CN113134090A - Anti-tumor medicine composition - Google Patents

Abstract

The invention discloses a pharmaceutical composition consisting of a deubiquitinase USP8 inhibitor and a PD-1/PD-L1 antibody. The invention discloses that deubiquitinase USP8 can regulate the abundance of PD-L1 protein in various tumor cells, and the expression of PD-L1 can be obviously improved by using an inhibitor of USP8 or knocking out USP8 in the tumor cells, and the expression of an antigen presenting gene is concomitantly regulated up. In a mouse tumor model, the combined use can significantly inhibit tumor growth and improve survival of mice compared to the use of either the USP8 inhibitor or the PD-1/PD-L1 antibody alone. Therefore, the invention not only discloses a molecular mechanism of USP8 for regulating the expression of PD-L1 and antigen presenting genes, but also provides that the combination of a USP8 inhibitor and a PD-1/PD-L1 antibody can obviously improve the clinical curative effect on tumor patients.

Description

Anti-tumor medicine composition

Technical Field

The invention belongs to the field of tumor treatment and pharmacy, and relates to an anti-tumor pharmaceutical composition and application thereof.

Background

Tumor immunotherapy, particularly immunotherapy targeting programmed death 1/programmed death ligand 1(PD-1/PD-L1), has achieved excellent therapeutic efficacy in some cancer types of patients. PD-1 (also known as CD279) is a cell surface co-inhibitory receptor that is expressed predominantly on activated T cells to regulate T cell apoptosis, exhaustion and tolerance^1-3. PD-L1 (also known as B7-H1 or CD274), often expressed on tumor cells and other immune cells in the tumor microenvironment, has been identified as a ligand for PD-1⁴. When PD-L1 on tumor cells or other immune cells in a tumor microenvironment is combined with PD-1 on T cells to cause T cell dysfunction and failure, the blocking of PD-1/PD-L1 interaction by using PD-1 or PD-L1 antibody can restore T cell function to eliminate cancer cells, thereby achieving the effect of treating cancer^5,6. Meanwhile, in clinical experiments, the expression level of PD-L1 in a tumor microenvironment is found to be positively correlated with the response rate of PD-L1 treatment⁷. Although anti-PD-1/PD-L1 immunotherapy alone has achieved good therapeutic results in some cancer types, only about 25% of tumor patients overall respond to treatment with the PD-1/PD-L1 antibody. Thus, the low response rate limits the use of anti-PD-1/PD-L1 therapy alone in most cancer patients.

Ubiquitination is an important protein posttranslational modification in cells, and regulates various important signal paths in cells mainly by regulating the stability, transportation, positioning of proteins, interaction among proteins and the like, thereby influencing the occurrence and development processes of diseases including tumors^8-10. One ubiquitin molecule has seven lysine (K) residues (K6, K11, K27, K29, K33, K48 and K63), and can be identified by covalently linking the C-terminal glycine of a second ubiquitin molecule to any one of the seven lysine residues on a first ubiquitin molecule or the amino-terminal methionine of a first ubiquitin moleculeAcid (Met1) to form eight different ubiquitin chains, and the different ubiquitin chains perform different functions in the cell^11,12. It is well known that K48 or K11 linked ubiquitin chains can trigger 26S proteasome mediated proteolysis as a degradation signal^13,14While K63 or Met1 linked ubiquitin chains play a key role in NF- κ B activation and immune response^15,16。

In contrast to the ubiquitin E3 ligase action of adding ubiquitin chain to target protein, the deubiquitinating protease can remove ubiquitin chain on substrate protein^17,18. In mammals, nearly 100 deubiquitinating proteases have been found, the ubiquitin-specific protease (USP) being the largest subfamily of deubiquitinating proteins, with about 54 members^18,19. USP8 (also known as UBPY) is one of the members of the ubiquitin-specific protease subfamily, and plays an important role in controlling endocytosis and protein trafficking by modulating intracellular protein stability and Endosomal Sorting Complex (ESCRT) required for trafficking, largely by its deubiquitinating enzyme activity^20,21. Moreover, previous studies have also shown that USP8 is frequently overexpressed in human cancers, and cancer patients with high USP8 expression show poor overall survival^22-24. Furthermore, the acquired functional mutant of USP8, i.e., the enhancement of deubiquitinating enzyme activity of USP8 after mutation, has been found in about 50% of Cushing's disease, which is mainly caused by pituitary adenomas secreting adrenocorticotropic hormone 25,²⁶. Therefore, inhibition of activity of USP8 may have a very good therapeutic effect on pituitary tumors induced by adrenocorticotropic hormone induced by USP8 mutation. In addition, previous studies found that USP8 is also involved in immune regulation in the body, and that T cell-specific knockout of USP8 was able to disrupt the activity of regulatory T cells (tregs), thereby recruiting large amounts of CD8 in the colon⁺T cells causing inflammatory bowel disease in mice²¹。

