Application of sodium cromoglycate in preparation of pharmaceutical composition for promoting breast cancer immunotherapy

Abstract

The invention discloses an application of sodium cromoglycate in preparation of a pharmaceutical composition for promoting immunotherapy of breast cancer, and belongs to the technical field of tumor therapy, and the breast cancer is breast cancer with immunotherapy failure or immunotherapy drug-resistant breast cancer. The invention provides an application of sensitizing an immunotherapy drug in treatment of breast cancer by inducing antigen presenting related functional molecules on antigen presenting mast cells to be up-regulated by using sodium cromoglycate, remarkably enhancing antigen presenting mast cell mediated T cell anti-tumor immunity and inhibiting tumor growth resistant to immunotherapy. The sensitization immunotherapy curative effect of a mouse experiment shows that the sodium cromoglycate combined immunotherapy inhibits the growth of mouse breast tumor resistant to immunotherapy, and the prospective II-stage clinical test proves that the immunotherapy combined sodium cromoglycate has the curative effect and safety on a patient suffering from the triple negative breast cancer resistant to immunotherapy, and the immunotherapy combined sodium cromoglycate can be used for treating the triple negative breast cancer resistant to immunotherapy. The preset main curative effect end point is reached, and the adverse reaction is controllable.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and particularly to the application of sodium cromoglycate in the preparation of a pharmaceutical composition for promoting breast cancer immunotherapy. Background Art

[0002] Breast cancer is the most common malignant tumor among women, seriously endangering people's lives and health. Triple-negative breast cancers (TNBC) refer to a type of breast cancer in which the expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are all negative, accounting for 15%-20% of all breast cancers. Compared with breast cancer patients with positive hormone receptors, TNBC patients are often older, prone to visceral metastasis, have a higher risk of postoperative recurrence, and a poorer prognosis. Since TNBC lacks the expression of key receptors, patients cannot benefit from targeted therapy, and chemotherapy remains the main treatment method at present. In recent years, immunotherapy represented by PD-1 / PD-L1 immune-checkpoint inhibitors (ICI) has brought breakthrough progress to the treatment of breast cancer, especially TNBC. However, many patients are resistant to ICI treatment. At the same time, many patients progress after immunotherapy, resulting in no significant effect in some patients after ICI treatment. How to use other methods in combination with ICI to enhance sensitivity and reverse drug resistance has become an important clinical issue.

[0003] Existing studies have found some methods to enhance immunotherapy in preclinical models, such as combining drugs targeting tumor metabolism, combining multiple ICIs, combining cytokine therapy, combining tumor vaccines, combining radiotherapy, combining anti-angiogenic drugs, etc. However, current methods for enhancing immunotherapy all have their limitations, such as large drug toxic and side effects, failure to achieve a sensitizing effect in clinical trials, or related drugs not being approved for clinical use. In addition, there is still a blank in how to select a more precise treatment method to reverse drug resistance after immunotherapy resistance. Therefore, exploring and developing immunotherapy sensitization methods with clinical accessibility and low drug toxic and side effects has important clinical significance.

[0004] Sodium cromoglycate is a mast cell stabilizer that can inhibit the release of pro-allergic active substances by mast cells, thereby playing an anti-allergic role in allergic diseases such as asthma. Mast cells are an important part of the immune system. Based on single-cell sequencing and multi-omics analysis, researchers at Fudan University Shanghai Cancer Center identified a subset of mast cells related to the benefit of immunotherapy - antigen-presenting mast cells (apMC). Sodium cromoglycate has good safety and rare side effects and has been widely used clinically, but its effects on breast tumor biology and therapeutic behavior are still unknown. This invention further explores the functional role of sodium cromoglycate in vivo. Sodium cromoglycate can slow down the tumor growth rate, and at the same time induce the upregulation of major histocompatibility complex (MHC) and co-stimulatory molecules on antigen-presenting mast cells, significantly enhancing the anti-tumor immunity of T cells mediated by antigen-presenting mast cells and inhibiting the growth of tumors resistant to immunotherapy. This invention provides a new effective treatment plan for breast cancer patients, especially those with triple-negative breast cancer who have failed immunotherapy or are resistant to immunotherapy, and has potential clinical promotion value. Summary of the Invention

[0005] The present invention provides an application of using sodium cromoglycate to induce the upregulation of antigen-presenting related functional molecules on antigen-presenting mast cells, significantly enhancing the anti-tumor immunity of T cells mediated by antigen-presenting mast cells, inhibiting the growth of tumors resistant to immunotherapy, so as to enhance the sensitivity of immunotherapy drugs in the treatment of breast cancer, especially solving the problem of poor treatment effect of triple-negative breast cancer with failed immunotherapy or triple-negative breast cancer resistant to immunotherapy.

