Method for treating systemic lupus erythematosus by targeted senescence B cells and application of targeted senescence B cells

Abstract

The invention relates to treatment of systemic lupus erythematosus by targeted senescence B cells, in particular to application of senescence B cells serving as targets or targeted senescence B cells in preparation of drugs for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis. The treatment of systemic lupus erythematosus or systemic lupus erythematosus nephritis is realized by taking senile B cells as targets and enabling the senile B cells to die or inactivate, wherein the senile B cells are CD19 + P21 + cells, CD19 + P16 + cells, CD19 + Spider-beta-gal+ cells, CD19 + CD11c + cells and CD19 + Tbet + cells. According to the invention, new cell subpopulation senescence B cells enriched in SLE patients are defined, and it is proved that the B cells can secrete autoantibodies and have causal association with disease occurrence and development. The combination of the Bcl-2 inhibitor and the Mcl-1 inhibitor which are designed on the basis of the disease mechanism and are targeted to the senescence B cells can remove the senescence B cells in a targeted manner, the senescence B cells have a synergistic effect, the removal effect is remarkably enhanced, and the systemic lupus erythematosus is treated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, it relates to the application of senescent B cells as targets or targeting senescent B cells in the preparation of drugs for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis, as well as the technical means and applications for achieving targeted action on senescent B cells. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Abnormal B cells and the resulting large number of autoantibodies play a crucial role in the development and progression of SLE. Currently, B-cell-targeting therapies are in clinical use or undergoing multiple clinical trials, including B-cell-clearing antibodies (rituximab), belimumab which inhibits B-cell maturation, and CAR-T therapy. However, most of these therapies cannot distinguish between pathogenic and non-pathogenic B cells, and while clearing autoantibodies, they also clear protective antibodies, posing a significant risk of infection.

[0003] The concept and characteristics of B lymphocyte aging, as a type of immune cell, have remained unclear. Over a decade ago, researchers discovered a specific group of B cells expressing CD11c and T-bet that aggregated in aged female mice, named age-associated B cells (ABCs). ABCs were later shown to accumulate in patients with autoimmune diseases and infectious diseases. Age-associated B cells (ABCs) have recently been found to be associated with autoantibody production, and clearing these cells can alleviate symptoms. However, current methods used in animal models to clear these cells all involve gene editing targeting their surface marker CD11c, and no such methods have yet been widely adopted for clinical application.

[0004] Furthermore, it remains unclear whether ABCs (autoimmune cells, autoantibodies, and alpha-12 cells) encompass all pathogenic B cells, or whether all ABCs are pathogenic B cells. The ultimate goal in the field of SLE is to precisely target pathogenic B cells, eliminating autoantibodies while preserving protective antibodies against various pathogens.

[0005] Therefore, further understanding the pathogenesis of systemic lupus erythematosus and designing more targeted treatments and novel drugs to precisely target and eliminate pathogenic B cells is an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides the use of senescent B cells as targets or targeting senescent B cells in the preparation of drugs for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis. By using the senescent B cells as targets and causing them to die or become inactive, the treatment of systemic lupus erythematosus or systemic lupus erythematosus nephritis is achieved.

[0007] As some embodiments of this application, the senescent B cells are CD19. + P21 + Cells, CD19 + P16 + Cells, CD19 + Spider-β-gal + Cells, CD19 + CD11c + Cells, CD19 + Tbet + One or more of them in the cell.

[0008] Age / autoimmunity-associated B cells (ABCs) in the prior art are a subset of B cells that expand with age (and are age-independent in some diseases). First discovered in mice in 2011, they may be a potential driver of autoimmune diseases. They are characterized by the expression of the transcription factor T-bet (Tbx21), traditionally associated with T cells, and their surface markers are CD21lowT-bet+CD11c+. In contrast to ABCs, this application first proposes that senescent B cells are enriched in patients with systemic lupus erythematosus (SLE), and this senescent B cell subset is defined as CD19... + P21 + / P16 + / Spider-β-gal + Cells. The applicant discovered that this group of senescent B cells highly expresses cell cycle-dependent kinase inhibitors P21 and P16, and exhibits elevated senescence-related β-galactosidase activity. Furthermore, it was found that these senescent B cells are larger than other B cells, highly express Bcl2 family anti-apoptotic molecules such as Bcl2 and Mcl1, and possess senescence-related secretory phenotypes (TNF-α, IL-6), etc.

[0009] B cells, also known as B lymphocytes, originate from pluripotent stem cells in the bone marrow. Progenitor cells of B lymphocytes are found in the islands of hematopoietic cells in the fetal liver (14 days into the embryonic stage of a mouse or 8-9 weeks into the human embryo). Subsequently, the bone marrow gradually takes over as the site of B lymphocyte production and differentiation. Mature B cells primarily reside in the superficial lymph nodes of the lymph node cortex and in the red and white pulp of the spleen. Under antigenic stimulation, B cells can differentiate into plasma cells, which synthesize and secrete antibodies (immunoglobulins), primarily performing humoral immunity. B cells have various membrane surface molecules that recognize antigens and interact with immune cells and molecules, serving as important criteria for the isolation and identification of B cells. The main surface molecules of B cells include leukocyte differentiation antigens, MHC, and various membrane surface receptors.

[0010] CD19 is a CD molecule (leukocyte differentiation antigen) expressed by B cells, belonging to the Ig superfamily. All B cell lines except plasma cells, as well as malignant B cells and free radical dendritic cells (FDCs), express this molecule. It is an important membrane antigen involved in B cell proliferation, differentiation, activation, and antibody production, and also promotes BCR signal transduction.

