Application of CD19 as diagnosis marker and treatment target of chronic obstructive pulmonary disease

Abstract

The invention relates to the technical field of biomedicine, in particular to application of CD19 serving as a chronic obstructive pulmonary disease diagnosis marker and a chronic obstructive pulmonary disease treatment target. The invention finds that the expression quantity of CD19 in the chronic obstructive pulmonary disease is increased and is in negative correlation with lung function indexes (FEV1 / FVC, FEV1% predicted); the number of CD19 positive cells in the lung of the patient with chronic obstructive pulmonary disease is increased for the first time; and it is found for the first time that the anti-CD19 antibody can relieve mouse emphysema and small airway collagen deposition caused by cigarette smoke exposure.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to the application of CD19 as a diagnostic marker and therapeutic target for chronic obstructive pulmonary disease. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by chronic respiratory symptoms (dyspnea, cough, sputum, acute exacerbation) caused by airway (bronchitis, bronchiolitis) and / or alveolar abnormalities (emphysema) leading to persistent and progressive airflow limitation. COPD is the most serious chronic respiratory disease in the world, with a mortality rate ranking third in the world, with more than 3 million deaths worldwide each year, seriously affecting people's life and health.

[0003] The pathogenesis of COPD is related to harmful exposure factors and host factors (i.e. environmental and genetic interaction). Studies have shown that a variety of factors can cause COPD, of which smoking is the most recognized environmental risk factor. A variety of mechanisms have been reported to play an important role in the initiation and evolution of COPD, including oxidative stress, lung innate cell damage, airway inflammation, imbalance between proteases and antiproteases, and autoimmunity. CD19 is a B cell surface antibody, and adaptive immune response is believed to be involved in the progression of COPD, but the role of CD19 in COPD is still unclear. SUMMARY

[0004] The present application encompasses the following technical solutions: One aspect of the present application relates to the use of a quantitative detection agent for CD19 in the preparation of a diagnostic kit for chronic obstructive pulmonary disease.

[0005] Another aspect of the present application relates to the use of an inhibitor of CD19 in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0006] The present application found that CD19 expression was increased in COPD, and was negatively correlated with lung function indicators (FEV1 / FVC, FEV1% predicted); for the first time, it was found that the number of CD19 positive cells in the lungs of COPD patients was increased; and for the first time, it was found that anti-CD19 antibody could alleviate emphysema and small airway collagen deposition in mice caused by cigarette smoke exposure.

[0007] These findings suggest that CD19 can become a target for the development of diagnostic and therapeutic products for COPD. BRIEF DESCRIPTION OF DRAWINGS

[0008] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings needed to be used in the specific embodiments or prior art description will be briefly introduced as follows. Obviously, the accompanying drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0009] Figure 1 Changes in CD19 expression in COPD and control lung tissues. (A) Volcano plot showing differentially expressed genes in COPD patients and controls. (B) CD19 expression in COPD and control lung tissues. (C) Receiver operating characteristic (ROC) curve for CD19 in lung tissue to predict COPD. (D-E) Spearman correlation analysis of CD19 expression in lung tissue with FEV1 / FVC and FEV1% predicted. (F) CD19 immunofluorescence staining and positive cell counts in lung tissue in normal and COPD patients. Scale bar represents 50 μm, data are presented as mean ± SEM, *p<0.05.

[0010] Figure 2 Therapeutic effects of anti-CD19 antibody on COPD mouse model. (A) Flow chart of COPD mouse modeling and anti-CD19 antibody administration. (B) Representative images of emphysema and mean linear intercept (MLI) statistical results. (C) Representative images and quantification of mouse small airway fibrosis. 6 mice per group, scale bar represents 100 μm, data are presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0011] Reference will now be made in detail to the embodiments of the present application, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the present application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0012] Unless otherwise indicated, all terms (including technical and scientific terms) used herein in the disclosure of the application have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. By further guidance, the following definitions are used to better define the present teachings. The terminology used in the specification of the application herein only for the purpose of describing specific embodiments and is not intended to limit the application.

[0013] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are the terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided as follows.

