CN112999346A - Application of BAFF antibody in preparation of medicine for treating inflammatory bowel disease - Google Patents
Application of BAFF antibody in preparation of medicine for treating inflammatory bowel disease Download PDFInfo
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- CN112999346A CN112999346A CN202110239481.1A CN202110239481A CN112999346A CN 112999346 A CN112999346 A CN 112999346A CN 202110239481 A CN202110239481 A CN 202110239481A CN 112999346 A CN112999346 A CN 112999346A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Abstract
The invention discloses an application of a BAFF antibody in preparing a medicament for treating inflammatory bowel disease, which comprises the following contents: the application of the BAFF antibody in preparing a medicament for treating inflammatory bowel disease; the application of the BAFF antibody in preparing a medicament for preventing inflammatory bowel disease; the application of the BAFF antibody in preparing a medicament for preventing and treating organ damage caused by inflammatory bowel disease; the application of the BAFF antibody in preparing a medicament for controlling inflammatory reaction caused by inflammatory bowel disease; a medicament for treating and preventing inflammatory bowel disease, wherein the effective component is a BAFF antibody. Through the arrangement, a novel therapeutic drug for inflammatory bowel diseases is provided, and related diseases such as organ injury, inflammation and the like caused by inflammatory bowel diseases can be effectively inhibited; also provides a new application of the BAFF antibody.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to application of a BAFF antibody in preparing a medicament for treating inflammatory bowel disease.
Background
With the development of economic globalization and the change of life style and living environment of people, the incidence of inflammatory bowel diseases in China rises year by year, the course of disease is not prolonged, and the life quality of patients is seriously influenced. The continuous activation of the intestinal mucosal immune system is thought to be the main cause of recurrent intestinal inflammatory diseases. In earlier studies, it was found that the levels of B cell activating factor (BAFF) in serum, feces and human colon tissue of patients with inflammatory bowel disease are closely related to clinical activity, C-reactive protein and the levels of various proinflammatory cytokines such as TNF-alpha, IL-1 in serum. And the concentration of BAFF in the stool of IBD patients is higher than that of IBS patients and healthy controls. We hypothesized that the role of BAFF in inflammatory bowel disease may exist as a pro-inflammatory factor, so the effect of BAFF in ulcerative colitis was observed using the antibody to antagonize BAFF.
The current agents for blocking the biological activity of BAFF mainly comprise two main classes of anti-BAFF antibodies and soluble receptors of BAFF, wherein the BAFF antibodies comprise lymphostat-B, EGFP/scFv F8, anti-BAFFscFv, anti-BAFF scFv-Fc and the like.
At present, no BAFF is applied to medicines for treating inflammatory bowel diseases and related diseases.
Disclosure of Invention
The invention aims to provide application of a BAFF antibody in preparing a medicament for treating inflammatory bowel diseases, and provides a novel therapeutic medicament for treating inflammatory bowel diseases, which can effectively inhibit related diseases such as organ injury, inflammation and the like caused by inflammatory bowel diseases; also provides a new application of the BAFF antibody.
In order to achieve the purpose, the invention provides the following technical scheme: application of BAFF antibody in preparing medicine for treating inflammatory bowel disease.
Preferably, the medicament for treating the inflammatory bowel disease comprises the BAFF antibody as an active ingredient.
Preferably, the application of the BAFF antibody in preparing the medicine for preventing inflammatory bowel diseases.
Preferably, the medicament for preventing inflammatory bowel disease comprises BAFF antibody as an active ingredient.
Preferably, the BAFF antibody is applied to preparing a medicament for preventing and treating organ injury caused by inflammatory bowel disease.
Preferably, the medicament for preventing and treating organ injury caused by inflammatory bowel disease is provided, and the effective component is BAFF antibody.
Preferably, the BAFF antibody is applied to the preparation of medicines for controlling inflammatory response caused by inflammatory bowel disease.
Preferably, the medicament for controlling inflammatory reaction caused by inflammatory bowel disease comprises BAFF antibody as an active ingredient.
