Composition for treating lung adenocarcinoma with ANXA6 + EGFR mutation

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a pharmaceutical composition in treating lung adenocarcinoma with ANXA6 + EGFR mutation. In the prior art, ANXA6 + fibroblasts (ANXA6 + CAFs) can cause drug resistance of lung adenocarcinoma cells sensitive to EGFR-TKI, at the same time, the ANXA6 + CAFs secretes high-level CCL20, and the high-level CCL20 promotes tumor angiogenesis, resulting in aggravation of the illness state and drug resistance of cells. The combined use of the EGFR-TKI targeting drug and the anti-CCL20 neutralizing antibody can achieve a good therapeutic effect and delay the resistance of a patient to the targeting drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and specifically relates to a pharmaceutical composition for treating ANXA6. + Application in EGFR-mutated lung adenocarcinoma. Background Art

[0002] Lung adenocarcinoma is the most common type of lung cancer, and its diagnosis and treatment have always been the focus of medical research. According to the multidisciplinary classification of the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS) in 2011, the classification of lung adenocarcinoma has been updated to better reflect its biological characteristics and clinical behavior (International Association for the Study of Lung Cancer / American Thoracic Society / European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma Travis, William D. et al. Journal of Thoracic Oncology, Volume 6, Issue 2, 244-285). In terms of treatment, molecular targeted therapy has become an important treatment for advanced lung adenocarcinoma. For example, patients with epidermal growth factor receptor (EGFR) mutations can benefit from EGFR tyrosine kinase inhibitors (Wang Cai, Wei Suju, Hong Lei, et al. Analysis of clinical prognostic factors in 319 patients with advanced lung adenocarcinoma [J]. Oncology Basics and Clinical Practice, 2016, 29(03): 240-246.). In addition, chemotherapy regimens such as pemetrexed combined with cisplatin have also shown efficacy in patients with advanced lung adenocarcinoma ([1] Chen Hua, Xu Haixia, Hu Qin. Clinical efficacy of different regimens for the treatment of advanced lung adenocarcinoma and their effects on tumor marker levels [J]. Journal of Clinical Military Medicine, 2020, 48(09): 1066-1068.).

[0003] Currently, about 50% of Asian lung adenocarcinoma patients have EGFR-sensitive mutations. A variety of targeted epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been approved for the treatment of patients with EGFR gene-sensitive mutations, which benefit patients. In particular, the third-generation EGFR-TKIs have significantly prolonged patients' survival and improved their quality of life. However, drug resistance is inevitable in the end, which greatly reduces the efficacy.

[0004] ANXA6 + Fibroblasts (ANXA6 +CAFs can cause resistance to EGFR-TKI-sensitive lung adenocarcinoma cells, and this resistance is related to ANXA6 + CAFs are closely related to the promotion of angiogenesis in lung cancer. + In patients with EGFR mutations in CAFs, the combined use of EGFR-TKI targeted drugs and anti-angiogenic drugs can achieve good therapeutic effects and delay the patient's resistance to targeted drugs.

[0005] CCL20 (CC motifchemokine ligand 20) is a β-chemokine that is mainly expressed in thymus, skin and intestinal epithelial cells and can specifically bind to and activate the CCR6 receptor (J. Zaballos et al. "NMR Solution Structure of Murine CCL20 / MIP-3α, a Chemokine That Specifically Chemoattracts Immature Dendritic Cells and Lymphocytes through Its Highly Specific Interaction with theβ-Chemokine Receptor CCR6*." The Journal of Biological Chemistry (2001).). CCL20 plays a role in a variety of physiological and pathological processes, including immune regulation, inflammatory response and tumor progression.