[0014] The selection range of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0015] The terms "containing", "including" and "comprising" used in the present application are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members, elements or method steps.

[0016] The numerical ranges in the present application expressed by endpoints include all the values and fractions contained in the range and the mentioned endpoints.

[0017] The term "about" or "approximately" used in the present application means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes specific numbers, for example, about 20 includes 20.

[0018] Furthermore, in describing representative embodiments of the present application, the specification can have presented the method and / or process of the present application as a particular sequence of steps. However, to the extent that the method or process depends on the performance of such steps, the

[0019] In the present application, the concentration values are intended to include fluctuations within a certain range. For example, there can be fluctuations within a corresponding range of accuracy. For example, 2% can be allowed to fluctuate within a range of ±0.1%. For values that are larger or do not need to be controlled too finely, it is also allowed that the meaning includes greater fluctuations. For example, 100 mM can be allowed to fluctuate within a range of ±1%, ±2%, ±5%, etc. For molecular weights, it is allowed that the meaning includes fluctuations of ±10%.

[0020] As used in the present application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] In the present application, the descriptions such as "a plurality of," "a plurality of kinds," etc. refer to greater than or equal to 2 in number unless otherwise specified.

[0022] In the present application, among the technical features described in an open-ended manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0023] In the present application, "preferably," "more preferably," "even more preferably," "suitably," and the like only describe embodiments or examples with better effects and should be understood as not constituting a limitation on the scope of protection of the present application. In the present application, "optionally," "optional," and "may" mean that it can or can not be present, i.e., it means that it is selected from either of the two parallel schemes "has" or "has not." If there are multiple "optionally" in a technical solution, each "optionally" is independent of each other unless otherwise specified, and there is no contradictory relationship or mutual restriction.

[0024] As used herein, the term "inhibitor" refers to a molecule that partially or completely inhibits the effects of another molecule through any mechanism. A CD19 inhibitor refers to a substance that reduces the bioavailability of CD19. As known to those skilled in the art, inhibitors can inhibit a molecule at the genomic level, transcriptional level, translational level, post-translational modification level, and other levels, as long as the level of a functional molecule is reduced. Examples of post-translational modifications include maturation of gene products or proteins, and post-translational modifications (e.g., glycosylation, alkylation, ubiquitination, etc.). Protein processing, transport, and release can also be modified, for example, by placement in storage organelles prior to release, or by binding to other proteins that affect release.

[0025] As used herein, the technical term "antibody" refers to a protein that binds to a specific antigen. It broadly refers to all proteins and protein fragments containing the complementarity determining regions (CDRs), particularly full-length antibodies or functional antibody fragments. The term "full-length antibody" encompasses both polyclonal and monoclonal antibodies. The term "antigen-binding fragment" refers to a substance that contains part or all of an antibody's CDRs, lacks at least some of the amino acids present in the full-length chain, and is still capable of specific antigen binding. "Antigen-binding fragments" include single-domain antibodies and fragments such as Fv, Fv', scFv, Fab, Fab', and F(ab')2.

[0026] For purposes of this invention, "diagnosis" refers to the identification or assessment of whether an individual has a specific disease, pathological condition, or abnormal physiological state, or is at risk of developing a disease, in the stage of disease progression, or in the phase of treatment response, by detecting, measuring, or analyzing one or more biomarkers, physiological parameters, clinical symptoms, behavioral indicators, or imaging results. Diagnosis encompasses both the initial diagnosis of a disease and the classification, staging, risk prediction, recurrence risk assessment, and treatment response assessment of the disease. Diagnosis can also be auxiliary diagnosis.

[0027] In the present invention, the term "treatment" refers to medical intervention on a disease, condition or pathological condition that an individual suffers from or is suspected of suffering from, with the purpose of achieving partial or complete relief, inhibition, improvement and / or alleviation, including but not limited to: alleviating or improving symptoms, delaying disease progression, reversing or stabilizing pathological processes, enhancing the individual's quality of life or functional status, and preventing the occurrence or aggravation of complications. The treatment is not limited to complete cure of the disease, but also includes symptomatic management, maintenance treatment and interventional control of disease risks.