Preferably, the BAFF antibody is lymphostat-B or EGFP/scFv F8 or anti-BAFF scFv-Fc.
Compared with the prior art, the invention has the beneficial effects that: provides a novel therapeutic drug for inflammatory bowel diseases, which can effectively inhibit the associated diseases caused by inflammatory bowel diseases, such as organ damage, inflammation and the like; also provides a new application of the BAFF antibody.
Drawings
FIG. 1 is a comparison of the expression of BAFF-associated receptors in mice of the model of chronic DSS colitis mediated by BAFF antibody of the present invention;
FIG. 2 is a graph comparing the effect of the BAFF antibody on body weight of mice model of chronic DSS colitis;
FIG. 3 is a graph comparing the colon length of mice model of chronic DSS colitis improved by BAFF antibody in the present invention;
FIG. 4 is a pathological section of the colon of three groups of mice in accordance with the present invention;
FIG. 5 is a comparison of inflammatory cytokines in distal colon tissue of three groups of mice in accordance with the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-5, an embodiment of the present invention is shown:
the present invention provides the following:
application of BAFF antibody in preparing medicine for treating inflammatory bowel disease. Provides a medicament for treating inflammatory bowel diseases, the effective component of which is BAFF antibody.
Application of BAFF antibody in preparing medicine for preventing inflammatory bowel disease. Provides a medicament for preventing inflammatory bowel diseases, the effective component of which is BAFF antibody.
Application of BAFF antibody in preparing medicine for preventing and treating organ injury caused by inflammatory bowel disease. Provides a medicine for preventing and treating organ injury caused by inflammatory bowel disease, and the effective component is BAFF antibody.
Application of BAFF antibody in preparing medicine for controlling inflammatory reaction caused by inflammatory bowel disease. A medicine for controlling inflammatory reaction caused by inflammatory bowel disease contains BAFF antibody as effective component.
The BAFF antibody is lymphostat-B or EGFP/scFv F8 or anti-BAFF scFv-Fc.
The verification process for BAFF effect is as follows:
1. test materials
1.1 Experimental animals
Male C57BL/6J mice, 6-8 weeks old, were purchased from the animal technology Co., Ltd, Viton, Beijing and raised in the SPF grade animal testing center.
1.2 Main Equipment, reagent
A cryogenic refrigerator (Panasonic), a fluorescence photometer (Thermo Fisher Scientific), an optical microscope (Nikon), a Mini-Cycler thermal Cycler (Roche), a multifunctional microplate reader (TECAN), a PCR instrument (Roche), a low temperature centrifuge (Eppendorf), a Western developing instrument (Azure Biosystem), a full automatic low temperature tissue homogenizer (shanghai yuming instruments ltd), a full automatic biochemical analyzer (shenzhe ledu life science ltd), a vortex oscillator (shanghai jing science ltd), and a shaker (shanghai wisi honesty analyzer ltd); DSS was purchased from american MP corporation; trizol (takara); reverse transcription and PCR kit (bio-technologies ltd, nunjin nuozokenza); tissue fixative (Wuhan Severer Biotech Co., Ltd.).
The BAFF neutralizing antibody (Sandy-2, Adipogen), (BAFF (D7I1U) Rabbit mAb #19944, Adipogen), (Human BAFF/TNFSF13B (hBAFF) #89628, Adipogen) all have similar experimental results, the results are slightly different and approximately the same results, and because the experimental results are similar, the results are not repeated and are represented by (Sandy-2, Adipogen) in the following results, and the results also represent the results of other types of antibodies.
2. Method of producing a composite material
2.1 construction of DSS-induced chronic inflammatory bowel disease model in mice and intervention of BAFF neutralizing antibody
1) Grouping experiments: 30 male C57BL/6 mice were randomly assigned to control, DSS + anti-BAFF groups. The mice were acclimatized for 7 days in an SPF-grade animal house before subsequent experiments.
