Application of combination of anti-CD73 monoclonal antibody and Adora1 inhibitor in treatment of intestinal fibrosis

Abstract

The invention relates to an application of an anti-CD73 monoclonal antibody combined with an Adora1 inhibitor in treatment of intestinal fibrosis. In the invention, the inventor finds that long-term WD generates GSDME-mediated pyroptosis by inducing intestinal epithelial cells and releases a large amount of intracellular molecules HMGB1 and ATP, the HMGB1 promotes CD39 + CD73 + CD8 + T cell amplification, the group of cells catalyzes extracellular ATP into Ado, and finally the Ado activates intestinal fibroblasts through a receptor Adara1 so as to participate in a complex intestinal fibrosis process. Based on this, the inventor finds that the CD-related intestinal fibrosis can be effectively improved by blocking the generation of intestinal adenosine or / and inhibiting the receptor of the intestinal adenosine, and a combined medicine, namely the anti-CD73 monoclonal antibody combined Adara1 inhibitor, is researched and developed, can be used for treating the CD-related intestinal fibrosis, and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically relates to the application of an anti-CD73 monoclonal antibody combined with an Adora1 inhibitor in the treatment of intestinal fibrosis. Background Art

[0002] Intestinal fibrosis is a chronic intestinal disease, characterized by the replacement of muscle cells in the intestinal wall by excessive fibrous tissue, resulting in slowed intestinal peristalsis, which may cause symptoms such as constipation, abdominal discomfort, and digestive problems.

[0003] Crohn's disease (CD) is an inflammatory bowel disease with unknown etiology. In the early stage, it is mainly manifested as inflammatory reactions and ulcers. With repeated disease activity, it gradually develops into a series of stenotic and penetrating lesions, including intestinal strictures and enterocutaneous fistulas. The essence of these lesions is intestinal fibrosis characterized by excessive deposition of extracellular matrix rich in collagen in the intestinal wall. Currently, the generally accepted view is that intestinal fibrosis is closely related to the abnormal activation of fibroblasts and the subsequent excessive deposition of collagen-rich extracellular matrix. Although the application of biological agents such as anti-TNF-α monoclonal antibodies and IL-12 / 23 inhibitors has significantly improved the enteritis and mucosal healing rate of CD patients, these drugs have little reversing effect on the already formed intestinal fibrosis lesions. Clinical studies have shown that up to 80% of CD patients are ultimately forced to undergo surgery due to intestinal strictures, but 70% of the patients still have restenosis after surgery. The overall prognosis of CD patients with intestinal fibrosis lesions is extremely poor, and intestinal fibrosis has become one of the greatest challenges in the prevention and treatment of CD. However, the current understanding of the pathogenesis of CD-related intestinal fibrosis is still unclear, making it difficult to find effective prevention and treatment drugs.

[0004] The Western diet (WD) is a diet rich in sugar and fat but low in fiber content. It has long been considered a risk factor for diseases such as atherosclerosis, diabetes, and CD. Some studies have reflected the fact that environmental factors such as WD have promoted the huge increase in the incidence of CD in developed countries. There are also studies showing that in underdeveloped countries where the incidence of CD was previously low, the incidence of this disease is increasing significantly, and this increase is parallel to the increasing popularity of WD in these countries. Since WD is a risk factor for CD, does it participate in the process of CD-related intestinal fibrosis? If it does, what is the internal mechanism of this CD-related intestinal fibrosis process? Can it provide a direction for the prevention and treatment of intestinal fibrosis? These are currently not clear. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide the application of an anti-CD73 monoclonal antibody combined with an Adora1 inhibitor in the treatment of intestinal fibrosis through the study of the factors and related molecular mechanisms inducing CD-related intestinal fibrosis.

[0006] The realization of the above object includes the following technical solutions.

[0007] In the first aspect of the present invention, there is provided an application of an anti-CD73 monoclonal antibody combined with an Adora1 inhibitor in the preparation of a drug for preventing and / or treating intestinal fibrosis.

