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] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of senescent B cells as targets or targeted senescent B cells in the preparation of drugs for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis, as well as technical means and applications for achieving targeted senescent B cells. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Abnormal B cells and the resulting production of numerous autoantibodies play a key role in the development and progression of SLE. Currently, therapies targeting B cells are being used clinically 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 are unable to distinguish between pathogenic and non-pathogenic B cells. While clearing autoantibodies, they also clear protective antibodies, posing a significant risk of infection.

[0003] As a type of immune cell, the concept and characteristics of aging of B lymphocytes have not been clearly understood. More than a decade ago, researchers discovered that a special group of B cells expressing CD11c and T-bet gathered in elderly female mice and were named age-associated B cells (ABCs). ABCs were later confirmed to be enriched in patients with autoimmune diseases and other infectious diseases. Age-associated B cells (ABCs) have recently been found to be associated with the production of autoantibodies, and eliminating this group of cells can alleviate the disease. However, the current elimination methods used in animal models all target their surface marker CD11c through gene editing, and there is no means that can be extended to clinical applications.

[0004] Furthermore, it remains unclear whether ABCs encompass all pathogenic B cells, or whether all ABCs are pathogenic B cells. The ultimate goal in the SLE field is to precisely target pathogenic B cells, eliminating autoantibodies while retaining protective antibodies against various pathogens.

[0005] Therefore, further understanding the pathogenesis of systemic lupus erythematosus and designing more targeted treatments and new drugs based on this to accurately target and eliminate pathogenic B cells are issues that urgently need to be addressed in this field. Summary of the Invention

[0006] To address the deficiencies of the prior art, the present application provides the use of senescent B cells as targets or targeted senescent B cells in the preparation of a drug for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis, wherein the treatment of systemic lupus erythematosus or systemic lupus erythematosus nephritis is achieved by targeting the senescent B cells and causing their death or inactivation.

[0007] As certain embodiments of the present 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 cells.

[0008] Age / autoimmunity-associated B cells (ABCs) in the prior art are a B cell subset that expands with age (in some diseases, it is not related to age). They were first discovered in mice in 2011 and 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 the surface markers are CD21lowT-bet+CD11c+. Different from ABCs, this application first proposed that senescent B cells are enriched in patients with systemic lupus erythematosus. 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 the cell cycle-dependent kinase inhibitors P21 and P16, and has the characteristics of increased senescence-related β-galactosidase activity. In addition, it was also 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 have a senescence-related secretory phenotype (TNF-α, IL-6).

[0009] B cells, also known as B lymphocytes, originate from pluripotent stem cells in the bone marrow. B lymphocyte progenitors are found in the islands of hematopoietic cells of the fetal liver (14 days after conception in mice or 8-9 weeks after conception in humans). Subsequently, the bone marrow gradually replaces the site of B lymphocyte production and differentiation. Mature B cells primarily reside in the lymph nodes of the superficial cortex of lymph nodes and in the red and white pulp of the spleen. In response to antigen stimulation, B cells differentiate into plasma cells, which synthesize and secrete antibodies (immunoglobulins) and primarily contribute to humoral immunity. B cells possess a variety of surface molecules that recognize antigens, interact with immune cells and molecules, and are crucial for the isolation and identification of B cells. These molecules primarily include leukocyte differentiation antigens, MHC, and various surface receptors.

[0010] CD19 is a CD molecule (leukocyte differentiation antigen) expressed by B cells and belongs to the Ig superfamily. It is expressed by all B cell lineages except plasma cells, as well as by malignant B cells and FDCs. It is a key membrane antigen involved in B cell proliferation, differentiation, activation, and antibody production, and also promotes BCR signaling.

