Application of PD-1 antibody in preparation of medicine for treating atherosclerosis diseases

Abstract

The invention relates to the field of biological medicines, and discloses application of a PD-1 antibody in preparation of a medicine for treating atherosclerosis diseases. The invention provides an application of a PD-1 antibody in preparation of a medicine for treating atherosclerosis. According to the invention, the surface-linked PD-1 antibody can inhibit activated and proinflammatory PD-1 + T cells in the atherosclerotic plaque and promote inflammation ablation of a plaque microenvironment, and a new theoretical basis and a potential intervening immune target are expected to be provided for immunotherapy of the atherosclerotic plaque.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a PD-1 antibody in the preparation of a drug for treating atherosclerotic diseases. Background Art

[0002] Atherosclerosis is a major cause of cardiovascular and cerebrovascular disease. Its characteristic manifestation is that lesions begin in the intima of the affected arteries, typically with the accumulation of lipids and complex carbohydrates, followed by fibrosis and calcification, forming atherosclerotic plaques. These lesions often involve large and medium-sized arteries. Once they progress sufficiently to obstruct the arterial lumen, the tissues or organs supplied by these arteries will suffer ischemia and necrosis. The etiology of atherosclerosis remains unclear, but studies have shown that it is driven by multiple risk factors, including age, gender, family history, dyslipidemia, hypertension, smoking, diabetes, impaired glucose tolerance, and obesity. Current treatments primarily include appropriate work and lifestyle habits, lipid-lowering medications, and atherectomy or vascular stenting. However, current conventional clinical treatments, such as statins and surgical interventions (such as endarterectomy and vascular interventions), still place a significant number of patients at high risk for cardiovascular and cerebrovascular events following treatment. Therefore, this prompts us to explore the immune mechanisms and immunotherapy targets of atherosclerotic diseases, so as to more effectively inhibit or even reverse the progression of atherosclerotic plaques.

[0003] Programmed death receptor-1 (PD-1) is a key immunosuppressive molecule that regulates immune cell function and is widely expressed on activated T cells, B cells, and natural killer cells. Tumor cells typically express programmed death receptor ligand 1 (PD-L1) and bind to the PD-1 receptor on T cells. The PD-1 / PD-L1 signaling pathway induces a state of functional exhaustion in tumor tissue-infiltrating T cells, causing them to lose the ability to effectively recognize tumor cells, thereby leading to immune escape of tumor cells. PD-1 inhibitors can block the PD-1 / PD-L1 signaling pathway between tumor cells and T cells, allowing suppressed effector T cells to restore their function and regain their ability to kill tumor cells, thereby promoting tumor regression.

[0004] PD-1 monoclonal antibodies (also known as PD-1 immune checkpoint inhibitors) are currently common PD-1 inhibitors, such as nivolumab (Opdivo, Bristol-Myers Squibb), pembrolizumab (Keytruda, Merck), camrelizumab (Erica, Hengrui Medicine), toripalimab (Toyi, Junshi Biosciences), sintilimab (Tyvyt, Innovent Biologics), and tislelizumab (Tislelizumab, BeiGene). Fcγ receptors (FcγRs) are receptors for the Fc region of IgG antibodies. They are primarily expressed on the membranes of immune cells and mediate interactions between immune cells and antigen-antibody complexes or other cells, triggering and regulating various immunological responses in the body. They serve as a bridge between specific antibodies and effector cells. FcγRI (CD64) has a high affinity for IgG4 antibodies (such as PD-1 monoclonal antibodies). In the treatment of cancer, it can capture and bind to PD-1 monoclonal antibodies, thereby weakening the ability of PD-1 monoclonal antibodies to block the PD-1 / PD-L1 signaling pathway, leading to hyperprogression of tumors. Currently, only tislelizumab is a PD-1 inhibitor specifically engineered within the Fc constant region. This blocks the binding of tislelizumab to the FcγRI receptor on the surface of macrophages, thereby enhancing the anti-tumor therapeutic efficacy of PD-1 inhibitors.

[0005] Currently, PD-1 immune checkpoint inhibitors are mainly used in the treatment of clinical tumor diseases (such as solid tumors such as lung cancer, melanoma, liver cancer, and nasopharyngeal carcinoma), but the effect of PD-1 inhibitors on the prognosis of human atherosclerotic plaques and their immune regulatory mechanism are unknown. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a use of a PD-1 antibody in the preparation of a drug for atherosclerotic diseases. The present invention proposes that "surface-linked PD-1 antibodies can inhibit activated, pro-inflammatory PD-1 in atherosclerotic plaques." + T cells, promoting the inflammatory ablation of the plaque microenvironment", which is expected to provide a new theoretical basis and potential interventional immune targets for the immunotherapy of atherosclerotic plaques.

