CN116063493A

CN116063493A - Pharmaceutical composition comprising antibody and mesenchymal stem cell exosomes and method for preparing the same

Info

Publication number: CN116063493A
Application number: CN202210870090.4A
Authority: CN
Inventors: 张化杰; 裴舜
Original assignee: Beijing Yiyuan Biotechnology Co ltd
Current assignee: Guizhou Saier Biotechnology Co ltd
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2023-05-05
Anticipated expiration: 2042-07-22
Also published as: CN116063493B

Abstract

The application provides a pharmaceutical composition for treating inflammatory bowel disease and application thereof, wherein the pharmaceutical composition comprises an extracellular body and an anti-IL-17R antibody, and the extracellular body is stem cell extracellular body induced by TGF-beta, IL-2, IGF-1 and bFGF. The stem cell exosome and the anti-IL-17R antibody are combined to treat IBD, so that IBD symptoms can be obviously relieved, and the in vivo immune environment is maintained stable; an anti-IL-17R antibody with medium affinity is selected, so that transient immune stress response can be inhibited, normal physiological functions of an organism can be maintained, and IBD symptoms are prevented from deteriorating; the exosomes derived from umbilical cord mesenchymal stem cells are selected, so that the ethical risk is overcome, the sources are stable and wide, and the clinical large-scale application is facilitated; exosomes generated by the umbilical cord mesenchymal stem cells after being induced by various immune factors can obviously improve the proliferation capacity of colonic mucosa epithelial cells, effectively resist oxidative stress and maintain normal physiological functions of digestive tracts.

Description

Pharmaceutical composition comprising antibody and mesenchymal stem cell exosomes and method for preparing the same

Technical field:

the invention belongs to the field of medicine research, and in particular provides a pharmaceutical composition containing an antibody and mesenchymal stem cell exosomes and a preparation method thereof.

The background technology is as follows:

inflammatory bowel disease (Inflammatory bowel disease, IBD) is a long-term recurrent inflammatory disease affecting various parts of the gastrointestinal tract, is widely distributed throughout the world, has approximately 500 tens of thousands of patients worldwide, and in recent years the prevalence of young people of the disease has increased significantly. IBD includes two common diseases, crohn's Disease (CD) and ulcerative colitis (Ulcerative colitis, UC), both of which have the same clinical course, the differences being limited only to the location and nature of inflammatory lesions, crohn's disease being a transmural disease affecting the entire intestinal wall, which can intermittently involve any part of the intestinal tract, but most cases are primarily limited to the distal end of the small intestine; ulcerative colitis is mainly a colonic disease that extends continuously from the rectum to the proximal end of the large intestine, unlike CD, where UC lesions are usually localized to the epithelial mucosa.

Clinically, IBD presents a variety of clinical symptoms including abdominal discomfort, diarrhea, bloody stool, fever, fatigue, weight loss, etc., and in addition to the above frequently reported symptoms, IBD patients often present with extra-intestinal complications, possibly related to skin diseases (erythema nodosum and pyoderma gangrenosum), ocular diseases (conjunctivitis, uveitis), rheumatic diseases (ankylosing spondylitis and peripheral arthritis), hepatobiliary diseases (primary sclerosing cholangitis), urinary system diseases (kidney stones), etc., which severely affect the quality of life of the patient, and the complications thereof also create a great economic burden.

The exact pathogenesis of IBD is not known and is often thought to be the result of a sustained inflammatory process against endogenous microorganisms in genetically susceptible individuals. This disease has been shown to be a genetic disease and twins studies have shown consistent incidence of IBD, especially in syngeneic twins, with positive family history in as many as 25% to 30% of cases. In addition to genetic factors, IBD is mediated by a variety of environmental factors including smoking, stress, oral contraceptives, non-steroidal anti-inflammatory drugs (NSAIDs), vitamin D deficiency, pollution, diet and early antibiotic use, among others. It has also been reported that the intestinal microbiome is obviously essential for the development of IBD, and that less diverse microorganisms are susceptible to the disease, in which case a drastic change in intestinal microbiome composition is associated with increased susceptibility to IBD. IBD is also attributed to an uncontrolled inflammatory state caused by deregulation of the immune response at the intestinal epithelial level, which arises from the up-regulation of various pro-inflammatory pathways and the parallel down-regulation of several regulatory pathways, firstly due to impaired intestinal barrier integrity, and secondly to the inability of the endoluminal pathogens to recognize and eventually eliminate invading bacteria by the intestinal wall, and increased expression of potent inflammatory molecules such as IL-1, IL-6, IL-12, IL-23 and TNF-a, leading to delayed clearance of microbial agents and subsequent excessive inflammatory responses.