[0011] The p21 protein is a cyclin-dependent kinase inhibitor (CDKI) with multiple biological functions. It degrades cyclins, inhibits cyclin-dependent kinase (CDK) activity, and binds to the cyclin / CDK complex or proliferating cell nuclear antigen (PCNA), thereby arresting cell cycle progression. p21 also regulates inflammatory responses through macrophage activation and polarization and neutrophil apoptosis. Furthermore, p21 regulates cellular apoptosis. It can inhibit apoptosis by interacting with apoptosis signal-regulating kinase 1 (ASK1) and pro-apoptotic factors, and promote apoptosis by upregulating the pro-apoptotic protein Bax or activating TNF family death receptors (TRAIL). p21 can also promote autophagy by upregulating microtubule-associated protein 1A / 1B light chain 3B (LC3) and Unc-51-like autophagy-activating kinase 1 (ULK1). Furthermore, p21 plays a crucial 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. The complex of CDK (cyclin-dependent kinase) and cyclin functions at different phases of the cell cycle, initiating DNA replication and inducing mitosis. CDKs may be central to the cell cycle machinery, regulating the cell cycle by phosphorylating a series of key substrates. The CDK4-cyclin complex participates in the regulation of the G1-S transition. The P16 protein inhibits Cdk4 activity, ultimately preventing cells from entering the S phase. If the P16 gene becomes dysfunctional due to deletion or mutation, it is unable to inhibit CDK4, ultimately leading to malignant cell proliferation and accelerated tumorigenesis. CDK4-cyclin can act on a variety of oncogene products, such as PP60c-sYc and C-abl, modulating their phosphorylation. It can also act on certain anti-cancer gene products. For example, phosphorylation of the Rb protein results in a loss of growth-inhibiting function. At the G1 / S junction, CDK4-G1cyclin phosphorylates and inactivates Rb, shifting cells from a quiescent state to a proliferative state. The P16 gene is a crucial anti-cancer gene; once inactivated, it can cause malignant cell proliferation.

[0013] β-galactosidase is a hydrolase found in lysosomes. Cellular senescence is the process by which cells gradually decline in their normal physiological functions and proliferation capacity over time or in response to external stress, leading to their exit from the cell cycle. Cell enlargement, pH-dependent expression of β-galactosidase activity, and altered gene expression patterns are further hallmarks of senescent cells. When cells undergo senescence, β-gal exhibits high enzymatic activity at pH 6.0.

[0014] Systemic lupus erythematosus (SLE) is a chronic, diffuse connective tissue disease caused primarily by abnormal immune system activation, which attacks the body's own tissues. It may be related to genetics, environmental factors, and estrogen. Long-term sun exposure, use of certain medications, infection, and oral estrogen use can all exacerbate SLE symptoms. The disease can damage organs throughout the body and may lead to a variety of complications, including kidney damage and neuropsychiatric symptoms. Commonly used drug treatments include nonsteroidal anti-inflammatory drugs, antimalarial drugs, immunosuppressants, and corticosteroids.

[0015] As certain embodiments of the present application, the present application provides the use of senescent B cells as a target in the preparation of a drug for treating systemic lupus erythematosus. As certain embodiments of the present application, the present application provides the use of senescent B cells as a target in the preparation of a drug for treating systemic lupus erythematosus nephritis. As certain embodiments of the present application, the present application provides the use of targeting senescent B cells in the preparation of a drug for treating systemic lupus erythematosus. As certain embodiments of the present application, the present application provides the use of targeting senescent B cells in the preparation of a drug for treating systemic lupus erythematosus nephritis.

[0016] As certain embodiments of the present application, the senescent B cells are CD19 + P21 + As certain embodiments of the present application, the senescent B cells are CD19 + P16 + As certain embodiments of the present application, the senescent B cells are CD19 + Spider-β-gal + cell.

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

[0018] As certain embodiments of the present application, the death or inactivation of senescent B cells includes one or more of transition to a senescent phenotype, apoptosis, clearance, and killing.

[0019] As certain embodiments of the present application, the death or inactivation of senescent B cells is achieved by treatment with B cell-targeted drugs that can convert, apoptose, clear, or kill senescent B cells to a senescent phenotype.

[0020] As certain embodiments of the present application, B cell-targeted drugs are used to eliminate the senescent B cells, and the B cell-targeted drugs include one or more of nanocarrier-based anti-aging drug delivery systems, anti-aging drug senolytics, anti-aging drug senomorphics, immune-based senescent cell clearance reagents, SASP neutralizing reagents, and biological macromolecules and cellular drugs targeted to eliminate senescent B cells.

[0021] As certain embodiments of the present 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 viral particle-based delivery system.

