Chemokine ligand 18 (CCL18) for diagnosis and treatment of inflammatory lung disease

Abstract

Compositions and use in the treatment of lung diseases and disorders include agents targeting chemokines, such as, CCL18.

Description

CHEMOKINE LIGAND 18 (CCL18) FOR DIAGNOSIS AND TREATMENT OF INFLAMMATORY LUNG DISEASE

[0001] The present application claims the benefit of U.S. provisional application no. 63 / 621,399 filed January 16, 2024 which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under grants HL 151530 and HL132055 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy in various solid malignancies, providing significant improvements in survival for patients with advanced stage cancer (1). Yet, ICIs are also associated with unique toxicities, termed immune related adverse events (irAEs) (2-4). irAEs can occur in any organ system, but one irAE in particular - checkpoint inhibitor pneumonitis (CIP) - has been recently shown to be a) of higher incidence than previously suspected, especially in non-small cell lung cancer (NSCLC) patients (5,6); and b) independently associated with significant morbidity and mortality (7). Clinically, patients present with shortness of breath, dyspnea and, in more severe cases, hypoxemia requiring supplemental oxygen therapy through non-invasive means or mechanical ventilation (8). CIP clinically resembles acute lung injury (ALI), and a subset of CIP patients (deemed steroid-refractory / resistant CIP) progress to develop chronic interstitial findings and prolonged use of steroids and other immunosuppressants (9).SUMMARY

[0005] Compositions and methods of treating ling diseases or disorders are provided herein.

[0006] Accordingly, in certain aspects, a method of preventing or treating a lung disease or disorder in a subject, comprises administering to the subject a pharmaceutical composition comprising one or more agents in a therapeutically effective dose to modulate CCL18 chemokine levels; thereby treating the subject. In certain embodiments, the method further comprises administering one or more agents which inhibit M2 macrophage secretion of chemokines. Incertain embodiments, the one or more agents which selectively inhibit M2 macrophage secretion of chemokines inhibit CCR1 binding of CCR1 ligands or inhibit IL-4 levels or inhibit CXCL-10 levels or combinations thereof. In certain embodiments, CCR1 ligands comprise CCL3, CCL5, or CCL8. In certain embodiments, the lung disease or disorder comprises checkpoint inhibitor pneumonitis (CIP), asthma, pneumonia, chronic obstructive pulmonary disease (COPD), bronchitis, acute respiratory distress syndrome (ARDS), interstitial lung disease (ILD), inflammation or lung cancer. In certain embodiments, the lung disease or disorder is checkpoint inhibitor pneumonitis (CIP). In certain embodiments, macrophages comprising markers CD45+FSC-AhlCD3 CD206+are increased in subjects having CIP as compared to a healthy subject. In certain embodiments, distinct macrophage subsets are detected in subjects with CIP. In certain embodiments, the macrophage subsets comprise alveolar macrophages and interstitial macrophages. In certain embodiments, the alveolar macrophages express CD1 lb+CD169hl. In certain embodiments, the interstitial macrophages express CDl lb+CD16910. In certain embodiments, the interstitial macrophages are increased in subjects having CIP as compared to a healthy subject. In certain embodiments, macrophages expressing M2 markers comprising CD163 and CD206 and low levels of Ml marker CD80 are increased in subjects having CIP as compared to a healthy subject. In certain embodiments, the one or more agents comprise small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds.

[0007] In another aspect, a method of treating a subject diagnosed with cancer, comprises administering to the subject a pharmaceutical composition comprising one or more agents in a therapeutically effective dose to modulate CCL18 chemokine levels; thereby treating the subject. In certain embodiments, the subject is undergoing immunotherapy In certain embodiments, the pharmaceutical composition comprising one or more agents decreases CCL18 chemokine levels. In certain embodiments, the pharmaceutical composition is administered prior to, during the course of, after immunotherapy or combinations thereof, has been administered to the subject. In certain embodiments, the pharmaceutical composition is administered as an aerosol or nebulized formulation. In certain embodiments, the agent is administered intra-subcutaneous (s.c ), intravenous (i.v .), intramuscular (i.m.), intravitreal (i. v.i.). intra-ci sterna magna (i.c.m.). or intrast ernal injection.

[0008] In another aspect, a method of identifying candidate agents which inhibit CC 18 comprise: contacting a cell expressing CCL18, a substrate comprising CCL18 or fragments thereof, or a solution comprising CCL18 or fragments thereof; conducting assays to measure decreases in CCL18 expression or binding to CCL18 or fragments thereof; thereby identifying candidate agents. In certain embodiments, the assays comprise immunoassays, : immunoassays, Southern blots, Western blots, polymerase chain reaction (PCR), Northern blots, sequencing, reverse-transcriptase PCR, microarray technology, immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry mass spectrometry, Forster resonance energy transfer, time-resolved fluorescence energy transfer, amplified luminescent proximity homogeneous assay, fluorescence polarization, cell-based assays or combinations thereof. In certain embodiments, the assay is a high throughput screening (HTS) assay.

[0009] Definitions

[0010] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, cell culture, molecular genetics, and biochemistry).

[0011] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0012] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical rangesby endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0013] As used herein, by “administering” is meant a method of giving a dosage of a composition described herein (e.g., CCL18 and / or a pharmaceutical composition thereof) to a subject. The compositions utilized in the methods described herein can be administered by any suitable route, including, for example, by inhalation, nebulization, aerosolization, intranasally, intratracheally, intrabronchially, orally, parenterally (e.g., intravenously, subcutaneously, or intramuscularly), orally, nasally, rectally, topically, or buccally. In some embodiments, a composition described herein is administered in aerosolized particles intratracheally and / or intrabronchially using an atomizer sprayer (e.g., with a MADgic® laryngo-tracheal mucosal atomization device).

[0014] As used herein, the terms “agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2 fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; hormones, oligonucleotides, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA- approval.

[0015] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0016] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0017] In the descriptions herein and in the claims, phrases such as “at least one of’ or “one or more of’ may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0018] As used herein, the term “cellular immune response” can be used interchangeably with the term “cell-mediated immune response” and refers to an immune response that does not predominantly involve antibodies. Instead, a cellular immune response involves the activation of different immune cells (e.g., phagocytes and antigen-specific cytotoxic T-lymphocytes) that produce various effector molecules (e.g., cytokines, perforin, granzymes) upon activation (e.g., via antigen stimulation). As used herein, the term “humoral immune response” refers to an immune response predominantly mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. The term “antibody- mediated immune response” refers to an aspect of a humoral immune response that is mediated by antibodies.

[0019] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements,thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements-or, as appropriate, equivalents thereof-and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0020] An “immune response,” as used herein, refers to a biological response within a vertebrate against foreign agents, e.g., virus, or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell. Accordingly, an immune response can comprise a humoral immune response (e.g., mediated by B-cells), cellular immune response (e.g., mediated by T cells), or both humoral and cellular immune responses. In some aspects, an immune response is an “inhibitory” immune response. An “inhibitory” immune response is an immune response that blocks or diminishes the effects of a stimulus (e.g., antigen). In certain aspects, the inhibitory immune response comprises the production of inhibitory antibodies against the stimulus. In some aspects, an immune response is a “stimulatory” immune response. A “stimulatory” immune response is an immune response that results in the generation of effectors cells (e.g., cytotoxic T lymphocytes) that can destroy and clear a target antigen (e.g., tumor antigen or viruses).

[0021] As used herein, the term “immune cells” refers to any cells of the immune system that are involved in mediating an immune response. Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof. In some aspects, an immune cell expresses CD3. In certain aspects, the CD3 -expressing immune cells are T cells (e.g., CD4+T cells or CD8+T cells). In some aspects, an immune cell that can be targeted with a targeting moiety (e.g., anti-CD3) comprises a naive CD4+T cell. In some aspects, an immune cell comprises a memory CD4+T cell. In some aspects, an immune cell comprises an effector CD4+T cell. In some aspects, an immune cell comprises a naive CD8+T cell. In some aspects, an immune cell comprises a memory CD8+T cell. In some aspects, an immune cell comprises an effector CD8+T cell. In some aspects, an immune cell is a dendritic cell. In certain aspects, a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDCl), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.

