Application of targeted stimulation immune molecule in preparation of medicine for synergistically treating tumor diseases

Abstract

The invention discloses an application of a targeted immune stimulation molecule in preparation of a medicine for synergistically treating tumor diseases, and relates to the field of biological medicines. 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. One end of the functional molecule for targeted senescence cell immune sensitization targets senescence cell membrane molecules, glutamic acid is marked on the surfaces of senescence cells, coupling between endogenous immune cells and the senescence cells is mediated, and adhesion and removal of the senescence cells by the immune cells are promoted. The molecule can regulate the immune microenvironment of senescent tissues, effectively reduce the number of positive senescent cells of senescence-related beta-galactosidase, reduce or avoid the metastasis risk in tumor treatment, and reduce side effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of targeted stimulation of immune molecules in the preparation of drugs for the treatment and synergistic treatment of tumor diseases. Background Technology

[0002] As the aging population continues to grow, achieving "healthy aging" has become a major social and scientific issue that urgently needs to be addressed in my country and the world. Therefore, breakthroughs are urgently needed in the intervention of aging and related diseases to further improve the "healthy lifespan" of the elderly population and respond to the needs of the Healthy China strategy.

[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 slow aging. Multiple studies have shown the benefits of injecting transgenic or young immune cells to clear senescent cells from aging individuals, manifested in improved survival rates and histopathological improvements. These findings highlight the feasibility and potential of using immune strategies to clear senescent cells. However, exogenous immunotherapy carries the risks of cytokine release syndrome and neurotoxicity. In 2020, Nature reported on a chimeric antigen receptor T-cell (CAR-T) immunotherapy for selectively clearing senescent cells in mice. However, researchers also noted that a common complication of CAR-T therapy is cytokine release syndrome (also known as a cytokine storm). When this toxicity occurs, a strong T-cell response can lead to fever, affecting blood pressure and respiration. In this new study, the authors observed that high doses of anti-aging CAR-T cells did indeed induce cytokine release syndrome. Therefore, finding new methods to activate endogenous immune cells to reverse aging warrants further investigation.

[0004] The interaction and integration of the neuroendocrine and immune systems play a crucial role in the multi-system regulatory hierarchy of life. This interaction can mediate immune synaptic signaling through neurotransmitters (dopamine, acetylcholine, and γ-aminobutyric acid) and metabolic receptors present in the immune system. In one study, researchers compared metabolites in lymph nodes near immune sites with those in lymph nodes on the opposite side of a mouse's body after immune exposure to exogenous proteins. They found significant differences in the levels of metabolites associated with glutamate pathway activation in lymph nodes near immune response sites. The glutamate, alanine, and aspartate pathways are the strongest metabolic signatures distinguishing resting from activated immune sites. Glutamate is considered a neurotransmitter and immunotransmitter involved in signal transduction in the nervous and immune systems, and the expression and activation of metabolic glutamate receptors in immune cells may affect immune cell function and lifespan. Glutamate and its levels decline with age, and age is negatively and significantly correlated with metabolic signatures (serine and glutamate). Targeting glutamate receptors on immune cells may be a candidate drug for regulating glutamate receptor-mediated immunosensitization in the aging microenvironment. Therefore, strategies that promote the presentation of new targets on the surface of the inflammatory microenvironment may enhance immune activity, thereby improving the efficacy of aging immunotherapy.

[0005] Furthermore, recent studies have shown that chemotherapy can induce lung fibroblasts to enter a senescent state and release senescence-associated secretory factors (SASP), which stimulate neutrophils to form extracellular traps (NETs). NETs can then promote the proliferation of dormant DTCs by remodeling the extracellular matrix, ultimately leading to distant metastases such as lung metastases. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the use of a molecule that targets senescent cells to stimulate endogenous immunity in the preparation of drugs that synergistically treat tumor diseases and reduce side effects. Specifically, this invention prepares a dual-ligand polypeptide that can act as a "mediator" and promoter to mediate the interaction between senescent cells and immune cells, enhance endogenous immune function, and achieve the goal of effectively clearing senescent cells and improving age-related diseases.

[0007] To achieve the objectives of this invention, the following technical solutions are provided:

[0008] This invention provides the use of a molecule that targets senescent cells to stimulate endogenous immunity in the preparation of drugs that synergistically treat tumor diseases and reduce side effects, wherein the molecule contains at least two basic units.

[0009] One of the basic units is a unit with targeting functions for senescent cells, senescent tissues, or the senescent microenvironment.

[0010] Another basic unit is the unit used to stimulate endogenous immunity;

[0011] Alternatively, the molecule of the drug contains at least one basic unit, which is a unit capable of simultaneously targeting senescent cells, senescent tissues, or senescent microenvironments and stimulating endogenous immunity.

[0012] Specifically, this invention provides a molecule that induces immune activation in senescent cells. One end of the peptide is a ligand for urokinase-type plasminogen activator receptor (uPAR), which is widely expressed on the cell membrane of senescent cells, while the other end binds to a glutamate receptor, which is 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. This polypeptide can target and bind to receptors on the surface of senescent cells and glutamate receptors, promoting the interaction between senescent cells and immune cells. By labeling senescent cells with glutamate, it activates glutamate receptors on immune cells, promoting their adhesion to immune cells and thus effectively clearing senescent cells.

[0013] There are no particular limitations on the target functional units in this invention, as long as they can be target functional molecular fragments for senescent cells / tissues / microenvironments.