[0006] CCL20 promotes tumor growth by interacting with the CXCR4 pathway (K. Beider, Michal Abraham et al. "Interaction between CXCR4 and CCL20 Pathways Regulates Tumor Growth." PLoS ONE (2009).), and also plays an important role in chemotherapy-induced breast cancer resistance (Weilong Chen, Yuanyuan Qin et al. "CCL20 triggered by chemotherapy hinders the therapeutic efficacy of breast cancer." PLoS Biology (2018).). Patients with paclitaxel-resistant breast cancer will have an increase in CCL20. In colorectal cancer, CCL20 promotes tumor progression by promoting cancer cell proliferation and migration and inducing further CCL20 production through autocrine HGF-c-Met and MSP-MSPR signaling pathways (B. Nandi, Jonathan Pastrana Del Valle et al. "CCL20induces colorectal cancer neoplastic epithelial cell proliferation, migration, and further CCL20production through autocrine HGF-c-Met and MSP-MSPR signaling pathways." Oncotarget (2021).).CCL20 is also involved in other types of cancer, such as hepatocellular carcinoma (Ke-zhu Hou, Z. Fu et al. “Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition..” World journal of gastroenterology (2015).), non-small cell lung cancer (S. Kirshberg, U. Izhar et al. “Involvement of CCR6 / CCL20 / IL-17 Axis in NSCLCDisease Progression.” PLoS ONE (2011).) and lung adenocarcinoma (Tao Fan, Shuofeng Li et al. “CCL20 promotes lung adenocarcinoma progression by driving epithelial-mesenchymal transition.” International Journal of Biological Sciences (2022).), promoting tumor progression by promoting epithelial-mesenchymal transition (EMT) and the activation of other signaling pathways. CCL20 plays a key role in a variety of physiological and pathological processes, including inflammatory responses, tumor progression, and immune regulation through its interaction with CCR6. These findings provide potential targets for the development of therapeutic strategies targeting the CCL20 / CCR6 axis.

[0007] Anti-CCL20 neutralizing antibodies are monoclonal antibodies against CCL20 (a chemokine ligand), which inhibit the inflammatory response and immune cell recruitment mediated by CCL20 by blocking the binding of CCL20 to its receptor CCR6. CCL20 plays an important role in a variety of inflammatory diseases and tumor development, so anti-CCL20 neutralizing antibodies may become potential drugs for the treatment of these diseases. Summary of the invention

[0008] The present invention discovered that ANXA6 + Fibroblasts (ANXA6 + The invention relates to the present invention that CAFs can lead to drug resistance in lung adenocarcinoma cells that are sensitive to EGFR-TKI. The combined use of EGFR-TKI targeted drugs and CCL20 inhibitors can achieve good therapeutic effects and delay the resistance of patients to targeted drugs.

[0009] In a first aspect, the present invention provides a method for treating ANXA6+ A pharmaceutical composition for lung adenocarcinoma resistant to EGFR-TKI caused by CAFs, comprising an EGFR-TKI targeted drug and a CCL20 inhibitor, and having at least one of the following effects:

[0010] Inhibit angiogenesis in lung cancer;

[0011] Reduce tumor volume;

[0012] Preventing and / or treating lung adenocarcinoma.

[0013] Furthermore, the CCL20 inhibitor is preferably an anti-CCL20 neutralizing antibody.

[0014] Furthermore, the EGFR-TKI targeted drugs include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vometinib and befortinib, preferably vometinib and / or osimertinib.

[0015] Furthermore, the gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vometinib and befortinib also include pharmaceutically acceptable salts or esters thereof.

[0016] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0017] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, injection preparation or aerosol; the above various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0018] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0019] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0020] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0021] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0022] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0023] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0024] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0025] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0026] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0027] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0028] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0029] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0030] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0031] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0032] In a second aspect, the present invention provides a method for treating ANXA6 by combining an EGFR-TKI targeted drug and a CCL20 inhibitor.+ Use of CAFs in a pharmaceutical composition for treating EGFR-TKI-resistant lung adenocarcinoma.

[0033] Furthermore, the CCL20 inhibitor is preferably an anti-CCL20 neutralizing antibody.

[0034] Furthermore, the EGFR-TKI targeted drugs include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vometinib and befortinib, preferably vometinib and / or osimertinib.

[0035] Furthermore, the gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vometinib and befortinib also include pharmaceutically acceptable salts or esters thereof.

