Pharmaceutical composition of Galectin-9 inhibitor and EGFR-TKI and application of pharmaceutical composition in tumor treatment

Abstract

The invention provides a pharmaceutical composition of a galectin-9 (Galectin-9) inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and an application of the pharmaceutical composition in tumor treatment. According to the pharmaceutical composition disclosed by the invention, the Galectin-9 inhibitor and the EGFR-TKI are combined for use, and infiltration of CD8 + T cells in a tumor microenvironment can be remarkably enhanced, so that tumor growth is effectively inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pharmaceutical composition of a Galectin-9 inhibitor and EGFR-TKI and use thereof in treating tumors. Background Art

[0002] Epidermal Growth Factor Receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC) and is abnormally overexpressed in a variety of malignancies, including colorectal cancer (CRC), head and neck squamous cell carcinoma, breast cancer, and pancreatic cancer. Existing EGFR inhibitors are primarily divided into two categories: small molecule tyrosine kinase inhibitors (TKIs) that target the mutant EGFR that is constitutively activated in NSCLC, and monoclonal antibodies (mAbs) that target EGFR overexpression in cancers such as CRC. Although clinically approved EGFR-TKIs (e.g., erlotinib, gefitinib, afatinib, and osimertinib) have demonstrated significant initial clinical responses in EGFR-mutant NSCLC (particularly in patients harboring exon 19 deletions or L858R substitution mutations), acquired resistance inevitably arises through various mechanisms, such as secondary EGFR mutations (e.g., T790M, C797S), compensatory pathway activation (e.g., mesenchymal epithelial transition factor amplification, MET amplification), or adaptive tumor plasticity. Current research on overcoming resistance primarily focuses on tumor-intrinsic pathways, but largely overlooks the immunosuppressive tumor immune microenvironment (TIME) shaped by EGFR inhibition.

[0003] Growing evidence suggests that EGFR-TKIs can modulate immune responses through multiple mechanisms, including enhancing cytotoxic T cell-mediated tumor killing, inducing antiviral immune responses, and regulating PD-L1 immune checkpoint expression. Although preclinical studies have shown that combining EGFR-targeted therapy with anti-PD-1 / PD-L1 immune checkpoint blockade (ICB) can enhance anti-tumor activity, its clinical efficacy remains limited, suggesting the existence of unresolved immune escape mechanisms. These limitations highlight the urgency of further analyzing the immune regulatory mechanisms driven by EGFR-TKIs and developing combination therapy strategies that can achieve durable clinical remissions.

[0004] Galectin-9 (Gal-9), a β-galactoside-binding lectin, serves as a key immunosuppressive checkpoint molecule in the tumor microenvironment (TME) and has become a highly sought-after therapeutic target in the field of tumor immunotherapy. Evidence accumulated by our group and others indicates that Gal-9 is highly expressed in antigen-presenting cells (APCs) and can be upregulated in tumor cells upon stimulation with type I / II interferons (IFN-β / γ). Structurally, Gal-9 contains two conserved carbohydrate-recognition domains (CRDs) that mediate specific β-galactoside binding. Although Gal-9 was initially identified as a ligand that induces T cell death by binding to T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), recent studies have revealed that it can also promote macrophage M2 polarization and regulatory T cell (Treg) maturation by binding to immunoregulatory receptors such as Dectin-1 and CD44, respectively. Gal-9 is abnormally overexpressed in various malignancies and is significantly positively correlated with advanced disease and shortened overall survival. Our preclinical studies suggest that Gal-9 blockade strategies have significant therapeutic potential, particularly when combined with immune checkpoint modulators (such as glucocorticoid-induced tumor necrosis factor receptor-related protein agonists, GITR agonists) or DNA damage response (DDR) inhibitors. Early clinical evaluation of the anti-Gal-9 monoclonal antibody LYT-200 has shown acceptable tolerability and potential clinical activity in patients with metastatic solid tumors, both as a single agent and in combination with chemotherapy and PD-1 inhibitors. However, detailed efficacy data are pending. Despite the expanding therapeutic applications of anti-Gal-9 antibodies, significant knowledge gaps remain regarding their mechanisms of action. Furthermore, the regulatory mechanisms of Gal-9 under therapeutic conditions and its role in mediating treatment resistance remain to be elucidated. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes a pharmaceutical composition of a Galectin-9 inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) and its use in treating tumors.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The present invention aims to provide a method for preparing a drug for treating tumors by combining a Galectin-9 inhibitor and EGFR-TKIs.

[0008] In some embodiments, the tumor is a solid tumor.

[0009] In some embodiments, the tumor is lung cancer or colorectal cancer.