[0028] All the documents mentioned in the present application are cited as references in the present application as if each document was individually cited as a reference. The documents mentioned in the present application are cited in their entirety, for all purposes, unless and except to the extent that such citation conflicts with the description of the present application or the patenting of the present application. The definitions of the relevant technical features, terms, names, phrases, etc. in the cited documents are also cited in the present application. The examples, preferred modes of the relevant technical features cited in the cited documents are also cited in the present application as references, if applicable. It should be understood that, when the cited content conflicts with the description in the present application, the present application is the correct one or the description in the present application is modified adaptively.

[0029] The first aspect of the present application relates to the use of a quantitative detection agent for CD19 for the manufacture of a diagnostic kit for chronic obstructive pulmonary disease.

[0030] In the present application, CD19 is used as a molecular marker for chronic obstructive pulmonary disease and is detected. In the present application, "CD19 mRNA" and "CD19 protein" can be replaced by "marker", "biomarker", "marker for chronic obstructive pulmonary disease" unless otherwise specified. It specifically refers to a molecule to be used as a target for analyzing patient test samples.

[0031] The molecular marker mRNA used as a marker in the present application is intended to include the full-length ribonucleotide sequence thereof, or naturally occurring variants, or fragments of the full-length sequence and variants, particularly fragments that can be detected and determined for the specific sequence, more preferably fragments that can be distinguished from other RNA sequences in blood. Preferably, at least 7, 8, 9, 10, 11, 12, 15, or 20 consecutive ribonucleotides of the full-length ribonucleotide sequence are included.

[0032] The molecular marker protein used as a marker in the present application is intended to include naturally occurring variants of the protein and fragments of the protein or the variants, particularly immunologically detectable fragments. The immunologically detectable fragments preferably include at least 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 consecutive amino acids of the marker polypeptide. For example, the expression "CD19 protein" includes the complete protein sequence of CD19, and the marker polypeptide as defined above.

[0033] It is also known that mRNA, protein or fragments thereof can also exist as part of a complex, and such a complex can also be used as a marker in the sense of the present application. In addition, in an alternative, the marker polypeptide or variant thereof can carry a post-translational modification. Non-limiting examples of post-translational modifications are glycosylation, acylation and / or phosphorylation. "Naturally occurring variants" are to be understood as meaning that the genes of higher animals are usually accompanied by a high frequency of polymorphisms. There are also a number of molecules which, in the course of splicing, give rise to isoforms which contain mutually different amino acid sequences.

[0034] In some embodiments, the quantitative detection agent comprises a reagent for detecting CD19 mRNA changes suitable for at least one of the following methods: nucleic acid sequencing, polymerase chain reaction, isothermal amplification reaction, resonance light scattering method, biological mass spectrometry, electrochemical analysis, gel electrophoresis, capillary electrophoresis, microarray, CRISPR-Cas detection system.

[0035] wherein the nucleic acid sequencing can be Maxammy-Gilbert sequencing, chain termination method, shotgun sequencing, bridge PCR, single molecule real-time sequencing, ion torrent sequencing, sequencing by synthesis, sequencing by ligation, chain termination, massively parallel tag sequencing, polymerase cloning sequencing, pyrosequencing, Illumina sequencing, DNA nanoball sequencing, single molecule real-time sequencing, nanopore sequencing, tunneling current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidic Sanger sequencing, RAP sequencing and in vitro viral high-throughput sequencing.

[0036] wherein the PCR is preferably RT-qPCR and digital PCR.

[0037] In some embodiments, the quantitative detection agent preferably comprises a probe and / or a primer capable of specifically binding to CD19 mRNA or cDNA. Preferably, the probe and / or the primer is labeled with a detectable label. The quantitative detection agent can be packaged in a kit in the form of a chip.

[0038] In some embodiments, the quantitative detection agent comprises a reagent for detecting CD19 protein changes suitable for at least one of the following detection methods: immunoassay, biological mass spectrometry, lectin-based detection method and nucleic acid aptamer-based detection method.

[0039] As a method for assaying the molecular marker of the present application, for example, as long as it is a method for specifically assaying the molecular marker protein, any method known as immunoassay, mass spectrometry, etc. can be used. Among the reagents used in assaying the marker protein of the present application, as a detection agent, an antibody, a lectin and an aptamer, etc. can be used.