[0007] The specific technical solution of the present invention is: the use of PD-1 antibodies in the preparation of drugs for atherosclerotic diseases, wherein: the PD-1 antibodies meet the following conditions: (a) are IgG1-IgG4 type antibodies; (b) carry an antibody Fc segment domain; and (c) can be linked to the cell surface or a pharmaceutically acceptable drug carrier.

[0008] The present invention has shown through research that, because of the loss of PD-L1 expression in the microenvironment of human atherosclerotic plaques, PD-1+ T cells are not functionally exhausted but remain in a functionally activated and pro-inflammatory state. The mechanism of action of the PD-1 antibody of the present invention is as follows: the FcγRI (CD64) receptor on the surface of macrophages in atherosclerotic plaques captures and binds to the free PD-1 antibody, causing the PD-1 antibody to link to the surface of macrophages; the PD-1 antibody linked to the cell surface can function as a non-natural ligand of the PD-1 receptor; the non-natural ligand of the PD-1 receptor binds to and interacts with the PD-1 receptor on the surface of T cells, inducing PD-1 + T cell exhaustion; by inducing PD-1 + T cell function depletion promotes inflammatory ablation in the local microenvironment of atherosclerotic plaques.

[0009] In summary, the present invention discloses PD-1 + T cells are activated in human atherosclerotic plaques and have pro-inflammatory functions. However, the current international research on PD-1 in human atherosclerotic plaques is still under investigation. + The phenotype and function of T cells have not been reported, so the present invention proposes that "surface-linked PD-1 monoclonal antibodies can inhibit the activated, pro-inflammatory PD-1 in human atherosclerotic plaques." + T cells, promoting inflammatory ablation of the plaque microenvironment" is expected to provide a new theoretical basis and potential immune target for immunotherapy of human atherosclerotic plaques.

[0010] Preferably, the PD-1 antibody is a PD-1 monoclonal antibody, a PD-1 polyclonal antibody or a PD-1 bispecific antibody.

[0011] Preferably, the PD-1 monoclonal antibody is a humanized antibody.

[0012] Preferably, the light chain of the PD-1 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1: eivltqspatlslspgeratlscrasqsvssylawyqqkpgqaprlliydasnratgiparfsgsgsgtdftltisslepedfavyycqqssnwprtfgqgtkveik rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec; The heavy chain of the PD-1 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 2: qvqlvesgggvvqpgrslrldckasgitfsnsgmhwvrqapgkglewvaviwydgskryyadsvkgrftisrdnskntlflqmnslraedtavyycatnddywgqgtlvtvss astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthhhhhh.

[0013] Preferably, the PD-1 monoclonal antibody is nivolumab.

[0014] Preferably, the cells are macrophages.

[0015] Preferably, the drug carrier is selected from a metal stent, a drug-coated stent or a bioabsorbable stent.

[0016] Preferably, the atherosclerotic disease is a disease caused by vascular atherosclerosis, including coronary heart disease, myocardial infarction, valvular heart disease, heart failure, ischemic cardiomyopathy, cerebral vascular infarction, stroke, aneurysm, and aortic dissection.

[0017] Preferably, the drug is a pharmaceutical preparation administered by injection or stent carrier. Preferably, the injection administration includes peripheral injection and local coronary intervention; the stent carrier administration includes coronary intervention stent carrier administration. Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has shown that due to the lack of expression of PD-L1 in the microenvironment of human atherosclerotic plaques, PD-1 + T cells are not functionally exhausted but remain in a functionally activated and pro-inflammatory state. In vitro experiments have shown that PD-1 antibodies captured by FcγRI on the surface of macrophages act as non-natural ligands for PD-1 receptors to inhibit PD-1. + T cell activation and pro-inflammatory function. In addition, a retrospective study of cancer patients treated with PD-1 antibodies found that the atherosclerotic plaques of cancer patients treated with PD-1 antibodies showed a clinical phenotype of significant reduction. Therefore, PD-1 antibodies can be used to prepare immunotherapy drugs for the treatment of human atherosclerotic diseases.

[0018] (2) The surface-immobilized PD-1 antibody of the present invention can promote the ablation of local inflammation and plaque reduction in human atherosclerotic plaques. The present invention has conducted research on the use of PD-1 monoclonal antibodies as therapeutic drugs for human atherosclerosis, providing sufficient theoretical and experimental basis for new immunotherapy for this disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Immunofluorescence staining of human atherosclerotic plaque tissue sections, scale bar = 20 μm. A: PD-1 expression in human atherosclerotic plaques. + Expression on T cells, where green is PD-1, red is CD3, gray is CD4, and blue is DAPI representing the cell nucleus. B: PD-1 in human atherosclerotic plaques CD8 + Expression on T cells, with PD-1 in green, CD3 in red, CD8 in gray, and DAPI in blue representing the cell nucleus. The large image is a composite of four protein channels, while the small images are confocal microscopy scans of individual protein channels.