[0010] In some embodiments, the lung cancer is non-small cell lung cancer.

[0011] In some embodiments, the Galectin-9 inhibitor is an anti-Galectin-9 antibody (α-Gal-9).

[0012] In some embodiments, the EGFR-TKIs are key targeted drugs for the treatment of non-small cell lung cancer. With the deepening of research on drug resistance mechanisms, EGFR-TKIs have developed into the third generation, with gradually optimized efficacy and safety. Representative first-generation drugs include gefitinib, erlotinib, and icotinib; second-generation drugs include afatinib and dacomitinib; and third-generation drugs include osimertinib, almonertinib, and furmonertinib.

[0013] In some embodiments, the EGFR-TKIs is one of gefitinib, afatinib, or osimertinib. In some embodiments, the EGFR-TKIs is gefitinib and / or afatinib.

[0014] Another object of the present invention is to provide a pharmaceutical composition for treating tumors, comprising a Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the tumor is a solid tumor.

[0016] In some embodiments, the tumor is lung cancer or colorectal cancer.

[0017] In some embodiments, the lung cancer is non-small cell lung cancer.

[0018] The drug composition can significantly enhance the expression of CD8 T cells in the tumor microenvironment by combining Galectin-9 inhibitors with EGFR-TKIs. +T cell infiltration, thereby effectively inhibiting tumor growth.

[0019] In some embodiments, the Galectin-9 inhibitor is an anti-Galectin-9 antibody (α-Gal-9).

[0020] In some embodiments, the Galectin-9 inhibitor is administered once every 3 days.

[0021] In some embodiments, the Galectin-9 inhibitor is administered at a dose of 100 ug per animal.

[0022] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered once daily.

[0023] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered at a dose of 10 mg / kg each time.

[0024] In some embodiments, the Galectin-9 inhibitor, the EGFR-TKIs, or pharmaceutically acceptable salts thereof are administered simultaneously or sequentially.

[0025] Another object of the present invention is to provide a method for preparing a drug for treating tumors by combining a Galectin-9 inhibitor, EGFR-TKIs and an immune checkpoint inhibitor.

[0026] In some embodiments, the Galectin-9 inhibitor is an anti-Galectin-9 antibody (α-Gal-9).

[0027] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0028] In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody (α-PD-1).

[0029] In some embodiments, the tumor is a solid tumor.

[0030] In some embodiments, the tumor is lung cancer or colorectal cancer.

[0031] In some embodiments, the lung cancer is non-small cell lung cancer.

[0032] Another object of the present invention is to provide a pharmaceutical composition for treating tumors, which comprises a Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor.

[0033] Furthermore, the Galectin-9 inhibitor is an anti-Galectin-9 antibody (α-Gal-9).

[0034] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0035] Furthermore, the immune checkpoint inhibitor is an anti-PD-1 antibody (α-PD-1).

[0036] Furthermore, the tumor is lung cancer or colorectal cancer.

[0037] In some embodiments, the Galectin-9 inhibitor is administered once every 3 days.

[0038] In some embodiments, the Galectin-9 inhibitor is administered at a dose of 100 ug per animal.

[0039] In some embodiments, the immune checkpoint inhibitor is administered once every 3 days.

[0040] In some embodiments, the anti-PD-1 antibody is administered at a dose of 100 ug per animal.

[0041] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered once daily.

[0042] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered at a dose of 10 mg / kg each time.

[0043] In some embodiments, the Galectin-9 inhibitor, the EGFR-TKIs or pharmaceutically acceptable salts thereof, and the immune checkpoint inhibitor are administered simultaneously or sequentially.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] The present invention first uses clinical specimens to find that the expression of galectin-9 (Galectin-9) in tumor tissues of patients with non-small cell lung cancer is upregulated after treatment with EGFR-TKIs, and the high expression of Galectin-9 is closely related to clinical progression and drug resistance. Subsequently, a tumor-bearing mouse model was used to prove that the expression of Galectin-9 in myeloid cells in tumor tissues and tumor-draining lymph nodes was upregulated after treatment with EGFR-TKIs. The above results suggest that Galectin-9 may be a potential therapeutic target. The results showed that in a colon cancer mouse model, the combination of Galectin-9 inhibitors and EGFR-TKIs can significantly inhibit mouse tumor growth and prolong mouse survival compared with the monotherapy group. In a lung cancer mouse model with poor immunogenicity, the tumor inhibition effect of the combination of EGFR-TKI and Galectin-9 inhibitors was more significant than that of the combination of EGFR-TKI / PD-1 inhibitors, and the combination of EGFR-TKI, Galectin-9 inhibitors and PD-1 inhibitors showed the best tumor inhibition effect with good safety. In terms of molecular mechanism, compared with the single-drug group, the combination of EGFR-TKI and Galectin-9 inhibitor significantly enhanced the expression of CD8 + T cell infiltration; CD8 depletion with CD8 inhibitor (α-CD8) + After T, the efficacy of the combined treatment was completely suppressed. In summary, the present invention provides a safe and non-toxic drug combination that can effectively inhibit tumor growth and enhance anti-tumor immunity, providing new research ideas and treatment strategies for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A is a representative image of Gal-9 immunohistochemical staining in tumor tissues of non-small cell lung cancer (NSCLC) patients before treatment and after EGFR-TKI resistance;

