Use of recombinant protein CCL11 in the treatment of malignant pleural effusions

Abstract

Disclosed in the present invention is the use of a recombinant protein CCL11 in the treatment of malignant pleural effusions. Specifically, the present invention provides a method for treating and / or relieving malignant pleural effusions by means of intrapleural injection of the recombinant protein CCL11. The present invention first found that CCL11 can relieve MPEs by means of increasing eosinophilic granulocytes in the thoracic cavity. An MPE animal model is constructed, and intrapleural injection of the recombinant protein CCL11 remarkably increases eosinophilic granulocytes in the thoracic cavity, and meanwhile, obviously relieves MPEs.

Description

Application of recombinant protein CCL11 in the treatment of malignant pleural effusion Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of recombinant protein CCL11 in treating malignant pleural effusion. Background Art

[0002] Malignant pleural effusions (MPE) are a common complication in cancer patients. MPE is caused by malignant pleural mesothelioma originating in the pleural cavity or metastasis from tumors in other locations, such as the lungs, gastrointestinal tract, and breast. MPE is a poor prognostic sign in patients with malignant tumors. The median survival of MPE patients is 3-12 months, with poor prognosis and high mortality rates, posing significant challenges to clinical treatment.

[0003] MPE patients primarily experience progressively worsening dyspnea, chest pain, and dry cough, severely impacting their quality of life. Current treatments for MPE primarily include closed chest drainage and chemical / surgical pleurodesis, both of which are palliative and fail to significantly improve patient survival.

[0004] In summary, patients with MPE complications have a short median survival, poor prognosis, and high mortality rate, and currently lack effective therapeutic drugs. Therefore, new drugs that effectively treat and alleviate MPE complications are urgently needed in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a medicine and its application for effectively treating and alleviating MPE complications.

[0006] In a first aspect of the present invention, there is provided a use of a recombinant protein CCL11 or a nucleic acid molecule encoding the protein CCL11 for preparing a medicament for treating and / or alleviating malignant pleural effusion (MPE) in a subject.

[0007] In another preferred embodiment, the subject has one or more MPE typing features selected from the following group:

[0008] (a) The amount of CCL11 protein is significantly increased in pleural effusion; and / or

[0009] (b) a significant decrease in the number of eosinophils in the pleural effusion; and / or

[0010] (c) expression of a marker selected from the group consisting of Cxcl13, Ccl3, Ccl4, or a combination thereof is significantly upregulated in eosinophils; and / or

[0011] (d) The expression of a marker selected from the group consisting of S100a9, Ear1, Ear6, or a combination thereof is significantly downregulated in eosinophils.

[0012] In another preferred embodiment, the "significant increase" refers to the ratio of the amount or concentration C1 of CCL11 protein in the pleural effusion of the subject to the amount or concentration C0 of CCL11 protein in the pleural transudate (i.e., C1 / C0) being ≥1.5, preferably ≥2.0, and more preferably ≥3.0.

[0013] In another preferred embodiment, the concentration of CCL11 protein in the pleural effusion is ≥800 pg / mL, preferably ≥1000 pg / mL; more preferably ≥1100 pg / mL.

[0014] In another preferred embodiment, the "significant decrease in the number of eosinophils" refers to the ratio of the number or concentration B1 of eosinophils in the subject's pleural effusion to the number or concentration B0 of eosinophils in the subject's own peripheral blood (i.e., B1 / B0) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0015] In another preferred embodiment, the "significant decrease in the number of eosinophils" refers to the ratio of the number or concentration D1 of eosinophils in the pleural effusion of the subject to the number or concentration D0 of eosinophils in the pleural transudate (i.e., D1 / D0) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0016] In another preferred embodiment, the "significantly upregulated expression" refers to the ratio of the expression level (mRNA or protein) M1 of the marker in the eosinophils of the subject to the expression level (mRNA or protein) M0 of the marker in the eosinophils in the subject's own bone marrow (i.e., M1 / M0), which is ≥1.5, preferably ≥2.0, and more preferably ≥3.0.

[0017] In another preferred embodiment, the "significantly downregulated expression" refers to the ratio of the expression level (mRNA or protein) N1 of the marker in the eosinophils of the subject to the expression level (mRNA or protein) N0 of the marker in the eosinophils in the subject's own bone marrow (i.e., N1 / N0), ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0018] In another preferred embodiment, the subject has one or more auxiliary typing features selected from the following group:

[0019] (Y1) The number of eosinophils in peripheral blood is normal or almost normal; and / or

[0020] (Y2) The number of T cells in peripheral blood is normal or almost normal.

[0021] In another preferred embodiment, the subject's immune T cell function is intact.

[0022] In another preferred embodiment, the recombinant protein CCL11 recruits eosinophils into the thoracic cavity, thereby alleviating MPE through eosinophils.

[0023] In another preferred embodiment, the recombinant protein CCL11 is exogenous recombinant CCL11 (rCCL11).

[0024] In another preferred embodiment, the recombinant protein CCL11 is of human or mouse origin.

[0025] In another preferred embodiment, the MPE refers to pleural effusion caused by an in situ tumor or a metastatic tumor.

[0026] In another preferred embodiment, the in situ tumor is selected from the group consisting of lung cancer, breast cancer, or a combination thereof.

[0027] In another preferred embodiment, the metastatic tumor is selected from the group consisting of colon cancer, gastric cancer, large intestine cancer, pancreatic cancer, or a combination thereof.

[0028] In another preferred embodiment, the expression level of Ki67 on CD3+ T cells in the pleural effusion of the subject is low; and / or the expression level of CD69 on CD3+ T cells in the pleural effusion of the subject is low.