[0022] As used herein, the term “modulate” includes to “increase” or “decrease” one or more quantifiable parameters, optionally by a defined and / or statistically significant amount. By “increase” or “increasing,” “enhance” or “enhancing,” or “stimulate” or “stimulating,” refers generally to the ability of one or more, e.g. CCL18 compositions, in accordance with the present disclosure to produce or cause a greater physiological response (i.e., downstream effects) in a cell or in a subject relative to the response caused by either no CCL18 composition or a control compound. Relevant physiological or cellular responses (in vivo or in vitro) upon administration of CCL18 compositions will be apparent to persons skilled in the art. An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times), including all integers and decimal points in between and above 1 (e.g., 1.5, 1.6, 1.7. 1.8), the amount produced by no CCL18 composition or a control compound. The term “reduce” or “inhibit” may relate generally to the ability of one or more CCL18 compositions to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of a disease, illness or condition, such as cancer, inflammation or pain. A “decrease” in a response may be “statistically significant” as compared to the response produced by no CCL18 composition or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0023] As used herein, the term “T cell” or “T-cell” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T-cells include all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg), and gamma-delta T cells. Among the sub-types and subpopulations of T cells and / or of CD4+and / or of CD8+T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCMX central memory T (TCM effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as THI cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0024] A “naive” T cell refers to a mature T cell that remains immunologically undifferentiated (i.e., not activated). Following positive and negative selection in the thymus, T cells emerge as eitherCD4+ or CD8+naive T cells. In their naive state, T cells express L-selectin (CD62L+), IL-7 receptor-a (IL-7R-a), and CD 132, but they do not express CD25, CD44, CD69, or CD45RO. As used herein, “immature” can also refer to a T cell which exhibits a phenotype characteristic of either a naive T cell or an immature T cell, such as a TSCM cell or a TCM cell. For example, an immature T cell can express one or more of L-selectin (CD62L+), IL-7Ra, CD 132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, and LFA-1. Naive or immature T cells can be contrasted with terminal differentiated effector T cells, such as TEM cells and TEFF cells.

[0025] As used herein, the term “effector” T cells or “TEFF” cells refers to a T cell that can mediate the removal of a pathogen or cell without requiring further differentiation. Thus, effector T cells are distinguished from naive T cells and memory T cells, and these cells often have to differentiate and proliferate before becoming effector cells.

[0026] As used herein, the term “memory” T cells refer to a subset of T cells that have previously encountered and responded to their cognate antigen. In some aspects, the term is synonymous with “antigen-experienced” T cells. In some aspects, memory T cells can be effector memory T cells or central memory T cells. In some aspects, the memory T cells are tissue-residentmemory T cells. As used herein, the term “tissue-resident memory T cells” or “TRM cells” refers to a lineage of T cells that occupies tissues (e.g., skin, lung, gastrointestinal tract) without recirculating. TRM cells are transcriptionally, phenotypically and functionally distinct from central memory and effector memory T cells which recirculate between blood, the T cell zones of secondary lymphoid organs, lymph and nonlymphoid tissues. One of the roles of TRM cells is to provide immune protection against infection in extra lymphoid tissues.

[0027] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0028] The terms “treatment”, or “treating” as used herein, is defined as the application or administration of, e.g. a CCL18 composition and / or combination of therapeutic agents (e.g., an anti-inflammatory agent, a cancer preventing and / or therapeutic agent (e.g., chemotherapeutic)) to a patient, or application or administration of, e.g. a CCL18 composition to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term “treatment” or “treating” is also used herein in the context of administering agents, e.g., a CCL18 composition prophylactically. The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0029] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Concentrations, amounts, cell counts, percentages and other numerical values may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to includenot only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0030] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, are intended to encompass homologous and / or orthologous genes and gene products from other species.

[0031] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic of sample collection for the study. * Patients were consented for samples collection at time of bronchoscopy by members of the study team (AW, AV, MG) not involved in the clinical adjudication process performed by the irTox team, and adjudication team was blinded to the research participation status.#Validation cohorts for scRNAseq - UZ-Leuven (Ctl: n=6, CIP: n=l 1); BALF chemokine analysis - MD Anderson (Ctl: n=3; CIP: n=3); Flow cytometry analyses - Gustave Roussy (CIP: n=5) ** After unblinding, n’s for Ctl (ICI+CIP-) and CIP groups by experiment type: scRNAseq - Ctl: 3, CIP: 3); Flow cytometry - Ctl: 4, CIP: 9; BALF chemokine analysis - Ctl: 5, CIP: 19.

[0033] FIGS. 2A-2D show increased macrophages in CIP BAL immune cells. FIG. 2A) UMAP plot showing scRNAseq clusters of BAL cells in ICI+CIP- (Ctl; n=3) and CIP (n=3) patients. FIG. 2B) Expression maps of macrophage markers FIG. 2C) Top 10 differentially expressed genes that identify each of the macrophage clusters shown in 1A. FIG. 2D) Schematic describing gating strategy for phenotyping myeloid cells in control and CIP BAL samples, and scatterplot showing mean (± SEM) of macrophages (% of CD45+cells) in BAL cell pellets of ICI+CIP- (Ctl) and CIP patients.

[0034] FIGS. 3A-3I show increased interstitial macrophages (IM) and IM subsets in CIP. FIG. 3A) Gating strategy for separating macrophages based on alveolar / interstitialdesignation or as Ml - or M2-like. FIGS. 3B-3C) Scatter plots showing % of alveolar (FIG. 3B) and interstitial (FIG. 3C) macrophages in the BAL samples of ICI+CIP- (Ctl) and ICI+CIP+ (CIP) patients. FIGS. 3D-3E) Scatterplot showing % of M2- like Macs (FIG. 3D) and M2-like interstitial macs (FIG. 3E) in Ctl and CIP BAL samples. FIG. 3F) Bar graphs showing relative contribution of ligand-receptor pairs involving the M2-associated receptor CCR1 (inset: cell-cell chord graph showing source-target relationship between T-cell clusters expressing the CCR1 ligand CCL5 and macrophage clusters (especially the C6 Macrophage cluster) FIG. 3G) Chord graph showing paracrine MIF- (CD74 / CXCR4) signaling targeting the CO Macrophage cluster. FIGS. 3H-3I) Validation of flow cytometric measurements in macrophages. FIG. 3H) Scatterplots showing FIG. 3H) BAL macrophage % and FIG. 31) BAL interstitial macrophage (IM) / alveolar macrophage (AM) ratio in controls as well as CIP patient samples from Hopkins (CIP- JHU) and CIP patient samples from Gustave-Roussy (CIP-GR).

[0035] FIGS. 4A-4G demonstrate that CCL18 transcript levels are increased in CIP. FIG. 4A) Expression plots showing expression of CCL18 transcript in CIP and control. FIG. 4B) Violin plots showing CCL18 transcript levels in various macrophage clusters in CIP and Control. FIGS. 4C-4D) Validation of CCL18 transcript expression in myeloid cells JHU: FIG. 4A. UZ-Leuven: FIG. 4C), Dotplot showing increased CCL18 expression in myeloid clusters and lack of CCL18 expression in other cell types FIG. 4D) Volcano plot of differentially expressed genes in myeloid clusters in the Leuven cohort showing CCL18 as a transcript that is differentially expressed in CIP samples. FIG. 4E) Scatterplot showing per-patient average CCL18 transcript expression in the combined scRNAseq dataset. FIG. 4F) Dotplot of 4C0CL18 expression in immune cells from various organs in the GT ex snRNAseq pilot study (n=16 donors) showing CCL18 expression restricted to immune cells in the lung. FIG. 4G) violin plot showing further restriction of CCL18 expression within the lung to activated macrophages.

[0036] FIGS. 5A-5F demonstrate that BALF CCL18 protein levels are increased in CIP and correlate with severity, especially in steroid-naive patients. Scatter plots showing mean±SEM BALF CCL18 protein levels in ICI+CIP- (Ctl) and ICI+CIP+ (CIP) patients in the Hopkins cohort. FIG. 5B) Scatterplot showing BALF CCL18 protein levels (measuring using identical ELISA method) in control and CIP patients the MD Anderson cohort. FIG. 5C) Receiver operating curve demonstrating performance of BALF CCL18 levels in discriminating between control and CIP. FIGS. 5D-5E) Scatterplots showing mean +SEM BALF CCL18protein levels in CIP patients in the Hopkins cohort stratified by FIG. 5C) type of ICI therapy and FIG. 5D) CIP grade (Low = less than Grade 2; High = greater than Grade 2). FIG. 5F) Scatterplot showing mean ±SEM CCL18 protein levels in CIP patients stratified by presence or absence of steroid therapy at time of bronchoscopy.