[0047] Figure 1 B shows quantitative analysis of Gal-9 immunohistochemical staining of tumor tissues from NSCLC patients in cohort #1 (n=15) before treatment and after EGFR-TKI resistance (left panel). Gal-9-positive areas were quantified using Image J software. The proportion of patients with high Gal-9 expression (Gal-9-positive areas >40%) in the short-term responder group (SR, progression-free survival <12 months) and the long-term responder group (LR, progression-free survival ≥12 months) is shown (right panel).

[0048] Figure 1 C is based on publicly available single-cell RNA sequencing data of NSCLC patients (cohort #2), analyzing the expression levels of Gal-9 and PD-L1 mRNA in biopsy tissues of treated patients (n=5) and osimertinib-resistant patients (n=2);

[0049] Figure 2 A is an immunoblot analysis of the expression levels of phosphorylated EGFR (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated ERK (p-ERK) in CT26 tumor tissues of BALB / c mice after 7 days of afatinib treatment;

[0050] Figure 2 B is an immunoblot analysis of Gal-9 protein expression in CT26 tumor tissues of BALB / c mice treated with afatinib for 7 days. Data show the relative fold change in Gal-9 protein expression (using β-actin as an internal reference).

[0051] Figure 2 C is the flow cytometric analysis of the Gal-9 expression levels (median fluorescence intensity, MFI) of dendritic cell (DC) subsets in tumor-draining lymph nodes (tdLN) of CT26 tumor-bearing BALB / c mice treated with afatinib for 7 days;

[0052] Figure 2 D is immunoblotting analysis of the expression levels of phosphorylated AKT (p-AKT) and Gal-9 proteins in LLC tumor tissues of C57BL / 6 mice treated with afatinib for 7 days;

[0053] Figure 2 E is the flow cytometric analysis of the Gal-9 expression levels of myeloid cell subsets in LLC tumor tissues of C57BL / 6 mice after 7 days of afatinib treatment;

[0054] Figure 3 A is a schematic diagram of the experimental scheme: a schematic diagram of the dosing schedule for CT26 tumor-bearing BALB / c mice receiving isotype control, afatinib alone, Gal-9 inhibitor alone, and combination therapy;

[0055] Figure 3 B is a typical tumor image: representative tumors isolated from CT26 tumor-bearing mice on day 15 after inoculation in different treatment groups and images of representative tumor-bearing mice on day 35;

[0056] Figure 3 C is individual tumor growth: dynamic monitoring of tumor volume in CT26 tumor-bearing mice in different treatment groups (n = 5 per group);

[0057] Figure 3 D is the tumor growth curve: the average tumor volume changes of CT26 tumor-bearing BALB / c mice in different treatment groups (error bars represent the standard error of the mean, SEM);

[0058] Figure 3 E is survival analysis: survival curves of CT26 tumor-bearing BALB / c mice in different treatment groups;

[0059] Figure 3 F is body weight monitoring: average body weight changes of CT26 tumor-bearing BALB / c mice in different treatment groups (n = 5 per group);

[0060] Figure 3 G Tumor growth curve: Mean tumor volume changes of CT26 tumor-bearing mice in different treatment groups (error bars represent standard error of the mean, SEM). Groups were: isotype control group, gefitinib monotherapy group, Gal-9 inhibitor monotherapy group, and combination therapy group;

[0061] Figure 3 H represents survival analysis: survival curves of CT26 tumor-bearing BALB / c mice in different treatment groups;

[0062] Figure 4 A is a schematic diagram of the experimental scheme: a schematic diagram of the dosing schedule for LLC tumor-bearing C57BL / 6 mice receiving isotype control, afatinib alone, Gal-9 inhibitor alone, PD-1 inhibitor alone, and combination therapy;

[0063] Figure 4 B shows tumor growth curves and mouse survival analysis: mean tumor volume changes (error bars indicate standard error of the mean, SEM) and survival curves of LLC tumor-bearing mice in different treatment groups. Groups were divided into: isotype control group, Gal-9 inhibitor monotherapy group, PD-1 inhibitor monotherapy group, and Gal-9 inhibitor and PD-1 inhibitor combination treatment group.