Taken together, these studies indicate that USP8 may be promoting tumorigenesis and inhibiting CD8⁺The key role played by the T cell function, revealing that USP8 may be a potential target in the treatment of human cancer. However, so far, it has not beenWhether USP8 is involved in tumor immunity and plays a role in tumor immunotherapy has not been reported. Our studies revealed that deubiquitinase USP8 is able to modulate the abundance of the tumor immune checkpoint PD-L1 protein in a variety of tumor cells. Moreover, knockout of USP8 in tumor cells using inhibitors of USP8 or CRISPR technology can significantly increase the expression of PD-L1, and is accompanied by a significant up-regulation of the expression of antigen presenting genes. IN a mouse tumor model, the combination significantly inhibited tumor growth and increased overall survival IN mice compared to the USP8 inhibitor DUBs-IN-2 alone or anti-PD-1 or anti-PD-L1 antibodies. Therefore, our studies not only revealed the molecular mechanism of USP8 in modulating PD-L1 and expression of antigen presenting genes, but also suggested that the combined use of a USP8 inhibitor and PD-1/PD-L1 antibody is likely to significantly improve the therapeutic effect on tumor patients clinically.

Reference to the literature

1.Chen,L.&Han,X.Anti-PD-1/PD-L1 therapy of human cancer:past,present,and future.J Clin Invest 125,3384-3391(2015).

2.Ishida,Y.,Agata,Y.,Shibahara,K.&Honjo,T.Induced expression of PD-1,a novel member of the immunoglobulin gene superfamily,upon programmed cell death.EMBO J 11,3887-3895(1992).

3.Keir,M.E.,Butte,M.J.,Freeman,G.J.&Sharpe,A.H.PD-1and its ligands in tolerance and immunity.Annu Rev Immunol 26,677-704(2008).

4.Freeman,G.J.et al.Engagement of the PD-1immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med 192,1027-1034(2000).

5.Hirano,F.et al.Blockade of B7-H1 and PD-1 by monoclonal antibodiespotentiates cancer therapeutic immunity.Cancer Res 65,1089-1096(2005).

6.Zou,W.,Wolchok,J.D.&Chen,L.PD-L1(B7-H1)and PD-1 pathway blockadefor cancer therapy:Mechanisms,response biomarkers,and combinations.Sci Transl Med 8,328rv324(2016).

7.Zhang,J.,Dang,F.,Ren,J.&Wei,W.Biochemical Aspects of PD-L1Regulation in Cancer Immunotherapy.Trends Biochem Sci 43,1014-1032(2018).

8.Komander,D.&Rape,M.The ubiquitin code.Annu Rev Biochem81,203-229(2012).

9.Martinez-Forero,I.,Rouzaut,A.,Palazon,A.,Dubrot,J.&Melero,I.Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation.Clin Cancer Res 15,6751-6757(2009).

10.Walsh,G.&Jefferis,R.Post-translational modifications in the context of therapeutic proteins.Nat Biotechnol 24,1241-1252(2006).

11.Akutsu,M.,Dikic,I.&Bremm,A.Ubiquitin chain diversity at a glance.J Cell Sci 129,875-880(2016).

12.Grabbe,C.,Husnjak,K.&Dikic,I.The spatial and temporal organization of ubiquitin networks.Nat Rev Mol Cell Biol 12,295-307(2011).

13.Ravid,T.&Hochstrasser,M.Diversity of degradation signals in the ubiquitin-proteasome system.Nat Rev Mol Cell Biol 9,679-690(2008).

