Systemic lupus erythematosus marker and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a systemic lupus erythematosus marker and application. And the biomarker is a CD71 + B cell. The invention provides application of a CD71 + B cell in prognosis evaluation, diagnosis or monitoring of systemic lupus erythematosus. The systemic lupus erythematosus specific diagnosis and / or curative effect evaluation marker provided by the invention has the advantages of noninvasive sampling, convenience, simplicity in operation, visual and clear result and easiness in popularization in detection, and is very suitable for early diagnosis and early treatment of systemic lupus erythematosus and curative effect evaluation of a treatment scheme.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a systemic lupus erythematosus marker and its use. Background Art

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of large amounts of autoantibodies and inflammatory cytokines. Clinical manifestations vary, including rash, arthralgia and arthritis, alopecia, mucosal ulcers, and fever. The most severe manifestations are often caused by end-organ involvement, such as kidney disease (lupus nephritis), neurological involvement, and heart and lung disease. Globally, the prevalence of SLE is 43.7 per 100,000 people, affecting 3.41 million people. In China, the prevalence is 47.53 per 100,000 people, affecting approximately 700,000 people. The incidence is higher in women than in men, with a particularly severe impact on women between adolescence and menopause. Currently, known SLE causative factors can be divided into two categories: 1) genetic factors, including rare single gene defects in complement components C1Q and C4, and mutations in interferon (IFN) regulatory factor 5 (IRF5), which are the most closely associated with SLE; and 2) environmental risk factors, including ultraviolet radiation exposure, Epstein-Barr virus (EBV) infection, immune system disorders, and various medications. The diverse clinical features and complications of SLE increase the complexity and mortality of the disease, leading to a significant impact on the physical and mental health and quality of life of the population.

[0003] The primary etiology of SLE is defective production and clearance of autoantibodies. Autoantibodies form immune complexes that accumulate in the bloodstream and tissues, activating complement and inflammatory cytokines, leading to an immune attack against the body's own tissues, resulting in systemic inflammation and tissue damage. These reactions are caused by dysregulation of both innate and adaptive immune responses. B lymphocytes are the primary producers of autoantibodies, and targeting abnormally activated B cells is a major new target for the treatment of SLE.

[0004] Iron, as a component of proteins such as Fe-S clusters, is essential for the proliferation, differentiation, and antibody production of B lymphocytes. The homeostasis of intracellular iron depends on the expression level and activity of iron carriers, iron transporters, and iron regulatory and storage proteins. Transferrin receptor 1 (TfR1, also known as CD71) is a transmembrane single-chain glycoprotein with a molecular weight of approximately 95 kDa and is also the main iron import receptor of cells. In peripheral blood, free Fe 3+It combines with apotransferrin (apo-Tf) to form an Fe-Tf complex (holotransferrin, holo-Tf). In target cells, holo-Tf binds to TfR1 on the cell membrane. The TfR1-Tf-Fe complex transports iron ions into the cell through receptor-mediated endocytosis. In the acidic environment of the endosome, Fe 3+ It is released from Tf-TfR1 and reduced to Fe by the metalloreductase STEAP3 (six-transmembraneepithelial antigen of the prostate 3). 2+ ‎, and then transported to the cytoplasm by divalent metal transporter 1 (DMT1), while Tf-TfR1 is recycled to the cell surface and apo-Tf is released into the circulation. 2+ Iron can enter the mitochondria for metabolic processes (such as the synthesis of hemoglobin and Fe-S clusters) or be stored in ferritin as cellular storage iron. Excess iron is transported out of the cell via ferroportin 1 (FPN1), which is located on the cell membrane.

[0005] Abnormal intracellular iron metabolism has been reported to be involved in the pathogenesis of lupus, particularly renal lesions. Administration of the iron chelator deferiprone or other measures to inhibit the accumulation of intracellular iron can significantly delay the onset of proteinuria and reduce blood urea nitrogen levels. Therefore, targeting cellular iron metabolism may provide a new treatment strategy for lupus patients. Summary of the Invention

[0006] The purpose of the present invention is to provide a systemic lupus erythematosus marker and its use, so as to achieve effective treatment of systemic lupus erythematosus.