[0006] The technical solution adopted by the present invention to achieve the above purpose is: the application of sodium cromoglycate in the preparation of a drug composition for promoting breast cancer immunotherapy.

[0007] Preferably, the breast cancer is breast cancer with failed immunotherapy or breast cancer resistant to immunotherapy.

[0008] Preferably, the breast cancer is triple-negative breast cancer.

[0009] The present invention discloses the application of sodium cromoglycate combined with immunotherapy drugs in the preparation of drugs for treating breast cancer.

[0010] Preferably, the immunotherapy drugs include anti-PD-1 antibody and anti-PD-L1 antibody.

[0011] The present invention discloses a drug composition for treating breast cancer, comprising sodium cromoglycate and a pharmaceutically acceptable carrier.

[0012] Compared with the prior art, the beneficial effects that can be achieved by at least one of the above technical solutions adopted in the embodiments of this specification at least include:

[0013] (1) By using sodium cromoglycate, the present invention induces the upregulation of major histocompatibility complex and costimulatory molecules on antigen-presenting mast cells, significantly enhances the T cell anti-tumor immunity mediated by antigen-presenting mast cells, inhibits the growth of tumors resistant to immunotherapy, and increases the sensitivity of triple-negative breast cancer that fails immunotherapy or triple-negative breast cancer resistant to immunotherapy to immunotherapy;

[0014] (2) The efficacy of sensitizing immunotherapy in mouse experiments shows that the combination of sodium cromoglycate and immunotherapy inhibits the growth of mouse breast tumors resistant to immunotherapy, and a prospective phase II clinical trial proves that the combination of immunotherapy and sodium cromoglycate for triple-negative breast cancer patients resistant to immunotherapy reaches the preset primary efficacy endpoint, and the adverse reactions are controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained according to the drawings without creative efforts.

[0016] Figure 1 It is a diagram of the clustering and population analysis of mast cell single-cell transcriptomes of triple-negative breast cancer patients. Among them, (A) is a cell population diagram after dimensionality reduction of mast cell single-cell transcriptome data by the uniform manifold approximation and projection method (UMAP); (B) is a heat map of the expression of marker genes of each major mast cell subset;

[0017] Figure 2 It is a diagram of the correlation analysis between apMC infiltration and the immune microenvironment in the FUSCC-TNBC multi-omics cohort. Among them, (A) is the correlation between apMC infiltration and the expression of immune stimulatory molecules and immune checkpoint molecules; (B) is the correlation between the abundance of each immune cell and apMC infiltration;

[0018] Figure 3 It is a flow cytometry analysis diagram of effector T cells in high and low apMC patients. Among them, (A) is the percentage of CD8 + T in all cells in high and low apMC patients; (B) is the percentage of cytotoxic GZMB + CD8 + T in CD8 + T cells in high and low apMC patients; (C) is the percentage of tumor-reactive PD-1 + CD8 +Percentage of T cells that are CD8 + Percentage of T cells;

[0019] Figure 4 Immunohistochemical staining analysis diagram of the tumor immune microenvironment of breast cancer patients. Among them, (A) is the immunohistochemical staining result diagram of apMC, and the cells double positive for Tryptase and CD74 are identified as apMC cells; (B) is the correlation analysis diagram of the immunohistochemical staining abundance of apMC and the expression of CD8, stromal TIL (tumor-infiltrating lymphocytes), and PD-L1 (TC: percentage of PD-L1 positive in tumor cells; IC: percentage of PD-L1 positive in immune cells); (C) is the spatial distribution diagram of apMC and tertiary lymphoid structures (TLS) / lymphoid aggregates (LA); (D) is the apMC infiltration percentage diagram of patients with and without TLS / LA-like structures;

