Application of sorafenib in the preparation of drugs for treating lung metastases

Abstract

This invention discloses the application of sorafenib in the preparation of drugs for treating lung metastases, relating to the field of biomedical technology. This invention focuses on CD4... + Sorafenib treatment of CD4 cells during T cell differentiation into Th9 cells + T cells were found to significantly improve the differentiation efficiency of Th9 cells in vitro, indicating that sorafenib can expand Th9 cells with anti-tumor effects in large quantities in vitro, providing a new idea and approach for ACT therapy in clinical tumors.

Description

[0001] This application is a divisional application of the parent application entitled "An in vitro treatment method to improve the differentiation efficiency of Th9 cells", application number "2024114799244", and application date "October 23, 2024". Technical Field

[0002] This invention relates to the field of biomedical technology, and in particular to the use of sorafenib in the preparation of drugs for treating lung metastases. Background Technology

[0003] Adoptive cell therapy (ACT), a cutting-edge branch of cancer immunotherapy, has made significant progress in tumor treatment in recent years, offering new hope to many patients. The basic principle of this therapy is to activate and expand the patient's own immune cells, especially tumor-specific T cells, in vitro, and then reinfuse these modified cells into the patient to recognize and destroy tumor cells. Traditionally, ACT has primarily focused on CD8+ cells. + Cytotoxic T cells (CTLs) are key effector cells in the tumor immune response, capable of directly killing recognized cancer cells. However, with further research, CD4+ cells... + The role of T cells in anti-tumor immunity is becoming increasingly prominent, especially their subsets such as Th1 and Th17 cells. They participate in the anti-tumor process indirectly or directly by regulating immune responses, promoting the activation and maintenance of CTLs, and regulating the tumor microenvironment.

[0004] Among many CD4 + Among T cell subsets, Th9 cells are a relatively new and promising group. Since their initial discovery and reporting in 2008, Th9 cells have attracted considerable attention due to their unique cytokine secretion pattern (primarily secreting IL-9, but also producing IL-21 and IL-3) and their important roles in various diseases. In tumor immunology, Th9 cells exhibit unique anti-tumor potential. Through the secretion of cytokines such as IL-9, they can not only directly act on tumor cells, inhibiting their growth and proliferation, but also activate and regulate other immune cells, such as dendritic cells (DCs) and CD8+ cells. + CTLs, natural killer cells (NK cells), and mast cells form a complex immune network that works together in the tumor microenvironment to enhance the anti-tumor immune response.

[0005] Of particular note is the recent research demonstrating that Th9 cell infusion can completely eradicate melanoma in mouse models. This achievement not only validates the effectiveness of Th9 cells in anti-tumor therapy but also reveals their potential for superior therapeutic efficacy compared to other T cell subsets (such as Th1 and Th17) in certain types of tumors, including melanoma, often considered "cold tumors." Cold tumors refer to those with minimal immune cell infiltration and weak immune responses, often exhibiting poor responses to traditional immunotherapies. The discovery of Th9 cells provides a new strategy for treating these tumors.

[0006] However, despite the enormous therapeutic potential of Th9 cells, effectively expanding these cells in vitro to meet the needs of clinical ACT therapy remains a major challenge. Traditional cell expansion methods may not efficiently induce and maintain Th9 cell differentiation, and may be accompanied by problems such as decreased cell function or deviation from the differentiation direction. Therefore, developing new and efficient Th9 cell expansion technologies is of great significance for promoting the application of ACT in cancer treatment.

[0007] On the other hand, sorafenib (Sora), as a multi-target, multi-kinase inhibitor, has demonstrated effective inhibitory effects against various tumors (including hepatocellular carcinoma, renal cell carcinoma, and certain types of thyroid cancer) in clinical practice. It achieves a dual effect of inhibiting tumor proliferation and angiogenesis by inhibiting key kinases on tumor cells and their blood vessels, such as serine / threonine kinases and receptor tyrosine kinases. Although sorafenib is primarily used as a monotherapy, recent studies have explored its potential for combination therapy with other treatments (such as immunotherapy) to achieve better therapeutic outcomes.

