Treatment or prevention of chronic lung allograft dysfunction by inhibition of met

Abstract

The present application provides the use of carmotinib or a pharmaceutically acceptable salt thereof as a MET inhibitor for treating or preventing chronic lung allograft dysfunction (CLAD) in a subject. The subject has received a lung transplant, particularly an allogeneic transplanted lung, and preferably, the subject is diagnosed with pleuroparenchymal fibroelastosis. The present application also provides a method of treating or preventing CLAD or improving long-term survival of a lung transplant subject.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and specifically relates to a novel use of MET inhibitors for the treatment or prevention of chronic lung allograft dysfunction (CLAD) in subjects. Background Technology

[0002] Solid organ transplantation has revolutionized the treatment of end-stage organ failure, but chronic allogeneic graft dysfunction remains a major obstacle to long-term graft survival. Among all solid organ transplants, lung transplantation exhibits the worst outcomes, with a five-year survival rate of less than 60% (Graham et al., 2022), primarily due to the high incidence and poor prognosis of chronic lung allogeneic graft dysfunction (CLAD). CLAD is a progressive disease characterized by an initial inflammatory phase of tissue damage triggered by an allogeneic immune response, followed by extensive pathological tissue remodeling, ultimately leading to irreversible graft failure. Despite advances in immunosuppressive therapy, CLAD remains a significant unresolved clinical challenge, with a median survival of less than five years after diagnosis, highlighting the urgent need for new mechanistic insights and treatment strategies.

[0003] The pathological changes in CLAD involve a series of tissue remodeling processes, including airway remodeling, alveolar fibrosis, and pleural fibrosis. Based on these pathological features, CLAD is divided into two main subtypes: bronchiolitis obliterans syndrome (BOS), which involves only airway remodeling; and restrictive allograft syndrome (RAS), in which fibrosis extends beyond the airways, involving both alveoli and pleural tissue. Patients with RAS generally have a worse prognosis than those with BOS, and pleural fibrosis is associated with accelerated disease progression and significantly poorer outcomes. Although pleural fibrosis plays a crucial role in driving disease progression, the underlying cellular and molecular mechanisms of this process remain unclear, highlighting a critical gap in our understanding of the mechanisms of tissue remodeling in CLAD.

[0004] The pathogenesis of CLAD has traditionally been attributed to a chronic alloimmune response, where the receptor immune system targets donor lung tissue. Current treatment strategies focus on suppressing lymphocytes through calcineurin inhibitors (such as tacrolimus), in vitro photosensitization therapy, and whole-body lymphatic irradiation. However, these strategies have proven insufficient to halt disease progression or reduce pleural remodeling, suggesting that non-allogeneic immune mechanisms, in addition to alloimmune responses, may be involved in the pathological process of advanced CLAD. Summary of the Invention

[0005] Chronic lung allogeneic graft dysfunction (CLAD) remains a major obstacle to long-term survival in lung transplant recipients, and current immunosuppressive therapies have failed to halt its progression. This invention reveals a previously unknown immune-receptor-derived mesothelial cell interaction driving CLAD pathogenesis. Studies using orthotopic mouse lung transplantation models and human CLAD specimens demonstrate that during CLAD pathogenesis, recipient-derived pleural mesothelial cells migrate to the surface of the allogeneic lung and evade allogeneic immune surveillance. Activated basophils in the allogeneic lung immune microenvironment produce HGF, which promotes recipient mesothelial cell proliferation and invasion of the transplanted lung parenchyma via MET signaling. This pathological pathway accelerates pleural fibrosis and CLAD progression in the allogeneic lung. Pharmacological inhibition of HGF / MET signaling disrupts this signaling axis, significantly blocking disease progression. These findings reveal the crucial role of recipient-derived pleural mesothelial cells in CLAD tissue remodeling and highlight the therapeutic potential of combining targeted tissue remodeling cellular responses with immunosuppression. This invention provides a variety of strategies to improve long-term transplant outcomes beyond targeting allogeneic immune responses.

[0006] Specifically, in this invention, the inventors discovered a previously unknown immune-receptor mesothelial cell interaction that has advanced the understanding of CLAD. Using an orthotopic mouse lung transplantation model and human CLAD specimens, the inventors identified a pathological pathway between receptor-derived mesothelial cells and basophils. Specifically, basophils secrete HGF, which promotes the proliferation and invasion of receptor mesothelial cells via MET signaling. Pharmacological inhibition of MET disrupts this signaling axis, significantly reducing pleural fibrosis.

[0007] By revealing the dynamic shift from allogeneic immune-mediated damage to a late-stage pathological process regulated by basophils and receptor mesothelial cells, this invention provides mechanistic insights and opens pathways for combined therapies targeting allogeneic immune responses and tissue remodeling pathways.

[0008] Therefore, in a first aspect, the present invention provides a MET inhibitor for the treatment or prevention of chronic lung allogeneic graft dysfunction (CLAD) in lung transplant recipients (especially allogeneic lung transplant recipients).

[0009] In some implementations, the subject is diagnosed with pleuroparenchymal fibroelastosis.

[0010] In some implementations, the treatment includes relieving, delaying, or preventing the progression of CLAD, or relieving or reducing pleural fibrosis.

[0011] In some embodiments, the inhibitor is any one of an antibody or its antigen-binding fragment, antigen-binding protein, or small molecule compound that binds to and reduces the activity of a target; or it is an siRNA / shRNA or CRISPR system that inhibits the expression and / or activity of a target, wherein the target is MET. The inhibitor can inhibit signal transduction via the HGF / MET pathway.

[0012] In some implementations, the inhibitor is a MET inhibitor.

[0013] In some embodiments, the MET inhibitor is an antibody that binds to and neutralizes MET.

[0014] In some embodiments, the antibody is prepared through genetic engineering. In some embodiments, the monoclonal antibody is human or humanized.

[0015] In some embodiments, the MET inhibitor comprises onartuzumab, emibetuzumab, 5D5, SU5416, SU11274, ARQ197 (tivantinib), GSK / 1363089 / XL880 (foretinib), XL184 (cabozotinib), HMPL-504 / AZD6094 / volitinib (Savolitinib), MSC2156119J (EMD) 1214063, Tepotinib, LY2801653 Merestinib, AMG337, INCB28060 Capmatinib, AMG458, PF-04217903, PF-02341066 Crizotinib, E7050 Golvatinib, MK-2461, BMS-777607, JNJ-38877605, SOMG-833, or any combination thereof.

[0016] In some implementations, the MET inhibitor is carmatinib.

[0017] In a second aspect, the present invention provides a method for treating or preventing chronic lung allogeneic graft dysfunction (CLAD) in lung transplant recipients (especially allogeneic lung transplant recipients), the method comprising administering a therapeutically effective amount of a MET inhibitor to the recipient.

[0018] In some embodiments, the inhibitor is any one of an antibody, an antigen-binding protein, a small molecule compound, or siRNA / shRNA that can inhibit signal transduction through the HGF / MET pathway.

[0019] In some implementations, the inhibitor is a MET inhibitor.

[0020] In a third aspect, the present invention provides a pharmaceutical composition or kit for treating or preventing chronic lung allogeneic graft dysfunction (CLAD) in lung transplant recipients (especially allogeneic lung transplant recipients), wherein the composition or kit comprises a MET inhibitor or an acceptable salt form thereof as an active ingredient.

[0021] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0022] In a fourth aspect, the present invention provides a MET inhibitor for improving the long-term survival rate of lung transplant recipients. Attached Figure Description

[0023] Figure 1A H&E-stained lung sections of the pleural region from human CLAD and healthy donor lungs are shown. Scale bar, 100 μm.

[0024] Figure 1B Images of WT1 immunofluorescence staining in human CLAD and healthy donor lungs are shown. Scale bar, 15 μm.

