Application of aerosol inhalation pyroptosis activator in enhancement of lung cancer immunotherapy effect

Abstract

The invention relates to the field of biological medicine, in particular to application of an aerosol inhalation pyroptosis activator in enhancing the lung cancer immunotherapy effect. The exogenous modR-p53 is delivered to the lung through the administration route of aerosol inhalation to induce pyroptosis of tumor-related cells, so that the immune microenvironment of the lung tumor is remodeled, and the purpose of enhancing the immunotherapy effect of the immune checkpoint inhibitor is achieved. The invention is expected to provide a theoretical basis for clinical development and application of pyroptosis and immunotherapy, provides a guidance basis for enriching non-small cell lung cancer treatment means and improving patient prognosis, and has great theoretical value and social practical significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of atomized inhaled pyroptosis activator in enhancing the immunotherapy effect of lung cancer. Background Art

[0002] Lung cancer is a tumor with high morbidity and mortality. Among them, non-small cell lung cancer (NSCLC) is the most common type, accounting for more than 80% of all lung cancer cases. In recent years, tumor immunotherapy has developed rapidly, and immune checkpoint inhibitors (ICIs) represented by PD-1 / PD-L1 inhibitors have been widely used in the treatment of lung cancer. However, due to tumor heterogeneity, the therapeutic response of ICIs is low, which limits its clinical treatment effect.

[0003] According to the state of anti-tumor immune response, tumors can be divided into three types: Inflammed, Immune-excluded, and Immune Desert. Inflammatory tumors, also known as "hot tumors," are characterized by high expression levels of PD-L1 in cancer cells and high infiltration of immune cells within the tumor, resulting in a more favorable response to immunotherapy. In contrast, immune-excluded and immune-desert tumors are called "cold tumors," characterized by the inability of immune cells to penetrate the stroma and the absolute lack of immune cells, resulting in poor immunotherapy effects. Therefore, converting cold tumors into hot tumors has become the key to improving the efficacy of ICIs.

[0004] Pyroptosis is a new form of programmed cell death, which is mainly triggered by inflammasomes, activating caspases (caspase-1 / -3 / -4 / -5 / -8 / -11) of the downstream Gasdermin protein family (GSDM) to cut. Subsequently, the N-terminal fragment of the GSDM protein can bind to the phospholipid molecules on the cell membrane to form pores and cause membrane rupture, thus leading to pyroptosis and the release of a large number of inflammatory factors. [5]. Pyroptosis plays an important role in the occurrence and development of many diseases. It is also closely related to the remodeling of the tumor immune microenvironment and the immunotherapy response of tumors. Pyroptosis can synergize with immune checkpoint inhibitors to exert anti-tumor effects in a variety of ways, including: ① Release of tumor-associated antigens: Pyroptosis can cause cell membrane rupture and release of cell contents to the outside of the cell. These cell contents include tumor-associated antigens, which can be recognized and activated by immune cells. ② Release of proinflammatory factors: Pyroptosis can lead to the release of proinflammatory factors, including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). These proinflammatory factors can recruit immune cells and promote inflammatory responses. ③ Inhibition of tumor microenvironment: The tumor microenvironment includes tumor cells, immune cells, stromal cells and blood vessels. Zhibin Zhang et al. found that the activation of pyroptosis can increase the infiltration of macrophages, NK cells and CD8+T cells, enhance the anti-tumor immune effect, and thus inhibit tumor growth.Therefore, the activation of tumor-related pyroptosis is expected to improve the effect of ICIs immunotherapy. How to effectively activate tumor-specific pyroptosis remains to be solved (see references MJ Zhang, YY Wang, LL Han, XY Liu, YY Xie, Z. Xu, ZJ Sun, Biomaterials Elicit Pyroptosis Enhancing Cancer Immunotherapy, Advanced Functional Materials (2023). P. Yu, X. Zhang, N. Liu, L. Tang, C. Peng, XJ St Chen, t. therapy, Pyroptosis: mechanisms and diseases, 6 (1) (2021) 128. Y. Wang, W. Gao, X. Shi, J. Ding, W. Liu, H. He, K. Wang, F. Shao, Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin, Nature 547(7661)(2017)99-103.Z. Zhang,Y. Zhang,S. anti-tumor immunity,Nature 579(7799)(2020)415-420.). Summary of the invention

[0005] The purpose of the present invention is to provide a strategy and application for enhancing the immunotherapy effect of lung cancer by atomized inhalation of a pyroptosis activator.