[0007] To this end, the present invention provides the following technical solutions.

[0008] A first aspect of the present invention provides an application as described in any one of the following: (1) Quantitative detection of CD71 ＋ Use of a reagent for measuring B cell levels in the preparation of a product for prognostic assessment, diagnosis or monitoring of systemic lupus erythematosus; (2) Inhibition of CD71 ＋ Use of a B cell level reagent in preparing a pharmaceutical composition for treating systemic lupus erythematosus; (3) CD71 ＋ Application of B cells in screening drug candidates for the treatment of systemic lupus erythematosus; and (4) CD71＋ Application of B cells in constructing a computational model for predicting systemic lupus erythematosus.

[0009] A second aspect of the present invention provides a pharmaceutical composition for treating systemic lupus erythematosus, comprising: (i) Inhibition of CD71 ＋ Reagents for B cell levels; and (ii) pharmaceutically acceptable carriers and / or excipients.

[0010] In a preferred embodiment of the present invention, the inhibition of CD71 ＋ Reagents for B cell levels include anti-human CD71 antibodies modified with different fluorescent groups.

[0011] In a preferred embodiment of the present invention, the inhibition of CD71 ＋ Reagents at the B cell level are CART cells targeting CD71 and / or bispecific T cell engagers targeting CD71.

[0012] The third aspect of the present invention provides a product for prognosis assessment, diagnosis or monitoring of systemic lupus erythematosus, said product comprising a method for quantitatively detecting CD71 in a sample. ＋ Chips, kits or nucleic acid membrane strips for B cell levels.

[0013] In a preferred embodiment of the present invention, the chip comprises a chip capable of detecting CD71 ＋ Reagents for B cell levels.

[0014] In a preferred embodiment of the present invention, the kit includes a method for detecting CD71 by flow cytometry or immunofluorescence staining. ＋ Reagents for B cell levels.

[0015] In a preferred embodiment of the present invention, the nucleic acid membrane strip comprises a nucleic acid membrane strip capable of detecting CD71 ＋ Reagents for B cell levels.

[0016] In a preferred embodiment of the present invention, the sample is derived from peripheral blood.

[0017] A fourth aspect of the present invention provides a method for screening candidate drugs for treating systemic lupus erythematosus, comprising: Treat cells expressing or containing CD71 with substances to be screened ＋ B cell culture system; and Detection of CD71 in the system ＋ B cell levels; Wherein, when the substance to be screened inhibits CD71 ＋When the expression of the antigen-binding protein is detected at the B cell level, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

[0018] By means of the above technical solution, the present invention has at least the following advantages: The present invention provides CD71 + Application of B cells in the prognosis assessment, diagnosis or monitoring of systemic lupus erythematosus. The inventors found that compared with healthy volunteers, the peripheral blood of patients with systemic lupus erythematosus had a higher level of CD71 + The proportion of B cells increased significantly. Therefore, the detection of CD71 + The reagent for measuring the content of B cells in a biological sample can be used as a detection reagent for systemic lupus erythematosus and for preparing a systemic lupus erythematosus detection kit.

