2 significantly inhibited the growth of LL / 2 tumors in immune-competent mice (P < 0.0001).

[0262] 1.7 Tumors in OTUB2-knockout B16-F10 cells and control B16-F10 cells grown in C57BL / 6J immune-competent mice Growth assessment

[0263] Six-week-old C57BL / 6J mice were selected as experimental animals and subcutaneously inoculated with 100 μL of 3×10 5 B16-F10 cell suspension (OTUB2 KO and Control), tumor size was monitored starting 7 days after tumor inoculation. Tumors were measured every other day and tumor volume was continuously measured until day 17.

[0264] Figure 1 The experimental results of H showed that compared with the control, knockout of OTUB2 significantly inhibited the growth of B16-F10 tumors in immune-competent mice (P=0.0036).

[0265] 1.8 In vitro tumor growth of OTUB2-overexpressing B16-F10 cells and control B16-F10 cells in C57BL / 6J immune-competent mice Tumor growth assessment

[0266] Six-week-old C57BL / 6J mice were selected as experimental animals and subcutaneously inoculated with 100 μL of 3×10 5 B16-F10 cell suspension (OTUB2 OE and Control), tumor size was monitored starting 7 days after tumor inoculation. Tumors were measured every two days and tumor volume was continuously measured until day 16.

[0267] Figure 1 The experimental results of I showed that compared with the control, overexpression of OTUB2 significantly promoted the growth of B16-F10 tumors in immune-competent mice (P=0.0097).

[0268] The above results indicate that OTUB2 serves as a potential negative regulatory target of tumor immunity, promoting tumor immune escape.

[0269] Example 2. Tumor-targeted OTUB2 enhances CD8+ T cell killing of tumor cells

[0270] This example uses T cell killing experiments in vitro to verify that targeting OTUB2 in tumor cells can increase CD8 + T cell response to tumor cells.

[0271] 2.1 Experiment on the specific killing of tumor cells by tumor-specific T cells

[0272] OT-1 T cells isolated from the spleen of OT-1 transgenic mice (Jackson Laboratory, Cat. No. 003831) were cultured at 2×10 6 The cells were cultured at a density of 1 μg / ml in RPMI 1640 medium containing 1 μg / ml SIINFEKL peptide (Sino Biological) and 50 U / ml mouse recombinant IL-2 protein (Sino Biological, 51061-MNAE-20). After 7 days of activation, OT-1T 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. Finally, flow cytometry was used to detect the survival rate of tumor cells.

[0273] Figure 2 The experimental results of A-2B showed that compared with the control cells, OTUB2 knockout enhanced the killing ability of tumor-specific OT-1T cells against B16-F10 and LL / 2 tumor cells.

[0274] 2.2 Experiment on specific killing of tumor cells by tumor-specific T cells

[0275] 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 TMHuman T-Activator CD3 / CD28 (Thermo Fisher Scientific, Catalog No. 11131D) and 50U / ml human recombinant IL-2 protein (STEMCELL, Catalog No. 78036). After PBMCs were stimulated for 3 days, they were added to tumor cells (shOTUB2 and control Scramble cells) pretreated with 1ng / ml IFN-γ and co-cultured for 120 hours. The ratio of activated cells (effector cells) to tumor cells (target cells) was 5:1. Finally, the survival rate of tumor cells was detected by flow cytometry.

[0276] Figure 2 The experimental results of C showed that compared with the control cells, knockdown of OTUB2 in tumor cells increased the killing ability of PBMCs against tumor cells.

[0277] In conclusion, targeting OTUB2 enhances the ability of tumor-specific T cells to kill tumor cells, thereby inhibiting tumor growth.

[0278] Example 3. OTUB2-IN-1, a novel inhibitor targeting OTUB2, can promote anti-tumor immunity

[0279] In this example, novel and specific inhibitors targeting OTUB2 are developed and the inhibitory effects on tumor growth are evaluated in vivo.

[0280] 3.1 Identification of substrate proteins

[0281] OTUB2 is a deubiquitinase that binds to substrate proteins and removes their ubiquitination, that is, it interacts with substrate proteins to stabilize substrate proteins from being ubiquitinated and degraded. Therefore, the enzymatic activity of OTUB2 can be determined by verifying the ubiquitination and degradation of OTUB2 substrate proteins. In this experiment, a substrate protein of OTUB2 was identified, and the effect of OTUB2 inhibitors on OTUB2 enzyme activity was verified using this substrate protein as an example.

