Application of IL-6 inhibitor in preparation of myocarditis treatment drug or tumor treatment drug enhancer

Abstract

The invention relates to the field of biological medicines, in particular to application of an IL-6 inhibitor in preparation of a myocarditis treatment medicine or a tumor treatment medicine enhancer. The IL-6 inhibitor is used as a medicine for treating myocarditis and is immune checkpoint inhibitor related myocarditis. The IL-6 inhibitor is targeted to an IL-6 / JAK2 / STAT3 signal channel, the potential of relieving myocarditis induced by the immune checkpoint inhibitor is achieved, and the anti-tumor immune response of the immune checkpoint inhibitor is enhanced when the IL-6 inhibitor and the immune checkpoint inhibitor are jointly applied. The invention also provides the first combined application of the IL-6 inhibitor and the immune checkpoint inhibitor in the animal model, and opens up a new way for the combined application of immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an IL-6 inhibitor in preparing a drug for treating myocarditis or in preparing a drug enhancer for treating tumors. Background Art

[0002] Immune checkpoint blockade has changed the landscape of cancer treatment, significantly improving the response rate and durability of disease control in patients. However, immune checkpoint inhibitors (ICIs) may induce potentially serious or even fatal immune-related adverse events (irAEs) involving any organ after activating the immune system, among which myocarditis has a mortality rate of up to 46%. The treatment of irAEs (including myocarditis) is mostly based on experience, and the underlying mechanism is assumed to be an autoimmune reaction. Currently, first-line treatment is usually high-dose glucocorticoid pulse therapy, and second-line treatment or hormone combination therapy is not recommended. However, long-term immunosuppression affects tumor prognosis, and hormone-related toxicity (such as osteoporosis, Cushing's syndrome, and infection) can also have a serious impact on patients' quality of life. In addition, some irAEs are ineffective against glucocorticoid treatment and require additional significant immunosuppressive therapy. Therefore, there is an urgent need for a precise treatment model that can reduce dependence on hormone therapy and potentially inhibit non-target autoimmune reactions while maintaining anti-tumor activity.

[0003] ICIs show significant anti-tumor effects, but only a small number of patients (10% to 30%) can produce a durable and strong response. The efficacy of ICIs alone is limited and they are prone to drug resistance, which limits their application. Combination therapy strategies based on ICIs are the direction of future exploration. Current research focuses on these innovative combination therapies, including combinations of dual immunosuppressants, anti-angiogenic drugs, targeted therapy, chemotherapy, radiotherapy, and adoptive cell transfer therapy. Although these multimodal treatments can enhance anti-tumor efficacy and durability, they also inevitably increase the risk of adverse reactions. Therefore, finding an ICIs combination therapy strategy that can both enhance anti-tumor efficacy and maintain safety has always been a research focus. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of an IL-6 inhibitor in the preparation of a drug for treating myocarditis or in the preparation of a drug enhancer for treating tumors, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention provides the use of an IL-6 inhibitor in preparing a drug for treating myocarditis or in preparing an enhancer for treating tumors.

[0006] Preferably, the myocarditis is immune checkpoint inhibitor-associated myocarditis.

[0007] The present invention also provides a composition for tumor treatment, which comprises the aforementioned IL-6 inhibitor and the aforementioned immune checkpoint inhibitor and a pharmaceutically acceptable excipient.

[0008] The present invention also provides use of the above composition in preparing tumor treatment products.

[0009] As described above, the use of the IL-6 inhibitor of the present invention in the preparation of a drug for treating myocarditis or in the preparation of a drug enhancer for treating tumors has the following beneficial effects:

[0010] The present invention targets the IL-6 / JAK2 / STAT3 signaling pathway through tocilizumab (TCZ), has the potential to alleviate myocardial inflammation induced by immune checkpoint inhibitors, and enhances the anti-tumor immune response of immune checkpoint inhibitors when administered in combination. This is also the first combined application of TCZ and immune checkpoint inhibitors in animal models, opening up a new path for the combined application of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown are the clinical characteristics of three lung cancer patients before and after immunotherapy in the present invention. (AF) Case 1: (AB) Electrocardiogram (ECG) changes before (A) and 35 days after (B) treatment with immune checkpoint inhibitors (ICIs). (C) Time series changes in brain natriuretic peptide (BNP) levels after ICI treatment. (D) Serum cytokine profile after 35 days of ICI treatment. (E) Changes in echocardiographic results before and after ICI treatment. (F) Chest CT images before (left) and 49 days after the start of ICI treatment (right); (GK) Case 2: (GH) Electrocardiogram changes before (G) and 18 days after (H) treatment with ICI. (I) Time series changes in cardiac troponin I (cTnI) levels after ICI treatment. (J) Serum cytokine profile after 18 days of ICI treatment. (K) Chest CT images before (left) and 49 days after ICI treatment (right); (LP) Case 3: (LM) Electrocardiogram changes before (L) and 43 days after (M) ICI treatment. (N) Temporal changes in brain natriuretic peptide levels after ICI treatment. (O) Temporal changes in serum cytokine IL-6 levels after ICI treatment. (P) Chest CT images before (left) and 47 days after (right) ICI treatment.

[0012] Figure 2Shown are the results of myocardial inflammatory damage induced by PD-1 inhibitor in tumor mice of the present invention. (A) Mouse model scheme and administration time points; analysis of cTnI (B) and TNF-α (C) in serum samples showed that the expression of inflammatory factors and myocardial injury markers was significantly increased in mice treated with PD-1 inhibitors; (D) Representative echocardiographic images showed changes in cardiac function in the control and PD-1 inhibitor groups; indicators at 4 weeks (LVEF, LVFS, IVS;d, IVS;s, LVID;d, and LVID;s) indicated impaired cardiac function; (E) HE staining of control mice did not show obvious abnormalities, but necrosis, myocardial structural disorder, edema and hypertrophy, nuclear eccentricity, and inflammatory cell infiltration occurred in mice treated with PD-1 inhibitors; (FJ) Representative immunohistochemistry (IHC) images of heart sections; myocardial interstitial macrophages (F4 / 80+); (F), neutrophils (Ly6G+) (G), T lymphocyte infiltration (H), and CD8+ T lymphocytes were predominant among T lymphocytes (IJ); original magnification, ×400.