[0019] The present invention provides a specific diagnostic and / or efficacy evaluation marker for systemic lupus erythematosus. The detection thereof is non-invasive and convenient in sampling, simple in operation, and the results are intuitive and clear, and easy to promote. It is very suitable for the early diagnosis and treatment of systemic lupus erythematosus, as well as the efficacy evaluation of treatment plans.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The following figure shows the expression of CD71 on B cells in the peripheral blood and spleen of mice in different treatment groups; A is the gating strategy for flow cytometry analysis of CD71 expression on B cells; B is the flow cytometry detection of CD71 expression on spleen and peripheral B cells in R848-induced lupus mice (B6+R848), control mice (B6+acetone), and fas gene knockout lupus mice (MRL / lpr); C is the flow cytometry detection of TLR7 expression on B cells in lupus mice; D is the purity of B cells after flow cytometry sorting; E is the RT-qPCR detection of CD71 gene expression on B cells in lupus mice (Tfrc); Figure 2 The figure shows the expression of CD71 on mouse spleen B cells stimulated by R848 in vitro; wherein, A shows the effect of different concentrations of R848 on the activity of mouse spleen cells; B shows the proportion of CD19+ B cells in mouse spleen cells treated with different concentrations of R848; C shows the expression of CD71 on B cells in mouse spleen cells treated with different concentrations of R848; D shows the effect of different concentrations of TLR7 inhibitor on cell activity; E shows the effect of different concentrations of TLR7 inhibitor on the proportion of B cells in spleen cells; F shows the effect of different concentrations of TLR7 inhibitor on CD71 expression on B cells; Figure 3The figure shows the production of iron and lipid peroxides in B cells mediated by CD71. A is the flow cytometric analysis of the expression of mouse B cells after CD71 blockade in vitro. B and B are the flow cytometric analysis of intracellular iron, ROS (reactive oxygen species), and lipid peroxide C11 after anti-CD71 blockade of R848-stimulated B cells. Figure 4 The effects of CD71-mediated peroxide production on B cell proliferation, activation, and differentiation are shown; A is the flow cytometry analysis of B cell proliferation in R848-induced lupus mice (proportion of Ki67-positive cells); B is the flow cytometry analysis of CD86 expression in B cells in R848-induced lupus mice; C is the flow cytometry analysis of the proportion of plasma cells in R848-induced lupus mice; D is the proliferation of mouse B cells after treatment with R848 in vitro; E is the expression of CD86 in mouse cells after treatment with R848 in vitro; F is the proportion of plasma cells in mouse cells after treatment with R848; Figure 5 The expression of TLR7 and CD71 on B cells of patients with active lupus is shown; A is the expression of TLR7 on B cells of healthy controls, inactive lupus patients and active lupus patients detected by flow cytometry; B is the expression of CD71 on B cells of healthy controls, inactive lupus patients and active lupus patients detected by flow cytometry; C is the correlation between the expression of CD71 and TLR7 on B cells of lupus patients; D is the expression of CD71 + ROC curve of B cell evaluation in lupus patients. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0023] Current treatment strategies for systemic lupus erythematosus (SLE), such as glucocorticoids, immunosuppressants, and anti-CD20 monoclonal antibodies, primarily rely on broad-spectrum immunosuppression or non-selective B cell depletion. However, these approaches suffer from the following causal chain flaws: 1. Existing treatments inadequately regulate pathogenic B cell subsets. ① Specific B cell subsets (such as DN2 B cells) are the primary source of autoantibodies in SLE pathology. ② Existing therapies are unable to precisely target pathogenic B cell subsets. ③ This can lead to treatment failure or relapse (e.g., inefficient depletion of DN B cells by anti-CD20 monoclonal antibodies). 2. The pathological mechanisms of the TLR7 signaling pathway have not been effectively addressed. SLE B cells exhibit TLR7 overactivation, abnormal upregulation of the P53 / CD71 signaling axis, iron overload and accumulation of oxidized lipids, and the promotion of DN2 B cell expansion and antibody secretion through ROS. Existing therapies lack specificity to block the CD71-ROS positive feedback loop. 3. Currently, there are limitations in the regulation of oxidative stress in the treatment of SLE patients. CD71-mediated iron influx is a key pathway for B cell ferroptosis and lipid peroxidation; broad-spectrum antioxidants cannot inhibit CD71-dependent ROS production; and oxidative damage continues to aggravate organ pathological changes.

[0024] To address the above shortcomings, the present invention provides a precise treatment strategy targeting CD71. It can target pathogenic B cell subsets and avoid the infection risk caused by broad-spectrum immunosuppression by selectively inhibiting DN2 B cells with high CD71 expression.

[0025] To this end, the present invention provides any of the following applications: (1) Quantitative detection of CD71 ＋ Use of a reagent for measuring B cell levels in the preparation of a product for prognostic assessment, diagnosis or monitoring of systemic lupus erythematosus; (2) Inhibition of CD71 ＋ Use of a B cell level reagent in preparing a pharmaceutical composition for treating systemic lupus erythematosus; (3) CD71 ＋ Application of B cells in screening drug candidates for the treatment of systemic lupus erythematosus; and (4) CD71 ＋ Application of B cells in constructing a computational model for predicting systemic lupus erythematosus.