[0006] TP53 is one of the most widely studied tumor suppressor genes. As a transcription factor, p53 can regulate cell growth, inhibit cell cycle progression and promote tumor cell apoptosis, thus playing a key role in the tumor suppression process. The latest studies have shown that there is a close correlation between p53 and pyroptosis levels. In NSCLC cells, the expression level of p53 is positively correlated with the degree of pyroptosis. Upregulating the level of p53 can promote pyroptosis and exert an anti-NSCLC effect. In addition, studies have confirmed that the delivery of exogenous p53 / liposomes can improve the tumor microenvironment and increase the infiltration of immune cells, thereby enhancing the immune efficacy against liver cancer and lung cancer (see references LJ Hernandez Borrero, WS El-Deiry, Tumor suppressor p53: Biology, signaling pathways, and therapeutic targeting, Biochim Biophys Acta Rev Cancer 1876(1)(2021)188556. X. Chen, T. Zhang, W. Su, Z. Dou, D. Zhao, X. Jin, H. Lei, J. Wang, X. Xie, B. Cheng, Q. Li, H. Zhang, C. Di, Mutant p53 in cancer: from molecular mechanism to therapeutic modulation, Cell Death & Disease 13(11)(2022). G. Guo, M. Yu, W. Xiao, E. Celis, Y. Cui, Local Activation of p53 in the Tumor Microenvironment OvercomesImmune Suppression and Enhances Antitumor Immunity, Cancer Res 77(9)(2017)2292-2305. Y.Cui, GJIjomsGuo, Immunomodulatory function of the tumorsuppressor p53 in host immune response and the tumor microenvironment, 17(11)(2016)1942.). The present invention uses exogenous TP53 as an example to verify that the delivery of exogenous p53 improves the immunotherapy effect of NSCLC by activating cell pyroptosis.

[0007] However, exogenous gene delivery still faces challenges such as in vivo degradation, off-target effects, and endosomal escape, making it difficult for drugs to effectively accumulate in tumor sites and tumor cells. Chemically synthesized modified message RNA (modRNA) refers to mRNA synthesized in vitro using chemically modified bases and is a new type of gene carrier.Compared with traditional plasmid or viral gene vectors, modRNA has unique advantages: it does not need to enter the cell nucleus or integrate into the host genome, and it has good controllability, stability and low immunogenicity, which enables the target gene to be expressed rapidly, efficiently and pulsed, thereby achieving therapeutic purposes (FWYAN Bingqian, modRNA Technology: A Newly-Developed Method of Protein Expression, 15 (2) (2019) 107-110. IC Turnbull, AA Eltoukhy, KM Fish, M. Nonnenmacher, K. Ishikawa, J. Chen, RJ Hajjar, DG Anderson, KDJ MT Costa, Myocardial delivery of lipid nanoparticle carrying modRNA induces rapid and transient expression, 24 (1) (2016) 66-75. C. Labonia, M. Estape Senti, M. Brans, C. Snijders, J. Sluijter, PJ C R Vader, Modified mRNA delivery to the heart using Lipid Nanoparticles,118(Supplement_1)(2022)cvac066.083.L.Roncati,L.Corsi,Nucleoside-modifiedmessenger RNA COVID-19vaccine platform,J Med Virol93(7)(2021)4054-4057.L.Chen,J.Li,Y.Fan,J.Qiu,L.Cao,R.Laurent,S.Mignani,A.-M.Caminade,J.-P.Majoral,X.Shi,Revisiting Cationic Phosphorus Dendrimers as a NonviralVector for Optimized Gene Delivery Toward Cancer Therapy Applications,Biomacromolecules 21(6)(2020)2502-2511.). Therefore, exogenous p53 modRNA (modR-p53) is expected to be used to achieve the purpose of improving the immunotherapy effect of NSCLC.