[0037] FIGS. 6A-6I demonstrate that PD-1 dependent CCL18 secretion by M2 macrophages exerts paracrine effects on Ml macrophages. FIG. 6A) Scatterplots showing secreted CCL18 levels in THP-1 cells treated with either isotype control, IL-4, Nivolumab or IL- 4+Nivolumab. FIG. 6B) Fold change (compared to either unstimulated or isotype control) in CCL18 secretion (i.e. supernatant CCL18 levels) in THP-1 cells following Nivo, IL-4 or Nivo+IL4 treatment. FIG. 6C) CD14 MFI in THP-1 cells following treatment with IFN-LPS, CCL18 or IFN-LPS+CCL18. FIG. 6D) Scatter plots showing CCR8+ Ml-like macs in Control (Ctl) or CIP BAL cell pellets. FIG. 6E) Fold change in IFN-LPS induced CXCL10 secretion in untreated (Ctl) or CCL18-treated THP-1 cells. FIG. 6F) Ligand-receptor analysis of CXCL family ligands / receptors showing significant interactions between CXCL 10 and its ligand CXCR3. FIG. 6G) Scatterplots showing BALF CXCL-10 levels in Ctl and CIP samples from Hopkins and Leuven cohorts. FIG. 6H) Correlation between BALF CXCL-10 and CCL18 levels in samples from Hopkins cohort where both chemokines were measured in the same sample. FIG. 61) Scatterplot showing relationship between BALF CCL18 levels and BAL lymphocyte % (FIG. 6F) in steroid-naive patients (i.e. patients who did not have steroid therapy on board at time of CIP diagnosis / bronchoscopy).

[0038] FIGS. 7A-7H demonstrate that exogenous CCL18 is sufficient to induce CIP-like phenotype in mice. FIGS. 7A-7J) Scatterplots showing mean mean±SEM % of FIGS. 7A, 7E) AM. FIGS. 7B, 7F) IM. FIGS. 7C, 7G) CD4+, and FIGS. 7D, 7H) CD8+ in BAL specimens (top row) and lung homogenate (bottom row) specimens of WT and PD1- / - treated with CCL18 (2mg / kg) for 48h.

[0039] FIG. 8A shows % live cells in control and CIP human BAL samples FIG. 8B) Gating strategy for THP-1 cells. FIG. 8C) Unstained controls and positive gates for THP-1 flow cytometry.

[0040] FIG. 9 shows the gating strategy for mouse BAL and lung homogenate samples.

[0041] FIG. 10A shows dotplots showing macrophage subpopulations in Control and CIP. FIG. 10B) UMAP plot showing Human Lung CellRef Map with adjudicated cell clusters.FIG. IOC) Mapping of the current scRNAseq dataset to the reference Lung CellRef dataset. FIG. 10D) UMAP plot and violin plot (inset) showing increased interstitial macrophages (IMs) in the reference-mapped data.

[0042] FIGS. 11A-11D show CCL18 flow cytometry CIP BALF. FIG. 11A) Gating strategy for defining CCL18+ gate. FIGS. 11B-11D) Gating strategy / scatterplots showing % CCL18+cells following gating for (FIG. 11B) Macrophages, FIG. 11C) alveolar macrophages and FIG. 11D) interstitial macrophages.

[0043] FIG. 12A shows BALF CCL18 concentrations normalized to BALF urea. FIG. 12 B) BALF CCL18 levels in ICI+CIP- (Ctl) patients compared to CIP patients not on steroids and CIP Patients on steroids.

[0044] FIG. 13A shows scatter plots demonstrating the secreted CCL18 levels in the media of THP-1 cells treated with either isotype control or Nivolumab, with and without IL-4 stimulation FIG. 13B) Effect of CCL18 treatment on CD206 and CD209 expression in unstimulated and IL-4 stimulated, differentiated THP-1 cells.

[0045] FIG. 14A shows the gating strategy for Ml -like macrophages based on CD209 and CD163 expression as well as expression of IL-ip and HLA-DR. FIG. 14B) % Ml-like Macs in Control and CIP samples.

[0046] FIG. 15A shows the CCR8 gate FIG. 15B) CCR8+ macrophages and CCR8+ M2 mac populations in Ctl and CIP patients.

[0047] FIG. 16A is a line plot showing percent weight in WT and PD1- / - mice treated intratracheally (i.t.) with either PBS (Control) or CCL18 (2mg / kg). FIG. 16B) Scatter plots showing weight change at 48h in WT and PD1- / - mice with and without i.t. CCL18 treatment. FIG. 16 C) Scatter plot showing BAL albumin values for WT and PD1- / - mice with and without CCL18 treatment.

[0048] FIG. 17 is a scatterplot showing correlation between BALF CCL18 and BAL monocyte % in steroid-naive CIP patients.

[0049] FIG. 18A is a heatmap and contour plots showing increased expression of pro- fibrotic genes in the macrophages from cluster 0. FIG. 18B) Scatter plot showing FN+ interstitial macrophages (IM) in Control and CIP BAL samples. FIG. 18C) Bar plots showing relative contribution of various FN1 ligand-receptor pairs. FIG. 18D) Chord plot showing source of (Macs, CO and C4) and targets (CD8 cells) of FN.

[0050] FIG. 19A a schematic of lentivirus constructs expressing either eGFP (LV-GFP) or co-expressing CCL18 and eGFP (LV-CCL18) and %GFP+macrophages in freshly isolated alveolar macrophages (AM) infected with LV-GFP. FIG. 19B is a histogram showing GFP transduction efficiency at 2.5 MOI of LV-GFP in freshly isolated alveolar macrophages. FIG. 19C shows IL-1|3 mRNA levels in CCL18 overexpressing AMs.

[0051] FIG. 20A depicts flow plots and histogram showing %GFP+macrophages isolated from the lungs of a mice 5d after i.t. LV-GFP treatment (compared to untreated control). GFP+absent in CD45" cells and robust in macrophages (CD45+F4 / 80+). Across CD45+cell types, the majority of GFP+cells were alveolar macrophages, showing tropism of the viral vector (LV) for CD45 cells, and more specifically alveolarjnacrophages. FIG. 20B shows a Western blot showing secreted CCL18 protein in the BAL fluid of LV-CCL18 treated mice at 5 days and FIG. 20C shows a graph showing % weight loss in mice on days 1-5 after i.t. LV-GFP and LV- CCL18 (n=7-9 animals per group) inset: Gross appearance of lungs at time of harvest (D5). FIG. 20D are bar graphs showing increased BAL total protein and cell count in LV-CCL18 treated mice at D5. FIG. 20E shows bar graphs showing decreased BAL Treg number and decreased BAL Treg CD25 MFI in mice at d5 after i.t. LV-CCL18

[0052] FIG. 21 depicts a series of bar graphs showing increased BAL levels of alveolar macrophages, interstitial macrophages, CD4+cells and CD8+cells in the BAL of LV-CCL18 treated mice at d5.DETAILED DESCRIPTION

[0053] The diagnosis of CIP is currently made by excluding alternate etiologies like infection and heart failure and is not guided by specific biomarkers. Furthermore, current treatment actions for CIP (i.e. discontinuation of ICI, high-dose steroids) risk significant sideeffects and adverse outcomes (including increased risk of tumor recurrence). The imprecision associated with diagnosing and managing CIP is in part due to the lack of understanding of the pathobiology of this morbid irAE.

[0054] Accordingly, this disclosure is directed in part to elucidating mechanisms of alveolar inflammation in CIP and is applicable in other lung diseases or disorders. As discussed in detail in the examples section which follows, in a prospective cohort of CIP patients and controls, bronchoalveolar lavage immune cell populations were examined to identify subsets ofpro-inflammatory macrophages as well as increases in the macrophage secreted chemokine CCL18. Briefly, the results obtained how link CCL18 and pro- inflammatory macrophage skewing in the pathobiology of CIP.

[0055] MACROPHAGE PLASTICITY AND POLARIZATION

[0056] Macrophages are cells that have the highest plasticity of the hematopoietic system. They are involved both in innate immunity (phagocytosis capacity) and in adaptive immunity (cell polarization), but also in ontogeny, in homeostasis and in tissue repair (Mantovani, A., et al. (2013). Macrophage plasticity and polarization in tissue repair and remodelling. J. Pathol. 229, 176-185; Wynn, T. A., etal. (2013). Macrophage biology in development, homeostasis and disease. Nature 496, 445-455). Macrophages are present in all tissues. They have a large phenotypic and functional diversity. During ontogeny, these cells also exhibit a diversity of origins which persists into adulthood. In the tissues, monocytes- macrophages respond to environmental stimuli (product from microbial infection, damaged cells, activated lymphocytes) and acquire distinct phenotypes. For a long time, these cells have been classified according to their function in a binary manner in connection with the inflammatory condition.

[0057] Depending on stimuli monocyte-macrophage received, they reprogram their transcriptome, resulting in distinct functional and phenotypic spectra. Macrophages are categorized simplistically into 2 sub-populations or states of polarization (or activation): classical activation phenotype Ml and the alternative activation phenotype M2 (Gabrilovich, D. I., et l. (2012). Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 12, 253-268.). The Ml classification is associated in vitro with the use of IFNy factor alone or in combination with microbial factors such as LPS or inflammatory cytokines such as TNF-a and GM-CSF. The polarization M2 is rather associated with the IL-4 or IL-13. Other cytokines are also identified as inducing M2 type polarization such as IL-33, which induces overexpression of Argl (arginase 1), CCL24 or CCL17 playing a role in inflammation. The IL-21 and more commonly CSF1 are major players in the polarization of macrophages. Macrophages may also acquire the status of “M2-like”, sharing common characteristics of M2. In fact, a large number of stimuli such as immune complexes associated with LPS, IL-1, glucocorticoids, TGF 0, Wnt5a and IL-10 result in a functional phenotype of type “M2 -like”.