[0029] In another preferred embodiment, the "low expression level of Ki67" refers to the ratio of the expression level E1 of Ki67 on CD3+T cells in the pleural effusion of the subject to the expression level E0 of Ki67 on CD3+T cells in the subject's own peripheral blood (i.e., E1 / E0), which is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0030] In another preferred embodiment, the “low expression of CD69” refers to the ratio of the expression level F1 of CD69 on CD3+ T cells in the subject's pleural effusion to the expression level F0 of CD69 on CD3+ T cells in the subject's own peripheral blood (i.e., F1 / F0), which is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0031] In another preferred embodiment, the pleural effusion of the subject has low levels of NK cells, DC cells and mononuclear macrophages.

[0032] In another preferred embodiment, the “low content of NK cells, DC cells, and monocyte macrophages” refers to the ratio Z1 of the number of NK cells (numerator) in the pleural effusion of the subject to the number of NK cells (denominator) in the pleural transudate of the subject ≤ 0.75, preferably ≤ 0.5, and more preferably ≤ 0.3;

[0033] The ratio Z2 of the number of DC cells (numerator) to the number of NK cells in the pleural transudate (denominator) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3; and

[0034] The ratio Z3 of the number of mononuclear macrophages (numerator) to the number of NK cells in the pleural effusion (denominator) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0035] In another preferred embodiment, the pleural effusion of the subject has low levels of γδT cells, Th1 cells, and Th17 cells.

[0036] In another preferred embodiment, the “low content of γδT cells, Th1 cells, and Th17 cells” refers to the ratio Z4 of the number of γδT cells (numerator) in the pleural effusion of the subject to the number of γδT cells (denominator) in the pleural transudate of the subject is ≤0.75, preferably ≤0.5, and more preferably ≤0.3;

[0037] The ratio Z5 of the number of Th1 cells (numerator) to the number of Th1 cells in the pleural transudate (denominator) is ≤0.75, preferably ≤0.5, more preferably ≤0.3; and

[0038] The ratio Z6 of the number of Th17 cells (numerator) to the number of Th17 cells in the pleural effusion (denominator) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0039] In another preferred embodiment, the subject has a high content of Treg cells in the pleural effusion.

[0040] In another preferred embodiment, the "high Treg cell content" refers to the ratio of the number of Treg cells in the pleural effusion of the subject (numerator) to the number of Treg cells in the pleural transudate (denominator) is ≥1.2, preferably ≥1.8, and more preferably ≥2.5.

[0041] In another preferred embodiment, the treatment and / or relief refers to a reduction in the volume of pleural tumors and a reduction in the amount of pleural effusion.

[0042] In another preferred embodiment, the reduction in pleural effusion volume refers to the restoration of the subject's pleural effusion to normal pleural fluid content (ie, no pleural effusion).

[0043] In another preferred embodiment, the pleural effusion of the subject has low levels of γδT cells, Th1 cells and Th17 cells, while high levels of Treg cells.

[0044] In another preferred embodiment, the treatment and / or relief refers to that the content of γδT cells, Th1 cells and / or Th17 cells in the subject's pleural lavage fluid is increased by ≥50% compared with that before treatment; preferably, ≥100%.

[0045] In another preferred embodiment, the treatment and / or relief refers to that the Treg cell content in the subject's pleural lavage fluid is 80% of the Treg cell content in the pleural lavage fluid before treatment; preferably, 50%.

[0046] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising (a) a recombinant protein CCL11 or a nucleic acid molecule encoding protein CCL11 as a first active ingredient; (b) an immunostimulant as a second active ingredient; and (c) optionally a pharmaceutically acceptable carrier.

[0047] In another preferred embodiment, the immunostimulant is selected from the group consisting of interleukin-2, immune checkpoint inhibitors, CAR-T cells, or a combination thereof.

[0048] In another preferred embodiment, the immune checkpoint inhibitor is selected from the group consisting of CTLA4, PD-1, PD-L1, or a combination thereof.

[0049] In another preferred embodiment, the pharmaceutical composition is an injection.

[0050] In another preferred embodiment, the pharmaceutical composition is in the form of an intrathoracic administration.

[0051] In the third aspect of the present invention, there is provided use of the pharmaceutical composition according to the second aspect of the present invention in the preparation of a medicament for treating and / or alleviating MPE.

[0052] In another preferred embodiment, the drug is an injection.

[0053] In another preferred embodiment, the drug is in the form of an intrathoracic administration dosage form.

[0054] In a fourth aspect of the present invention, a method for preventing and / or treating and / or alleviating MPE is provided, comprising administering a therapeutically effective amount of recombinant protein CCL11 or a nucleic acid molecule encoding protein CCL11 to a subject in need thereof.

[0055] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0056] In another preferred embodiment, the subject is a mammal; preferably a mouse, rat, rabbit, monkey or human.

[0057] In another preferred embodiment, the expression level of Ki67 on CD3+ T cells in the pleural effusion of the subject is low; and / or the expression level of CD69 on CD3+ T cells in the pleural effusion of the subject is low.

[0058] In another preferred embodiment, the "low expression level of Ki67" refers to the ratio of the expression level E1 of Ki67 on CD3+T cells in the pleural effusion of the subject to the expression level E0 of Ki67 on CD3+T cells in the subject's own peripheral blood (i.e., E1 / E0), which is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0059] In another preferred embodiment, the “low expression of CD69” refers to the ratio of the expression level F1 of CD69 on CD3+ T cells in the subject's pleural effusion to the expression level F0 of CD69 on CD3+ T cells in the subject's own peripheral blood (i.e., F1 / F0), which is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0060] In another preferred embodiment, the eosinophil function of the subject is impaired or suppressed.

