Medicine for targeting senescent cells to stimulate endogenous immunity and application thereof

Abstract

The invention discloses a medicine for targeting senescent cells to stimulate endogenous immunity and application of the medicine, and relates to the field of biological medicine. The medicine provided by the invention can mediate the interaction between senescence cells and immune cells, enhance the endogenous immune function, and achieve the purposes of effectively removing senescence cells and improving senescence-related diseases. According to the functional molecule for targeted aging cell immune sensitization, one end of the molecule targets an aging cell membrane molecule, glutamic acid is used for marking the surface of an aging cell, coupling between an endogenous immune cell and the aging cell is mediated, and adhesion and removal of the aging cell by the immune cell are promoted. The molecule can regulate the immune microenvironment of senescence tissues, effectively reduce the number of positive senescence cells of senescence-related beta-galactosidase, reverse fibrosis and improve natural senescence phenotypes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug for stimulating endogenous immunity by targeting senescent cells and an application thereof. Background Art

[0003] The accumulation of senescent cells in tissues can lead to diseases such as diabetes, osteoarthritis, liver and lung fibrosis, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), intestinal inflammation, and atherosclerosis. Immune clearance of senescent cells can delay aging. Several studies have shown that injecting genetically modified or young immune cells to clear senescent cells in aging individuals is beneficial, as reflected in improved survival and improved tissue pathology. These findings highlight the feasibility and potential of using immune strategies to clear senescent cells. However, exogenous immunotherapy poses the risk of cytokine release syndrome and neurotoxicity. In 2020, Nature magazine reported that an anti-cancer chimeric antigen receptor T cell (CAR-T) immunotherapy was used to selectively clear senescent cells in mice. However, the researchers also pointed out that a common complication of using CAR-T therapy is cytokine release syndrome (also known as cytokine storm). When this toxic effect occurs, the strong T cell response can cause fever, affect blood pressure and breathing. In this new study, the authors observed that high doses of anti-aging CAR-T cells did induce cytokine release syndrome. Therefore, finding new methods to activate endogenous immune cells to reverse aging deserves further study.

[0004] The interaction and integration of the neuroendocrine and immune systems plays an important role in the multi-system regulatory hierarchy throughout life, and this interaction can occur through neurotransmitters (dopamine, acetylcholine, and γ-aminobutyric acid) and metabolic receptors present in the immune system, thereby mediating signals at the immune synapse. In one study, researchers compared the metabolites of lymph nodes near the immune site with those of lymph nodes on the other side of the mouse body after exposure to exogenous proteins through immunization. They found that there were significant differences in the levels of metabolites associated with glutamate pathway activation in lymph nodes near the site of immune response. The glutamate, alanine, and aspartate pathways were the strongest metabolic features that distinguished between resting and activated immune sites. Glutamate should be considered a neurotransmitter and immunotransmitter involved in signaling in the nervous and immune systems, and the expression and activation of metabotropic glutamate receptors in immune cells may affect the function and lifespan of immune cells. As individuals age, glutamate and glutamate levels decrease, and age is negatively and significantly correlated with metabolic features (serine and glutamate). Targeting immune cell glutamate receptors may become a candidate drug for regulating glutamate receptor-mediated immune sensitization in the aging microenvironment. Therefore, strategies that promote the presentation of novel targets on the surface of the inflammatory microenvironment may enhance immune activity and thus improve the efficacy of senescence immunotherapy. We are addressing this question and determining whether immunotherapy targeting senescent cells can reverse chemotoxicity and tissue homeostasis in aging-related diseases. Summary of the invention

[0005] Based on the deficiencies in the prior art, the present invention provides a drug for stimulating endogenous immunity by targeting senescent cells and its application. Specifically, the present invention prepares a dual-ligand polypeptide, which can act as a "matchmaker" and promoter to mediate the interaction between senescent cells and immune cells, enhance endogenous immune function, and achieve the goal of effectively removing senescent cells and improving aging-related diseases.