[0013] Figure 3 The results of IL-6 expression in the mouse model of myocardial inflammatory injury induced by PD-1 inhibitors in the present invention are shown. (A) Enzyme-linked immunosorbent assay (ELISA) showed that the serum IL-6 level of mice treated with PD-1 inhibitors was increased compared with the control group; (BC) Immunohistochemical staining showed that IL-6 was positively expressed in mice treated with PD-1 inhibitors; original magnification, ×400; (D) Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) showed that the expression level of IL-6 mRNA in mice treated with PD-1 inhibitors was increased; (EF) Western blot (WB) test showed that the expression of IL-6 protein in the heart tissue of mice treated with PD-1 inhibitors was increased.

[0014] Figure 4 The results of M1 polarization of macrophages and IL-6 synthesis and secretion in the mouse model of myocardial inflammatory injury induced by PD-1 inhibitors in the present invention are shown. (A) Co-localization of macrophages (F4 / 80+), T lymphocytes (CD3+) and neutrophils (Ly6G+) with the inflammatory factor IL-6 showed that IL-6 was mainly secreted by macrophages in mice treated with PD-1 inhibitors; (B) Co-localization of macrophages and iNOS showed that M1 polarization occurred in macrophages in mice treated with PD-1 inhibitors; (CD) Western blot (WB) detection showed that the iNOS protein expression level in mice treated with PD-1 inhibitors was higher than that in the control group; (EG) WB detection showed that after adding PD-1 inhibitors to the cell model, the protein expression of IL-6 and iNOS was enhanced.

[0015] Figure 5 The results of TCZ in the present invention that alleviates PD-1 inhibitor-induced myocarditis in mice are shown. (A) Model scheme and time point of TCZ administration of anti-IL-6R antibody in mice with myocarditis; (B) Ultrasound examination of cardiac function of mice in the control group, PD-1 inhibitor group and PD-1 inhibitor + TCZ group after 28 days of administration showed that the cardiac function of mice in the PD-1 inhibitor + TCZ group was basically restored; (CD) Analysis of inflammatory factors such as (C) IL-6 and (D) cTnI in serum samples showed that compared with the PD-1 inhibitor group, the myocardial damage and inflammation levels of mice in the PD-1 inhibitor + TCZ group were reduced; (EI) Real-time fluorescence quantitative polymerase chain reaction qRT-PCR experiments showed that compared with the PD-1 inhibitor group, the expressions of IL-1β, IL-6, IL-18, TNF-α and iNOS in the mice in the PD-1 inhibitor + TCZ group were significantly decreased; (J) HE staining showed that the PD-1 inhibitor + TCZ caused less structural damage to the heart and less inflammatory infiltration; (KM) Immunohistochemistry (IHC) staining showed that the positive expressions of IL-6, F4 / 80 and iNOS in the mice in the PD-1 inhibitor + TCZ group were reduced; original magnification, ×400.

[0016] Figure 6 This is shown as a result of the present invention in which TCZ alleviates inflammation by inhibiting the IL6-JAK2-STAT3 pathway. (A) Gene set enrichment analysis (GSEA) of IL6-JAK2-STAT3 signaling pathway enrichment in normal and immune myocarditis heart tissues; (B) In the RAW264.7 macrophage model, the protein expressions of IL-6, p-JAK2 / JAK2 and p-STAT3 / STAT3 were detected by WB experiment; (C) In the RAW264.7 macrophage model in the control group, LPS group, PD-1 inhibitor group and PD-1 inhibitor + TCZ group, the protein expressions of IL-6, p-JAK2 / JAK2, p-STAT3 / STAT3 and iNOS were detected by WB experiment; (D) M1 polarization of macrophages in the RAW264.7 macrophage model in the control group, LPS group, PD-1 inhibitor group and PD-1 inhibitor + TCZ group was detected by flow cytometry; (E) The protein expressions of IL-6, p-JAK2 / JAK2, p-STAT3 / STAT3 and iNOS in heart tissues were detected by WB experiment.

[0017] Figure 7The results show that TCZ enhances the anti-tumor effect of PD-1 inhibitor in the mouse model. (AB) In vivo imaging measurement of the tumor region of interest (ROI) value of each group; (C) Tumor growth rate of the control group, PD-1 inhibitor group, and PD-1 inhibitor + TCZ group; (D) Positive expression of IL-6 in tumor tissues of the three groups; (EG) Detection of tumor proliferation and invasion indicators including Ki67 (E), N-cadherin (F), and vimentin (G) by immunohistochemistry (IHC) staining; Original magnification, ×400.

[0018] Figure 8 The enhanced anti-tumor effect of TCZ in the cell model of the present invention is shown. (A) Transwell experiment shows that after adding TCZ, the migration ability of LLC is inhibited, and the inhibitory effect is more obvious after adding TCZ and RAW264.7 supernatant; (B) Scratch experiment shows that compared with the control group, the cell scratch healing is significantly delayed after adding TCZ, and the delay is more obvious after adding TCZ and RAW264.7 supernatant; (C) Compared with the control group, the cell proliferation rate is reduced after adding EdU stained TCZ, and the reduction is more obvious after adding TCZ and RAW264.7 supernatant. Scale bar: EdU, 50 microns; Transwell and scratch experiment, 200 microns. (D) Protein expressions of IL-6, p-JAK2 / JAK2, p-STAT3 / STAT3 and iNOS in the TCZ group, RAW264.7 supernatant group and TCZ+RAW264.7 supernatant group (detected by WB experiment); **p<0.01, ***p<0.001; (E) TCZ effectively inhibited tumor growth and significantly reduced cardiac inflammation by acting on the IL-6 receptor signaling pathway of tumor cells and macrophages.

[0019] Fig. 9 This shows the results of the present invention that patients with tumors with high IL-6 expression have a poor prognosis. DETAILED DESCRIPTION

[0020] The present invention provides use of an IL-6 inhibitor in preparing a drug for treating myocarditis or in preparing a drug enhancer for treating tumors.

[0021] In some specific embodiments, the myocarditis is immune checkpoint inhibitor (ICIs)-related myocarditis, wherein ICIs-related myocarditis can be understood as myocarditis induced by immune checkpoint inhibitors.