[0064] Figure 4 C is individual tumor growth: Dynamic monitoring of tumor volume in LLC tumor-bearing mice under different treatment groups (n = 5 per group). Groups were divided into: isotype control group, afatinib combined with PD-1 inhibitor group, afatinib combined with Gal-9 inhibitor group, and afatinib + Gal-9 inhibitor + PD-1 inhibitor triple-drug combination group;

[0065] Figure 4 D is the tumor growth curve and mouse survival analysis: the average tumor volume changes (error bars represent the standard error of the mean, SEM) and survival curves of LLC tumor-bearing mice in different treatment groups;

[0066] Figure 4 E is body weight monitoring: average body weight changes of LLC tumor-bearing mice in different treatment groups (n = 5 per group);

[0067] Figure 5 A is the flow cytometric analysis of tumor-infiltrating GB in CT26 tumor-bearing BALB / c mice in different treatment groups on day 15 after inoculation. + CD8 + Changes in the number of T cells;

[0068] Figure 5 B is a schematic diagram of the experimental scheme: Treatment plan for CT26 tumor-bearing mice. CD8+ T cell depleting antibody was injected 1 day before afatinib treatment;

[0069] Figure 5 C is the flow cytometry analysis of the total T cells and CD8 + Changes in the number of T cells (n = 3 per group);

[0070] Figure 5 D is a typical tumor sample: a physical picture of individual tumors isolated from CT26 tumor-bearing BALB / c mice in different treatment groups on day 15 after inoculation;

[0071] Figure 5 E is individual tumor growth: Tumor volume dynamics of CT26 tumor-bearing BALB / c mice in different treatment groups (n=7 per group). Groups were divided into: control group, group treated with EGFR-TKIs combined with Gal-9 inhibitor, and group treated with combination therapy plus CD-8 inhibitor (α-CD-8);

[0072] Figure 5 F and 5G show tumor growth and survival analysis: mean tumor volume and survival curves of CT26 tumor-bearing BALB / c mice in different treatment groups, with error bars representing the standard error of the mean (SEM);

[0073] Figure 5 H is survival analysis: survival curves of LLC tumor-bearing C57BL / 6 mice in different treatment groups (n=5 per group). DETAILED DESCRIPTION

[0074] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0075] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0076] Where values are described herein as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values falling within that range, regardless of whether a specific value or sub-range is explicitly stated.

[0077] In this document, the word "further" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0078] In this article, the term "and / or" is used to describe the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0079] As used herein, the term "about" means + / - 10%, preferably + / - 5%, more preferably + / - 1% of the specified value.

[0080] In this document, the terms “include,” “including,” “have,” “contain,” etc. are open-ended terms, meaning including but not limited to.

[0081] As used herein, "combination therapy group," "combination drug," or "combination drug" refers to a drug used in combination with one or more other drugs to treat a disease, including both combinations of a drug with one or more other drugs and combinations of a drug with instructions indicating that the drug can be used in combination with one or more other drugs. As used herein, a "combination group" refers to an experimental group in which a drug is used in combination with another drug, a dual-drug or triple-drug combination, or a dual-drug regimen is used in combination with a CD-8 inhibitor.

[0082] Gal-9 inhibitors, EGFR-TKIs, immune checkpoint inhibitors, and CD-8 inhibitors can be administered orally or by intraperitoneal injection. EGFR-TKIs are administered orally, and can be administered orally with a pharmaceutically acceptable carrier (CMC-Na). Gal-9 inhibitors, immune checkpoint inhibitors, and CD-8 inhibitors are administered by intraperitoneal injection.

[0083] In some embodiments, the amount of the Gal-9 inhibitor in the pharmaceutical combination is a daily dose.

[0084] In some embodiments, the Gal-9 inhibitor is included in the pharmaceutical combination in a once-daily dose.

[0085] In some embodiments, the Gal-9 inhibitor in the pharmaceutical combination is present in a uniform dose.

[0086] In some embodiments, the content of the immune checkpoint inhibitor in the drug combination is a daily dose.

[0087] In some embodiments, the immune checkpoint inhibitor in the pharmaceutical combination is administered in a once-daily dose.

[0088] In some embodiments, the immune checkpoint inhibitor in the drug combination is contained in a uniform dose.

[0089] In some embodiments, the content of EGFR-TKIs or pharmaceutically acceptable salts thereof in the pharmaceutical combination is a daily dose.