[0036] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0037] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, injection preparation or aerosol; the above various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0038] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0039] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0040] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0041] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0042] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0043] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0044] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0045] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0046] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0047] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0048] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0049] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0050] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0051] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0052] Beneficial Effects

[0053] In ANXA6 + In patients with EGFR mutations in CAFs, the combined use of EGFR-TKI targeted drugs and anti-CCL20 neutralizing antibodies can achieve good therapeutic effects and delay patients' resistance to targeted drugs, providing precise and effective treatment for patients with lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 ANXA6 + CAFs promote angiogenesis and cell resistance in EGFR-mutated lung adenocarcinoma cells.

[0055] Note: A: ANXA6 +CAFs cell supernatant treated HCC827 cells, HCC827 cells response to AZD9291; B: ANXA6 + PC-9 cells were treated with CAFs cell supernatant, and the response of PC-9 cells to AZD9291; C: angiogenesis in ANXA6+CAFs+HCC827 group; D: angiogenesis in ANXA6+CAFs+PC-9 group.

[0056] Figure 2 HCC827-3 cell resistance experiment.

[0057] Note: A: AZD9291-treated HCC827 cells, HCC827 cell drug sensitivity test; B: AZD9291-treated HCC827-3 cells, HCC827-3 cell drug sensitivity test; C, D: The therapeutic effect of vometinib on lung metastatic lesions formed by HCC827 / HCC827-3 cells.

[0058] Figure 3 ANXA6 + CAFs secrete high levels of CCL20.

[0059] Note: A: Knockdown of ANXA6 + Cytokine chip detection of cell supernatants before and after ANXA6 in CAFs; B: Knockdown of ANXA6 + Different cytokines in the cell supernatant before and after ANXA6 in CAFs; C: 13 differential molecules were verified by RT-PCR in HCC827 cells; D: 13 differential molecules were verified by RT-PCR in PC-9 cells; E: Levels of CCL20 and CGA in lung cancer serum.

[0060] Figure 4 ANXA6 + CCL20 secreted by CAFs promotes angiogenesis.

[0061] Note: A: In HUVEC cells, ANXA6 + CCL20 secreted by CAFs promotes angiogenesis; B: In MAEC cells, ANXA6 + CCL20 secreted by CAFs promotes angiogenesis; C:ANXA6 + CCL20 secreted by CAFs can attract HUVEC cells; D: ANXA6 + CCL20 secreted by CAFs can chemotactic MAEC cells.

[0062] Figure 5 The therapeutic effect of anti-CCL20 neutralizing antibody combined with vometinib in EGFR-TKI-resistant cell lines. DETAILED DESCRIPTION

[0063] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0064] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0065] Test materials

[0066] The basal culture medium used in the example is: serum-free RPMI-1640 / DMEM culture medium, fetal bovine serum is added to a concentration of 10%, penicillin is added to a concentration of 1%, and streptomycin is added to a concentration of 1%.

[0067] Example 1 ANXA6 + CAFs promote angiogenesis in HCC827 / PC-9 cell transplanted tumors

[0068] (1) HCC827 / PC-9 cells and ANXA6 + CAFs cells were digested into single cells and counted;

[0069] (2) HCC827 / PC-9 cell suspension and ANXA6 + CAFs cell suspension was mixed at a ratio of 1:1;

[0070] (3) Prepare 4- to 6-week-old BALB / c nude mice and inoculate the cell suspension into the axilla of the nude mice, injecting 2 million cells in each site;

[0071] (4) When nude mice have subcutaneous tumors, the mice are killed, and the transplanted tumors are dissected and fixed;

[0072] (5) HE staining observation and record.

[0073] Example 2 Immunohistochemistry

[0074] (1) Tissue processing: slice the tissue to be examined and dry it to ensure that the immunohistochemical reagent can bind to the tissue;

[0075] (2) Antigen repair: heating or enzymatic hydrolysis, etc.