[0008] The present invention provides an application of an anti-CD73 monoclonal antibody combined with an Adora1 inhibitor in the preparation of a drug for preventing and / or treating intestinal fibrosis or intestinal fibrosis-related diseases.

[0009] In some embodiments, the Adora1 inhibitor is Derenofylline.

[0010] In some embodiments, the intestinal fibrosis-related disease is Crohn's disease (CD)-associated intestinal fibrosis.

[0011] In some embodiments, the application includes blocking intestinal adenosine production and / or inhibiting intestinal adenosine receptors.

[0012] In the second aspect of the present invention, there is provided a drug for preventing and / or treating intestinal fibrosis, the drug comprising an anti-CD73 monoclonal antibody and an Adora1 inhibitor, the anti-CD73 monoclonal antibody and the Adora1 inhibitor being separate administration units or an administration unit formed jointly by the anti-CD73 monoclonal antibody and the Adora1 inhibitor.

[0013] In some embodiments, the drug is formulated into an injection.

[0014] In some embodiments, the anti-CD73 monoclonal antibody and the Adora1 inhibitor in the drug are respectively injection preparations.

[0015] In the first aspect of the present invention, there is provided an application of an anti-CD73 monoclonal antibody in the preparation of a drug for preventing and / or treating intestinal fibrosis or intestinal fibrosis-related diseases.

[0016] In some embodiments, the intestinal fibrosis-related disease is Crohn's disease (CD)-associated intestinal fibrosis.

[0017] In the present invention, the inventors found that long-term WD induces GSDME-mediated pyroptosis in intestinal epithelial cells and releases a large amount of intracellular molecules HMGB1 and ATP, wherein HMGB1 promotes CD39 + CD73 + CD8 +T cell expansion. This group of cells catalyzes extracellular ATP into Ado, and finally Ado activates intestinal fibroblasts through the receptor Adora1, thereby participating in the complex process of intestinal fibrosis. Based on this, the inventors found that blocking intestinal adenosine production and / or inhibiting its receptor can effectively improve CD-related intestinal fibrosis, and developed a combination drug, namely anti-CD73 monoclonal antibody combined with Adora1 inhibitor, which can be used to treat CD-related intestinal fibrosis and has important clinical application value. Description of the Drawings

[0018] Figure 1 Shows the effects of long-term regular diet (RD) and western diet (WD) on the development of enteritis and intestinal fibrosis in mice. Among them, (A-K) Wild-type mice from the same litter (6 mice per group) were fed with regular diet (RD) and western diet (WD) for 8 weeks, 12 weeks, and 16 weeks respectively. (B) Changes in mouse body weight. (C, F, I) On the left are representative colon pictures, and on the right is the colon length. (D, G, J) Representative pictures of H&E staining of colon sections (scale bars: 50μm) and histological scores of colon tissues. (E, H, K) Representative pictures of Masson staining of colon tissues (scale bars: 50μm) and quantitative scores of collagen area. Data are expressed as mean ± standard error; ns: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0019] Figure 2 Shows that western diet exacerbates intestinal fibrosis in TNBS-induced mouse models. Among them, (A-F) Wild-type mice from the same litter (8 mice per group) were fed with regular diet (RD) and western diet (WD) for 12 weeks. The CD model of mice was induced by 2.5% TNBS according to the reference method, that is, TNBS pre-sensitization was performed in the 6th week, and TNBS enema was performed once a week from the 7th week, while the control group was enema with PBS. (B) Representative colon picture; (C) Colon length, (D) Representative pictures of H&E staining and Masson staining of colon sections (scale bars: 50μm), (E) Histological score of colon tissue, (F) Quantitative score of collagen area of colon tissue. Data are expressed as mean ± standard error. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0020] Figure 3 Shows that knockout of GSDME in intestinal epithelial cells improves intestinal fibrosis in WD+TNBS mice. Among them, (A-E) GSDME from the same litter ΔIEC and GSDME fl / flMice were fed a Western diet (WD) for 12 weeks (8 mice per group), pre-sensitized with TNBS at week 6, and induced with TNBS enema once a week from week 7 to establish a CD model in mice. (B) Left: Representative pictures of the colon, Right: Colon length. (C) Representative pictures of H&E staining of colon sections (scale bars: 50 μm) and histological scores of the colon. (D) Representative pictures of Masson staining of the colon (scale bars: 50 μm) and quantitative scores of collagen area. (E) WB images of full-length and cleaved GSDME in colon tissues. Data are expressed as mean ± SEM. ns: No significant difference; *P < 0.05; ****P < 0.0001.