[0011] p21 protein is a cyclin-dependent kinase inhibitor (CDKI) with multiple biological functions. p21 protein can degrade cyclins, inhibit cyclin-dependent kinase (CDK) activity, and bind to cyclin / CDK complexes or proliferating cell nuclear antigen (PCNA), thus arresting cell cycle progression. p21 protein can also participate in regulating the body's inflammatory response through macrophage activation and polarization and neutrophil apoptosis. Furthermore, p21 protein regulates apoptosis. It can both interact with apoptosis signal-regulated kinase 1 (ASK1) and pro-apoptotic factors to inhibit apoptosis, and upregulate the pro-apoptotic protein Bax or activate the TNF family death receptor (TRAIL) to promote apoptosis. p21 protein can upregulate microtubule-associated protein 1A / 1B light chain 3B (LC3) and Unc-51-like autophagy-activated kinase 1 (ULK1) proteins to promote autophagy. In addition, p21 protein also plays an important role in various viral infections.

[0012] The P16 protein, encoded by the P16 gene, is an inhibitor of CDK4, a key enzyme in the cell division cycle. CDKs (cyclin-dependent kinases) and cyclins function at different phases of the cell cycle, initiating DNA replication and inducing mitosis. CDKs are likely central to the cell cycle regulation mechanism, modulating the cell cycle through phosphorylation of a series of key substrates. The CDK4-cylin complex participates in the regulation of the G1-S phase transition. The P16 protein inhibits Cdk4 activity, ultimately preventing cells from entering the S phase. If the P16 gene loses function due to deletion, mutation, or other reasons, it cannot inhibit CDK4, ultimately leading to malignant cell proliferation and accelerated tumorigenesis. CDK4-cyclin can act on various oncogene products, such as PP60c-sYc and C-abl products, to regulate their phosphorylation. It can also act on certain anti-cancer gene products; for example, phosphorylation of Rb protein leads to loss of growth inhibition. At the G1 / S junction, CDK4-G1 cyclin phosphorylates and inactivates Pb, causing cells to transition from a quiescent to a proliferative state. The P16 gene is a crucial anti-cancer gene; its inactivation can induce malignant cell proliferation.

[0013] β-galactosidase is a hydrolytic enzyme found in lysosomes. Cellular senescence is the gradual decline in normal physiological functions and proliferative capacity of cells over time or under external stress, leading to detachment from the cell cycle. Cell enlargement, pH-dependent expression of β-galactosidase activity, and altered gene expression patterns are further characteristics of senescent cells. During cellular senescence, β-galactosidase exhibits high enzymatic activity at pH 6.0.

[0014] Systemic lupus erythematosus (SLE) is a chronic, diffuse connective tissue disease primarily caused by abnormal activation of the immune system, leading to the attack of the body's own tissues. It may be related to genetics, environmental factors, and estrogen. Prolonged sun exposure, certain medications, infections, and oral estrogen can all exacerbate SLE symptoms. This disease can damage various organs throughout the body, potentially causing a range of complications, including kidney damage and neuropsychiatric symptoms. Commonly used drug treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, immunosuppressants, and corticosteroids.

[0015] As some embodiments of this application, this application provides the use of senescent B cells as a target in the preparation of a medicament for treating systemic lupus erythematosus (SLE). As some embodiments of this application, this application provides the use of senescent B cells as a target in the preparation of a medicament for treating systemic lupus erythematosus nephritis. As some embodiments of this application, this application provides the use of targeting senescent B cells in the preparation of a medicament for treating systemic lupus erythematosus nephritis. As some embodiments of this application, this application provides the use of targeting senescent B cells in the preparation of a medicament for treating systemic lupus erythematosus nephritis.

[0016] As some embodiments of this application, the senescent B cells are CD19. + P21 + Cells. In some embodiments of this application, the senescent B cells are CD19. + P16 + Cells. In some embodiments of this application, the senescent B cells are CD19. + Spider-β-gal + cell.

[0017] As some embodiments of this application, the senescent B cells are CD19. + P21 + Cells or CD19 + Spider-β-gal + cell.

[0018] As some embodiments of this application, the death or inactivation of senescent B cells includes one or more of the following: transformation of their senescent phenotype, apoptosis, clearance, and killing.

[0019] As some embodiments of this application, the death or inactivation of senescent B cells is achieved by treating them with a B cell-targeting drug that can cause the senescent phenotype of senescent B cells to change, undergo apoptosis, be cleared, or be killed.

[0020] As some embodiments of this application, B-cell targeted drugs are used to eliminate senescent B cells. The B-cell targeted drugs include one or more of the following: nanocarrier-based anti-aging drug delivery systems, anti-aging drugs senolytics, anti-aging drugs senomorphics, immune-based senescent cell elimination agents, SASP neutralizing agents, and biomacromolecules and cell drugs that target and eliminate senescent B cells.

[0021] As some embodiments of this application, the nanocarrier-based anti-aging drug delivery system includes a silicon nanoparticle-based delivery system, a gold nanoparticle-based delivery system, an iron oxide nanoparticle-based delivery system, a liposome-based delivery system, a polymer-based delivery system, and a virus particle-based delivery system.

