CN117210554A - Use of substances specifically binding to Dectin-2 for the preparation of diagnostic products for asthma - Google Patents

Abstract

The application relates to the field of biological medicine, in particular to application of a substance specifically binding to Dectin-2 in preparation of an asthma diagnosis product. The application can obviously distinguish asthma patients from non-asthma patients, in particular to Th2 cell-driven asthma, namely, the cytokine IL-4 secreted by Th2 cells of the asthma patients, the IL-13 is obviously increased compared with a healthy control group, and the Dectin-2 expression level is obviously increased; as a molecular target, the targeting inhibition of Dectin-2 activity can obviously improve inflammation of Th2 cell mediated asthma, and provides a direction for developing new medicines for treating Th2 cell mediated asthma.

Description

Use of substances specifically binding to Dectin-2 for the preparation of diagnostic products for asthma

Technical Field

The application relates to the field of biological medicine, in particular to application of a substance specifically binding to Dectin-2 in preparation of an asthma diagnosis product.

Background

Bronchial asthma is a chronic allergic inflammatory gas involving a variety of cells and cellular components, and the disease, airway hyperresponsiveness, is an essential feature of asthma, airway inflammation often causes subepithelial fibrosis, smooth muscle hyperplasia, vascular hyperplasia, goblet cell proliferation. Patients with asthma often show impaired immunomodulation, mainly with Th2 hyperactivity. The prevalence rate of asthma in China is 4.2% according to statistics, about 4750 thousands of asthmatics exist in China at present, and the good control rate of definite patients is only 30%.

Diagnosis of asthma relies primarily on clinical symptoms combined with airflow-limited laboratory tests, lacking effective biomarkers. The therapeutic drugs for asthma mainly comprise spasmolytic antiasthmatic drugs and anti-inflammatory drugs, and have more adverse reactions and are easy to tolerate after long-term use.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a use of a substance that specifically binds to Dectin-2 for the preparation of an asthma diagnostic product for solving the problems of prior art diagnosis of asthma patients mainly depending on clinical symptoms and lack of diagnostic molecules for lung function detection; the acute attack and the long-term control of asthma mainly depend on hormone medicines, and the problems of more side effects and easy tolerance of the hormone medicines for long-term use are solved.

To achieve the above and other related objects, the present application provides the use of a substance that specifically binds to Dectin-2 in the preparation of an asthma diagnostic product.

Preferably, the substance that specifically binds to Dectin-2 is selected from one or more of an antibody, a primer or a probe

The application also provides an asthma diagnosis kit which comprises a substance specifically binding to Dectin-2 in the application and a pharmaceutically acceptable carrier or auxiliary material.

The application also provides the use of a Dectin-2 inhibitor in the manufacture of a product for the treatment of asthma.

The present application also provides a composition for preventing or treating asthma, the active substance of the composition comprising: dectin-2 inhibitors for the aforementioned uses and pharmaceutically acceptable carriers, diluents or excipients

As described above, the use of the substance specifically binding to Dectin-2 of the present application for the preparation of an asthma diagnostic product has the following beneficial effects:

the application can obviously distinguish asthma patients from non-asthma patients, in particular to Th2 cell-driven asthma, namely, the cytokine IL-4 secreted by Th2 cells of the asthma patients, the IL-13 is obviously increased compared with a healthy control group, and the Dectin-2 expression level is obviously increased; as a molecular target, the targeting inhibition of Dectin-2 activity can obviously improve inflammation of Th2 cell mediated asthma, and provides a direction for developing new medicines for treating Th2 cell mediated asthma.

Drawings

FIG. 1 shows schematic representations of Dectin-2, IL-4 and IL-13 expression in asthmatic patients and healthy volunteers according to the application.

FIG. 2 is a schematic representation showing the results of the changes in cytokine secretion by T lymphocytes in the Dectin-2 knockout asthma model of the present application.

Detailed Description

The present application provides the use of a substance that specifically binds to Dectin-2 in the preparation of a diagnostic product for asthma.