[0014] Furthermore, the unit having the function of targeting senescent cells, senescent tissues, or senescent microenvironment is one or more of the following: antibodies targeting specific antigens on the surface of senescent cells, ligand molecules targeting receptors highly expressed on senescent cells, polypeptides targeting senescent cells, nucleic acid aptamers targeting senescent cells, polysaccharides targeting senescent cells, and molecules targeting the senescent microenvironment.

[0015] The polypeptides with specific targeting properties to senescent cells in this invention can be in linear or cyclic form.

[0016] Furthermore, the antibody targeting the specific antigen on the surface of senescent cells is an antibody against urokinase-type plasminogen activator receptor (uPAR).

[0017] The ligand molecule that targets the receptor highly expressed in senescent cells is urokinase-type plasminogen activator (uPA).

[0018] The polypeptide targeting senescent cells is a urokinase-type plasminogen activator (PURA)-binding domain polypeptide. In some embodiments of this invention, animal experiments were first conducted using mice. Therefore, when conducting experiments with mice, the urokinase-type PURA-binding domain polypeptide was CQNGGVCVSYKYFSRIRRCSCPRK (as shown in SEQ ID NO:1), i.e., a mouse-derived sequence. However, the human urokinase-type PURA-binding domain polypeptide differs from this sequence. Therefore, when conducting experiments targeting humans, the urokinase-type PURA-binding domain polypeptide was LNGGTCVSNKYFSNIHWCNCPKKF (as shown in SEQ ID NO:2), i.e., a human-derived sequence. Of course, the sequences mentioned above in this invention can be all or part of the sequence, such as SNKYFS (as shown in SEQ ID NO:3), VSNKYFSNI (as shown in SEQ ID NO:4), and VSNKYFSNIHW (as shown in SEQ ID NO:5), as long as they satisfy the targeting function.

[0019] Peptides can be linear molecules or cyclic peptides.

[0020] Nucleic acid aptamers that target senescent cells are nucleic acid aptamers with specific targeting properties for senescent cells;

[0021] The polysaccharide that targets senescent cells is β-galactose, which targets β-galactosidase, a marker of senescent cells.

[0022] Molecules that target the aging microenvironment are molecules that target specific microenvironments related to aging.

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

[0024] Furthermore, the molecule capable of activating metabolic glutamate receptors is one or more of a polyglutamate repeating unit and a glutamate agonist.

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

[0026] Furthermore, the targeting unit of the molecule used to target senescent cells and stimulate endogenous immunity is selected as a urokinase-type plasminogen activator binding domain polypeptide, and the unit used to stimulate endogenous immunity is selected as a repeating unit of polyglutamic acid. At this time, the murine amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. x CQNGGVCVSYKYFSRIRRCSCPRK, where x is any integer.

[0027] A molecule designed to target senescent cells and stimulate endogenous immunity; its human amino acid sequence is E. y LNGGTCVSNKYFSNIHWCNCPKKF is a sequence of all or part of the sequence, where y is any integer.

[0028] Furthermore, the molecule used to target senescent cells and stimulate endogenous immunity is E. y SNKYFS, E y VSNKYFSNI, E y One of the VSNKYFSNIHW.

[0029] If a targeted peptide molecule contains one or more glutamates, it may simultaneously fulfill both dual targeting and endogenous immune stimulation functions, independently achieving the purpose of this invention. However, targeted peptide molecules are often affected by the hydrophilicity or hydrophobicity of different glutamates, which may influence the targeting affinity of the targeted peptide.

[0030] The targeting portion of the polypeptide molecule is responsible for binding to membrane proteins highly expressed on the surface of senescent cell membranes, preventing off-target effects. Therefore, due to space limitations, not all of them are listed exhaustively. However, other targeting molecules not described in this invention can also play the same role. The immune-stimulating portion can be a repeating unit containing polyglutamate (En, n≥1) or a glutamate receptor activator. The immune-stimulating portion is responsible for activating endogenous immune cells. Other glutamate receptors that can activate class I metabolic glutamate receptors, not described in detail in this invention, can also play the same role.

[0031] Furthermore, in our experiments, we found that the mouse-derived polypeptide molecules shown in SEQ ID NO:1 above have low solubility, and increasing the amount of glutamate can increase their solubility. However, the amount of glutamate is negatively correlated with the targeting of the molecule. Therefore, considering all factors, 8≤x≤24.

[0032] The human-derived peptide molecules disclosed above have relatively good solubility, so there is no need to consider increasing glutamate to improve their solubility. Instead, we should focus on the molecular targeting specificity and the effect of stimulating immunity. Taking all factors into account, 1≤y≤24.

[0033] In this invention, the two functional regions (targeting + immunosensitization) that target senescent cells to stimulate endogenous immunity can be achieved by two different molecular units (targeting functional domain + immunosensitization functional domain), such as uPA-polyglutamic acid crosslinking molecules; or they can be combined into one, that is, one functional unit has both of these functions (targeting + immunosensitization), such as huPA.

[0034] Specifically, our further research revealed that the molecule may contain only one basic unit, which is a polypeptide molecule with an amino acid sequence of all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

[0035] Furthermore, the amino acid sequence of the polypeptide molecule can be one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, or VSNKYFSNIHW.

[0036] When applied, the administration methods include intravenous administration, interventional administration to tissue lesions, oral administration, nasal inhalation administration, or intraperitoneal injection.