[0022] In some embodiments, the immune checkpoint inhibitor may be selected from one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0023] Further, the immune checkpoint inhibitor can be selected from one or more of Cemiplimab, Nivolumab, Pembrolizumab, Tislelizumab, Sintilimab, Camrelizumab, Tislelizumab, Atezolizumab, Avelumab, Durvalumab, Ipilimumab or BE1046 (Bio X Cell).

[0024] In the present invention, IL-6 inhibitor refers to a molecule that has an inhibitory effect on IL-6 gene expression or IL-6 protein activity. The inhibitory effect on a gene includes, but is not limited to, inhibiting the expression or activity of a gene. Taking the IL-6 gene as an example, an IL-6 gene inhibitor refers to a molecule that has an inhibitory effect on IL-6, that is, the target gene IL-6 of the IL-6 gene inhibitor. The inhibitory effect on IL-6 includes, but is not limited to, inhibiting the expression or activity of IL-6.

[0025] Inhibiting gene activity means reducing gene activity and reducing the biological function of the gene. Preferably, the gene activity is reduced by at least 10% compared to before inhibition, such as at least 30%, 50%, 70% or 90%.

[0026] Inhibiting gene expression may be inhibiting the transcription or translation of the gene. Specifically, it may mean: preventing the gene from being transcribed, or reducing the transcriptional activity of the gene, or preventing the gene from being translated, or reducing the translation level of the gene.

[0027] Those skilled in the art can use conventional methods to regulate gene expression, such as gene knockout, homologous recombination, interfering RNA, etc.

[0028] The inhibition of gene expression can be verified by detecting the expression level through PCR and Western Blot.

[0029] Preferably, compared to the wild type, gene expression is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, and most preferably, the gene is not expressed at all.

[0030] The myocarditis treatment drug product must include an IL-6 inhibitor, and uses the IL-6 inhibitor as the active ingredient for the aforementioned efficacy.

[0031] In the product, the active ingredient that exerts the aforementioned function may be only the gene inhibitor, or may contain other molecules that can exert the aforementioned function.

[0032] That is, the IL-6 inhibitor is the only active ingredient or one of the active ingredients of the product.

[0033] The product can be a single-component substance or a multi-component substance.

[0034] The product is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0035] The IL-6 inhibitor can be selected from one or more of nucleic acid molecules, polypeptides, proteins, chemical small molecules or viruses.

[0036] In one embodiment, the IL-6 inhibitor can be a nucleic acid molecule that reduces gene expression in tumor cells.

[0037] The nucleic acid molecule is selected from any one or more of an antisense oligonucleotide, a nucleic acid construct comprising a nucleic acid fragment encoding a guide RNA and a Cas protein, a double-stranded RNA (dsRNA) or a short hairpin RNA (shRNA).

[0038] The double-stranded RNA contains a nucleotide sequence that can hybridize with a gene.

[0039] The shRNA contains a nucleotide sequence capable of hybridizing with a gene.

[0040] Furthermore, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to the target sequence in the target gene (eg, IL-6 gene).

[0041] The target sequence in the target gene is the fragment in the target gene corresponding to the mRNA fragment recognized and silenced by the nucleic acid molecule.

[0042] Furthermore, the double-stranded RNA is small interfering RNA (siRNA).

[0043] In some embodiments, the protein can be an antibody.

[0044] Further, the antibody can be selected from one or more of Tocilizumab, Siltuximab, Sarilumab, Satralizumab or Olokizumab. Preferably, the antibody is Tocilizumab.

[0045] In some embodiments, the virus is selected from a lentivirus, an adenovirus, or an adeno-associated virus.

[0046] The composition for treating tumors is a composition for treating lung cancer.

[0047] The composition may be a pharmaceutical composition.

[0048] When the composition is used to prevent or treat lung cancer in a subject, an effective dose of the composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and / or metastasis of the lung cancer is inhibited. Further, at least 10% to 30%, 30% to 50%, 50% to 70%, 70% to 90%, 90% to 100% of the growth, proliferation, recurrence and / or metastasis of the lung cancer is inhibited.

[0049] The form of the composition is not particularly limited, and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0050] The composition is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0051] In some specific embodiments, the tumor therapeutic drug is selected from the aforementioned immune checkpoint inhibitors.

[0052] In the present invention, the tumor therapeutic drug enhancer is a substance that does not have tumor therapeutic function or has weak tumor therapeutic function itself but can enhance the efficacy of the tumor therapeutic drug when used in combination with the tumor therapeutic drug.

[0053] In some specific embodiments, the tumor is selected from one or more of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma. Preferably, the tumor is lung cancer.

[0054] The present invention also provides a composition for tumor treatment, which comprises the aforementioned IL-6 inhibitor, the aforementioned immune checkpoint inhibitor and pharmaceutically acceptable excipients.

[0055] In some embodiments, the adjuvant includes various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after administration. The adjuvant includes sterile water or saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or adhesives. The adjuvant also includes other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. The adjuvant is selected from saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannoses or sugar alcohol isotonic solution when used for injection of aqueous solution. The injection aqueous solution includes a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and / or nonionic surfactant (Tween 80 or HCO-50). In the tumor treatment product provided by the present invention, the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned T cell is a single effective ingredient, and can also be combined with one or more other active components useful for tumor treatment to form a combined preparation. The active component is other various drugs used for tumor treatment. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient of the aforementioned composition depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the composition as the active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0056] In some embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0057] In one embodiment, the tumor is lung cancer. The L-6 inhibitor is an enhancer for the treatment of tumors with immune checkpoint inhibitors. Further, when the drug is used to prevent or treat lung cancer in a subject, an effective dose of the aforementioned IL-6 inhibitor and immune checkpoint inhibitor needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and / or metastasis of the lung cancer is inhibited. Further, at least 10% to 30%, 30% to 50%, 50% to 70%, 70% to 90%, 90% to 100% of the growth, proliferation, recurrence and / or metastasis of the lung cancer is inhibited.

[0058] The present invention also provides use of the above composition in preparing tumor treatment products.