[0090] In some embodiments, the content of EGFR-TKIs or pharmaceutically acceptable salts thereof in the pharmaceutical combination is a once-daily dose.

[0091] In some embodiments, the content of EGFR-TKIs or pharmaceutically acceptable salts thereof in the drug combination is a uniform dose.

[0092] Another object of the present invention is to provide a kit for treating tumors, which comprises a Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof, and instructions for using the Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof in combination to treat tumors.

[0093] Another object of the present invention is to provide a kit for treating tumors, comprising a Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof, an immune checkpoint inhibitor, and instructions for the combined use of the Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor and for treating tumors.

[0094] In some embodiments, the Galectin-9 inhibitor in the kit is in the form of a liquid formulation. In some specific embodiments, the Galectin-9 inhibitor is in the form of an injection. The administration method is generally intraperitoneal injection.

[0095] In some embodiments, the immune checkpoint inhibitor in the kit is in the form of a liquid formulation. In some specific embodiments, the immune checkpoint inhibitor is in the form of an injection. The administration method is generally intraperitoneal injection.

[0096] In some embodiments, the dosage form of EGFR-TKIs or pharmaceutically acceptable salts thereof is a suspension preparation, and the administration method is generally oral gavage.

[0097] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is colorectal cancer, lung cancer (non-small cell lung cancer).

[0098] In some embodiments, the Galectin-9 inhibitor, PD-1 immune checkpoint inhibitor, EGFR-TKIs, or pharmaceutically acceptable salts thereof are administered in the same or different dosing regimens, respectively.

[0099] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered in a regimen of 10 consecutive days.

[0100] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered once daily at a dose of 10 mg / Kg.

[0101] In some embodiments, the EGFR-TKIs or pharmaceutically acceptable salts thereof are administered at a dose of 10 mg / Kg per time, once daily for 10 consecutive days.

[0102] In some embodiments, Galectin-9 inhibitors, PD-1 immune checkpoint inhibitors, EGFR-TKIs, or pharmaceutically acceptable salts thereof, respectively, have the same or different treatment cycles.

[0103] In some specific embodiments, Galectin-9 inhibitors and PD-1 immune checkpoint inhibitors have the same treatment cycle, for example, one treatment cycle is every 3 days.

[0104] In some embodiments, every 3 days is a treatment cycle, a Galectin-9 inhibitor is administered on the first day of each treatment cycle, an immune checkpoint inhibitor is administered on the first day of each treatment cycle, and EGFR-TKIs or pharmaceutically acceptable salts thereof are administered daily.

[0105] In some embodiments, the Galectin-9 inhibitor, immune checkpoint inhibitor, EGFR-TKIs, or pharmaceutically acceptable salts thereof are administered as a single dose in each treatment cycle.

[0106] In some embodiments, the Galectin-9 inhibitor is administered to tumor-bearing mice at a uniform dose of 100 μg each time.

[0107] In some embodiments, the PD-1 immune checkpoint inhibitor is administered to tumor-bearing mice at a uniform dose of 100 μg each time.

[0108] In some embodiments, EGFR-TKIs or pharmaceutically acceptable salts thereof are administered continuously for 10 days. In some embodiments, EGFR-TKIs or pharmaceutically acceptable salts thereof are administered orally once daily at a dose of 10 mg / kg each time for 10 consecutive days.

[0109] In some embodiments, the α-CD8 (CD8 inhibitor) is administered once every 3 days. In some embodiments, the α-CD8 is administered at a dose of 200 μg each time.

[0110] In an embodiment of the present invention, EGFR-TKIs afatinib and gefitinib were purchased from Selleck. Gal-9 inhibitor (α-Gal-9 antibody), immune checkpoint inhibitor (α-PD-1 antibody) and α-CD8 were all purchased from BioXCell.

[0111] Statistical methods

[0112] All statistical analyses were performed using GraphPad Prism statistical software. Data are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). Comparisons between two groups were performed using an unpaired two-tailed t-test or one-way analysis of variance. P values < 0.05 were considered statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0114] The present invention will be described in detail below with reference to the embodiments.

[0115] Example 1

[0116] Gal-9 expression is upregulated in tumor tissues of patients with non-small cell lung cancer after treatment with EGFR-TKIs.

[0117] Experimental Methods: Paired tumor samples were collected from 15 patients with EGFR-mutant non-small cell lung cancer (NSCLC) before and after first-line EGFR-TKI treatment and disease progression. Immunohistochemistry (IHC) staining was performed to analyze changes in Gal-9 expression in tumor tissues before and after treatment and its correlation with EGFR-TKI efficacy. Furthermore, using a publicly available single-cell sequencing dataset of NSCLC patients (5 patients before treatment and 2 patients after osimertinib resistance), we further analyzed changes in Gal-9 expression in different immune cell subsets in tumor tissues after EGFR-TKI treatment.