The treatment means of IBD has advanced to some extent, and for mild IBD, aminosalicylate type drugs represented by mesalamine are the preferred therapeutic drugs, mesalamine is administered rectally to alleviate UC or CD symptoms; oral administration of glucocorticoids or immunomodulators such as TNF- α monoclonal antibodies (infliximab) is a popular treatment regimen for patients with moderate disease that are resistant to mescalin; steroid drugs such as budesonide are also an alternative therapeutic agent, but long term administration is prone to systemic adverse effects, and therefore it is often necessary to truncate the steroid drug within 6 weeks. In addition to drug treatment, surgical therapy is also a conventional treatment, with bypass ostomy for patients with severe fistulae and total colectomy for patients with severe UC, but it creates a serious economic burden and life inconvenience, with a risk of recurrence. Thus, there is a great clinical need to develop new therapeutic drugs or treatment techniques to effectively and chronically alleviate IBD symptoms. The main mechanism of action of IBD therapy is to correct immune disorder and inhibit inflammatory reaction in intestinal mucosa, and exosomes are one of the research hot spots in cell biology in recent years, and the substances can participate in body immune regulation through various ways, thus becoming a new break-through in the field of IBD treatment.

In 1983, pan and Johnstone have found for the first time that during sheep reticulocyte maturation, the release of transferrin receptor into the extracellular space is associated with a vesicle, which is the first report on extracellular exosomes (Pan BT, johnstone RM. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983;33: 967-78); in 1989, johnstone defined such functional vesicles as exosomes (Johnstone RM, biankhini A, teng K. Reticlectobyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions. Blood 1989; 74:1844-51). In the last three decades, studies on exosomes were in a state of stasis, nor was there a unified concept of extracellular vesicles proposed, called "microvesicles", "exosomes", "microparticles" appearing in different research reports; currently, researchers have classified extracellular vesicles into two major classes according to the manner in which they secrete. One type is known as microbubbles, which are shed directly from the cell membrane; the other is called exosomes, which are released by exocytosis when the multivesicular bodies fuse with the plasma membrane. Exosomes may be released from a number of cell types, such as blood cells, endothelial cells, immune cells, platelets and smooth muscle cells, and it is widely believed that exosomes may modulate the biological activity of receptor cells by transporting lipids, proteins and nucleic acids while circulating in the extracellular space, and that exosomes play an important role in immune responses, tumor progression and neurodegenerative diseases.

Exosomes are rich and complex in composition, mainly comprising proteins, micrornas (mirnas), messenger RNAs (mrnas) and lipids, and have been found to include the Ras superfamily of monomeric G proteins (rabs), adhesion molecules, cytokines, heat shock proteins (such as HSC73, HSC 90), annexins I, II, V and VI, cytoskeletal proteins (such as actin, moesins, albumin), tetraspanins (CD 9, CD63, CD81, CD 82) and GTPases, and the like; exosome lipids include sphingolipids such as cholesterol, phospholipids (e.g., phosphatidic acid, phosphatidylserine), diglycerides, glycerophospholipids, arachidonic acid (polyunsaturated omega-6 fatty acids), ceramides, and sphingomyelins; the exosome RNA species are quite abundant, and the exosome RNA database reports 1833 mrnas, 58330 circular RNAs (circrnas) and 15501 long non-coding RNAs (lncrnas) that are involved in regulating gene expression associated with stem cell differentiation, hematopoiesis, organogenesis, tumorigenesis and tumor metastasis and play a key role in the protein transport process.