14.Zhang,J.,Wan,L.,Dai,X.,Sun,Y.&Wei,W.Functional characterization of Anaphase Promoting Complex/Cyclosome(APC/C)E3 ubiquitin ligases in tumorigenesis.Biochim Biophys Acta 1845,277-293(2014).

15.Chen,Z.J.Ubiquitination in signaling to and activation of IKK.Immunol Rev 246,95-106(2012).

16.Spit,M.,Rieser,E.&Walczak,H.Linear ubiquitination at a glance.J Cell Sci 132(2019).

17.Clague,M.J.,Urbe,S.&Komander,D.Publisher Correction:Breaking the chains:deubiquitylating enzyme specificity begets function.Nat Rev Mol Cell Biol 20,321(2019).

18.Harrigan,J.A.,Jacq,X.,Martin,N.M.&Jackson,S.P.Deubiquitylating enzymes and drug discovery:emerging opportunities.Nat Rev Drug Discov17,57-78(2018).

19.Cheng,J.et al.Functional analysis of deubiquitylating enzymes in tumorigenesis and development.Biochim Biophys Acta Rev Cancer 1872,188312(2019).

20.Crespo-Yanez,X.et al.CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis.PLoS Genet 14,e1007456(2018).

21.Dufner,A.et al.The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells.Nat Immunol 16,950-960(2015).

22.Byun,S.et al.USP8 is a novel target for overcoming gefitinib resistance in lung cancer.Clin Cancer Res 19,3894-3904(2013).

23.Shin,S.et al.Deubiquitylation and stabilization of Notch1 intracellular domain by ubiquitin-specific protease 8enhance tumorigenesis in breast cancer.Cell Death Differ 27,1341-1354(2020).

24.Jeong,M.et al.USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability.Oncogene 36,458-470(2017).

25.Reincke,M.et al.Mutations in the deubiquitinase gene USP8 cause Cushing's disease.Nat Genet 47,31-38(2015).

26.Ma,Z.Y.et al.Recurrent gain-of-function USP8 mutations in Cushing's disease.Cell Res 25,306-317(2015).

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for treating tumors, which improves the curative effect of immunotherapy on tumor patients by combining a deubiquitinase USP8 inhibitor and an anti-PD-1/PD-L1 antibody.

The invention is realized on the basis of the following research findings:

1. it was found that the up-regulation of the expression of the immune checkpoint protein PD-L1 following inhibition or knock-out of USP8 in a variety of tumor cells including lung cancer (H460 and PC9), osteosarcoma (U2OS) and colon cancer (CT26 and MC38)

1) Following treatment with the small molecule inhibitor DUBs-IN-2 of USP8 IN tumor cell lines: the expression of PD-L1 was detected to be significantly upregulated by the Western blot method.

2) Upon removal of USP8 by CRISPR Knock Out (KO) in tumor cell lines: a significant upregulation in PD-L1 expression was detected by Western blot.

2. Found that after USP8 is inhibited or knocked out in tumor cells, the antigen presentation related factor involved in anti-tumor immunity is remarkably up-regulated

1) Following treatment with the small molecule inhibitor DUBs-IN-2 of USP8 IN tumor cell lines: significant up-regulation of antigen presentation-related factors was detected by fluorescent quantitative pcr (qpcr).

2) Upon knockout of USP8 in tumor cell lines: the significant up-regulation of the antigen presentation related factor was detected by qPCR.

Based on these studies of molecular mechanisms, we speculate that the combination of an inhibitor of USP8 and an anti-PD-1/PD-L1 antibody may improve the efficacy of tumor therapy. To validate this hypothesis, we further performed preclinical experiments on mouse tumor models.

3. It was found that IN animal models of transplantable tumors with normal immune systems, the combination significantly inhibited tumor growth and prolonged survival IN mice compared to the use of the USP8 inhibitor DUBs-IN-2 or anti-PD-1/PD-L1 antibody alone.

The inhibitors of deubiquitinase USP8 include, but are not limited to, DUBs-IN-2.

When the deubiquitinase USP8 inhibitor is DUBs-IN-2, the weight ratio of the inhibitor to PD-1/PD-L1 antibody is 1: 1-5.

The invention has the following advantages:

1) it is found for the first time that inhibition of USP8 by a small molecule inhibitor or gene knockout method can significantly up-regulate the expression of PD-L1.