[0037] The molecule for targeting senescent cells and stimulating immunity provided by this invention has been experimentally verified to specifically target senescent cells, stimulate immune cells, including NK cells, and eliminate senescent cells. Therefore, in principle, this molecule for targeting senescent cells and stimulating immunity can be used to prepare drugs suitable for treating all fibrosis and age-related diseases. This invention primarily targets cancers and other diseases caused by cellular senescence following tumor development and chemotherapy.

[0038] In addition, the present invention provides a drug for synergistic treatment of tumor diseases and reduction of side effects, the drug comprising the above-mentioned molecules that target senescent cells and stimulate immunity.

[0039] The synergistic therapeutic drug for tumor diseases and reduction of side effects contains, in addition to the molecule of the present invention that targets senescent cells to stimulate immunity, necessary excipients, and may also contain other therapeutic drugs.

[0040] Furthermore, the synergistic treatment of tumor diseases and the reduction of side effects drugs can deliver the molecules that target senescent cells to stimulate immunity via oral, intravenous, intralesional, or intraperitoneal injection.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] This invention provides a class of functional molecules that target senescent cells to stimulate immunity. One end of this molecule targets the membrane molecules of senescent cells and labels the surface of senescent cells with glutamate, mediating a "coupling" between endogenous immune cells and senescent cells, promoting the adhesion and clearance of senescent cells by immune cells. This molecule can regulate the immune microenvironment of senescent tissues, effectively reducing the number of senescence-associated β-galactosidase-positive senescent cells, while simultaneously improving cellular senescence. Therefore, it can be used as an adjunct to tumor chemotherapy. By clearing senescent cells, it can reduce or avoid the risk of tumor metastasis by eliminating senescent tumor cells, and also alleviate the side effects of chemotherapy, such as organ and tissue fibrosis. Attached Figure Description

[0043] Figure 1 This section shows the uPAR expression in the replicated senescence and chemotherapy-induced cellular senescence model in Example 1. a represents SA-β-gal staining of expanded mouse embryonic fibroblasts (MEF) cells from generations 2, 7, and 9, and the detection of uPAR protein levels in MEF cells of different generations. Blue-green indicates positive staining. b represents SA-β-gal staining of LLC cells in the quiescent, proliferative, and bleomycin (BLM) treatment phases, and the detection of uPAR protein levels.

[0044] Figure 2 E16-uPA in Example 1 24 It specifically binds to uPAR on the membranes of senescent cells. a, Chimeric polypeptide E16-uPA 24 b. Dynamic binding sensing diagram of uPAR recombinant protein. Flow cytometry analysis of the target peptides binding on the surface of young and senescent MEF cells. As shown in the figure, the rightward shift of the peak indicates that the chimeric peptide molecules selectively bind to the surface of uPAR-positive senescent cells, while not binding to uPAR-negative young cells.

[0045] Figure 3 The co-focused immunofluorescence staining image in Example 1 shows that the chimeric peptide and uPAR immunofluorescence co-localize in senescent sites in liver tissue sections of mice with liver fibrosis.

[0046] Figure 4 Mass cytometry analysis of immune cells in carbon tetrachloride (CCl4)-induced liver fibrosis in Example 2. a, E16-uPA in CCl4-induced liver fibrosis. 24 The treatment diagram shows the normal control group, the CCl4 model group, and the E16-uPA group. 24 Mass cytometry visualization of t-SNE data of immune cells in liver tissue. b, normal control group, CCl4 model group and E16-uPA group. 24 The proportion of different immune cell subsets in the group. T-SNE represents t-distributed random neighbor embedding. DNT represents double-negative T cells, CD4+. - CD8 - TCRb + Cells. DPT represents double-positive T cells, CD4 + CD8 + TCRb + cell.

[0047] Figure 5 E16-uPA in Example 3 24Stimulation activates NK cells. a. E16-uPA was quantified by flow cytometry. 24 Incubated LLC senescent cells with CD45 + CD3 in cell co-culture system + CD4 - T cells and CD3 - NK1.1 + The proportion of CD107a-positive NK cells. CD45 cells incubated with solvent. + Cells shown are a negative control. CD45 cells incubated with PMA / ion + Cells served as a positive control. b, purified NK cells in E16-uPA... 24 The release of interferon-gamma after incubation exhibits cytotoxicity.

[0048] Figure 6 E16-uPA in Example 3 24 Activates NK cells to specifically kill senescent cells. (a) Compared with different concentrations of E16-uPA... 24 NK cell cytotoxicity analysis after co-culturing young and senescent MEF cells with pre-incubated cells. b, Purified NK cells with different concentrations of uPA. 24 E16 and E16-uPA 24 Cytotoxicity of pre-incubated senescent MEF cells after co-culture.

[0049] Figure 7 E16-uPA in Example 4 24 It exerts its anti-fibrotic effect by clearing senescent cells in CCl4-induced liver fibrosis. a, b, compare the severity of liver fibrosis in different treatment groups at 0, 6, and 12 weeks of induction based on the mean echogenicity of the liver. c, compare the groups (healthy control group, D+Q group, PBS group, uPA group) using scatter histograms. 24 Group, E16 group and E16-uPA 24 The statistical differences in Sirius red staining of liver tissue from each group were used to determine the treatment efficacy. (d) The groups (healthy control group, D+Q group, PBS group, uPA group) were compared. 24 Group, E16 group and E16-uPA 24 Scatter histogram comparing SA-β-gal staining of liver tissue from different groups. e, healthy control group, D+Q group, PBS group, uPA group 24 Group, E16 group and E16-uPA 24 Quantitative analysis of the NK cell marker CD161c in paraffin sections of liver tissue in the group.