[0025] Figure 1C This is a schematic diagram of the experimental design for an orthotopic single lung transplantation model in mice, including allogeneic grafts (allograft) and isografts (isograft).

[0026] Figure 1D Showing unprocessed (na) H&E stained lung sections of B6 lung (control), syngeneic graft 35 days post-transplantation (Iso_D35), allogeneic graft 14 days post-transplantation (Allo_D14), and allogeneic graft 35 days post-transplantation (Allo_D35). Scale bar, 100 μm.

[0027] Figure 1EImages of WT1 and Ki67 immunofluorescence staining of control lung, Iso_D35 lung, Allo_D14 lung, and Allo_D35 lung are shown. Scale bar, 15 μm.

[0028] Figure 1F CD45 isolated from control lung, Allo_D14 lung, and Allo_D35 lung + Cells and CD45 - A schematic diagram of the workflow for single-cell RNA sequencing (scRNA-seq) analysis.

[0029] Figure 1G The bar chart shows the increased CD45 levels in Allo_D14 and Allo_D35 lungs compared to the control lung. + The relative proportions of immune cell subsets.

[0030] Figure 1H and Figure 1I The image shows basophils (CD45medFcεR1α) in the control lung, Allo_D14 lung, and Allo_D35 lung. + c-Kit - CD49b + Flow cytometry analysis of cells. The number and proportion of basophils were quantified using bar charts. p<0.05, p<0.01, p<0.001, Student's t test.

[0031] Figure 1J Images of ENPP3 immunofluorescence staining in basophils from human CLAD lungs and donor lungs are shown. Scale bar, 15 μm.

[0032] Figure 1K Spatial transcriptomic analysis results showing ENPP3 expression in human CLAD lungs and donor lungs are presented.

[0033] Figure 2A This is a schematic diagram of DT-mediated basophil depletion during CLAD progression (n=5 per group). Allogeneic lungs were collected on day 35 post-transplantation.

[0034] Figure 2B Showing data from DTR F / + and Mcpt8 -Cre、DTR F / + H&E stained Allo_D35 lung sections of the receptor. Scale bar, 100 μm.

[0035] Figure 2C Showing data from DTR F / + and Mcpt8 -Cre、DTR F / + Quantitative analysis of pleural thickness in the lungs of Allo_D35 mice (n=5 per group). p<0.001, Student's t test.

[0036] Figure 2D and Figure 2E Showing data from DTR F / + and Mcpt8 -Cre、DTR F / + Immunofluorescence staining images of WT1 and Ki67 in the lungs of Allo_D35 receptors (n=5 per group) and Ki67 + WT1 + Quantitative analysis of mesothelial cells. p<0.001, Student's t Inspection. Scale bar, 15 μm.

[0037] Figure 3A The image shows CD45 in the control lung and Allo_D35 lung. - UMAP diagram of cells.

[0038] Figure 3B The image shows CD45 in the control lung and Allo_D35 lung. - UMAP diagram of cell type distribution.

[0039] Figure 3C The results show that, compared to the control lung, Allo_D35 lungs contain CD45 - A bar chart showing the changes in the proportion of cell types.

[0040] Figure 3D Shown from Wt1 -CreER、Ai14 F / + Cases of donor lung transplantation into wild-type recipients.

[0041] Figure 3E The diagram shows WT1 in the lungs of Allo_D0, Allo_D14, and Allo_D35 (n=3 per group). + RFP + Immunofluorescence staining image of cells. Scale bar, 15 μm.

[0042] Figure 3FThe diagram shows WT1 in the lungs of Allo_D0, Allo_D14, and Allo_D35 (n=3 per group). + RFP + Quantitative analysis of cells. p<0.001, Student's t test.

[0043] Figure 3G Showing lung transplantation from wild-type donors to Wt1 -CreER、Ai14 F / + The situation within the receptor.

[0044] Figure 3H The diagram shows WT1 in the lungs of Allo_D0, Allo_D14, and Allo_D35 (n=3 per group). + RFP + Immunofluorescence staining image of cells. Scale bar, 15 μm.

[0045] Figure 3I The diagram shows WT1 in the lungs of Allo_D0, Allo_D14, and Allo_D35 (n=3 per group). + RFP + Quantitative analysis of cells. p<0.001, Student's t test.

[0046] Figure 3J This is a schematic diagram illustrating that the dilated mesothelial cells in the lungs of Allo_D35 are of recipient origin rather than donor origin.

[0047] Figure 4A Cell-cell interaction analysis is shown, revealing the interaction between basophils and tissue-resident cells in the lungs of Allo_D35.

[0048] Figure 4B This study illustrates the ligand-receptor interaction analysis between basophils and mesothelial cells in the lungs of Allo_D35.

[0049] Figure 4C The image shows CD45 in the control lung and Allo_D35 lung. + Cell subsets Hgf A bubble chart for expression.

[0050] Figure 4D The image shows CD45 in the control lung and Allo_D35 lung. - Cell subsets Met A bubble chart for expression.

[0051] Figure 4E Images of WT1 and pMet immunofluorescence staining are shown for control lung, Allo_D14 lung, and Allo_D35 lung (n=5). Scale bar, 15 μm.

[0052] Figure 4F Images of WT1 and pERK1 / 2 immunofluorescence staining are shown for control lung, Allo_D14 lung, and Allo_D35 lung (n=5). Scale bar, 15 μm.

[0053] Figure 4G and Figure 4H Mesothelial cells with RFP lineage markers cultured in lower chambers are shown co-cultured with IL-33-activated basophils with or without carmatinib. As controls, mesothelial cells were cultured in medium only or medium containing recombinant HGF. Mesothelial cell proliferation was quantified by RFP and Ki67 immunofluorescence staining (n=3). Scale bar, 6 μm. NS, no significant difference. p<0.01, p<0.001, Student's t test.

[0054] Figure 4I and Figure 4J Mesothelial cells with RFP lineage markers in the upper transwells are shown co-cultured with IL-33-activated basophils in the lower transwells, with or without carmatinib. As a control, mesothelial cells were cultured in the lower transwells in medium only or medium containing recombinant HGF. Invading mesothelial cells (n=3) were stained with crystal violet. Scale bar, 30 μm. NS, no significant difference. p<0.01, p<0.001, Student's t test.

[0055] Figure 5A Images of WT1 and pMet immunofluorescence staining of lung sections from donors and CLAD patients are shown. Scale bar, 15 μm.

[0056] Figure 5B Images of WT1 and pERK1 / 2 immunofluorescence staining of lung sections from donors and CLAD patients are shown. Scale bar, 15 μm.

[0057] Figure 5CRecipient mice treated with solvent or carmatinib from day 14 to day 35 post-transplantation are shown. Allogeneic lungs were harvested on day 35 (Allo_D35).

[0058] Figure 5D and Figure 5E H&E staining images show reduced pleural thickness in the Allo_D35 lungs of the receptor treated with carmatinib compared to the receptor treated with solvent (n=5 per group). Scale bar, 100 μm.

[0059] Figure 5F Images of WT1 and pMet immunofluorescence staining of Allo_D35 lung cells from solvent-treated or carmatinib-treated receptors are shown. Scale bar, 15 μm.

[0060] Figure 5G Immunofluorescence staining images of WT1 and pERK1 / 2 in Allo_D35 lungs from solvent-treated or carmatinib-treated receptors are shown. p<0.001, Student's t test.

[0061] Figure 5H and Figure 5I Immunofluorescence staining images of WT1 and Ki67 in the lungs of Allo_D35 receptors treated with solvent or carmatinib, and WT1. + Ki67 + Quantitative analysis of cells (n=5 per group). Scale bar, 15 μm. p<0.001, Student's t test.

[0062] Figure 6 A schematic diagram illustrating the pathological pathway by which basophils secreting HGF promote mesothelial cell proliferation and invasion, thereby accelerating pleural fibrosis and the progression of CLAD.