[0020] Figure 2 PD-1 from human atherosclerotic plaques + Protein phenotypes of T cells detected by CyTOF. A: T cell activation-related protein molecules (Fas, CD27, CD28, ICOS, CD38, HLA-DR, Ki67 and CD161) in PD-1 + CD4 + T cell subsets (T09, T11, T12, T13) and PD-1 + CD8 + Histogram of T cell subsets (T23, T24) expression. B: T cell activation (CD7, CD69, CD161, CD27, CD28, HLA-DR) and functional exhaustion (TIGIT, Tim-3, LAG-3) related protein molecules in PD-1 + CD4 + (PT02, PT05, PD11, PT12) and PD-1 + CD8 + (PT27) expression histogram.

[0021] Figure 3 .Origin of human atherosclerotic plaques PDCD1 + T cell signaling pathway enrichment analysis in single-cell transcriptome sequencing. A, left; B, left: respectively CD4 + PDCD1 + T cells and CD8 + PDCD1 + Volcano plot of differentially expressed genes in T cells, red and blue represent PDCD1 + T and PDCD1 - Genes expressed by T cells, typical genes are marked in the figure; A, right; B, right: respectively CD4 + PDCD1 + T cells and CD8 + PDCD1 + Signaling pathways highly enriched in T cells.

[0022] Figure 4 .CD4 from human atherosclerotic plaques and lung cancer tissues + / CD8 + PD-1 + T cell activation experiment in vitro. A: CD4 T cells from plaques and lung tumor tissues were activated by CD3 / CD28 stimulation beads. + / CD8 + PD-1 + After T cells were stimulated, the release of IL-2 and IFN-γ cytokines (pg / ml); B: CD4 + / CD8 + PD-1 + The activation degree of IL-2 and IFN-γ cytokine release ability of T cells after in vitro stimulation (%), unpaired t test, * p <0.05,** p <0.01,*** p <0.001; C: plaque-derived PD-1 + CD4 + and PD-1 + CD8 + The release of cytokines (IL-2, TNF-α, IFN-γ, IL-1β, IL-6) by T cells before and after activation with CD3 / CD28 stimulation beads (pg / ml) was compared with paired t-test and marked. p Value; D: plaque-derived PD-1 + CD4 + and PD-1 + CD8 + The activation degree (%) of cytokines (IL-2, TNF-α, IFN-γ, IL-1β, IL-6) after T cell stimulation.

[0023] Figure 5.Flow cytometry analysis and immunohistochemical staining of PD-L1 expression. A: PD-L1 expression in immune cells and non-immune cells of human lung tumors (n=4) and plaques (n=3). + Representative images of flow cytometry analysis of cells (top) and statistical results of the two groups (bottom); B: Representative images of PD-L1 immunohistochemistry (clone number: 22C3, top; clone number: SP26, bottom) in human lung tumor and plaque tissue sections.

[0024] Figure 6 FcγRI in plaque tissue + (CD64) expression in macrophages. A: Mass spectrometry flow cytometry detection of CD64 + The proportion of macrophages in myeloid cells in plaque tissue (n=20); B: Typical representative images of immunohistochemical co-localization of PD-L1, PD-1, and CD64 on consecutive paraffin sections of plaque tissue, scale bar represents 200μm.

[0025] Figure 7 .FcγRI + HEK293 cells can capture PD-1 monoclonal antibodies in vitro and interact with PD-1 + Jurkat T cells interact. A: PE-labeled PD-1 monoclonal antibody (left; 0.00005, 0.0005, 0.005, 0.05, 0.5, 5 μg / ml) and FcγRI + Mean fluorescence intensity (MFI) of the PE channel signal after binding to HEK293 cells; B: GFP fluorescent protein-labeled PD-1 + Jurkat T cells (green) in vitro with FcγRI + HEK293 cells undergo cell-cell interaction with PD-1 monoclonal antibody (red).