[0076] (3) Blocking nonspecific binding: adding nonspecific antiserum, protein solution or other blocking reagents;

[0077] (4) Primary antibody incubation: CD31 primary antibody was coated on the tissue sections and incubated;

[0078] (5) Secondary antibody incubation: Apply the labeled secondary antibody to the tissue section and incubate;

[0079] (6) Colorimetric observation: Observe the distribution and expression of the target antigen in the tissue.

[0080] like Figure 1 As shown in C and 1D, compared with the HCC827 / PC-9 group, ANXA6 + CAFs+HCC827 / ANXA6 + The tumor angiogenesis in the CAFs+PC-9 group was significantly increased. The detection of endothelial cell marker CD31 confirmed that compared with the HCC827 / PC-9 group, ANXA6 + CAFs+HCC827 / ANXA6 + The tumor angiogenesis in the CAFs+PC-9 group was significantly increased.

[0081] Example 3 Using ANXA6 + Treatment of EGFR-mutated lung cancer cells with CAFs supernatant and drug sensitivity test

[0082] (1)ANXA6 + CAFs cells were cultured in DMEM medium, and the cell culture supernatant was collected;

[0083] (2) HCC827 and PC-9 cells were inoculated in 10% RPMI-1640 medium, passaged, digested into single cells, and counted;

[0084] (3) Inoculate HCC827 and PC-9 cells in well plates;

[0085] (4) ANXA6 + CAFs cell culture supernatant was mixed with 10% RPMI-1640 medium at a volume ratio of 1:1, and different concentrations of AZD9291, including 6 nM, 12 nM, 120 nM, 250 nM, 500 nM, 1200 nM, 6000 nM, 12000 nM, and 24000 nM, were prepared and added to the well plate inoculated with HCC827 cells;

[0086] (5) ANXA6 + The CAFs cell culture supernatant was mixed with 10% RPMI-1640 medium at a volume ratio of 1:1, and different concentrations of AZD9291, including 1 nM, 2 nM, 4 nM, 40 nM, 400 nM, 4000 nM, 6000 nM, 8000 nM, and 16000 nM, were prepared and added to the well plate inoculated with PC-9 cells;

[0087] (6) Draw the drug sensitivity curve of cells to AZD9291 based on the data.

[0088] like Figure 1 As shown in A and 1B, ANXA6 + CAFs promote angiogenesis and cell resistance in lung adenocarcinoma cells with EGFR mutations. + After HCC827 and PC-9 cells were treated with CAFs cell supernatant, the cells became resistant to AZD9291.

[0089] Example 4 Obtaining AZD9291-tolerant cells HCC827-3

[0090] A. Obtaining cells

[0091] (1) HCC827 / PC-9 cells and ANXA6 + CAFs cells were digested into single cells and counted;

[0092] (2) HCC827 / PC-9 cell suspension and ANXA6 + CAFs cell suspension was mixed at a ratio of 1:1;

[0093] (3) Prepare 4- to 6-week-old BALB / c nude mice and inoculate the cell suspension into the axilla of the nude mice, injecting 2 million cells in each site;

[0094] (4) After nude mice have subcutaneous tumors, the mice are killed and the tumors are dissected out;

[0095] (5) Cut the tumor into pieces, wash them, add DMEM medium and culture them;

[0096] (6) After the tumor cells adhered to the wall and grew, they were digested, subcultured and named HCC827-3 cells.

[0097] B. Verify tolerance

[0098] (1) Inoculate HCC827 and HCC827-3 cells in a well plate;

[0099] (2) AZD9291 at different concentrations of 0 nM, 3 nM, 6 nM, 12 nM, 120 nM, 250 nM, 500 nM, 1200 nM, 6000 nM, 12000 nM, and 24000 nM was prepared and added to the well plate inoculated with HCC827 and HCC827-3 cells;

[0100] (3) Draw the drug sensitivity curves of the two cell types to AZD9291 based on the data;

[0101] (4) HCC827 and HCC827-3 were injected into lung metastatic lesions through the tail vein and treated with vometinib to observe their response to the drug.