There are numerous studies showing that exosomes can be involved in the physiological processes of IBD, exerting immunomodulatory functions, including: (1) Systemic administration of exosomes involved in cellular immunomodulation, such as obtained from bone marrow mesenchymal stem cells, can greatly reduce colitis in various IBD models, which treatment leads to polarization of M2b macrophages without causing intestinal fibrosis, maintains the integrity of the intestinal barrier and down regulates inflammatory responses, these exosomes also being involved in modulating a variety of proteins that are active against colitis, in particular metallothionein-2 involved in inhibiting inflammatory responses; exosome-induced cellular regulation of the immune system; administration of exosomes to the Inflammatory Bowel Disease (IBD) environment can promote an anti-inflammatory response to polarize macrophages to the M2 phenotype, inhibit dendritic cell activation and induce immune tolerance, trigger regulatory T cell (Treg) activation while inhibiting T helper type 1 (Th 1) cells; (2) In connection with gut microbiome modulation, exosomes may utilize gram-negative (e.g., toll like receptor, TLR 4) and gram-positive (e.g., TLR 2) receptors to stimulate pro-inflammatory responses, and exosome HSP72 has been studied for its role in IEC function, and total 1963 proteins, including common EV-related proteins such as CD9, flotillin-1 and Annexin A5, and 633 unidentified proteins, have been reported, which may be involved in gut microbiome modulation in normal and IBD mucosal microenvironments, altering the diversity and composition of the mouse gut microbiome; (3) Exosomes derived from bone marrow mesenchymal stem cells are involved in intestinal barrier modulation, significantly restoring abnormal intestinal barrier function by decreasing intestinal permeability and decreasing infiltration of intestinal microbiota into the lamina propria, and mRNA levels of colonic antibacterial proteins such as lysozyme 1 (Lyz 1), angiopoietin-4 and defensins Defa29 and Defa20 are also restored.

Stem cell derived exosomes are increasingly being considered as potent bioregulators that elicit their therapeutic activity by delivering cargo consisting of potentially therapeutic proteins and RNAs to recipient cells. Numerous studies have shown that stem cell derived exosomes can alleviate IBD symptoms, such as those obtained in human umbilical cord Mesenchymal Stem Cells (MSCs), by up-regulating the levels of IL10 and IFN-gamma-inducible protein-10 (IP 10), while down-regulating the levels of IL-1β, TNF- α, IL-6, ubiquitin-binding enzyme (E2M), significantly alleviating IBD; reduced expression levels of K48, K63, FK2 and other proteins in exosome-treated IBD mice indicate their involvement in regulating ubiquitin modification; MSC-derived exosome treatment can also down regulate inflammatory responses, maintain intestinal mucosal barrier integrity and polarize macrophages to the M2b phenotype, while inhibiting intestinal fibrosis; bone marrow MSC derived exosomes also prevent IBD, oxidation-induced factors such as MPO and Malondialdehyde (MDA) from being down-regulated, while antioxidant factors such as Glutathione (GSH) and superoxide dismutase (SOD) are increased by decreasing mRNA and protein levels of nuclear factors kappa B (NF-. Kappa.B), p65, iNOS, COX2, TNF-. Alpha.and IL-1β, but increasing IL-10 levels. Although stem cell exosomes play an important role in the IBD treatment process, the preparation process and the structural components are complex, the instability is high, and the clinical application is limited.

The occurrence and development of IBD are also related to the unbalance of inflammatory factors in vivo, IL-17 is one of important inflammatory factors, IL-17 is a main cytokine secreted by Th17 cells and is divided into 6 subtypes of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, wherein IL-17A is the most common, and IL-17 refers to IL-17A unless specified in the prior researches. Clinical studies have found that inflammatory bowel disease, including UC and CD patients, has far higher levels of Th17 cells and IL-17 than healthy individuals in the colonic mucosa, and that other studies have found that IL-17A and IL-17F are in the R of the intestinal tract of inflammatory bowel disease patientsNA expression was also far higher than normal, and these results suggest that increased IL-17 expression is closely related to the onset of IBD. However, the present research results show that IL-17 seems to have dual effects of anti-inflammation and pro-inflammation, and contradictory results also appear in clinical and animal experiments, on the one hand, research shows that IL-17 neutralizing antibodies or IL-17 inhibitors can relieve IBD symptoms, if researchers administer IL-17 antibodies to T cell transformed mouse colonitis models can obtain similar therapeutic effects as IL-6 antibodies, and IL-17 and IL-6 antibodies can also obtain overlapped therapeutic effects when used together; inhibition of IL-17A or IL-17F alone, blocking the signaling pathway of both cytokines, can inhibit CD4/CD25 metastasis induced colitis (Wedebye Schmidt EG, larsen HL, kristensen NN, et al TH17cell induction and effects of IL-17A and IL-17F blockade in experimental colitis).

Bowel Dis2013; 19:1567-76); on the other hand, overuse of IL-17 inhibitors appears to exacerbate the colonic validation response, disrupting intestinal mucosal structures, as in clinical trials with approved marketed anti-IL-17 antibodies ixekizumab and brodalumab for the treatment of psoriasis, symptoms of IBD were observed in some patients; in IL-17 knockout mice, dextran sulfate (DDS) was used to induce a model of colitis, and symptoms of the knockout mice were aggravated. The above evidence is difficult to give a clear mechanism of action of IL-17 in IBD, making IL-17 antibodies a dilemma in the treatment of IBD. IL-17R is a receptor for IL-17, and after the IL-17R and the receptor are combined, a series of in-vivo biological signals can be mediated to realize an immunoregulation effect, and evidence shows that the IL-17R plays a crucial role in the immunoregulation of IBD, so that the IL-17R is a potential therapeutic target.