[0063] Figure 7A The study described how recipient mice were treated with solvent or U0126 from day 14 to day 35 post-transplantation, and how allogeneic lungs were harvested on day 35.

[0064] Figure 7B Images of pERK1 / 2 immunofluorescence staining of Allo_D35 lung cells from solvent-treated and U0126-treated receptors are shown. Scale bar, 15 μm.

[0065] Figure 7C and Figure 7DThe H&E staining images and quantitative analysis show that pleural thickness was decreased in Allo_D35 lungs from U0126-treated Allo_D35 lungs compared to Allo_D35 lungs from solvent-treated Allo_D35 lungs (n=5 per group). Scale bar, 100 μm. p<0.01, Student's t test.

[0066] Figure 7E and Figure 7F Immunofluorescence staining images of WT1 in Allo_D35 lungs from solvent-treated and U0126-treated receptors are shown. + Quantitative analysis of cells (n=5 per group). Scale bar, 15 μm. p<0.01, Student's t test. Detailed Implementation

[0067] The description of specific implementations and embodiments is provided by way of illustration rather than limitation. Those skilled in the art will readily recognize that many non-critical parameters can be changed or modified to achieve substantially similar results.

[0068] As used in this article, the articles “a” and “an” refer to one or more grammatical objects (e.g., at least one).

[0069] As used herein, the term “or” means “and / or” and is used interchangeably with “and / or” unless the context clearly indicates otherwise.

[0070] "About" and "approximately" typically refer to the acceptable range of error for a measured value, given the nature or precision of the measurement. An exemplary range of error is within 20% of a given value or range of values, typically within 10%, and more typically within 5%.

[0071] As used herein, all numerical values ​​or ranges include integers within or covering the range, as well as fractions of numerical values ​​or integers within or covering the range, unless the context clearly indicates otherwise. Thus, for example, references to a range of 90%–100% include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, the range of 1-5000 times includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, 2.1, 2.2, 2.3, 2.4, 2.5 times, and so on.

[0072] The terms “include,” “includes,” “including,” “have,” “has,” “having,” “contains,” or “containing,” and their grammatically equivalent expressions, should generally be understood as open-ended and non-restrictive, for example, not excluding other elements or steps not mentioned unless explicitly stated otherwise or understood from the context.

[0073] As used herein, the terms “subject” and “patient” are used interchangeably and refer to an organism treated by the methods and compositions of the present invention. Such organisms are preferably mammals (e.g., humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, or non-human primates such as monkeys, chimpanzees, baboons, and rhesus monkeys), more preferably, humans.

[0074] As used herein, the term "pharmaceutical composition" refers to a combination of an active ingredient with a carrier, diluent, and / or adjuvants (inert or active) that makes the composition particularly suitable for in vivo or in vitro diagnostic or therapeutic use.

[0075] Pharmaceutically acceptable carriers that may be included in a pharmaceutical composition may be those commonly used in pharmaceutical formulations, and may include, but are not limited to, at least one selected from the group consisting of: lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Examples of other carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow 10. In addition to the above-mentioned components, the pharmaceutical composition may further include at least one selected from the group consisting of: diluents, adjuvants, lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, and preservatives.

[0076] The pharmaceutical compositions of the present invention may be in the form of a solution, suspension, syrup or emulsion in an oil or water medium, or may be formulated as extracts, powders, granules, tablets or capsules, and may further contain dispersants or stabilizers for formulation processes.

[0077] In particular, when the inhibitor is an antibody or its antigen-binding fragment, a pharmaceutically effective amount of the inhibitor as the active ingredient can be formulated into immunoliposomes. Liposomes containing antibodies can be prepared using any method known in the relevant art. Immunoliposomes are lipid compositions comprising phosphatidylcholine, cholesterol, and polyethylene glycol-derived phosphatidylethanolamine, which can be prepared by reverse-phase evaporation. For example, the Fab' fragment of an antibody can be coupled to the liposome via a disulfide exchange reaction.

[0078] As used herein, the term "therapeutic" means an agent intended to treat, counteract, alleviate, prevent, or improve an undesirable condition or disease in a patient. In some implementations, a therapeutic agent (such as an HGF / MET signaling pathway inhibitor) is used for the treatment and / or prevention of chronic lung allogeneic graft dysfunction (CLAD).

[0079] When used in conjunction with a therapeutic agent, "application" means the systemic or local administration of the therapeutic agent, such as direct application to the interior or surface of a target tissue, or administration to a patient, whereby the therapeutic agent has a positive effect on the targeted tissue. Therefore, as used herein, when the term "application" is used in conjunction with the compositions described herein, it may include, but is not limited to, providing the composition to the interior or surface of a target tissue; or systemically administering the composition to a patient, for example, via oral administration, whereby the therapeutic agent reaches the target tissue or cells. The "application" of the composition may be achieved by injection, local application, and oral administration, or by other methods alone or in combination with other known techniques.

[0080] As used herein, the term “effective amount” refers to an amount of an active agent (e.g., an HGF / MET signaling pathway inhibitor) sufficient to produce a beneficial or desired result, such as, for example, producing a clinical benefit in a subject. An effective amount can be administered by single or multiple applications, application, or dosing, and is not limited to a particular formulation or route of administration.

[0081] As used herein, the terms “treat,” “treated,” “treatment,” or “treating” refer both to therapeutic therapies in some implementations and preventative or preventative measures in others, aimed at preventing or delaying (mitigating) undesirable physical conditions, impairments, or diseases, or achieving beneficial or desired clinical outcomes. For the purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, symptom relief: reduction in the severity of the condition, impairment, or disease; stabilization (i.e., non-deterioration) of the condition, impairment, or disease state; delay in the onset or progression of the condition, impairment, or disease; improvement of the condition, impairment, or disease; and relief (whether partial or complete), whether detectable or undetectable, or enhancement or improvement of the condition, impairment, or disease. Treatment includes inducing a clinically significant response without producing excessive levels of side effects. Treatment also includes prolonged survival compared to expected survival without treatment. Preventative benefits of treatment include prevention of disease, delay in disease progression, stabilization of disease, or reduction in the likelihood of disease occurrence. As used herein, “treat,” “treated,” “treatment,” or “treating” may include preventative measures in some implementations.

[0082] As used herein, “inhibitors” of the HGF / MET signaling pathway include antibodies, antigen-binding proteins, nucleic acid-based therapeutic agents, aptamers and mirror images of target molecules (such as components in the HGF / MET pathway) that bind to them, or antisense or siRNA molecules or CRISPR systems that inhibit the expression and / or activity of target sites (such as components in the HGF / MET pathway), or small molecule inhibitors, such as small molecule inhibitors of components in the HGF / MET pathway, or combinations thereof. Such components include, but are not limited to, hepatocyte growth factor (HGF) and MET. In embodiments of the invention, the inhibitor binds to the target site and / or reduces the signal transduction output of the target site.

[0083] In some implementations, the dissociation constant (K) of the inhibitor binding to the target site DThe Kc of the inhibitor is 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, 0.05 nM, 0.01 nM, 0.0075 nM, 0.005 nM, 0.001 nM or lower, and is determined by standard binding assays, such as surface plasmon resonance or biolayer interferometry. In some embodiments, the Kc of the inhibitor when it binds to the target site is... D The range is from approximately 20 nM to approximately 0.001 nM, determined by standard combination detection methods, such as surface plasmon resonance or biolayer interferometry.

[0084] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Therefore, the term is used in the broadest sense to specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, etc., provided they exhibit the desired biological activity. As used herein, unless otherwise stated, an antibody or its antigen-binding fragment is an antibody or fragment capable of specifically binding to any component of the HGF / MET pathway. This component includes, but is not limited to, MET.