[0008] However, there are no reports on whether sorafenib is related to Th9 cells. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention provides the application of sorafenib in the preparation of drugs for treating lung metastatic tumors.

[0010] Initial CD4 + In vitro, T cells can be induced by TGF-β combined with IL-4 to differentiate into Th9 cells, which secrete IL-9, IL-10, and IL-21. The applicant aims to further improve the differentiation efficiency of Th9 cells using this method. The applicant screened clinically commonly used kinase inhibitors to see if they could improve Th9 cell differentiation efficiency and found that sorafenib can significantly promote Th9 cell differentiation.

[0011] The applicant's preliminary research data showed that sorafenib can significantly improve the differentiation efficiency of Th9 cells in vitro. Analysis of the relevant mechanisms revealed that sorafenib's promotion of Th9 cell differentiation depends on the ERK signaling pathway.

[0012] Therefore, when Th9 cells treated and untreated in vitro were reinfused via the tail vein into mice with other cold tumors, such as breast cancer and osteosarcoma lung metastases, the results showed that, compared with mice with breast cancer and osteosarcoma lung metastases infused with untreated Th9 cells, mice with breast cancer and osteosarcoma lung metastases infused with treated Th9 cells had enhanced anti-tumor immunity and slower tumor progression.

[0013] First, this invention provides an in vitro treatment method to improve the differentiation efficiency of Th9 cells, specifically in CD4... + Sorafenib treatment of CD4 cells during T cell differentiation into Th9 cells + T cells.

[0014] Preferably, sorafenib is used at a concentration of 0.2–2 μM. The treatment also includes inducing differentiation in Th9 cells using the following conditions: plating with 10 μg / ml anti-IFNγ, 20 ng / ml IL4, and 10 ng / ml TGF-β.

[0015] The present invention also provides the use of sorafenib in the preparation of a medicament for treating lung metastases, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt.

[0016] Sorafenib is clinically used to treat unresectable hepatocellular carcinoma, advanced renal cell carcinoma, and locally recurrent or metastatic, progressive, differentiated thyroid cancer that is difficult to treat with radioactive iodine. Therefore, sorafenib, as an readily available drug, has a very high safety profile for treating lung metastases. Furthermore, in vivo studies have shown that sorafenib-treated Th9 cells significantly prolonged the survival time of mouse 4T1-OVA (mouse breast cancer cells) or K7M2-OVA (mouse osteosarcoma cells) lung metastasis models. The mechanism lies in sorafenib's ability to significantly increase the expression level of protein kinase p-ERK in Th9 cells.

[0017] Preferably, the lung metastases are caused by breast cancer or osteosarcoma.

[0018] In CD4 + Sorafenib is added when T cells differentiate into Th9 cells, and the concentration of sorafenib used is 0.2-2 μM.

[0019] In vivo experiments showed that infusion of sorafenib-treated Th9 cells significantly increased CD8+ levels in the spleen of mouse 4T1-OVA or K7M2-OVA lung metastasis tumor models.+ CD45 + The proportion of cells reduced the tumor area of ​​4T1-OVA and K7M2-OVA in the mouse lungs, thereby prolonging the survival time of the mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model.

[0020] The present invention also provides a drug for treating metastatic lung tumors, the active ingredient of which comprises Th9 cells, wherein the Th9 cells are CD4 cells. + T cells were differentiated into Th9 cells by stimulation with sorafenib, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt. The concentration of sorafenib used was 0.2–2 μM.

[0021] Preferably, the drug is in the form of a liquid injection. In this application, Th9 cells are reinfused into mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model mice via the tail vein. The dosage of the liquid injection is 3 × 10⁻⁶ cells per mouse. 6 One Th9 cell.