In order to solve the difficulties encountered in the development of the IBD drugs, the present invention provides a pharmaceutical composition comprising exosomes derived from mesenchymal stem cells and an anti-IL-17R antibody with a moderate affinity, which can effectively alleviate IBD devices, inhibit the over-expression of inflammatory factors, inhibit apoptosis of intestinal mucosal epithelial cells, combat oxidative stress, maintain the normal immune microenvironment of the intestinal tract, and improve intestinal function.

Disclosure of Invention

In order to solve the technical problems, the invention provides a pharmaceutical composition for treating inflammatory bowel disease, which is characterized by comprising an extracellular body and an anti-IL-17R antibody, wherein the extracellular body is stem cell extracellular body induced by TGF-beta, IL-2, IGF-1 and bFGF; the heavy chain variable region of the anti-IL-17R antibody comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2 and HCDR3 shown as SEQ ID NO. 3, and the light chain variable region of the anti-IL-17R antibody comprises LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6.

On one hand, in the invention, mesenchymal stem cell exosomes are used for treating IBD, the mesenchymal stem cells have multidirectional differentiation capacity, and the exosomes contain abundant active ingredients such as regulatory proteins, cytokines, immune factors, lipid components, long-chain non-coding RNA, miRNA and the like, so that the exosomes secreted by umbilical cord source stem cells are reported to effectively relieve IBD symptoms; in addition, when the mesenchymal stem cell exosome is prepared, the immunoregulatory factors such as TGF-beta, IL-2, IGF-1, bFGF and the like are adopted for induction, so that the cell proliferation promoting capacity and the immunoregulatory capacity of the exosome are enhanced.

On the other hand, in the present invention, anti-IL-17R antibody is used for treating IBD, IL-17 is an immune factor with dual functions of anti-inflammation and pro-inflammation, and over-high or under-expression of IL-17 content in the body has adverse effects on IBD patients; IL-17, by binding to IL-17R, inhibits the increase of Th1 cell level, exerts an anti-inflammatory effect, promotes the expression of Th17 cells and neutrophil chemokines, exerts a pro-inflammatory effect, which is one of the immunoregulatory mechanisms of IL-17. Therefore, the invention selects the anti-IL-17R antibody with medium affinity, which can neutralize the over-expressed IL-17R and prevent the excessive inhibition of IL-17R signal path, thereby effectively relieving IBD symptoms.

Furthermore, the amino acid sequence of the heavy chain variable region of the anti-IL-17R antibody is shown as SEQ ID NO. 7.

Furthermore, the amino acid sequence of the light chain variable region of the anti-IL-17R antibody is shown as SEQ ID NO. 8.

Further, the stem cell exosome is at least one selected from the group consisting of a bone marrow mesenchymal stem cell exosome, an umbilical cord mesenchymal stem cell exosome, an adipose mesenchymal stem cell exosome, an endothelial progenitor cell exosome, and a neural stem cell exosome.

Further, the stem cell exosome is umbilical cord mesenchymal stem cell exosome, and the preparation method comprises: culturing umbilical cord mesenchymal stem cells, adding exosome induction culture medium when cell fusion degree reaches more than 80%, and adding 5% CO ₂ Culturing in an incubator at 37 ℃ for 48 hours; collecting stem cell exosomes by gradient centrifugation, collecting cell supernatant after induction culture, filtering with 0.22 μm needle filter, centrifuging at 300×g for 10min at low temperature, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; the pellet was resuspended by centrifugation at 100000 Xg for 1h with sterile PBS to obtain stem cell exosomes.

According to the invention, umbilical cord-derived mesenchymal stem cells are used, and stem cell exosomes are obtained through induced expression, so that the stem cells have strong multidirectional differentiation capacity, are abundant in source, have no ethical risk and limitation, and can be conveniently used in a large scale clinically.

Further, the exosome induction medium is an alpha MEM medium containing 10% FBS, 50. Mu.g/mL TGF-beta, 10. Mu.g/mL IL-2, 10. Mu.g/mL IGF-1, 10. Mu.g/mLbFGF.