[0006] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0007] The present invention provides a drug for stimulating endogenous immunity by targeting senescent cells.

[0008] The drug molecule contains at least two basic units.

[0009] One of the basic units is a unit with the function of targeting senescent cells, senescent tissues or senescent microenvironments.

[0010] Another basic unit is the unit for stimulating endogenous immunity;

[0011] Or the drug molecule contains at least one basic unit,

[0012] The basic unit is a unit that can simultaneously have the function of targeting senescent cells, senescent tissues or senescent microenvironment and stimulating endogenous immunity.

[0013] Specifically, the present invention provides a molecule that induces senescent cells to activate immunity. One end of the peptide serves as a ligand targeting the urokinase-type plasminogen activator receptor (uPAR) widely expressed on the cell membrane of senescent cells, and the other end binds to the glutamate receptor widely expressed on the cell membrane of immune cells. A chimeric polypeptide consisting of 40 amino acids is synthesized by adding polyglutamic acid to the amino terminus of the uPAR-specific ligand fragment. It can target and bind to senescent cell surface receptors and glutamate receptors, promote the interaction between senescent cells and immune cells, label senescent cells with glutamate, activate glutamate receptors on immune cells to promote their adhesion to immune cells, and thus effectively eliminate senescent cells.

[0014] There is no particular limitation on the targeting functional unit in the present invention, as long as it can be a targeting functional molecular fragment of senescent cells / tissues / microenvironments.

[0015] Furthermore, the unit having the function of targeting senescent cells, senescent tissues or senescent microenvironments is one or more of antibodies targeting specific antigens on the surface of senescent cells, ligand molecules targeting receptors highly expressed in senescent cells, polypeptides targeting senescent cells, nucleic acid aptamers targeting senescent cells, polysaccharides targeting senescent cells, and molecules targeting senescent microenvironments.

[0016] The polypeptides with specific senescent cell targeting properties of the present invention may be in linear or circular form.

[0017] Furthermore, the antibody targeting the specific antigen on the surface of senescent cells is an antibody against urokinase-type plasminogen activator receptor (uPAR);

[0018] The ligand molecule targeting the receptor highly expressed in senescent cells is urokinase-type plasminogen activator (uPA);

[0019] The polypeptide targeting senescent cells is a urokinase-type plasminogen activator binding domain polypeptide; such as one or more of CQNGGVCVSYKYFSRIRRCSCPRK, a linear molecule or a cyclic peptide form thereof.

[0020] The nucleic acid aptamer targeting senescent cells is a nucleic acid aptamer with specific senescent cell targeting properties;

[0021] The polysaccharide targeting senescent cells is β-galactose targeting senescent cell marker β-galactosidase.

[0022] Molecules targeting the aging microenvironment are molecules that target the specific microenvironment of aging.

[0023] Preferably, the unit for stimulating endogenous immunity is a molecule capable of activating metabotropic glutamate receptors or capable of stimulating immune cells in aging tissues or aging microenvironments.

[0024] Furthermore, the molecule capable of activating metabotropic glutamate receptors is one or more of a repeating unit of polyglutamic acid and a glutamate agonist;

[0025] The immune cells are natural killer cells, macrophages, T cells or dendritic cells.

[0026] Preferably, the drug for targeting senescent cells to stimulate endogenous immunity is a senescent cell-targeting chimeric polypeptide having an amino acid sequence of E x CQNGGVCVSYKYFSRIRRCSCPRK, where x is any integer.

[0027] If the number of glutamic acid contained in the targeting polypeptide molecule is greater than 2, it may satisfy both dual targeting and endogenous immune stimulation functions and independently achieve the purpose of the present invention. However, the targeting polypeptide molecule is usually affected by the hydrophilicity of different glutamic acids, which may affect the targeting affinity of the targeting polypeptide.