[0026] In one embodiment of the present invention, a pharmaceutical composition for treating systemic lupus erythematosus is provided, comprising: (i) Inhibition of CD71 ＋ Reagents for B cell levels; and (ii) pharmaceutically acceptable carriers and / or excipients.

[0027] In some embodiments of the present invention, the inhibition of CD71 ＋ Reagents for B cell levels include anti-human CD71 antibodies modified with different fluorescent groups.

[0028] In some embodiments of the present invention, the inhibition of CD71 ＋ Reagents at the B cell level are CART cells targeting CD71 and / or bispecific T cell engagers targeting CD71.

[0029] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes oral preparations such as tablets, powders, granules, capsules, pills, sustained-release pellets, solid dispersions, inclusion compounds, liquid preparations such as suspensions, emulsions, melts, syrups, mixtures, solutions, injectable preparations such as injection solutions, aqueous or oily suspensions, emulsions, liposomes, microcapsules, microspheres, nanoparticles, sustained-release or controlled-release preparations, preferably injectable preparations.

[0030] The pharmaceutically acceptable carrier or excipient described in the present invention refers to additives commonly used in the pharmaceutical field other than active ingredients, including but not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants. Among them, diluents such as lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and bentonite, etc.; lubricants such as zinc stearate, monostearate, etc. Fatty acid glycerides, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides.

[0031] The pharmaceutical composition of the present invention may further include additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants. Stabilizers include human serum albumin, L-amino acids, sugars, and cellulose derivatives. L-amino acids may also include any one of glycine, cysteine, and glutamic acid. Carbohydrates include monosaccharides such as glucose, mannose, galactose, and fructose; sugar alcohols such as mannitol, inositol, and xylitol; disaccharides such as sucrose, maltose, and lactose; and polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, and their derivatives. Cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium hydroxymethylcellulose. Surfactants include ionic or nonionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, and fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and their corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium salts, potassium salts, calcium salts, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars, and glycerol. Chelating agents include sodium edetate and citric acid.

[0032] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or injection is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, excipient, or vehicle.

[0033] In another embodiment of the present invention, a product for prognosis assessment, diagnosis or monitoring of systemic lupus erythematosus is provided, wherein the product comprises a method for quantitatively detecting CD71 in a sample. ＋ Chips, kits or nucleic acid membrane strips for B cell levels.

[0034] In some embodiments of the present invention, the chip comprises a chip capable of detecting CD71 ＋ Reagents for B cell levels.

[0035] In some embodiments of the present invention, the kit includes detecting CD71 by flow cytometry or immunofluorescence staining. ＋ Reagents for B cell levels.

[0036] In some embodiments of the present invention, the nucleic acid membrane strip includes a nucleic acid capable of detecting CD71 ＋ Reagents for B cell levels.

[0037] In some embodiments of the present invention, the sample is derived from peripheral blood.

[0038] Yet another embodiment of the present invention provides a method for screening candidate drugs for treating systemic lupus erythematosus, comprising: Treat cells expressing or containing CD71 with substances to be screened ＋ B cell culture system; and Detection of CD71 in the system ＋ B cell levels; Wherein, when the substance to be screened inhibits CD71 ＋ When the expression of the antigen-binding protein is detected at the B cell level, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

[0039] The culture system includes, but is not limited to, a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, rabbit, sheep or monkey).

[0040] In an embodiment of the present invention, the method further comprises: further testing the candidate drug obtained in the above step for its effect of inhibiting systemic lupus erythematosus; if the test compound has a significant inhibitory effect on systemic lupus erythematosus, it indicates that the candidate drug is a candidate drug for treating systemic lupus erythematosus.