[0282] 3.1.1 Detection of the interaction between OTUB2 and substrate proteins by co-immunoprecipitation

[0283] The pLenti-OTUB2-Flag or pLenti and pLenti-PD-L1 plasmids were co-transfected into 293T cells using lipofectamine 2000 transfection reagent. Total protein samples were collected 72 hours later and M2Affinity Gel (Sigma-Aldrich, Catalog No. A2220) was used for co-immunoprecipitation experiment, and then the co-precipitated protein and total protein samples were detected by protein immunoblotting. Finally, PD-L1 and OTUB2 were detected with corresponding antibodies. The PD-L1 antibody was Abcam, ab213480; OTUB2 was detected using tag antibody anti-Flag (Sigma-Aldrich, F1804). Figure 3 The experimental results of A showed that compared with the control, capturing OTUB2 protein was able to detect PD-L1 protein, indicating that there was a direct interaction between OTUB2 and PD-L1.

[0284] 3.1.2 Knockout of OTUB2 in tumor cells can reduce substrate protein levels

[0285] OTUB2 knockout and control tumor cells in the logarithmic growth phase were cultured at 5×10 5 The cells were seeded at a density of 100 μg / well in a 6-well culture plate. 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 system, catalog number 285-IF). After 24 hours, the cells were collected and stained with APC-labeled anti-mouse PD-L1 antibody (BioLegend, catalog number 124312) and APC-labeled anti-human PD-L1 antibody (BioLegend, catalog number 329708). Finally, the expression level of PD-L1 protein was detected by flow cytometry. Figure 3 The experimental results of B-3D showed that compared with the control, knocking out OTUB2 in B16-F10 cells or knocking down OTUB2 in NCI-H358 and LoVo cells would reduce the expression level of PD-L1 in tumor cells.

[0286] The above results indicate that PD-L1 is a substrate protein of OTUB2 and can be used to detect the enzyme activity of OTUB2.

[0287] 3.2 OTUB2-IN-1 inhibitor can target the catalytic center of OTUB2

[0288] The recombinant OTUB2 protein was coupled to a CM5 chip (GE Healthcare, Catalog No. BR100530), and 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM OTUB2-IN-1 inhibitor (Lifechemical, Catalog No. F0444-0064) were used as mobile phases, respectively. The assay was performed using Biacore 8K and the experimental data were processed using Biacore InsightEvaluation Software.

[0289] Figure 4A shows that through virtual screening, we identified an OTUB2-IN-1 inhibitor that can specifically bind 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.

[0290] Figure 4 B shows a schematic diagram of the docking interaction between the OTUB2-IN-1 inhibitor and the amino acid residues in the catalytic pocket of OTUB2.

[0291] Figure 4 C shows that the interaction affinity between OTUB2-IN-1 inhibitor and OTUB2 was 12 μM as determined by SPR method.

[0292] 3.3 Effects of OTUB2-IN-1 inhibitors on substrate protein ubiquitination

[0293] 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, and the experimental data were processed using Image J software (NIH).

[0294] Figure 4 D and 4E show that as the concentration of OTUB2-IN-1 inhibitor was increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0295] 3.4 Effects of OTUB2-IN-1 inhibitors on substrate protein degradation in cells

[0296] LL / 2 and B16-F10 cells in the logarithmic growth phase were selected and cultured at 5 × 10 5 The cells were inoculated at a density of 10 cells / well in a 6-well culture plate. When the cell density reached 70%, the OTUB2-IN-1 inhibitor (0, 0.15, 0.6, 2.5, 10, 40 μM) was gradiently diluted with fresh culture medium and the cells were treated with drugs. After 24 hours of drug treatment, the corresponding samples were collected for protein immunoblotting. The expression of substrate proteins PD-L1 and OTUB2 was detected with specific antibodies.

[0297] Figure 4F and 4G show that, whether in LL / 2 cells or B16-F10 cells, as the concentration of OTUB2-IN-1 inhibitor increased, the content of substrate protein 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 the stability of OTUB2, thereby playing a role in degrading substrate protein.