[0061] In another preferred embodiment, the pleural effusion of the subject has low levels of γδT cells, Th1 cells, and Th17 cells.

[0062] In another preferred embodiment, the “low content of γδT cells, Th1 cells, and Th17 cells” refers to the ratio Z4 of the number of γδT cells (numerator) in the pleural effusion of the subject to the number of γδT cells (denominator) in the pleural transudate of the subject is ≤0.75, preferably ≤0.5, and more preferably ≤0.3;

[0063] The ratio Z5 of the number of Th1 cells (numerator) to the number of Th1 cells in the pleural transudate (denominator) is ≤0.75, preferably ≤0.5, more preferably ≤0.3; and

[0064] The ratio Z6 of the number of Th17 cells (numerator) to the number of Th17 cells in the pleural effusion (denominator) is ≤0.75, preferably ≤0.5, and more preferably ≤0.3.

[0065] In another preferred embodiment, the subject has a high content of Treg cells in the pleural effusion.

[0066] In another preferred embodiment, the "high Treg cell content" refers to the ratio of the number of Treg cells in the pleural effusion of the subject (numerator) to the number of Treg cells in the pleural transudate (denominator) is ≥1.2, preferably ≥1.8, and more preferably ≥2.5.

[0067] In another preferred embodiment, the treatment and / or relief refers to a reduction in the volume of pleural tumors and a reduction in the amount of pleural effusion.

[0068] In another preferred embodiment, the pleural effusion of the subject has low levels of γδT cells, Th1 cells, and Th17 cells, while high levels of Treg cells.

[0069] In another preferred embodiment, the treatment and / or relief refers to that the content of γδT cells, Th1 cells and Th17 cells in the subject's pleural lavage fluid is increased by ≥50% compared with that before treatment; preferably, ≥100%.

[0070] In another preferred embodiment, the treatment and / or relief refers to that the Treg cell content in the subject's pleural lavage fluid is 80% of the Treg cell content in the pleural lavage fluid before treatment; preferably, 50%.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 shows the status of eosinophils in the pleural lavage fluid after LLC modeling.

[0073] Panel A shows representative confocal microscopy images of eosinophils in pleural lavage fluid on day 3, stained with DAPI and EPX; scale bar: 20 μm.

[0074] Panel B shows the flow cytometry gating strategy for mouse eosinophils. Eosinophils were labeled with DAPI-CD45+Gr1- / lowCD11c-CD11b+F4 / 80+SiglecF+SSChi;

[0075] Panel C shows representative flow cytometric images of eosinophils in pleural lavage fluid at different time points after intrapleural injection of LLC;

[0076] Figure D shows the proportion of white blood cells at different time points after intrathoracic injection of LLC.

[0077] Figure 2 shows the cytokine profiles in pleural lavage fluid (PLF) of mice with MPE. Panel A shows a heatmap of cytokine microarray differential analysis results (Eos-null group, n=3; WT group, n=3). Panel B shows CCL11 concentrations in mouse peripheral blood (PB) and PLF (PLF), n=3-7. Data in A were analyzed for differential expression using the limma R package, and statistically analyzed in B using two-way ANOVA. P<0.05 indicates a statistically significant difference, and ns indicates no statistically significant difference. In the figure, pleural lavage fluid represents pleural lavage fluid. Panel C shows the transcriptional profile of eosinophils in PLF.

[0078] Figure 3 shows that rmCCL11 promotes eosinophil recruitment and alleviates MPE. Figure A shows the in vivo experimental design; Figure B shows a representative image of eosinophil FC; Figure C shows the percentage of eosinophils / total cells. The experimental groups are as follows: WT mice + PBS group: n = 3, WT mice + rmCCL11 group: n = 3, LLC-MPE mice + PBS group: n = 3, LLC-MPE mice + rmCCL11 group: n = 3; Figure D shows a representative image of in vivo imaging of LLC-MPE mice. The unit of bioluminescence is photons / second / square centimeter / steradian (photons / second / cm 2 / steradian); where Eos-null indicates transgenic mice lacking eosinophils. Panel E shows the statistical graph of small animal in vivo imaging, grouped as follows: LLC-MPE mice + PBS group: n = 5, LLC-MPE mice + rmCCL11 group: n = 4, LLC-MPE Eos-null mice + PBS group: n = 5, LLC-MPE Eos-null mice + rmCCL11 group: n = 3; Panel F shows the statistical graph of pleural effusion volume, grouped as in Panel E; Panel G shows the statistical graph of mouse survival time, grouped as follows: LLC-MPE mice + PBS group: n = 8, LLC-MPE mice + rmCCL11 group: n = 11, LLC-MPE Eos-null mice + PBS group: n = 8, LLC-MPE Eos-null mice + rmCCL11 group: n = 12. Figures C, E, and F were statistically analyzed using two-way ANOVA, and survival analysis was performed using the Kaplan-Meier method. P < 0.05 indicated a statistically significant difference, and ns indicated no statistically significant difference.