[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and Examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Simple modifications to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0042] The following embodiments relate to and mention: 1. Data Analysis Unpaired t-tests were used to analyze differences between the HC and SLE groups; P < 0.05 was considered statistically significant. All clinical data are presented as mean ± standard deviation (m ± s). Pearson correlation analysis was used to investigate correlations between clinical parameters. Correlations were considered significant when r values > 0.3 and P < 0.05. Statistical analysis and graphics were performed using GraphPad Prism 9 software.

[0043] Example 1: CD71 expression in peripheral blood and spleen B cells of R848-induced lupus mice A systemic lupus erythematosus mouse model was established by inducing R848, and the expression of CD71 in peripheral blood and spleen B cells of normal and model mice was compared. The specific steps included: The substance used to induce the model mice was R848, whose chemical name is Resiquimod and whose Chinese name is Ruiqunmod. R848 is an agonist of Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8).

[0044] The R848-induced lupus model in mice was established by feeding 7-week-old SPF female C57BL / 6 (B6) mice for one week. The mice were then randomly divided into a B6+acetone solvent group (B6+acetone) and a B6+R848 disease modeling group (5 mice in each group). The mice in each group were treated as follows: B6+R848 disease model group (B6+R848): 35 μL of R848 (2 mg / ml) dissolved in acetone was applied to the outer ear edge of one side of the mouse, once every three days, for 12 consecutive treatments; B6+acetone solvent group (B6+acetone): 35 μL of acetone was applied to the outer ear edge of one side of the mouse, once every three days, for 12 consecutive treatments; MRL / lpr group: Five non-TLR7 / 8 activated MRL / lpr lupus mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were also selected.

[0045] Mouse peripheral blood mononuclear cells (PBMCs) were then isolated as follows: 1-2 mL of mouse blood was collected and centrifuged at 1650 rpm for 5 minutes. The upper plasma layer was transferred to a 1.5 mL EP tube, numbered, and stored at -80°C. An equal volume of 1× PBS was added to the lower blood cell layer, mixed and diluted, and then slowly added along the wall of the centrifuge tube to the upper layer of mouse lymphocyte separation solution, ensuring a 1:1 ratio of blood cell diluent to mouse lymphocyte separation solution. The tube was then placed in a centrifuge and centrifuged at 2000 rpm for 20 minutes at room temperature. At this point, the centrifuge tube was divided into four layers from top to bottom: the plasma layer, the cloudy mononuclear cell layer (PBMCs layer), the transparent separation layer, and the red blood cell layer. The cloudy cells in the second layer were carefully aspirated and placed in a new centrifuge tube. The tube was resuspended in an appropriate amount of 1× PBS and centrifuged at 1650 rpm for 5 minutes at room temperature. This operation was repeated twice. After centrifugation, the supernatant was discarded and the tube was resuspended in 1 mL of 1× PBS and counted using a cell counter.

[0046] Flow cytometry was used to detect the expression of CD71 and TLR7 in B cells of mice in each group. 6After centrifugation, resuspend the cells in 90 μL flow cytometry buffer, add 10 μL mouse Fc receptor blocker, and incubate at 4°C in the dark for 10 minutes; then wash with flow cytometry buffer, centrifuge at 1650 rpm, 4°C for 5 minutes, and discard the supernatant; resuspend the cells in 100 μL flow cytometry buffer, add Fixable Viability Dye eFluor™ 506, incubate at 4°C in the dark for 10 minutes, repeat the above washing and centrifugation steps; resuspend the cells in 100 μL flow cytometry buffer, add anti-mouse CD45 APC (Biolgend, USA, cat. no. 103112) diluted 1:400, anti-mouse CD19 percp-Cy5.5 (Biolgend, USA, cat. no. 115534) diluted 1:300, and anti-mouse CD71 BV421 (Biolgend, USA, Cat. No. 113813), incubated at 4°C in the dark for 25 minutes; after washing, resuspend the cells in 100 μL of cell membrane permeabilization fixative and fix and permeabilize at 4°C for 30 minutes; add 100 μL of 1× cell membrane permeabilization wash to each well, centrifuge at 700g and 4°C for 5 minutes, and discard the supernatant; add 100 μL of anti-mouse TLR7 PE antibody (Biolgend, USA, Cat. No. 160004) diluted 1:200 in 1× cell membrane permeabilization wash to each well, resuspend the washed cells, incubate at 4°C in the dark for 30 minutes, repeat the above centrifugation and discard the supernatant operation; finally, resuspend the cells in 200 μL of flow staining buffer in each well into a flow tube, detect using a Fortessa flow cytometer, and the obtained data are analyzed using FlowJo software.