[0050] Figure 8 E16-uPA in Example 5 24It can eliminate senescent cells and alleviate lung damage. a) For the first 5 days of treatment, mice were administered BLM (2 mg / kg) or saline (normal control group) via intratracheal injection. Mice with lung injury were then intravenously injected with E16-uPA for two consecutive weeks. 24 (50 mg / kg) or physiological saline (carrier group), 3 times a week, scatter histograms of statistical comparison of Masson staining of lung tissue in the three groups (n=4). b, Scatter histogram showing healthy group, control group or E16-uPA 24 Comparison of SA-β-Gal staining in lung tissue of the treatment group. c, healthy group, control group or E16-uPA 24 Quantitative analysis of the NK cell marker CD161c in paraffin sections of lung tissue from the treatment group. (d. Healthy group, control group, or E16-uPA) 24 Expression of aging markers in lung tissue of group (n=3). E16-uPA was analyzed using the Mantel-Cox log-rank test. 24 Kaplan-Meier survival curves of lung-injured mice treated with saline or physiological saline (n=10).

[0051] Figure 9 E16-uPA in Example 5 24 NK cell therapy for pulmonary fibrosis in mice. a, b, control group and E16-uPA. 24 CD45 in the treatment group + Quantitative analysis of the percentage of NK cells in lung immune cells and flow cytometry cell count.

[0052] Figure 10 E16-uPA in Example 6 24 Eliminates senescent cells in the liver, kidneys, and inguinal fat of aged mice. (a) Young mice, solvent group, and E16-uPA. 24 Quantitative analysis of SA-β-Gal staining area in liver tissue sections of aged mice in groups D+Q and D+Q. b. Young mice and solvent group, E16-uPA... 24 Quantitative analysis of SA-β-Gal staining area in renal tissue sections of aged mice in groups D+Q and young mice, as well as those in the solvent group and E16-uPA group. 24 Quantitative analysis of SA-β-Gal staining in inguinal white fat (iWAT) of aged mice in the D+Q group and the D+Q group.

[0053] Figure 11 Ey-huPA in Example 7 24 Targeted sensitization of human senescent cells triggers NK cell killing. a, compared with different concentrations of E1-huPA 24NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with pre-incubated cells. b, Comparison with cells cultured with different concentrations of E2-huPA. 24 NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with different concentrations of E4-huPA. 24 Cytotoxicity analysis of NK92 cells co-cultured with pre-incubated young and senescent IMR90 cells. (d, compared with cells cultured with different concentrations of E8-huPA) 24 NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with different concentrations of E16-huPA. 24 NK92 cytotoxicity analysis after co-culturing pre-incubated young and senescent IMR90 cells. f, Ey-huPA 24 Quantitative analysis of IFN-γ in cell culture supernatant after co-incubation with NK92. (g, different Ey-huPA) 24 Flow cytometry analysis of binding affinity to young IMR90 cells. h, different Ey-huPA 24 Flow cytometry quantitative analysis of binding ability with senescent IMR90 cells. i. Flow cytometry detection of senescent and young cells in huPA. 24 Quantitative statistics on cell mortality after co-treatment with NK92 cells.

[0054] Figure 12 To illustrate the sensitization of human senescent cells with E4-huPAz in Example 8, triggering NK cell killing: a) NK92 cytotoxicity analysis after co-culturing with young and senescent IMR90 cells pre-incubated with different concentrations of E4-huPA6. b) NK92 cytotoxicity analysis after co-culturing with young and senescent IMR90 cells pre-incubated with different concentrations of E4-huPA9. c) NK92 cytotoxicity analysis after co-culturing with different concentrations of E4-huPA9. 11 d. Quantitative analysis of IFN-γ in cell culture supernatant after co-incubation of pre-incubated young and aged IMR90 cells. e. Flow cytometry quantitative analysis of the binding affinity of different E4-huPAz to aged IMR90 cells. f. Flow cytometry quantitative analysis of the binding affinity of different E4-huPAz to young IMR90 cells.

[0055] Figure 13 E16-uPA in Example 9 24Delaying tumor progression and reducing chemotherapy toxicity. a) Quantitative statistical analysis of tumor volume in mice. b) Quantitative statistical analysis of senescence-related β-galactosidase-positive areas in mouse tumor tissue. c) Quantitative statistical analysis of pulmonary fibrosis areas in mice. d) Statistical analysis of mouse survival curves. e) Quantitative statistical analysis of CD161c staining intensity in mouse tumor tissue. Detailed Implementation

[0056] Example 1

[0057] To verify the molecular effects of this invention, animal experiments were first conducted. Mice, a common laboratory animal, were selected as the research subject. Therefore, the mouse senescent cell-targeting chimeric peptide (E) was chosen. x CQNGGVCVSYKYFSRIRRCSCPRK (x is any integer) reverses chemical toxicity and tissue homeostasis associated with age-related diseases by stimulating immune cells through targeting senescent cells.