[0102] C. Test Results

[0103] like Figure 2 As shown in A and 2B, the IC50 of HCC827 cells to AZD9291 is 11.75 nM, and the IC50 of HCC827-3 cells to AZD9291 is 10232 nM, which is 870.81 times that of HCC827 cells. HCC827-3 cells are resistant to AZD9291.

[0104] like Figure 2 As shown in C and 2D, the therapeutic effect of vometinib on lung metastases formed by HCC827 cells was significantly better than that on lung metastases formed by HCC827-3 cells. These results showed that HCC827-3 cells were resistant to the third-generation EGFR-TKI.

[0105] Example 5 ANXA6 + CAFs promote the screening of key molecules for drug resistance in EGFR-mutated lung cancer cells

[0106] (1) Expanding the culture of ANXA6 + CAFs cells, knockdown of ANXA6 using lentiviral vector + ANXA6 in CAFs and expansion

[0107] Proliferate cells;

[0108] (2) Collection of ANXA6 before and after knockdown + CAFs cell supernatant was used for cytokine chip detection;

[0109] (3) Analyze the significantly differentially expressed cytokines and verify them in HCC827 / PC-9 cells;

[0110] (4) Detect differentially expressed molecules in lung cancer serum.

[0111] like Figure 3 As shown, knockdown of ANXA6 + ANXA6 in CAFs screened out 13 cytokines with significant differences, among which CCL20 and CGA showed consistent change trends in two HCC827 / PC-9 cell lines, and the expression of CCL20 in lung cancer serum was significantly different.

[0112] Example 6 ANXA6 + Study on the promotion of tumor angiogenesis by CCL20 in CAFs

[0113] (1) Cell culture: Culture HUVE / MAEC cells, subculture, and inoculate an appropriate amount of cells to achieve a cell confluence of 70-90%;

[0114] (2) Prepare gel: Take out Matrigel gel, thaw it completely, and mix it;

[0115] (3) Matrigel gel coating of 12-well plate: Add Matrigel gel into the well plate and incubate to form a Matrigel gel base;

[0116] (4) adding different concentrations of anti-CCL20 neutralizing antibodies to the supernatant of ANXA6+CAFs cells to prepare conditioned medium containing different concentrations of anti-CCL20 neutralizing antibodies;

[0117] (5) The HUVEC / MAEC cells cultured in (1) were digested, centrifuged and counted, and the culture medium of (4) was used to prepare 40

[0118] 10,000 / ml single cell suspension, inoculated at 200,000 / 500μl / well;

[0119] (6) culturing, staining with calcein at different time points, taking photos and observing the formation of tubular structures;

[0120] (7) Image analysis: Image J was used for quantitative analysis.

[0121] like Figure 4 As shown in A and 4B, ANXA6 was neutralized by CCL20 neutralizing antibody in two endothelial cell lines HUVEC and MAEC. + When CCL20 in the supernatant of CAFs cells was expressed, ANXA6 + The angiogenesis-promoting effect of CAFs cell supernatant was significantly reduced. With the increase of CCL20 neutralizing antibody concentration, ANXA6 + The more obvious the effect of CAFs cell supernatant on promoting angiogenesis.

[0122] Example 7 ANXA6 + CCL20 in CAFs recruits endothelial cells

[0123] (1) Cell culture: culture HUVEC / MAEC cells, subculture, and inoculate to achieve a cell confluence of 70-90%;

[0124] (2) In ANXA6 + Different concentrations of anti-CCL20 neutralizing antibodies were added to the supernatant of CAFs cells to prepare conditioned medium containing different concentrations of anti-CCL20 neutralizing antibodies;

[0125] (3) digesting, centrifuging and counting the HUVEC / MAEC cells cultured in (1) to prepare a single cell suspension of 10 million / ml;

[0126] (4) Add the conditioned medium containing different concentrations of anti-CCL20 neutralizing antibody in (2) to the lower chamber of the transwell chamber, and

[0127] The cell suspension was added into the upper chamber at 2 million / well and cultured;

[0128] (5) Wash the chamber, add gentian violet solution, observe the cells that pass through the membrane and grow, and take pictures for analysis.