[0021] Figure 4 Is the Western diet significantly increased Effector CD8 in the intestine + T cells and HMGB1 in IECs. Among them, (A) UMAP dimensionality reduction analysis map and proportion of various intestinal cells. (B) Effector CD8 + T cell subset characterization. (C) Average expression of marker genes enriched in major cell populations. (D-G) Cell-chat analysis of signals from other cells to Effector CD8 + T cells, where D and F are the number and weight analyses of the RD group, respectively, and E and G are the number and weight analyses of the WD group, respectively. (H) Differences in the expression of DAMPs in intestinal epithelial cells between the two groups. (I) Differences in the expression of AR in intestinal fibroblasts between the two groups. Data are expressed as mean ± SEM. ns: No significant difference; ****P < 0.0001.

[0022] Figure 5 Is the knockout of HMGB1 in IECs reduced CD8 in the intestine + CD73 + T cells and Ado levels. Among them, (A) Representative pictures of immunohistochemical staining of tissue sections (scale bars: 20 μm). (B) Double-staining cell count of CD8 and CD73. (C) Ado concentration in tissues. Data are expressed as mean ± SEM. *P < 0.05; ***P < 0.001.

[0023] Figure 6 Is the knockout of HMGB1 in intestinal epithelial cells improved intestinal fibrosis in WD + TNBS mice. Among them, (A-D) HMGB1 of the same litter origin ΔIEC and HMGB1 fl / flMice were fed a Western diet (WD) for 12 weeks (8 mice per group), pre-sensitized with TNBS at week 6, and induced with a CD model in mice by TNBS enema once a week starting from week 7. (B) Left: Representative pictures of the colon; Right: Colon length. (C) Representative pictures of H&E staining of colon sections (scale bars: 50 μm) and histological scores of the colon. (D) Representative pictures of Masson staining of the colon (scale bars: 50 μm) and quantitative scores of the collagen area. Data are expressed as mean ± SEM. *P<0.05; ****P<0.0001.

[0024] Figure 7 It is the Adora1 inhibitor rather than the Adora2b inhibitor that blocks adenosine from activating intestinal fibroblasts. Human colonic fibroblasts CCD-18Co were cultured in vitro and stimulated with 50 μM Adora1 inhibitor Derenofylline and 5 μM Adora2b inhibitor MRS1754 respectively. After 1 hour, 50 μM Ado was added, and the cells were harvested after 48 hours. The mRNA levels of α-SMA, TGF-β, Vimentin, Col1a1, Col1a2, Col3a1, Fibronectin and FAP genes were detected by qRT-PCR. Data are expressed as mean ± SEM. **P<0.01; ***P<0.001; ****P<0.0001.

[0025] Figure 8 Supplementation with an adenosine A1 receptor inhibitor improves intestinal fibrosis in WD+TNBS mice. Among them, (A-E) Wild-type mice of the same litter origin were fed a Western diet (WD) for 12 weeks (8 mice per group), pre-sensitized with TNBS at week 6, and induced with a CD model in mice by TNBS enema once a week starting from week 7. During this period, 2 mg / kg of Derenofylline was injected intraperitoneally once every 48 hours, and the control group was injected with an equal volume of normal saline. (B) Representative pictures of the colon and colon length. (C) Representative pictures of H&E staining and Masson staining of colon sections (scale bars: 50 μm). (D) Histological scores of the colon. (E) Quantitative scores of the collagen area of colon tissue. Data are expressed as mean ± SEM. ns: No significant difference; ****P<0.0001.