[0118] Research results: Immunohistochemistry results showed that the expression of Gal-9 in tumor tissues after EGFR-TKI treatment resistance was significantly increased compared with that before treatment ( Figure 1 A and Figure 1 B). Compared with patients with longer progression-free survival (PFS>12 months, Long Response, LR), patients with shorter survival (PFS<12 months, Short Response, SR) had a higher proportion of tumor tissues with high Gal-9 expression (defined as positive tumor area >40%) ( Figure 1 B). Further analysis of single-cell RNA sequencing data from NSCLC tumors before and after osimertinib treatment revealed that Gal-9 expression was specifically upregulated in myeloid cells, especially monocytes and dendritic cells (DCs), after TKI treatment, while PD-L1 expression levels did not change significantly ( Figure 1 C). Data from two independent cohorts showed that both Gal-9 mRNA and protein levels were significantly upregulated after EGFR-TKI treatment, and high Gal-9 expression was closely associated with clinical progression and drug resistance.

[0119] Example 2

[0120] Gal-9 expression was upregulated in tumor tissues and tumor-draining lymph nodes of mice after treatment with EGFR-TKIs.

[0121] Experimental Methods: BALB / c mice and C57BL / 6 mice (6-8 weeks old) were purchased from Beijing Weitong Lihua Co., Ltd. All animal experiments were approved by the Animal Experimental Ethics Committee of Tianjin Medical University General Hospital. Subcutaneous mouse homograft tumor models bearing CT26 colon cancer and Lewis Lung Carcinoma (LLC) were established. Seven days after treatment with the EGFR-TKI (afatinib), tumor tissue and tumor-draining lymph nodes (tdLN) were harvested for the following analyses: immunoblotting was used to determine the protein levels of P-EGFR, p-ERK, p-AKT, and Gal-9 in tumor tissue; single-cell suspensions were prepared, and flow cytometry was used to determine the expression of Gal-9 in myeloid cells, including dendritic cells, in tumor-draining lymph nodes and tumor tissue.

[0122] Experimental results: In the CT26 colon cancer mouse model, immunoblotting results showed that EGFR phosphorylation and downstream AKT / ERK signaling pathways were significantly weakened ( Figure 2 A), confirming the effective inhibition of EGFR pathway by afatinib; compared with the control group, the level of Gal-9 protein in tumor tissues of the afatinib-treated group was significantly increased ( Figure 2 B); Flow cytometry analysis showed that after afatinib treatment, Gal-9 expression was widely upregulated in dendritic cell subsets (including classical type 1 dendritic cells cDC1 and type 2 dendritic cells cDC2) in tumor-draining lymph nodes (tdLNs) compared with the control group ( Figure 2 C). In the LLC lung cancer mouse model, immunoblotting results showed that compared with the control group, the phosphorylation level of AKT downstream of EGFR in tumor tissues of the afatinib-treated group was significantly weakened, and the level of Gal-9 protein was significantly increased ( Figure 2D); Flow cytometry analysis showed that after afatinib treatment, Gal-9 expression was generally upregulated in tumor-infiltrating myeloid cell subsets (including macrophages, dendritic cells, mononuclear-derived dendritic cells (MoDCs), neutrophils, and monocytes) ( Figure 2 E) These results indicate that Gal-9 is a unique EGFR-TKI-responsive immune checkpoint molecule that is upregulated in both tumor cells and host myeloid cells.

[0123] Example 3

[0124] Combination therapy of Gal-9 inhibitors and EGFR-TKIs significantly inhibited tumor growth in a mouse model of colon cancer.

[0125] Experimental methods: BALB / c mice (6-8 weeks old) were purchased from Beijing Weitonglihua Company. All animal experiments were approved by the Animal Experiment Ethics Committee of Tianjin Medical University General Hospital. A total of 5×10 5 The CT26 cell suspension was inoculated into the inguinal region of mice to establish a subcutaneous homograft tumor model of CT26 colon cancer in mice. The mice were randomly divided into four groups: control group, Gal-9 inhibitor monotherapy group, EGFR-TKI monotherapy group (afatinib or gefitinib), and Gal-9 inhibitor and EGFR-TKI combination treatment group. The mice were given drugs by intraperitoneal injection or gavage according to the grouping. The tumor volume was measured every two days and the calculation formula was: tumor volume = 0.5 × length (L) × width (W) 2 The mice in each group were weighed every three days. According to animal ethics requirements, mice were euthanized when the tumor volume reached 2000 mm3.