[0020] Figure 5 Results diagram of in-situ co-transplantation animal experiment of apMC-breast tumors in mast cell-deficient mice. Among them, (A) is the co-transplanted tumor growth curve diagram of the E0771 cell line; (B) is the co-transplanted tumor growth curve diagram of the AT3 cell line;

[0021] Figure 6 Results diagram of animal experiments on mice with conditional knockout of mast cell-specific CD74. Among them, (A) is the schematic diagram of the construction of conditional knockout mice; (B) is the expression of CD74 in mast cells and control cells in conditional knockout mice and control mice; (C) is the tumor growth curve and tumor weight of E0771 transplanted tumors in conditional knockout mice and control mice; (D) is the treatment-responsive CXCL13 in conditional knockout mice and control mice + CD4 + T cells and treatment-responsive CXCL13 + GZMB + PD-1 + CD8 + Immunofluorescence analysis diagram of CD8 T cells; (E) is GZMB in conditional knockout mice and control mice + Number of effector cells and Perforin+ effector cells per high-power field of view;

[0022] Figure 7 Results diagram of the experiment of using cromolyn sodium combined with sensitizing anti-PD-1 monoclonal antibody in a mouse model of immunotherapy-resistant breast tumors. Among them, (A) is the tumor growth curve and tumor weight diagram after using cromolyn sodium combined with anti-PD-1 monoclonal antibody in a 4T1 immunotherapy-resistant mouse model; (B) is immunohistochemical staining analysis of CD8 + T cells and GZMB+ Effector cell infiltration; (C) shows the tumor growth curve and tumor weight graph after treatment with cromolyn sodium combined with anti-PD-1 monoclonal antibody in a 66cl4 immunotherapy-resistant mouse model; (D) shows immunohistochemical staining analysis of CD8 + T cells and GZMB + Effector cell infiltration; (E) shows the tumor growth curve and tumor weight graph after treatment with cromolyn sodium combined with anti-PD-1 monoclonal antibody in an AT3 immunotherapy-resistant mouse model; (F) shows immunohistochemical staining analysis of CD8 + T cells and GZMB + Effector cell infiltration;

[0023] Figure 8 It is the effect diagram of cromolyn sodium reversing immunotherapy resistance in TNBC patients. Among them, (A) is the efficacy evaluation of 10 immunotherapy-resistant patients after receiving cromolyn sodium + immunotherapy; (B) is the maximum percentage change in the long diameter of the tumor compared to the pre-treatment baseline level of the patients; (C) is the percentage change in the long diameter of the tumor compared to the pre-treatment baseline level of the patients over the treatment time; (D) is the immunohistochemical staining analysis diagram of the infiltration of mast cells and antigen-presenting mast cells in the tissue sections of the patients before treatment. Detailed implementation manners

[0024] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0025] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0026] It should be noted that the following description relates to various aspects of embodiments within the scope of the appended claims. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art should understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement a device and / or practice a method. Additionally, this device can be implemented and this method can be practiced using other structures and / or functionality in addition to one or more of the aspects set forth herein.

[0027] Example 1: Analyzing the mast cell subsets in human triple-negative breast cancer through a single-cell atlas of triple-negative breast cancer

[0028] Mast cells are an evolutionarily conserved myeloid cell type, mainly distributed around barrier tissues that contact the external environment and are an important part of the body's immune system. However, in tumors, the function of mast cells remains unconfirmed. In breast cancer, its prognostic value for patients still shows differences, indicating its obvious heterogeneity. To systematically reveal the heterogeneity of mast cells in human triple-negative breast cancer, tumor samples from 26 untreated triple-negative breast cancer patients were first collected for single-cell transcriptome sequencing (scRNA-seq). Using the Seurat package in R language, a collection of 1064 high-quality mast cells sorted based on typical cell markers (KIT and CPA3) was obtained. UMAP dimensionality reduction clustering divided the sorted 1064 high-quality mast cells into three distinct subsets, each subset having specific markers, named after the most prominent marker, namely NDUFA4 + mast cells with upregulated glycolysis and energy metabolism, ZFP36 + mast cells with a potential allergic phenotype, CD74 + antigen-presenting mast cells. Figure 1 The data indicate the heterogeneity of mast cells in triple-negative breast cancer, among which CD74 + mast cells are enriched in antigen presentation-related pathways, suggesting their potential important role in regulating the immune microenvironment and responding to immunotherapy.