2) And (3) experimental intervention: control group: freely drinking high-pressure distilled water for 30 days, and injecting 200ul PBS solution into abdominal cavity on days 1 and 15; and (4) DSS group: freely drinking 2.5% DSS solution on days 1-5, 11-15, 21-25, and injecting control antibody (2mg/kg and 1mg/kg) into abdominal cavity on days 1, 15; DSS + anti-BAFF group: freely drinking 2.5% DSS solution on days 1-5, 11-15, 21-25, freely drinking high pressure distilled water for the rest of molding time, and injecting BAFF antibody (2mg/kg and 1mg/kg) into abdominal cavity on days 1, 15.
3) Recording of general conditions in mice: after the start of modeling, the weight, stool characteristics (normal, pasty stool or watery stool), bloody condition (no, occult blood or obvious bloody stool), activity condition (active or burnout) and the like of the mice were recorded every day.
4) Mouse sacrifice and specimen collection: mice in a control group, a DSS group and a DSS + anti-BAFF group are killed at 30 days of model building, blood is taken from eyeballs after the mice are anesthetized in an abdominal cavity by 0.5 percent sodium pentobarbital, the mice are kept stand for 2 hours at room temperature, are centrifuged at 3000rpm and 4 ℃ for 15 minutes, and then supernatant serum is taken and stored at-80 ℃ for later use. The abdominal cavity and the thoracic cavity were opened along the midline of the abdomen, and the colon and spleen were removed. Taking a section of colon tissue, fixing the colon tissue in tissue fixing liquid at normal temperature for 24 hours, and then carrying out paraffin section and H & E staining; the rest tissues were removed, washed and quickly stored in a-80 ℃ refrigerator for subsequent RT-qPCR detection.
2.2 hematoxylin-eosin staining (hematoxylin-eosin staining, H & E)
1) Fixing ileum tissue in tissue fixing solution for 24h, dehydrating with ethanol with gradient concentration, clearing in xylene, embedding with paraffin, and slicing;
2) dewaxing: sequentially placing the glass slide in dimethylbenzene, 100% ethanol, 95% ethanol and 80% ethanol for dewaxing;
3) dyeing: adding hematoxylin staining solution for 10 minutes until the cell nucleus is purple red, washing the cell nucleus with running water for 1 minute until the cell nucleus is bright blue, 0.5% hydrochloric acid alcohol is subjected to color separation for 3-10 seconds, observing under a mirror, washing the cell nucleus with the running water for 5-10 seconds, rewetting the saturated lithium carbonate aqueous solution for 15-20 seconds, washing the cell nucleus with the running water for 1 minute-70% ethanol for 5 minutes, washing the cell nucleus with 80% ethanol for 5 minutes, 1% eosin staining solution for 1-3 minutes-95% ethanol for 5 minutes, 100% ethanol for 5 minutes, xylene and ethanol (1: 1) for 5 minutes, and pure xylene for 5 minutes;
4) sealing: a small drop of neutral resin was added dropwise and mounted with a cover slip and air dried.
2.3 real-time quantitative polymerase chain reaction (RT-qPCR)
2.3.1 extraction of tissue RNA
1) Using an autoclaved ophthalmic scissors and tweezers to cut about 20mg of ileum tissue of the mouse, quickly placing the tissue into an autoclaved 2ml EP tube containing 1ml of Trizol, adding 3 small magnetic beads, homogenizing for 2 times at a low temperature in a grinding instrument at 65Hz and 120 sec;
2) to the tissue homogenate was added 200ul chloroform, the EP tube was shaken vigorously upside down for 15s, allowed to stand at room temperature for 5min, and centrifuged at low temperature for 15min (12000 rpm). After centrifugation, the liquid is divided into three layers, wherein the upper colorless solution is an RNA phase;
3) sucking 300ul of the colorless upper layer solution by a pipette, adding 300ul of isopropanol, slightly inverting and mixing, standing at room temperature for 5min, and centrifuging at low temperature for 15min (12000 rpm);
4) carefully removing all the solution in the EP tube by using a pipettor, wherein the sediment at the bottom of the EP tube is the extracted RNA sediment, adding 1ml of precooled 75% ethanol into the EP tube, gently blowing and cleaning the RNA sediment by using the pipettor, and centrifuging at low temperature for 5min (12000 rpm);
5) discarding the supernatant, inverting the EP tube on clean filter paper, and drying for 1min to volatilize the residual ethanol.