[0022] Nanoparticle-based drug delivery systems utilize nanotechnology to precisely deliver drugs to the lesion site or application site. By using nanomaterials as drug carriers, the system can control the release rate and dosage of drugs, achieving precise therapeutic effects. Nanoparticle-based drug delivery systems can precisely deliver drugs to the application site, increasing drug concentration and efficacy, thereby improving therapeutic efficacy. Simultaneously, because the drug is more precisely distributed in the body, it can reduce damage to other sites and decrease side effects. Furthermore, nanomaterials as drug carriers can protect drugs from degradation by enzymes and acid-base environments in the body, improving drug stability.

[0023] As some embodiments of this application, the anti-aging drug in the nanocarrier-based anti-aging drug delivery system is used to eliminate or inactivate the senescent B cells.

[0024] As some embodiments of this application, the anti-aging drug delivery system based on nanocarriers is modified with antibodies targeting senescent B cells on its surface; by modifying the surface of the delivery system with antibodies targeting senescent B cells to precisely target senescent B cells, the loaded anti-aging drugs can directly act on senescent B cells.

[0025] As some embodiments of this application, the polymer includes one or more of poly-(L-glutamic acid) (PGA), N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA), polyethylene glycol (PEG), poly-(L-aspartic acid) (PAA), poly-(L-lactide) (PLA), poly-(amidoamine) (PAMAM), albumin, chitosan, and heparin.

[0026] As some embodiments of this application, the viral particles include one or more of cowpea mosaic virus (CPMV), cowpea green spot virus, canine parvovirus, heat shock protein (HSP) cages, and bacteriophages.

[0027] As some embodiments of this application, the liposomes include one or more of phospholipids, ionizable lipids, cholesterol, and polyethylene glycol-modified lipids.

[0028] As some embodiments of this application, the anti-aging drugs senolytics include one or more of drugs that interfere with senescent cell signaling pathways, thereby disabling the senescent cell anti-apoptotic pathway (SCAP), and drugs that target and clear ANT2.

[0029] Anti-aging drugs, specifically senolytics, selectively eliminate senescent cells; their mechanism of action in delaying aging is the removal of these cells. First-generation senolytics target various SCAPs, including tyrosine kinase receptors (TKRs), growth factor receptors (GFRs), Ephrin receptor B1 (EFNB1), SRC kinases, PI3K-AKT, HSP90, BCL-2 family members, caspase inhibitors, and p53. Second-generation senolytics target prodrugs and nanomedicines mediated by lysosomes and SA-β-Gal activation, sodium-potassium pump (Na+ / K+-ATPase)-dependent apoptosis, SASP inhibition and CAR-T cells, antibody-drug conjugates, or vaccine-mediated immune clearance.

[0030] As some embodiments of this application, anti-aging drugs senolytics include one or more of dasatinib, quercetin, Fisetin, luteolin, curcumin, Navitoclax (ABT-263), A1331852, and A1155463.

[0031] As some embodiments of this application, anti-aging drugs senolytics include a combination of dasatinib and quercetin.

[0032] As some embodiments of this application, the drug that targets and clears ANT2 includes tamoxifen (MitoTam).

[0033] As some embodiments of this application, the drugs that disable the anti-apoptotic pathway (SCAP) of senescent cells include one or more of the following: anti-apoptotic Bcl-2 protein inhibitors, p53-related pathway regulators, anti-apoptotic transcription factor FOXO4 inhibitors, and molecular chaperone HSP90 inhibitors.

[0034] As some embodiments of this application, the biomacromolecules and cellular drugs that target and eliminate senescent B cells include one or more of siRNA, shRNA, microRNA, piRNA, ASO, CAR-T reagent, and TCRT reagent.

[0035] As some embodiments of this application, the anti-apoptotic Bcl-2 protein inhibitors include one or more of Bcl-2 inhibitors, Bcl-xl inhibitors, Bcl-w inhibitors, Mcl-1 inhibitors, Bfl1 / A-1 inhibitors, and Bc-B inhibitors.

[0036] Anti-apoptotic Bcl-2 protein inhibitors include at least one inhibitor of one or more anti-apoptotic proteins in the Bcl-2 family. Specifically, the Bcl-2 inhibitors of the present invention selectively kill senescent cells. Methods for determining whether a compound inhibits one or more anti-apoptotic proteins in the Bcl-2 family are known in the art. For example, the nucleic acid expression, protein expression, or activity of Bcl-2 family proteins can be measured as described in the detailed description below. Methods for determining whether a compound selectively kills senescent cells are known in the art.

[0037] Members of the B-cell lymphoma 2 (Bcl-2) family control the integrity of the outer mitochondrial membrane (OMM) and are therefore crucial in determining cellular sensitivity to intrinsic pathway-induced apoptosis. Based on structural and functional characteristics, Bcl-2 family members can be divided into three subfamilies: the anti-apoptotic family, the multi-domain pro-apoptotic family, and the -BH3-only pro-apoptotic family. The anti-apoptotic subfamily inhibits apoptosis and promotes cell survival but does not inhibit cell proliferation. Therefore, anti-apoptotic proteins in the Bcl-2 family can also be referred to as pro-survival proteins. Non-limiting examples of anti-apoptotic Bcl-2 family proteins include Bcl-2, Bcl-x1, Bcl-w, Mcl-1, Bfl 1 / A-1, and Bcl-B. Anti-apoptotic Bcl-2 family proteins are characterized by the presence of up to four relatively short sequence motifs, less than 20 amino acids in length, called Bcl-2 homology 1 (BH1), BH2, BH3, and BH4 domains. They also possess C-terminal membrane anchoring sequences and similar three-dimensional structures. Inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family can induce apoptosis and promote cell death by antagonizing the pro-survival function of the Bcl-2 protein family. The inhibitors of this invention can inhibit one or more anti-apoptotic proteins in the Bcl-2 family.