[0059] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0061] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0062] The experimental materials and methods used in the embodiments of the present invention are as follows:

[0063] 1. Bioinformatics data analysis: Gene expression (ribonucleic acid-sequencing V2, RNAseqV2) and survival data were classified and downloaded from the Cancer Genome Atlas (TCGA), downloaded using UCSC Cancer Browser, and parsed in GraphPad Prism 7.0. Data on human immune myocarditis were obtained from the Sequence Read Archive (SRA) database (ERP120456 and SRP201972) and organized into the Trusted Platform Module (TPM) format. Myocarditis-related pathways were ranked and used in Gene Set Enrichment Analysis (GSEA), and pathways with Padj values ​​< 0.05 were considered as pathways with significant enrichment of differentially expressed genes.

[0064] 2. Patient data: This study included NSCLC patients who received ICIs treatment in our center between February 2019 and August 2021. Inclusion criteria: (1) Patients diagnosed with lung cancer by pathology in our center, regardless of the pathological type of lung cancer; (2) Patients treated with ICIs, regardless of the type of drug; (3) Patients diagnosed and treated for ICIs-related myocarditis who met the diagnostic criteria of the National Comprehensive Cancer Network (NCCN), the Chinese Society of Clinical Oncology (CSCO) guidelines, and the Chinese Expert Consensus (2020 edition); (4) Electrocardiogram, echocardiogram, and laboratory tests were completed before, during, and after treatment, including cardiac troponin I (cTNI), brain natriuretic peptide (BNP), and blood routine tests; (5) When ICIs-related inflammatory injury occurred, IL-6 levels increased compared with baseline levels. The clinical part of this study was approved by the Biomedical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University, and informed consent was obtained from the three patients.

[0065] 3. Mice and cell lines: Male C57BL / 6 mice (56 weeks old, weighing 1921 g) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Changsha, China) and raised at the Experimental Animal Center of Nanchang University. Mouse Lewis lung cancer cells (Lewismurine-derived lung adenocarcinoma cells, LLC) and mouse Raw264.7 macrophages were provided by Wuhan Pronosai Life Science Technology Co., Ltd. All experimental procedures involving animals have been approved by the Experimental Animal Ethics Review Committee of Nanchang University, and the ethics project approval number is NCULAE-20221031141.

[0066] 4. Chemical reagents and materials: Programmed death receptor-1 (PD-1) inhibitor (BE1046) was purchased from Bio X Cell. Lipopolysaccharide (LPS) (L2880) was purchased from Sigma. Tocilizumab (TCZ) was purchased from Roche. D-Luciferin potassium salt (D8390) was provided by Solebio. The main antibodies used for Western blot (WB) detection are as follows: anti-inducible nitric oxide synthase (iNOS) antibody was purchased from Abmart Biotechnology, Shanghai, China; anti-phosphorylated signal transducer and activator of transcription 3 (STAT3), pSTAT3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Wuhan ProteinTech Co., Ltd. (ProteinTech); anti-Janus kinase 2 (JAK2), phosphorylated JAK2 (pJAK2) and interleukin-6 (IL-6) antibodies were purchased from Wanleibio, Shenyang, China. The main antibodies used for immunohistochemistry (IHC) staining are as follows: anti-F4 / 80, Ly6G, CD3, CD8, CD4, Ki67, E-cadherin, vimentin, IL-6, and iNOS antibodies were purchased from Servicebio, Wuhan, China. The antibodies used for immunofluorescence (IF) were the same as those used for IHC staining.

[0067] 5. Animal model construction: 0.1 ml of mouse Lewis lung cancer cell (LLC) suspension containing 8×10^6 / ml stably transfected with luciferase GV260 vector (Shanghai Gene Gene, China) was subcutaneously injected 2 cm below the right posterior axillary line of mice. Then, the tumor-bearing mice were randomly divided into groups 7 days after tumor implantation (average starting volume = 40 cubic millimeters). The PD-1 inhibitor treatment group was intraperitoneally injected with 200 micrograms of InVivoPlus anti-mouse PD-1 inhibitor on days 0, 2, and 4 after tumor formation. The control group mice were injected with an equal volume of IgG. In the tocilizumab (TCZ) combined with PD-1 inhibitor administration group, TCZ was intraperitoneally injected once a week at a dose of 8 mg / kg for a total of four times, and PD-1 inhibitors were administered as described above. At the beginning of administration and 3 weeks after administration, the tumor bioluminescence signal was measured using a live imaging system (lumina3, PE) to assess tumor size. Four weeks after the first administration, the mice were killed and heart tissue and blood were collected.

[0068] 6. Establishment of cell model: RAW264.7 macrophages in the logarithmic growth phase were inoculated into T25 culture flasks (approximately three million cells / flask) and incubated in an incubator for 12 hours. According to the experimental design, lipopolysaccharide (LPS) with a final concentration of 100 ng / mL was added to the corresponding culture flask, and the culture medium was supplemented to 5 ml. After 6 hours of culture, cell protein or RNA was extracted for subsequent experiments. One hour after the addition of LPS, a mouse-derived PD-1 inhibitor was added thereto at a final concentration of 20 μg / mL to establish a PD-1 inhibitor inflammatory cell model, and the rest of the steps were the same as above. In the TCZ+PD-1 inhibitor cell model, TCZ was added to the culture medium at a final concentration of 150 μg / mL 24 hours before the addition of the PD-1 inhibitor, and the rest of the steps were the same as above.

[0069] 7. Enzyme-linked immunosorbent assay (ELISA): ELISA tests for cardiac troponin I (cTnI), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were performed according to the manufacturer's instructions (Shenzhen New Industry Biomedical Co., Ltd., China). All experiments were repeated three times.

[0070] 8. Echocardiography: Short-axis M-mode echocardiography was performed on the hearts of mice before tumor inoculation and PD-1 administration, and 4 weeks after PD-1 administration. The mice were anesthetized by inhalation of isoflurane after depilation of the anterior chest. Echocardiographic evaluation parameters were used to evaluate cardiac function (Vevo TM 2100, Visual Sonics, Canada). The left ventricular end-diastolic dimension (LVID; d), left ventricular end-systolic dimension (LVID; s), interventricular septal-diastolic thickness (IVS; d), and interventricular septal-systolic thickness (IVS; s) were measured using short-axis sections. The ejection fraction (EF) and fraction shortening (FS) values ​​were calculated according to the following formulas: EF% = [(LVEDV-LVESV) / LVEDV] × 100%, FS% = [(LVID; d-LVID; s) / LVID; d] × 100%.