[0026] Figure 8 PD-1 monoclonal antibodies can significantly inhibit the intensity of inflammatory responses in the plaque microenvironment. A: PD-1 monoclonal antibodies treat CD45 from plaques and lung tumors. + After the cells were treated with PD-1 monoclonal antibody, the release capacity of pro-inflammatory cytokines (IL-2, IFN-γ and TNF-α) (pg / ml); B: After the PD-1 monoclonal antibody was treated, the release capacity of CD45 + Inhibition degree of cellular inflammatory factor release (%), unpaired t-test, *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 9Effect of PD-1 monoclonal antibody therapy on the prognosis of atherosclerotic plaques in cancer patients (regardless of cancer type). A: Ultrasound scans of atherosclerotic plaques in cancer patients at different time points before and after PD-1 monoclonal antibody treatment. Plaque areas are marked with white dashed lines, and the scale bar represents 5 mm. B: Changes in atherosclerotic plaque size in cancer patients (patients, n=8; plaques, n=13) before and after PD-1 monoclonal antibody treatment. Paired t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the embodiments.

[0029] Overall embodiment A PD-1 antibody for preparing a drug for atherosclerotic disease, wherein the PD-1 antibody meets the following conditions: (a) being an IgG1-IgG4 antibody; (b) carrying an antibody Fc segment domain; and (c) being linkable to a cell surface or a pharmaceutically acceptable drug carrier.

[0030] In some specific embodiments, the PD-1 antibody is a PD-1 monoclonal antibody, a PD-1 polyclonal antibody, or a PD-1 bispecific antibody; in some more specific embodiments, the PD-1 monoclonal antibody is a humanized antibody.

[0031] In some specific embodiments, the light chain of the PD-1 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1: Eivltqspatlslspgeratlscrasqsvssylawyqqkpgqaprlliydasnratgiparfsgsgsgtdftltisslepedfavyycqqssnwprtfgqgtkveik rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec.

[0032] The heavy chain of the PD-1 monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2: qvqlvesgggvvqpgrslrldckasgitfsnsgmhwvrqapgkglewvaviwydgskryyadsvkgrftisrdnskntlflqmnslraedtavyycatnddywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthhhhhh.

[0033] In some specific embodiments, the PD-1 monoclonal antibody is nivolumab.

[0034] In some specific embodiments, the cells are macrophages and the like.

[0035] In some specific embodiments, the drug carrier is selected from a metal stent, a drug-coated stent, a bioabsorbable stent, and the like.

[0036] In some specific embodiments, the atherosclerotic disease is a disease caused by vascular atherosclerosis, including coronary heart disease, myocardial infarction, valvular heart disease, heart failure, ischemic cardiomyopathy, cerebral vascular infarction, stroke, aneurysm, and aortic dissection.

[0037] In some specific embodiments, the drug is a drug preparation administered by injection or stent carrier; in some more specific embodiments, the injection administration includes peripheral injection, local coronary intervention, etc.; the stent carrier administration includes coronary intervention stent carrier administration, etc. Specific embodiments The technical solution of the present invention is further described below through specific embodiments. In order to better understand the purpose, technical solution and advantages of the present invention, it is clearer and more obvious. The embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] Example 1: PD-1 from human atherosclerotic plaques + Characterization of T cell phenotype and function 1 Materials and Methods 1.1 Clinical samples, reagents, and instruments 1.1.1 Clinical samples included: Patients with atherosclerosis underwent manual plaque removal, and patients with lung cancer underwent surgical resection of tumor tissue. Patients of all genders, aged 18 and above, were eligible. All samples were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine, under clinical ethics approval number #2017-102. Sample collection was approved by the patients or their guardians, who signed informed consent forms. Sample handling and procedures were approved by the Clinical Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0040] 1.1.2 Reagents: Human atherosclerotic plaque tissue was digested with type IV collagenase (purchased from Sigma-Aldrich), hyaluronidase (purchased from Sigma-Aldrich), and DNase (purchased from Sigma-Aldrich). Protein targets were detected by mass cytometry (CyTOF): CD45, CD3, CD47, CD56, TCRγδ, CD19, CCR6, CD95, CD123, CD66b, CD33, CD25, CD14, CD38, CD39, PD-L1, Ki- 67, CD45RA, CD11c, CD68, CCR7, GITR, CD28, CTLA-4, FoxP3, CXCR3, RORγ, CD161, CD27, ICOS, T-bet, CD15, IL-7Rα, GATA-3, BTLA, GranzymeB, PD-1, CD16, HLA-DR, CD4, CD8a, CD11c, LAG-3, Tim-3, TIGIT, CD69, CD7; single-cell sequencing detection reagents: Single Cell 5'Library and Gel Bead Kit (purchased from 10×Genomics), Chromium Single Cell A Chip Kit (purchased from 10×Genomics). T cell in vitro stimulation beads: Human T-Activator CD3 / CD28 Dynabeads (purchased from Thermo Fisher); inflammatory factor detection kit: CBA (Cytometric Bead Array) flow cytometry multiplex protein quantitative detection kit for IL-2, IFN-γ, TNF-a, IL-1β and IL-6 (purchased from BD Biosciences).