[0047] See the results Figure 1 .like Figure 1 As shown in the figure, by comparing the expression of CD71 in B cells of spleen cells and peripheral PBMCs of R848-induced lupus mice (B6+R848) and control mice, the results showed that compared with the control group, the expression of CD71 in spleen and PBMCs of R848-induced lupus mice was significantly higher than that of the control group. + CD19 cells + The proportion of B cells increased. Flow cytometry sorting of B cells in mouse spleens and RT-qPCR detection revealed that the transcription level of the CD71-expressing gene Tfrc was significantly increased in R848-induced lupus mice.

[0048] Example 2: In vitro R848 stimulation can also induce B cell CD71 expression In order to investigate whether in vitro R848 stimulation would produce similar phenomena as in vivo, this example further conducted an in vitro R848 stimulation cell experiment. First, to determine the optimal concentration of R848 to promote CD71 expression, B6 + acetone mouse spleen cells (B6 + acetone) were treated with 100, 200, 500, and 1000 ng / mL of R848, and the effects of different concentrations of R848 on cell viability and other factors were tested. The results are shown in Table 1. Figure 2 .like Figure 2 The results showed that compared with the blank control group, the cell viability of the 100 ng / mL R848 treatment group increased (e.g. Figure 2 A), there was no significant difference in the ratio of CD19⁺ B cells to CD45⁺ cells (as shown in Figure 2 B), and the expression of CD71 membrane protein on the surface of B cells increased by about 4 times (as shown in Figure 2 Based on this result, 100 ng / mL was selected as the concentration of R848 in subsequent in vitro experiments.

[0049] To verify whether the expression of CD71 is regulated by TLR7, this example further examined the changes in CD71 expression after treatment of each group of cells with different concentrations (250, 500, 750, and 1000 nM) of the TLR7 inhibitor Enpatoran (M5049). Flow cytometry revealed that compared with the blank control, the cell viability (as shown in Figure 2D) and the ratio of CD19⁺ B cells to CD45⁺ cells (as shown in Figure 2D) in the M5049 treatment groups at 250, 500, 750, and 1000 nM were significantly increased. Figure 2 E) showed a downward trend, and the expression of CD71 in B cells decreased in a concentration-dependent manner (as shown in Figure 2 F), where the most significant reduction was observed at 1000 nM. Therefore, 1000 nM was selected as the concentration of M5049 used in subsequent experiments.

[0050] In addition, spleen B cells from B6+acetone mice were sorted and divided into control group, R848 group, and M5049 group, with 3×10 cells in each group. 5 Each group was processed as follows: Control group: cultured with only RPMI 1640 +10% FBS complete medium; M5049 group: 100 ng / mL R848 and 1000 nM M5049 were added to the control group; R848 group: 100 ng / mL R848 was added to the control group.

[0051] After 24 hours of culture, the Tfrc mRNA levels in each group were detected. Figure 2 G. As Figure 2 G The results showed that compared with the control group, the Tfrc mRNA level increased approximately 4-fold after R848 stimulation (R848 group), while M5049 could reverse this phenomenon (M5049 group).

[0052] Example 3: CD71 mediates B cell iron and lipid peroxide production To investigate whether the increase in B cell peroxides induced by TRL7 is mediated by CD71, this example further conducted an in vitro CD71 blocking experiment on B cells isolated from mice in the B6+acetone group in Example 1. The sorted B cells were divided into two groups, namely the B cell+R848+isotype control group and the B cell+R848+anti-CD71 group, with the number of cells in each group being 3×10 5 Each group of cells was treated as follows: B cell + R848 + isotype control group (isotype group): B cells were added with 100 ng / mL R848 and 2 μg / mL IgG2a,κ isotype control; B cell + R848 + anti-CD71 group (anti-CD71 group): 100 ng / mL of R848 and 2 μg / mL of anti-mouse CD71 antibody were added to B cells.