[0029] Example 2: Antigen-presenting mast cells are related to the immune therapy-responsive microenvironment

[0030] To understand the molecular characteristics and biological functions of antigen-presenting mast cells in TNBC patients, the association between antigen-presenting mast cells and the activated immune microenvironment was analyzed by immunofluorescence flow cytometry, omics analysis, and immunohistochemistry. In the FUSCC-TNBC multi-omics cohort (n = 360), the antigen-presenting mast cell infiltration score was calculated using the antigen-presenting mast cell signature gene set extracted from single-cell transcriptome data, and the infiltration of 22 cell populations in the tumor microenvironment of each patient was analyzed using CIBERSORTx. From Figure 2 the results, it can be seen that antigen-presenting mast cells are associated with higher expression of immune-stimulatory molecules, immune checkpoint molecules, and higher CD8 + T cell infiltration. Further, immunofluorescence flow cytometry analysis was performed using fresh tumor tissue samples from breast cancer patients. Specifically, the patient tumor tissues were digested into single-cell suspensions, and each immune cell subset was labeled with targeted antibodies to determine the abundance of each immune cell subset in the patient tumor immune microenvironment. From Figure 3 it can be known that an increase in CD8 + T cells was observed in tumors rich in antigen-presenting mast cells, along with higher cytotoxicity (GZMB+) and tumor reactivity (PD-1+) markers. Further, immunohistochemical staining analysis was used to evaluate the tumor immune microenvironment characteristics of breast cancer patients. Specifically, the patient tumor tissues were sectioned, and each immune cell subset was labeled with targeted antibodies, and finally, the distribution of each immune cell subset in the patient tumor tissues was observed under a microscope. Figure 4 showed that antigen-presenting mast cell infiltration was positively correlated with CD8, stromal TIL (tumor-infiltrating lymphocytes), PD-L1, and tertiary lymphoid structures (TLS) / lymphoid aggregates (LA).

[0031] All of the above data indicate that the infiltration of antigen-presenting mast cells in TNBC suggests overall activation of anti-tumor immunity and induces an immune therapy response through interaction with T cells.

[0032] Example 3 Antigen-presenting mast cells enhance T cell function through antigen presentation and thus sensitize immune therapy

[0033] To verify the sensitizing effect of antigen-presenting mast cells, antigen-presenting mast cells and other mast cells were sorted from mouse mammary tumors, and antigen-presenting mast cells (1×10 5 cells) or other mast cells (1×10 5 cells) were separately mixed with 1×10 6 E0771 cells and 3×10 5 AT3 triple-negative breast cancer mouse cell lines and injected in situ into mast cell-deficient B6.Cg-Kit W-shIn the fourth pair of fat pads of mice, when the tumor volume reached 50 - 100 mm 3 intraperitoneal injection of 10 mg / kg of mouse monoclonal antibody (BioXcell, catalog number BE0146) was started twice a week. Figure 5 The results showed that co - transplantation of antigen - presenting mast cells significantly improved the efficacy of anti - PD - 1 monoclonal antibody in inhibiting tumor growth in E0771 and AT3 tumors, while co - transplantation of other mast cells did not have the same effect.

[0034] CD74 is an important functional molecule in the MHC complex and is involved in antigen presentation and cross - presentation functions. To verify whether the impaired immunotherapy effect in mice lacking antigen - presenting mast cells is mediated by the antigen - presenting function of mast cells, a conditional knockout mouse model (Cpa3 CreERT2 Cd74 fl / fl ) was constructed. Tamoxifen at 120 mg / kg was administered continuously for 5 days to specifically ablate the key antigen - presenting molecule CD74 in mast cells. As Figure 6 could be seen, conditional knockout of CD74 in mast cells was found to greatly disrupt the efficacy of anti - PD - 1 in the E0771 mouse tumorigenesis model, resulting in a decrease in immunotherapeutic reactive T cells and a reduction in T - cell cytotoxicity.

[0035] The above data indicate that antigen presentation by intratumoral mast cells plays a crucial role in triggering anti - tumor T - cell immune responses and enhancing the efficacy of anti - PD - 1.