[0038] Members of the B-cell lymphoma 2 (Bcl-2) family control the integrity of the outer mitochondrial membrane (OMM) and are therefore crucial in determining cellular sensitivity to intrinsic pathway-induced apoptosis. Based on structural and functional characteristics, Bcl-2 family members can be divided into three subfamilies: the anti-apoptotic family, the multi-domain pro-apoptotic family, and the -BH3-only pro-apoptotic family. The anti-apoptotic subfamily inhibits apoptosis and promotes cell survival but does not inhibit cell proliferation. Therefore, anti-apoptotic proteins in the Bcl-2 family can also be referred to as pro-survival proteins. Non-limiting examples of anti-apoptotic Bcl-2 family proteins include Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Bfl 1 / A-1, and Bcl-B. Anti-apoptotic Bcl-2 family proteins are characterized by the presence of up to four relatively short sequence motifs, less than 20 amino acids in length, called the Bcl-2 co-domains (BH1), BH2, BH3, and BH4. They also possess C-terminal membrane anchoring sequences and similar three-dimensional structures. Inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family can induce apoptosis and promote cell death by antagonizing the pro-survival function of the Bcl-2 protein family. The inhibitors of the present invention can inhibit one or more anti-apoptotic proteins in the Bcl-2 family. Therefore, the inhibitors of the present invention can inhibit one or more anti-apoptotic proteins selected from Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Bfll / A-1, and Bcl-B.

[0039] As some embodiments of this application, the Bcl-2 inhibitor includes one or more of venetoclax (ABT-199), BGB-11417 (sonrotoclax), ABT737, ABT-263, Lisaftoclax (APG-2575), ICP-248, Navitoclax, APG-1252 (Pelcitoclax), FCN-338, LP-108, LP-118, BP-1002, BRD-K20733377, BRD-K56819078, and BRD-K44839765.

[0040] As some embodiments of this application, the Bcl-xl inhibitor includes one or more of BH3I-1, WEHI-539hydrochloride, A1331852, and A1155463.

[0041] As some embodiments of this application, the Mcl-1 inhibitor includes one or more of AZD5991, UMI-77, AZD5991, BRD810, S63845, ABBV-467, and APG-3526.

[0042] As some embodiments of this application, the anti-apoptotic Bcl-2 protein inhibitor includes one or more of ABT737, ABT-263, S63845, BRD-K20733377, BRD-K56819078, and BRD-K44839765.

[0043] As some embodiments of this application, the anti-apoptotic Bcl-2 protein inhibitor includes a Bcl-2 inhibitor and a Mcl-1 inhibitor, and when the Bcl-2 inhibitor is ABT-263, the Mcl-1 inhibitor is not S63845.

[0044] As some embodiments of this application, the B-cell targeted drug also includes an mTOR inhibitor and metformin.

[0045] As some embodiments of this application, the mTOR inhibitor includes rapamycin and its analogues.

[0046] As some embodiments of this application, the mTOR inhibitor includes rapamycin.

[0047] As some embodiments of this application, the application has any of the following effects: a. treating systemic lupus erythematosus (SLE); b. treating systemic lupus erythematosus nephritis; c. treating cutaneous lupus erythematosus; d. treating systemic lupus erythematosus encephalopathy; e. treating pulmonary complications of SLE, preferably including pulmonary hypertension; f. treating hematologic complications of SLE, preferably including cytopenia; g. treating digestive complications of SLE; h. reducing glomerular hyperplasia and peritubular and perivascular lymphocyte infiltration caused by SLE; i. reducing renal immune complex deposition caused by SLE; j. reversing renal tissue aging caused by SLE; and k. alleviating renal fibrosis caused by SLE.

[0048] This application provides a B-cell targeted drug that targets senescent B cells, wherein the B-cell targeted drug is the aforementioned B-cell targeted drug.

[0049] This application also provides a medicament for treating systemic lupus erythematosus, the medicament comprising an effective amount of the B-cell targeted drug and a pharmaceutically acceptable carrier.

[0050] As some embodiments of this application, the medicament for treating systemic lupus erythematosus further includes a second medicament, which includes one of a B-cell-targeting clearance antibody, an antibody that inhibits B-cell maturation, or a CAR-T agent.

[0051] As some embodiments of this application, the B-cell-targeting clearance antibody includes rituximab, and the B-cell-inhibiting antibody includes belimumab.

[0052] This application provides the B-cell targeted drug that targets senescent B cells, or the use of the drug in the preparation of a drug that eliminates senescent B cells.

[0053] This application also provides the B-cell targeted drug and its use in the preparation of products for treating systemic lupus erythematosus (SLE), its complications, and symptoms, wherein the product is selected from any of the following: a. a product for treating systemic lupus erythematosus; b. a product for treating systemic lupus erythematosus nephritis; c. a product for treating cutaneous lupus erythematosus; d. a product for treating systemic lupus erythematosus encephalopathy; e. a product for treating pulmonary complications of systemic lupus erythematosus, preferably including pulmonary hypertension; f. a product for treating hematologic complications of systemic lupus erythematosus, preferably including cytopenia; g. a product for treating digestive complications of systemic lupus erythematosus; h. a product for reducing glomerular hyperplasia and peritubular and perivascular lymphocyte infiltration caused by systemic lupus erythematosus; i. a product for reducing renal immune complex deposition caused by systemic lupus erythematosus; j. a product for reversing renal tissue aging caused by systemic lupus erythematosus; and k. a product for alleviating renal fibrosis caused by systemic lupus erythematosus.