[0041] 1.1.3 Instruments: Mass spectrometry flow cytometer (CyTOF), Gem Code Single Cell Platform (10×Genomics), NovaSeq6000 Platform (Illumina), BD FACS AriaII flow sorter (BD Biosciences), Beckman flow analyzer (Beckman Coulter), confocal scanning electron microscope (Leica).

[0042] 1.2 Experimental plan: 1.2.1 Immunofluorescence staining Human atherosclerotic plaque tissue was collected after surgery, embedded and frozen, and PD-1, CD3, CD4, DAPI or PD-1, CD3, CD8, DAPI multicolor fluorescence staining was used to evaluate the expression of PD-1 in CD4. + and CD8 + Expression on T cells.

[0043] Immunofluorescence results of human atherosclerotic plaques are shown in Figure 1 As shown; Figure 1 Middle: A: PD-1 in human atherosclerotic plaques CD4 + Expression on T cells, where green is PD-1, red is CD3, gray is CD4, and blue is DAPI representing the cell nucleus. B: PD-1 in human atherosclerotic plaques CD8 + Expression on T cells, where green is PD-1, red is CD3, gray is CD8, and blue is DAPI representing the cell nucleus. The large picture is a composite picture of four protein channels, and the small pictures are confocal microscope scans of single protein channels. Figure 1 It can be seen that there are PD-1 positive CD4 + Helper T cells and CD8 + Killer T cells.

[0044] 1.2.2 Mass cytometry (CyTOF) detection Fresh plaque tissue was sampled and dissociated into a single-cell suspension using 4 mg / mL type IV collagenase, 250 μg / mL hyaluronidase, and 20 μg / mL type I DNase. Simultaneously, more than 40 heavy metal-conjugated antibodies were labeled. High-throughput mass cytometry was used to detect plaque-infiltrating PD-1. + Functional protein expression levels of T cells.

[0045] Plaque-derived PD-1 + T cell protein phenotype Figure 2 As shown; Figure 2Middle: A: T cell activation-related protein molecules (Fas, CD27, CD28, ICOS, CD38, HLA-DR, Ki67 and CD161) in PD-1 + CD4 + T cell subsets (T09, T11, T12, T13) and PD-1 + CD8 + Histogram of T cell subsets (T23, T24) expression. B: T cell activation (CD7, CD69, CD161, CD27, CD28, HLA-DR) and functional exhaustion (TIGIT, Tim-3, LAG-3) related protein molecules in PD-1 + CD4 + (PT02, PT05, PD11, PT12) and PD-1 + CD8 + (PT27) expression histogram. Figure 2 It is known that PD-1 + T cells in the plaque microenvironment still express protein molecules related to T cell activation (Fas, CD28, ICOS, CD38, HLA-DR, CD7, CD69), but almost do not express protein molecules related to T cell exhaustion (TIGIT, Tim-3, LAG-3).

[0046] 1.2.3 Single-cell transcriptome sequencing After fresh sampling of human atherosclerotic plaque tissue, single cell suspension was dissociated according to the above digestion method, and CD45 + Immune cells were captured and reacted using the Gem Code Single Cell platform, and transcriptome libraries were constructed and deep sequenced using the NovaSeq6000 platform.

[0047] PDCD1 + T cell signaling pathway enrichment analysis see Figure 3 As shown; Figure 3 Middle: A, left; B, left: respectively CD4 + PDCD1 + T cells and CD8 + PDCD1 + Volcano plot of differentially expressed genes in T cells, red and blue represent PDCD1 + T and PDCD1 - Genes expressed by T cells, typical genes are marked in the figure; A, right; B, right: respectively CD4 + PDCD1 + T cells and CD8 + PDCD1 + Signaling pathways highly enriched in T cells. Figure 3 It can be seen that CD4 + PDCD1 + T cells and CD8 + PDCD1 + T cells exhibit an activated functional phenotype at the transcriptional level.

[0048] 1.2.4 PD-1 + T cell activation assay PD-1 from lung tumors obtained by flow cytometry + T cells, lung tumor-derived PD-1 - T cells and atherosclerotic plaque-derived PD-1 + T cells were placed in RPMI complete medium and placed in a 37°C, 5% CO2 incubator for 2 hours. 2×10 4 The cells were divided into two groups and stimulated with CD3 / CD28 beads at a ratio of 1:3 in 200 μL RPMI-1640 medium and incubated at 37°C in a 5% CO2 incubator for 48 hours. The supernatants were collected and the release of cytokines (IL-2, IFN-γ, TNF-α, IL-1β, and IL-6) was quantified by flow cytometry.