[0022] Nanocarrier-based drug delivery systems utilize nanotechnology to precisely deliver drugs to the site of lesions or medication. By utilizing nanomaterials as drug carriers, the system can be designed to control the release rate and dosage of drugs, achieving precise drug treatment. Nano drug delivery systems can precisely deliver drugs to the site of application, increasing drug concentration and efficacy, thereby improving efficacy. Furthermore, due to the more defined distribution of drugs within the body, damage to other parts of the body can be reduced, minimizing side effects. Furthermore, nanomaterials as drug carriers can protect drugs from degradation by enzymes and the acid-base environment within the body, thereby improving drug stability.

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

[0024] As certain embodiments of the present application, the surface of the nanocarrier-based anti-aging drug delivery system is modified with antibodies targeting senescent B cells; by modifying the surface of the delivery system with antibodies targeting senescent B cells, senescent B cells are precisely targeted, so that the loaded anti-aging drugs can directly act on senescent B cells.

[0025] As certain embodiments of the present 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 certain embodiments of the present application, the virus particles include one or more of cowpea mosaic virus (CPMV), cowpea green spot virus, canine parvovirus, heat shock protein (HSP) cage, and bacteriophage.

[0027] As certain embodiments of the present application, the liposome comprises one or more of phospholipids, ionizable lipids, cholesterol and PEGylated lipids.

[0028] As certain embodiments of the present application, the anti-aging drugs senolytics include one or more drugs that interfere with the senescent cell signaling pathway, disable the senescent cell anti-apoptosis pathway (SCAP), and drugs that target the clearance of ANT2.

[0029] Senolytics are drugs that selectively eliminate senescent cells. Their principle of delaying aging is to eliminate senescent cells. The first generation of senolytic drugs targets different SCAPs, including tyrosine kinase receptors (TKRs), growth factor receptors (GFRs), Ephrin receptor B1 (EFNB1), SRC kinase, PI3K-AKT, HSP90, BCL-2 family members, caspase inhibition, and p53. The second generation of senolytic drugs targets prodrugs and nanomedicines, including lysosomal and SA-β-Gal-activated prodrugs and nanomedicines, sodium-potassium pump (Na+ / K+-ATPase)-dependent apoptosis, SASP inhibition, and immune clearance mediated by CART cells, antibody-drug conjugates, or vaccines.

[0030] As certain embodiments of the present application, the anti-aging drugs senolytics include one or more of Dasatinib, Quercetin, Fisetin, Luteolin, Curcumin, Navitoclax (ABT-263), A1331852, and A1155463.

[0031] As certain embodiments of the present application, the anti-aging drug senolytics includes a combination of Dasatinib and Quercetin.

[0032] As certain embodiments of the present application, the drug targeting the clearance of ANT2 includes tamoxifen (MitoTam).

[0033] As certain embodiments of the present application, the drug that disables the senescent cell anti-apoptotic pathway (SCAP) includes one or more of an anti-apoptotic Bcl-2 protein inhibitor, a p53-related pathway regulator, an anti-apoptotic transcription factor FOXO4 inhibitor, and a molecular chaperone HSP90 inhibitor.

[0034] As certain embodiments of the present application, the biological macromolecules and cellular drugs targeted at eliminating senescent B cells include one or more of siRNA, shRNA, micro RNA, piRNA, ASO, CAR-T reagents, and TCRT reagents.

[0035] As certain embodiments of the present application, the anti-apoptotic Bcl-2 protein inhibitor includes one or more of a Bcl-2 inhibitor, a Bcl-xl inhibitor, a Bcl-w inhibitor, a Mcl-1 inhibitor, a Bfl1 / A-1 inhibitor, and a Bc-B inhibitor.