[0071] 9. Tissue and cell immunofluorescence (IF): Paraffin sections were first dewaxed with xylene, then dehydrated with different concentrations of alcohol, and washed three times with phosphate buffered saline (PBS) after antigen retrieval. After the cell slides in the 24-well plate were directly washed three times with PBS, they were incubated with 3% bovine serum albumin (BSA) at room temperature for 1 hour. After that, the samples were incubated with specific primary antibodies at 4°C overnight, and then incubated with fluorescent secondary antibodies at room temperature for 1 hour. For heterologous double labeling, two different primary antibodies were mixed in a certain ratio and incubated with sections or slides at room temperature. In the case of heterologous double labeling, two primary antibodies from different sources were mixed in a certain ratio and incubated with samples at 4°C overnight. In addition, they were incubated with secondary antibodies of the corresponding species labeled with the primary antibody at a certain ratio for 1 hour at room temperature. After rinsing the sections or slides with running water, they were sealed with anti-fluorescence quenching tablets and observed under a fluorescence microscope.

[0072] 10. Construction of tumor cell model: LLCs in the logarithmic growth phase were taken and inoculated in a 10 cm cell culture dish. Four groups were set up: control group, TCZ group, RAW264.7 cell supernatant group, and TCZ+RAW264.7 cell supernatant group. They were placed in an incubator and incubated for 12 hours. According to the experimental design, the control group added culture medium; the TCZ group added TCZ with a final concentration of 150 μg / mL; the RAW264.7 cell supernatant group added the supernatant of RAW264.7 macrophages incubated in culture medium containing a final concentration of 100 ng / mL LPS and 20 μg / mL PD-1 inhibitor for 6 hours, and added a two-fold diluted culture medium; the TCZ+RAW264.7 cell supernatant group added the supernatant of the above concentration and a final concentration of 150 μg / mL TCZ. Each group was incubated for another 24 hours for tumor cell function detection.

[0073] 11. Scratch assay: The migration ability of the control group, TCZ group, RAW264.7 cell supernatant group and TCZ+RAW264.7 cell supernatant group was determined by Transwell migration assay and scratch assay. After digestion, centrifugation and resuspension of LLCs, they were seeded in a 6-well plate at a density of 2×10^6 cells per well and then incubated in an incubator. After the cells adhered to the wall, a scratch assay was performed using a 200 μL sterile pipette, and then the cells were washed three times with PBS, culture medium was added and placed in a 37°C incubator. Finally, images were taken with a microscope at 0 hours and 48 hours and analyzed using ImageJ.

[0074] 12. Transwell experiment: Four groups of cell models were inoculated in Transwell chambers, each containing 2×10^4 cells. The upper layer of the chamber used serum-free culture medium, and the lower layer used culture medium containing 10% fetal bovine serum (FBS). After being placed in a 37°C incubator for 24 hours, they were washed, fixed, and stained. Finally, images were taken using a microscope and analyzed using ImageJ.

[0075] 13. EdU proliferation assay: To observe the proliferation capacity of the control group, TCZ group, RAW264.7 supernatant group, and TCZ+RAW264.7 supernatant group, the EdU assay was used. The four groups of cells were seeded in 96-well plates, with 1.5×10^4 cells per well. EdU was diluted to 50 μmol / L with complete medium according to the instructions of the YF594 Click-iT EDU (Shanghai Union Biotech Co., Ltd., China) staining kit. Then, 100 μL of the diluted EdU was added to each well and incubated for 2 hours. After that, the culture medium was removed, the cells were fixed with 4% paraformaldehyde, neutralized with 2 mg / mL glycine solution, and washed twice with 3% bovine serum albumin. Next, the necessary Click-iT working solution was prepared using 0.5% Triton X-100 as a penetration enhancer and incubated in the dark for 30 minutes. Then, nuclear staining was performed using 1× Hoechst 33342 solution. Finally, images were captured using a fluorescence microscope and analyzed using ImageJ.

[0076] 14. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR): Total RNA was isolated from cardiac tissue using TRIzol reagent (Beijing Quanshijin Biotechnology Co., Ltd.). 2 μg of total RNA was used to synthesize complementary deoxyribonucleic acid (cDNA) using a reverse transcription kit (Takara Biomedicals, Shiga, Japan). Amplification and qRT-PCR analysis were performed using TAKARA SYBR Premix EX Tag II in a CFX96 RT-PCR detection system (Bio-Rad, Hercules, California (CA), the United States of America (USA)). The relative messenger RNA (mRNA) expression level of the target gene was normalized with GAPDH as a reference. Then, three independent experiments were performed. The specific primer pairs used were as follows: iNOS, forward primer 5′-GAGACAGGGAAGTCTGAAGCAC-3′, reverse primer 5′-CCAGCAGTAGTTGCTCCTCTTC-3′; IL-1β, forward primer 5′-GAAATGCCACCTTTTGACAGTG-3′, reverse primer 5′-TGGATGCTCTCATCAGGACAG-3′; IL-6, forward primer 5′-TACCACTTCACAAGTCGGAGGC-3′, reverse primer 5′-CTGCAAGTGCATCATCG TTGTTC-3′; TNF-α, forward primer 5′-CCTGTAGCCCACGTCGTAG-3′, reverse primer 5′-GGGAGTAGACAAGGTACAACCC-3′; IL-18, forward primer 5′-GACTCTTGCGTCAACTTCAAGG-3′, reverse primer 5′-CAGGCTGTCTTTTGTCAACGA-3′; GAPDH, forward primer 5′-ACGGATTTGGCCGTATTGG-3′, reverse primer 5′-CATTCTCGGCCTTGACTGTG-3′.

[0077] 15. Western blot analysis: Cells and cardiac tissues were lysed in phosphatase inhibitors, protease inhibitors and radioimmunoprecipitation assay (RIPA) lysis buffer at a ratio of 1:1:100. The supernatant was retained by centrifugation. The protein concentration was determined by the bicinchoninic acid (BCA) method and diluted to the same level. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 5% skim milk at room temperature for 1 hour. The PVDF membranes were cut according to the target molecular weight and placed in various specific primary antibodies and incubated overnight at 4°C. After washing with Tris-buffered saline with Tween 20 (TBST), they were placed in mouse or rabbit secondary antibodies corresponding to the primary antibodies and incubated for 1 hour at room temperature. The Bio-Rad gel imaging system was used to expose and photograph, and the images were analyzed using ImageJ software.