[0058] As mentioned earlier, the polypeptide molecule in mice has poor solubility. Therefore, in the examples, experiments were conducted by linking more glutamic acid molecules. In the following studies on mice, the examples of the present invention mainly use 8, 16, and 24 glutamic acid molecules as representatives. This does not mean that other numbers of glutamic acid molecules cannot achieve the corresponding function.

[0059] Example of polypeptide sequence abbreviation: E 16 CQNGGVCVSYKYFSRIRRCSCPRK(E16-uPA 24 ),E8CQNGGVCVSYKYFSRIRRCSCPRK(E8-uPA 24 E 24 CQNGGVCVSYKYFSRIRRCSCPRK(E24-uPA 24 ).

[0060] (1) uPAR is highly expressed in senescent cells

[0061] To establish a suitable cell senescence model, for replication-induced senescence, primary MEF cells (first generation) were cultured using normal cell passages until they lost their proliferative capacity and became senescent. MEF cells typically enter senescence after approximately 7 passages or 10 population doublings. For bleomycin-induced senescent Lewis lung carcinoma (LLC) cells, LLC cells were treated with fresh BLM medium at a concentration of 30 μg / mL for approximately 24 to 48 hours, with the medium being replaced with fresh medium, until the cells ceased proliferation and entered senescence. The results showed that MEF cells began to senescent at the 7th generation, and by the 9th generation, they had essentially entered senescence and ceased proliferation. uPAR expression gradually increased with increasing MEF cell passage number, with low-passage MEF cells showing almost no uPAR expression. Figure 1 a) BLM treatment induced senescence in mouse LLC cells, with serum starvation serving as a control to induce quiescent states in mouse LLC cells. After 5 days of drug treatment, most cells were successfully induced into senescence. Quiescent LLC cells expressed almost no uPAR, but after 24 hours of drug treatment to induce senescence, the expression of uPAR on the cell surface gradually increased over time. Figure 1 (b) This indicates that cells exhibit high uPAR expression after senescence induced by chemotherapy or replication passages.

[0062] (2)E16-uPA 24 Specific binding of uPAR

[0063] Based on surface plasmon resonance (SPR) to detect the affinity of peptides for uPAR, a commercially available His-tagged recombinant uPAR protein was first immobilized on a CM5 sensor chip with a carboxymethyl dextran matrix covalently linked to a gold surface. Subsequently, a series of E16-uPAR proteins at different dilution ratios were... 24 Injected onto the sensor surface, the chimeric polypeptide exhibited concentration-dependent binding activity with the uPAR protein. Based on the results, E16-uPAR... 24 Suitable for kinetic fitting calculations. As shown in the figure, the response signal (Response, RU) increases with increasing sample concentration, and E16-uPA can be calculated accordingly. 24 The KD value is 7.310 × 10 -8 M( Figure 2 a) Chimeric polypeptide E16-uPA 24 It has a relatively high affinity for uPAR.

[0064] To establish a suitable model of cellular senescence, we induced MEF cells to replicate and senescence through cell expansion. Figure 1a) Flow cytometry results also showed the chimeric peptide E16-uPA. 24 The number of senescent MEF cells was significantly higher than that of young MEF cells. Figure 2 b).

[0065] To determine the chimeric peptide E16-uPA 24 Specific targeting of aging tissues, further confirmed by tissue immunofluorescence staining, revealed E16-uPA. 24 Co-localization with uPAR-positive cells. Normal mice and CCl4-treated mice were intravenously injected with E16-uPAR conjugated with 5-FAM. 24 E16 and liver tissue were dissected and frozen sections were prepared after 4 hours of metabolism. Colocalization analysis of the liver tissue after 4 hours of metabolism revealed ( Figure 3 E16-uPA 24 The fluorescence (green) of the chimeric polypeptide E16-uPA clearly overlaps with the fluorescence (red) of the uPAR antibody. This indicates that the chimeric polypeptide E16-uPAR... 24 It can be targeted to senescent cell membranes and senescent tissue sites by combining with uPAR.

[0066] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK all found that tail vein injection of uPAR-targeting peptides can significantly target senescent cells.

[0067] Example 2

[0068] Balance the immune microenvironment in liver fibrosis tissue.

[0069] Liver fibrosis was induced in C57BL / 6N mice by intraperitoneal injection of 1 ml / kg CCl4 twice weekly. After six weeks, mice with similar degrees of liver fibrosis were randomly divided into a control group and a treatment group. Subsequently, the mice were injected via tail vein with the peptide E16-uPA. 24 Mice were given 50 mg / kg of PBS three times a week for six weeks, while continuing CCl4 treatment at the same dose and intervals. Normal liver tissue and PBS or E16-uPA were collected from mice after treatment. 24 Post-treatment fibrotic tissue was analyzed, and immune cells were characterized by clustering using mass cytometry. Figure 4 Analysis identified approximately 12 distinct, conventional clusters. Figure 4 b). The t-SNE plot shows the locational clustering of large immune cell subsets: dendritic cells (DCs), macrophages, B cells, eosinophils, basophils, neutrophils, and CD4+. + T cells, CD4 +Treg cells, CD8 + T cells, CD4 - CD8 - TCRb + Cells, NK cells and CD4 + CD8 + TCRb + cell( Figure 4 a) Eosinophils, basophils, NK cells, and CD4+ + T cells and other large cell subsets in the CCl4 model group and E16-uPA 24 The numerical changes between the treatment groups were statistically significant. We found that, compared with the CCl4 group, the treatment groups had a decreased proportion of pro-inflammatory granulocytes (eosinophils), a decreasing trend in neutrophils, and a decrease in cytotoxic lymphocytes (NK cells and CD4+). + The proportion of T cells increased significantly. Figure 4 b). These findings indicate that E16-uPA 24 It can regulate the microenvironment of organ aging by enhancing the cytotoxic killing effect of immune cells.