[0129] like Figure 4 C and 4D, two endothelial cell lines HUVEC and MAEC were then used to study the chemotactic effect of CCL20 on endothelial cells. The results showed that CCL20 from ANXA6 + CCL20 of CAFs can chemotacticly recruit endothelial cells. When CCL20 neutralizing antibodies are used, ANXA6 + The chemotaxis of CAFs cell supernatant to endothelial cells was inhibited, and with the increase of CCL20 neutralizing antibody concentration, ANXA6 + The weaker the chemotactic effect of CAFs cell supernatant on endothelial cells, the weaker the effect, suggesting that it comes from ANXA6 + CCL20 of CAFs can chemotacticize vascular endothelial cells, thereby promoting tumor angiogenesis.

[0130] Example 8 Efficacy of anti-CCL20 neutralizing antibody combined with vometinib in AZD9291-resistant strains

[0131] (1) HCC827-3 cells were digested into single cells, counted, and resuspended at 10 million / ml;

[0132] (2) Prepare 4- to 6-week-old BALB / c nude mice and inoculate the cell suspension into the axilla of the nude mice, injecting 2 million cells in each site;

[0133] (3) When the tumor grows to 1000 mm 3 The patients were divided into a vumetinib treatment group and an anti-CCL20 neutralizing antibody and vumetinib combined treatment group. Vumetinib was administered orally at a dose of 5 mg / kg once a day; the anti-CCL20 neutralizing antibody was injected into the tail vein at a dose of 1.25 mg / kg once a week.

[0134] (4) Observe the tumor formation in the axilla of nude mice, measure the tumor size, and draw the growth curve of each group of transplanted tumors.

[0135] like Figure 5As shown, in HCC827-3 cells resistant to AZD9291, anti-CCL20 neutralizing antibody combined with vometinib had a good therapeutic effect. Six days before treatment, the tumor volume was 1161.37±4.73mm 3 Reduced to 33.96±19.06mm 3 , and then slowly decreased. By the 20th day, the tumor had completely disappeared. In the first 6 days of treatment, although the therapeutic effect of the vometinib monotherapy group was not as good as that of the combination group, the tumor was also significantly reduced, from 1017.66±91.53mm 3 Reduced to 61.19±7.39mm 3 , and maintained at a slowly decreasing level until 12 days, and then showed a slowly increasing trend, indicating that in HCC827-3 cells resistant to AZD9291, anti-CCL20 neutralizing antibody combined with vometinib treatment is better than vometinib alone and can reduce tumor recurrence.

Claims

1. A treatment for ANXA6 + A pharmaceutical composition for lung adenocarcinoma resistant to EGFR-TKI caused by CAFs, the pharmaceutical composition comprising an EGFR-TKI targeted drug and a CCL20 inhibitor, the pharmaceutical composition having at least one of the following effects: inhibiting angiogenesis of lung cancer; Reduce tumor volume; Preventing and / or treating lung adenocarcinoma.

2. Combination of an EGFR-TKI targeted drug and a CCL20 inhibitor in the treatment of ANXA6 + Use of CAFs in a pharmaceutical composition for treating EGFR-TKI-resistant lung adenocarcinoma.

3. The CCL20 inhibitor according to any one of claims 1 or 2 is preferably an anti-CCL20 neutralizing antibody.

4. The EGFR-TKI targeted drug according to any one of claims 1 or 2 comprises gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vometinib, befortinib and pharmaceutically acceptable salts or esters thereof, preferably vometinib and / or osimertinib.

5. The pharmaceutical composition according to any one of claims 1 or 2, further comprising one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to any one of claims 1 or 2, which can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

7. The pharmaceutical composition according to any one of claims 1 or 2 can be prepared into various forms such as injection, tablets, powders, granules, capsules, oral solutions, injection preparations or aerosols; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

8. The preparation as claimed in claim 6 can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

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