[0008] So, how can exogenous p53 modRNA (modR-p53) be effectively delivered to the recipient's lungs? Inhaled modRNA vaccines have been widely developed and used, and have achieved ideal results. Compared with ordinary mRNA vaccines, inhaled modRNA vaccines have higher safety and stability: compared with systemic administration (such as intramuscular injection or intravenous infusion), nebulized administration can achieve instant, efficient and dose-controlled pulmonary administration, achieving therapeutic levels while reducing systemic toxicity, and has the unique advantage of non-invasive administration (AY Jiang, J. Witten, IORaji, F. Eweje, C. MacIsaac, S. Meng, FAO Ladimeji, Y. Hu, RS Manan, RJ NN Langer, Combinatorial development of nebulized mRNA delivery formulations for the lungs, (2023) 1-12. SAAhrendt, Y. Hu, M. Buta, MPMcDermott, N. Benoit, SCY, L. Wu, DJ JotNCI Sidransky, p53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study,95(13)(2003)961-970.H.Sun,SYLiu,JYZhou,JTXu,HKZhang,HHYan,JJHuan,PPDai,CRXu,J.Su,YFGuan,X.Yi,RSYu,WZZhong,YLWu,Specific TP53 subtype as biomarker for immune checkpointinhibitors in lung adenocarcinoma,EBioMedicine 60(2020)102990.MD Hellmann,T.Nathanson,H.Rizvi,BCCreelan,F.Sanchez-Vega,A.Ahuja,A.Ni,JBNovik,LMB Mangarin,M.Abu-Akeel,C.Liu,JLSauter,N.Rekhtman,E.Chang,MKCallahan,JEChaft,MHVoss,M.Tenet,XMLi,K.Covello,A.Renninger,P.Vitazka,WJGeese,H.Borghaei,CMRudin,SJAntonia,C.Swanton,J.Hammerbacher,T.Merghoub,N.McGranahan,A.Snyder,JDWolchok,Genomic Features of Response to CombinationImmunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer,CancerCell 33(5)(2018)843-852e4.Q.Feng,W.Fang,Y.Guo,P.Hu,J.Shi,Nebulized Therapy ofEarly Orthotopic Lung Cancer by Iron-Based Nanoparticles:Macrophage-RegulatedFerroptosis of Cancer Stem Cells,J Am Chem Soc 145(44)(2023)24153-24165.). The above advantages make aerosol inhalation of modR-p53 an ideal way to activate tumor-specific pyroptosis.

[0009] A first aspect of the present invention provides the use of a pyroptosis activator in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, wherein the pyroptosis activator is an aerosol inhalant.

[0010] Furthermore, the pyroptosis activator is exogenous p53 modRNA (modR-p53).

[0011] Furthermore, the lung cancer is non-small cell lung cancer (NSCLC).

[0012] Furthermore, the immunotherapy is immunotherapy with immune checkpoint inhibitors (ICIs) represented by PD-1 / PD-L1 inhibitors.

[0013] The second aspect of the present invention provides a pharmaceutical preparation for enhancing the immunotherapy effect of lung cancer, wherein the pharmaceutical preparation is an aerosol inhalation agent and uses exogenous p53 modRNA as an active ingredient.

[0014] Furthermore, the active ingredient of the pharmaceutical preparation is modR-p53@CPD nanocomplex.

[0015] Furthermore, the preparation method of the modR-p53@CPD nanocomplex is as follows: the positively charged cationic carrier CPD is electrostatically adsorbed with 5 μg mod p53 under different N / P ratios (the molar ratio of the number of positively charged groups in the dendrimer to the number of phosphates in the pDNA backbone; the preliminary experiment of the present invention set three ratios of 10:1, 20:1, and 30:1, and finally 20:1 was selected based on cytotoxicity and transfection efficiency), and incubated at room temperature for 30 minutes to finally form a modR-p53@CPD nanocomplex.

[0016] The third aspect of the present invention provides a use of the pharmaceutical preparation as described above in the preparation of a drug for enhancing the immunotherapy effect of lung cancer.

[0017] The advantages of the present invention are:

[0018] The present invention delivers exogenous modR-p53 by atomization inhalation, regulates the process of macrophage pyroptosis, reshapes the tumor immune microenvironment, and thus improves the therapeutic effect of immune checkpoint inhibitors (ICIs). The present invention is expected to provide a theoretical basis for the clinical expansion application of pyroptosis + immunotherapy, provide a guiding basis for enriching NSCLC treatment methods and improving patient prognosis, and provide a new theoretical basis for the development of more effective tumor treatment strategies. It is expected to provide a new treatment turnaround for patients with advanced non-small cell lung cancer resistant to ICIs treatment and improve prognosis. It provides a new method for targeted drug delivery for tumor treatment, which has great theoretical value and social practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 . Schematic diagram of phosphorus-containing dendrimers loaded with modR-p53.

[0020] Figure 2 .Expression of target genes, cytokine release and pyroptosis-related pathways in cells after transfection with modR-p53.

[0021] Figure 3 .Organ enrichment of CPD / modR-p53 after aerosol inhalation at different times.

[0022] Figure 4 . Schematic diagram of establishment of small animal in situ lung cancer model and drug administration scheme.