[0026] Figure 9It is to target CD73 to improve intestinal fibrosis and adenosine levels in WD+TNBS mice. Among them, (A-F) Wild-type mice from the same litter were fed a Western diet (WD) for 12 weeks (8 mice per group), pre-sensitized with TNBS at the 6th week, and induced with a CD model in mice by TNBS enema once a week from the 7th week. During this period, 100 μg of anti-CD73 monoclonal antibody was intraperitoneally injected once every 72 hours, and the control group was injected with an equal volume of normal saline. (B) Representative pictures of the colon and colon length. (C) Representative pictures of H&E staining and Masson staining of colon sections (scale bars: 50 μm). (D) Histological score of the colon. (E) Quantitative score of the collagen area in colon tissue. (F) Ado concentration in colon tissue. Data are expressed as mean ± standard error. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0027] Figure 10 Blocking CD73 combined with inhibiting Adora1 significantly improves intestinal fibrosis in WD+TNBS mice. Among them, (A-D) The experiment was divided into four groups (6 mice per group). All four groups induced a CD model under the background of a Western diet (WD), that is, wild-type mice from the same litter were fed WD for 12 weeks, pre-sensitized with TNBS at the 6th week, and induced with a CD model in mice by TNBS enema once a week from the 7th week. In the second group, 100 μg of anti-CD73 monoclonal antibody was intraperitoneally injected once every 72 hours during TNBS enema. In the third group, 2 mg / kg of Derenofylline was intraperitoneally injected once every 48 hours during TNBS enema. In the fourth group, 50 μg of anti-CD73 monoclonal antibody was intraperitoneally injected once every 72 hours and 1 mg / kg of Derenofylline was intraperitoneally injected once every 48 hours during TNBS enema. (A) Representative pictures of Masson staining of colon sections (scale bars: 50 μm). (B) Quantitative score of the collagen area in colon tissue. (C,D) The mRNA levels of α-SMA and Col1a1 genes in the colon were detected by qRT-PCR. Data are expressed as mean ± standard error. **P<0.01; ****P<0.0001. Detailed implementation manners

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0029] For the experimental methods without specific conditions noted in the following examples, they are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0031] The present invention will be further described in detail below in conjunction with specific embodiments.

[0032] Example 1

[0033] 1. Materials and Instruments

[0034] Table 1 Materials Used in the Experiment

[0035]

[0036]

[0037] Table 2 Primer Series Used in qRT-PCR Experiment

[0038]

[0039] Table 3 Instruments Used in the Experiment

[0040]

[0041] 2. Research on the Intrinsic Mechanism of CD-Related Intestinal Fibrosis

[0042] 2.1 Effects of Long-Term Western Diet (WD) on Enteritis and Intestinal Fibrosis in Mice

[0043] To explore the effect of WD on CD-related intestinal fibrosis, wild-type mice from the same litter were first divided into two groups of 6 each and fed a normal diet (RD) and a Western diet (WD) for 8 weeks, 12 weeks, and 16 weeks, respectively. Here, the normal diet refers to drinking pure water and eating conventional mouse feed, while the Western diet refers to drinking pure water containing 10% sucrose and eating high-fat food with 60 kcal% fat. The feeding methods of RD and WD hereafter are the same. The results are as Figure 1 shown.

[0044] Wild-type mice from the same litter were divided into two groups of 8 mice each and fed RD and WD for 12 weeks, respectively. According to the reference method (Wirtz S, Popp V, Kindermann M, Gerlach K, Weigmann B, Fichtner-Feigl S, Neurath MF: Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc 2017, 12(7): 1295-1309.), a mouse CD model was induced with 2.5% Trinitrobenzenesulfonic acid (TNBS), that is, TNBS pre-sensitization was performed at the 6th week, and TNBS enema was given once a week from the 7th week, while the control group was given PBS enema. The results are as Figure 2 shown.