It has been reported that various cytokines and inflammatory factors can affect secretion of exosomes in stem cells, and in the face of a huge number of candidate factors, the invention creatively proposes induction by four factors of TGF-beta, IL-2, IGF-1, bFGF and the like by combining physiological characteristics of mesenchymal stem cells and pathogenesis of IBD, and optimizes the applicable proportion so as to achieve optimal combination effect.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Provides an application of the pharmaceutical composition in preparing medicines for treating inflammatory bowel diseases.

Further, the inflammatory bowel disease includes crohn's disease and ulcerative colitis.

Advantageous effects

The application provides a pharmaceutical composition for treating inflammatory bowel disease and application thereof, and the pharmaceutical composition has the following advantages:

(1) The stem cell exosome and the anti-IL-17R antibody are combined to treat IBD, so that IBD symptoms can be obviously relieved, and the in vivo immune environment is maintained stable;

(2) An anti-IL-17R antibody with medium affinity is selected, so that transient immune stress response can be inhibited, normal physiological functions of an organism can be maintained, and IBD symptoms are prevented from deteriorating;

(3) The exosomes derived from umbilical cord mesenchymal stem cells are selected, so that the ethical risk is overcome, the sources are stable and wide, and the clinical large-scale application is facilitated;

(4) Exosomes generated by the umbilical cord mesenchymal stem cells after being induced by various immune factors can obviously improve the proliferation capacity of colonic mucosa epithelial cells, effectively resist oxidative stress and maintain normal physiological functions of digestive tracts.

Drawings

Fig. 1: NCM460 cell proliferation capacity;

fig. 2: rat DAI score;

fig. 3: serum TNF- α expression levels;

fig. 4: serum IL-1 beta expression levels;

fig. 5: serum IL-6 expression levels.

Detailed Description

The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.

The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.

EXAMPLE 1 preparation of anti-IL-17R antibodies

Recombinant human IL-17R protein (Peprotech, USA) was fully emulsified with complete Freund's adjuvant and BALB/c mice were subcutaneously injected in multiple injections at a dose of 50. Mu.g/mouse, once every two weeks for three total immunizations. Tail vein blood is taken after 10 days of third immunization, the antibody titer is measured, the highest antibody titer reaches 1:32000, and the tail vein of the mouse with the highest antibody titer is selected for intravenous injection to strengthen the immunization once; after 3 days, mice were sacrificed, spleens of the mice were removed, spleen lymphocytes were isolated by a pressure water injection method, the cells were centrifugally washed and resuspended in DMEM medium, and the SP2/0 cells of the myeloma cells of the mice were collected, and the cells were collected using 10% bovine serum in DMEM medium, 5% CO ₂ SP2/0 myeloma cells were primary cultured in an incubator at 37℃and cell fusion was performed after the cells entered the logarithmic growth phase. Adding mouse spleen lymphocytes and mouse myeloma cells SP2/0 into the cell culture plate according to the ratio of 10:1, uniformly mixing, adding DMEM culture medium containing 10% bovine serum and 5% CO ₂ Culturing in a 37 ℃ incubator, and changing a cell culture medium according to the growth condition of cells; after 10 days of culture, positive clones were screened using ELISA.

Positive hybridoma cells were cultured, 8-10 weeks BALB/C healthy mice were selected after the cells had grown to logarithmic growth phase, and each abdomen was inoculated with 5X 10 cells ⁶ The positive hybridoma cell PBS buffer solution, the abdomen of the mouse is obviously enlarged after inoculating cells for 7-10 days, the abdomen signs of the health condition of the mouse are closely observed, and the ascites of the mouse is collected when the ascites is as much as possible. The monoclonal antibody in the ascites is purified by adopting an affinity purification method (protein G agar gel), and the purity of the antibody obtained by measurement reaches more than 95 percent, thereby meeting the requirements of subsequent experiments.

The affinity of the antibodies obtained by screening with human IL-17R of the target protein was detected by using a molecular interaction analysis platform Biacore, and the results are shown in Table 1. The prior studies show that IL-17R factor has dual effects of anti-inflammation and pro-inflammation, and over-high or under-low expression of the factor can aggravate the pathogenesis symptoms of inflammatory bowel disease, so that intestinal immune dysfunction is caused, and the expression level of IL-17R in IBD organisms is observed to be increased compared with that of normal individuals in existing human or animal experiments, so that the inventor realizes that the normal level of IL-17R is critical for maintaining the immune balance of organisms, and the expression of IL-17R cannot be excessively inhibited or promoted, and the 5C6 antibody with medium affinity is selected for subsequent experiments in the invention so as to effectively inhibit the high expression of IL-17R and simultaneously maintain the immune balance of organisms, thereby providing a favorable immune environment for treating IBD.