[0078] 16. Flow cytometry: Flow cytometry was used to detect the M1 polarization of macrophages in the RAW264.7 macrophage model in the control group, lipopolysaccharide (LPS) group, PD-1 inhibitor group, and PD-1 inhibitor + TCZ group. The induction concentrations of LPS, PD-1 inhibitor, and TCZ were consistent with the above description. RAW264.7 cells were scraped with a cell scraper and counted after resuspending in phosphate buffered saline (PBS). In addition, 1×10^6 cells were taken and resuspended with 100 μl PBS. Then, 1 μl of CD16 / 32 blocker was added on ice for incubation for 10 minutes and stained with 1.25 μl of fluorescently labeled CD86 (APC) antibody for 30 minutes at 4°C. Next, 100 μl of membrane disruptor A was added for fixation, and 100 μl of membrane disruptor B was added for membrane rupture. Finally, 2.5 μl of CD206 (PE) was added to stain the cells at room temperature for 30 minutes. Subsequently, cells were resuspended in 500 μl of flow staining buffer and subjected to data analysis on a CytoFLEX flow cytometer (Beckman, CA).

[0079] 17. Data statistics and analysis: All data are expressed as mean ± standard error. For comparison of two samples, independent sample T test was used. For comparison between three or more groups, one-way analysis of variance (ANOVA) was used. Data analysis was performed using SPSS (Statistical Product and Service Solutions) 25.0 software. When P value < 0.05, the difference was considered statistically significant. All experiments were repeated three times.

[0080] Example 1 IL-6 levels are elevated in ICI-related myocarditis and are a poor prognostic factor for tumors.

[0081] Case 1: The patient was a 62-year-old male who was diagnosed with squamous cell lung cancer. Thirty-five days after the first treatment with 200 mg of tislelizumab, he developed symptoms of chest tightness and shortness of breath. At the initial visit, the electrocardiogram (ECG) showed sinus rhythm and the BNP level was within the normal range. However, after the onset of the above symptoms, the ECG showed sinus tachycardia with a heart rate as high as 149 beats / min, accompanied by ST segment and T wave (ST-T) changes. The BNP level was significantly elevated, reaching 1,224.59 pg / mL (normal range: 1-300 pg / mL). In addition, cardiac ultrasound results showed that the left atrium was significantly enlarged and the left ventricular function was decreased during the onset of myocarditis, while the baseline echocardiogram suggested mild valvular regurgitation. Based on these findings, the patient was diagnosed with ICI-related myocarditis. Notably, his serum IL-6 level was higher than normal, at 7.62 pg / mL (reference range: 0-5.4 pg / mL). Computed tomography (CT) re-examination images showed that the lung lesions were significantly reduced ( Figure 1 AF).

[0082] Case 2: The patient was a 42-year-old male with squamous cell lung cancer. Eighteen days after the first treatment with 200 mg of tislelizumab, he developed symptoms such as low-grade fever, ptosis of the left eyelid, dyspnea, and confusion. At the initial visit, the electrocardiogram showed normal sinus rhythm, and the levels of troponin, creatine kinase (CK), CK-MB, and lactate dehydrogenase (LDH) were within the normal range. However, after the onset of the above clinical symptoms, the patient's electrocardiogram changed significantly, including ST segment elevation of 0.05-0.15 mV in leads I, avL, and V2-V3, embryonic r waves in leads avF and III, complete right bundle branch block, and ST-T abnormalities. Further laboratory examinations revealed that the high-sensitivity troponin level was significantly elevated, reaching 30.50 ng / mL (normal range: 0-0.78 ng / mL). The echocardiogram results at the onset of myocarditis were not significantly different from those before ICI treatment. In summary, the patient was diagnosed with ICI-related myocarditis with myositis. It is noteworthy that his IL-6 level was also elevated to 15.93 pg / mL. Despite receiving methylprednisolone and other symptomatic treatments, the patient's condition continued to deteriorate and he was eventually discharged from the hospital. During this period, chest CT reexamination showed that the lesion had enlarged ( Figure 1 GK).

[0083] Case 3: The patient was a 73-year-old male who was diagnosed with non-small cell lung cancer (with sarcomatoid carcinoma components) and received two doses of tislelizumab. 43 days after the first dose, he began to experience chest tightness and shortness of breath. Before the onset of these symptoms, the baseline electrocardiogram showed sinus rhythm and troponin levels were within the normal range. At the time of the onset of chest tightness and dyspnea, the electrocardiogram showed signs of sinus arrhythmia, atrial premature beats, complete right bundle branch block, and right ventricular overload. Troponin levels were significantly elevated to 2.47 ng / mL, and BNP levels were also high. Echocardiogram results showed no significant difference before and after ICI treatment. These findings led to the patient being diagnosed with ICI-related myocarditis. Importantly, his IL-6 level was significantly elevated to 247.21 pg / mL. The patient's condition improved after receiving methylprednisolone treatment. Subsequent chest CT scans showed a significant reduction in the lesions ( Figure 1 LP).

[0084] In the three patients with ICI-induced myocarditis, the IL-6 levels were observed to be elevated in this example. When anti-inflammatory treatment is effective, myocardial inflammation is alleviated and the tumor regresses. On the contrary, when anti-inflammatory treatment is ineffective, the tumor progresses. In addition, the information on IL-6 expression levels and prognosis of lung cancer patients was collected based on the TCGA database in this example, and it was found that patients with high IL-6 expression had a poor prognosis ( Fig. 9 ).