[0045] As Figure 1 can be seen, although 8-week WD can induce enteritis, it does not induce obvious intestinal fibrosis, manifested as a significant increase in the shortening of the colon length and the histological score of the intestinal mucosa, while the Masson staining score of the intestinal mucosa does not increase significantly. After 12 weeks of WD feeding, not only the shortening of the colon length and the histological score of the intestinal mucosa increase significantly, but the Masson staining score of the intestinal mucosa also increases significantly. These results indicate that long-term WD is a risk factor for inducing intestinal fibrosis.

[0046] Because the intestinal pathological manifestations of the TNBS-induced chronic enteritis model are similar to those of human CD, subsequent experiments were all carried out under the background of WD feeding to explore the effect of WD on CD intestinal fibrosis. It was found that WD would exacerbate TNBS-induced enteritis and intestinal fibrosis, manifested as a significant increase in the shortening of the colon length and the histological score of the intestinal mucosa in the WD+TNBS group of mice, and the Masson staining score of the intestinal mucosa also increased significantly ( Figure 2 ). These findings indicate that long-term WD is involved in the process of CD-related intestinal fibrosis.

[0047] 2.2 Knockout of GSDME in intestinal epithelial cells improves intestinal fibrosis in WD+TNBS mice

[0048] Our previous studies have found that WD induces GSDME-mediated intestinal epithelial cell (IEC) pyroptosis by producing high concentrations of bile acid, thereby causing enteritis. It is known that long-term and repeated intestinal inflammatory response is the key to the formation of intestinal fibrosis. Therefore, these findings suggest that WD may cause enteritis by inducing IEC pyroptosis and then lead to CD intestinal fibrosis. To explore the role of GSDME-mediated IEC pyroptosis in CD-related intestinal fibrosis, a GSDME knockout gene in intestinal epithelial cells was constructed. fl / fl Villincre mice (GSDME ΔIEC ) and control GSDME fl / fl Mouse.

[0049] GSDME from littermates ΔIEC and GSDME fl / fl Mice, 8 in each group, were fed with WD for 12 weeks, pre-sensitized with TNBS in the 6th week, and induced the mouse CD model by TNBS enema once a week from the 7th week. Figure 3 shown.

[0050] In the context of long-term WD feeding, TNBS-induced chronic intestinal inflammation model was used and it was found that compared with GSDME fl / fl Mouse, GSDME ΔIEC The degree of enteritis and intestinal fibrosis in mice was significantly reduced. fl / fl The GSDME functional fragment GSDME-NT (GSDME-NT) that can induce pyroptosis was detected in the colon tissue of mice. Figure 3 These results suggest that WD participates in intestinal fibrosis by inducing GSDME-mediated pyroptosis of intestinal epithelial cells. 2.3

[0052] Wild-type mice from the same littermates were fed RD and WD for 12 weeks and then their intestinal tissues were extracted for single-cell sequencing and analysis. Figure 4 .

[0053] HMGB1 gene knockout in intestinal epithelial cells fl / fl Villin cre Mouse (HMGB1 ΔIEC ) and control HMGB1 fl / fl The colon tissue of mice was double-stained with CD8 and CD73 fluorescence, and the level of adenosine (Ado) in the tissue was detected by ELISA. Figure 5 .

[0054] HMGB1 from littermates ΔIEC and HMGB1fl / fl Mice were fed a Western diet (WD) for 12 weeks (8 mice per group), pre-sensitized with TNBS at week 6, and CD models were induced in mice by TNBS enema once a week starting from week 7. The results are as follows Figure 6 .