[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, nucleic acid expression, protein expression, or activity of a Bcl-2 family protein can be measured as described in detail 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 key in determining the sensitivity of cells to apoptosis induced by the intrinsic pathway. Based on structural and functional characteristics, Bcl-2 family members can be divided into three subfamilies: the anti-apoptotic family, the multidomain 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, the 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 can 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, referred to as Bcl-2 homology 1 (BH1), BH2, BH3, and BH4 domains. They also have a C-terminal membrane anchor sequence and a similar three-dimensional structure. Inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family can promote cell death by antagonizing the pro-survival function of the Bcl-2 protein family, thereby inducing apoptosis. The inhibitors of the present 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 a cell's sensitivity to intrinsic pathway-induced apoptosis. Based on their structural and functional characteristics, Bcl-2 family members can be divided into three subfamilies: the anti-apoptotic family, the multidomain 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, termed the Bcl-2 homolog 1 (BH1), BH2, BH3, and BH4 domains. They also possess a C-terminal membrane anchor sequence and a similar three-dimensional structure. Inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family can promote cell death by antagonizing the pro-survival function of the Bcl-2 family of proteins, thereby inducing apoptosis. The inhibitors of the present invention can inhibit one or more anti-apoptotic proteins in the Bcl-2 family. Thus, 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 certain embodiments of the present application, the Bcl-2 inhibitors include 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 certain embodiments of the present application, the Bcl-xl inhibitor includes one or more of BH3I-1, WEHI-539 hydrochloride, A1331852, and A1155463.

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

[0042] As certain embodiments of the present 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 certain embodiments of the present 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 certain embodiments of the present application, the B cell targeting drug further includes an mTOR inhibitor and metformin.

[0045] As certain embodiments of the present application, the mTOR inhibitor includes rapamycin and its analogs.

[0046] As certain embodiments of the present application, the mTOR inhibitor includes rapamycin.

[0047] As certain embodiments of the present application, the application has any 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 pulmonary complications of systemic lupus erythematosus, preferably, pulmonary complications of systemic lupus erythematosus include pulmonary hypertension; f. treating hematological complications of systemic lupus erythematosus, preferably, hematological complications of systemic lupus erythematosus include hematocytopenia; g. treating digestive system complications of systemic lupus erythematosus; h. reducing glomerular proliferation and lymphocyte infiltration around renal tubules and renal blood vessels caused by systemic lupus erythematosus; i. reducing renal immune complex deposition caused by systemic lupus erythematosus; j. reversing renal tissue aging caused by systemic lupus erythematosus; and k. alleviating renal fibrosis caused by systemic lupus erythematosus.

[0048] The present application provides a B cell targeting drug targeting senescent B cells, wherein the B cell targeting drug is the above-mentioned B cell targeting drug.

[0049] The present application also provides a drug for treating systemic lupus erythematosus, which comprises an effective amount of the B cell targeting drug and a pharmaceutically acceptable carrier.

[0050] As certain embodiments of the present application, the drug for treating systemic lupus erythematosus also includes a second drug, which includes one of a B cell-targeting clearance antibody, an antibody that inhibits B cell maturation, and a CAR-T reagent.

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

[0052] The present application provides the B cell targeting drug targeting senescent B cells, or the use of the drug in preparing a drug for eliminating senescent B cells.

[0053] The present application also provides the B cell targeted drug and the use of the drug in the preparation of products for treating systemic lupus erythematosus, 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, pulmonary complications of systemic lupus erythematosus include pulmonary hypertension; f. a product for treating hematological complications of systemic lupus erythematosus, preferably, hematological complications of systemic lupus erythematosus include hematocytopenia; g. a product for treating digestive system complications of systemic lupus erythematosus; h. a product for reducing glomerular proliferation and lymphocyte infiltration around renal tubules and renal blood vessels 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] The present 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 cells.

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

[0056] As certain embodiments of the present application, the substances include flow cytometers, immunofluorescence reagents, and immunocytochemistry reagents.

[0057] The present application also provides a use of a substance for detecting senescent B cells in preparing a kit, wherein the use is to evaluate the therapeutic effect of systemic lupus erythematosus and / or determine the prognosis of 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 cells.

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

[0059] As certain embodiments of the present application, the substances include flow cytometers, immunofluorescence reagents, and immunocytochemistry reagents.

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

[0061] This application first demonstrated the enrichment of a large number of senescent B cells in SLE patients, specifically defined as P21 + B cell or β-gal + B cells; senescent B cells are enriched in the periphery of lupus patients, characterized by high expression of the cell cycle-dependent kinase inhibitors P21 and P16, increased activity of senescence-associated β-galactosidase (SA-β-gal), and high expression of anti-apoptotic molecules of the BCL2 family. The proportion of senescent B cells is positively correlated with serum autoantibody levels and negatively correlated with complement levels. Furthermore, renal biopsies from patients with lupus nephritis also show a large infiltration of P21+ senescent B cells.