[0058] Similarly, in vivo studies have shown the existence of Ml, M2 and M2 -like macrophages. These subtypes represent only the extremes on a continuum of functional states that must be integrated in an environmental complex system .CD64, a high-affinity Fc-y receptor, is known as a marker of Ml macrophages, and CD163 and CD206 have been identified as major markers of M2 macrophages. The expression of these surface makers is closely associated with pathogen phagocytosis and inflammatory responses.

[0059] Generally, Ml macrophages present IL-12hlgh, IL-23hlghand IL-10lowphenotype and produce molecular effectors such as reactive oxygen species (ROS) and intermediates of Nitric Oxide (NO) and inflammatory cytokines (IL-10, TNF-a, IL-6). Ml macrophages participate in Thl responses, play a role in resistance against intracellular parasites and are key effectors in the elimination of tumor cells. In contrast, M2 macrophages have an IL-12low, IL- 23lowand IL-10hlghphenotype with a variability in the production of inflammatory cytokines according to stimuli present in the environment. M2 cells display on their surface a strong expression of scavenger, mannose and galactose-type receptors. The metabolism of arginine is changed to an ornithine and polyamines metabolism. M2 macrophages are generally associated to a Th2 type response, to a parasite clearance, to a decrease of inflammation, to tissue repair promotion, angiogenesis, tumor growth and immune regulation.

[0060] Ml and M2 also have distinct expression profdes of chemokines. Ml macrophages express CXCL9 and CXCL10 chemokines which are known for attracting Thl, while M2 macrophages express CCL17, CCL22 and CCL24. Chemokines such as CCL2 and CXCL4 can also polarize macrophages to an M2-like phenotype.

[0061] Depending on their polarization state, macrophages have different characteristics in terms of iron, folate and glucose metabolisms. For example, Ml express large amounts of proteins involved in iron storage, such as Ferritin, while they express only weakly Ferroportin, involved in the iron exportation to the extracellular medium. In contrast, M2 macrophages express low levels of Ferritin but high levels of Ferroportin. This difference can result in functional outcomes, such as a bacteriostatic effect of Ml (protection against infection) and an effect promoting tissue repair by M2 macrophages, which also promote the tumor growth, as observed in some studies. The management of iron by macrophages according to their polarity is an important element underlining the importance of controlling the polarization of macrophages according to the condition of an individual.

[0062] Similarly, macrophages face an oxygen gradient in tissues under normal or pathological conditions. Macrophages or monocytes adapt to this gradient by modifying their glycolytic metabolism. The HIF1 and 2 are transcriptional factors leaders of these changes, including expression of chemokines or chemokine receptor CXCR4 or CXCL12 and VEGF (an angiogenic factor). Macrophages are involved in the tissue response to hypoxic conditions.

[0063] MACROPHAGES AND INFLAMMATORY LUNG DISEASES

[0064] Macrophages play an important role in the innate and adaptive immune responses of organ systems, including the lungs, to particles and pathogens. Cumulative results show that macrophages contribute to the development and progression of acute or chronic inflammatory responses through the secretion of inflammatory cytokines / chemokines and the activation of transcription factors in the pathogenesis of inflammatory lung diseases, such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ARDS related to COVID- 19 (coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), allergic asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (LPF).

[0065] Macrophages are abundantly present in the lung microenvironment, where they are mainly found as alveolar macrophages (AMs) and interstitial macrophages (IMs) (Saradna, A. etal., Macrophage polarization and allergic asthma. Transl. Res. 2018, 191, 1-14). Circulating monocytes are known to act as a new source of macrophages when AMs are damaged (Okuma, T. et al., C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin- induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinases. J. Pathol. 2004, 204, 594-604. Tsou, C.-L. et al. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J. Clin. Investig. 2007, 117, 902-909) [5,6], AMs are polarized into Ml and M2 phenotype macrophages, whereby Ml-type macrophages play a pivotal role in the pro-inflammatory reactions of host defense, and M2 -type macrophages contribute to anti-inflammatory responses and tissue remodeling (Liu, Y.-C. et al., Macrophage Polarization in Inflammatory Diseases. Int. J. Biol. Sei. 2014, 10, 520-529). Thus, regulation of macrophages is important for both pro-inflammatory and anti-inflammatory effects. Accumulating evidence has shown that the polarization of macrophages, which is regulated by cytokines, chemokines, and transcription factors, is closely related to the initiation anddevelopment of pulmonary inflammatory diseases, such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), COVID-19 (coronavirus disease 2019)-related ARDS, allergic asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) (Lee J-W., et al. The Role of Macrophages in the Development of Acute and Chronic Inflammatory Lung Diseases. Cells. 2021; 10(4):897. doi.org / 10.3390 / cellsl0040897).

[0066] Macrophages in ALI / ARDS: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS, known as ALI clinical symptoms) are recognized as serious public health issues as they result in respiratory failure and high mortality rates. ALLARDS has been known to originate from various factors, such as bacteria, chemicals, and viruses, and are characterized by leukocyte aggregates and alveolar epithelial damages [12,13], Various pharmacologic treatments, including glucocorticoids, anti-inflammatory agents, and antioxidants, have been tested in clinical trials for ARDS (Yang, C.-Y et al. New Insights into the Immune Molecular Regulation of the Pathogenesis of Acute Respiratory Distress Syndrome. Int. J. Mol. Sci. 2018, 19, 588). However, there is currently no effective drug therapy for these disorders.

[0067] Neutrophils and macrophages exert host defense against microbial invasion. A neutrophil influx into the lungs is a hallmark of ALLARDS (Zemans, R.L. and Matthay, M.A. What drives neutrophils to the alveoli in ARDS? Thorax 2017, 72, 1-3), and activated neutrophils cause inflammatory responses and tissue damage by producing toxic molecules and cytokines (Yang, S. C., et al. (2021). Understanding the role of neutrophils in acute respiratory distress syndrome. Biomedical Journal, 44(4), 439-446). In the pathogenesis of ALLARDS, alveolar macrophages (AMs) are polarized into M1 / M2 macrophages and participate in the exudative phase, rehabilitation phase, and, finally, the fibrotic phase.

[0068] Macrophages in COVID-19-Related ARDS: Macrophage Toll-like receptors (TLRs) recognize single-stranded RNA (ssRNA) fragments of SARS-CoV-2 (Booz, G.W.; et al. Macrophage responses associated with COVID-19: A pharmacological perspective. Eur. J. Pharmacol. 2020, 887, 173547. Ojo, A.S. et al. Pulmonary Fibrosis in COVID-19 Survivors: Predictive Factors and Risk Reduction Strategies. Pulm. Med. 2020, 2020, 1-10), and this recognition could increase the expression of inflammatory cytokines, chemokines, and growth factors, including TNF-a, IL-10, IL-6,7,8,9,10, MCP-1, and granulocyte-macrophage colonystimulating factor (GMCSF) in cases of COVID-19 (Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497-506).

[0069] Recent studies have indicated that increased levels of macrophage-associated cytokines lead to lung inflammation and cytokine storm, and these increases in cytokines are closely correlated with increased disease severity (Chua, R.L. et al. COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis. Nat. Biotechnol. 2020, 38, 970-979. Wang, J. et al. Cytokine storm and leukocyte changes in mild versus severe SARS-CoV-2 infection: Review of 3939 COVID-19 patients in China and emerging pathogenesis and therapy concepts. J. Leukoc. Biol. 2020, 108, 17-41. Hojyo, S. et al. How COVID-19 induces cytokine storm with high mortality. Inflamm. Regen. 2020, 40, 1-7. Bonam, S.R et al. Potential immuno-nanomedicine strategies to fight COVID-19 like pulmonary infections. Nano Today 2021, 36, 101051). The high increase in pro-inflammatory cytokines, including TNF-a and IL-10, and in chemokines, such as IL-8, have been confirmed in the upper respiratory tract (Liao, M. et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nat. Med. 2020, 26, 842-844). Furthermore, it has been reported that macrophages exert a strong inflammatory reaction in the lower airways compared to the upper respiratory tract. These results suggest that macrophages-derived pro-inflammatory cytokines play an essential role in cytokine storms, and thus these molecules are thought to be associated with COVID-19-associated ARDS mortality. Pulmonary fibrosis, along with cytokine storms, accompany SARS-CoV-2 (Gracia-Hernandez, M. et al. Targeting Macrophages as a Therapeutic Option in Coronavirus Disease 2019. Front. Pharmacol. 2020, 11, 577571). M2 -type macrophages are thought to play an important role in the fibrosis of COVID-19.