[0085] Example 2 Construction of a mouse model of myocardial inflammatory injury induced by PD-1 inhibitor therapy in lung cancer

[0086] In order to further explore the specific role of IL-6 in mediating ICIs-related myocarditis and its impact on anti-tumor therapy, a mouse model of myocardial inflammatory injury induced by lung cancer PD-1 inhibitor treatment was constructed in this example (see Figure 2 A). The results showed that after mice were treated with PD-1 inhibitors for 4 weeks, the level of cardiac troponin I (cTNI), a marker of myocardial injury, in their serum was significantly increased (see Figure 2 B). At the same time, the level of tumor necrosis factor-α (TNF-α) in serum also showed an upward trend, which is consistent with the inflammatory damage (see Figure 2 C). Echocardiographic analysis further confirmed that the cardiac function of mice in the PD-1 inhibitor group was significantly worse than that in the control group (see Figure 2 D). Hematoxylin-eosin (HE) staining of the heart tissues of mice in the PD-1 inhibitor group showed that compared with the control group, this group of mice had cardiac structural disorder, myocardial cell edema, hypertrophy, necrosis, nuclear condensation, and inflammatory cell infiltration (see Figure 2E). Consistent with the myocardial histological results of patients with ICIs-related myocarditis, an increase in the number of macrophages and T lymphocytes was observed in the mouse model in this example. The results of immunohistochemistry (IHC) of the heart tissue of mice treated with PD-1 inhibitors showed that compared with the control group, the heart tissue of this group of mice showed positive expression of macrophages (F4 / 80+), neutrophils (Ly6G+) and T lymphocytes (CD3+), and T lymphocytes were mainly CD8+ (see Figure 2 FJ).

[0087] Example 3 Increased IL-6 expression in the mouse model of myocardial inflammatory injury induced by PD-1 inhibitor

[0088] Using the mouse model established in Example 2, the expression of IL-6 in mouse heart tissue and serum was evaluated in this example. The results showed that the serum IL-6 level of mice in the PD-1 inhibitor group was significantly increased compared with that in the control group (see Figure 3 In addition, tissue immunohistochemistry (IHC) staining showed that IL-6 was positively expressed in the heart tissue of mice in the PD-1 inhibitor group (see Figure 3 BC). Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis showed that the mRNA expression level of IL-6 in the heart tissue of these mice was higher (see Figure 3 D). Similarly, Western blot (WB) experiments also revealed that the expression of IL-6 protein in the heart tissue of mice in the PD-1 inhibitor group increased (see Figure 3 EF). In summary, IL-6 expression was increased in the mouse model of myocardial inflammatory injury induced by PD-1 inhibitor, which is consistent with the observation results in clinical patients.

[0089] Example 4 PD-1 inhibitors promote IL-6 secretion by mediating macrophage M1 polarization

[0090] In order to clarify which inflammatory cell type is associated with IL-6 secretion, the present example used immunofluorescence (IF) double staining to co-localize IL-6 with different inflammatory cells. The results showed that IL-6 was mainly localized in macrophages, while neutrophils and T lymphocytes were less correlated with high IL-6 expression (see Figure 4 A), which indicates that the synthesis and secretion of IL-6 are closely related to macrophages. Macrophages can be transformed into M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages through polarization. In order to determine whether the secretion of IL-6 is mainly related to the M1 macrophage subtype, the IF double staining technique was used in this example to observe that in the PD-1 inhibitor group, F4 / 80 co-localized with inducible nitric oxide synthase (iNOS) (see Figure 4B), which indicates that macrophages have undergone significant M1 polarization. Similarly, WB experiments also showed that in the mouse model with the addition of PD-1 inhibitors, M1 macrophage polarization existed in the heart tissue (see Figure 4 CD). In addition, RAW264.7 macrophage model experiments showed that the addition of PD-1 inhibitors can enhance the M1 polarization of macrophages, accompanied by an increase in IL-6 protein expression (see Figure 4 EG). In summary, M1 polarization of macrophages is closely related to the secretion of IL-6, and both are closely related to myocardial inflammatory injury induced by PD-1 inhibitors.

[0091] Example 5: The alleviating effect of tocilizumab on myocardial inflammatory injury induced by PD-1 inhibitors

[0092] In order to explore whether the increased IL-6 level plays a promoting role in myocardial inflammatory injury induced by PD-1 inhibitors, a mouse model of PD-1 inhibitors combined with IL-6R receptor inhibitor tocilizumab (TCZ) was constructed in this example, as shown in the schematic diagram Figure 5 A. It is noteworthy that the cardiac function of mice in the TCZ-treated group was significantly restored compared with that in the PD-1 inhibitor group ( Figure 5 B), and the levels of IL-6 and cardiac troponin I (cTNI) in mouse serum also decreased ( Figure 5 CD). Similarly, in the heart tissues of mice treated with TCZ, the mRNA levels of IL-1β, IL-6, IL-18, TNF-α, and inducible nitric oxide synthase (iNOS) were significantly decreased ( Figure 5 EI). In pathological level, HE staining ( Figure 5 J) showed that the structural disorder of cardiac tissue in mice treated with TCZ was improved and the infiltration of inflammatory cells was reduced. Immunohistochemistry (IHC) staining ( Figure 5 KM) further revealed that the positive expressions of IL-6, F4 / 80 and iNOS in the heart tissue sections of mice treated with TCZ were reduced. In summary, tocilizumab can alleviate myocardial inflammatory damage induced by PD-1 inhibitors.

[0093] Example 6 Tocilizumab reduces macrophage M1 polarization and IL-6 production by inhibiting the IL-6-JAK2-STAT3 inflammatory pathway

[0094] To further explore the potential regulatory mechanism of IL-6 in myocardial inflammatory injury mediated by PD-1 inhibitors, the sequencing data of normal human heart tissue and ICIs-related myocarditis heart tissue were analyzed in this example, and it was found that the IL6-JAK2-STAT3 signaling pathway was significantly enriched in the ICIs-related myocarditis group ( Figure 6A). Based on published literature, this example assumes that PD-1 inhibitors may promote inflammation through the IL-6-JAK2-STAT3 signaling pathway. To verify this hypothesis, this example conducted experiments in the RAW264.7 macrophage model, and the results showed that after the addition of PD-1 inhibitors, the protein expressions of p-JAK2 / JAK2, p-STAT3 / STAT3, and IL-6 increased ( Figure 6 B). However, after the addition of tocilizumab, the protein expressions of p-JAK2 / JAK2, p-STAT3 / STAT3, iNOS, and IL-6 were decreased ( Figure 6 C). In addition, flow cytometry analysis was performed in this example to evaluate the effect of tocilizumab on macrophage M1 polarization. Using the RAW264.7 macrophage model, the macrophage model was divided into a lipopolysaccharide (LPS) group, an LPS+PD-1 inhibitor group, an LPS+PD-1 inhibitor+tocilizumab intervention group, and a control group. The results showed that under the induction of LPS and PD-1 inhibitors, the level of M1 polarized macrophages increased significantly. However, under the intervention of tocilizumab, the number of M1 polarized macrophages decreased significantly ( Figure 6 D). Similarly, in the mouse model, the tocilizumab group also showed a decrease in the expression of p-JAK2 / JAK2, pSTAT3 / STAT3, and iNOS proteins ( Figure 6 E). In summary, both mouse and cell models showed that tocilizumab reduced inflammation by inhibiting the IL-6-JAK2-STAT3 pathway and attenuated the M1 polarization level and IL-6 production of macrophages.