[0028] The targeting portion in the polypeptide molecule is responsible for binding to the membrane protein highly expressed on the surface of the aging cell membrane to prevent off-target. Therefore, due to the limited space of the present invention, it is not exhaustively listed, but even other targeting molecules not described in the present invention can also play the same role. The stimulating immune function end can be a repeating unit containing polyglutamic acid (En, n≥2), or a glutamate receptor activator. The stimulating immune function portion is responsible for activating endogenous immune cells, and other activating class I metabolic glutamate receptors not described in detail in the present invention can also play the same role.

[0029] Most preferably, 8≤x≤24.

[0030] In the examples of the present invention, it was verified that when x=8, 16, and 24, tail vein injection of uPAR targeting peptide can significantly target senescent cells.

[0031] In the present invention, the two functional areas of targeting senescent cells to stimulate endogenous immunity (targeting + immunosensitization) can be achieved through two different molecular units (targeting functional domain + immunosensitization functional domain), such as uPA-polyglutamic acid cross-linking molecules; or they can be combined into one, that is, one functional unit has both functions (targeting + immunosensitization).

[0032] The present invention also provides the use of the drug in preparing a drug for reversing aging-related diseases.

[0033] When used, the administration method is intravenous administration, tissue lesion intervention administration, oral administration, nasal inhalation administration or intraperitoneal injection.

[0034] The molecules provided by the present invention for stimulating immunity by targeting senescent cells are applicable to all aging-related diseases, including Alzheimer's disease, Parkinson's disease, atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by some genotoxic chemotherapy), tumorigenesis, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, liver fibrosis, renal fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, intestinal inflammation, diabetes, osteoarthritis and osteoporosis.

[0035] The present invention also provides the use of the above-mentioned senescent cell-targeted immune-stimulating molecule in the preparation of senescent clearance drugs.

[0036] In addition, the present invention also provides an aging clearance drug, which contains the above-mentioned molecule targeting senescent cell immune sensitization.

[0037] The aging clearance drug contains not only the molecules targeting senescent cell immunosensitization of the present invention, but also necessary auxiliary materials and may also contain other therapeutic drugs.

[0038] Furthermore, the aging clearance drug can administer the immune sensitization molecule orally, intravenously, intralesionally, or by intraperitoneal injection.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides a class of functional molecules that target senescent cell immune sensitization. One end of the molecule targets senescent cell membrane molecules and marks the surface of senescent cells with glutamate, mediates the "coupling" between endogenous immune cells and senescent cells, and promotes the adhesion and removal of senescent cells by immune cells. The molecule can regulate the immune microenvironment of senescent tissues, effectively reduce the number of senescent-associated-β-galactosidase-positive senescent cells, and reverse fibrosis and improve the natural aging phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1The expression of uPAR in the replicative senescence and chemotherapy drug-induced cell senescence model in Example 1. a represents the SA-β-gal staining of mouse embryonic fibroblasts (MEF) cells at the 2nd, 7th, and 9th generations of amplification and the detection of uPAR protein levels in MEF cells of different generations. Blue-green represents positive staining. b represents the SA-β-gal staining of LLC cells in the resting phase, proliferation phase, and after bleomycin (BLM) treatment and the detection of uPAR protein levels.

[0042] Figure 2 E16-uPA in Example 1 24 Specific binding to uPAR on the membrane of senescent cells. a, Chimeric peptide E16-uPA 24 Dynamic binding sensor diagram with uPAR recombinant protein. b, Flow cytometry detection of the targeting peptide binding to the surface of young MEF and senescent MEF cells. As can be seen from the figure, the right shift of the peak indicates that the chimeric peptide molecule selectively binds to the surface of uPAR-positive senescent cells, but has no binding to uPAR-negative young cells.

[0043] Figure 3 The cofocal immunofluorescence staining images in Example 1 show that the chimeric polypeptide and uPAR immunofluorescence co-localize in the senescent site in the liver tissue sections of mice with liver fibrosis.