[0040] As the immunoassay, various enzyme immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay, double monoclonal antibody sandwich immunoassay, monoclonal polyclonal antibody sandwich immunoassay, immunostaining method, immunofluorescence method, Western blotting method, biotin-avidin method, immunoprecipitation method, colloidal gold agglutination method, immunochromatography method, latex agglutination method, and immunoturbidimetry method, etc. can be cited.

[0041] As the reagent used in the immunoassay, an antibody or an antigen-binding fragment thereof against CD19 which has been already marketed can be used, or an antibody or an antigen-binding fragment thereof can be prepared based on the known amino acid sequence of CD19 by a conventional method.

[0042] As long as it is an antibody which can detect CD19, the animal species of origin and cloning are not particularly limited. Antibodies derived from rabbit, goat, mouse, rat, guinea pig, horse, sheep, camel, chicken, etc. can be cited, and both monoclonal antibodies and polyclonal antibodies can be used. In addition, an antibody which is suitable for specifically binding to all subclasses of CD19 protein can be used. Of course, a fragment such as a recombinant antibody, Fab, Fab', or F(ab')2 fragment can also be used.

[0043] In some embodiments, the quantitative detection agent comprises an antibody or an antigen-binding fragment thereof against CD19.

[0044] In some embodiments, the quantitative detection agent is preferably selected from the group consisting of an ELISA reagent, a Western blotting reagent, or an immunohistochemical reagent.

[0045] In some embodiments, the detection sample of the kit is selected from the group consisting of blood, sputum, cell or tissue extract, tracheal aspirate, bronchoalveolar lavage fluid.

[0046] A second aspect of the present application relates to use of an inhibitor of CD19 for the manufacture of a medicament for the treatment of chronic obstructive pulmonary disease.

[0047] According to the understanding of those skilled in the art, the function of the inhibitor includes one or more of the following: reducing the expression of CD19, reducing the translation of CD19, inhibiting the activity of CD19.

[0048] The above should be understood in accordance with the usual understanding of those skilled in the art, and since CD19 is a B cell-specific membrane protein, "inhibiting the activity of CD19" is generally understood to mean "eliminating B cells expressing CD19 or modulating B cell activity" or blocking the physiological function mediated by CD19 after binding to CD19.

[0049] In some embodiments, the inhibitor comprises at least one of an antisense oligonucleotide, a dsRNA, a siRNA, a shRNA, a miRNA, a RNAi, and a ribozyme, which is designed to reduce or block the expression of the CD19 gene.

[0050] In one embodiment of the present disclosure, as the inhibitor of CD19, a substance that specifically binds to the CD19 protein can be cited, and at least one of an antibody or an antigen-binding fragment thereof, a small molecule compound, an aptamer, an immune cell comprising a chimeric antigen receptor targeting CD19 (e.g., CAR-T), which is designed to reduce or offset the activity of the CD19 protein, can be cited.

[0051] In some embodiments, the inhibitor knocks down or knocks out the TCN2 gene. The knockdown or knockout can employ any known prior art, and knockdown or knockout of the CD19 gene by ZFN technology, TALEN technology, or CRISPR technology can be cited.

[0052] In some embodiments, the inhibitor comprises a gRNA and a Cas enzyme, or a vector capable of expressing the same. In some embodiments, the antagonist is a CRISPR-CAS gene editing composition / combination product. In some embodiments, it comprises a gRNA and a Cas enzyme (e.g., Cas9) or a vector capable of expressing the same.

[0053] In some embodiments, the inhibitor is composed of a pharmaceutically acceptable adjuvant to form a pharmaceutical composition.

[0054] The pharmaceutical composition can be formulated by a method well known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution or suspension of a sterile solution or suspension of a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. It is considered to be formulated by mixing in a unit dosage form required for generally accepted drug implementation. The amount of the active ingredient in these formulations is set to obtain an appropriate capacity within the indicated range.

[0055] The sterile composition for injection can be prescribed according to the usual formulation using an excipient such as distilled water for injection, etc.