[0062] Furthermore, the present invention found that β-gal also exists in lupus mice. + Senescent B cells, and in vitro culture, β-gal + Senescent B cells, not 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 progression by secreting autoantibodies and inducing B cell senescence in recipient mice, confirming a strong causal relationship between senescent B cells and the onset of lupus.

[0063] The present application screened out a drug for treating systemic lupus erythematosus in vitro, wherein the drug includes an effective amount of the B cell targeting drug, which can target and eliminate P21 + Senescent B cells. When a combination of Bcl-2 and Mcl-1 inhibitors is used, because they act on different sites, a synergistic effect occurs, significantly enhancing the clearance function. In both the spontaneous SLE mouse model MRL / lpr and the humanized SLE mouse model that is more similar to humans, this drug combination effectively cleared senescent B cells, thereby greatly improving the disease;

[0064] The drugs screened out in this application for the treatment of systemic lupus erythematosus can not only treat systemic lupus erythematosus, but also treat systemic lupus erythematosus nephritis, reduce glomerular hyperplasia and lymphocyte infiltration around the renal tubules and renal blood vessels caused by systemic lupus erythematosus, reduce the deposition of renal immune complexes caused by systemic lupus erythematosus, reverse renal tissue aging and alleviate renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0066] Figure 2 It shows that the proportion of senescent B cells in peripheral blood is correlated with the patient's clinical indicators;

[0067] Figure 3 It was shown that senescent B cells are also enriched in the kidney tissues of patients with lupus nephritis;

[0068] Figure 4 In vitro culture showed that senescent B cells are enriched in self-reactivity and secrete a large amount of autoantibodies;

[0069] Figure 5 It was shown that β-gal + senescent B cells secrete more autoantibodies than CD11c + ABCs;

[0070] Figure 6 In vivo transplantation experiments demonstrated that senescent B cells accelerate disease progression and are causally related to the development of SLE.

[0071] Figure 7 It shows that the anti-aging drug combination screened in vitro can effectively eliminate senescent B cells;

[0072] Figure 8 The study showed that a combination of anti-aging drugs eliminated senescent B cells in a spontaneous SLE mouse model, demonstrating significant therapeutic efficacy.

[0073] Figure 9 The study showed that a combination of anti-aging drugs reduced renal pathological damage and reversed renal tissue aging in a spontaneous SLE mouse model.

[0074] Figure 10 The results showed that the anti-aging drug combination was also effective in a humanized SLE mouse model. DETAILED DESCRIPTION

[0075] 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.

[0076] [Experimental Materials]

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

[0078] Table 1 Conventional 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] markers clone Fluorescein supplier Item No. CD19 1D3 / CD19 FITC Biolegend 152404 IgD 11-26c.2a APC-Cy7 Biolegend 405744 CD138 281-2 PE Biolegend 142504 CD45 30-F11 PE-Cy7 Biolegend 103114 IgM RMM-1 PE-Cy5 Biolegend 406544 CD11c HL3 AF700 BD Biosciences 560583 CD3 500A2 PE Biolegend 152310 CD19 6D5 APC Biolegend 115512 m / h CD45R RA3-6B AF488 BD Biosciences 557669 CD45.2 104 FITC Biolegend 109805 CD45.1 A20 APC Biolegend 110714 IgD 11-26c.2a BV786 BD Biosciences 563618

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

[0087] Example 1 Phenotypic Identification of Senescent B Cells

[0088] Mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy controls (HCs) and patients (SLE). Flow cytometry was used to detect the expression of the cell cycle-dependent kinase inhibitor P21 on B cells in the peripheral blood of healthy controls (HCs) and SLE patients. The activity of β-galactosidase (β-gal) dye was used to detect its activity in B cells of HCs and SLE patients. Peripheral blood B cells were purified using magnetic bead separation, and RNA was extracted. qPCR was used to detect the expression levels of P16 and P21 genes in B cells. The detection of these characteristic markers of aging demonstrated that B cell senescence occurs in SLE patients, and the proportion of senescent B cells is much higher than that in healthy controls.