[0044] Figure 4 This is the mass spectrometry analysis of immune cells in liver fibrosis induced by carbon tetrachloride (CCl4) in Example 2. a, E16-uPA in liver fibrosis induced by CCl4 24 Schematic diagram of treatment and normal control group, CCl4 model group and E16-uPA 24 Mass spectrometry visualization t-SNE data of immune cells in liver tissue of the group. b, Normal control group, CCl4 model group and E16-uPA 24 The proportion of different immune cell subsets in the group. T-SNE stands for t-distributed stochastic neighbor embedding. DNT stands for double negative T cells, CD4-CD8-TCRb + DPT stands for double positive T cells, CD4 + CD8 + TCR + cell.

[0045] Figure 5 E16-uPA in Example 3 24 Stimulation and activation of NK cells. a, Quantification of E16-uPA by flow cytometry 24 Incubate LLC senescent cells with CD45 + CD3 in cell co-culture system+ The ratio of CD107a positive cells in CD4-T cells and CD3-NK1.1+NK cells. + Cells are shown as negative control. CD45 + Cells were used as positive control. b, Purified NK cells were incubated with E16-uPA 24 Cytotoxicity of IFN-γ released after incubation.

[0046] Figure 6 E16-uPA in Example 3 24 Activated NK cells specifically kill senescent cells. a, with different concentrations of E16-uPA 24 Cytotoxicity analysis of purified NK cells after co-culture with pre-incubated young MEF cells and senescent MEF cells. b, Purified NK cells were incubated with different concentrations of uPA 24 , E16 and E16-uPA 24 Cytotoxicity after co-culture with pre-incubated senescent MEF cells.

[0047] Figure 7 E16-uPA in Example 4 24 It exerts anti-fibrotic effects by removing senescent cells in CCl4-induced liver fibrosis. a, b, Comparison of the severity of liver fibrosis in different treatment groups at 0, 6, and 12 weeks of induction based on the average echo intensity of the liver. c, Scatter histogram comparison of the groups (healthy control group, D+Q group, PBS group, uPA group 24 Group, E16 group and E16-uPA 24 The statistical difference of Sirius red staining of liver tissue in each group was used to judge the therapeutic effect of each group. d, The statistical difference of Sirius red staining of liver tissue in each group (healthy control group, D+Q group, PBS group, uPA group 24 Group, E16 group and E16-uPA 24 e, healthy control group, D+Q group, PBS group, uPA 24 Group, E16 group and E16-uPA 24 Quantitative analysis of NK cell marker CD161c in paraffin sections of liver tissue in the two groups.

[0048] Figure 8 E16-uPA in Example 5 24 Eliminate senescent cells and reduce lung damage. a, Five days before treatment, mice were injected with BLM (2 mg / kg) or saline (normal control group) by intratracheal administration. Mice with lung damage were intravenously injected with E16-uPA for 2 consecutive weeks. 24(50 mg / kg) or saline (vehicle group), 3 times a week, and the scatter plot histogram of Masson staining of lung tissues of the three groups was compared (n=4). b, Scatter plots showing the healthy group, control group or E16-uPA 24 Comparison of SA-β-Gal staining in lung tissues of the treatment groups. c, Healthy group, control group or E16-uPA 24 Quantitative analysis of NK cell marker CD161c in paraffin sections of lung tissues in the treatment groups. d, healthy group, control group or E16-uPA 24 Expression of senescence markers in lung tissue of the two groups (n=3). e, Mantel-Cox test was used to analyze the expression of E16-uPA 24 Kaplan-Meier survival curves of lung-injured mice treated with 4% paraformaldehyde or saline (n=10).

[0049] Fig. 9 E16-uPA in Example 5 24 Treatment of mouse pulmonary fibrosis by NK cells. a, b, control group and E16-uPA 24 CD45 + Quantification and flow cytometry of the percentage of NK cells in lung immune cells.