[0023] Figure 5 On days 7, 14, and 22, the fluorescence intensity of tumors in mice was observed using a small animal in vivo imaging device (× represents the death of mice). DETAILED DESCRIPTION

[0024] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0025] Example 1: Synthesis of modR-p53

[0026] The CDS sequence of TP53 gene (NM_000546.5) was found from the gene database of NCBI website; 5' and 3' UTR sequences were added before and after the CDS sequence to synthesize the gene, and then inserted into PUC57BL / 6 plasmid vector; the PUC57BL / 6 plasmid was transformed into TOP10 host bacteria, preserved and amplified; the plasmid amplified for 16 hours was extracted and purified; the plasmid was linearized using XhoI restriction endonuclease, and then the upstream and downstream primers corresponding to the sequence were designed, the required fragments were PCR amplified and the position and size were identified by running gel; the product of the PCR was transcribed in vitro and the mRNA was chemically modified at the same time; the product of in vitro transcription was further purified and anti-phosphorylation treated, and finally purified and the RNA concentration and quality were controlled by Nanodrop and bioanalyzer.

[0027] Upstream primer: 5'-ATGGAGGAGCCGCAGTCAG-3' (SEQ ID NO.1)

[0028] Downstream primer: 5'-TCAGTCTGAGTCAGGCCCTTC-3' (SEQ ID NO. 2).

[0029] Example 2: Preparation of modR-p53@CPD Nanocomplex

[0030] The positively charged cationic carrier CPD was incubated with 5 μg mod p53 at room temperature for 30 minutes through electrostatic adsorption under different N / P ratios (the molar ratio of the number of positively charged groups in the dendrimer to the number of phosphates in the pDNA backbone was 10:1, 20:1, and 30:1) to form a CPD / mod p53 complex ( Figure 1 ).

[0031] Example 3: Exogenous modR-p53 transfection and induction of cell pyroptosis

[0032] Immunofluorescence staining of CPD / mod p53 transfected RAW264.7 cells, with 1×10 5LLC cells were seeded in a 12-well plate at a density of cells / well and cultured overnight in a 37°C, 5% CO2 incubator. Subsequently, the cells were replaced with CPD / mod p53 complex (control, N / P=10, 20, 30, messenger-max), 1 μgmood p53 per well. Mixed with 1 ml of fresh cell culture medium (serum-free), incubated for 4 hours, and then replaced with complete culture medium. Cultured at 37°C, 5% CO2 for 48 hours. The expression of p53 was observed by fluorescence microscopy, the levels of IL-1β and IL-18 in the culture supernatant of RAW264.7 cells were determined by ELISA, and the expression levels of c-caspase-1 and GSDMD were detected by western blotting.

[0033] Expression of target genes, cytokine release and pyroptosis-related pathways in cells after transfection with modR-p53 Figure 2 As shown, A observed the expression of p53 gene in transfected cells by immunofluorescence, and it can be seen that the expression of p53 protein (red fluorescence) in the N / P ratio 10, 20, and 30 groups was significantly higher than that in the control group (naked p53 incubation), among which the p53 expression in the 30 group was the highest, and the p53 expression fluorescence intensity in the 20 group was also high, significantly higher than that in the max group (P < 0.05). It was confirmed that CPD / modR-p53 can successfully transfer exogenous modR-p53 into cells and translate it into protein. B was measured by protein immunoblotting after CPD / mod p53 transfection, and the expression of GSDMD and c-caspase-1 proteins in cells. Wb results showed that the c-caspase-1 bands of c-caspase-1 in N / P=10, 20, and 30 groups were darker than those in the control group, while the expression of GSDMD gradually decreased. The expression of GSDMD in the N / P=20 and 30 groups was significantly lower than that in the control group, indicating that GSDMD was cleaved and may be converted into GSDMD-N. Therefore, after CPD / mod p53 transfection, the expression of c-caspase-1 in cells increased and GSDMD was cleaved, mediating the caspase-1-GSDMD cell pyroptosis pathway. CD measured IL-1β and IL-18 cytokines by ELISA, and found that after CPD / mod p53 treatment, the cytokine content in the cell supernatant of 10, 20, and 30 groups was nearly twice that of the control group (P<0.0001), and the cell membrane was ruptured, releasing a large number of inflammatory factors.

[0034] Example 4: Organ-specific enrichment of modR-p53@CPD nanocomplexes by aerosol inhalation

[0035] 2 ml of CPD / CY3 solution (5 μg gene, N / P=20) was prepared and administered to 4 mice by nebulization inhalation: the nebulization inhalation rate was 1 ml / 10 min. After the inhalation was completed, the mice were dissected at 1 h, 2 h, 4 h and 6 h, and the heart, liver, spleen, lungs and kidneys were removed. At the same time, one mouse was given an intraperitoneal injection of CPD / CY3 solution, and the heart, liver, spleen, lungs and kidneys were removed after 6 h. The mouse tissues and organs were placed in a small animal in vivo imager (In Vivo IVIS spectrum Imaging System (PerkinElmer, USA)) for optical imaging, and the tissue distribution of nanoparticles in the mouse body at different time periods after nebulization inhalation was observed by fluorescence.