[0070] Macrophages in Allergic Asthma: Airway inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR) are the major symptoms of allergic asthma and are profoundly impacted by Th2 cytokines, such as IL-4, IL-5, and IL-13, and chemokines (Becerra- Diaz, M. et al. Androgen and Androgen Receptor as Enhancers of M2 Macrophage Polarization in Allergic Lung Inflammation. J. Immunol. 2018, 201, 2923-2933). The increased infiltration of eosinophils in the lungs is recognized as a hallmark of allergic asthma, resulting in excessive mucus secretion and AHR (Yang, H. et al. Protective effect of lolkinolide B on LPS-induced mouse acute lung injury. Int. Immunopharmacol . 2015, 26, 119-124). C-C motif cytokine ligand (CCL)l 1 / eotaxin, specifically eosinophil chemokine, is crucial for eosinophil recruitment into inflammatory sites in allergic asthma (Kim, J. etal. Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust ContainingCockroach Allergens. J. Immunol. 2001, 167, 2808-2815. Pope, S.M.; et al. The Eotaxin Chemokines and CCR3 Are Fundamental Regulators of Allergen-Induced Pulmonary Eosinophilia. J. Immunol. 2005, 175, 5341-5350). CCL11 is thought to be produced by Th2 cells via IL-4 / IL-13 signaling [Kuperman, U.A. etal. Interleukin-4, interleukin- 13, signal transducer and activator of transcription factor 6, and allergic asthma. Curr. Mol. Med. 2008, 8, 384-392), and the suppression of IL-4 and IL-13 results in decreased not only CCL11 expression but also reduced pulmonary eosinophilia (Hosokawa, Y. et al. IL-4 Modulates CCL11 and CCL20 Productions from IL- I P-Stimulated Human Periodontal Ligament Cells. Cell. Physiol. Biochem. 2016, 38, 153-159. Miyagawa, Y.; Murakami, A.; Ebihara, N. The proteolytic effect of mast cell tryptase to eotaxin-l / CCLl 1 eotaxin-2 / CCL24 and eotaxin-3 / CCL26 produced by conjunctival fibroblasts. Jpn. J. Ophthalmol. 2019, 63, 215-220).

[0071] Th2 cytokines, such as IL-4 and IL- 13, are major inducers of M2 macrophage polarization, and M2 -type macrophages produce inflammatory cytokines and eosinophilrecruiting chemokines affecting airway inflammation, remodeling, and mucus hypersecretion and resulting in decreased lung function (Athari, S.S. Targeting cell signaling in allergic asthma. Signal Transduct. Target. Ther. 2019, 4, 1-19). Airway epithelial cell-derived IL-33 following exposure to an allergen was shown to induce airway inflammatory response with increased cytokines / chemokines and M2 macrophage polarization. Collectively, the increase in M2 macrophage polarization in the lungs reflects asthma severity, leading to the development of an allergic inflammatory response.

[0072] Macrophages in COPD: COPD is characterized by chronic bronchitis, oxidative stress, mucus hypersecretion, emphysema, and persistent airflow limitation and respiratory infection makes breathing much more difficult leading to further lung damage in the pathogenesis of COPD (Guiedem, E. et al. Chronic obstructive pulmonary disease (COPD): Neutrophils, macrophages and lymphocytes in patients with anterior tuberculosis compared to tobacco related COPD. BMC Res. Notes 2018, 11, 192).

[0073] Cumulative evidence indicates that increased numbers of macrophages are found in the sputum and lungs of COPD patients, suggesting this is related to the severity of the disease (Kohler, J.B. et al. Microenvironmental stimuli induce different macrophage polarizations in experimental models of emphysema. Biol. Open 2019, 8, bio040808. Grashoff, W.F. et al. Chronic obstructive pulmonary disease: Role of bronchiolar mast cells and macrophages. Am. J.Pathol. 1997, 151, 1785-1790. Finkelstein, R. et al. Alveolar inflammation and its relation to emphysema in smokers. Am. J. Respir. Crit. Care Med. 1995, 152, 1666-1672. Hogg, J.C.; etal. The Nature of Small-Airway Obstruction in Chronic Obstructive Pulmonary Disease. N. Engl. J. Med. 2004, 350, 2645-2653). AMs have been found in damaged regions of the lungs and play an important role in the development of chronic bronchitis and emphysema by regulating monocyte and neutrophil recruitment in the pathogenesis of COPD (Yamasaki, K. et al. Macrophage Phenotypes and Functional Responses: Role in the Pathogenesis of COPD. Int. J. Mol. Sci. 2018, 19, 582. Kapellos, T.S. et al. Dysregulated Functions of Lung Macrophage Populations in COPD. J. Immunol. Res. 2018, 2018, 1-19). Ml and M2 phenotype macrophages have been found in the lungs of COPD patients (Kapellos, T.S. et al. Dysregulated Functions of Lung Macrophage Populations in COPD. J. Immunol. Res. 2018, 2018, 1-19).

[0074] LPS, a contaminant of cigarette smoke, may induce Ml macrophage polarization (Boorsma, C.E. et al. Macrophage Heterogeneity in Respiratory Diseases. Medial. Inflamm. 2013, 2013, 1-19). Elevated levels of inducible nitric oxide synthase (iNOS) expression were found in the AMs of COPD patients (Ichinose, M. et al. Increase in Reactive Nitrogen Species Production in Chronic Obstructive Pulmonary Disease Airways. Am. J. Respir. Crit. Care Med. 2000, 162, 701-706). iNOS-induced NO and ROS are able to induce oxidative stress. Thus, Ml polarization-mediated iNOS promotes oxidative stress in COPD. Furthermore, accumulating evidence from research shows that the levels of IL- 1 p, IL-6, IL-8, and TNF-a are upregulated in COPD (Bucchioni, E. et al. High levels of interleukin-6 in the exhaled breath condensate of patients with COPD. Respir. Med. 2003, 97, 1299-1302. Daldegan, M. et al. Concentration of CCL11, CXCL8 and TNF -alpha in sputum and plasma of patients undergoing asthma or chronic obstructive pulmonary disease exacerbation. Braz. J. MedBiol. Res. 2005, 38, 1359-1365). These results are a reflection of Ml -type macrophage-derived cytokines being related to the development of COPD. These results indicate that Ml macrophage-induced cytokines contribute to the pathogenesis of COPD.

[0075] A recent study showed that M2 phenotype macrophages are dominant in the BALF of COPD patients with increased cytokines such as IL-4, IL- 13, IL-8, and IL 10 (Eapen, M.S. et al. Abnormal M1 / M2 macrophage phenotype profiles in the small airway wall and lumen in smokers and chronic obstructive pulmonary disease (COPD). Sci. Rep. 2017, 7, 1-12. Wang, Y. et al. Role of inflammatory cells in airway remodeling in COPD. Int. J. Chronic Obstr. Pulm.Dis. 2018, 13, 3341-3348). Several groups reported that smoking promotes M2 polarization of AMs and the expression of MMP12 (Boorsma, C.E. et al. Macrophage Heterogeneity in Respiratory Diseases. Medial. Inflamm. 2013, 2013, 1-19. Kumar, R. et al. Role of MAPK / MNK1 signaling in virus replication. Virus Res. 2018, 253, 48-61, Mohanta, T.K. etal. Molecular Insights into the MAPK Cascade during Viral Infection: Potential Crosstalk between HCQ and HCQ Analogues. BioMed Res. Int. 2020, 2020, 1-9). IL-4-induced M2 macrophages produce MMP12, which has been found to play a pivotal role in emphysema.