[0085] In some embodiments, the inhibitor is a small molecule inhibitor or a pharmaceutically acceptable salt thereof.

[0086] A pharmaceutically acceptable salt is one that, within reasonable medical judgment, is suitable for contact with human and lower animal tissues without causing excessive toxicity, irritation, or allergic reactions, and has a reasonable benefit / risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds and pharmaceuticals are well known in the art. For example, pharmaceutically acceptable salts are described in detail by SM Berge et al. in J. Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference. Such salts may be prepared in situ during the final separation and purification of the pharmaceuticals described in this disclosure, or individually by reacting a free base or free acid functional group with a suitable reagent, as outlined below. For example, a free base functional group may react with a suitable acid. Furthermore, when the pharmaceuticals described in this disclosure contain an acidic group, suitable pharmaceutically acceptable salts may include metal salts, such as alkali metal salts, like sodium or potassium salts; and alkaline earth metal salts, such as calcium or magnesium salts. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts formed from amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts prepared by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptyl sulfate, glycerol phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, p-valerate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. In addition, where appropriate, pharmaceutically acceptable salts also include non-toxic ammonium ions, quaternary ammonium ions, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates.

[0087] MET inhibitors The HGF receptor MET (also known as c-Met, an abbreviation for interstitial epithelial transformation factor; or hepatocyte growth factor receptor, abbreviated as HGFR) is a 145 kDa receptor tyrosine kinase (RTK) typically expressed by epithelial cells in the brain, kidneys, liver, and other tissues. When its ligand, hepatocyte growth factor (HGF), binds to it, it triggers receptor autophosphorylation and activates multiple downstream effector molecules, including mitogen-activated protein kinases ERK-1 and ERK-2, as well as PLC-γ. Activation of the c-Met signaling pathway elicits a variety of cellular responses, including cell motility, dispersion, proliferation, survival, and angiogenesis.

[0088] MET antagonists may exert their effects by binding to MET and competitively blocking the binding or activation of HGF. Exemplary agents include truncated HGF proteins such as NK1, NK2, and NK4 (see above), and anti-MET monoclonal antibodies (anti-MET mAbs). A preferred example is a genetically engineered anti-MET antibody with only one "arm" (i.e., binding domain), such as OA-5D5 (Martens et al., 2006) and onartuzumab (Merchant et al., 2013). Onartuzumab is a single-arm, recombinant, humanized, monoclonal monovalent antibody that binds to the extracellular domain of the receptor tyrosine kinase MET, thereby blocking the binding of hepatocyte growth factor (HGF) and subsequent receptor activation. Other anti-MET antibodies include emibetuzumab (LY2875358), developed by Eli Lilly & Company in 2014. Emibetuzumab is a humanized MET-specific IgG4 antibody that blocks the binding of HGF to MET and causes MET degradation, thereby inhibiting HGF-dependent and non-dependent signaling cascades.

[0089] In some embodiments, (i) the c-Met inhibitor selectively binds to and inhibits MET kinase (e.g., selectively binds to and inhibits dephosphorylated MET kinase); (ii) the c-Met inhibitor is an ATP-noncompetitive inhibitor of MET kinase; and / or (iii) the c-Met inhibitor has cytotoxic activity independent of its ability to bind to MET kinase. In some embodiments, the c-Met inhibitor simultaneously possesses (i)-(iii).

[0090] Examples of small molecule c-Met inhibitors are provided below. The methods or uses disclosed herein cover each of these compounds, as well as their pharmaceutically acceptable salts and derivatives.

[0091] In some embodiments, the c-Met inhibitor is SU5416 (also known as Semaxinib; CAS No. 204005-46-9). In some embodiments, the c-Met inhibitor is SU11274 (CAS No. 658084-23-2). In some embodiments, the c-Met inhibitor is ARQ 197 (also known as Tivantinib; CAS No. 905854-02-6). In some embodiments, the c-Met inhibitor is EMD1214063 (MSC2156119J; Tepotinib). In some embodiments, the c-Met inhibitor is GSK / 1363089 / XL880 (Foretinib). In some embodiments, the c-Met inhibitor is XL184 (Cabozantinib). In some embodiments, the c-Met inhibitor is HMPL-504 / AZD6094 / volitinib (Savolitinib). In some embodiments, the c-Met inhibitor is SOMG-833. In some embodiments, the c-Met inhibitor is MSC2156119J (EMD 1214063, Tepotinib). In some embodiments, the c-Met inhibitor is LY2801653 (Merestinib). In some embodiments, the c-Met inhibitor is AMG 337. In some embodiments, the c-Met inhibitor is INCB28060 (Capmatinib). In some embodiments, the c-Met inhibitor is AMG 458. In some embodiments, the c-Met inhibitor is PF-04217903. In some embodiments, the c-Met inhibitor is PF-02341066 (crizotinib). In some embodiments, the c-Met inhibitor is E7050 (golvatinib). In some embodiments, the c-Met inhibitor is MK-2461. In some embodiments, the c-Met inhibitor is BMS-777607. In some embodiments, the c-Met inhibitor is JNJ-38877605.

[0092] In some implementations, the c-Met inhibitor is ARQ197 (tivantinib). Tivantinib has the IUPAC name (3R,4R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-pyrrolidinedione.

[0093] In some implementations, the c-Met inhibitor is EMD1214063 (MSC2156119J; Tepotinib). Tepotinib has the IUPAC name 3-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)benzyl)-1,6-dihydro-6-oxopyridazin-3-yl)benzonitrile (3-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)benzyl)-1,6-dihydro-6-oxopyridazin-3-yl)benzonitrile).

[0094] In some implementations, the c-Met inhibitor is GSK / 1363089 / XL880 (Foretinib). Foretinib has the IUPAC name N1'-[3-fluoro-4-[[6-methoxy-7-(3-morpholinopropoxy)-4-quinolyl]oxy]phenyl]-N1-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide.

[0095] In some implementations, the c-Met inhibitor is XL184 (Cabozantinib). Cabozantinib has the IUPAC name N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide.

[0096] In some implementations, the c-Met inhibitor is HMPL-504 / AZD6094 / volitinib (Savolitinib). Volitinib has the IUPAC name (S)-1-(1-(imidazo[1,2-a]pyridin-6-yl)ethyl)-6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine ((S)-1-(1-(imidazo[1,2-a]pyridin-6-yl)ethyl)-6-(1-methyl-1H-pyrazol-4-yl)-1H-[1,2,3]triazolo[4,5-b]pyrazine).

[0097] In some implementations, the c-Met inhibitor is MSC2156119J (EMD 1214063, Tepotinib). Tepotinib has the IUPAC name 3-[1,6-dihydro-1-[[3-[5-[(1-methyl-4-piperidinyl)methoxy]-2-pyrimidinyl]phenyl]methyl]-6-oxo-3-pyridazinyl]-.

[0098] In some implementations, the c-Met inhibitor is LY2801653 (Merestinib). Merestinib has the IUPAC name N-(3-fluoro-4-{[1-methyl-6-(1H-pyrazol-4-yl)-1H-indazol-5-yl]oxy}phenyl)-1-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide.

[0099] In some implementations, the c-Met inhibitor is AMG 337. AMG 337 has the IUPAC name 7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine.

[0100] In some implementations, the c-Met inhibitor is INCB28060 (capmatinib). Capmatinib has the IUPAC name 2-fluoro-N-methyl-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl]benzamide.

[0101] In some implementations, the c-Met inhibitor is AMG 458. AMG 458 has the IUPAC name 1-(2-hydroxy-2-methylpropyl)-N-(5-((7-methoxyquinolin-4-yl)oxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide.

[0102] In some implementations, the c-Met inhibitor is PF-04217903. PF-04217903 has the IUPAC name 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol.

[0103] In some implementations, the c-Met inhibitor is PF-02341066 (crizotinib). Crizotinib has the IUPAC name (R)-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine.