[0036] Example 4 Therapeutic effect of cromolyn sodium + immunotherapy in triple - negative breast cancer resistant to previous immunotherapy

[0037] (1) Establishment of an immunotherapy - resistant mouse model: 3×10 5 immunotherapy - resistant 4T1, 66cl4, and AT3 triple - negative breast cancer cells were respectively injected into the fourth pair of fat pads of female mice to construct an immunotherapy - resistant mouse model.

[0038] (2) Drug administration treatment: The drug administration treatment was divided into the following 4 groups, namely:

[0039] i) Control group: Intraperitoneal injection of IgG isotype control antibody into mice twice a week, with a dosage of 10 mg / kg; Intraperitoneal injection of control solvent into mice three times a week, with a dosage of 20 mg / kg;

[0040] ii) Anti - PD - 1 antibody treatment group: Intraperitoneal injection of anti - PD - 1 monoclonal antibody into mice twice a week, with a dosage of 10 mg / kg; Intraperitoneal injection of control solvent into mice three times a week, with a dosage of 20 mg / kg;

[0041] iii) Cromolyn sodium treatment group: Mice were intraperitoneally injected with IgG isotype control antibody twice a week at a dosage of 10 mg / kg; mice were intraperitoneally injected with cromolyn sodium three times a week at a dosage of 20 mg / kg;

[0042] iv) Cromolyn sodium + anti-PD-1 antibody treatment group: Mice were intraperitoneally injected with anti-PD-1 monoclonal antibody twice a week at a dosage of 10 mg / kg; mice were intraperitoneally injected with cromolyn sodium three times a week at a dosage of 20 mg / kg.

[0043] (3) Experimental results

[0044] It can be seen from Figure 7 that in the three immune therapy-resistant mouse models, there was no significant difference in tumor size between the anti-PD-1 antibody treatment group and the control group, further verifying the resistance of these three models to immune therapy. However, when anti-PD-1 antibody was combined with cromolyn sodium, tumor growth was significantly inhibited. In addition, through immunohistochemical staining analysis, it was found that when anti-PD-1 antibody was combined with cromolyn sodium, CD8 + T cells and GZMB + effector cell infiltration increased significantly. The above results suggest that cromolyn sodium can effectively sensitize anti-PD-1 therapy and reverse immune therapy resistance.

[0045] Example 5 Cromolyn sodium reverses immune therapy resistance in TNBC patients

[0046] Ten metastatic TNBC patients who had progressed after anti-PD-1 treatment (immune therapy-resistant) were enrolled in a phase II clinical trial (NCT05076682). Patients were administered 5 mg of cromolyn sodium in each nostril four times a day. On days 1 and 15, patients were intravenously injected with 200 mg of camrelizumab, and on days 1, 8, and 15, patients were intravenously injected with 100 mg / m 2 albumin-bound paclitaxel. Each four-week period was a treatment cycle. The primary study endpoint was the objective tumor response rate.

[0047] Figure 8 The results showed that among the 10 enrolled patients, the target lesion size decreased in 7 patients compared to the baseline. Confirmed partial or complete tumor remission was observed in 5 patients, and the confirmed objective response rate was 50.0%, reaching the preset primary efficacy endpoint, and there were no new serious adverse reactions. Immunohistochemical staining was performed on pre-treatment sections of the patients, and it was found that compared with patients with stable disease or disease progression, patients who achieved an objective response had a higher baseline level of apMC infiltration, which further demonstrated that cromolyn sodium promoted the efficacy of immune therapy and reversed the immune therapy resistance of patients by regulating the function of apMC.

[0048] The above embodiments are only used to illustrate the technical concept and features of the present invention, and the purpose is to enable those of ordinary skill in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the essence of the content of the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of sodium cromoglycate in the preparation of a pharmaceutical composition for promoting breast cancer immunotherapy.

2. The use according to claim 1, characterized in that The breast cancer is breast cancer that has failed immunotherapy or is immunotherapy-resistant breast cancer.

3. The use according to claim 1, characterized in that The breast cancer is triple-negative breast cancer.

4. Application of sodium cromoglycate combined with immunotherapy drugs in the preparation of drugs for the treatment of breast cancer.

5. The use according to claim 4, characterized in that The immunotherapy drugs include anti-PD-1 antibodies and anti-PD-L1 antibodies.

6. A pharmaceutical composition for treating breast cancer, characterized in that: The invention comprises sodium cromoglycate and a pharmaceutically acceptable carrier.

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