Table 1 antibody affinity assay

Antibodies to	K _D Value (M)
		2B3	27.14E-10
2E4	36.41E-10
		3A2	11.54E-11
5C6	29.54E-9
		6D3	28.54E-10
6E7	18.45E-8

The heavy chain variable region of the anti-IL-17R antibody comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2 and HCDR3 shown as SEQ ID NO. 3, and the light chain variable region of the anti-IL-17R antibody comprises LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6; furthermore, the amino acid sequence of the heavy chain variable region of the anti-IL-17R antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

Example 2 preparation of umbilical cord mesenchymal Stem cell exosomes

2.1 Primary culture of umbilical cord mesenchymal Stem cells

Collecting umbilical cord specimen of healthy neonate, washing umbilical cord with sterile PBS solution, removing blood stain and other impurities, removing umbilical artery and umbilical vein, separating umbilical cord Wharton's jelly tissue, cleaning, and cutting into pieces of about 0.5-1mm ³ Adding 0.2% type II collagenase and hyaluronidase into the tissue block, digesting at 37deg.C for 1-2h, centrifuging 500g for 5min, and collecting cells; the cell sediment is resuspended by adding an alpha-MEM culture medium containing 10% FBS+1% of streptomycin, inoculated in a culture flask, placed in a 37 ℃ and 5% CO2 incubator for culture, fresh culture solution is replaced every 2-3 days according to the cell growth condition, and subculture can be carried out when the cell fusion degree reaches more than 80%.

2.2 preparation of umbilical cord mesenchymal Stem cell exosome

Subculturing umbilical mesenchymal stem cells obtained in section 2.1 for 3-5 generations, and adding exosome induction culture medium when cell fusion degree reaches more than 80%, wherein the exosome induction culture medium is alpha MEM culture medium containing 10% FBS, 50 μg/mL TGF-beta, 10 μg/mL IL-2, 10 μg/mL IGF-1, 10 μg/mLbFGF, and 5% CO ₂ Culturing in an incubator at 37 ℃ for 48 hours; the method for collecting stem cell exosomes by adopting a gradient centrifugation method comprises the following specific steps: collecting cell supernatant after induction culture, filtering with 0.22 μm needle filter, centrifuging at 300×g for 10min, and collecting supernatant; centrifuging the supernatant at 2000 Xg for 10min, and collecting the supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 1 hr, re-suspending the precipitate with sterile PBS solution, and storing in-80 deg.C refrigerator for a long period.

The non-induced umbilical mesenchymal stem cell exosome is prepared by adopting a similar method, the culture medium is alpha MEM culture medium containing 10% FBS, the rest steps are the same, the obtained exosome is recorded as EXO, and the exosome is stored in a refrigerator at the temperature of minus 80 ℃ after being resuspended by PBS.

2.3 umbilical cord mesenchymal Stem cell exosome proliferation-promoting experiment

To verify the proliferation promoting effect of stem cell exosomes on human intestinal cells, human colorectal mucosal epithelial cell line NCM460, which was preserved by the inventors, was selected as the subject in this example.

Measurement of proliferation potency of stem cell exosomes Using CCK8, 1×10 cell line was seeded in 96 well plates ⁵ After incubation at 37℃for 12h at NCM460,5% CO2 per well, 20. Mu.g/mL of EXO and 20. Mu.g/mL of in-EXO and an equal amount of DMEM medium (control) were added, respectively, after 24h 10ul of CCK8 detection solution per well was added, and the plates were placed in an incubator for incubation for 3h, and the OD of each well was determined by an microplate reader at a wavelength of 450 nm.

As shown in fig. 1, the stem cell exosomes provided by the invention can effectively promote proliferation of NCM460 cells, but the proliferation promoting capacity of the stem cell exosomes induced by no cytokine is limited, and is only slightly improved compared with the control group; while the proliferation of cells is markedly enhanced upon induction with cytokines such as TGF-beta, IL-2, IGF-1, bFGF, etc., which mediate a variety of physiological responses in the body, it is reported that TGF-beta is a major regulator of fibrosis in all organs including the intestinal tract, and that the expression of TGF-beta and its receptor is increased in the intestinal cells of IBD patients, particularly CD patients, but blocking TGF-beta signaling pathway with inhibitors is rather dangerous, because TGF-beta induces tissue fibrosis in addition to that, the invention also participates in various immune regulation and cell proliferation and differentiation promotion effects, which are necessary for maintaining normal life activities, and factors such as IL-2, IGF-1, bFGF and the like are also related to various physiological activities.