[0056] As an aqueous solution for injection, an isotonic solution containing, for example, a physiological saline solution, lactose, glucose, and other supplements (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be cited. A suitable dissolution aid such as an alcohol (ethanol, etc.), a polyol (propylene glycol, polyethylene glycol, etc.), and a nonionic surfactant (polysorbate 80, etc.) can be used in combination.

[0057] As an oily liquid, sesame oil and soybean oil can be cited, and benzyl benzoate and / or benzyl alcohol can be used in combination as a dissolution aid. The composition can be mixed with a buffer (e.g., a phosphate buffer solution and a sodium acetate buffer solution), an analgesic (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled in a suitable ampoule.

[0058] The pharmaceutical composition is preferably administered by parenteral administration. For example, in some embodiments, the pharmaceutical composition is in the form of one or more of a solution, an injection, a spray, a nose drop, an aerosol, a powder mist, a tablet, a capsule, and a granule. For example, the pharmaceutical composition can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

[0059] In some embodiments, the subject of the pharmaceutical composition is a mammal.

[0060] In some embodiments, the subject of the pharmaceutical composition is a primate, such as a human.

[0061] Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In the following examples, the experimental methods not specified in the specific conditions can be preferably referred to the guidelines given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or can be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturers.

[0062] In the following specific examples, the measurement parameters of the raw material components may, without specific instructions, have slight deviations within the weighing accuracy range. With respect to the temperature and time parameters, acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed.

[0063] In the embodiments of the present application, the lung tissues of 12 patients with chronic obstructive pulmonary disease and 14 control persons were subjected to transcriptome sequencing, it was found that CD19 gene was highly expressed in patients with chronic obstructive pulmonary disease, the ROC curve showed that it had high specificity and sensitivity as a detection variable; and the results of Spearman correlation analysis showed that the expression level of CD19 gene was negatively correlated with the measured lung function indexes (FEV1 / FVC, FEV1% predicted); at the same time, it was proved by animal experiments that anti-CD19 antibody could relieve emphysema and small airway remodeling caused by cigarette smoke, suggesting that CD19 can become an important target for the development of drugs for treating chronic obstructive pulmonary disease. The present application can be applied to the fields of chronic obstructive pulmonary disease diagnostic reagent development, prevention and treatment drug research and development, etc.

[0064] Embodiments 1 Experimental design and experimental methods 1.1 Study population A total of 14 lung tissues from control group and 12 lung tissues from COPD group were included in this study. Lung tissues from COPD group were obtained from patients with chronic obstructive pulmonary disease who underwent lung transplantation. Normal lung tissues were obtained from patients who underwent lung surgery in the department of thoracic surgery. This study was approved by the ethics committee of China-Japan Friendship Hospital.

[0065] (1) Inclusion criteria of COPD group: 1) Patients who underwent single or double lung transplantation due to end-stage COPD in our hospital; 2) Patients who underwent pulmonary function test within 6 months: FEV1 / FVC <0.7 after inhalation of bronchodilators; 3) Patients who underwent chest CT and excluded diseases that could lead to respiratory failure such as pneumonia, interstitial pneumonia, bronchiectasis, etc.; 4) Patients who signed the informed consent form.

[0066] (2) Inclusion criteria of control group: 1) Patients who underwent lobectomy or wedge resection due to lung nodules in our hospital; 2) Patients who underwent pulmonary function test within 6 months: FEV1 / FVC ≥0.7 after inhalation of bronchodilators; 3) Patients who had no history of respiratory diseases; 4) Patients who signed the informed consent form.

[0067] (3) Exclusion criteria: 1) Patients who had allergic rhinitis, allergic conjunctivitis, urticaria, or other allergic diseases; 2) Patients who were taking hormone drugs; 3) Patients who were participating in clinical trials; 4) Patients who refused to sign the informed consent form.

[0068] 1.2 Transcriptome sequencing Total RNA was extracted using Trizol reagent according to the manufacturer's instructions. The quantity and purity of total RNA were analyzed using Bioanalyzer 2100, and RIN >7.0 was considered to be qualified. After removing ribosomal RNA, RNA fragments were reverse transcribed using Superscript II reverse transcriptase (Invitrogen, cat. 1896649, USA) to generate cDNA. Then, RNA sequencing was performed on Illumina Novaseq™6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) according to the recommended protocol of the supplier.