[0095] Example 7 Inhibitory effect of tocilizumab on tumor growth in tumor-bearing mice

[0096] In order to evaluate the anti-tumor therapeutic potential of tocilizumab in the context of ICIs treatment, this example summarizes the evaluation of tumor growth in different groups of mouse models. The results showed that compared with the group using PD-1 inhibitors alone, the tumor growth of mice in the group using tocilizumab combined with PD-1 inhibitors was significantly slower ( Figure 7 C). Similarly, in vivo imaging of mice at different time points visually showed that the tumor growth rate of mice in the combination group was slower ( Figure 7 AB). Immunohistochemistry (IHC) staining of mouse heart tissues showed that the positive expression of IL-6 in tumor tissues in the tocilizumab combined with PD-1 inhibitor group was reduced compared with that in the PD-1 inhibitor group ( Figure 7 D). In addition, compared with the control group, the tumor proliferation index Ki67, tumor invasion and metastasis-related molecule N-cadherin, and vimentin ( Figure 7In the tocilizumab combined with PD-1 inhibitor group, the expression levels of these molecules were even lower.

[0097] Example 8 Tocilizumab fights tumor growth by inhibiting the JAK2-STAT3 pathway

[0098] This example further verified the tumor inhibitory effect of tocilizumab by establishing an in vitro tumor cell culture model, including a control group, a tocilizumab group, a RAW264.7 cell supernatant group, and a tocilizumab + RAW264.7 cell supernatant group. Transwell experiment ( Figure 8 A) and scratch test ( Figure 8 B) showed that compared with the control group, the migration ability of the tocilizumab group was significantly reduced, and the reduction in migration ability was more obvious in the tocilizumab combined with RAW264.7 cell supernatant group. Figure 8 C) The effect of tocilizumab on LLC cell proliferation was further analyzed, and it was found that the proliferation ability of the tocilizumab group was significantly reduced compared with the control group, and the reduction in this regard was more significant in the tocilizumab combined with RAW264.7 cell supernatant group. This indicates that tocilizumab can inhibit IL-6 autocrine secreted by tumor cells and IL-6 paracrine secreted by macrophages, thereby achieving anti-tumor growth. In summary, both in vivo and in vitro experiments showed that the tocilizumab combined with PD-1 inhibitor group had a better effect in anti-tumor growth.

[0099] Western blot (WB) experiments using cell models in this example confirmed that Tocilizumab fights tumor growth by inhibiting IL-6R and IL6-JAK2-STAT3 pathways: compared with the control group, the protein expressions of IL-6, p-JAK2 / JAK2, and p-STAT3 / STAT3 in the Tocilizumab group were all reduced ( Figure 8 D). In addition, the protein expressions of IL-6, p-JAK2 / JAK2, and p-STAT3 / STAT3 decreased more significantly in the tocilizumab combined with RAW264.7 cell supernatant group. Therefore, tocilizumab may inhibit tumor growth through the IL6-JAK2-STAT3 pathway.

[0100] In summary, the present invention demonstrates elevated IL-6 levels in the serum of patients with ICI-related myocarditis and in a mouse model of ICI-induced myocardial inflammation injury, and highlights the potential of tocilizumab (TCZ) to alleviate ICI-induced myocardial inflammation by targeting the IL-6 / JAK2 / STAT3 signaling pathway while enhancing anti-tumor immune responses. This is also the first combined application of TCZ and ICI in an animal model, opening up a new avenue for the combined application of immunotherapy.

[0101] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of IL-6 inhibitors in the preparation of drugs for treating myocarditis or in the preparation of enhancers for treating tumors.

2. The use according to claim 1, characterized in that The myocarditis is immune checkpoint inhibitor-associated myocarditis.

3. The use according to claim 2, characterized in that: The immune checkpoint inhibitor may be selected from one or more of a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

4. The use according to claim 2, characterized in that: The immune checkpoint inhibitor can be selected from one or more of Cemiplimab, Nivolumab, Pembrolizumab, Tislelizumab, Sintilimab, Camrelizumab, Tislelizumab, Atezolizumab, Avelumab, Durvalumab, Ipilimumab or BE1046.

5. The use according to claim 1, characterized in that: The IL-6 inhibitor is selected from one or more of nucleic acid molecules, polypeptides, proteins, chemical small molecules or viruses.

6. The use according to claim 5, characterized in that The nucleic acid molecule is selected from one or more of an antisense oligonucleotide, a nucleic acid construct comprising a nucleic acid fragment encoding a guide RNA and a Cas protein, a double-stranded RNA or a short hairpin RNA; and / or the protein is an antibody; and / or the virus is selected from a lentivirus, an adenovirus or an adeno-associated virus.

7. The use according to claim 6, characterized in that When the IL-6 inhibitor is an antibody, the antibody is selected from one or more of Tocilizumab, Siltuximab, Sarilumab, Satralizumab or Olokizumab; preferably, the antibody is Tocilizumab.

8. The use according to claim 1, characterized in that The tumor is selected from one or more of adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma; preferably, the tumor is lung cancer.

9. The use according to claim 1, characterized in that The tumor treatment drug is selected from one or more of Tocilizumab, Siltuximab, Sarilumab, Satralizumab or Olokizumab; preferably, the antibody is Tocilizumab.

10. A composition for tumor treatment, characterized in that: The composition comprises an IL-6 inhibitor, an immune checkpoint inhibitor and pharmaceutically acceptable excipients.

11. Use of the composition according to claim 10 in preparing a tumor treatment product.

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