[0076] Macrophages in Idiopathic Pulmonary Fibrosis (IPF): IPF is a chronic lung disease that is characterized by progressive pulmonary scarring, fibrosis, and shortness of breath. Misharin et al. reported that profibrotic genes (Argl and MMP13) are increased in monocyte- derived AM in the progression of lung fibrosis (Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. J. Exp. Med. 2017, 214, 2387-2404). Cumulative evidence has shown that the M2 phenotype rather than the Ml phenotype is dominantly discovered in the lungs during IPF progression (Zhang, L. et al. Macrophages: Friend or foe in idiopathic pulmonary fibrosis? Respir. Res. 2018, 19, 170. Van Dyken, S.J.; Locksley, R M. Interleukin-4- and interleukin- 13 -mediated alternatively activated macrophages: Roles in homeostasis and disease. Annu. Rev. Immunol. 2013, 31, 317-343. Yao, Y et al. Chop Deficiency Protects Mice Against Bleomycin-induced Pulmonary Fibrosis by Attenuating M2 Macrophage Production. Mol. Ther. 2016, 24, 915-925. Wynn, T.A.; Vannella, K.M. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity 2016, 44, 450-462). M2 macrophages promote lung fibrosis progression. M2 macrophage-derived TGF-0 leads to promote lung fibrosis, and M2 macrophage depletion leads to amelioration of fibrosis. M2 macrophage polarization is induced by IL-4, IL- 13 and IL-33 (Duru, N.; Wolfson, B.; Zhou, Q. Mechanisms of the alternative activation of macrophages and non-coding RNAs in the development of radiation-induced lung fibrosis. World J. Biol. Chem. 2016, 7, 231-239. Mora, A.L. et al. Activation of Alveolar Macrophages via the Alternative Pathway in Herpesvirus- Induced Lung Fibrosis. Am. J. Respir. Cell Mol. Biol. 2006, 35, 466-473. Drake, L.Y.; Kita, H. IL-33: Biological properties, functions, and roles in airway disease. Immunol. Rev. 2017, 278, 173-184).

[0077] Accordingly, in certain embodiments, a method of preventing or treating a lung disease or disorder in a subject, comprises administering to the subject a pharmaceuticalcomposition comprising one or more agents in a therapeutically effective dose to modulate CCL18 chemokine levels. In certain embodiments, one or more agents which inhibit M2 macrophage secretion of chemokines are also administered. These agents selectively inhibit M2 macrophage secretion of chemokines inhibit CCR1 binding of CCR1 ligands or inhibit IL-4 levels or inhibit CXCL-10 levels or combinations thereof. In certain embodiments, the lung disease or disorder comprises checkpoint inhibitor pneumonitis (CIP), asthma, pneumonia, chronic obstructive pulmonary disease (COPD), bronchitis, acute respiratory distress syndrome (ARDS), interstitial lung disease (ILD), inflammation or lung cancer.

[0078] METHODS OF TREATMENT

[0079] In certain embodiments, the one or more agents modulate CCL18 chemokine levels; selectively inhibit M2 macrophage secretion of chemokines inhibit CCR1 binding of CCR1 ligands or inhibit IL-4 levels or inhibit CXCL-10 levels or combinations thereof.

[0080] The agents identified by the methods embodied herein can be formulated and compositions of the present disclosure may be administered in conjunction with one or more additional active ingredients, pharmaceutical compositions, or other compounds. The therapeutic agents of the present disclosure may be administered to an animal, preferably a mammal, most preferably a human.

[0081] The pharmaceutical formulations may be for administration by oral (solid or liquid), parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using ionophoresis or electroporation), transmucosal and systemic (nasal, vaginal, rectal, or sublingual), or inhalation routes of administration, or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0082] The compositions of the invention may be administered to animals by any conventional technique. The compositions may be administered directly to a target site by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. Other methods of delivery, e.g., liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form.

[0083] The compounds identified by this disclosure invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg / kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg / kg.

[0084] An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg / kg; preferably between 0.1 and 20 mg / kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine protease. The compounds may be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg / kg / day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect. Prodrugs of compounds of the present invention may be prepared by any suitable method. For those compounds in which the prodrug moiety is a ketone functionality, specifically ketals and / or hemiacetals, the conversion may be effected in accordance with conventional methods.

[0085] No unacceptable toxicological effects are expected when compounds, derivatives, salts, compositions etc., of the present invention are administered in accordance with the present invention. The compounds of this invention, which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.

[0086] Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0087] Dose: An effective dose of a composition of the presently disclosed subject matter is administered to a subject in need thereof. A “therapeutically effective amount” or a “therapeutic amount” is an amount of a therapeutic composition sufficient to produce a measurable response (e.g., a biologically or clinically relevant response in a subject being treated). The response can be measured in many ways, e.g. cytokine profiles, cell types, cell surface molecules, etc. Actual dosage levels of active ingredients in the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon the activity of the therapeutic composition, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The potency of a composition can vary, and therefore a “treatment effective amount” can vary. However, using the assay methods described herein, one skilled in the art can readily assess the potency and efficacy of a candidate compound of the presently disclosed subject matter and adjust the therapeutic regimen accordingly.

[0088] PHARMACEUTICAL COMPOSITIONS

[0089] Pharmaceutical compositions according to the present disclosure can be prepared in a variety of ways known to one of ordinary skill in the art. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscularinjection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable

[0090] This disclosure also includes pharmaceutical compositions which contain, as the active ingredient, antibodies, small molecules, nucleic acids, vectors and the like , in combination with one or more pharmaceutically acceptable carriers. The terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance. In making the compositions of the disclosure, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. In some embodiments, the carrier can be, or can include, a lipid-based or polymer-based colloid. In some embodiments, the carrier material can be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. As noted, the carrier material can form a capsule, and that material may be a polymer-based colloid.

[0091] The nucleic acid sequences of the disclosure can be delivered to an appropriate cell of a subject. This can be achieved by, for example, the use of a polymeric, biodegradablemicroparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 pm in diameter can be used. The polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell. A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5pm and preferably larger than 20pm). Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The nucleic acids can be incorporated alone into these delivery vehicles or co incorporated with tissue-specific antibodies. Alternatively, one can prepare a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI.

[0092] The therapeutic agents embodied herein, may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. They can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g, physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP / NF (United States Pharmacopeia and the National Formulary).

[0093] For administration by inhalation, the compositions described herein can conveniently be delivered in the form of an aerosol (e.g., through liquid nebulization, dry powder dispersion or meter-dose administration The aerosol can be delivered from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator canbe formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0094] For aqueous and other non-pressurized liquid systems, a variety of nebulizers (including small volume nebulizers) can be used to aerosolize the formulations. Compressor- driven nebulizers can utilize jet technology and can use compressed air to generate the liquid aerosol. Such devices are commercially available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pan Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic nebulizers generally rely on mechanical energy in the form of vibration of a piezoelectric crystal to generate respirable liquid droplets and are commercially available from, for example, Omron Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely upon either piezoelectric or mechanical pulses to respirable liquid droplets generate Commercial examples of nebulizers that RESPIRGARD II®, AERONEB®, AERONEB® Pro, and AERONEB® Go produced by Aerogen; AERX® and AERX ESSENCE™ produced by Aradigm; PORTA- NEB®, FREEWAY FREEDOM™, Sidestream, Ventstream and I-neb produced by Respironics, Inc.; and PARI LC-PLUS®, PARI LC-STAR®, and e-Flow7m produced by PARI, GmbH.

[0095] CANDIDATE THERAPEUTIC GENTS

[0096] In certain embodiments, a method of identifying candidate agents which inhibit CCL18 comprise: contacting a cell expressing CCL18, a substrate comprising CCL18 or fragments thereof, or a solution comprising CCL18 or fragments thereof; conducting assays to measure decreases in CCL18 expression or binding to CCL18 or fragments thereof.

[0097] Non limiting assays comprise immunoassays, immunoassays, Southern blots, Western blots, polymerase chain reaction (PCR), Northern blots, sequencing, reversetranscriptase PCR, microarray technology, immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry mass spectrometry, Forster resonance energy transfer, time-resolved fluorescence energy transfer, amplified luminescent proximity homogeneous assay, fluorescence polarization, cell-based assays or combinations thereof. In certain embodiments, the assay is a high throughput screening (HTS) assay.

[0098] Candidate / Test Agents'. Various candidate agents can be employed in the screening methods of the invention, including any naturally existing or artificially generatedagents. They can be of any chemistry class, such as antibodies, proteins, peptides, small organic compounds, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids, and various structural analogs or combinations thereof. In some embodiments, the screening methods utilize combinatorial libraries of candidate agents. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, beta-turn mimetics, nucleic acids, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL). Peptide libraries can also be generated by phage display methods.

[0099] Candidate agents include numerous chemical classes, though typically they are organic compounds including small organic compounds, nucleic acids including oligonucleotides, peptides or antibodies. Small organic compounds suitably may have e.g. a molecular weight of more than about 40 or 50 yet less than about 2,500. Candidate agents may comprise functional chemical groups that interact with proteins and / or DNA.

[0100] Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of e.g. bacterial, fungal and animal extracts are available or readily produced.

[0101] Chemical Libraries'. Developments in combinatorial chemistry allow the rapid and economical synthesis of hundreds to thousands of discrete compounds. These compounds are typically arrayed in moderate-sized libraries of small molecules designed for efficient screening. Combinatorial methods can be used to generate unbiased libraries suitable for the identification of novel compounds. In addition, smaller, less diverse libraries can be generated that are descended from a single parent compound with a previously determined biological activity.