2) It was first found that inhibition of USP8 by small molecule inhibitors or gene knock-out methods can significantly up-regulate the level of antigen presentation, a key molecule in anti-tumor immunity.

3) It was first discovered that when a USP8 inhibitor is combined with an anti-PD-1/PD-L1 antibody, it can significantly inhibit tumor growth and simultaneously improve survival of mice.

Drawings

FIG. 1: inhibition of USP8 with DUBs-IN-2 was able to upregulate PD-L1 expression IN a variety of tumor cell lines (lung cancer cell lines H460 and PC 9; osteosarcoma cell line U2 OS; colon cancer cell lines CT26 and MC 38).

FIG. 2: knockout of Usp8 in the mouse colon cancer CT26 cell line was able to significantly up-regulate the expression of PD-L1.

FIG. 3: inhibition of USP8 with DUBs-IN-2 was able to significantly up-regulate the levels of factors associated with antigen presentation IN lung cancer cell lines H460 and PC 9.

FIG. 4: the expression level of the antigen presentation related factor was significantly up-regulated after knockout of Usp8 in the mouse colon cancer cell line CT 26.

FIG. 5: knockout of USP8 in the lung cancer cell line PC9 significantly upregulated the levels of PD-L1 and antigen presentation related factors.

FIG. 6: IN an immune normal C57BL/6 mouse transplantation tumor model, the USP8 inhibitor DUBs-IN-2 and PD-1/PD-L1 antibodies combined significantly inhibited mouse tumor growth.

FIG. 7: IN the case of the immune normal C57BL/6 IN mouse transplantable tumors, the survival time of mice is significantly improved when DUBs-IN-2 is combined with PD-1/PD-L1 antibody.

The specific implementation mode is as follows:

the present invention will be described in detail below with reference to specific examples.

Test materials:

h460 cells, PC9 cells, U2OS cells, CT26 cells, MC38 cells: h460 and PC9 cells were given to professor Schuxus Schwarzschia of chemoradiotherapy department in the Central & south Hospital, Wuhan university, and U20S, CT26 and MC38 cells were purchased from ATCC company;

c57BL/6 female: purchased from college drug recovery corporation;

DUBs-IN-2: purchased from MedChemExpress;

PD-L1 antibody (for Western blot): available from Cell Signaling Technology, inc;

PD-L1 antibody (for mouse tumor therapy): purchased from Bio X Cell Co.

Example 1 inhibition of USP8 using USP8 inhibitor DUBs-IN-2 or a gene knockout approach significantly upregulated the levels of PD-L1 and antigen presentation related factors IN tumor cells.

1) By treating various tumor cells with different concentrations of the USP8 inhibitor DUBs-IN-2, the expression level of PD-L1 was found to be up-regulated with drug concentration gradients.

Human lung cancer cell lines H460 and PC9 and human osteosarcoma cell line U2OS were seeded IN 6-well plates, and when the cell density was about 70-80%, DUBs-IN-2 (0. mu.M, 2. mu.M, 4. mu.M) was added to the culture medium of H460 and PC9, and the drug concentrations IN U2OS cell line were 0. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, and the drug treatment time was 6 hours for H460 cells, 24 hours for PC9 cells, and 24 hours for U2OS cells, respectively. After drug treatment, cells were harvested and a significant increase in PD-L1 levels was detected by Western blot (fig. 1). Mouse colon cancer cell lines CT26 and MC38 were treated IN the same manner with DUBs-IN-2 administered at concentrations of 0. mu.M, 4. mu.M, and 6. mu.M, and cells were harvested after 24 hours of treatment, and the level of PD-L1 was significantly up-regulated as measured by Western blot assay (FIG. 1). PC9 and H460 cells were lysed simultaneously by TRIzol, PC9 cells were treated with DUB-IN-2 at concentrations of O μ M and 2 μ M for 24 hours, H460 cells were treated with 0 μ M and 1 μ M for 24 hours, intracellular RNA was extracted, after reverse transcription of the RNA, significant upregulation of anti-tumor associated antigen presentation related molecules was observed by fluorescent quantitative PCR (qPCR) detection (FIG. 3).

2) Changes in PD-L1 expression levels were verified by knocking out the Usp8 gene in the CT26 cell line.