[0069] 1.3 Differential gene screening After filtering out unqualified sequences, the raw data obtained by sequencing was compared with the reference genome, and finally transcript assembly and quantitative analysis were performed. The p-value calculation model based on negative binomial distribution was used for p-value calculation and DESeq2 was used for differential gene analysis. The threshold criteria for screening differentially expressed genes were |log2FC| >=1 & adj.p <0.05.

[0070] 1.4 CD19 immunofluorescence staining Human lung sections were subjected to CD19 immunofluorescence staining and analysis. The specific experimental steps were as follows: after paraffin sections were deparaffinated with xylene, they were rehydrated with ethanol. Then, the sections were subjected to antigen retrieval with EDTA buffer (pH = 9.0) (Solarbio, Beijing, China). After blocking endogenous peroxidase activity, the sections were incubated with anti-CD19 primary antibody at 4°C overnight. Subsequently, Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody was added, and the sections were incubated at room temperature for 1 hour. Then, the sections were mounted with mounting medium containing DAPI and stored at 4°C in the dark. Images were acquired using a laser confocal microscope, and the number of CD19-positive cells was calculated.

[0071] 1.5 Animal experiments 6-8-week-old wild-type C57BL / 6J mice were purchased from Beijing Sibeier Biotechnology Co., Ltd. All mice were raised in a specific pathogen free (SPF) level animal room, the temperature was maintained at 24-26°C, the humidity was maintained at 60-70%, the mice were given a 12 h light / 12 h dark cycle every day, and the mouse feed and bedding were changed regularly. The mice were divided into indoor air control group (n = 6), cigarette smoke exposure + isotype control group (n = 6), and cigarette smoke exposure + anti-CD19 antibody treatment group (n = 6).

[0072] A mouse model of COPD was constructed using the classic cigarette smoke exposure method. Specifically, the mice were divided into a COPD group and a control group. The mice in the COPD group were exposed to cigarette smoke twice a day, each time for 2 hours. Twenty Marlboro cigarettes were lit at a uniform speed, and the cigarette smoke was introduced into the mouse exposure chamber to maintain the concentration of the cigarette smoke. After the exposure, the mice were returned to their cages. The exposure was performed 5 days a week. The mice in the control group were placed in clean indoor air under the same conditions as the COPD mice. The modeling period was 8 months. Then, the mice in the cigarette smoke exposure group were divided into an isotype control group and an anti-CD19 antibody treatment group, which were given rat IgG2a isotype control or anti-CD19 (1D3) antibody once a week, respectively, at a dose of 200 μg / mouse / time, for a total of 4 times.

[0073] 1.6 Lung tissue fixation and embedding The mouse abdominal cavity was exposed with scissors, and the inferior vena cava and renal vein were cut. The inferior vena cava was clamped at a higher position with a vascular clamp. A certain amount of physiological saline was injected into the right ventricle with a 1 mL syringe until the edges of the lung lobes turned white. The left main bronchus was clamped, and an appropriate amount of 4% paraformaldehyde fixing solution was injected into the left lung through the tracheal cannula. Subsequently, the left lung was fixed in neutral formalin for more than 72 h. The fixed tissue was dehydrated with gradient alcohol and xylene, embedded with paraffin, and prepared into 5 μm thick paraffin sections. After baking, the sections were used for H&E and Masson staining.

[0074] 1.7 H&E staining H&E staining procedure: (1) De-waxing and hydration: paraffin sections were immersed in xylene for 15 min x 2 times, after removing the excess liquid, immersed in anhydrous ethanol for 2 min x 2, then immersed in 95% ethanol, 90% ethanol, 85% ethanol, 75% ethanol for 2 min, respectively, and finally washed with distilled water for 1 min. (2) Cell nuclei were dyed with hematoxylin solution for 7 min and immersed in tap water. (3) Differentiated with 1% hydrochloric acid alcohol solution for 30 s and immersed in tap water. (4) Cytoplasm was dyed with eosin solution for 7 min and immersed in tap water. (5) Dehydration: 75% ethanol, 85% ethanol, 90% ethanol, 95% ethanol were used for 30 s, respectively, and 100% ethanol for 2 times, each for 30 s. (5) Transparency and mounting: xylene transparency for 2 times, each for 5 min, and neutral balsam sealing.