[0022] In summary, this invention has shown that sorafenib can significantly improve the differentiation efficiency of Th9 cells in vitro, indicating that sorafenib can expand Th9 cells with anti-tumor effects in large quantities in vitro, providing a new idea and approach for ACT therapy in clinical tumors. Attached Figure Description

[0023] Figure 1 Normal C57 mice CD4 + The ability of T cells to polarize into Th9 cells was assessed in vitro with or without sorafenib treatment, specifically by flow cytometry analysis of the proportion of Th9 cells. Figure a shows the flow cytometry results of Th9 cells, and figure b shows the statistical analysis of the results in figure a. "**" indicates P < 0.005; "***" indicates P < 0.001.

[0024] Figure 2 The expression level of cytokine IL9 in the supernatant of Th9 cells was detected by enzyme-linked immunosorbent assay (ELISA). "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0025] Figure 3 To detect the expression level of the Il9 gene in Th9 cells using real-time quantitative PCR. "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0026] Figure 4 Normal C57 mice CD4 +The expression levels of ERK signaling pathway proteins during T cell polarization into Th9 cells were measured in vitro with or without sorafenib treatment, specifically by Western blotting (WB) to detect the expression levels of p-ERK, ERK, and β-Actin proteins in Th9 cells.

[0027] Figure 5 Normal C57 mice CD4 + The ability of T cells to polarize into Th9 cells was assessed in vitro with and without sorafenib or U0126 (a MEK1 / MEK2 inhibitor). Specifically, the proportion of Th9 cells was detected by flow cytometry. Figure a shows the flow cytometry results for Th9 cells, and figure b shows the statistical analysis of the results in figure a. ns indicates no significant difference; "*" indicates P < 0.05; "**" indicates P < 0.005.

[0028] Figure 6 A lung metastasis model was induced in normal C57 mice by tail vein injection of 4T1-OVA cells. Sorafenib-treated or untreated Th9 cells were then reinfused via tail vein. Specifically, the tumor area percentage and survival curves of lung sections from the 4T1-OVA lung metastasis model were detected using hematoxylin-eosin staining. Image a shows a hematoxylin-eosin stained lung section; image b shows the tumor area percentage statistics for lung sections in image a; and image c shows the survival curves of the tumor-bearing mice. "*" indicates P < 0.05; "**" indicates P < 0.005; and "***" indicates P < 0.001.

[0029] Figure 7 A lung metastasis model was induced in normal C57 mice by tail vein injection of K7M2-OVA cells. Sorafenib-treated or untreated Th9 cells were then reinfused via tail vein. Specifically, the tumor area percentage and survival curves of lung sections from the K7M2-OVA lung metastasis model were detected using hematoxylin-eosin staining. Image a shows a hematoxylin-eosin stained lung section; image b shows the tumor area percentage statistics for lung sections in image a; and image c shows the survival curves of the tumor-bearing mice. "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0030] Figure 8 To detect CD8 in the tumor microenvironment of mice by flow cytometry + T cell ratio, treatment conditions: normal C57 mice were used to induce a lung metastasis model by tail vein injection of 4T1-OVA cells, followed by tail vein reinfusion of Th9 cells with or without sorafenib treatment or untreated cells. "*" indicates P < 0.05; "**" indicates P < 0.005.

[0031] Figure 9 To detect CD8 in the tumor microenvironment of mice by flow cytometry+ T cell ratio, treatment conditions: normal C57 mice were used to induce a lung metastasis model by tail vein injection of K7M2-OVA cells, followed by tail vein reinfusion of Th9 cells with or without sorafenib treatment or untreated cells. "**" indicates P < 0.005; "***" indicates P < 0.001. Detailed Implementation

[0032] The C57 mice used in this invention were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0033] Example 1

[0034] T cell sorting:

[0035] (1) Take the spleen and lymph nodes of C57 mice and squeeze them with a syringe plunger to make a tissue suspension;

[0036] (2) Transfer to a 15ml conical test tube and allow the large precipitate to settle to the bottom of the test tube or filter through a nylon filter to obtain a single-cell suspension;

[0037] (3) Centrifuge at 1500 rpm and 4℃ for 5 min to precipitate the cell suspension, and discard the supernatant;

[0038] (4) Resuspend the sample in 2 ml of PBS buffer, dilute with 10 μl of 3% glacial acetic acid, and count the cells.