[0079] Figure 4 shows that eosinophil reinfusion did not affect pleural tumor growth and pleural effusion formation in MPE nude mice. Figure A shows a representative image of eosinophils in the pleural lavage fluid of LLC-MPE nude mice on day 2; Figure B shows a statistical graph of the absolute number of eosinophils in the pleural cavity of the LLC-MPE model at different time points, with n = 3 nude mice at each time point; Figure C shows a representative image of in vivo imaging of LLC-MPE nude mice, with bioluminescence measured in photons / second / cm2 / steradian; Figure D shows a statistical graph of in vivo imaging of small animals, with n = 9 for the PBS group and n = 9 for the eosinophil group; Figure E shows a statistical graph of pleural effusion volume, with the same grouping as in Figure D; Figure F shows a representative image of in vivo imaging of LLC-MPE nude mice, with bioluminescence measured in photons / second / cm2 / steradian; Figure G shows a statistical graph of in vivo imaging of small animals, with n = 5 for the PBS group and n = 8 for the rmCCL11 group; and Figure H shows a statistical graph of pleural effusion volume, with the same grouping as in Figure G. Figures D, E, G, and H were statistically analyzed using unpaired t-tests. P < 0.05 indicated statistically significant differences, and ns indicated no statistically significant differences.

[0080] Figure 5 shows the changes in CD4+T cells and CD8+T cells in the chest cavity of MPE mice over time, where A is a representative FC picture of CD4+T cells and CD8+T cells; B is the change in the absolute value of CD4+T cells in the chest cavity of LLC-MPE mice, n=3; C is the change in the absolute value of CD8+T cells in the chest cavity of LLC-MPE mice, n=3; dpi represents days post intrapleural injection.

[0081] Figure 6 shows immune cell phenotype analysis in the pleural microenvironment of WT and Eos-null mice after intrathoracic injection of LLC. Panel A shows a tSNE plot of CD45+ cells in the pleural cavities of WT and Eos-null mice; Panel B shows the frequency of immune cell subsets; Panel C shows a tSNE plot of CD3+ T cells in the pleural cavities of WT and Eos-null mice; Panel D shows the frequency of T cell subsets; and Panel E shows the expression of Ki67 and CD69 on CD3+ T cells in the two groups. Data are shown as mean ± standard error.

[0082] Figure 7 shows the effects of eosinophils on tumor cells and tumor tissues. Figures AD show the evaluation of eosinophil killing of tumor cells. Figure A shows representative DAPI flow cytometric histograms of LLC, 4T1, and MC38 tumor cells co-cultured with eosinophils at varying ratios in a non-contact co-culture system. Figure B shows the survival rate of LLC, 4T1, and MC38 tumor cells in a non-contact co-culture system. Figure C shows the gating strategy for the contact co-culture system (top). Figure D shows representative 7-AAD flow cytometric histograms of LLC, 4T1, and MC38 tumor cells in contact co-culture (bottom). Figure 4 shows the survival rate of LLC, 4T1, and MC38 tumor cells in a contact co-culture system. Figure 5 E shows pleural tumor tissue from the LLC-MPE model of WT and Eos-null mice, using immunohistochemical staining for cleaved-caspase 3 and Ki-67. Figure E shows representative IHC images of cleaved-caspase 3 and Ki-67 in pleural tumors. Scale bar: 100 μm. Panel F shows the number of cleaved-caspase 3+ cells per mm². Panel G shows the number of Ki-67+ cells per mm². Data are expressed as mean ± standard error. DETAILED DESCRIPTION

[0083] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the recombinant protein CCL11 (or a corresponding preparation) can be used to treat malignant pleural effusion (MPE). Experiments have shown that upon administration of exogenous CCL11 protein, CCL11 recruits eosinophils. These recruited eosinophils and T cells work together to effectively treat and / or alleviate MPE by, among other things, improving the tumor microenvironment. This is the basis for the present invention.

[0084] the term

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art.

[0086] As used herein, the term "active ingredient of the present invention" refers to the recombinant protein CCL11, a nucleic acid molecule encoding the recombinant protein CCL11 (such as mRNA, nucleic acid vector), or a combination thereof.

[0087] As used herein, the term "composition of the present invention" refers to a composition, especially a pharmaceutical composition, containing the active ingredients of the present invention.

[0088] As used herein, the term "MPE" is used interchangeably with "MPE complication" to refer to pleural effusion caused by malignant pleural mesothelioma originating in the pleural cavity or metastasis of tumors from other sites such as the lungs, gastrointestinal tract, and breast to the pleural cavity.

[0089] As used herein, the term "pleural transudate" refers to pleural effusion caused by heart failure or the like, which is different from malignant pleural effusion and can serve as a negative control for malignant pleural effusion.

[0090] Recombinant protein CCL11 or its coding sequence

[0091] CCL11 is an important chemokine in the human body and a member of the CC chemokine family. CCL11 can bind to the chemokine receptor CCR3 expressed on the surface of eosinophils.

[0092] In the present invention, preferred CCL11 proteins are derived from mammals, such as humans, rodents, or non-human primates, such as mouse, rabbit, monkey, pig, or human CCL11 proteins. The CCL11 proteins of the present invention include wild-type and mutant CCL11 proteins, as long as the mutant protein retains the function of recruiting eosinophils.

[0093] In addition, the recombinant CCL11 of the present invention also includes active fragments or fusion proteins of the CCL11 protein, as long as the active fragment or fusion protein retains the function of recruiting eosinophils. In the present invention, retaining the function of recruiting eosinophils means having ≥50%, preferably ≥70%, ≥90% (e.g., 50-300%, or 70-150%) of the eosinophil recruitment function of the wild-type CCL11 protein.

[0094] Recombinant protein CCL11 treats and / or alleviates MPE

[0095] The inventors have pioneered the use of the recombinant protein CCL11 for treating and / or alleviating complications of MPE. Specifically, the inventors have demonstrated through experiments that recombinant CCL11 recruits eosinophils and reshapes the immune microenvironment of MPE, thereby achieving treatment or alleviation of MPE.

[0096] On the one hand, as the number of eosinophils increased in the pleural effusion, the concentration of CCL11 in the pleural effusion of MPE mice also increased, and there was a difference between the WT group and the Eos-null group, suggesting that CCL11 is involved in the recruitment of eosinophils in this process.