[0102] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks,” such as reagents. For example, a linear combinatorial chemicallibrary, such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in a large number of combinations, and potentially in every possible way, for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

[0103] A “library” may comprise from 2 to 50,000,000 diverse member compounds. Preferably, a library comprises at least 48 diverse compounds, preferably 96 or more diverse compounds, more preferably 384 or more diverse compounds, more preferably, 10,000 or more diverse compounds, preferably more than 100,000 diverse members and most preferably more than 1,000,000 diverse member compounds. By “diverse” it is meant that greater than 50% of the compounds in a library have chemical structures that are not identical to any other member of the library. Preferably, greater than 75% of the compounds in a library have chemical structures that are not identical to any other member of the collection, more preferably greater than 90% and most preferably greater than about 99%.

[0104] The screening assays of the disclosure suitably include and embody, animal models, cell-based systems and non-cell based systems. Identified genes, variants, fragments, or oligopeptides thereof are used for identifying agents of therapeutic interest, e.g. by screening libraries of compounds or otherwise identifying compounds of interest by any of a variety of drug screening or analysis techniques. The gene, allele, fragment, or oligopeptide thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The measurements will be conducted as described in detail in the examples section which follows.

[0105] In some embodiments, a method of identifying candidate therapeutic agents comprises screening a sample containing the specific target molecule in a high-throughput screening assay.

[0106] In another embodiment, a method of identifying therapeutic agents comprises contacting: (i) a target molecule with a candidate therapeutic agent; determining whether (i) the agent modulates a function of the peptide or interaction of the peptide with a partner molecule; or (ii) the agent modulates expression and / or function of the nucleic acid sequence of the target as measured by the light emission assays embodied herein.

[0107] In another embodiment, a method of identifying candidate therapeutic agents for treatment of disease, comprises culturing an isolated cell expressing a target molecule, administering a candidate therapeutic agent to the cultured cell; correlating the target molecules expression, activity and / or function in the presence or absence of a candidate therapeutic agent as compared to control cells, wherein a drug is identified based on desirable therapeutic outcomes. For example, a drug which modulates levels of the target molecule whereby such levels are responsible for the disease state or the target molecule modulates the activity or amount of another molecule whether upstream or downstream in a pathway. In other examples the assays measure kinase activity. In other examples, the assay measure binding partners. In other examples, the assay measures amounts of candidate therapeutic agents which provide a desired therapeutic outcome.

[0108] Another suitable method for diagnosis and candidate drug discovery includes contacting a test sample with a cell expressing a target molecule and detecting interaction of the test agent with the target molecule, an allele or fragment thereof, or expression product of the target molecule an allele or fragment thereof.

[0109] In another embodiment, a sample, such as, for example, a cell or fluid from a patient is isolated and contacted with a candidate therapeutic molecule. The genes, expression products thereof, are monitored to identify which genes or expression products are regulated by the drug.

[0110] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their invention.EXAMPLES

[0111] EXAMPLE 1: MACROPHAGE CCL18 PROMOTES ALVEOLAR INFLAMMATION IN CHECKPOINT INHIBITOR PNEUMONITIS (CIP).

[0112] The imprecision associated with diagnosing and managing CIP is in part due to the lack of understanding of the pathobiology of this morbid irAEh. To address this gap, immune cells subsets in the bronchoalveolar lavage fluid (BALF) of CIP patients and controls (ICI- treated cancer patients without evidence of CIP) were examined. It was noted that the BALF inCIP patients was enriched for a variety of pro-inflammatory immune cell subsets (10). Globally, an increase in CD4 lymphocytes and a decrease in CD14+and CD16+monocytes was observed. Within the T-cell population, increased levels of conventional T-cell subsets (specifically central memory T-cells) was observed. Within the myeloid population, increased subsets of IL1 [3111myeloid cells were observed. This finding of pro-inflammatory myeloid cells was also recently observed in a second independent cohort (11). However, the granular details of which specific myeloid cell populations (e.g. macrophages) and the molecular mechanisms sustaining such pro- inflammatory immune cell states in CIP remained unknown. In particular, whether specific subsets of lung macrophages contribute to inflammation and lung injury / impaired lung repair in CIP was not clear.

[0113] Pro-inflammatory macrophages play key roles in acute lung injury as well as fibrosis. When examined by compartment (i.e. alveolar vs. interstitial), interstitial macrophages (IMs) have been shown to produce pro-inflammatory messengers (including IL-10, which is increased in CIP BAL cells) that participate in lung injury and fibrosis (13). Macrophages can also be phenotyped by polarization state (i.e. Ml-like vs. M2 -like). While M2-like macrophages in the lung have traditionally been considered to participate in repair from lung injury (14), recent data suggests that certain subsets M2-like macrophages can actually promote inflammation and fibrosis in the lung. It is likely that IMs can develop a pro-fibrotic phenotype by acquiring M2 -like markers (including mannose receptor - CD206) (16). Interestingly, this finding was noted in clinical samples and pre-clinical models of radiation-induced lung injury / fibrosis, an entity that bears many similarities to CIP including presentation, responsiveness to steroids, and progression to fibrosis in steroid-refractory cases. However, the specific roles played by these various macrophages subtypes (AM vs. IM; Ml- vs. M2 -like) in CIP was not known.

[0114] To further elucidate mechanistic underpinnings of pro-inflammatory macrophages in CIP, a prospective collection protocol of bronchoalveolar lavage samples in CIP patients and controls (i.e. ICI-treated patients without evidence of CIP; Figure 1) was undertaken. In this cohort, all cases of CIP were independently adjudicated by the Hopkins immune related toxicity team (irTox team) consisting of a pulmonologist, two oncologists and a radiologist. Flow cytometric analyses (using monocyte / macrophage targeted flow cytometric panels) were pairedwith cytokine assays of the bronchoalveolar lavage fluid as well as in vitro and in vivo models to more clearly understand the details of macrophage dysfunction in CIP. Importantly, validation of the key human BALF findings was performed in additional independent cohorts of CIP patients, as part of a partnership with three other institutions with expertise in diagnosing and managing CIP (The University of Texas MD Anderson Cancer Center - Houston, TX; Gustave Roussy Cancer Center- Paris, France; University Hospitals Leuven - Leuven, Belgium). Using the multi-center cohort, it was sought to determine the specific macrophage subsets that were enriched in CIP and specific proteins that may be playing a role in skewing macrophages towards a pro-inflammatory state in this disease.

[0115] It was hypothesized that macrophages would be increased in CIP BALF, and that CIP macrophages would be more skewed towards an M2 -like phenotype. It was further hypothesized that specific chemokines produced by pro-inflammatory macrophages in CIP may be promoting further alveolar inflammation by recruiting other cell types (including T-cells) to the lung. In addition to shedding light on the pathobiology of CIP, it was reasoned that elucidating these specifics of macrophage-secreted chemokines that are elevated in CIP might allow development of potential biomarkers for the diagnosis of CIP.

[0116] RESULTS

[0117] Clinical cohort: The details of the clinical cohort are presented in Table 1. Most patients in both the control (ICUCIP') and CIP groups were NSCLC patients receiving anti-PDl therapy (either Nivolumab or Pembrolizumab). Approximately 60% of patients were either former or active smokers in both groups, and there were no large imbalances in age or sex between the two groups.

[0118] Macrophages are increased in CIP BALF: To broadly delineate the extent of myeloid cell dysfunction in CIP, single cell RNA sequencing was performed on a subset of control and CIP samples (n=3 / group). It was observed that CIP samples were enriched for several clusters of myeloid cells (FIG. 2A). Examination of expression maps of common macrophage markers revealed that the clusters contained primarily macrophages, expressing markers such as FABP4, C1QB, C12QC and APOCI, particularly in cluster 0 (FIGS. 2B-2C). To validate this finding of increased macrophages in CIP BALF, flow cytometry of BALF samples from control and CIP patients, was performed using an optimized macrophage-focusedpanel. Using the gating strategy outlined in Figure 2D, it was observed that macrophages (defined here as CD45+FSC-AhiCD3 CD206+) were increased in CIP samples compared to control.