[0126] Experimental results: The elevated Gal-9 levels induced by EGFR-TKI treatment suggest that it may serve as a potential therapeutic target. Therefore, we evaluated the antitumor efficacy of EGFR-TKI combined with Gal-9 inhibitors in the CT26 colon cancer mouse model ( Figure 3 A). The results showed that compared with afatinib or Gal-9 inhibitor alone, the combination of the two could induce significant tumor regression and prolong the survival of mice ( Figure 3 B- Figure 3 E), that is, the anti-tumor efficacy of the combination group was significantly better than that of the monotherapy group. The safety of the treatment was verified by the stability of body weight in each group ( Figure 3 F). This synergistic effect also applies to the first-generation EGFR-TKI. Compared with the single-drug group, gefitinib combined with Gal-9 inhibitor treatment can significantly improve tumor control effect ( Figure 3 G- Figure 3 H). The above experimental results show that in the colon cancer mouse model, the combination of Gal-9 inhibitors and EGFR-TKIs can significantly inhibit tumor growth and prolong mouse survival.

[0127] Example 4

[0128] Combination therapy of Gal-9 inhibitor, EGFR-TKI and α-PD1 significantly inhibited tumor growth in lung cancer mouse model.

[0129] Experimental methods: C57BL / 6 mice (6-8 weeks old) were purchased from Beijing Weitonglihua Company. All animal experiments were approved by the Animal Experiment Ethics Committee of Tianjin Medical University General Hospital. A total of 5×10 5 LLC cell suspension was inoculated into the inguinal area of mice to construct a subcutaneous homograft tumor model of LLC lung cancer mice. The mice were randomly divided into seven groups: control group, Gal-9 inhibitor monotherapy group, EGFR-TKI monotherapy group (afatinib), Gal-9 inhibitor and EGFR-TKI combination treatment group, Gal-9 inhibitor and PD-1 inhibitor combination treatment group, EGFR-TKI and PD-1 inhibitor combination treatment group, and Gal-9 inhibitor + EGFR-TKI + PD-1 inhibitor triple treatment group. Depending on the grouping, the mice were intraperitoneally injected or gavage-treated. The tumor volume was measured every two days, and the calculation formula was: tumor volume = 0.5 × length (L) × width (W) 2 The mice in each group were weighed every three days. According to animal ethics requirements, mice were euthanized when the tumor volume reached 2000 mm3.

[0130] Experimental results: We further evaluated the efficacy of the combined therapy in the low immunogenic LLC lung cancer model ( Figure 4 A), which is unresponsive to existing immune checkpoint blockade therapies. The results showed that in the LLC mouse tumor model, PD-1 inhibitors, Gal-9 inhibitors, and their combination did not show significant anti-tumor activity ( Figure 4 B). Notably, the combination of afatinib and a Gal-9 inhibitor significantly inhibited tumor growth and prolonged mouse survival, with superior efficacy compared to the combination of afatinib and a PD-1 inhibitor ( Figure 4 C- Figure 4 D). More importantly, compared with the two-drug combination group, the three-drug combination group (afatinib + Gal-9 inhibitor + PD-1 inhibitor) showed a more powerful tumor suppression effect ( Figure 4 C- Figure 4 D), and the body weight of mice in each group remained stable during the treatment period ( Figure 4 E), indicating a good safety profile. These experimental results demonstrate that the combination of EGFR-TKI and Gal-9 inhibitor significantly inhibits tumor growth and prolongs survival in a low-immunogenic mouse lung cancer model, and that the three-drug combination (EGFR-TKI + Gal-9 inhibitor + PD-1 inhibitor) is superior to the two-drug combination.

[0131] Example 5

[0132] The anti-tumor efficacy of the combination of Gal-9 inhibitors and EGFR-TKIs depends on CD8 + T cells.

[0133] Experimental methods: BALB / c and C57BL / 6 mice (6-8 weeks old) were purchased from Beijing Weitonglihua Company. All animal experiments were approved by the Animal Experiment Ethics Committee of Tianjin Medical University General Hospital. A total of 5×10 5 The CT26 cell suspension was inoculated into the inguinal region of mice to construct a subcutaneous homologous transplant tumor model of CT26 colon cancer mice. The mice were randomly divided into four groups: control group, Gal-9 inhibitor monotherapy group, EGFR-TKI monotherapy group (afatinib), and combined treatment group. The mice were intraperitoneally injected or gavaged according to the grouping. To study the effect of the combined treatment of Gal-9 inhibitor and EGFR-TKI on tumor-infiltrating T cells, the tumor tissue of the mice was removed on the 15th day after inoculation, and single cell suspension was prepared. CD8 + T cell activation.