[0097] On the other hand, results from intrathoracic injection of rmCCL11 in MPE model mice showed that recombinant CCL11 protein efficiently recruited eosinophils, thereby alleviating MPE in the presence of eosinophils. Furthermore, the effect of CCL11 protein on MPE was weakened in the absence of eosinophils, suggesting that CCL11 protein alleviates MPE complications through eosinophils.

[0098] Therefore, in the present invention, recombinant CCL11 protein can be administered to reach the chest cavity, thereby recruiting eosinophils in the chest cavity or surrounding tissues, reshaping the MPE immune microenvironment, and thereby achieving the treatment or relief of MPE complications.

[0099] Pharmaceutical compositions and applications

[0100] The present invention also provides a pharmaceutical composition containing the recombinant protein CCL11, which can be used to treat and / or alleviate MPE.

[0101] Preferably, the pharmaceutical composition of the present invention may further comprise other active ingredients, such as additional immunostimulants, anticancer drugs, or a combination thereof.

[0102] Generally, the pharmaceutical composition of the present invention comprises (a) a recombinant protein CCL11 or a nucleic acid molecule encoding the recombinant protein CCL11 as an active ingredient; and (b) a pharmaceutically acceptable carrier.

[0103] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0104] As used herein, the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0105] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be compatible with the route of administration. The pharmaceutical compositions of the present invention are available in the form of injections, transdermal formulations, and sustained-release formulations. For example, they can be prepared using conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0106] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0107] In the present invention, representative administration methods include (but are not limited to): intrathoracic administration, such as intrathoracic injection.

[0108] Typically, when the pharmaceutical composition of the present invention is administered intrathoracically or intravenously, the average daily dose for a 60 kg body weight subject (human) is generally 1-1000 mg, preferably 5-500 mg, and more preferably 10-250 mg. The daily dose can be administered once, twice, or multiple times, or once every 1, 2, 3, 4, 5, or 6 days.

[0109] In addition, the dosage can also be adjusted based on the volume of malignant pleural effusion. When the pharmaceutical composition of the present invention is administered intrapleurally or intravenously, the typical daily dose is 0.1-10 mg CCL11 protein / 100 mL, preferably 0.5-5 mg CCL11 protein / 100 mL. The daily dose can be administered once, twice, or multiple times, or once every 1, 2, 3, 4, 5, or 6 days.

[0110] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0111] Compared with the prior art, the main advantages of the present invention include:

[0112] (a) For the first time, we found that the expression of CCL11 protein was significantly correlated with eosinophil infiltration and eosinophil improvement in MPE complications.

[0113] (b) The present invention proposes and verifies for the first time that intrathoracic injection of recombinant CCL11 protein can significantly and rapidly alleviate MPE, and that this process is mainly achieved through eosinophils.

[0114] (c) The present invention demonstrates that, when administered with recombinant CCL11 protein, eosinophils effectively treat or alleviate MPE mainly through multiple effects such as reshaping the MPE immune microenvironment.

[0115] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, the test reagents (such as rmCCL11 protein) and kits in the examples were commercially available.

[0116] General experimental methods

[0117] I. Establishment of MPE model mice

[0118] The mice to be treated with MPE were given an intraperitoneal injection of 1% sodium pentobarbital solution for general anesthesia. When the mice entered a deep anesthesia state, they were fixed on the experimental table, the hair on the right chest of the mice was shaved with a shaver, and the skin was disinfected with 75% alcohol.

[0119] Aspirate 50 μL of 1.5 × 10 5 A single-cell suspension of 100 tumor cells (LLC cells, MC38 cells, and 4T1 cells) in PBS was prepared. An incision was made in the right anterolateral chest wall at the level of the xiphoid process of the mouse to expose the iliac crest. The single-cell suspension was then injected into the mouse's pleural cavity through the intercostal space under direct visualization. After removing the syringe, the injection site was disinfected. The muscle and epidermal layers were sutured with surgical suture, and the mouse was placed in a warm cage. This was designated the LLC-MPE model mouse. The normal control group received 50 μL of PBS solution as the injection solution. All other procedures were the same as those for the LLC-MPE model group.

[0120] For mice, the criteria for judging MPE complications are as follows: when the volume of pleural effusion in mice is ≥100 μL, the mice are considered to have MPE (fluid aspiration can be performed); when the volume of pleural effusion in mice is <100 μL, the mice are considered to not have MPE (pleural lavage can be performed).

[0121] II. Mouse Handling Methods

[0122] On day 14, mice were anesthetized with an intraperitoneal injection of 0.2 mL of 1.5% sodium pentobarbital solution and sacrificed. Venous blood was drawn from the retroorbital vein into heparinized tubes. The mice were secured on a laboratory bench. The chest and abdomen were disinfected with 75% alcohol. A T-shaped incision was made in the midline of the abdomen, exposing the diaphragm without disrupting the integrity of the thoracic cavity.

[0123] If the mouse has not yet formed pleural effusion, inject 1 mL of PBS into the pleural cavity through the diaphragm. After standing for 30 seconds to 1 minute, withdraw the PBS and place it in a sterile 1.5 mL EP tube for subsequent experimental testing. If bloody pleural effusion can be seen through the diaphragm, use a 1 mL syringe to aspirate the pleural effusion and place it in a sterile 1.5 mL EP tube. Use a micropipette to accurately measure its volume and record the data. Use straight scissors to cut the sternum, open the chest cavity, and use surgical forceps to remove the tumor tissue in the mouse chest cavity and place it in 4% formaldehyde tissue fixative (the fixative should be at least 20 times the volume of the tumor tissue).