[0054] This application also provides the use of a substance for detecting senescent B cells in the preparation of a kit for detecting systemic lupus erythematosus, wherein the senescent B cells are CD19. + P21 + Cells, CD19 + P16 + Cells, CD19 + Spider-β-gal + Cells, CD19 + CD11c + Cells, CD19 + Tbet + One or more of them in the cell.

[0055] As some embodiments of this application, the senescent B cells are CD19. + P21 + Cells or CD19 + Spider-β-gal + cell.

[0056] As some embodiments of this application, the substances include flow cytometers, immunofluorescence reagents, and immunocytochemical reagents.

[0057] This application also provides the use of a substance for detecting senescent B cells in the preparation of a kit, wherein the use is for evaluating the treatment efficacy of systemic lupus erythematosus (SLE) and / or determining the prognosis of SLE, wherein the senescent B cells are CD19. + P21 + Cells, CD19 + P16 + Cells, CD19 + Spider-β-gal + Cells, CD19 + CD11c + Cells, CD19 + Tbet + One or more of them in the cell.

[0058] As some embodiments of this application, the senescent B cells are CD19. + P21 + Cells or CD19 + Spider-β-gal + cell.

[0059] As some embodiments of this application, the substances include flow cytometers, immunofluorescence reagents, and immunocytochemical reagents.

[0060] As described above, the method and application of the present invention for treating systemic lupus erythematosus by targeting senescent B cells have the following beneficial effects:

[0061] This application is the first to demonstrate the enrichment of a large number of senescent B cells in SLE patients, specifically defined as P21. + B cell or β-gal + B cells; peripheral blood of lupus patients is enriched with senescent B cells, characterized by high expression of cell cycle-dependent kinase inhibitors P21 and P16, elevated senescence-associated β-galactosidase (SA-β-gal) activity, and high expression of BCL2 family anti-apoptotic molecules. The proportion of senescent B cells is positively correlated with the patient's serum autoantibody level and negatively correlated with complement level. Furthermore, a large number of P21+ senescent B cells are also found to infiltrate renal biopsy tissues from lupus nephritis patients.

[0062] Furthermore, this application found that β-gal is also present in lupus mice. + Senescent B cells, and in vitro culture, β-gal + Senescent B cells rather than CD11c +ABCs produce a large number of autoantibodies. By transplanting senescent or non-senescent B cells into non-lupus-susceptible mice and inducing them with the TLR7 agonist R848, this application demonstrates that the presence of senescent B cells can significantly accelerate the disease process by secreting autoantibodies and inducing B cell senescence in recipient mice, thus confirming a strong causal relationship between them and the pathogenesis of lupus.

[0063] This application has screened drugs in vitro for the treatment of systemic lupus erythematosus, said drugs comprising an effective amount of the B-cell-targeting drug, capable of specifically clearing P21. + Senescent B cells, when treated with a combination of Bcl-2 inhibitors and Mcl-1 inhibitors, exhibit synergistic effects due to their respective action on different sites, significantly enhancing clearance function. In the spontaneous SLE mouse model MRL / lpr and the more humanized SLE mouse model, this combination of drugs effectively cleared senescent B cells, thereby greatly improving the condition.

[0064] The drugs screened in this application for the treatment of systemic lupus erythematosus (SLE) can not only treat SLE, but also treat SLE nephritis, reduce glomerular hyperplasia and peritubular and perivascular lymphocyte infiltration caused by SLE, reduce renal immune complex deposition caused by SLE, reverse renal tissue aging, and alleviate renal fibrosis. Attached Figure Description

[0065] Figure 1 This study showed that senescent B cells were enriched in the peripheral blood of SLE patients;

[0066] Figure 2 The study showed a correlation between the proportion of senescent B cells in peripheral blood and patients' clinical indicators;

[0067] Figure 3 This study showed that senescent B cells were also enriched in the kidney tissue of patients with lupus nephritis.

[0068] Figure 4 The in vitro culture demonstrated that senescent B cells were enriched with autoreactivity and secreted large amounts of autoantibodies;

[0069] Figure 5 This study showed that β-gal+ senescent B cells secreted more autoantibodies compared to CD11c+ABCs;

[0070] Figure 6 The in vivo transplantation experiment demonstrated that senescent B cells accelerate disease progression and are causally associated with the occurrence and development of SLE;

[0071] Figure 7 This study demonstrates that a combination of anti-aging drugs screened in vitro can effectively eliminate senescent B cells;

[0072] Figure 8 The study demonstrated that the combination of anti-aging drugs cleared senescent B cells in a spontaneous SLE mouse model, exhibiting significant therapeutic effects.

[0073] Figure 9 The study demonstrated that the combination of anti-aging drugs alleviated kidney pathological damage and reversed kidney tissue aging in a spontaneous SLE mouse model.

[0074] Figure 10 The study demonstrated that the combination of anti-aging drugs also showed promising efficacy in a humanized SLE mouse model. Detailed Implementation

[0075] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0076] [Experimental Materials]

[0077] The sources of the reagents used in the examples are shown in Tables 1-3.