[0039] (5) Centrifuge the cells again, discard the supernatant, and resuspend the cells in a sorting buffer to adjust the density to 1×10⁻⁶ cells based on the counting results. 8 pcs / ml;

[0040] (6) Use the CD4 negative selection kit (EasySep™ Mouse CD4) + T Cell Isolation Kit (#19765) sorts out CD4 + T cells were prepared for use in subsequent experiments.

[0041] Example 2

[0042] In vitro differentiation of Th9 cells: In 96-well plates, anti-CD3 (145-2C11, Bio X cell) and anti-CD28 (PV-1, Bio X cell) were diluted with autoclaved PBS to a final concentration of 2 μg / ml, and coated at a rate of 200 μl / well. The plates were then incubated at 37°C for at least 2 hours.

[0043] CD4 was obtained by sorting according to Example 1. + T cells, 4 × 10 5Th9 cells were induced to differentiate using 200 μl / well. The differentiation conditions were as follows: 10 μg / ml anti-IFNγ (BE0054, Bio X cell), 20 ng / ml IL4 (130-094-061, Miltenyi Biotec), and 10 ng / ml TGF-β (130-095-067, Miltenyi Biotec). Sorafenib (HY-10201, MedChemExpress) was added or not added, with concentrations of 0.2 μM, 0.5 μM, 1 μM, and 2 μM, respectively. Plating was performed on day 0 of differentiation. Cells were cultured until day 4, at which point they were Th9 cells, ready for subsequent experiments.

[0044] Example 3

[0045] Flow cytometry: To detect the intracellular cytokine IL-9 in Th9 cells, cells need to be treated before staining. Stimulation with 50 ng / mL phorbol ester (PMA, IP1010, Solarbio), 1 μg / mL ionomycin (I8800, Solarbio), and 3 μg / mL Brefeldin A (00-4506-51, Thermofisher) for 4 h is performed before collection. Cells to be tested are washed once with PBS, centrifuged, and resuspended in PBS. CD4-PE (12-0041-82, Thermofisher) flow cytometry antibody is added, and the cells are incubated on ice or at 4°C in the dark for 30 min. After neutralization with PBS and centrifugation twice, the supernatant is discarded. 200 μl of 1× flow cytometry intracellular fixation buffer (00-8222-49, Thermofisher) is added to each tube, and the cells are incubated at room temperature in the dark for 20 min. After neutralization with flow cytometry permeabilization buffer (00-8333-56, Thermofisher), centrifuge. Discard the supernatant after centrifugation, resuspend the cells in 100 μl of 1× flow cytometry permeabilization buffer, add IL9-APC (50-8091-82, Invitrogen) flow cytometry antibody, incubate at 4°C in the dark for 30 min, then neutralize with 1× flow cytometry permeabilization buffer and wash twice. Discard the supernatant, resuspend the cells in an appropriate amount of PBS, and perform flow cytometry analysis using a NovoCyte flow cytometer (ACEA). Data were analyzed using FlowJo software.

[0046] To detect T cell surface antigens CD45 and CD8, wash the cells once with PBS, centrifuge, and resuspend in PBS. Add CD45-PB (MCD4528, Thermofisher) and CD8-APC (MHCD0805, Thermofisher) flow cytometry antibodies, incubate on ice or at 4°C in the dark for 30 min, neutralize with PBS, centrifuge twice, discard the supernatant, resuspend the cells in an appropriate amount of PBS, and then perform flow cytometry analysis. Figure 1 The results showed that sorafenib significantly increased IL-9. + CD4 + Number of T cells (Th9 cells). Figure 5 The results showed that the addition of U0126 (0.5 μM, HY-12031A, MedChemExpress) eliminated the effect of sorafenib on IL9. + CD4 + The effect of the number of T cells (Th9 cells).