Example 3 treatment of animal models of inflammatory bowel disease

3.1 preparation of animal models of inflammatory bowel disease

Taking clean SD rats, wherein the weight of the male and female rats is 200+/-20 g, the relative humidity is 40-60% at the room temperature of 20-24 ℃, and carrying out modeling IBD experiments after 1 week of cage-division adaptive breeding. SD rats were anesthetized by intraperitoneal injection with 2% sodium pentobarbital, a rubber catheter of 2mm diameter was inserted into the colon about 6cm from the anal orifice, and TNBS solution (5% TNBS: absolute ethanol=3:2) was slowly introduced at a concentration of 30mg/mL in an administration volume of 3 mL/kg. Weighing the weight and observing the stool characters every day, and modeling the conditions of loose stool, bloody stool, weight reduction and poor activity of the IBD rats on the 3 rd day of modeling, thus indicating successful modeling.

3.2 grouping and administration treatments of animals

After successful modeling, IBD model rats were randomly divided into 4 groups of 10 animals each, each treated as follows: in-EXO group: every two days, tail intravenous injection is 50mg/Kg in-EXO; an anti-IL-17R antibody group, which was injected with 50mg/Kg anti-IL-17R antibody every two days by tail vein; combination group, tail intravenous injection of 50mg/Kg in-EXO and 50mg/Kg anti-IL-17R antibody every two days; in the control group, equal amount of sterile physiological saline is injected into tail vein every two days for total treatment for 10 days.

3.3DAI scoring

During the dosing period, mice were observed every 2 days for body weight changes, drinking water intake, hair gloss, mental state, activity, etc., and were scored according to the criteria established by Ganta et al, with the DAI scoring criteria shown in table 2. As shown in fig. 2, the DAI scores of the model group were fluctuating, but were always maintained at a higher level; after treatment with in-EXO or anti-IL-17R antibodies, IBD symptoms are reduced, the treatment effects of the two are close, and no significant difference is seen in the observation period; the co-administration of in-EXO and anti-IL-17R antibodies significantly increased efficacy at the late stage of treatment, and the DAI score of the combination group significantly decreased from day 4.

TABLE 2DAI scoring benchmark

Remarks: DAI = weight loss fraction + fecal occult blood/macroscopic blood stool fraction.

3.4 oxidative stress detection

Intestinal epithelium has organ-specific and immune functions and has been thought to play a central role in the pathogenesis of IBD, and once excessive Reactive Oxygen Species (ROS) are produced in the gut, oxidative stress accelerates cell damage by altering protein function and causing lipid peroxidation. During mucosal inflammation, neutrophils and macrophages produce superoxide and nitric oxide by activating Nitrogen Oxides (NOX) and Inducible Nitric Oxide Synthase (iNOS), respectively, both of which are induced by inflammatory cytokines, and by NOX and iNOS activation produce more ROS/RNS that destroy cytoskeletal proteins and lead to tight junctions and changes in epithelial permeability in IEC, ultimately leading to barrier destruction and thus to IBD (Tian, zining Wang, and Jinhua, pathomechanisms of Oxidative Stress in Inflammatory Bowel Disease and Potential Antioxidant Therapies, oxid Med Cell longev.2017; 2017:4535194). SOD is the only antioxidant enzyme capable of specifically scavenging superoxide radicals, MDA is the final product of lipid peroxidation, and both can comprehensively reflect oxidative stress conditions in the body or tissues.

In this example, rats were sacrificed 10 days after treatment, colon tissues of the rats were taken, a 9-fold amount of cold physiological saline was added to prepare homogenate, freeze thawing was repeated 3 times after completion of the homogenate, cells were broken, and the supernatant was measured for SOD and MDA contents (purchased from nanjing built biochemistry limited) according to the kit. As shown in table 3, the levels of SOD expression were elevated and the levels of MDA were reduced to varying degrees in each treatment group, and the stem cell exosomes appeared to be stronger than the anti-IL-17R antibodies in terms of antioxidant stress, which were similar in terms of enhancement of SOD, but the in-EXO effect was significantly stronger than the anti-IL-17R antibodies in terms of MDA inhibition, suggesting that in-EXO might also inhibit MDA expression through pathways other than SOD, thereby inhibiting oxidative stress; in the combination group, MDA levels were similar to in-EXO groups, but SOD levels were significantly higher than in the other two treatment groups.