In some embodiments, the substance that specifically binds to Dectin-2 is selected from one or more of an antibody, a primer, or a probe. Preferably, the substance that specifically binds to Dectin-2 is a primer. More preferably, the nucleotide sequence of the primer is shown as SEQ ID NO.1 and/or SEQ ID NO. 2.

In some embodiments, the asthma is T lymphocyte mediated asthma. More specifically, the T lymphocytes are selected from one or more of Th1 cells, th2 cells, th17 cells, or Treg cells. Preferably, the T lymphocyte mediated asthma is Th2 cell mediated asthma.

The application also provides an asthma diagnosis kit which comprises the substance specifically binding to Dectin-2 and a pharmaceutically acceptable carrier or auxiliary material.

The application also provides the use of a Dectin-2 inhibitor in the manufacture of a product for the treatment of asthma.

In some embodiments, the Dectin-2 inhibitor is selected from one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical, an antibody, a polypeptide, a protein, or a virus.

In some embodiments, the small molecule chemical comprises R406 or fosamatinib (R788) discodium. Wherein R406 or Fostamatinib (R788) discodium is an inhibitor of the Dectin-2 pathway.

In some embodiments, the asthma treatment product is a product for treating inflammation caused by asthma.

The nucleic acid molecule is selected from one or more of siRNA, shRNA or sgRNA. Preferably, the nucleic acid molecule is sgRNA or shRNA.

The virus is selected from one or more of lentivirus, adenovirus or adeno-associated virus.

The protein is selected from an anti-Dectin-2 antibody or an antibody that specifically binds to a Dectin-2 target.

Such antibodies are to be understood in their broadest sense and include monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) that contain at least two different antigen-binding regions. Antibodies may contain additional modifications such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins comprising an epitope of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

Further, the asthma treatment product treats asthma by inhibiting the expression or activity of Dectin-2 or a target thereof.

Inhibiting the expression or activity of Dectin-2 or its target refers to decreasing the expression activity of Dectin-2 or its target. Preferably, the expression activity of Dectin-2 or a target thereof is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90% compared to that prior to inhibition.

Inhibition of expression of Dectin-2 or a target thereof may specifically be inhibition of transcription or translation of a gene of Dectin-2 or a target thereof, and specifically may refer to: the method comprises the steps of preventing transcription of Dectin-2 or a target gene thereof, reducing the transcriptional activity of Dectin-2 or a target gene thereof, preventing translation of Dectin-2 or a target gene thereof, or reducing the translation level of Dectin-2 or a target gene thereof.

The person skilled in the art can use conventional methods to regulate the gene expression of Dectin-2 or its target, such as gene knockout, homologous recombination, interfering RNA, etc.

The inhibition of the gene expression of Dectin-2 or its target point can be verified by PCR and Western Blot detection.

Preferably, the gene expression of Dectin-2 or its target is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, yet more preferably by at least 90% compared to the wild type, and most preferably the gene of Dectin-2 or its target is not expressed at all.

Specifically, the asthma treatment product treats asthma by: cytokine secretion by Th2 cells is modulated by inhibiting expression of Dectin-2 or its target. More specifically, the asthma therapeutic product down-regulates one or more of the following cytokines secreted by Th2 cells by inhibiting the expression of Dectin-2 or its target: IL-4, IL-5, IL-9 and IL-13.

The application also provides a Dectin-2 gene or a gene interference nucleic acid construct of a target point thereof, wherein the construct is a viral vector.

The vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the carrier is further selected from one or more of the following: (1) a plasmid; (2) phagemid; (3) a cosmid; (4) Artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes, or artificial chromosomes derived from P1; (5) phage, such as lambda phage or M13 phage; (6) Animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, sporangia viruses, poxviruses, baculoviruses.

In some embodiments, the asthma is T lymphocyte mediated asthma. More specifically, the T lymphocytes are selected from one or more of Th1 cells, th2 cells, th17 cells, or Treg cells. Preferably, the T lymphocyte mediated asthma is Th2 cell mediated asthma.

The present application also provides a composition for preventing or treating asthma, the active substance of which comprises:

the aforementioned Dectin-2 inhibitors and pharmaceutically acceptable carriers, diluents or excipients.

The composition may be a pharmaceutical composition.