[0047] Example 4

[0048] Enzyme-linked immunosorbent assay (ELISA): Collect the supernatant from Th9 cells obtained in Example 2, centrifuge, and use the supernatant as the sample. Using the kit (88-8092-88, Thermofisher), equilibrate the pre-coated plate and required reagents to room temperature. Add 350 μl of washing buffer to each well, let stand for 40 seconds, discard the supernatant, and tap the plate on absorbent paper. Repeat three times. Perform serial dilutions of the lyophilized standard powder with sample diluent. Add 100 μl of different concentrations of standard and prepared sample to each well. Add 100 μl of sample diluent to each blank well. Perform three replicates for each sample. Seal the wells with sealing film and incubate at 37°C for 2 hours. Discard the liquid in the wells, wash three times, add 100 μl of streptomycin-avidin-horseradish peroxidase working solution to each well, seal with sealing film, and incubate at 37°C for 30 minutes. Discard the liquid in the wells and wash three times. Add 100 μl of tetramethylbenzidine substrate to each well and incubate at 37°C in the dark for 15-20 min. Once the liquid in the well turns blue, add 50 μl of stop solution and measure the OD value using a microplate reader at room temperature. Calculate the cytokine content in the sample based on the measured OD value. Figure 2 The results showed that sorafenib significantly increased the IL9 content in the supernatant of Th9 cells, and the increase was related to the increase of sorafenib concentration.

[0049] Example 5

[0050] RNA reverse transcription: Total RNA from sorafenib-treated (0.2 μM, 0.5 μM, 1 μM, 2 μM) and untreated cells was extracted according to the TaKaRa RNAiso Plus instructions. Using 1 μg of RNA as a template, cDNA was synthesized via reverse transcription according to the TaKaRa Reverse Transcription Kit instructions (#RR037A). The reverse transcription system was prepared as follows: 5×PrimeScript Buffer (2 μl), Oligo dT Primer (0.5 μl), Random 6mers (0.5 μl), PrimeScript RT EnzymeMix I (0.5 μl), Total RNA (500 ng), and RNase-Free H2O (total to 10 μl). The reverse transcription reaction was performed at 37℃ for 15 min, followed by heat inactivation of enzyme activity at 85℃ for 5 s.

[0051] Real-time quantitative PCR: The synthesized cDNA can be used as a template for the reaction. Quantitative PCR amplification is performed using SYBR Premix Ex Taq™ (#RR041A) with the corresponding quantitative PCR primers. The quantitative PCR system is 10 μl: SYBR Premix (5 μl), cDNA template (1 μl), primers (0.6 μl), ddH2O (total to 10 μl). The following two-step PCR amplification standard procedure is used: Step 1: Pre-denaturation, 95℃ for 30 s; Step 2: PCR reaction (40 cycles), 95℃ for 5 s, 60℃ for 30 s.

[0052] The PCR primer sequences are as follows:

[0053] Actb-F:

[0054] Actb-R:

[0055] Il9-F:

[0056] Il9-R:

[0057] Figure 3 The results showed that sorafenib significantly increased the expression level of Il9 in Th9 cells, and the level increased with increasing sorafenib concentration.

[0058] Example 6

[0059] Protein extraction: Collect 2 μM sorafenib-treated and untreated Th9 cells into 1.5 mL EP tubes, wash twice with PBS, add 30 μL of RIPA lysis buffer (R0020, Solarbio) containing the protease inhibitor PMSF (P0100, Solarbio), mix well, and incubate on ice for lysis for 30 min. Centrifuge at 12000×g for 10 min at 4 °C, transfer the supernatant to a new EP tube, add 5×SDS Loading Buffer (P1040, Solarbio), and incubate in boiling water for 10 min. The obtained protein sample is ready for use.