[0070] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were all found to regulate the organ aging microenvironment by enhancing the cytotoxic killing effect of immune cells.

[0071] Example 3

[0072] It sensitizes senescent cells, triggering NK cell killing.

[0073] Senescent LLC cells from Example 1 were cultured at 4000 cells / well using E16-uPA. 24 After 0.5 h of pre-incubation, CD45 isolated from mouse spleen + Immune cells were co-cultured for 6 hours. Flow cytometry analysis showed that, compared with T cells, CD3-NK1.1... + NK cells exposed to E16-uPA 24 Subsequently, the degranulation marker CD107a was significantly induced ( Figure 5 a) CD3-NK1.1 + The expression of CD107a, a marker of NK cell degranulation, was significantly increased, by approximately 22.12%. This indicates that NK cells respond to E16-uPA. 24 The main effector cells. When the targeting peptide uPA... 24 Polypeptide E16 and chimeric polypeptide E16-uPA 24 After co-incubation with NK cells for 48 hours, E16-uPA24 It can significantly stimulate the production of interferon-γ, while E16 has only a weak activating effect. Figure 5 b). Next, young MEF cells and senescent MEF cells were compared with different concentrations of E16-uPA. 24 After incubation, the cells were co-cultured with NK cells isolated and purified from the spleen. Results showed that the NK cells eliminated the cytotoxicity of senescent cells over a broad E16-uPA spectrum. 24 Improvement was observed at drug concentrations, but no such effect was observed in young cells. Figure 6 a). Additionally, compared to E16 and uPA 16 Only E16-uPA 24 It can significantly promote the cytotoxicity of NK cells against senescent cells. Figure 6 b). E16 can also activate the cytotoxic killing effect of NK cells to some extent. The above results indicate that E16-uPA... 24 It can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0074] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK can all effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0075] Example 4

[0076] Recruit NK cells to eliminate senescent cells and reduce fibrosis in a liver fibrosis model.

[0077] Liver fibrosis was induced in C57BL / 6N mice by intraperitoneal injection of 1 ml / kg CCl4 twice weekly. After six weeks, mice with similar degrees of liver fibrosis were randomly divided into a model vector group and a treatment group. Subsequently, the treatment group was injected with the peptide E16-uPA via the tail vein. 24 (50 mg / kg), polypeptide uPA 24 Mice receiving PBS (50 mg / kg), peptide E16 (50 mg / kg), D+Q (50 mg / kg dasatinib and 5 mg / kg quercetin), and the model vector group were treated three times a week for six weeks, while continuing CCl4 treatment at the same dose and intervals. Liver ultrasound and histopathological sections were performed on normal mice and treated mice at weeks 0, 6, and 12. Imaging results showed E16-uPA 24 The single treatment achieved the same effect as the synergistic treatment with D+Q anti-aging agents, using E16-uPA. 24Both treatment with anti-aging agents reversed liver fibrosis after 6 weeks, characterized by reduced and more homogeneous echogenicity. Figure 7 a, b); Simultaneously, the efficacy of single-drug E16-uPA was assessed by SA-β-Gal and Sirius red staining. 24 Liver samples obtained after D+Q treatment showed fewer senescent cells and less fibrosis. Figure 7 c, d). Immunohistochemical staining of mouse liver tissue sections for the detection of the NK cell marker CD161c was performed. The results showed that CD161c was present in E16-uPA. 24 The staining intensity was significantly increased in the treatment group, indicating significant NK cell infiltration in the liver fibrosis area. Figure 7 e). Explanation of E16-uPA 24 It can effectively eliminate senescent cells in the fibrotic liver of mice.

[0078] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were all found to effectively eliminate senescent cells in the fibrotic liver of mice.

[0079] Example 5

[0080] Recruit NK cells to eliminate senescent cells and reduce fibrosis in a mouse lung injury model.

[0081] Male mice (8-12 weeks old) were administered bleomycin (Selleck, S1214) via intratracheal injection at a dose of 2 mg / kg. Following inoculation, mice were randomly divided into a control group and a treatment group based on their body weight changes over 5 days. The treatment group received E16-uPA intravenously three times weekly starting on day 5. 24 (50 mg / kg) or physiological saline, treatment lasting 2 weeks. Lung tissue was collected from treated mice for pathological staining including Masson's staining, β-galactosidase staining, and CD161c immunohistochemistry. Figure 8 As shown, via E16-uPA 24 After treatment, the area of ​​lung collagen fiber staining and the percentage of SA-β-gal positive cells were significantly reduced, while NK cell infiltration increased. Figure 8 (a, b, c), and at the same time, the mRNA levels of aging-related markers such as Col1a1, Serpine1, and P21 genes were also significantly reduced. Figure 8 d). Because there was a statistically significant increase in survival rate compared to mice injected with the vehicle, it can be concluded that removing senescence and inhibiting fibrosis are beneficial to survival. Figure 8 e). These results indicate that through E16-uPA 24Treatment of activated NK cells in the lungs can attenuate the accumulation of senescent cells during bleomycin-induced pulmonary fibrosis, thereby producing an anti-fibrotic effect and improving survival rate. E16-uPA 24 Treatment can reduce the secretion of aging inflammatory factors to a certain extent.