[0134] To study CT8 + To investigate the contribution of CD8 T cells to tumor control, mice were randomly divided into three groups: a control group, a Gal-9 inhibitor and EGFR-TKI (afatinib) combination group, and a combination group plus a CD-8 inhibitor (α-CD8). On day 15 after inoculation, the spleens of the mice were removed, single cell suspensions were prepared, and flow cytometry was used to detect CD8 T cells. + T cell clearance. Tumor volume was measured every two days and calculated using the formula: tumor volume = 0.5 × length (L) × width (W) 2 In addition, the experiment was repeated in LLC tumor-bearing C57BL / 6 mice. According to animal ethics requirements, mice were euthanized when the tumor volume reached 2000 mm3.

[0135] Experimental results:

[0136] To elucidate the anti-tumor immunological mechanism of combined therapy with Gal-9 inhibitors and EGFR-TKIs, we performed flow cytometry on day 15 after inoculation of CT26 tumor-bearing BALB / c mice to analyze the effects of different treatment groups on tumor-infiltrating T cells. The results showed that compared with the single-drug group, the combination of the two drugs significantly increased the number of tumor-infiltrating CD8 + Functional killing of T cells, specifically granzyme B (GB) + CD8 + The proportion of T cells increased ( Figure 5A) The results showed that the combination of Gal-9 inhibitor and EGFR-TKI significantly promoted the proliferation of tumor-infiltrating CD8 + T cell activation.

[0137] Given that CD8 + T cells increased, we used anti-CD8 antibodies to eliminate this cell subset before treatment in the CT26 tumor model (experimental design see Figure 5 B) Flow cytometry analysis confirmed the presence of CD8 + Successful elimination of T cells ( Figure 5 C) As expected, CD8 + T cells completely abolished the effect of combination therapy on tumor growth ( Figure 5 D- Figure 5 F) and mouse survival ( Figure 5 G). Similar phenomenon was also observed in LLC tumor model ( Figure 5 H). The above results indicate that T cells (especially CD8 + T cells) are the core immune cell subset that mediates the efficacy of this combination therapy.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a Galectin-9 inhibitor and EGFR-TKIs in combination in the preparation of a drug for treating tumors.

2. The use according to claim 1, characterized in that: The tumor is a solid tumor; and / or, The tumor is lung cancer or colorectal cancer; and / or, The lung cancer is non-small cell lung cancer.

3. The use according to claim 1, characterized in that: The EGFR-TKIs are selected from afatinib, gefitinib, and osimertinib, and the Galectin-9 inhibitor is an anti-Galectin-9 antibody.

4. A pharmaceutical composition for treating tumors, characterized in that: Including Galectin-9 inhibitors, EGFR-TKIs or pharmaceutically acceptable salts thereof.

5. The pharmaceutical composition according to claim 4, characterized in that: The tumor is a solid tumor; and / or, The tumor is lung cancer or colorectal cancer; and / or, The lung cancer is non-small cell lung cancer; and / or, The Galectin-9 inhibitor is an anti-Galectin-9 antibody, and the EGFR-TKIs or a pharmaceutically acceptable salt thereof are administered simultaneously or sequentially.

6. Use of a Galectin-9 inhibitor, EGFR-TKIs and an immune checkpoint inhibitor in combination in the preparation of a drug for treating tumors; and / or, The immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor; and / or, The immune checkpoint inhibitor is an anti-PD-1 antibody; and / or, The tumor is a solid tumor; and / or, The tumor is lung cancer or colorectal cancer; and / or, The lung cancer is non-small cell lung cancer.

7. A pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition includes a Galectin-9 inhibitor, EGFR-TKIs or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor.

8. The pharmaceutical composition for treating tumors according to claim 7, characterized in that: The immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor; and / or, The immune checkpoint inhibitor is an anti-PD-1 antibody; and / or, The tumor is a solid tumor; and / or, The tumor is lung cancer or colorectal cancer; and / or, The lung cancer is non-small cell lung cancer.

9. The pharmaceutical composition for treating tumors according to claim 7, characterized in that: The Galectin-9 inhibitor, the EGFR-TKIs or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor are administered simultaneously or sequentially.

10. The pharmaceutical composition for treating tumors according to claim 7, characterized in that: The Galectin-9 inhibitor is a liquid preparation; and / or, The dosage form of EGFR-TKIs or pharmaceutically acceptable salts thereof is a suspension preparation; and / or, Immune checkpoint inhibitors are liquid preparations.

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