6) And adding a proper amount of DEPC water to dissolve the precipitate by using a pipette according to the RNA precipitation amount, and gently blowing and beating to fully dissolve the precipitate.
7) And (3) RNA concentration detection: the RNA concentration was determined spectrophotometrically by first washing the cuvette at high pressure and then zeroizing it by adding 100ul ddH 2O. 2ul of the extracted RNA solution and 98ul of ddH2O were added to the cuvette and the RNA concentration was determined at an absorbance of 280nm and the purity and concentration of the RNA was recorded.
2.3.2 reverse transcription
1) cDNA synthesis was performed according to the reverse transcription kit instructions, and the amount of RNA required was calculated based on the amount of cDNA to be synthesized and the concentration of RNA extracted.
2) Preparing a reaction system: take 10ul reaction system as an example
3) Reverse transcription: reverse transcription is carried out in a mini PCR instrument, the procedure is 37 ℃, 15 min-85 ℃ and 15 sec-4 ℃, and then the corresponding cDNA can be obtained.
2.3.3 real-time quantitative PCR
1) Preparing a PCR reaction system: 10ul of amplification system: TB Green 5ul, primer 1ul, cDNA 1ul, ddH2O ul;
2) sample adding: and adding the prepared reaction system into a 96-well PCR plate, sealing the 96-well plate by using a sealing plate membrane after the sample is added, and centrifuging.
3) And (3) amplification procedure: 10min at 95 ℃ to 30s at 60 ℃ to 1min at 72 ℃, 40 cycles of amplification-dissolution curve, 0s at 95 ℃, 15s at 65 ℃ and 0s at 95 ℃.
4) And (4) analyzing results: beta-actin is used as an internal reference, and 2 is adopted-△△CTThe method performs calculations and analysis.
3. Data statistics
Statistical analysis and plotting of experimental data was performed using GraphPad Prism 8.0, with statistical results expressed as mean ± standard error. Analysis of differences between groups was performed using One-way ANOVA or Mann-Whitney test, depending on the type of experimental data. Differences between groups were considered statistically significant when P < 0.05.
4. Results and analysis
Influence of BAFF antibody on interference of mouse BAFF-associated receptor in chronic DSS colitis model
The expression of BAFF related receptor in the mice is detected by the RT-qPCR method and the mean value and standard deviation are shown by a bar chart. As shown in figure 1, compared with a control group, the expression level of BAFF-associated receptors BAFF-R and TACI in colon tissues of mice in a DSS group is remarkably increased, the BAFF-R and TACI are remarkably reduced after BAFF antibody intervention, the difference has statistical significance, while BCMA has a reduction trend in gene expression in colon tissues of mice with chronic colitis, and the prognosis of BAFF antibody is reduced compared with that of the DSS group, but has no obvious statistical significance, which indicates that the inflammatory BAFF-associated receptors caused by the DSS-induced chronic colitis model mice and BAFF are mainly BAFF-R and TACI but not BCMA.
Effect of BAFF antibody on body weight of mice model of chronic DSS colitis
As shown in fig. 2, the body weight of the mice in the blank control group increases with the age of the mice, the body weight of the mice with chronic DSS colitis decreases significantly, and the body weight of the mice in the clear water period without DSS solution can not be restored to the level of the mice in the blank control group. The DSS intervention is simultaneously given to BAFF antibody intervention, the weight of mice is reduced by taking DSS in each period, but the weight of a BAFF antibody intervention group is slightly lower than that of a DSS group, and after the recovery of a clear water period, the weight of the BAFF antibody intervention group mice is gradually higher than that of chronic DSS colitis mice (P is less than 0.05), and the distribution and the trend of a weight change rate curve can still indicate that the weight reduction and the recovery of the chronic DSS colitis mice are improved by BAFF antibody intraperitoneal injection.