[0104] In some implementations, the c-Met inhibitor is E7050 (Golvatinib). Golvatinib has the IUPAC name N-(2-fluoro-4-((2-(4-(4-methylpiperazin-1-yl)piperidine-1-carboxamido)pyridin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide.

[0105] In some implementations, the c-Met inhibitor is MK-2461. MK-2461 has the IUPAC name N-((2R)-1,4-dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide.

[0106] In some implementations, the c-Met inhibitor is BMS-777607. BMS-777607 has the IUPAC name N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide.

[0107] In some implementations, the c-Met inhibitor is JNJ-38877605. JNJ-38877605 has the IUPAC name 6-(difluoro(6-(1-methyl-1H-pyrazol-3-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)quinoline.

[0108] The descriptions of specific implementations and examples are provided in an illustrative rather than restrictive manner. Those skilled in the art will readily recognize that many non-critical parameters can be changed or modified to achieve substantially similar results.

[0109] Example Materials and Methods mice The mouse strains used in this study include Mcpt8-cre (B6.129S4-) Mcpt8 tm1(cre)Lky / J), Wt1 -CreER, Rosa26-Ai14, and Rosa26-DTR mice all have a C57BL / 6 (B6) genetic background. Specifically, Wt1 -CreER、Ai14 F / + The mice were from a B6D2F1 background, and were transmitted through the B6 background. Wt1 -CreER、Ai14 F / + F1 offspring were obtained by crossing mice with wild-type DBA2 / n mice. Wild-type mice carrying B6 / n, DBA2 / n, and B6D2F1 backgrounds were purchased from VitalRiver Laboratory Animal Technology Co., Ltd. Unless otherwise stated, all donor and recipient mice were age-matched (8–10 weeks old). In this study, both donor and recipient mice were male, and all experimental animals were untreated prior to analysis. All mouse strains were housed in the NIBS animal facility under specific pathogen-free (SPF) conditions, strictly adhering to institutional guidelines and ethical regulations. All experiments were conducted in accordance with the National Guidelines for the Housing and Care of Laboratory Animals (Ministry of Health, China), and the operating procedures were reviewed and approved by the Institutional Animal Protection and Use Committee of the Beijing Institute of Life Sciences.

[0110] Human specimen All experiments involving human tissue samples were conducted in accordance with the operating procedures approved by the Institutional Review Boards of the Beijing Institute of Life Sciences, the Second Affiliated Hospital of Zhejiang University School of Medicine, and Wuxi People's Hospital. Informed consent was obtained from all participants. Chronic lung allogeneic graft dysfunction (CLAD) tissue was obtained from lung grafts of CLAD patients who received lung transplants (n=3; 2 males and 1 female). Donor lung tissue was obtained from healthy adult donors (n=5; 4 males and 1 female; age >40 years) whose lungs were not used for clinical transplantation due to volume mismatch or deemed unsuitable for transplantation. No sex-related effects were observed in this study.

[0111] Orthotopic mouse lung transplantation The procedure for orthotopic mouse lung transplantation has been previously described (Krupnick et al., 2009). A brief overview is provided here: Donor operating procedures: The donor mouse was anesthetized and connected to a mechanical ventilator. Positioned supine, a thoracotomy was performed to expose the thoracic cavity. Heparin (100 U / kg) was injected into the inferior vena cava to prevent clotting. Adhesions between the heart and the left chest wall were carefully dissected, and the thymus and covering adipose tissue were removed to clearly expose the roots of the ascending aorta and left pulmonary artery. The inferior vena cava was transected, followed by the auricle. A needle was inserted parallel to the surgical plane near the base of the right atrium, and 2 mL of cold lactated Ringer's solution was injected through the right ventricle until the lungs were completely decolorized. Ventilation was stopped, and the heart-lung mass was removed and preserved in 4°C Ringer's solution.

[0112] The excised heart and lung masses were wrapped in sterile gauze moistened with Ringer's solution. Under a dissecting microscope, the bronchus, vein, and artery of the left lung were carefully dissected, ensuring that sufficient length of the trachea and bronchus were preserved. The connections between the left and right lungs and the heart were severed. A suitably sized cannula was applied to the donor's bronchus, vein, and artery: the distal end was folded over to cover the cannula and secured with 10-0 nylon sutures. Subsequently, the prepared donor lung was wrapped in moistened sterile gauze and stored on ice until transplantation.

[0113] Receptor operating procedures: The recipient mouse was anesthetized, cannulated with a 20-gauge catheter, and connected to a small animal ventilator delivering oxygen mixed with 0.5% isoflurane. After shaving the hair from the left chest region and disinfecting with povidone-iodine, a thoracotomy was performed between the third and fourth ribs. Using a retractor, the ribs were separated, and the left lung was gently externalized with blunt forceps and cotton swabs, aligning the bronchus, veins, and arteries parallel to the surgical plane. A microvascular clip was placed at the hilum to block the bronchus, veins, and arteries. Adhesive tissue was carefully separated, and 5-0 nylon sutures were placed around these structures in preparation for anastomosis. Small incisions were made in the recipient's arteries, veins, and bronchi. The previously prepared donor lung was removed from ice, and its cannulated arteries, veins, and bronchi were inserted into the corresponding incisions in the recipient. The cannulas were advanced to establish a stable connection and secured with 10-0 nylon sutures. The microvascular clips were then removed to restore blood flow to the transplanted lung, and the recipient's own left lung was excised. The donor lung was gently repositioned into the thoracic cavity using a cotton swab moistened with lactated Ringer's solution. The ribs were aligned, and the chest incision was closed with 5-0 nylon sutures, followed by skin suturing. Postoperatively, the mouse remained connected to the ventilator and placed on a 42°C warming pad until it regained consciousness.

[0114] Immunofluorescence staining procedure Lung tissues from mice and humans were fixed with 4% paraformaldehyde at 4°C for 36 hours. After washing with phosphate-buffered saline (PBS), the tissues were immersed in 30% sucrose solution overnight at 4°C. Subsequently, the tissues were embedded in an OCT compound and stored at -80°C. Frozen tissue sections with a thickness of 10 μm were prepared for immunofluorescence staining.

[0115] Prior to staining, antigen retrieval was performed by heating the sections in citrate buffer at 100°C for 15 minutes, followed by cooling to room temperature. Subsequently, the sections were incubated in blocking buffer (PBS containing 3% bovine serum albumin) at room temperature for 1 hour. Primary antibody was applied, and the sections were incubated overnight at 4°C. The primary antibodies are as follows: CC10 (1:200, 07-623, Sigma Aldrich), α-SMA (1:200, C6198, Sigma), IL-33 (1:200, AF3626 and AF3625, R&D System), RFP (1:200, 600-401-379, Rockland), WT1 (1:200, ab89901, Abcam), Ki67 (1:200, ab15580, Abcam), phosphorylated Met (1:500, 3077, Cell signaling), phosphorylated ERK1 / 2 (1:500, 4370T, Cell signaling), MCP-8 (1:400, 647401, Biolegend), and ENPP3 (1:400, HPA043772, Sigma Aldrich). The samples were washed three times with PBST (PBS containing 0.1% Triton X-100) for 10 minutes each time, followed by incubation with secondary antibody at room temperature for 3 hours. After an additional PBST wash, the cell nuclei were stained with DAPI at room temperature for 15 minutes. The samples were mounted with antifluorescence quenching mounting medium, and images were acquired using a Zeiss 980 confocal microscope and analyzed using ImageJ software (National Institutes of Health).

[0116] For tyrosine signal amplification (TSA), after incubation with the primary antibody, the sections were incubated with biotin-labeled secondary antibody, followed by incubation for 30 minutes using the VECTASTAIN Elite ABC kit (1:100, PK-6100, Vector Laboratories). Fluorophores were then added and incubated for 5 minutes to complete the staining.