[0082] E16-uPA 24 NK cell therapy for pulmonary fibrosis in mice. a, b, control group and E16-uPA. 24 CD45 in the treatment group + Quantitative analysis of the percentage of NK cells in lung immune cells and flow cytometry cell counting. In the quantitative results of flow cytometry, E16-uPA... 24 The proportion of NK cells in the lungs was also significantly higher in the treatment group than in the solvent group. Figure 9 This indicates that E16-uPA 24 Treatment can effectively recruit NK cells to aggregate in the lung injury area.

[0083] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 Treatment with CQNGGVCVSYKYFSRIRRCSCPRK can effectively recruit NK cell aggregation in the lung injury area.

[0084] Example 6

[0085] Eliminate senescent cells in naturally aging mice.

[0086] To further investigate the effects of chimeric peptides on naturally occurring senescent cells in aged mice, we used E16-uPA on 22-month-old C57 mice. 24 (50 mg / kg), D+Q (50 mg / kg dasatinib and 5 mg / kg quercetin) or blank solution, injected three times weekly for 2 months, followed by assessment by SA-β-Gal staining for chimeric peptide E16-uPA. 24 Both treatment and D+Q combined treatment significantly reduced SA-β-gal activity in the liver, kidney, and inguinal adipose tissue of aged mice, suggesting a significant reduction in senescent cells in these regions. Figure 10 These results indicate that the chimeric peptide E16-uPA 24 It can systematically improve the aging pathological characteristics of naturally aging mice. The above experimental methods were repeated, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E... 24 CQNGGVCVSYKYFSRIRRCSCPRK can also systematically improve the aging pathological characteristics of naturally aging mice.

[0087] Example 7

[0088] E y -huPA 24 It targets and sensitizes human senescent cells, triggering NK cell killing.

[0089] To investigate the stimulatory effect of different amounts of glutamate on NK cells, five human senescent cell chimeric peptides were synthesized, with the sequence E. y LNGGTCVSNKYFSNIHWCNCPKKF, where y = 1, 2, 4, 8, 16.

[0090] Young and senescent IMR90 cells were mixed at 4000 / well with different concentrations of E. y -huPA 24 After incubation, the cells were co-cultured with NK92 cells. Results showed that NK92 cells eliminated the cytotoxicity of senescent cells over a relatively wide E50 range. y -huPA 24 Improvement was observed at drug concentrations, but no such effect was observed in young cells. Figure 11 ae). Explanation of E y -huPA 24 It can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0091] E at a concentration of 5 μM y -huPA 24 After co-incubating with NK92 cells for 12 hours, the cell culture supernatant was collected, and the IFN-γ content secreted by NK cells was detected by ELISA. The results showed that E y -huPA 24 It can effectively stimulate NK cells to release IFN-γ ( Figure 11 f) The effect is particularly significant when the number of glutamic acid is greater than 4.

[0092] E with a concentration of 5 μM coupled with 5-FAM y -huPA 24 After co-incubating with young and senescent IMR90 cells for 20 minutes, the interaction between young and senescent cells and E was detected by flow cytometry. y -huPA 24 The binding situation of E. The results show that E y -huPA 24 The binding to senescent cells is specific ( Figure 11 gh), and E y -huPA 24 The targeting specificity to senescent cells is negatively correlated with the number of glutamate molecules.

[0093] In addition, the study investigated whether a functional unit can simultaneously possess two functions (targeting + immune sensitization).

[0094] Young or senescent cells were stained with the fluorescent dye CFSE and then inoculated with 0, 5, and 50 μM huPA. 24 In the presence of [a specific ingredient], NK92 cells were co-incubated for 6 hours, followed by the addition of propidium iodide dye. The content of dead cells in young or senescent cells was detected by flow cytometry. The results showed that ([the following text is incomplete and requires further context to translate accurately]). Figure 11 i), in huPA 24 Under the influence of huPA, NK92 cells showed increased killing ability against senescent cells, and the killing effect of NK92 cells on senescent cells was significantly stronger than that on young cells. These results suggest that huPA... 24 It can simultaneously have both targeting and immune-sensitizing functions.

[0095] Example 8

[0096] E4-huPAz (4≤z≤24) targets and sensitizes human senescent cells, triggering NK cell killing.

[0097] In order to study huPA 24 Whether partial fragments in the sequence are sufficient to meet the targeting function, three senescent cell chimeric peptides (E4-huPA6, E4-huPA9, E4-huPA) were synthesized. 11 The sequences are E4SNKYFS, E4VSNKYFSNI, and E4VSNKYFSNIHW. The targeting regions of the three chimeric peptides consist of 6, 9, and 11 amino acid residues, respectively, all derived from huPA. 24 Part of the sequence. The activation function of the four glutamate repeat units for NK cells has been verified in Example 7.

[0098] Young and senescent IMR90 cells were incubated at 4000 cells / well with different concentrations of E4-huPAz and then co-cultured with NK92 cells. The results showed that NK92 cells exhibited improved cytotoxicity against senescent cells at a wider range of E4-huPAz concentrations, but this effect was not observed in young cells. Figure 12 (ac). This indicates that the above three types of E4-huPAz can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0099] NK92 cells were co-incubated with 5 μM E4-huPAz for 12 hours, and the cell culture supernatant was collected. The IFN-γ content secreted by NK cells was detected by ELISA. The results showed that E4-huPAz could effectively stimulate NK cells to release IFN-γ (… Figure 12 d).