[0124] III. Pleural permeability test

[0125] MPE model mice were injected with 0.1 mL of Evans Blue working solution (10 mg / mL) through the tail vein. The mice were euthanized 30 minutes later, and the pleural lavage fluid or pleural effusion and corresponding peripheral blood (PB) were collected. After centrifugation at 400 g for 10 minutes and 4°C, the supernatant was collected and diluted appropriately. The OD value of each sample was measured at a wavelength of 630 nm using a microplate reader. The ratio of the OD value of the pleural lavage fluid or pleural effusion supernatant to the OD value of the corresponding PB supernatant of each mouse was calculated.

[0126] IV. Small Animal In Vivo Imaging

[0127] Mouse autoluminescence was detected using the PerkinElmer IVIS Spectrum in vivo imaging system. Beforehand, click the Living Imaging software to start the program and initialize the IVIS system. During initialization, the temperature status light in the IVIS Acquisition Control Panel will be red. Imaging can only be performed when the temperature status light turns green.

[0128] MPE mice were intraperitoneally injected with D-luciferin sodium salt (15 mg / mL, 100 μL / mouse). Timer was started after 6 minutes of isoflurane gas anesthesia (generally, mice were fully anesthetized within 3 minutes). At 10 minutes, the anesthetized mice were placed in the imaging chamber of the PerkinElmer Small Animal Live Imager and the door was closed. The imaging mode was set to luminescent, and optimal exposure parameters were adjusted for each mouse. After standardizing these parameters across all experiments, images were acquired. After acquisition, quantitative values ​​within the ROI region were obtained for subsequent statistical analysis.

[0129] V. Plotting of mouse survival curves

[0130] The MPE model mice were numbered and their vital signs were observed at the same time every 2 days starting from the intrathoracic injection of tumor cells. Death was considered to have occurred when the mouse's breathing and heartbeat stopped. The number and time of death of the mouse were recorded, and a mouse survival curve was generated.

[0131] VI. CCL11 Administration and Treatment

[0132] MPE model mice were injected intrathoracically with recombinant mouse CCL11 starting on day 4 at a dose of 0.5 μg / mouse. Mouse CCL11 was diluted in 100 μL of PBS and injected intrathoracically into the MPE mice every two days. A control group of mice received 100 μL of PBS intrathoracically. The number of eosinophils in the pleural cavity of the mice was monitored on day 7. Pleural tumor burden and pleural effusion volume were measured on day 14, and mouse survival was monitored (see Figure 3, Panel A).

[0133] Example 1: Testing the cytokine profile of pleural lavage fluid in MPE mice

[0134] First, an LLC-MPE model was constructed in WT mice, and it was found that in the early stage of MPE formation, the number of eosinophils in the chest cavity increased significantly. The results are shown in Figure 1, indicating that eosinophils are involved in the occurrence and development of MPE.

[0135] Next, an LLC-MPE model was established in Eos-null and WT littermate mice. Pleural lavage fluid was obtained on day 2 and analyzed using a Mouse Cytokine Array GS4000 cytokine array. As shown in Figure 2A and Table 1, CCL11 levels were significantly elevated in Eos-null mice, while other eosinophil-related factors, such as IL-4, IL-5, IL-13, IL-33, and TSLP, showed no significant changes.

[0136] Table 1. Cytokines associated with eosinophil function measured using cytokine arrays

[0137] As shown in Table 1, the protein chip detection results showed that among the many cytokines related to eosinophil function, the level of CCL11 in the pleural lavage fluid showed extremely significant changes.

[0138] Furthermore, ELISA testing further confirmed that CCL11 levels in pleural lavage fluid increased significantly by at least 2-fold (Figure 2B). Furthermore, CCL11 levels in peripheral blood did not show significant changes. This suggests that CCL11 levels in pleural lavage fluid can serve as a biomarker for the classification of MPE complications suitable for the present invention's therapy (administration of CCL11 protein).

[0139] In addition, RNA sequencing of eosinophils in the pleural effusion of LLC-MPE model mice (before administration) revealed upregulation of markers such as Cxcl13, Ccl3, and Ccl4, and downregulation of markers such as S100a9, Ear1, and Ear6.

[0140] Example 2: CCL11 promotes eosinophil recruitment and alleviates MPE

[0141] To examine the role of eosinophils, an MPE model was constructed using eosinophil-deficient mice (Eos-null mice obtained by transgenic means) and their littermate control WT mice.

[0142] Referring to Figure 3A, MPE modeling and drug administration were performed on WT and Eos-null mice. After intrathoracic injection of CCL11 on day 7, the number of eosinophils was measured by flow cytometry on day 8. Figure 3B shows a representative flow cytometry diagram. As shown in Figure 3B, the experimental results show that rmCCL11 administration can recruit more eosinophils than the control group rmCCL11. In particular, the number of eosinophils increased by more than 2 times in the CCL11 group.

[0143] The present invention constructed an LLC-MPE model in WT and Eos-null mice. Starting on the 4th day, mouse recombinant CCL11 (0.5 μg / mouse) was injected into the thoracic cavity, and the injection was repeated every 2 days. The mice were killed on the 14th day, the pleural effusion and tumor tissue of the mice were collected, and the survival of the mice was calculated. As shown in Figures 3C-D, compared with the control group, the eosinophils in the experimental group were significantly increased; as shown in Figures 3E-G, after 14 days, the pleural tumor burden of the WT mouse treatment group was significantly reduced, the pleural effusion volume was significantly decreased, and the survival time was significantly prolonged, while no significant MPE relief was observed in the Eos-null treatment group. This shows that rmCCL11 can promote the recruitment of eosinophils into the thoracic cavity in vivo and inhibit the formation of MPE through eosinophils.