When the composition is used for preventing or treating asthma neoplasms in a subject, an effective amount of the composition is required to be administered to the subject.

The form of the composition is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.

The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.

By "pharmaceutically acceptable" is meant that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

In the present application, the pharmaceutical dosage form is not particularly limited unless otherwise specified, and may be formulated into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by conventional methods. The choice of the pharmaceutical dosage form should be compatible with the mode of administration.

The treatment is administration of a pharmaceutical agent or a procedure to obtain an effect. The foregoing treatments may include treating a disease or disorder (e.g., cancer) in a mammal, particularly in a human, including: (a) Preventing the occurrence of a disease or disease symptom (e.g., including diseases that may be associated with or caused by a primary disease) in a subject susceptible to the disease that has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any indication of success in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as elimination; relief; reducing symptoms or making the disease condition more tolerable to the patient; slowing the rate of deterioration or decay; or to reduce end point debilitation of deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of the physician's examination.

In the present application, the terms "Th1 cells", "Th2 cells", "Th17 cells" or "Treg cells" are subpopulations of T lymphocytes, th1 cells mainly secreting IFN- γ, IL-12 and TNF- β, wherein IFN- γ is a cytokine which is characteristic of its secretion; th2 cells mainly secrete IL-4, IL-5, IL-9 and IL-13, with IL-4 being a cytokine that is characteristic of its secretion; th17 cells are a subset of CD4+ T lymphocytes that specifically secrete IL17, IL-17A, IL-17F, and IL-23, non-specifically secrete IL-21, where IL17 is a cytokine that is characteristic of its secretion; treg cells are a subset of cd4+ T lymphocytes, typically divided into 2 subsets, namely a nTreg (natural Treg) subset and an iTreg (adaptive Treg) subset, which develop in the thymus, whereas iTreg develop peripherally from naive or other differentiated cells induced by antigen.

Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.

Before the embodiments of the application are explained in further detail, it is to be understood that the application is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the application is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the application; in the description and claims of the application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present application may be used to practice the present application according to the knowledge of one skilled in the art and the description of the present application.

Example 1Dectin-2 as a marker to distinguish between asthmatic patients and healthy people

Peripheral blood was collected from outpatient, confirmed asthma patients and healthy volunteers according to GINA guidelines, PBMCs were isolated and total RNA was extracted. RT-QPCR experiments were performed on the RNAs using the following primers: dectin-2 ((Forward) SEQ ID NO.1:5'-GCTTTCAGACCCACAAGGTAAT-3' and (Reverse) SEQ ID NO.2:5'

-GCAGAATGATTGGGCTCACCTA-3 ') and cytokine expression by Th2 cells (IL-4 (Forward) SEQ ID NO.3:5'-CCAACTGCTTCCCCCTCTG-3' and (Reverse) SEQ ID NO.4:5' -TCTGTTACGGT-CAACTCGGTG-3', IL-13 (Forward) SEQ ID NO.5:5'-CCTCATGGCGCTTTTGTTGAC-3' and (Reverse) SEQ ID NO.6:5'-TCTGGTTCTG GGTGATGTTGA-3').

The specific experimental process is as follows:

1. collecting peripheral blood (5 ml, anticoagulation tube) of each asthmatic patient and healthy control group;

2. carefully adding peripheral blood above Ficoll-Paque (GE healthcare) (mixing is reduced as much as possible) in a ratio of 1:1, and centrifuging by using a density gradient machine (2000 rpm. Times.20 min, note that the rising speed and the falling speed of the centrifuge are both 1); 3. after centrifugation, carefully aspirate the intermediate layer of cell suspension;

4. washing 1-2 times with PBS to obtain PBMC;

5. obtaining total RNA of PBMC by using Trizol (Invitrogen);

6. reverse transcription of the RNA using PrimeScriptTM RT Master Mix for RT-PCR (RR 036A, takara) reagent to obtain cDNA;

reaction conditions: preserving at 37 deg.C for 15min to 85 deg.C for 5sec to 4 deg.C

7. RT-PCR was performed using the cDNA obtained above with TB Green Premix Ex Taq II (CN 830S, takara);

8. RT-PCR was performed using a 7500Real Time PCR apparatus (Thermo Fisher) under the following reaction conditions:

as shown in FIG. 1, the results of the experiment show that the levels of Dectin-2 expressed by asthmatic patients are obviously increased compared with that of healthy human control groups, and the cytokines IL-4 and IL-13 expressed by Th2 cells of asthmatic patients are obviously increased (as shown in FIG. 1, the average levels of Dectin-2, IL-4 and IL-13 expressed by peripheral blood PBMCs of asthmatic patients are more than 2 times higher than those of healthy patients), which indicates that Dectin-2 is related to asthma and Th2 cells.