[0055] It is known that the process of intestinal fibrosis involves the abnormal activation of myofibroblasts from multiple sources, including resident fibroblasts, circulating fibrocytes, endothelial cells, and epithelial-mesenchymal transition, leading to the production and secretion of a large amount of extracellular matrix. However, the cellular components involved in this process are very complex and may be far more than these. To explore the specific mechanism of WD in this process, single-cell analysis of intestinal mucosal tissues of mice fed WD for a long time was performed, and it was found that long-term WD feeding would change the intestinal mucosal microenvironment of mice, which was prominently manifested as a significant increase in Effector CD8 + T cells, and such cells were mainly of the CD39 + CD73 + CD8 + T subset. In addition, it was found that WD would cause a significant increase in the signal communication from IECs to Effector CD8 + T cells ([[]] Figure 4 ). Previous studies found that after pyroptosis of IECs, by releasing damage-associated molecular patterns (DAMPs), especially HMGB1 among them, it was involved in the pathological processes of enteritis and enteritis-associated colorectal cancer. Further analysis of single-cell data found that WD could significantly increase the expression level of HMGB1 in intestinal epithelial cells, while the expression levels of several other DAMP molecules did not increase ([[]] Figure 4 ). Knockout of HMGB1 in IECs significantly reduced CD73 + CD8 + T cells and Ado in the intestine ([[]] Figure 5 ), and alleviated TNBS-induced intestinal fibrosis ([[]] Figure 6 ). Therefore, these findings indicate that WD promotes intestinal fibrosis by increasing and releasing HMGB1 in IECs to increase CD73 + CD8 + T cells in the intestine.

[0056] 2.4 Adenosine activates intestinal fibroblasts through Adora1

[0057] Human colon fibroblasts CCD-18Co were cultured in vitro and stimulated with 50 μM Adora1 inhibitor Derenofylline and 5 μM Adora2b inhibitor MRS1754 respectively. After 1 hour, 50 μM Ado was added. After 48 hours, the cells were harvested and the mRNA levels of α-SMA, TGF-β, Vimentin, Col1a1, Col1a2, Col3a1, Fibronectin and FAP genes were detected by qRT-PCR. The results are as Figure 7 .

[0058] Analysis: It is known that extracellular ATP can be successively catalyzed by CD39 and CD73 to generate Ado. Ado exerts its physiological effects by acting on adenosine receptors (ARs) on the cell membrane. ARs belong to the G-protein coupled receptor superfamily and can be divided into four subtypes: Adora1, Adora2a, Adora2b and Adora3. Some studies have found that knocking out the Adora2a gene in mice can inhibit CCL4-induced liver fibrosis. However, knocking out this gene exacerbates unilateral ureteral obstruction-induced renal interstitial fibrosis in mice, while activating Adora2a can effectively relieve renal interstitial fibrosis. Other studies have found that knocking out the Adora2b gene in mice can reduce bleomycin-induced pulmonary fibrosis and unilateral ureteral obstruction-induced renal fibrosis, but activating Adora2b plays a role in reducing myocardial fibrosis, indicating that the functions of ARs in different tissues are different. Through research, it has been found that intestinal fibroblasts mainly express Adora1 and Adora2b ( Figure 4 ), but Ado mainly activates intestinal fibroblasts through Adora1 ( Figure 7 ). In summary, based on these findings, it is suggested that WD induces GSDME-mediated pyroptosis in intestinal epithelial cells and releases a large amount of intracellular molecules HMGB1 and ATP. Among them, HMGB1 promotes CD39 + CD73 + CD8 + T cell expansion. These cells catalyze extracellular ATP into Ado, and finally Ado activates intestinal fibroblasts through the receptor Adora1 to participate in the complex intestinal fibrosis process. However, it is still unclear whether blocking intestinal adenosine production and inhibiting its receptor have the function of treating CD-related intestinal fibrosis.

[0059] Example 2 Exploration of treating CD-related intestinal fibrosis

[0060] 1. Wild-type mice from the same litter were fed a Western diet (WD) for 12 weeks (8 mice per group). TNBS pre-sensitization was performed at week 6, and from week 7, TNBS enema was performed once a week to induce a CD model in mice. During this period, 2 mg / kg of the Adora1 inhibitor Derenofylline was intraperitoneally injected once every 48 hours, and the control group was injected with an equal volume of normal saline. The results are as Figure 8 shown. It was found that compared with the control group, there were no significant changes in the colon length, colon H&E staining, and colon histological score in the Derenofylline treatment group. However, the colon Masson staining and the quantitative score of the collagen area in the colon tissue were significantly reduced, indicating that although Derenofylline could not alleviate the enteritis in CD model mice, it could improve their intestinal fibrosis level.