[0078] Table 1 Common Reagents

[0079]

[0080]

[0081] Table 2. Anti-human antibodies used in flow cytometry analysis

[0082]

[0083]

[0084] Table 3. Mouse antibodies used in flow cytometry analysis

[0085]

[0086] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0087] Example 1: Phenotypic Identification of Senescent B Cells

[0088] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (HC) and patients with severe leukemia (SLE). The expression of the cell cycle-dependent kinase inhibitor P21 in PBMCs of both HC and SLE patients was detected by flow cytometry. The activity of senescence-associated β-galactosidase (β-gal) in HC and SLE patient B cells was detected using β-gal dye. PBMCs were purified using magnetic bead separation, and RNA was extracted. The expression levels of P16 and P21 genes in B cells were detected by qPCR. The detection of these characteristic biomarkers of senescence demonstrated the presence of B cell senescence in SLE patients, with a significantly higher proportion of senescent B cells compared to healthy controls.

[0089] Figure 1 This study showed that senescent B cells were enriched in the peripheral blood of SLE patients. Figure 1 A shows a schematic diagram of blood samples collected from a human body for analysis. Figure 1 B cells showed higher expression of β-gal+ in B cells of SLE patients compared to healthy controls. Figure 1 Image C shows β-gal reagent detection images of SLE patients and healthy controls. It is evident that SLE patients have higher aging-related β-gal activity in B cells compared to healthy controls. Figure 1 Figure D shows the expression of P21 and P16 genes in SLE patients and healthy controls. The expression of P21 and P16 genes in B cells of SLE patients was significantly higher than that in healthy human cells. Figure 1 E showed that the expression of P21 protein was significantly increased in B cells of SLE patients compared to healthy human cells.

[0090] Figure 2 The study showed that the proportion of senescent B cells in peripheral blood was correlated with patients' clinical indicators; the proportion of senescent B cells in peripheral blood was positively correlated with the level of anti-dsDNA antibodies in serum and negatively correlated with the levels of complement C3 and C4.

[0091] B cells were detected in the kidney tissues of patients with non-lupus nephritis such as IgA nephritis and patients with lupus nephritis. The results are shown in [the table below]. Figure 3 , Figure 3 The study showed that senescent B cells were also enriched in the kidney tissue of patients with lupus nephritis, and the P21 protein was significantly expressed in these senescent B cells.

[0092] Example 2: In vitro detection of antibody secretion capacity of senescent B cells

[0093] β-gal- and β-gal+ B cells from the spleen of lupus mice were sorted by flow cytometry using senescence-associated β-galosidase staining and cultured in vitro for 7 days under R848 induction. Cells were harvested to assess cell differentiation; cell culture supernatants were collected, and total antibody and autoantibody secretion were detected by ELISA. The results demonstrated that β-gal+ B cells had a stronger ability to differentiate into plasma cells and secreted significantly more autoantibodies than β-gal- B cells.

[0094] Figure 4 The in vitro culture demonstrated that senescent B cells were enriched with autoreactivity and secreted large amounts of autoantibodies; Figure 4 A demonstrates that senescent B cells are also enriched in lupus mice. Figure 4 B shows a flowchart of flow cytometry sorting and in vitro culture. Figure 4 Figures C through D show that β-gal+ B cells have a stronger ability to differentiate into plasma cells, and Figure E shows that β-gal+ B cells secrete more anti-dsDNA autoantibodies and are enriched with autoreactivity.

[0095] Furthermore, to compare antibody secretion in senescent B cells with ABCs, four B cell subsets were obtained by flow cytometry staining and sorting: CD11c- / β-gal-, CD11c+ / β-gal-, CD11c- / β-gal+, and CD11c+ / β-gal+. These subsets were then cultured in vitro for 7 days under the induction of R848 and cytokines (IL-2 and IL-10) (see [link to study]). Figure 5 A).

[0096] Cell culture supernatants were collected, and total antibody and autoantibody secretion were detected by ELISA. In in vitro cultures of the above-mentioned B cell subsets from the spleen of lupus mice and peripheral blood of SLE patients, the CD11c+ / β-gal+ subset showed the strongest autoantibody secretion capacity, the CD11c- / β-gal+ subset was slightly stronger than the other two subsets, and the CD11c+ / β-gal- subset showed virtually no difference from the CD11c- / β-gal- subset, both being very low (see...). Figure 5 (B~5E). Therefore, β-gal+ is better able to distinguish pathogenic B cell subsets that secrete autoantibodies than CD11c+.

[0097] Example 3: In vivo transplantation of senescent B cells to investigate the causal relationship between senescent B cells and the pathogenesis of SLE.

[0098] Similarly, β-gal- and β-gal+ B cells were sorted by senescence-associated β-galosidase staining and flow cytometry, and an equal number of cells were transplanted into non-lupus-susceptible mice. SLE was induced by ear administration of the TLR7 agonist R848 (see [link to article]). Figure 6 A).

[0099] The rate of disease progression and severity were monitored in two groups of recipient mice, including autoantibody levels, B cell senescence, and kidney damage. Results showed that recipient mice transplanted with β-gal+ senescent B cells developed splenomegaly (see...). Figure 6 B), elevated serum autoantibody levels, enhanced B cell differentiation into plasma cells, increased proportion of senescent B cells, and more severe kidney damage demonstrate a causal relationship between senescent B cells and the development of SLE (see [link to relevant documentation]). Figure 6 C~H).

[0100] Example 4: Screening for novel anti-aging drug combinations to specifically eliminate senescent B cells for the treatment of SLE

[0101] First, B cells from healthy controls and SLE patients were separated and cultured for 3 days in a medium containing different anti-aging drugs or combinations thereof. The proportion of P21+ senescent B cells was measured to screen for the drugs or drug combinations with the highest clearance efficiency (see...). Figure 7 ).