[0144] Example 3: Infusion of eosinophils into MPE nude mice does not affect pleural tumor growth and pleural effusion formation

[0145] Previous studies have shown that activated eosinophils in the TME significantly improve T cell infiltration into tumors, enhancing tumor rejection and survival. In the absence of T cells, eosinophils do not exhibit substantial anti-tumor activity.

[0146] First, we examined the pleural microenvironment of MPE mice. The results, shown in Figures 4A-B, show that after LLC cell stimulation, a large number of eosinophils accumulated in the pleural cavity of nude mice, indicating that T cell depletion does not affect the migration of eosinophils into the pleural cavity.

[0147] In nude mice, the LLC-MPE model was constructed (the procedure was the same as for the LLC-MPE model, with the same dosage and time as above). After eosinophils were reinfused into the tail vein of the LLC-MPE nude mice, as shown in Figures 4C-E, the pleural tumors did not decrease, nor did the pleural effusion volume. This suggests that T cell depletion does not affect the migration of eosinophils into the pleural cavity. However, as shown in Figures 4F-H, after intrathoracic injection of rmCCL11 into the nude mice, the pleural tumors did not decrease, nor did the pleural effusion volume.

[0148] The above results show that the presence or absence of T cells does not affect the recruitment of eosinophils by CCL11. Eosinophils will still migrate to the thoracic cavity, but eosinophils alone cannot alleviate / treat MPE, indicating that the treatment / alleviation effect of eosinophils on MPE requires the participation of T cells.

[0149] Example 4: Changes of CD4+ T cells and CD8+ T cells in the chest cavity of MPE mice over time

[0150] This example further analyzes the timing of the emergence of adaptive immune cells in the MPE thoracic microenvironment.

[0151] The operation was the same as that of LLC-MPE model. Flow cytometry was used to detect CD4 + T cells and CD8 + The number of T cells.

[0152] The results are shown in Figure 5B-C. The numbers of CD4+ T cells and CD8+ T cells gradually increased on the 7th day.

[0153] Example 5: CD45+ cell cyTOF results

[0154] According to the results of Example 4, pleural lavage fluid was obtained on the 7th day of the LLC-MPE model of WT mice and Eos-null mice, and after in vitro stimulation with phorbol ester (PMA) / ionomycin (Ion) / Brefeldin A (BFA) for 4 hours, mass spectrometry flow detection was performed. The acquired data were circled using Flowjo to select single, living, and complete CD45+ immune cells, and the CD45+ cells were subjected to non-biased automatic dimensionality reduction cluster analysis. 32 cell subsets were analyzed and manually annotated. By merging similar cells, the differences between the major cell types in the WT group and the Eos-null group were statistically analyzed.

[0155] The results are shown in Figures 6A-B . In the Eos-null group, almost no eosinophils were detected, there was no difference in B cells and T cells, and NK cells, DC cells and monocytes and macrophages decreased significantly.

[0156] FlowJo was used to further identify CD3+ T cells for T cell subset analysis. Automatic dimensionality reduction clustering analysis of CD3+ T cells without bias revealed the distribution of eight subpopulations.

[0157] Statistical analysis results are shown in Figures 6C-D. In the Eos-null group, γδT cells, Th1 cells, and Th17 cells were significantly decreased, while Treg cells were significantly increased. These results are consistent with previous literature. Previous literature suggests that γδT cells, Th1 cells, and Th17 cells have a protective effect against MPE, while Treg cells promote the development of MPE. Notably, no Th2 cells were detected in these results.

[0158] As shown in Figure 6E , decreased expression of Ki67 and CD69 was found on CD3 + T cells in the Eos-null group, indicating that both the proliferation and activation abilities of CD3 + T cells were decreased.

[0159] The immune cell phenotypes in the thoracic microenvironment of WT mice and Eos-null mice after intrathoracic injection of LLC were analyzed. The results showed that the absence of eosinophils would affect the immune cell composition and T cell function of the thoracic microenvironment of MPE mice, thereby promoting the occurrence and development of MPE.

[0160] Example 6: Eosinophils themselves do not affect the proliferation of tumor cells or tissues

[0161] Under contact and non-contact conditions, eosinophils were co-cultured with three different tumor cells in different proportions to observe the effects of eosinophils on tumor cells and tumor tissues.

[0162] Extraction of eosinophils from the peripheral blood of Il5Tg mice: Granulocytes were first separated from the peripheral blood of Il5Tg mice using Percoll, and then eosinophils were isolated using immunomagnetic bead sorting. Antibodies used in the immunomagnetic bead sorting experiment included: biotin anti-mouse CD4, CD8a, B220, NK1.1, Ly6G, CD115, CD11c, Ter119 antibodies and anti-biotin magnetic beads. FC analysis of the sorted eosinophils confirmed a purity of >95%. Non-contact culture system for eosinophils and tumor cells: Using a Transwell chamber with a 4μm pore size, 500μL of 5×10 5 LLC, 4T1, and MC38 cells at 400 μL / mL were seeded in the lower chamber. Eosinophils suspended in 400 μL of RPMI-1640 medium were seeded in the upper chamber at varying ratios of eosinophils to tumor cells (0:1, 1:1, 2:1, and 5:1). After a 4-hour incubation, tumor cell death was detected using DAPI.