[0075] 1.8 Average linear intercept statistics Quantitative assessment based on average linear intercept was used to assess alveolar enlargement and emphysema in mice. MLI counting was performed on parenchymal tissue fields at 40x magnification (fields with airways or large vessels were excluded), with 10 evenly spaced horizontal lines overlaid on random fields, and the number of intersections between parenchymal tissue and horizontal lines was recorded. The MLI of each field was equal to the ratio of line length to the number of intersections, and the average number of 10 fields was recorded as the MLI of the section.

[0076] 1.9 Masson staining 2) Masson staining: (1) Paraffin sections were deparaffmized to water: paraffin sections were placed in xylene for 15 min x 2 times, after removing the excess liquid, they were immersed in two cylinders of absolute ethanol for 2 min each, then they were immersed in 95% ethanol, 90% ethanol, 85% ethanol, 75% ethanol for 2 min each, and finally washed with distilled water for 1 min. (2) The sections were placed in 2.5% potassium dichromate medium and immersed at room temperature overnight. (3) Weigert iron hematoxylin staining solution A and B were mixed in equal proportions, and the sections were immersed in the staining solution for 3 min, and the excess staining solution was washed off with tap water. (4) 1% hydrochloric acid alcohol differentiation for about 1 min until the cell nucleus was dark gray and the background was almost colorless or light gray. (5) Washed with tap water, drained the excess water on the sections, and immersed in acid fuchsin solution for 6 min, at which time the tissue appeared bright red. (6) Washed with tap water, drained the water, and differentiated with 1% phosphomolybdic acid solution for 1 min, until the collagen fibers were light red and the fibers were red. (7) Remove the 1% phosphomolybdic acid solution without water washing, and dye with 2.5% aniline blue solution for 15 s. (8) The sections were rinsed with 1% glacial acetic acid for 3 times, 8 s each time. (9) The sections were dehydrated with 3 cylinders of absolute ethanol for 5 s, 10 s, and 30 s respectively, then cleared with 2 cylinders of xylene for 5 min each, and mounted with neutral resin.

[0077] 1.10 Basement membrane thickness statistics Airway remodeling was evaluated by calculating the airway basement membrane thickness and normalizing to the perimeter of the basement membrane (Pbm). Specifically, 6 small airways (Pbm < 1000 pm) were randomly selected from the lungs of each mouse, and the collagen deposition area (represented in blue) around the small airways was normalized to the Pbm, and the average value was calculated.

[0078] 1.11 Statistical analysis Experimental data were analyzed using GraphPad Prism (v9.0.0). Normality test was performed using D'Agostino & Pearson method and Shapiro-wilk method. If the data were normally distributed, a non-paired t-test was used to compare the differences between the two groups; otherwise, a Mann-Whitney test was used. Spearman correlation analysis was used to test the relationship between two continuous variables. In all comparisons, a p-value less than 0.05 was considered significant, *p < 0.05, **p < 0.01, ***p < 0.001.

[0079] 2 Experimental results 2.1 Clinical and demographic characteristics of the study population The clinical and demographic characteristics of all subjects are shown in Table 1. There were no statistically significant differences in age, BMI, or smoking status between the COPD and control groups. However, there were statistically significant differences in FEV1 / FVC and FEV1% predicted, with the FEV1 / FVC and FEV1% predicted in the COPD group being significantly lower than those in the control group.