[0050] Fig.10 E16-uPA in Example 6 24 Elimination of senescent cells in the liver, kidney, and inguinal fat of old mice. a, young mice and solvent group, E16-uPA 24 Quantitative analysis of SA-β-Gal staining area in liver tissue sections of old mice in the D+Q group and the solvent group. b, young mice and E16-uPA 24 Quantitative analysis of SA-β-Gal staining area in renal tissue sections of old mice in the D+Q group and the solvent group. c, young mice and E16-uPA 24 Quantitative analysis of gross SA-β-Gal staining in the inguinal white fat (iWAT) of aged mice in group D and group D+Q. DETAILED DESCRIPTION

[0051] Example 1

[0052] Senescent cell targeting chimeric peptides (E x CQNGGVCVSYKYFSRIRRCSCPRK (x is an arbitrary integer), stimulates immune cells by targeting senescent cells, thereby reversing chemical toxicity and tissue homeostasis of aging-related diseases.

[0053] In the following experiments of this embodiment, unless otherwise specified, the polypeptide molecule sequence used is E 16CQNGGVCVSYKYFSRIRRCSCPRK(E16-uPA 24 ).

[0054] E16-uPA 24 It can specifically bind to uPAR on the membrane of aging cells.

[0055] (1) uPAR is highly expressed in senescent cells

[0056] In order to establish a suitable cell senescence model, for replication-induced senescence, the first generation MEF cells extracted from the primary culture were cultured according to normal cell subculture until they lost their proliferation ability and became senescent. MEF will enter the senescence period after about 7 cell subcultures or 10 population doublings. For bleomycin-induced aging mouse Lewis lung carcinoma (LLC) cells, a fresh culture medium containing 30 μg / mL of BLM was prepared to treat LLC cells for about 24 to 48 hours, during which time the fresh culture medium was replaced and cultured until the cells no longer proliferated and entered the senescence period. From the results, it can be seen that the 7th generation MEF cells began to age, and by the 9th generation they had basically entered the senescence period and no longer proliferated. With the increase in the number of MEF cell subcultures, the expression of uPAR gradually increased, among which the low-generation MEF cells almost did not express uPAR ( Figure 1 a); BLM was used to induce mouse LLC cell senescence, and serum starvation was used to induce mouse LLC cells into quiescence as a control. After 5 days of drug treatment, most cells were successfully induced into senescence; LLC cells in quiescence hardly expressed uPAR, but after 24 hours of drug treatment to induce senescence, the expression of uPAR on the cell surface gradually increased with time ( Figure 1 b) This indicates that uPAR is highly expressed after cells are induced to become senescent by chemotherapy or replicative passaging.

[0057] (2) E16-uPA 24 Specific binding to uPAR

[0058] Based on surface plasmon resonance (SPR) detection of peptide-uPAR affinity, commercial His-tagged recombinant uPAR protein was first immobilized on a CM5 sensor chip with a carboxymethyl dextran matrix covalently linked to a gold surface. 24 The chimeric polypeptides were injected into the sensor surface respectively, and the chimeric polypeptides showed binding activity with uPAR protein in a concentration-dependent manner. 24It is suitable for kinetic fitting calculation. As can be seen from the figure, the response signal (Response, RU) increases with the increase of sample concentration, and E16-uPA 24 The KD value is 7.310×10 -8 M( Figure 2 a) Chimeric polypeptide E16-uPA 24 The affinity for uPAR is relatively high.

[0059] In order to establish a suitable cell senescence model, we induced MEF cell replicative senescence by cell expansion ( Figure 1 a) Flow cytometry results also showed that the chimeric peptide E16-uPA 24 The number of combined senescent MEF cells was significantly higher than that of young MEF cells ( Figure 2 b).