[0163] As shown in Figures 7A and 7B , under non-contact co-culture conditions, eosinophils did not produce a killing effect on the three tumor cells (LLC cells, MC38 cells, and 4T1 cells);

[0164] Eosinophil-tumor cell contact culture system: LLC, 4T1, and MC38 cells were pretreated with CFSE according to the manufacturer's instructions and then seeded in 96-well plates at a concentration of 1 × 10 per well. 4 Eosinophils were then co-cultured with LLC, 4T1, and MC38 cells at varying ratios (0:1, 1:1, 2:1, and 5:1). After 4 hours of co-culture, cell death was detected using 7-AAD staining, and flow cytometry analysis was performed using CFSE to distinguish tumor cells from eosinophils.

[0165] As shown in Figure 7C and Figure 7D, after co-culture under contact conditions, eosinophils did not kill the three tumor cells (LLC cells, MC38 cells, and 4T1 cells) nor inhibit the proliferation of tumor cells.

[0166] Pleural tumors were obtained from WT mice and Eos-null mice on day 14 of the LLC-MPE model, and the expressions of cleaved-caspase3 and Ki67 in the tumor tissues were detected by immunohistochemistry.

[0167] As shown in Figures 7E and 7F, eosinophils themselves did not affect the proliferation of tumor tissue.

[0168] discuss

[0169] The pleural cavity is a potential space between the visceral and parietal pleura. In a normal person, there is 5 to 15 mL of fluid in the pleural cavity, which acts as a lubricant during breathing.

[0170] MPE is caused by tumor cells blocking lymphatic vessels, which hinders fluid reflux. Furthermore, tumor cells release vasoactive substances such as vascular endothelial growth factor (VEGF) and angiopoietin, which increase vascular permeability and fluid production. Recent evidence suggests that the formation of MPE is regulated by interactions between tumor cells, host blood vessels, and the immune system, leading to vascular hyperpermeability, angiogenesis, and inflammatory responses.

[0171] The tumor microenvironment (TME) of patients with MPE has a strong immunosuppressive effect and is rich in immunosuppressive cells and mediators. Mediators in MPE can drive immune cells to a hypofunctional state. Therefore, reprogramming immune cells or removing immunosuppressive cells from the TME can enhance the immunotherapy effect of MPE. Intrapleural injection of anti-programmed cell death protein 1 (PD-1) monoclonal antibodies and chimeric antigen receptor T-cells (CAR-T) can control the formation of pleural effusions and the growth of pleural tumors, suggesting that targeting the local immune system may be an ideal strategy for treating MPE and is currently a hot topic in MPE research.

[0172] A variety of chemokines can induce eosinophil migration into the tumor microenvironment (TME), and these chemokines are regulated by dynamic changes within the specific TME. The IL-5 and eotaxin-CCR3 signaling pathways play an important role in eosinophil recruitment in normal mucosal tissues, allergic inflammation, and tumors. In necrotic and hypoxic tumor types, damage-associated molecular patterns (DAMPs) such as HMGB1 and IL-33 promote eosinophil recruitment. The microbiome also influences eosinophil recruitment in the TME. Eosinophil recruitment pathways in the TME also include CCL3 and CCL5 (CCR1 ligands).

[0173] The inventors unexpectedly discovered for the first time that the recombinant protein CCL11 (or a corresponding preparation) can be used to treat complications of malignant pleural effusion (MPE). Experiments have shown that after administration of exogenous CCL11 protein, CCL11 recruits eosinophils. These recruited eosinophils and immune T cells work together to effectively treat and / or alleviate MPE complications by, among other things, improving the tumor microenvironment.

[0174] All documents mentioned herein are incorporated herein by reference as if each document were individually incorporated by reference. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the appended claims.

Claims

1. Use of a recombinant protein CCL11 or a nucleic acid molecule encoding protein CCL11, characterized in that, For the preparation of a medicament for treating and / or alleviating malignant pleural effusion (MPE) in a subject.

2. The use according to claim 1, characterized in that, The subject has one or more MPE classification characteristics selected from the group consisting of: (a) In the pleural effusion, the amount of CCL11 protein is significantly increased; and / or (b) In the pleural effusion, the number of eosinophils is significantly decreased; and / or (c) In eosinophils, the expression of a marker selected from the group consisting of Cxcl13, Ccl3, Ccl4, or a combination thereof is significantly upregulated; and / or (d) In eosinophils, the expression of a marker selected from the group consisting of S100a9, Ear1, Ear6, or a combination thereof is significantly downregulated.

3. The use according to claim 1, characterized in that, The MPE refers to pleural effusion caused by in-situ tumors or metastatic tumors.

4. The use according to claim 1, characterized in that, The expression level of Ki67 on CD3+ T cells in the pleural effusion of the subject is low; and / or the expression level of CD69 on CD3+ T cells in the pleural effusion of the subject is low.

5. The use according to claim 1, characterized in that, The content of NK cells, DC cells, and mononuclear macrophages in the pleural effusion of the subject is low.

6. The use according to claim 1, characterized in that, The content of γδT cells, Th1 cells, and Th17 cells in the pleural effusion of the subject is low.

7. The use according to claim 1, wherein The content of Treg cells in the pleural effusion of the subject is high.

8. Use of a pharmaceutical composition in the preparation of a medicament for treating and / or alleviating MPE, characterized in that, The pharmaceutical composition comprises (a) recombinant protein CCL11 or a nucleic acid molecule encoding protein CCL11 as a first active ingredient; (b) an immunopotentiator as a second active ingredient; and (c) optionally a pharmaceutically acceptable carrier.

9. The use according to claim 8, characterized in that, The pharmaceutical composition is in a dosage form for intrathoracic administration.

10. A method for preventing and / or treating and / or alleviating MPE, characterized in that, Administer a therapeutically effective amount of recombinant protein CCL11 or a nucleic acid molecule encoding protein CCL11 to a subject in need.

Images