Example 2Dectin-2 Change in T lymphocyte secretion cytokines in a model of knock-out asthma

Asthma mouse model was constructed using HDM: female Dectin-2 ^+/+ Mice and Dectin-2 ^-/- Mice were given PBS or HDM extract (50 μg/50 μl, D.ptronysinus, XPB70D3A2.5, greer Laboratories) by nasal drops on days 0, 7, 14-18, respectively. 48 hours after the last challenge, mice were anesthetized with ketamine hydrochloride and thiazine, respectively, and alveolar lavage fluid from the mice was collected.

Alveolar lavage fluid from wild type asthmatic mice and Dectin-2 deficient asthmatic mice were collected, respectively, and cytokine expression levels in alveolar lavage fluid were detected with ELISA kits (IL-4 (Thermo Fisher, # BMS 613), IL-5 (Thermo Fisher, # BMS 610), IFN-gamma (Thermo Fisher, # BMS 606-2), IL-17 (Thermo Fisher, # BMS 6001) for different cytokines, respectively, wherein Dectin-2 ^-/- Asthma mice were given a professor An Maomao to the university of Shanghai, medical college.

The above results are shown in FIG. 2, dectin-2 ^-/- Cytokine IL-4 and IL-13 secreted by Th2 cells of asthma mice are obviously reducedLow, but no obvious change in cytokine IFN-gamma secreted by Th1 cells and cytokine IL-23 secreted by Th17 cells, the knockout of Dectin-2 can improve inflammatory manifestation in Th2 cell-mediated asthma.

The above examples are provided to illustrate the disclosed embodiments of the application and are not to be construed as limiting the application. Further, various modifications of the methods set forth herein, as well as variations of the methods of the application, will be apparent to those skilled in the art without departing from the scope and spirit of the application. While the application has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the application should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in the art are intended to be within the scope of the present application.

Claims (10)

1. Use of a substance that specifically binds Dectin-2 for the preparation of a diagnostic product for asthma.

2. The use according to claim 1, wherein the substance that specifically binds Dectin-2 is selected from one or more of an antibody, a primer or a probe.

3. Use according to claim 2, characterized in that the nucleotide sequence of the primer is shown in SEQ ID No.1 and/or SEQ ID No. 2.

4. The use according to claim 1, wherein the asthma is T lymphocyte mediated asthma.

5. The use according to claim 4, wherein the T lymphocytes are selected from one or more of Th1 cells, th2 cells, th17 cells or Treg cells; preferably, the T lymphocyte mediated asthma is Th2 cell mediated asthma.

6. An asthma diagnosis kit, which is characterized by comprising a substance specifically binding to Dectin-2 and a pharmaceutically acceptable carrier or auxiliary material.

Use of a dectin-2 inhibitor for the preparation of an asthma treatment product.

8. The use according to claim 7, wherein the Dectin-2 inhibitor is selected from one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical, an antibody drug, a polypeptide, a protein or a virus; and/or, the asthma treatment product is a product for treating inflammation caused by asthma.

9. The use of claim 8, further comprising one or more of:

1) The nucleic acid molecule is selected from one or more of siRNA, shRNA or sgRNA;

2) The virus is selected from one or more of lentivirus, adenovirus or adeno-associated virus;

3) The protein is selected from an anti-Dectin-2 antibody or an antibody that specifically binds to a Dectin-2 target.

10. A composition for preventing or treating asthma, wherein an active substance of the composition comprises: dectin-2 inhibitors and pharmaceutically acceptable carriers, diluents or excipients.