TABLE 3 SOD and MDA levels in colon tissue of rats in each group

3.5 detection of TNF- α, IL-1β and IL-6 in serum

Inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 play an important role in inducing IBD onset, and secretion of these pro-inflammatory cytokines not only causes tissue damage, but also activates the adaptive immune system, resulting in chronic inflammation. In clinical trials, researchers observed varying degrees of up-regulation of the expression of the inflammatory factors described above, whereas administration of related inhibitors could alleviate IBD symptoms to some extent. For example, anti-TNF- α therapies that have been used in the clinical treatment of IBD, using TNF- α antagonists such as infliximab, adalimumab, golimumab, cetuximab, etanercept, etc., can produce a sustained anti-inflammatory response that promotes mucosal healing and restoration of intestinal epithelial barrier function. In the embodiment, ELISA is adopted to detect the expression level of inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like in rat serum so as to examine whether the medicine provided by the invention can effectively inhibit inflammatory reaction.

After 10 days of treatment, the rat orbit was bled, serum was collected by centrifugation at 3500g for 5min at 4℃and the levels of TNF- α, IL-1 β, and IL-6 in the rat serum were detected according to ELISA kit instructions (purchased from Meter Cunninghamia sinensis Biotechnology Co., ltd.). The results are shown in FIGS. 3-5, with anti-IL-17R antibodies having a greater inhibitory effect on TNF- α than in-EXO, and this immunomodulatory effect was enhanced in the combination group; for IL-1β, the IL-1β expression levels were reduced in all three treatment groups, but did not show significant differences; for IL-6, the anti-IL-17R antibodies still have a stronger effect than in-EXO, and similar therapeutic effects were obtained in the combination. The results demonstrate that both the stem cell exosomes and the anti-IL-17R antibodies provided in the present invention are effective in inhibiting the overexpression of inflammatory factors, and that the combined use of both therapeutic agents is better in a comprehensive view.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. An anti-IL-17R antibody, characterized in that: the heavy chain variable region of the anti-IL-17R antibody comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2 and HCDR3 shown as SEQ ID NO. 3, and the light chain variable region of the anti-IL-17R antibody comprises LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6.

2. The antibody of claim 1, wherein: the amino acid sequence of the heavy chain variable region of the anti-IL-17R antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region of the anti-IL-17R antibody is shown as SEQ ID NO. 8.

3. A pharmaceutical composition for treating inflammatory bowel disease, characterized in that the pharmaceutical composition comprises an extracellular body and an anti-IL-17R antibody, the extracellular body being a stem cell extracellular body induced by TGF- β, IL-2, IGF-1, bFGF.

4. The pharmaceutical composition according to claim 3, wherein the stem cell exosomes are at least one selected from the group consisting of bone marrow mesenchymal stem cell exosomes, umbilical cord mesenchymal stem cell exosomes, adipose mesenchymal stem cell exosomes, endothelial progenitor cell exosomes, and neural stem cell exosomes.

5. The pharmaceutical composition of claim 4, wherein the stem cell exosome is umbilical cord mesenchymal stem cell exosome, and the method of preparing the same comprises: culturing umbilical cord mesenchymal stem cells, adding exosome induction culture medium when cell fusion degree reaches more than 80%, and adding 5% CO ₂ Culturing in an incubator at 37 ℃ for 48 hours; collecting stem cell exosomes by gradient centrifugation, collecting cell supernatant after induction culture, filtering with 0.22 μm needle filter, centrifuging at 300×g for 10min at low temperature, and collecting supernatant; centrifuging at 2000 Xg at low temperatureCollecting supernatant after 10 min; centrifuging at 10000 Xg for 30min, and collecting supernatant; the pellet was resuspended by centrifugation at 100000 Xg for 1h with sterile PBS to obtain stem cell exosomes.

6. The pharmaceutical composition of claim 5, wherein the exosome induction medium is an alpha MEM medium comprising 10% fbs, 50 μg/mL TGF- β, 10 μg/mL IL-2, 10 μg/mL IGF-1, 10 μg/mL bFGF.

7. The pharmaceutical composition of any one of claims 3-6, further comprising a pharmaceutically acceptable carrier.

8. Use of a pharmaceutical composition according to any one of claims 3-7 for the preparation of a medicament for the treatment of inflammatory bowel disease.

9. The use of claim 8, wherein the inflammatory bowel disease comprises crohn's disease and ulcerative colitis.