[0061] 2. Wild-type mice from the same litter were fed a Western diet (WD) for 12 weeks (8 mice per group). TNBS pre-sensitization was performed at week 6, and from week 7, TNBS enema was performed once a week to induce a CD model in mice. During this period, 100 μg of anti-CD73 monoclonal antibody was intraperitoneally injected once every 72 hours, and the control group was injected with an equal volume of normal saline. The results are as Figure 9 shown. It was found that compared with the control group injected with normal saline, the colon length, colon H&E staining, colon histological score, colon Masson staining, and the quantitative score of the collagen area in the colon tissue in the group injected with anti-CD73 monoclonal antibody were all significantly reduced, indicating that anti-CD73 monoclonal antibody could not only alleviate the enteritis in CD model mice but also improve their intestinal fibrosis level.

[0062] 3. The experiment was divided into four groups (6 mice per group). All four groups induced a CD model under the background of a Western diet (WD) feeding, that is, wild-type mice from the same litter were fed WD for 12 weeks, TNBS pre-sensitization was performed at week 6, and from week 7, TNBS enema was performed once a week to induce a CD model in mice. In the second group, 100 μg of anti-CD73 monoclonal antibody was intraperitoneally injected once every 72 hours during TNBS enema. In the third group, 2 mg / kg of Derenofylline was intraperitoneally injected once every 48 hours during TNBS enema. The fourth group was intraperitoneally injected with 50 μg of anti-CD73 monoclonal antibody once every 72 hours and 1 mg / kg of Derenofylline once every 48 hours during TNBS enema. The results are as Figure 10, it was found that compared with the first group without using drugs, the quantitative scores of colon Masson staining and collagen area in the colon tissue, and the mRNA levels of α-SMA and Col1a1 genes in the colon of the second group using anti-CD73 monoclonal antibody and the third group using Derenofylline were all significantly reduced. In the fourth group with the combined use of both, although the drug injection amount was halved, these indicators were further reduced compared with the second and third groups, indicating that both anti-CD73 monoclonal antibody and Derenofylline can reduce the intestinal fibrosis level in CD model mice, and the combined use of both has a more obvious effect on anti-CD-related intestinal fibrosis.

[0063] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of an anti-CD73 monoclonal antibody in combination with an Adora1 inhibitor in the preparation of a medicament for preventing and / or treating intestinal fibrosis or an intestinal fibrosis-related disease.

2. The application according to claim 1, wherein The Adora1 inhibitor is Derenofylline.

3. The application according to claim 1, wherein The intestinal fibrosis-related disease is Crohn's disease (CD)-associated intestinal fibrosis.

4. The application according to claim 1, wherein The use includes blocking intestinal adenosine production and / or inhibiting intestinal adenosine receptors.

5. A drug for preventing and / or treating intestinal fibrosis, wherein, The medicament includes an anti-CD73 monoclonal antibody and an Adora1 inhibitor, and the anti-CD73 monoclonal antibody and the Adora1 inhibitor are each an independent dosing unit or the anti-CD73 monoclonal antibody and the Adora1 inhibitor together form a dosing unit.

6. The medicament according to claim 5, wherein, The Adora1 inhibitor is Derenofylline.

7. The medicament for preventing and / or treating intestinal fibrosis according to claim 5 or 6, wherein, In the medicament, the anti-CD73 monoclonal antibody and the Adora1 inhibitor are each an injection.

8. Use of an anti-CD73 monoclonal antibody in the preparation of a medicament for preventing and / or treating intestinal fibrosis or an intestinal fibrosis-related disease.

9. The application according to claim 8, wherein, The intestinal fibrosis-related disease is Crohn's disease (CD)-associated intestinal fibrosis.

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