[0117] Basophil Isolation and Culture Bone marrow progenitor cells were isolated from 8-week-old C57BL / 6 mice and cultured at 0.5 × 10⁻⁶. 6 Cells were cultured at a density of [number] cells / mL. To induce differentiation of mouse bone marrow-derived basophils, bone marrow cultures were maintained for 10 days in the presence of IL-3 (30 ng / mL; PeproTech). CD117 was then enriched using magnetically activated cell sorting (Miltenyi Biotec). - (c-Kit) -Cell populations were collected and reseeded into 96-well flat-bottomed tissue culture plates. Cells were then stimulated with IL-33 and IL-18 for 16 hours. All bone marrow cultures were performed in standard RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL antibiotic / antifungal agent (anti / anti). To maintain differentiation, the medium was replaced every three days with fresh IL-3 (30 ng / mL).

[0118] Isolation and culture of pleural mesothelial cells From B6 background Wt1 Pleural mesothelial cells were isolated from CreER, Ai14F / mice following a previously described method for rabbits (Antony et al., 1989). Mice were administered tamoxifen four times two weeks prior to euthanasia. Immediately after CO2 asphyxiation, an abdominal incision was made to expose the diaphragm. The pleural cavity was flushed with 2 mL of 0.25% trypsin-EDTA solution using a syringe, and the animals were incubated at room temperature for 30 minutes. The flushing fluid containing mesothelial cells was aspirated and centrifuged at 400 × g for 6 minutes. The resulting cell pellet was treated with erythrocyte lysis buffer for 2 minutes, followed by centrifugation and removal of the supernatant. Subsequently, the cells were washed twice with DMEM supplemented with 10% fetal bovine serum (FBS). The isolated cells were seeded into six-well plates and cultured at 37°C with 5% CO2 humidification in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. After 24 hours, the medium was changed to remove non-adherent cells. Adherent mesothelial cells proliferated to near confluence in the culture dish after one week of culture. The purity of the cultured mesothelial cells was verified by observing RFP expression under a fluorescence microscope.

[0119] Analysis of mesothelial cell proliferation and invasion To assess mesothelial cell proliferation, Transwell chambers with 0.4 μm polyester membranes (Corning) were used. RFP-labeled mesothelial cells were seeded into the lower chamber, and basophils were placed in the upper chamber. Co-culture was performed in DMEM supplemented with 50 ng / mL IL-33, 10% fetal bovine serum (FBS), and an antibiotic-antifungal agent (1×), with or without the addition of 1 μM carmatinib. After 48 hours of co-culture, mesothelial cell proliferation was quantitatively analyzed by immunofluorescence staining for RFP and Ki67.

[0120] For invasion analysis, Transwell chambers with 8 μm polyester membranes were used. A total of 2 × 10⁻⁶ cells were used. 4RFP-labeled mesothelial cells were seeded into the upper chamber, while IL-33-activated basophils were placed in the lower chamber. Co-culture was performed in DMEM supplemented with 50 ng / mL IL-33, 10% FBS, and 100 U / mL antibiotic / antifungal agent (anti / anti), with or without 1 μM carmatinib. After 24 hours, cells that had migrated to the submembrane surface were stained with crystal violet and quantified.

[0121] Tissue isolation and single-cell preparation After perfusion with cold phosphate-buffered saline (PBS), lung tissue from both the transplanted and control groups was excised and transferred to 2.0 mL Eppendorf tubes containing ice-cold digestion buffer. The digestion buffer consisted of DMEM supplemented with 10 U / mL neutral protease (Worthington, LS02111), 400 U / mL collagenase type I (GIBCO, 17100-017), 8 U / mL elastase (Worthington, 2294), and 0.66 U / mL DNase I (Roche, 10104159001). The tissue was cut directly into small pieces in the ice-cold tubes using surgical scissors and then digested at 37°C for 30 minutes.

[0122] After digestion, the cell suspension was filtered through a 100 μm cell filter and washed with DMEM containing 10% fetal bovine serum (FBS). The filtrate was collected and centrifuged to precipitate the cells, and the supernatant was discarded. The pellet was resuspended in RBC lysis buffer and incubated at room temperature for 2 minutes to lyse the red blood cells, followed by centrifugation. The resulting cell pellet was washed and resuspended in MACS buffer to obtain a single-cell suspension.

[0123] FACS sorting Single-cell suspensions from different samples were added to 96-well plates on ice, ensuring approximately equal cell counts in each well. The plates were centrifuged at 400×g for 5 minutes at 4°C, followed by removal of the supernatant. The single-cell suspensions were incubated with anti-CD16 / CD32 antibody and the following labeled antibodies at specified dilutions in MACS buffer (PBS containing 0.5% FBS and 2 mM EDTA): 1:400 anti-CD45 APC-Cy7, 1:400 anti-CD3 FITC, 1:400 anti-CD19 FITC, 1:400 anti-FcεR1α PE-Cy7, 1:200 anti-CD49b APC, and 1:300 anti-CD117 PE. The cells in each well were resuspended in 100 μL of staining solution and incubated on ice in the dark for 30 minutes. The cells were then washed twice with MACS buffer and centrifuged. The cell suspension was then filtered through a 40 μm cell filter to remove cell aggregates. The filtered sample was then analyzed using a Fusion 2 flow cytometer.

[0124] scRNA sequencing Single-cell RNA sequencing and computational analysis CD45 was isolated from control group and transplanted lung tissue using fluorescence-activated cell sorting (FACS). - and CD45 + Cells were then used to construct libraries using standard procedures from 10× Genomics. Multiple libraries were sequenced on the Illumina HiSeq X10 platform. Raw sequencing reads were aligned to the GRCh38 reference genome using CellRanger Count v3.1 (10× Genomics). Raw single-cell RNA-seq data have been stored in the GEO database.

[0125] Quantitative statistical analysis All data are presented as mean ± standard error (sem), as shown in the figure caption. The data shown in the figure are from multiple independent experiments conducted on different dates using different mice. Unless otherwise stated, most data in the figure are from at least three independent experiments. Statistical significance of differences between sample means was assessed using two-tailed unpaired Student's... t Evaluation and assessment. For animal experiments, the Kaplan-Meier survival curves were analyzed using the log-rank (Mantel-Cox) test in conjunction with Prism 9.5.0 software (GraphPad Software, LaJolla, CA).

[0126] Example 1 Allogeneic mouse lung transplantation model reproduces key pathological features of CLAD Pleural fibrosis is strongly associated with poor clinical outcomes and accelerated disease progression. Histological analysis of lung tissue from patients undergoing re-transplantation for end-stage CLAD revealed significant pleural fibrosis, characterized by multilayered mesothelial cell structures. Figure 1A and Figure 1B Despite immunosuppressive therapy, CLAD progressed to end-stage disease, highlighting the limited effectiveness of current therapies in halting disease progression.

[0127] To investigate the mechanisms driving CLAD pathogenesis, an orthotopic, major histocompatibility complex (MHC) semi-mismatched single lung transplantation model was established in mice. Figure 1C (Mimura et al., 2015). In this model, the left lung from a B6D2F1 (H-2b / d) or B6 (H-2b) donor mouse was transplanted into a B6 (H-2b) recipient mouse. The transplanted allogeneic grafts were analyzed at multiple time points post-transplantation. Figure 1C ).

[0128] The transplanted lung remained structurally intact 35 days post-transplantation, exhibiting a single-layer WT1 structure. + Mesothelial cells, comparable to those in untreated B6 mice ( Figure 1D and Figure 1E In contrast, allogeneic grafts produced progressive CLAD-like pathological changes. By day 14 post-transplantation (D14), immune cell infiltration and mild pleural fibrosis were observed. By day 35 (D35), large-scale tissue remodeling was observed, characterized by a significant increase in mesothelial cell proliferation and number, transforming from a single layer of mesothelial cells to a multilayered structure. Figure 1D and Figure 1E These findings suggest that pleural fibrosis and mesothelial cell proliferation are closely associated with the progression of CLAD.