[0089] Figure 1 showed that senescent B cells are enriched in the peripheral blood of SLE patients. Figure 1 A shows a schematic diagram of collecting blood samples from a human body for analysis. Figure 1 B shows that SLE patients' B cells have higher expression of β-gal+ B cells than healthy controls. Figure 1 C shows the β-gal reagent detection images of SLE patients and healthy controls. It can be seen that the aging-related β-gal activity of B cells in SLE patients is higher than that in healthy controls. Figure 1 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 is significantly higher than that in healthy controls. Figure 1 E shows that the expression of P21 protein in B cells of SLE patients is significantly higher than that in healthy subjects.

[0090] Figure 2 The results showed that the proportion of senescent B cells in peripheral blood was correlated with the clinical indicators of patients; 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 in the kidney tissues of patients with IgA nephritis and other non-lupus nephritis and B cells in the kidney tissues of patients with lupus nephritis were detected. The test results are shown in Figure 3 , Figure 3 It was shown that senescent B cells were also enriched in the kidney tissues 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 were flow cytometrically isolated from the spleens of lupus mice using senescence-associated β-galactosidase staining. These cells were then cultured in vitro for 7 days under R848 induction. Cell differentiation was assessed, and the culture supernatant was analyzed for total antibody and autoantibody secretion using ELISA. The results demonstrated that β-gal+ B cells were more capable of differentiating into plasma cells and secreting significantly more autoantibodies than β-gal- B cells.

[0094] Figure 4 In vitro culture showed that senescent B cells are enriched in self-reactivity and secrete a large amount of autoantibodies; Figure 4 A demonstrated that senescent B cells are also enriched in lupus mice. Figure 4 B shows the flow chart of flow cytometry sorting and in vitro culture. Figure 4 Figures C to 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 for self-reactivity.

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

[0096] The cell culture supernatant was collected and the secretion of total antibodies and autoantibodies was detected by ELISA. In the in vitro culture of the above-mentioned B cell subsets in the spleen of lupus mice and the peripheral blood of SLE patients, the CD11c+ / β-gal+ subset had the strongest ability to secrete autoantibodies, the CD11c- / β-gal+ subset was slightly stronger than the remaining two subsets, and there was basically no difference between the CD11c+ / β-gal- subset and the CD11c- / β-gal- subset, both of which were very small (see Figure 5 B-5E). Thus, β-gal+ can better distinguish pathogenic B cell subsets that secrete autoantibodies than CD11c+.

[0097] Example 3 In vivo transplantation of senescent B cells to explore the causal relationship between senescent B cells and the onset of SLE

[0098] Similarly, β-gal- and β-gal+ B cells were isolated by senescence-associated β-galactosidase staining and flow cytometry sorting, and equal numbers of cells were transplanted into non-lupus-prone mice. SLE was induced by ear administration of the TLR7 agonist R848 (see Figure 6 A).

[0099] The speed and severity of the disease in the two groups of recipient mice were monitored, including the levels of autoantibodies, B cell aging, and kidney damage. The results showed that the recipient mice transplanted with β-gal+ aging B cells developed splenomegaly (see Figure 6 B), increased serum autoantibody levels, enhanced B cell differentiation into plasma cells, increased proportion of senescent B cells, and more severe renal damage, all of which demonstrate a causal relationship between senescent B cells and the occurrence and development of SLE (see 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 sorted and cultured in a medium containing different anti-aging drugs or their combinations for 3 days. The proportion of P21+ senescent B cells was detected, and the drug or drug combination with the highest clearance efficiency was screened (see Figure 7 ).

[0102] MRL / lpr mice, a mouse model of spontaneous SLE, were treated with a 4-week sequential drug regimen. The specific regimen was: ABT263 (10 mg / kg) was administered for two consecutive days, followed by S63845 (25 mg / kg) for two consecutive days after a one-day interval, and then the next round of treatment was administered two days later. One round of treatment lasted for 7 days, and four rounds of treatment were administered (see the administration procedure). Figure 8 A). New anti-aging drug combinations can eliminate senescent B cells, thereby significantly alleviating lymphadenopathy, reducing serum autoantibody titers, lowering inflammatory factor TNF-α levels, reducing urinary protein production, and fully alleviating the condition (see Figure 8 B~8G).