BAFF antibody for improving colon inflammation of mice with chronic DSS (direct sequence of interstitial lung disease) colitis model
And (3) killing the mice after the modeling, taking out the colons of the mice, observing the gross change of the colons by naked eyes, measuring the length of the colons of each mouse, reserving colons, taking out the sections after the colons are embedded by paraffin, performing HE staining, and judging the damage degree of the colons. The same as the report in the literature, the colon length of the DSS chronic colitis model mouse is obviously shortened compared with that of the blank control group, the difference has statistical significance (P is less than 0.01), and the colon length of the BAFF antibody is obviously prolonged compared with that of the chronic DSS colitis model mouse after the prognosis of the BAFF antibody abdominal cavity injection, and the difference has statistical significance (P is less than 0.05). The results are shown in FIG. 3.
After HE staining of a mouse colon specimen, compared with a blank control group, the colon mucosa of a mouse in a DSS chronic colitis group is obviously damaged, a crypt structure is damaged, a large amount of inflammatory cells infiltrate into a submucosa, and the damage degree of the colon of the mouse after intervention of BAFF antibody is obviously reduced compared with that of the mouse in the DSS chronic colitis group. The results show that the BAFF monoclonal antibody can effectively relieve intestinal inflammation in DSS-induced colitis mice. Representative pathological sections are shown in FIG. 4.
As shown in FIG. 5, the inflammatory cytokines IL-1. beta. TNF-. alpha., IL-6, and TGF-. beta. of the terminal colon tissue of the three groups of mice were detected by RT-qPCR. The height of the column indicates the average value of the ratio of the mRNA level to the reference gene beta-actin, and the distance between the horizontal line above the column and the column indicates the standard deviation. The results show that the mRNA expression of IL-1 beta, TNF-alpha, IL-6 and TGF-beta is reduced to different degrees in the BAFF antibody intervention group and the DSS group mice, and further prove that the BAFF antibody can reduce the intestinal inflammation of the mice from the aspect of gene expression.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
- The application of the BAFF antibody in preparing a medicament for treating inflammatory bowel disease.
- 2. A medicament for the treatment of inflammatory bowel disease, characterized by: the effective component is BAFF antibody.
- Application of the BAFF antibody in preparing a medicament for preventing inflammatory bowel disease.
- 4. A medicament for preventing inflammatory bowel disease, characterized by: the effective component is BAFF antibody.
- Application of the BAFF antibody in preparing a medicine for preventing and treating organ injury caused by inflammatory bowel disease.
- 6. A medicine for preventing and treating organ injury caused by inflammatory bowel disease is characterized in that: the effective component is BAFF antibody.
- Application of the BAFF antibody in preparing a medicament for controlling inflammatory response caused by inflammatory bowel disease.
- 8. A medicament for controlling inflammatory response caused by inflammatory bowel disease, characterized by: the effective component is BAFF antibody.
- 9. The use according to claim 1 or 3 or 5 or 7, wherein the BAFF antibody is lymphostat-B or EGFP/scFv F8 or anti-BAFF scFv-Fc.
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Cited By (1)
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| CN114288386A (en) * | 2022-01-25 | 2022-04-08 | 华中科技大学同济医学院附属协和医院 | Application of Del-1 as new biomarker for inflammatory bowel disease and therapeutic drug |
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Inventor after: Fu Yu Inventor after: Zhang Ying Inventor before: Zhang Ying Inventor before: Fu Yu Inventor before: Chen Chaoyue Inventor before: Zhao Xi Inventor before: Tao Meihui Inventor before: Feng Qinyu |
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Application publication date: 20210622 |