[0100] Young and senescent IMR90 cells were co-incubated with 5 μM 5-FAM-conjugated E4-huPAz for 20 minutes. The binding of 5-FAM-E4-huPAz to young and senescent cells was detected by flow cytometry. The results showed that E4-huPAz specifically bound to senescent cells. Figure 12 Furthermore, the targeting of E4-huPAz to senescent cells is positively correlated with the sequence length of the targeted region.

[0101] Example 9

[0102] It slows tumor progression and reduces chemotherapy toxicity.

[0103] Clinical chemotherapy for tumors induces senescence in both tumor cells and normal cells. Senescent tumor cells exhibit drug resistance and metastasis-promoting properties; senescence in normal cells disrupts tissue homeostasis and affects patient health. This process utilizes E16-uPA... 24 Eliminating senescent cells may enhance anti-cancer treatment and reduce the side effects of chemotherapy.

[0104] Female C57BL / 6N mice aged 8-12 weeks were subcutaneously injected with 5×10⁻⁵ mg / L on their backs. 6 Mouse lung cancer LLC cell lines expressing luciferase. Five days later, mice were divided into four groups for treatment. Treatment included PBS, BLM (10 mg / kg), and E16-uPA. 24 (50 mg / kg) or BLM (10 mg / kg) and E16-uPA 24 Mice were treated with a combination therapy of 50 mg / kg. Starting on day 6, for 6 weeks, mice received two intraperitoneal injections of BLM weekly to establish a tumor chemotherapy-induced aging model, while simultaneously receiving three tail vein injections of E16-uPA weekly. 24 .

[0105] Figure 13 a shows that, although only E16-UPA 24 The therapeutic effect on tumor size was not significant, but compared with BLM alone, BLM and E16-UPA showed better therapeutic effects. 24 The combination therapy significantly enhanced tumor regression. Figure 13 Figures b and c show that the combination therapy significantly reduced the aging-related β-galactosidase staining areas in tumor tissue increased by BLM treatment, as well as the fibrotic areas in the lungs caused by BLM treatment. Figure 13 The results showed that the combination therapy prolonged the survival of tumor-bearing mice. Figure 13 The results showed that the number of CD161c-positive NK cells in mouse tumor tissues significantly increased after combination therapy. These results suggest that E16-uPA... 24 It slows tumor progression and reduces chemotherapy toxicity.

[0106] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK was also found to slow tumor progression and reduce chemotherapy toxicity.

[0107] Furthermore, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of targeted stimulation of immune molecules in the preparation of drugs for the treatment and synergistic therapy of tumor diseases, characterized in that, The targeted stimulating immune molecule contains at least two basic units. One of the basic units is a unit with targeting functions for senescent cells, senescent tissues, or the senescent microenvironment. Another basic unit is the unit used to stimulate endogenous immunity; Or the 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 microenvironments and stimulate endogenous immunity.

2. The use as described in claim 1, characterized in that, The unit having the function of targeting senescent cells, senescent tissues or senescent microenvironment is one or more of the following: antibody targeting specific antigens on the surface of senescent cells, ligand molecule targeting receptors highly expressed on senescent cells, polypeptide targeting senescent cells, nucleic acid aptamer targeting senescent cells, polysaccharide targeting senescent cells, and molecule targeting senescent microenvironment.

3. The use as described in claim 2, characterized in that, The polypeptide targeting senescent cells is a urokinase-type plasminogen activator binding domain polypeptide.

4. The use as described in claim 1, characterized in that, The unit used to stimulate endogenous immunity is a molecule that can activate metabolic glutamate receptors or a molecule that can stimulate immune cells in aging tissues or the aging microenvironment. The molecule that can activate metabolic glutamate receptors is one or more of a repeating unit of polyglutamate and a glutamate agonist. The immune cells are natural killer cells, macrophages, T cells, or dendritic cells.

5. The use as described in claim 4, characterized in that, The amino acid sequence of the urokinase-type plasminogen activator binding domain polypeptide molecule is all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

6. The use as described in claim 5, characterized in that, The amino acid sequence of the urokinase-type plasminogen activator binding domain polypeptide molecule is one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, and VSNKYFSNIHW.

7. The use as described in claim 5, characterized in that, The urokinase-type plasminogen activator binding domain polypeptide molecule is a linear molecule or its cyclic peptide form.

8. The use as described in claim 1, characterized in that, The molecule contains only one basic unit, which is a polypeptide molecule with an amino acid sequence of all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

9. The use as described in claim 8, characterized in that, The amino acid sequence of the polypeptide molecule is one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, and VSNKYFSNIHW.

10. The use as described in claim 5, characterized in that, The amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. y LNGGTCVSNKYFSNIHWCNCPKKF is a sequence or a partial sequence of which y is any integer.

11. The use as described in claim 10, characterized in that, 1≤y≤24。 12. The use as described in claim 10, characterized in that, The amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. y LNGGTCVSNKYFSNIHWCNCPKKF, E y SNKYFS, E y VSNKYFSNI, E y One of the VSNKYFSNIHW.

Images