[0102] The spontaneous SLE mouse model, MRL / lpr mice, underwent a 4-week sequential drug treatment regimen. The specific regimen was as follows: ABT263 was administered for two consecutive days (10 mg / kg), followed by a one-day interval and then S63845 for two consecutive days (25 mg / kg), followed by another two-day interval before the next round of treatment. One round of treatment lasted 7 days, and four rounds were administered consecutively (administration procedure see [link to administration protocol]). Figure 8 A). A novel combination of anti-aging drugs can eliminate senescent B cells, thereby significantly alleviating lymphadenopathy, reducing serum autoantibody titers, lowering TNF-α levels, reducing proteinuria, and comprehensively alleviating the condition (see [link]). Figure 8 B~8G).

[0103] Since the kidneys are the most commonly affected organ in lupus, kidney tissue damage is a primary focus of attention. Treatment with anti-aging drugs as described above significantly alleviated glomerular hyperplasia and peritubular and perivascular lymphocyte infiltration. Figure 9 A). Simultaneously, due to the decrease in circulating autoantibody levels, renal immune complex deposition was also significantly reduced ( Figure 9 B). Interestingly, in addition to immunosenescence, renal tissue aging was also observed in lupus mice. Treatment with a combination of anti-aging drugs could also reverse renal tissue aging. Figure 9 C), reducing kidney fibrosis ( Figure 9 D) has multiple effects.

[0104] Furthermore, considering the differences between the mouse and human immune systems, further validation was conducted using a novel, self-developed humanized SLE mouse model. The specific dosing regimen was consistent with that in the MRL / lpr model; see [link to dosing regimen]. Figure 10A. In the human immune system, we also observe the clearance of senescent B cells and the remission of SLE (see...). Figure 10 B~F).

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of senescent B cells as a target or targeting senescent B cells in the manufacture of a medicament for the treatment of systemic lupus erythematosus or lupus nephritis, characterized in that, The treatment of systemic lupus erythematosus or systemic lupus erythematosus nephritis is achieved by targeting and killing or inactivating the senescent B cells.

2. Use according to claim 1, characterized in that, the aged B cells are CD19 + P21 + cells, CD19 + P16 + cells, CD19 + Spider-β-gal + cells, CD19 + CD11c + cells, CD19 + Tbet + one or more of the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cells.

3. Use according to claim 1, characterized in that, The killing or inactivation of the senescent B cells includes one or more of the transformation of the senescent phenotype, apoptosis, clearance, and killing of the senescent B cells; Preferably, the killing or inactivation of the senescent B cells is by treatment with a B cell-targeting drug that can transform the senescent phenotype, induce apoptosis, clear, or kill the senescent B cells; Preferably, the B cell-targeting drug includes one or more of a nanocarrier-based anti- senolytic drug delivery system, an anti-senolytic drug senolytic, an anti-senolytic drug senomorphic, an immune-based senescent cell clearance agent, a neutralizing agent for SASP, and a biological macromolecule or cellular drug that targets the clearance of senescent B cells.

4. Use according to claim 3, characterized in that, The nanocarrier-based anti-senolytic drug delivery system includes a silicon nanoparticle-based delivery system, a gold nanoparticle-based delivery system, an iron oxide nanoparticle-based delivery system, a liposome-based delivery system, a polymer-based delivery system, and a virus particle-based delivery system; Or, the anti-senolytic drug in the nanocarrier-based anti-senolytic drug delivery system is used to clear or inactivate the senescent B cells; Or, the nanocarrier-based anti-senolytic drug delivery system is surface-modified with an antibody that targets senescent B cells.

5. Use according to claim 4, characterized in that, The polymer includes one or more of poly-(L-glutamic acid), N-((2-hydroxypropyl)- methacrylamide copolymer, polyethylene glycol, poly-(L-aspartic acid), poly-(L-lactide), poly-((amide-amine), albumin, chitosan, heparin; Or, the virus particle includes one or more of cowpea mosaic virus, cowpea green mosaic virus, canine parvovirus, heat shock protein cage, bacteriophage, adeno-associated virus; Or, the liposome includes one or more of phospholipid, ionizable lipid, cholesterol, and pegylated lipid.

6. Use according to claim 3, characterized in that, The anti-senolytic drug senolytic includes one or more of a drug that interferes with the signaling pathway of senescent cells and disables the senescent cell anti-apoptotic pathway (SCAP), and a drug that targets the clearance of ANT2; Preferably, the anti-senolytic drug senolytic includes one or more of Dasatinib, Quercetin, Fisetin, Luteolin, Curcumin, Navitoclax (ABT-263), A1331852, A1155463; Preferably, the anti-senolytic drug senolytic includes a combination of Dasatinib and Quercetin; Preferably, the drug that targets the clearance of ANT2 includes MitoTam; The drug for disabling the senescent cell anti-apoptotic pathway (SCAP) comprises one or more of an anti-apoptotic Bcl-2 protein inhibitor, a p53-associated pathway modulator, an anti-apoptotic transcription factor FOXO4 inhibitor, a chaperone HSP90 inhibitor; The biological macromolecule targeting the clearance of senescent B cells and the cell drug comprises one or more of an antibody, siRNA, shRNA, microRNA, piRNA, ASO, CAR-T, TCR-T.