[0053] The cells were cultured for one day and three days respectively. To ensure the survival of B cells in the in vitro environment, two stimulators, recombinant CD40L antibody and IL-21, were added to the culture system of both groups. After one day of culture, the expression of CD71 on the cell surface of each group was detected by flow cytometry. The results are shown in Figure 2. Figure 3 .like Figure 3 As shown, compared with the isotype group, CD71 in the anti-CD71 group was basically blocked ( Figure 3 A). Furthermore, the experiment found that blocking R848-stimulated B cells with anti-CD71 antibodies reduced intracellular levels of ferrous ions (Fe²⁺), reactive oxygen species (ROS), and lipid peroxide C11 (Figure 3 BD). These results suggest that CD71 promotes Fe²⁺ accumulation in B cells of lupus mice, thereby inducing the production of lipid peroxides.

[0054] Example 4: Plasma cell differentiation and autoantibody secretion are dependent on CD71 In the adaptive immune response, B cell proliferation, activation, and differentiation into autoantibody-secreting cells are key mechanisms in the pathogenesis of lupus. Iron and reactive oxygen species (ROS) play important roles in B cell function and activation and differentiation, respectively. This study aimed to investigate the effects of CD71-mediated peroxide production on B cell proliferation, activation, and differentiation.

[0055] To understand the function of B cells in lupus mice, flow cytometry was used to detect the related functions of B cells in lupus mice induced by R848 in Example 1. By comparing the B6+acetone group mice and the B6+R848 group mice, it was found that the proportion of Ki-67⁺ cells in B cells of R848-induced mice (B6+R848 group) increased nearly threefold ( Figure 4 A), the proportion of CD86⁺ cells increased approximately twofold ( Figure 4 B), indicating that R848 significantly promoted B cell proliferation and activation; the proportion and absolute number of CD19⁻CD138⁺ plasma cells increased several times ( Figure 4 C) confirmed that R848 promoted the differentiation of B cells into plasma cells, and the in vitro experimental results were similar to those in vivo ( Figure 4 DF). These results indicate that B cells in R848-induced lupus mice are functionally active, providing a reference for exploring the abnormal mechanisms of B cells in lupus.

[0056] Flow cytometry analysis showed that blocking CD71 in vitro did not significantly affect B cell proliferation ( Figure 4 G), activation marker CD86 showed an upward trend ( Figure 4 H), the proportion of plasmablasts decreased ( Figure 4 I). The secretion of antibodies in the culture supernatant was detected. The results showed that compared with the isotype group, anti-CD71 treatment reduced the levels of anti-ANA antibodies and anti-ds-DNA antibodies in the B cell culture supernatant ( Figure 4 JK).

[0057] Example 5: CD71 expression on B cells of lupus patients activated by TLR7 In this example, flow cytometry was used to detect CD71 in peripheral blood samples from patients with systemic lupus erythematosus and healthy controls. + The proportion of B cells and the detection of CD71 on B cells of lupus patients after TLR7 activation + changes in the situation.

[0058] 1. Sample Source 1.1 Systemic lupus erythematosus patient group (patient group) Inclusion criteria: Patients met the diagnostic criteria for SLE revised by the American College of Rheumatology (ACR) in 1997.

[0059] Exclusion criteria: patients with other autoimmune diseases, critical illness, concurrent infection, tumor and other serious diseases, pregnant or breastfeeding patients, and those who refused to participate.

[0060] 1.2 Healthy control group Inclusion criteria: Gender and age matched healthy subjects.

[0061] Exclusion criteria: history or family history of autoimmune diseases.

[0062] According to the above criteria, a total of 23 patients with systemic lupus erythematosus and 14 healthy controls were collected. There was no statistical difference in age and gender between the two groups of samples.