[0049] PD-1 + T cell activation experiments were performed as follows Figure 4 As shown; Figure 4 Middle: A: CD3 / CD28-stimulated beads activate CD4 from plaques and lung tumor tissues + / CD8 + PD-1 + After T cells were stimulated, the release of IL-2 and IFN-γ cytokines (pg / mL); B: CD4 + / CD8 + PD-1 + The activation degree of IL-2 and IFN-γ cytokine release ability of T cells after in vitro stimulation (%), unpaired t test, * p <0.05,** p <0.01,*** p <0.001; C: plaque-derived PD-1 + CD4 +and PD-1 + CD8 + The release of cytokines (IL-2, TNF-α, IFN-γ, IL-1β, IL-6) by T cells before and after activation with CD3 / CD28 stimulation beads (pg / mL) was compared with paired t-test and marked. p Value; D: plaque-derived PD-1 + CD4 + and PD-1 + CD8 + After T cell stimulation, the activation degree (%) of cytokines (IL-2, TNF-α, IFN-γ, IL-1β, IL-6). Figure 4 It is known that plaque-derived CD4 + / CD8 + PD-1 + T cells can still be reactivated in vitro and have the ability to release cell activation and pro-inflammatory cytokines, among which CD4 + PD-1 + T cells compared to CD8 + PD-1 + T cells have a stronger ability to release cytokines than T cells.

[0050] 1.3 Conclusion PD-1 in plaques + T cells behave as non-functionally exhausted, activated T cells at both the protein and transcriptome levels; at the same time, they can still release T cell activating factors (IL-2) and pro-inflammatory cytokines (IFN-γ and TNF-α) at ​​the functional level.

[0051] Example 2: Use of PD-1 monoclonal antibodies to inhibit the inflammatory response of immune cells in human atherosclerotic plaques 2 Materials and Methods 2.1 Inclusion of clinical samples, drugs, reagents and instruments.

[0052] 2.1.1 Medicines: Nivolumab monoclonal antibody (Bristol-Myers Squibb, 209-408) Lot No.: ABT4285.

[0053] The light chain of the nivolumab monoclonal antibody (PD-1 monoclonal antibody) contains the amino acid sequence shown in SEQ ID NO: 1: Eivltqspatlslspgeratlscrasqsvssylawyqqkpgqaprlliydasnratgiparfsgsgsgtdftltisslepedfavyycqqssnwprtfgqgtkveik rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec.

[0054] The heavy chain of the nivolumab monoclonal antibody (PD-1 monoclonal antibody) contains the amino acid sequence shown in SEQ ID NO: 2: qvqlvesgggvvqpgrslrldckasgitfsnsgmhwvrqapgkglewvaviwydgskryyadsvkgrftisrdnskntlflqmnslraedtavyycatnddywgqgtlvtvss astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthhhhhh.

[0055] 2.1.2 Clinical samples included: Patients with atherosclerosis underwent atherectomy, and patients with lung cancer underwent surgical resection of tumor tissue.

[0056] 2.1.3 Reagents: Human arteriosclerotic plaque tissue digestion: type IV collagenase, hyaluronidase and type I DNase; cytokine determination kit: CBA (Cytometric Bead Array) flow cytometry multiplex protein quantitative detection kit for IL-2, IFN-γ and TNF-a.

[0057] 2.1.4 Instruments: BD FACS AriaII flow sorter; Beckman flow analyzer; Nikon A1RSi confocal scanning electron microscope.

[0058] 2.2 Experimental plan: 2.2.1 PD-L1 expression Human atherosclerotic plaque tissue and lung cancer were collected after surgery, dissociated into single-cell suspensions, and labeled with PD-L1. Flow cytometry was used to detect and evaluate the expression of PD-L1 in immune cells and non-immune cells derived from plaques and lung cancer tissues. Plaques and lung cancer tissues were embedded and paraffin-sectioned, and PD-L1 immunohistochemical staining was used to evaluate the expression of PD-L1 in the plaque and lung cancer microenvironment.

[0059] Figure 5 Figure 1 shows the flow cytometry analysis and immunohistochemical staining results of PD-L1 expression. A: PD-L1 expression in immune cells and non-immune cells of human lung tumors (n=4) and plaques (n=3). + Representative images of flow cytometry analysis of cells (top) and statistical results of the two groups (bottom); B: Representative images of PD-L1 (clone number: 22C3, top; clone number: SP26, bottom) immunohistochemistry in human lung tumor and plaque tissue sections. Figure 5 As shown in the Figure 3, both flow cytometric analysis and immunohistochemical staining showed that both immune cells and non-immune cells in plaque tissues hardly expressed PD-L1.