Plasmid pX458-sgUSP8 is transiently transfected in CT26 cells of a colon cancer cell line of a mouse, single cells are sorted into a 96-well plate by a flow sorter 48 hours after transfection, after 2 weeks, after the single cells grow clone colonies in the 96-well plate, the cell clones are expanded and cultured, and the knockout effect is verified by Western blot. As a result, it was found that the expression level of PD-L1 was significantly up-regulated in Usp8 knock-out cells (FIG. 2).

Meanwhile, TRIzol is used for cracking Usp8 knockout CT26 cells, intracellular RNA is extracted, after the RNA is subjected to reverse transcription, a qPCR detection method is used, and the anti-tumor related antigen presentation related molecules are found to be obviously up-regulated (figure 4).

In the human lung cancer cell line PC9, after USP8 is knocked out by CRISPR knock-out (KO), PD-L1 can be detected to be obviously up-regulated by Western blot (shown in a lower part of a figure 5), meanwhile, after RNA is extracted by a TRIzo method and is reversely transcribed to obtain DNA, the anti-tumor related antigen presenting factor is detected to be obviously up-regulated by qPCR (shown in a figure 5).

Example 2 IN an immune normal mouse graft tumor, the USP8 inhibitor DUBs-IN-2, IN combination with the PD-1/PD-L1 antibody, significantly inhibited tumor growth and improved mouse survival.

Normal C57BL/6 female mice immunized at 6 weeks of age were injected subcutaneously with 2X 10⁵MC38 cells. Firstly, cells are cultured in vitro, when the cell density reaches 70-80%, the cells are digested by pancreatin, after the pancreatin is removed by centrifugation, the cells are resuspended by PBS, then the cell suspension is subjected to cell counting, the cell suspension is diluted by PBS according to the number of the injected cells and is respectively packed in an EP tube, and finally the cell suspension is placed on ice for standby. The mice were randomly and evenly divided into 4 groups of 8 mice each, four groups were: 1) control treatment group; 2) DUBs-IN-2 group; 3) PD-L1 antibody panel; 4) PD-L1 antibody and DUBs-IN-2 combination. The administration by intraperitoneal injection was started 7 days after tumor implantation, the PD-L1 antibody was administered at 5 μ g/g mouse, the DUBs-IN-2 was administered at 3 μ g/g mouse, the mice were injected with drug every 3 days, the tumor size of the mice was measured at the beginning of the drug treatment, twice weekly, and the calculation formula for the tumor size of the mice: length x width²X 0.5, and the number of growing days was counted for each group of mice. According to the ethics of the animal experiments and the welfare of the experimental animals, the observation is terminated when the tumor volume of the mice is more than 2000 cubic millimeters or the tumor ulcer diameter approaches 1cm, and the mice are immediately sacrificed and the death is defaulted. Wherein, the date when the average value of the tumor size of the control group mice reached 2000 cubic millimeters is taken as an ending point, the day is 16 days after the injection of the tumor cells, and the average values of the tumor sizes of the other three groups of mice are respectively calculated. Finally, to facilitate comparison of the differences in mean tumor size of four groups of mice at the same time, we calculated and analyzed the mean tumor size of each group of mice at the four time points, day 7, day 10, day 14 and day 16, for statistical differences between groups. As can be seen from the results of the experiments, the combination of the PD-L1 antibody and the USP8 inhibitor DUBs-IN-2 showed thatTumor growth was significantly inhibited (FIG. 6) and survival time was significantly increased in mice (FIG. 7).

Claims (6)

1. A pharmaceutical composition comprises a deubiquitinase USP8 inhibitor and a PD-1/PD-L1 antibody.

2. The pharmaceutical composition of claim 1, wherein: the inhibitor of deubiquitinase USP8 is DUBs-IN-2.

3. The pharmaceutical composition of claim 2, wherein: the weight ratio of the deubiquitinase USP8 inhibitor to the PD-1/PD-L1 antibody is 1: 1-5.

4. Use of the pharmaceutical composition of any one of claims 1-3 for the preparation of an anti-tumor medicament.

5. An antitumor agent whose active ingredient is the pharmaceutical composition according to any one of claims 1 to 3.

6. The antitumor agent as claimed in claim 5, which is an injectable drug.

Images