[0060] To determine the chimeric peptide E16-uPA 24 Specific targeting of senescent tissues was further confirmed by tissue immunofluorescence staining of E16-uPA 24 Colocalization with uPAR-positive cells. Normal mice and CCl4-treated mice were intravenously injected with 5-FAM-coupled E16-uPAR 24 and E16, after 4 hours of metabolism, the liver tissue was dissected out for frozen section and stained with uPAR antibody. Co-localization analysis of the liver tissue after 4 hours of metabolism revealed that ( Figure 3 ), E16-uPA 24 The fluorescence (green) of the chimeric polypeptide E16-uPAR antibody (red) overlaps significantly. 24 It can be targeted to the membrane of senescent cells and senescent tissue sites by binding to uPAR.

[0061] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK, E 16 CQNGGVCVSYKYFSRIRRCSCPRK and E 24 CQNGGVCVSYKYFSRIRRCSCPRK, both found that tail vein injection of uPAR-targeted peptides can significantly target senescent cells.

[0062] Example 2

[0063] E16-uPA 24 Balancing the immune microenvironment in liver fibrosis.

[0064] Liver fibrosis was induced in C57BL / 6N mice by intraperitoneal injection of 1 ml / kg CCl4 twice a week. Six weeks later, mice with similar degrees of liver fibrosis were randomly divided into a control group and a treatment group. Subsequently, the mice were injected with peptide E16-uPA24 (50 mg / kg) and PBS three times a week for six consecutive weeks, while continuing to receive CCl4 treatment at the same dose and interval. After the treatment, normal liver tissues and PBS or E16-uPA24 were collected from the mice. 24 Fibrotic tissue after treatment and immune cell population characterization using mass cytometry analysis ( Figure 4 Analysis, roughly identified 12 conventional different clusters ( Figure 4 b) t-SNE plot showing the location clustering of large immune cell subsets: DCs, macrophages, B cells, eosinophils, basophils, neutrophils, CD4 + T cells, CD4 + Treg cells, CD8 + T cells, CD4-CD8-TCRb + Cells, NK cells and CD4 + CD8 + TCR + cell( Figure 4 a). Eosinophils, basophils, NK cells and CD4 + T cells and other large cell subsets in CCl4 model group and E16-uPA 24 The quantitative changes between the treatment groups were statistically significant. We found that compared with the CCl4 group, the proportion of proinflammatory granulocytes (eosinophils) decreased in the treatment group, neutrophils showed a downward trend, and cytotoxic lymphocytes (NK cells and CD4 + The proportion of T cells increased significantly ( Figure 4 b) These findings suggest that E16-uPA 24 The microenvironment of organ aging can be regulated by enhancing the cytotoxic killing effect of immune cells.

[0065] Example 3

[0066] E16-uPA24 sensitizes senescent cells and triggers NK cell killing.

[0067] The senescent LLC cells in Example 1 were cultured at 4000 cells / well and treated with E16-uPA 24 After pre-incubation for 0.5 h, CD45 + Immune cells were co-cultured for 6 h, and flow cytometry analysis showed that compared with T cells, CD3-NK1.1 + NK cells exposed to E16-uPA 24 After that, the degranulation marker CD107a was significantly induced ( Figure 5 a), CD3-NK1.1 + The expression of NK cell degranulation marker CD107a increased significantly, with an increase of about 22.12%. This indicates that NK cells are responding to E16-uPA 24 When the targeting peptide uPA24, polymer peptide E16 and chimeric peptide E16-uPA24 were co-incubated with NK cells for 48 hours, E16-uPA24 could significantly stimulate the production of interferon-γ, while E16 had only a weak activation effect ( Figure 5 b) Next, young MEFs and aged MEFs were cultured in the presence of different concentrations of E16-uPA. 24 After incubation, the cells were co-cultured with NK cells isolated and purified from the spleen. The results showed that NK cells eliminated the cytotoxicity of senescent cells in a wide range of E16-uPA 24 drug concentrations, but not in young cells ( Figure 6 a). In addition, compared with E16 and uPA 16 , only E16-uPA 24 Can significantly promote the cytotoxicity of NK cells to senescent cells ( Figure 6 b) E16 can also activate the cytotoxic killing effect of NK cells to a certain extent. The above results show that E16-uPA 24 It can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0068] Example 4

[0069] E 16-uPA 24 Recruiting NK cells eliminates senescent cells and reduces fibrosis in a liver fibrosis model.