[0103] Since the kidney is the organ most easily affected by lupus, kidney tissue damage is the preferred focus. After treatment with the anti-aging drug according to the above method, glomerular hyperplasia and lymphocyte infiltration around the renal tubules and renal blood vessels were significantly alleviated ( 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 immune aging, renal tissue aging also occurs in lupus mice, and treatment with a combination of anti-aging drugs can also reverse renal tissue aging ( Figure 9 C), reduce renal fibrosis ( Figure 9 D), with multiple effects.

[0104] In addition, considering the differences between mouse and human immune systems, a novel humanized SLE mouse model developed independently was used for further verification. The specific dosing regimen is consistent with that in the MRL / lpr model. Figure 10A. In the human immune system, we have also seen the elimination of senescent B cells and the remission of SLE disease (see Figure 10 B~F).

[0105] 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. Use of senescent B cells as a target or targeting senescent B cells in the preparation of a drug for treating systemic lupus erythematosus or systemic lupus erythematosus nephritis, characterized in that: The treatment of systemic lupus erythematosus or systemic lupus erythematosus nephritis is achieved by targeting the senescent B cells and causing their death or inactivation.

2. The use according to claim 1, characterized in that 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 the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cell.

3. The use according to claim 1, characterized in that The death or inactivation of senescent B cells includes one or more of the following: transformation of senescent phenotype, apoptosis, clearance, and killing; Preferably, the death or inactivation of senescent B cells is caused by treatment with B cell-targeted drugs that can cause senescent B cells to undergo senescent phenotype transformation, apoptosis, clearance, or killing; Preferably, the B cell-targeted drug includes one or more of a nanocarrier-based anti-aging drug delivery system, anti-aging drug senolytics, anti-aging drug senomorphics, immune-based senescent cell clearance reagents, SASP neutralizing reagents, and biological macromolecules and cellular drugs that target the clearance of senescent B cells.

4. The use according to claim 3, characterized in that 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 viral particle-based delivery system; Or, the anti-aging drug in the nanocarrier-based anti-aging drug delivery system is used to eliminate the senescent B cells or inactivate the senescent B cells; Alternatively, the surface of the nanocarrier-based anti-aging drug delivery system is modified with antibodies targeting senescent B cells.

5. The use according to claim 4, characterized in that The polymer comprises 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, and heparin; Or, the virus particles include one or more of cowpea mosaic virus, cowpea green spot virus, canine parvovirus, heat shock protein cage, bacteriophage, and adeno-associated virus; Alternatively, the liposomes include one or more of phospholipids, ionizable lipids, cholesterol, and pegylated lipids.

6. The use according to claim 3, characterized in that The anti-aging drugs senolytics include one or more drugs that interfere with the senescent cell signaling pathway, disable the senescent cell anti-apoptosis pathway (SCAP), and drugs that target and eliminate ANT2; Preferably, the anti-aging drug senolytics include one or more of dasatinib, quercetin, fisetin, luteolin, curcumin, navitoclax (ABT-263), A1331852, and A1155463; Preferably, the anti-aging drug senolytics comprises a combination of Dasatinib and Quercetin; Preferably, the drug targeting ANT2 clearance includes tamoxifen (MitoTam); The drug that disables the senescent cell anti-apoptosis pathway (SCAP) includes one or more of an anti-apoptotic Bcl-2 protein inhibitor, a p53-related pathway regulator, an anti-apoptotic transcription factor FOXO4 inhibitor, and a molecular chaperone HSP90 inhibitor; The biological macromolecules and cellular drugs targeted at eliminating senescent B cells include one or more of antibodies, siRNA, shRNA, microRNA, piRNA, ASO, CAR-T, and TCR-T.