7. Use according to claim 6, characterized in that, The anti-apoptotic Bcl-2 protein inhibitor comprises one or more of a Bcl-2 inhibitor, a Bcl-xl inhibitor, a Bcl-w inhibitor, a Mcl-1 inhibitor, a Bfl 1 / A-1 inhibitor, a Bcl-B inhibitor; The Bcl-2 inhibitor comprises one or more of venetoclax (ABT-199), BGB-11417 (sonrotoclax), ABT737, ABT-263, Lisaftoclax (APG-2575), ICP-248, Navitoclax, APG-1252 (Pelcitoclax), FCN-338, LP-108, LP-118, BP-1002, BRD-K20733377, BRD-K56819078, BRD-K44839765; The Bcl-xl inhibitor comprises one or more of BH3I-1, WEHI-539 hydrochloride, A1331852, A1155463; The Mcl-1 inhibitor comprises one or more of AZD5991, UMI-77, AZD5991, BRD810, S63845, ABBV-467, APG-3526; Preferably, the anti-apoptotic Bcl-2 protein inhibitor comprises one or more of ABT737, ABT-263, S63845, BRD-K20733377, BRD-K56819078, BRD-K44839765; Preferably, the anti-apoptotic Bcl-2 protein inhibitor comprises a Bcl-2 inhibitor and a Mcl-1 inhibitor, and when the Bcl-2 inhibitor is ABT-263, the Mcl-1 inhibitor comprises one or more of AZD5991, UMI-77, AZD5991, BRD810, ABBV-467, APG-3526.

8. Use according to claim 3, characterized in that, The B cell targeting drug further comprises an mTOR inhibitor, metformin; Preferably, the mTOR inhibitor comprises rapamycin and analogs thereof; Preferably, the mTOR inhibitor comprises rapamycin.

9. The use according to claim 1, characterized in that, The application has any one of the following effects: a. treating systemic lupus erythematosus; b. treating systemic lupus erythematosus nephritis; c. treating cutaneous lupus erythematosus; d. treating systemic lupus erythematosus encephalopathy; e. treating systemic lupus erythematosus pulmonary complications, preferably, the systemic lupus erythematosus pulmonary complications comprise pulmonary arterial hypertension; f. treating systemic lupus erythematosus hematological complications, preferably, the systemic lupus erythematosus hematological complications include cytopenia; g. treating systemic lupus erythematosus digestive system complications; h. reducing glomerular hyperplasia and tubular, perivascular lymphocyte infiltration of the kidney caused by systemic lupus erythematosus; i. reducing kidney immune complex deposition caused by systemic lupus erythematosus; j. reversing kidney tissue aging caused by systemic lupus erythematosus; and k. reducing kidney fibrosis caused by systemic lupus erythematosus.

10. A B cell targeting drug targeting senescent B cells, characterized in that, The B cell targeting drug is the B cell targeting drug of any one of claims 1-9.

11. A medicament for treating systemic lupus erythematosus, characterized by, The drug comprises an effective amount of the B cell targeting drug of claim 10, and a pharmaceutically acceptable carrier; Preferably, the drug for treating systemic lupus erythematosus further comprises a second drug, the second drug comprising one of a B cell targeting depletion antibody, an antibody inhibiting B cell maturation, a CAR-T agent; Preferably, the B cell targeting depletion antibody comprises rituximab, and the antibody inhibiting B cell maturation comprises belimumab.

12. Use of the B cell targeting drug of claim 10, or the drug of claim 11 in the preparation of a drug for depleting senescent B cells.

13. The use of a B-cell targeting agent according to claim 10, or a medicament according to claim 11, for the manufacture of a product for the treatment of systemic lupus erythematosus disease, its complications, signs, characterized in that, The product is selected from any one of the following: a. a product for treating systemic lupus erythematosus; b. a product for treating systemic lupus erythematosus nephritis; c. a product for treating cutaneous lupus erythematosus; d. a product for treating systemic lupus erythematosus encephalopathy; e. a product for systemic lupus erythematosus pulmonary complications, preferably, the systemic lupus erythematosus pulmonary complications include pulmonary arterial hypertension; f. a product for systemic lupus erythematosus hematological complications, preferably, the systemic lupus erythematosus hematological complications include cytopenia; g. a product for systemic lupus erythematosus digestive system complications; h. a product for reducing glomerular hyperplasia and tubular, perivascular lymphocyte infiltration of the kidney caused by systemic lupus erythematosus; i. a product for reducing kidney immune complex deposition caused by systemic lupus erythematosus; j. a product for reversing kidney tissue aging caused by systemic lupus erythematosus; and k. a product for reducing kidney fibrosis caused by systemic lupus erythematosus.

14. Use of a substance that detects senescent B cells in the preparation of a kit for detecting systemic lupus erythematosus, wherein, the senescent B cells are CD19 + P21 + the cells, CD19 + P16 + the cells, CD19 + Spider-β-gal + the cells, CD19 + CD11c + the cells, CD19 + Tbet + one or more of the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cells.

15. Use of a substance that detects senescent B cells for the manufacture of a kit for assessing the effectiveness of a treatment of systemic lupus erythematosus and / or for judging the prognosis of systemic lupus erythematosus, wherein, the senescent B cells are CD19 + P21 + the cells, CD19 + P16 + the cells, CD19 + Spider-β-gal + the cells, CD19 + CD11c + the cells, CD19 + Tbet + one or more of the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cells.

16. Use according to claim 14 or 15, characterized in that, The substance comprises a flow cytometer, an immunofluorescence reagent, an immunocytochemical reagent.

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