[0063] 2. Sample Analysis Laboratory and clinical data of the patient group were compared with those of the healthy control group, and no significant sex or age differences were observed between the two groups. Clinical data were collected from electronic medical records in a standardized form, including demographic data, laboratory data, and clinical symptoms. The clinical characteristics of the study population are shown in Table 1. Disease activity in SLE patients was assessed using the SLEDAI score, which includes a range of clinical manifestations and examination items, including psychiatric symptoms, arthritis, rash, hematological abnormalities, proteinuria, pulmonary pleural disease, and renal disease. The SLEDAI score allows the assessment of various aspects of disease activity through 24 weighted clinical manifestations and examination items.

[0064] Table 1 Main clinical data of lupus patients Table 1 Main clinical data of lupus patients 1.3 Experimental methods and results Peripheral blood PBMCs were isolated and obtained from the control and patient groups according to the method described in Example 1. The samples were divided into a healthy control group, an inactive lupus erythematosus group with a disease activity score (SLEDAI) of 4 or less, and an active lupus erythematosus group with a SLEDAI of 4 or more (see Table 1).

[0065] The expression of TLR7 in peripheral blood PBMC B cells of active and inactive lupus patients and healthy controls was detected. Figure 5 A. As shown in the figure, TLR7 expression is higher in patients with active lupus compared with healthy controls and patients with inactive lupus. Figure 5As shown in Figure B, compared with the healthy control group and inactive lupus patients, the expression of CD71 (TFR1) was higher in active lupus patients, and the proportion of CD71⁺ cells was positively correlated with TLR7 (MFI) (r = 0.5179, p = 0.0017), suggesting that the expression of CD71 in B cells of lupus patients is closely related to the activation of TLR7 ( Figure 5 C). To verify the CD71 + Validation of the efficacy of cells as markers for systemic lupus erythematosus, drawing ROC curves ( Figure 5 D), the results showed that AUC = 0.736, indicating high specificity and sensitivity.

[0066] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Any of the following applications: (1) Quantitative detection of CD71 ＋ Use of a reagent for measuring B cell levels in the preparation of a product for prognostic assessment, diagnosis or monitoring of systemic lupus erythematosus; (2) Inhibition of CD71 ＋ Use of a B cell level reagent in preparing a pharmaceutical composition for treating systemic lupus erythematosus; (3) CD71 ＋ Application of B cells in screening drug candidates for the treatment of systemic lupus erythematosus; and (4) CD71 ＋ Application of B cells in constructing a computational model for predicting systemic lupus erythematosus.

2. A pharmaceutical composition for treating systemic lupus erythematosus, characterized in that: The pharmaceutical composition comprises: (i) Inhibition of CD71 ＋ Reagents for B cell levels; and (ii) pharmaceutically acceptable carriers and / or excipients.

3. The pharmaceutical composition according to claim 2, characterized in that The inhibition of CD71 ＋ Reagents for B cell levels include anti-human CD71 antibodies modified with different fluorescent groups.

4. The pharmaceutical composition according to claim 3, characterized in that The inhibition of CD71 ＋ Reagents at the B cell level are CART cells targeting CD71 and / or bispecific T cell engagers targeting CD71.

5. A product for prognosis assessment, diagnosis or monitoring of systemic lupus erythematosus, characterized in that: The product includes a method for quantitatively detecting CD71 in a sample ＋ Chips, kits or nucleic acid membrane strips for B cell levels.

6. The product according to claim 5, characterized in that The chip includes a chip capable of detecting CD71 ＋ Reagents for B cell levels.

7. The product according to claim 5, characterized in that The kit includes detecting CD71 by flow cytometry or immunofluorescence staining ＋ Reagents for B cell levels.

8. The product according to claim 5, characterized in that The nucleic acid membrane strip includes a nucleic acid capable of detecting CD71 ＋ Reagents for B cell levels.

9. The product according to any one of claims 5 to 8, characterized in that The samples are derived from peripheral blood.

10. A method for screening candidate drugs for treating systemic lupus erythematosus, characterized in that: include: Treat cells expressing or containing CD71 with substances to be screened ＋ B cell culture system; and Detection of CD71 in the system ＋ B cell levels; Wherein, when the substance to be screened inhibits CD71 ＋ When the expression of the antigen-binding protein is detected at the B cell level, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

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