[0129] In summary, these findings demonstrate that the allogeneic mouse model of the present invention faithfully reproduces the main pathological features of human CLAD, including pleural fibrosis and multilayered mesothelial cells.

[0130] Example 2 Progressive basophilic infiltration is associated with CLAD progression. CLAD is caused by a dysregulated allogeneic immune response, but the dynamics of immune cells involved in late tissue remodeling remain unclear. To elucidate the dynamic changes in immune cells during CLAD progression, CD45 antibodies isolated from lung grafts and from untreated control (B6) lungs were analyzed on days 14 (Allo-D14) and 35 (Allo-D35) after allogeneic transplantation. + Immune cells were subjected to single-cell sequencing (scRNA-seq) Figure 1FAnalysis of the proportion of immune cells showed that, compared with the control group, the number of T lymphocytes, monocytes, neutrophils, macrophages, mast cells, and basophils in allogeneic lung transplants were significantly increased. Figure 1G ).

[0131] Basophils became the most significant immune population in terms of dynamic expansion, with their proportion steadily increasing from day 14 (D14) to day 35 (D35). Figure 1G In addition to the increased number of basophils, interstitial macrophages and mast cells also showed a significant increase. Flow cytometry confirmed that the number and proportion of basophils in allogeneic lungs progressively increased during the progression of CLAD. Figures 1H to 1I ).

[0132] Notably, these findings are consistent with those in human CLAD: compared to healthy donors, lung tissue from CLAD patients contains higher levels of ENPP3. + A significant increase in the number of basophils / mast cells ( Figure 1J This finding was further validated by spatial transcriptomic analysis of the published CLAD dataset. Figure 1K (Khatri et al., 2023). In summary, these results identify basophils as a previously unrecognized immune subset enriched in the lungs of CLAD patients, suggesting they may serve as a potential driver of CLAD progression.

[0133] Example 3 Activated basophils drive tissue remodeling in CLAD To directly test its contribution to CLAD, an inducible diphtheria toxin (DT)-mediated conditional basophil clearance model was used, and the left lung of a B6D2F1 mouse was transplanted into... Mcpt8 -Cre; DTR F / + or DTR F / + B6 receptor mice in vivo ( Figure 2A ).

[0134] By day 35 (D35), Mcpt8 -Cre; DTR F / + Allogeneic lung transplantation showed a significant reduction in pleural fibrosis, and Ki67 + The proportion of mesothelial cells decreased ( Figures 2B-2E These results collectively suggest that basophils are a key driver of CLAD progression, especially during the tissue remodeling phase.

[0135] Example 4 Recipient-derived mesothelial cells proliferate and invade the allogeneic lung. While a dysregulated immune response is central to CLAD, progressive tissue remodeling is another well-defined pathological feature. To characterize the dynamic changes in non-immune cells in the lungs of CLAD patients, CD45... - Single-cell RNA sequencing (scRNA-seq) analysis was performed on the cells. Figure 3A and Figure 3B CLAD progression is associated with a significant reduction in the population of alveolar epithelial cells and club cells. Figure 3C This may be due to allogeneic immune-mediated damage. Conversely, the proportions of mesothelial cells, ciliated cells, endothelial cells, and smooth muscle cells are increased ( Figure 3C (Among them, mesothelial cells underwent the most significant expansion.)

[0136] To determine the origin of these expanded mesothelial cells, a lineage tracing experiment was performed. Wt1 -CreER; Ai14 F / + Donor lung transplantation to wild-type recipients showed that over 95% of WT1 cells were transplanted. + Mesothelial cells are RFP + ( Figures 3D to 3F However, by day 14, less than 50% of WT1... + The cells are RFP + And by day 35, almost all WT1 + All cells were RFP - ( Figure 3E and Figure 3F This indicates that the loss of donor-derived mesothelial cells is likely due to cell damage and death caused by an allogeneic immune response.

[0137] Conversely, when transplanting B6D2F1 lungs to Wt1 -CreER; Ai14 F / + B6 receptor ( Figure 3G In the reverse experiment, WT1 in the allogeneic lung at transplantation + Mesothelial cells do not express RFP, and by day 35 almost all WT1 cells... + The cells are RFP + No RFP during transplantation + Cell formation contrast ( Figure 3H and Figure 3I These results indicate that the expanded mesothelial cells were recipient-derived, rather than donor-derived. Figure 3J This receptor origin may enable mesothelial cells to evade allogeneic immune damage and promote their proliferation and invasion into the lung parenchyma.

[0138] Example 5 HGF derived from basophils drives the proliferation and invasion of mesothelial cells via MET signaling. After confirming that activated basophils drive CLAD progression (especially in the late stages of tissue remodeling), the interaction between basophils and tissue structural cells was further investigated, revealing an increase in these cells in Allo_D35 lungs. Analysis of potential cell-cell interactions showed that, among various tissue cell types, mesothelial cells exhibited the strongest interaction potential with basophils. Figure 4A ).

[0139] Systematic ligand-receptor interaction analysis identified hepatocyte growth factor (HLB) as a key component of HLB. Hgf )-Hepatocyte growth factor receptor ( Met The basophil-mesothelial cell crosstalk axis is the most prominent signaling pathway mediating this crosstalk. Figure 4B In allogeneic lung transplants, basophils showed a significant upregulation. Hgf Express( Figure 4C Mesothelial cells showed an increase in number compared to control mice in the lungs. Met Express( Figure 4D ).

[0140] The receptor tyrosine kinase MET, upon binding to HGF, undergoes phosphorylation at Tyr 1234 and Tyr 1235, triggering a downstream signaling cascade that regulates cell proliferation and migration. Immunofluorescence staining revealed the presence of phosphorylated MET (p-MET) in mesothelial cells from allogeneic lungs at days 14 and 35, while it was absent in control lungs. Figure 4E Similarly, phosphorylation of ERK1 / 2, a key downstream effector molecule in MET signaling (p-ERK1 / 2), was specifically detected in mesothelial cells from Allo_D14 and Allo_D35 lungs, consistent with the observed p-MET pattern. Figure 4F ).

[0141] To investigate the functional role of basophil-derived HGF in mesothelial cell proliferation, HGF derived from basophils was used... Wt1- CreER; Ai14 F / + RFP isolated from mice + A Transwell co-culture system was established using mesothelial cells. Basophils were seeded in the upper chamber of the Transwell and co-cultured with mesothelial cells in the lower chamber. Treatment with recombinant HGF protein or co-culture with activated basophils significantly increased Ki67 levels. + The proportion of mesothelial cells ( Figure 4G and Figure 4H This effect was blocked by capmatinib (a selective MET inhibitor). Figure 4G and Figure 4H Similarly, in Transwell invasion assays, when mesothelial cells seeded in the upper chamber of a Transwell were co-cultured with HGF or activated basophils in the lower chamber, enhanced mesothelial cell invasion was observed, an effect that was significantly inhibited by carmatinib. Figure 4I and Figure 4J ).

[0142] In summary, these results indicate that basophil-derived HGF drives receptor mesothelial cell proliferation and invasion via MET signaling, thereby participating in mesothelial cell remodeling during CLAD progression.

[0143] Example 6 Inhibiting HGF / MET signaling slows the progression of CLAD. Given that the results indicated that basophil-derived HGF activates MET signaling in mesothelial cells, the next step was to investigate whether this signaling axis is conserved in human CLAD lungs. Immunostaining results showed that, compared with donor lungs, mesothelial cells from human CLAD lungs exhibited significantly increased expression of p-MET and p-ERK1 / 2. Figure 5A and Figure 5B These results support the idea that HGF / MET signaling drives the proliferation of human CLAD mesothelial cells.