[0080] Table 1 Clinical and demographic characteristics of COPD patients and controls

[0081] Abbreviations: FEV1(forced expiratory volume in 1 second); FVC(forced vital capacity); BMI (body mass index). Data were presented as mean ± standard deviation. 2.2 CD19 expression is elevated in COPD and negatively correlated with lung function Through transcriptome analysis based on lung tissue, we found that the expression of CD19 in lung tissue of COPD patients was significantly increased (FC = 6.72, adj.p<0.001) ( Figure 1 A, B in the figure), and the sensitivity and specificity of CD19 for predicting COPD were 83.33% and 100%, respectively, with an AUC value of 0.893 ( Figure 1 C in the figure). Spearman correlation analysis showed that CD19 expression was negatively correlated with FEV1 / FVC (r = -0.56, p = 0.0028) and FEV1% predicted (r = -0.43, p = 0.029). Figure 1 These results suggest that CD19 has good specificity and sensitivity as a biomarker for predicting COPD, and that high CD19 expression is associated with the risk of decreased lung function.

[0082] 2.3 Increased proportion of CD19-positive cells in COPD CD19 immunofluorescence staining analysis of lung tissues from controls and COPD patients showed that the number of CD19-positive cells in COPD patients' lung tissues increased significantly, and they were mainly present in the form of lymphoid follicles ( Figure 1 F in ).

[0083] 2.4 Anti-CD19 antibodies slow the progression of COPD in mice To clarify the role of CD19 in COPD, we treated the chronic obstructive pulmonary disease model (cigarette smoke exposed mice) with anti-CD19 antibody and isotype control, respectively, 200 μg each time, 1 time per week, a total of 4 times, and then evaluated the severity of COPD in mice (A in Figure 2 The results show that: compared with the isotype control, the disease severity of mice is significantly alleviated after anti-CD19 antibody treatment in the cigarette smoke induced in vivo animal model, which is manifested as: 1) the emphysema of mice is alleviated, and the lung structure is restored to a certain extent (B in Figure 2 2) the collagen deposition of mouse small airway is inhibited, and the small airway structure is restored to normal (C in Figure 2 The above results show that CD19 is an effective therapeutic target for COPD, which is of great significance.

[0084] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of protection of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of a quantitative detection agent of CD19 for the preparation of a diagnostic kit for chronic obstructive pulmonary disease.

2. Use according to claim 1, wherein the quantitative detection agent comprises reagents for detecting CD19 mRNA variations, suitable for at least one of the following methods: nucleic acid sequencing, polymerase chain reaction, isothermal amplification reaction, resonance light scattering, biological mass spectrometry, electrochemical analysis, gel electrophoresis, capillary electrophoresis, microarray, CRISPR-Cas detection system; preferably the quantitative detection agent comprises probes and / or primers capable of specifically binding to CD19 mRNA or cDNA.

3. Use according to claim 1, wherein the quantitative detection agent comprises reagents for detecting CD19 protein variations, suitable for at least one of the following detection methods: immunoassay, biological mass spectrometry, lectin-based detection methods and nucleic acid aptamer-based detection methods; preferably the quantitative detection agent is selected from the group consisting of: ELISA reagents, Western blotting reagents or immunohistochemistry reagents.

4. Use according to any one of claims 1-3, wherein the detection sample of the kit is selected from the group consisting of: blood, sputum, cell or tissue extract, tracheal aspirate, bronchoalveolar lavage.

5. Use of an inhibitor of CD19 for the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.

6. Use according to claim 5, wherein the inhibitor comprises at least one of an antisense oligonucleotide, dsRNA, siRNA, shRNA, miRNA, RNAi and ribozyme designed to reduce or block the expression of the CD19 gene.

7. Use according to claim 5, wherein the inhibitor comprises at least one of an antibody or antigen-binding fragment thereof, a small molecule compound, an aptamer, an immune cell comprising a chimeric antigen receptor targeting CD19, designed to reduce or counteract the activity of the CD19 protein.

8. Use according to claim 5, wherein the inhibitor knocks down or knocks out the CD19 gene by ZFN technology, TALEN technology or CRISPR technology; preferably, the inhibitor comprises gRNA and Cas enzyme, or vectors capable of expressing them.

9. Use according to any one of claims 5-8, wherein the inhibitor is in a pharmaceutical composition with pharmaceutically acceptable excipients.

10. Use according to claim 9, wherein the pharmaceutical composition is in a form selected from one or more of the group consisting of solutions, injections, sprays, nose drops, aerosols, powder sprays, tablets, capsules and granules.

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