[0070] C57BL / 6N mice were induced to develop liver fibrosis by intraperitoneal injection of 1 ml / kg CCl4 twice a week. Six weeks later, mice with similar liver fibrosis were randomly divided into a model vehicle group and a treatment group. Subsequently, the mice in the treatment group were injected with peptide E16-uPA into the tail vein. 24 (50 mg / kg), peptide uPA 24 The mice were treated with PBS (50 mg / kg), peptide E16 (50 mg / kg), D+Q (50 mg / kg Dasatinib and 5 mg / kg quercitrin) and the model vehicle group three times a week for six weeks, while continuing to receive CCl4 treatment at the same dose and interval. Liver ultrasound and pathological tissue sections were performed on normal mice and treated mice at 0, 6 and 12 weeks. Imaging results showed that E16-uPA 24The single treatment achieved the same effect as the synergistic treatment with D+Q senescence agents. 24 Both senolytic and senolytic treatments for 6 weeks reversed liver fibrosis, characterized by decreased and more homogeneous echogenicity ( Figure 7 a, b); E16-uPA alone was evaluated by SA-β-Gal and Sirius red staining 24 Liver samples obtained after treatment with D+Q showed fewer senescent cells and less fibrosis ( Figure 7 c, d). Immunohistochemical staining of NK cell marker CD161c was performed on the pathological sections of mouse liver tissue. The results showed that CD161c was expressed in E16-uPA 24 The staining intensity in the treatment group was significantly increased, indicating that there was significant NK cell infiltration in the liver fibrosis tissue area ( Figure 7 e). Description of E16-uPA 24 It can effectively eliminate senescent cells in mouse fibrotic liver.

[0071] Example 5

[0072] E16-uPA 24 Recruiting NK cells eliminates senescent cells and reduces fibrosis in a murine lung injury model.

[0073] Male mice (8-12 weeks) were injected intratracheally with 2 mg / kg bleomycin (Selleck (S1214). After inoculation, the mice were randomly divided into a control group and a treatment group based on the weight changes of the mice for 5 days. After the 5th day, the treatment group began to receive E16-uPA intravenously 3 times a week. 24 (50 mg / kg) or saline, and the treatment lasted for 2 weeks. The lung tissues of the treated mice were taken for Masson staining, β-galactosidase staining, and CD161c immunohistochemistry and other pathological staining. Figure 8 As shown, E16-uPA 24 After treatment, the collagen fiber staining area and the percentage of SA-β-gal positive cells in the lungs were significantly reduced, and NK cell infiltration increased ( Figure 8 a, b, c), and the mRNA levels of aging-related markers such as Colla1, Serpinel, and P21 genes also decreased significantly ( Figure 8 d). Because there is a statistically significant increase in survival rate compared to vehicle-injected mice, it can be seen that removing senescence and inhibiting fibrosis are beneficial to survival. ( Figure 8 e) These results suggest that E16-uPA 24 Treatment with activated NK cells in the lung attenuates the accumulation of senescent cells during bleomycin-induced pulmonary fibrosis, resulting in antifibrotic effects and improved survival, E16-uPA 24Treatment can reduce the secretion of aging inflammatory factors to a certain extent.

[0074] E16-uPA 24 Treatment of mouse pulmonary fibrosis by NK cells. a, b, control group and E16-uPA 24 CD45 + Quantification and flow cytometry of the percentage of NK cells in lung immune cells. In the quantitative results of flow cytometry, E16-uPA 24 The proportion of NK cells in the lungs in the treatment group was also significantly higher than that in the solvent group ( Fig. 9 ), which indicates that E16-uPA 24 The treatment effectively recruited NK cells to areas of lung damage.