[0144] Based on these findings, it was hypothesized that inhibiting HGF / MET signaling could alleviate tissue remodeling and CLAD progression. To test this hypothesis, recipient mice were administered carmatinib daily, an FDA-approved selective MET inhibitor (Wolf et al., 2024), starting on day 14 post-transplantation (a time point when mesothelial cell proliferation and tissue remodeling had become apparent). Figure 5C As detected by immunofluorescence staining, carmatinib treatment effectively blocked MET phosphorylation in mesothelial cells. Figure 5D Furthermore, downstream ERK1 / 2 activation was significantly reduced in mesothelial cells from mice treated with carmatinib. Figure 5E By day 35, compared with the solvent control group, mice treated with carmatinib showed a significant reduction in CLAD progression, accompanied by decreased pleural fibrosis and reduced mesothelial cell proliferation. Figures 5F to 5I Furthermore, the invasion of mesothelial cells into the donor lung parenchyma was significantly reduced, with WT1 cells within the lung parenchyma decreasing. + The reduction in mesothelial cells was confirmed.

[0145] To further verify the role of downstream signal transduction, mice were treated with ERK1 / 2 inhibitors starting on day 14 post-transplantation. Similar to carmatinib, ERK1 / 2 inhibition significantly reduced CLAD progression. Figures 7A-7F These results highlight the crucial role of the HGF / MET signaling axis in driving mesothelial cell-mediated lung tissue remodeling and underscore the therapeutic potential of targeting this pathway to halt CLAD progression.

[0146] discuss CLAD remains a major obstacle to long-term survival in lung transplant recipients, and current immunosuppressive therapies have failed to extend median survival beyond five years after diagnosis. In this invention, the inventors reveal the dynamic transformation of the immune landscape during CLAD progression, specifically the transition from an allogeneic immune injury phase to a late basophil-dominated tissue remodeling phase. In the late basophil-dominated tissue remodeling phase, HGF-secreting basophils drive receptor-derived mesothelial cell proliferation and invasion, establishing a pathological pathway that accelerates pleural fibrosis and CLAD progression. Figure 6 Importantly, pharmacological inhibition of the HGF / MET signaling axis significantly slowed the progression of CLAD, thus providing a promising therapeutic strategy for CLAD.

[0147] Traditionally, the pathogenesis of CLAD has been attributed to a chronic allogeneic immune response. However, lineage tracing results reveal that the expanded mesothelial cells leading to pleural fibrosis are receptor-derived, potentially enabling them to evade allogeneic immune surveillance. Activated basophils secrete HGF to drive mesothelial cell proliferation and invasion, establishing a self-sustaining feedback pathway that accelerates CLAD progression. These findings redefine mesothelial cells as active drivers of CLAD pathogenesis, shifting the focus from donor allogeneic immune damage to receptor-driven tissue remodeling and opening new therapeutic opportunities targeting non-allogeneic immune mechanisms.

[0148] This invention identified a persistent and progressive accumulation of activated basophils in the lungs of mice with CLAD, peaking at day 35 post-transplantation, consistent with extensive tissue remodeling. While basophils are traditionally associated with allergic inflammation and antiparasitic immunity, emerging evidence suggests their involvement in tissue remodeling and fibrosis. This invention expands this paradigm by demonstrating that basophils not only amplify the immune response but also directly promote mesothelial cell remodeling through HGF secretion. Furthermore, mast cells and Trem2 cells were observed at day 35 post-transplantation. + A significant increase in macrophages and monocytes, accompanied by elevated HGF expression, highlights the role of these cell populations as key mediators in the progression of late-stage CLAD, coordinating immune-driven inflammation and tissue remodeling.

[0149] This invention reveals a previously unknown driver of CLAD (claustrophobic leukemia), demonstrating that combination therapies targeting allogeneic immune responses and tissue remodeling pathways may offer a more effective strategy for improving long-term outcomes in lung transplant recipients. By elucidating the interactions between innate immune cells and tissue-resident cells, this work opens new avenues for therapeutic intervention.

[0150] References 1. Graham, CN, Watson, C., Barlev, A., Stevenson, M., andDharnidharka, VR (2022). Mean lifetime survival estimates following solidorgan transplantation in the US and UK. J Med Econ 25, 230-237. 2. Krupnick, AS, Lin, X., Li, W., Okazaki, M., Lai, J., Sugimoto, S., Richardson, SB, Kornfeld, CG, Garbow, JR, Patterson, GA, et al .(2009). Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology. Nat Protoc 4, 86-93. 3. Antony, VB, Owen, CL, and Hadley, KJ (1989). Pleuralmesothelial cells stimulated by asbestos release chemotactic activity for neutrophils in vitro. Am Rev Respir Dis 139, 199-206. Christie, JD, Van Raemdonck, D., and Fisher, AJ (2024). LungTransplantation. N Engl J Med 391, 1822-1836. 4. Martens, T., Schmidt, N.O., Eckerich, C., Fillbrandt, R.,Merchant, M., Schwall, R., Westphal, M., Lamszus, K. (2006). A Novel One-Armed Anti-c-Met Antibody Inhibits Glioblastoma Growth In vivo. Clin. CancerRes. 12 (20): 6144-6152. 5. Merchant M., Ma X., Maun H.R., Zheng Z., Peng J., Romero M., etal. Monovalent antibody design and mechanism of action of onartuzumab, a METantagonist with anti-tumor activity as a therapeutic agent. Proc Natl AcadSci U S A. 2013; 110: E2987–96. 10.1073 6. Mimura, T., Walker, N., Aoki, Y., Manning, C.M., Murdock, B.J.,Myers, J.L., Lagstein, A., Osterholzer, J.J., and Lama, V.N. (2015). Localorigin of mesenchymal cells in a murine orthotopic lung transplantation modelof bronchiolitis obliterans. Am J Pathol 185, 1564-1574. 7. Khatri, A., Todd, J.L., Kelly, F.L., Nagler, A., Ji, Z., Jain, V.,Gregory, S.G., Weinhold, K.J., and Palmer, S.M. (2023). JAK-STAT activationcontributes to cytotoxic T cell-mediated basal cell death in human chroniclung allograft dysfunction. JCI Insight 8. 8. Wolf, J., Hochmair, M., Han, J.Y., Reguart, N., Souquet, P.J.,Smit, E.F., Orlov, S.V., Vansteenkiste, J., Nishio, M., de Jonge, M., et al .(2024). Capmatinib in MET exon 14-mutated non-small-cell lung cancer: finalresults from the open-label, phase 2 GEOMETRY mono-1 trial. Lancet Oncol 25,1357-1370.

Claims

1. Use of carmatinib or a pharmaceutically acceptable salt thereof as a MET inhibitor in the preparation of a medicament for the treatment or prevention of chronic allogeneic lung graft dysfunction (CLAD) in lung transplant recipients or for the promotion of long-term survival of said recipients.

2. The use according to claim 1, wherein the CLAD is restricted allogeneic graft syndrome (RAS), pleural pulmonary parenchymal fibroelastosis (PPFE)-related CLAD, or CLAD accompanied by pleural fibrosis.

3. The use according to claim 1 or 2, wherein the treatment includes relieving, delaying or preventing the progression of CLAD, or relieving or reducing pleural fibrosis.

4. The use according to claim 1 or 2, wherein the drug further comprises one or more additional MET inhibitors selected from HMPL-504 / AZD6094 / voritinib (cevoritinib), MSC2156119J (EMD 1214063, terpoxtinib) and pharmaceutically acceptable salts thereof, and any combination thereof.

5. The use according to claim 1 or 2, wherein the subject has an allogeneic lung transplant.

6. The use according to claim 1 or 2, wherein the subject is a mammal, preferably a human.

Images