7. The use according to claim 6, characterized in that Anti-apoptotic Bcl-2 protein inhibitors include one or more of Bcl-2 inhibitors, Bcl-xl inhibitors, Bcl-w inhibitors, Mcl-1 inhibitors, Bfl 1 / A-1 inhibitors, and Bcl-B inhibitors; The Bcl-2 inhibitors include 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; The Bcl-xl inhibitor includes one or more of BH3I-1, WEHI-539 hydrochloride, A1331852, and A1155463; The Mcl-1 inhibitor includes one or more of AZD5991, UMI-77, AZD5991, BRD810, S63845, ABBV-467, and APG-3526; Preferably, the anti-apoptotic Bcl-2 protein inhibitor includes one or more of ABT737, ABT-263, S63845, BRD-K20733377, BRD-K56819078, and BRD-K44839765; Preferably, 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 includes one or more of AZD5991, UMI-77, AZD5991, BRD810, ABBV-467, and APG-3526.

8. The use according to claim 3, characterized in that The B cell targeting drugs also include mTOR inhibitors and metformin; Preferably, the mTOR inhibitor includes rapamycin and its analogs; Preferably, the mTOR inhibitor comprises rapamycin.

9. The use according to claim 1, characterized in that The application has any of the following functions: a. Treatment of systemic lupus erythematosus; b. Treatment of systemic lupus erythematosus nephritis; c. Treatment of cutaneous lupus erythematosus; d. Treatment of systemic lupus erythematosus encephalopathy; e. Treatment of pulmonary complications of systemic lupus erythematosus, preferably, pulmonary complications of systemic lupus erythematosus including pulmonary hypertension; f. Treating hematologic complications of systemic lupus erythematosus, preferably, hematologic complications of systemic lupus erythematosus including cytopenia; g. Treatment of digestive system complications of systemic lupus erythematosus; h. Reduce glomerular proliferation and lymphocyte infiltration around renal tubules and renal blood vessels caused by systemic lupus erythematosus; i. Reduce renal immune complex deposition caused by systemic lupus erythematosus; j. Reversing renal tissue aging caused by systemic lupus erythematosus; and k. Alleviating renal 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 according to any one of claims 1 to 9.

11. A drug for treating systemic lupus erythematosus, characterized in that: The drug comprises an effective amount of the B cell targeting drug according to claim 10, and a pharmaceutically acceptable carrier; Preferably, the drug for treating systemic lupus erythematosus further comprises a second drug, which comprises one of a B cell-targeting clearance antibody, an antibody that inhibits B cell maturation, and a CAR-T agent; Preferably, the B cell-targeting depletion antibody includes rituximab, and the B cell maturation-inhibiting antibody includes belimumab.

12. Use of the B cell targeting drug according to claim 10 or the drug according to claim 11 in the preparation of a drug for eliminating senescent B cells.

13. Use of the B cell targeting drug according to claim 10 or the drug according to claim 11 in the preparation of a product for treating systemic lupus erythematosus, its complications, and symptoms, characterized in that: The product is selected from any of the following: a. Products for the treatment of systemic lupus erythematosus; b. Products for the treatment of systemic lupus erythematosus nephritis; c. Products for the treatment of cutaneous lupus erythematosus; d. Products for the treatment of systemic lupus erythematosus encephalopathy; e. Products for pulmonary complications of systemic lupus erythematosus, preferably, pulmonary complications of systemic lupus erythematosus include pulmonary hypertension; f. Products for hematological complications of systemic lupus erythematosus, preferably, hematological complications of systemic lupus erythematosus include cytopenia; g. Products for digestive system complications of systemic lupus erythematosus; h. Products that reduce glomerular proliferation and lymphocyte infiltration around renal tubules and renal blood vessels caused by systemic lupus erythematosus; i. Products that reduce immune complex deposition in the kidneys caused by systemic lupus erythematosus; j. Products for reversing renal tissue aging caused by systemic lupus erythematosus; and k. Products for alleviating renal fibrosis caused by systemic lupus erythematosus.

14. 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 the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cell.

15. Use of a substance for detecting senescent B cells in preparing a kit for evaluating the therapeutic effect of systemic lupus erythematosus and / or determining the prognosis of 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 the cells; Preferably, the senescent B cells are CD19 + P21 + cells or CD19 + Spider-β-gal + cell.

16. The use according to claim 14 or 15, characterized in that The substances include flow cytometers, immunofluorescence reagents, and immunocytochemical reagents.

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