[0075] Example 6

[0076] E16-uPA 24 Elimination of senescent cells in naturally aged mice.

[0077] In order to further study the chimeric peptide E16-uPA 24 To investigate the effects of naturally occurring senescent cells in aged mice, we treated 22-month-old C57 mice with E16-uPA 24 (50 mg / kg), D+Q (50 mg / kg dasatinib and 5 mg / kg quercitrin) or blank solvent were injected 3 times a week for 2 months of treatment, followed by evaluation by SA-β-Gal staining, chimeric peptide E16-uPA 24 Treatment and D+Q combined treatment can significantly reduce the SA-β-gal activity in the liver, kidney, and inguinal fat of old mice, indicating that senescent cells in the liver, kidney, and inguinal fat areas are significantly reduced ( Fig.10 These results indicate that the chimeric peptide E16-uPA 24 It can systematically improve the aging pathological characteristics of naturally aged mice.

Claims

1. A drug for stimulating endogenous immunity by targeting senescent cells, characterized in that: The drug molecule contains at least two basic units. One of the basic units is a unit with the function of targeting senescent cells, senescent tissues or senescent microenvironments. Another basic unit is the unit for stimulating endogenous immunity; Or the drug molecule contains at least one basic unit, The basic unit is a unit that can simultaneously have the function of targeting senescent cells, senescent tissues or senescent microenvironment and stimulating endogenous immunity.

2. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 1, characterized in that: The unit having the function of targeting senescent cells, senescent tissues or senescent microenvironments is one or more of antibodies targeting specific antigens on the surface of senescent cells, ligand molecules targeting receptors highly expressed in senescent cells, polypeptides targeting senescent cells, nucleic acid aptamers targeting senescent cells, polysaccharides targeting senescent cells, and molecules targeting senescent microenvironments.

3. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 2, characterized in that: The antibody targeting the specific antigen on the surface of senescent cells is an antibody against the urokinase-type plasminogen activator receptor; The ligand molecule targeting the receptor highly expressed in senescent cells is urokinase-type plasminogen activator; The polypeptide targeting senescent cells is a urokinase-type plasminogen activator binding domain polypeptide; The polysaccharide targeting senescent cells is β-galactose targeting senescent cell marker β-galactosidase.

4. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 1, characterized in that: The unit for stimulating endogenous immunity is a molecule capable of activating metabotropic glutamate receptors or capable of stimulating immune cells in aging tissues or aging microenvironments.

5. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 4, characterized in that: The molecule capable of activating metabotropic glutamate receptors is one or more of a repeating unit of polyglutamic acid and a glutamate agonist; The immune cells are natural killer cells, macrophages, T cells or dendritic cells.

6. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 5, characterized in that: The drug for targeting senescent cells to stimulate endogenous immunity is a senescent cell-targeted chimeric polypeptide, and the amino acid sequence is ExCQNGGVCVSYKYFSRIRRCSCPRK, wherein x is an arbitrary integer.

7. The drug for stimulating endogenous immunity by targeting senescent cells according to claim 6, characterized in that: 8≤x≤24。 8. Use of the drug according to any one of claims 1 to 7 in the preparation of a drug for reversing aging-related diseases or eliminating senescent cells.

9. The use according to claim 8, characterized in that When used, the administration method is intravenous administration, tissue lesion intervention administration, oral administration, nasal inhalation administration or intraperitoneal injection.

10. The use according to claim 8, characterized in that The aging-related disease is any one of the following: Alzheimer's disease, Parkinson's disease, atherosclerosis, cardiovascular dysfunction, tumorigenesis, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, liver fibrosis, renal fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, intestinal inflammation, diabetes, osteoarthritis and osteoporosis.

Images