[0036] The enrichment of CPD / modR-p53 in organs after aerosol inhalation at different times Figure 3 As shown. CPD / modR-p53 uses CY3-siRNA as a fluorescent indicator gene. CPD loaded with CY3-siRNA is inhaled by aerosol, and the fluorescence intensity is observed in a small animal in vivo imager. Each C57BL / 6 mouse inhaled CPD / CY3 through a small animal aerosol inhalation instrument. The mouse was killed 1 hour, 2 hours, 4 hours and 6 hours after inhaling CPD / CY3, and the heart, lungs, liver, spleen and kidneys were collected to detect the fluorescence intensity. At the same time, one mouse was intraperitoneally injected with the same dose of CPD / CY3. The mouse was killed 6 hours later, and the heart, lungs, liver, spleen and kidneys were collected to detect the fluorescence intensity. The results are shown in Figure 3 The results showed that the fluorescence intensity in the lungs was obvious at 1 hour, significantly higher than that of mice injected intraperitoneally 1 hour later. The fluorescence intensity was highest at 2 hours, indicating that the accumulation of CPD / CY3 in the lungs was the highest. At the same time, the accumulation of inhaled drugs in the lungs can last up to 6 hours. However, 6 hours after intraperitoneal injection, it mainly accumulated in the liver and kidneys, and the fluorescence intensity in the lungs was extremely low. Therefore, we can prove that CPD / CY3 can be significantly accumulated in the lungs through aerosol inhalation, achieving the effect of targeting the lungs.

[0037] Example 5: Improving the immunotherapy effect of NSCLC by aerosol inhalation of modR-p53@CPD nanocomplex (animal experiment)

[0038] The mice were divided into 4 groups: control group, a-PD-1 group, nebulized inhalation CPD / mod p53 group (hereinafter referred to as CPD / modp53 group), and nebulized inhalation CPD / mod p53 combined with a-PD-1 group (hereinafter referred to as combined group). There were 4 mice in each group. The intervention was carried out according to the dosing regimen in method 4.3.4. In the combined group, CPD / mod p53 was first nebulized and then a-PD-1 was intraperitoneally injected one day later, which was one course of treatment, for a total of 5 courses ( Figure 4) Meanwhile, during the drug administration period, the tumor growth in mice was observed by small animal in vivo imaging.

[0039] Tumor formation experiment and intervention results Figure 5 As shown, fluorescence was observed in the lungs of mice 7 days after tumor inoculation, indicating that the model was successfully established. Drug intervention was then performed. On the 14th day, one mouse in the control group died. The tumor fluorescence intensity in the combination group was significantly lower than that in the a-PD-1 group. There was no significant difference between the CPD / mod p53 group and the a-PD-1 group. After 5 courses of treatment, in vivo imaging of small animals was performed on the 22nd day. The results showed that aPD-1 treatment alone showed anti-tumor effects at early time points, but failed to achieve sustained tumor suppression. Although one mouse died in each group, the fluorescence intensity of the combination group changed less, and the fluorescence intensity was significantly lower than that of the a-PD-1 group, indicating that the combination of ICI and aerosol inhalation of CPD / mod p53 can promote the anti-tumor efficacy of ICI.

[0040] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of a pyroptosis activator in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, characterized in that: The pyroptosis activator is an aerosol inhalation agent.

2. The use of the pyroptosis activator according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, characterized in that: The pyroptosis activator is exogenous p53 modRNA.

3. The use of the pyroptosis activator according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, characterized in that: The lung cancer is non-small cell lung cancer.

4. The use of the pyroptosis activator according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, characterized in that: The immunotherapy is immunotherapy with immune checkpoint inhibitors.

5. Use of the pyroptosis activator according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of lung cancer, characterized in that: The immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor.

6. A pharmaceutical preparation for enhancing the immunotherapy effect of lung cancer, characterized in that: The pharmaceutical preparation is an aerosol inhalation agent, which uses exogenous p53 modRNA as an active ingredient.

7. The pharmaceutical preparation according to claim 6, characterized in that The active ingredient of the pharmaceutical preparation is the modR-p53@CPD nanocomplex.

8. Use of the pharmaceutical preparation according to claim 6 in the preparation of a drug for enhancing the immunotherapy effect of lung cancer.

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