[0298] 3.5 Antitumor effect of OTUB2-IN-1 inhibitor in tumor-bearing mice

[0299] Six-week-old C57BL / 6J mice were selected as experimental animals and inoculated subcutaneously with 100 μL of 5×10 5 LL / 2 cell suspension, wait until the tumor grows to 50mm 3 The OTUB2-IN-1 inhibitor and control drug were used for treatment once a day for a total of 6 times until the end of the experiment. After treatment, the tumor size was monitored and the tumor was measured every two days. The tumor volume was continuously measured with a tumor volume of 1000 mm 3 The end point of ethics.

[0300] Figure 4 H and 4I showed that compared with the control group, the OTUB2-IN-1 inhibitor could significantly inhibit tumor growth (P<0.001) without significant effect on the body weight of mice, indicating that OTUB2-IN-1 had a good anti-tumor effect in vivo and showed a certain degree of safety.

[0301] Example 4. Other novel inhibitors targeting OTUB2 can inhibit the activity of OTUB2

[0302] In this example, a series of novel and specific inhibitors targeting OTUB2 were developed, and the inhibitory effect of the inhibitors on the enzyme activity of OTUB2 was evaluated in vitro. The experimental method was the same as that of Example 3.

[0303] 4.1OTUB2-IN-2 can effectively inhibit the activity of OTUB2

[0304] Figure 5 A shows that through virtual screening, we identified the inhibitor OTUB2-IN-2, which can specifically bind 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}aceticacid (obtained from OTAVA chemicals, catalog number 1663785).

[0305] Figure 5 B shows that the interaction affinity between OTUB2-IN-2 inhibitor and OTUB2 was 41 μM as determined by SPR method.

[0306] Figure 5 C and Figure 5 D shows that as the concentration of OTUB2-IN-2 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0307] Figure 5 E shows that in B16-F10 cells, as the concentration of OTUB2-IN-2 inhibitor increased, the content of substrate protein decreased gradually, while the content of OTUB2 did not change significantly. This shows that the OTUB2-IN-2 inhibitor inhibits the function of OTUB2 by binding to OTUB2 without changing the stability of OTUB2, thereby playing a role in degrading substrate protein.

[0308] 4.2OTUB2-IN-3 can effectively inhibit the activity of OTUB2

[0309] Figure 6 A shows that through virtual screening, we identified the inhibitor OTUB2-IN-3 that can specifically bind 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 number F3224-0128).

[0310] Figure 6 B shows that the interaction affinity between OTUB2-IN-3 inhibitor and OTUB2 was 32.6 μM as determined by SPR method.

[0311] Figure 6 C and Figure 6 D shows that as the concentration of OTUB2-IN-3 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0312] 4.3OTUB2-IN-4 inhibitor can effectively inhibit the activity of OTUB2

[0313] Figure 7A shows that through virtual screening, we identified the inhibitor OTUB2-IN-4 that can specifically bind 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 OTAVAchemicals, catalog number 4349982).

[0314] Figure 7 B shows that the interaction affinity between OTUB2-IN-4 inhibitor and OTUB2 was 25.9 μM as determined by SPR method.

[0315] Figure 7 C and Figure 7 D shows that as the concentration of OTUB2-IN-4 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0316] 4.4OTUB2-IN-5 inhibitor can effectively inhibit the activity of OTUB2

[0317] Figure 8 A shows that through virtual screening, we identified the inhibitor OTUB2-IN-5, which can specifically bind 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 OTAVAchemicals, catalog number 4349908).

[0318] Figure 8 B shows that the interaction affinity between OTUB2-IN-5 inhibitor and OTUB2 was 3.91 μM as determined by SPR method.

[0319] Figure 8 C and Figure 8 D shows that as the concentration of OTUB2-IN-4 inhibitor increased, the level of ubiquitinated substrate protein increased significantly, indicating that the activity of OTUB2 enzyme was significantly inhibited.

[0320] Above Figure 5-Figure 8The results showed that these small molecule compounds can act as OTUB inhibitors to regulate tumor immunity.

[0321] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached 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) detecting the deubiquitinase activity of the OTUB2 protein on the substrate protein in the presence of the test agent obtained in step (i); (iii) comparing the result of step (ii) with the activity measured in the absence of the test agent.

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