[0060] 2.2.2 PD-1 + T cells and FcγRI + Spatial localization of macrophages in plaque tissue Plaque tissue was collected after surgery, embedded and paraffin-sectioned, and PD-1, PD-L1 and CD64 staining were used on the consecutive tissue sections to evaluate PD-1. + T cells and FcγRI + Spatial distribution of (CD64) macrophages in plaque tissue.

[0061] Figure 6 FcγRI in plaque tissue + (CD64) expression in macrophages; A: Mass spectrometry flow cytometry detection of CD64 + The proportion of macrophages in myeloid cells in plaque tissue (n=20); B: Typical representative images of immunohistochemical co-localization of PD-L1, PD-1 and CD64 on consecutive paraffin sections of plaque tissue, scale bar represents 200μm. Figure 6 As shown, a large number of macrophages in plaques (n=20) expressed FcγRI + (CD64) and PD-1 + T cells are spatially adjacent; this result is that FcγRI captures PD-1 monoclonal antibodies and binds to PD-1 + It provides favorable physical spatial location and conditions for T cell interaction.

[0062] 2.2.3 FcγRI+ Study on the interaction between HEK293 cells and PD-1 monoclonal antibody in vitro Different concentrations of PD-1 monoclonal antibodies were used in FACS buffer to + HEK293 cells or FcγRI - HEK293 cells were incubated for 30 minutes and labeled with PE-conjugated sheep F(ab')2 anti-human IgG (F(ab')2) fragments for flow cytometry analysis to evaluate FcγRI + HEK293 cells or FcγRI - The binding capacity of HEK293 cells to PD-1 monoclonal antibody; 1×10 6 GFP fluorescent protein-tagged PD-1 + Jurkat T cells were incubated with 1 μg / mL Alexa Fluor 405-labeled PD-1 monoclonal antibody for 30 minutes, and free PD-1 monoclonal antibody was washed away with PBS buffer. 6 FcγRI + HEK293 cells and the above-mentioned PD-1 + Jurkat T cells were mixed in a 1.5 mL EP tube. The mixed cells were centrifuged at 300 g for 5 minutes at 4°C to initiate cell-cell connections, then gently suspended and transferred to a confocal imaging dish. PD-1 was evaluated using a Nikon A1RSi confocal microscope. + Jurkat T cells and FcγRI + Interaction of HEK293 cells.

[0063] like Figure 7 As shown, FcγRI + HEK293 cells can capture PD-1 monoclonal antibodies in vitro and interact with PD-1 + Jurkat T cells interacted with each other. Among them: A: PE-labeled PD-1 monoclonal antibody (left; 0.00005μg / mL, 0.0005μg / mL, 0.005μg / mL, 0.05μg / mL, 0.5μg / mL, 5μg / mL) and FcγRI + Mean fluorescence intensity (MFI) of the PE channel signal after binding to HEK293 cells; B: GFP fluorescent protein-labeled PD-1 + Jurkat T cells (green) in vitro with FcγRI + HEK293 cells undergo cell-cell interaction with PD-1 monoclonal antibody (red).

[0064] 2.2.4 Regulatory Effects of PD-1 Monoclonal Antibodies on Immune Cell Inflammatory Responses in Plaques and Lung Cancer Tissues CD45 was isolated from single cell suspensions of human plaque and lung tumor tissues. + 5×10 cells were taken from each sample. 4 CD45 + As a negative control, 5×10 4 CD45 + The cells (experimental group) were incubated with 10 μg / mL PD-1 monoclonal antibody in RPMI-1640 complete medium (37°C, 5% CO2) for 1 hour, washed twice to remove the free PD-1 monoclonal antibody, and then stimulated with CD3 / CD28 to stimulate the beads with CD45 + The cells were incubated for 48 hours. The supernatants from the negative control group and the experimental group were collected and the release of cytokines (IL-2, IFN-γ, and TNF-α) was quantitatively detected by flow cytometry.

[0065] Figure 8 The results show that PD-1 monoclonal antibodies can significantly inhibit the intensity of inflammatory response in the plaque microenvironment; A: PD-1 monoclonal antibodies treat plaques and lung tumor-derived CD45 + After the cells were treated with PD-1 monoclonal antibody, the release capacity of pro-inflammatory cytokines (IL-2, IFN-γ and TNF-α) (pg / mL); B: After the PD-1 monoclonal antibody was treated, the release capacity of CD45 + Inhibition degree of inflammatory factor release in cells (%), unpaired t-test, * p <0.05,** p <0.01,*** p <0.001. Figure 8 As shown, PD-1 can be induced in vitro by capturing PD-1 monoclonal antibodies using FcγRI on the surface of macrophages. + T cell function depletion significantly downregulates the pro-inflammatory response of immune cells in plaques.