[0060] Western blotting: Place the prepared gel on the electrophoresis core, pour in the electrophoresis buffer (Tris 3g / L, glycine 14.4g / L, SDS 1g / L), and add the protein ladder (26616, Solarbio) and the prepared protein sample one by one into the wells. Start the run at 90V, and after the protein ladder separates, adjust the voltage to 180V. After completion, remove the gel, cut it into appropriate sizes, and soak it in transfer buffer (Tris 3g / L, glycine 14.4g / L, 20% methanol). Cut a PVDF membrane (IPVH00010, Millipore) of the appropriate size and activate it in methanol for at least 30 seconds. Arrange the membranes in the transfer clamp in the following order: black clamp - black sponge - thick filter paper - gel - PVDF membrane - thick filter paper - black sponge - transparent clamp. Place the clamp on the rack, pour in the transfer buffer, and transfer at 220mA for 90 minutes. Remove the PVDF membrane and incubate it in 5% skim milk on a shaker at room temperature for 1 hour. Wash the membrane three times with PBST for 5 minutes each time. Then incubate it overnight at 4°C with the corresponding primary antibody (p-ERK, #4370; ERK, #9102; β-Actin, #4967, Cell Signaling Technology). The next day, discard the primary antibody, wash the membrane three times with PBST for 10 minutes each time, and incubate it with the secondary antibody (#7074P2, Cell Signaling Technology) on a shaker at room temperature for 1 hour. Discard the secondary antibody, wash the membrane three times with PBST for 10 minutes each time. Gently wipe the membrane dry with absorbent paper, add ECL (4AW011-500A / B, Sizhengbai Biotechnology), and expose it using an exposure apparatus.

[0061] Figure 4 The results showed that sorafenib significantly increased the expression level of protein kinase p-ERK in Th9 cells.

[0062] Example 7

[0063] Mouse lung metastatic tumor model: On Day 0, 4T1-OVA and K7M2-OVA tumor cells were collected, washed twice with PBS, and counted under a microscope. The cell density was adjusted to 1×10⁻⁶.7 100 μl of tumor cell suspension was injected into each C57 mouse via the tail vein, and the cells were mixed by pipetting before injection.

[0064] T-cell infusion therapy: Th9 cells treated with and untreated with 2 μM sorafenib were obtained as described in Example 2, resuspended in PBS, and infused via the tail vein of lung metastasis model mice using insulin injection on Day 7 and Day 14, respectively. 6 Mice were observed daily using one cell or an equal volume of PBS. Figure 6 c and Figure 7 The results showed that reinfusion of Th9 cells significantly prolonged the survival time of mouse 4T1-OVA or K7M2-OVA lung metastasis tumor models; reinfusion of Th9 cells stimulated with sorafenib significantly enhanced this effect.

[0065] Example 8

[0066] A mouse lung metastasis model was established according to Example 7. The mice were treated on day 17, and the lungs of the mice were taken for H&E staining. Figure 6 a and b in Figure 7 The results in a and b indicate that reinfusion of Th9 cells significantly reduced the tumor area of ​​4T1-OVA and K7M2-OVA in the lungs of mice and prolonged survival time; reinfusion of Th9 cells stimulated by sorafenib further enhanced this effect.

[0067] Example 9

[0068] Lung flow cytometry: A mouse lung metastasis model was established according to Example 7. On day 17, the mice were treated, lungs were taken, ground, filtered through a filter, and the filtrate was centrifuged and resuspended in 1 mL of erythrocyte lysis buffer (R1010, Solarbio). The filtrate was allowed to stand at room temperature for 5 min, centrifuged and the supernatant was discarded. Flow cytometry was performed according to Example 3. Figure 8 and Figure 9 The results showed that the infusion of Th9 cells significantly increased CD8+ levels in the spleen of mouse 4T1-OVA or K7M2-OVA lung metastasis tumor models. + CD45 + The proportion of cells; reinfusion of Th9 cells stimulated by sorafenib significantly enhanced this effect.

Claims

1. Use of sorafenib in the preparation of a medicament for adoptive cell therapy for the treatment of lung metastases, wherein sorafenib is sorafenib itself or a pharmaceutically acceptable salt, and the medicament comprises CD4 cells treated in vitro with sorafenib. + Th9 cells differentiated from T cells, with sorafenib used in CD4... + When T cells differentiate into Th9 cells, sorafenib is added at a concentration of 0.2–2 μM.

2. The application as described in claim 1, characterized in that, The lung metastases were caused by breast cancer or osteosarcoma.

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