[0066] 2.2.5 Effect of PD-1 monoclonal antibodies on the prognosis of atherosclerotic plaques in cancer patients We selected patients with tumors and atherosclerotic plaques who received regular (every 2-3 weeks) PD-1 monoclonal antibody therapy at our hospital between 2018 and 2021. We observed changes in atherosclerotic plaque size before and after PD-1 monoclonal antibody treatment. Plaque size was quantified and assessed using ImageJ software using arterial ultrasound images according to previously reported methods.

[0067] Figure 9The effect of PD-1 monoclonal antibody therapy on the prognosis of atherosclerotic plaques in cancer patients (regardless of cancer type); A: Ultrasound scans of plaques in cancer patients at different time points before and after PD-1 monoclonal antibody treatment. The plaque area is marked with a white dotted line, and the scale bar represents 5 mm; B: Changes in atherosclerotic plaque size in cancer patients (patients, n=8; plaques, n=13) before and after PD-1 monoclonal antibody treatment. Paired t-test, * p <0.05,** p <0.01,*** p <0.001, **** p <0.0001. Figure 9 As shown in the results, atherosclerotic plaques in cancer patients showed a significant trend of shrinkage after treatment with PD-1 monoclonal antibodies.

[0068] 2.3 Conclusion In human atherosclerotic plaque tissue, the loss of expression of PD-1 receptor's natural ligand, PD-L1, leads to the activation of PD-1. + T cells are activated and have a pro-inflammatory functional state in terms of protein phenotype, transcriptome level and in vitro function; by using FcγRI on the surface of macrophages to capture PD-1 monoclonal antibodies to form non-natural PD-1 ligands, activated PD-1 can be induced in vitro + T cells enter a state of functional exhaustion, thereby significantly downregulating the inflammatory response of plaque immune cells; and the results of the clinical phenotype also verified that PD-1 monoclonal antibody treatment also has the effect of promoting plaque reduction.

[0069] In summary, the present invention has shown through research that, because of the lack of PD-L1 expression in the microenvironment of human atherosclerotic plaques, PD-1+T cells are not functionally exhausted but remain in a functionally activated and pro-inflammatory state. The mechanism of action of the PD-1 antibody of the present invention is as follows: the FcγRI (CD64) receptor on the surface of macrophages in atherosclerotic plaques captures and binds to the free PD-1 antibody, causing the PD-1 antibody to link to the surface of macrophages; the PD-1 antibody linked to the cell surface can function as a non-natural ligand of the PD-1 receptor; the non-natural ligand of the PD-1 receptor binds to the PD-1 receptor on the surface of T cells and interacts, inducing PD-1 + T cell exhaustion; by inducing PD-1 + T cell function depletion promotes inflammatory ablation in the local microenvironment of atherosclerotic plaques.

[0070] The present invention provides an application of a PD-1 antibody in the preparation of a drug for atherosclerotic diseases. The present invention proposes that "surface-linked PD-1 antibodies can inhibit activated, pro-inflammatory PD-1 in atherosclerotic plaques." +T cells, promoting the inflammatory ablation of the plaque microenvironment", which is expected to provide a new theoretical basis and potential interventional immune targets for the immunotherapy of atherosclerotic plaques.

[0071] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of a PD-1 antibody in the preparation of a drug for treating atherosclerosis, characterized in that: The PD-1 antibody is an IgG1-IgG4 antibody; The PD-1 antibody carries an antibody Fc segment domain; The PD-1 antibody can be linked to the cell surface or a pharmaceutically acceptable drug carrier.

2. The use according to claim 1, characterized in that: The PD-1 antibody is a PD-1 monoclonal antibody, a PD-1 polyclonal antibody or a PD-1 bispecific antibody.

3. The use according to claim 2, characterized in that: The PD-1 monoclonal antibody is a humanized antibody.

4. The use according to claim 2, characterized in that: The light chain of the PD-1 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

2.

5. The use according to claim 4, characterized in that: The PD-1 monoclonal antibody is nivolumab.

6. The use according to claim 1, characterized in that: The cells are macrophages.

7. The use according to claim 1, characterized in that: The drug carrier is selected from a metal stent, a drug-coated stent or a bioabsorbable stent.

8. The use according to claim 1, characterized in that: The atherosclerotic disease is a disease caused by vascular atherosclerosis, including coronary heart disease, myocardial infarction, valvular heart disease, heart failure, ischemic cardiomyopathy, cerebral vascular infarction, stroke, aneurysm, and aortic dissection.

9. The use according to claim 1, characterized in that: The drug is a pharmaceutical preparation administered by injection or stent carrier.

10. The use according to claim 9, characterized in that: The injection administration includes peripheral injection and local coronary intervention; the stent carrier administration includes coronary intervention stent carrier administration.

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