[0119] Distinct macrophage subsets are increased in CIP: To better understand the phenotype of CIP macrophages, macrophage phenotypes were examined using multi-parametric flow cytometry. Following gating for macrophages (as shown in Figure 2D), macrophage subsets were gated based on anatomic classifications (alveolar macrophage: Macs / CDl lb+CD169hlinterstitial macrophage: Macs / CDl lb+CD16910). The total macrophage pool and the AM / IM compartments were gated for M2 -like macrophages, based on CD163 / CD209 expression. This gating strategy is outlined in FIG. 3A. Differences were not observed in the % of alveolar macrophages in CIP BALF compared to control (FIG. 3B). However, IMs were significantly higher in CIP compared to control (FIG. 3C). When scRNAseq data was examined or M2- associated genes, it was noted that clusters upregulated in CIP (i.e. clusters 0,2, 4, 6) expressed M2 markers CD163 and MRC1 (CD206) and low levels of canonical Ml marker CD80 (FIG. IB). Indeed, when macrophages (regardless of AM / IM classification) were subdivided based on M1 / M2 likeness, there was a trend towards higher M2-like macrophages in CIP (FIG. 3E; p = 0.1). However, we observed increased M2-like macrophages in the IM subpopulation (FIG. 3E); in fact, M2-like IMs were virtually absent in the control BAL specimens. These data provide evidence that M2-polarized IMs may be more prominent in CIP BAL. Since CIP BAL specimens exhibit both lymphocyte and myeloid abnormalities, it was reasoned that M2 polarization in CIP may be fueled in part by secretion, either by macrophages themselves or other cell types, of chemokines known to promote and / or sustain inflammatory M2 skewing. For example, macrophage receptors like CCR1 (17), CD74 (18) and CXCR4 (19), when bound to their respective ligands, have been shown to initiate and / or sustain both M2 polarization and perpetuate a pro-inflammatory macrophage phenotype. To this end, cell-cell interactions were examined in the scRNASeq data using ligand-receptor analyses. As shown in FIG. 3F, among all CCL family ligand-receptor pairs, three significant interactions were observed, all involving the receptor CCR1, which has been previously shown to be associated with M2- skewed macrophages (20). The source of CCR1 ligands CCL3, CCL5 and CCL8 appears to be various T-cell cluster as well as other macrophage clusters (inset, FIG. 3F). Another chemokine associated with both cancer and pro-inflammatory macrophage signaling is macrophagemigration inhibitory factor (MIF), which is a known ligand for receptors CD74 and CXCR4. Significant sources of MIF were observed across a variety of clusters, with the target being cluster 0, a large cluster of macrophages found almost exclusively in CIP (FIG. 2A). These data provide evidence that paracrine signaling via chemokines such as CCL5 and MIF may be contributing towards the a) M2 skewing and b) continued alveolar persistence of a significant population of pro-inflammatory macrophages.

[0120] Next, external validation was sought for the macrophage phenotype findings. To accomplish this, the findings of increased macrophage % in the BAL of CIP patients were compared with an external cohort of CIP samples from Gustave-Roussy (CIP-GR) that were subjected to flow cytometry I mass cytometry. As shown in FIG. 3H, macrophages were similarly increased in the CIP BAL of patients from the Gustave-Roussy cohort (CIP-GR). The IM / AM ratio in the samples as well as in the validation cohort (CIP-GR) were examined. As shown in FIG. 31, an increase in the BAL IM / AM ratio was observed across two different cohorts of CIP patients.

[0121] Increased macrophage CCL18 transcript levels in CIP: The scRNAseq data was examined to more crisply define the transcriptional phenotype of the macrophages observed in the most prominently upregulated macrophage cluster in CIP - cluster CO. When the top differentially expressed genes in the macrophage clusters enriched in cluster CO were examined, significant expression of CCL18 (FIG. 2C) was observed. CCL18 is a chemokine previously observed to be associated with macrophages (in particular, M2-like macrophages). This was in line with observations of increased M2 skewing in CIP macrophages at the flow cytometry and scRNAseq cell -cell communication analyses (FIG. 3). Expression maps of CCL18 showed robust expression of CCL18 in multiple macrophage clusters that are increased in CIP. Notably, these clusters were also noted to be increased in CIP (FIGS. 4A, 4B). Before attempting to validate these findings at the protein levels, it was first sought to validate the scRNAseq data in an independent cohort. For this, we turned to the Leuven cohort (which included scRNAseq data on 11 CIP patients and 6 controls samples). This dataset was probed for CCL18 transcript levels. It was observed that CCL18 expression was largely restricted to myeloid cells (FIG. 4C). Differential expression (DE) of myeloid cell clusters in CIP samples (compared to controls) again showed a significant upregulation of CCL18 transcript in CIP (FIG. 3D). Since DE and cluster analyses of scRNAseq data relies on pooled cell populations from multiple patients,CCL18 transcript levels per biologic replicate (i.e. per patient) were quarried. To do this, CCL18 expression levels were averaged in myeloid clusters for each control and CIP patient in both the Hopkins and Leuven scRNAseq datasets. As shown in FIG. 4E, CCL18 levels (on a per patient basis) were higher in CIP patients compared to controls in the combined dataset. These findings provided evidence that, at least at a transcript level, macrophage CCL18 was increased in CIP. The specificity of CCL18 was determined and whether this could be a global phenomenon in all immune cells in CIP. To determine the specificity of CCL18 for lung immune cells, the GT ex consortium single cell immune atlas was leveraged, which provides organ and cell specific expression data in a cohort of 16 patients in whom single nuclear RNA sequencing had been performed. As shown in FIGS. 4F-4G, CCL18 expression is restricted to lung immune cells, and furthermore, is additionally restricted to the activated macrophages in the lung.

[0122] CCL18 protein levels are increased in CIP, and associate with clinical outcomes: Next, we wanted to gain further insight into CCL18 expression in macrophages using flow cytometry. For this, we optimized a CCL18 flow cytometry antibody for use in human macrophages (FIG. 11A). The population of CCL18+ cells was not significantly different in the total macrophage pool or in AM / IM subsets (FIGS. 1 IB- 1 ID). However, CCL18 is a secreted chemokine, and our BAL samples were prepared without the use of agents to block secretion of intracellularly produced chemokines (e.g. golgistop / golgiplug) due to concerns for effect on cell viability. Therefore, we questioned whether a) CCL18 secretion in the BAL supernatant (as opposed to intracellular expression in cells) was increased in CIP and b) whether M2-skewed macrophages were a source of secreted CCL18 production in CIP.

[0123] To examine CCL18 secretion, we measured CCL18 concentrations in the BAL supernatant of CIP patients (and controls). As shown in FIG. 5A, we observed a significant increase in BAL CCL18 levels in CIP BALF compared to controls. To account for variability in BALF dilution, we also normalized these results to BAL urea levels and again noted that CCL18 levels were significantly increased in CIP, even after this correction (FIG. 12A). To validate these findings, we measured CCL18 levels in a second, independent cohort of AML patients with and without CIP (MD Anderson cohort), and again noted increased BALF CCL18 levels in CIP. (FIG. 5B). Using the combined Hopkins / MD Anderson dataset, we examined the ability of BALF CCL18 levels to discriminate between CIP and controls, and noted good performance(FIG. 5C), with an AUC of > 0.9 (p=0.0002). A CCL18 cutoff of 14,879 pg / mL was associated with a sensitivity of 86% and a specificity of 100% for CIP in this combined dataset.

[0124] Given evidence suggesting that combination (i.e. PD-1 + CTLA4) therapy may associated with worse toxicity, we examined differences in BALF CCL18 levels in CIP patients receiving mono- vs. combination ICI therapy, but did not observe any significant differences. However, when CCL18 levels were stratified by CIP grade, we did note that high grade CIP (i.e. > Grade 2) was associated with higher BALF CCL18 levels (FIGS. 4D-4E).

[0125] The abovementioned findings of increased CCL18 in CIP BALF included patients with and without steroid therapy at the time of sample collection. Reasoning that steroid therapy may affect chemokine secretion and noting the significant variance in our CCL18 BALF data, we next examined CCL18 levels in CIP patients stratified by presence or absence of steroids at time of bronchoscopy. We noted that CCL18 levels were higher in steroid-naive patients (FIG. 5F). That said, CCL18 levels in both steroid-naive and steroid-treated CIP patients were still significantly higher than ICI+CIP- controls ( FIG. 12B).

[0126] Macrophage-derived CCL18 promotes paracrine pro-inflammatory signaling inT-cells and macrophages: Next, we explored the source of CCL18 secretion in the lung. First, we questioned whether M2 macrophages may be a source of CCL18 secretion, given that CCL18+macrophage clusters also expressed M2-associated markers (FIGS. 2A-2D, 5A-5F) showing that IL-4 stimulation is sufficient to increase CCL18 transcript levels (21). To examine this, we turned to differentiated THP1 cells as a simple system of examining the isolated role of macrophage polarization on CCL18 production. We examined the effect of M2-polarization (using IL-4) on media supernatant CCL18 levels in THP1 cells. Interestingly, IL-4 stimulation alone was sufficient to increase CCL18 protein levels in the supernatant. Importantly, this effect of IL-4 on CCL18 secretion was accentuated when PD-1 was inhibited pharmacologically with Nivolumab (FIG. 6A). We examined the effect of Nivolumab on CCL18 secretion by measuredfold change in media CCL18 levels in unstimulated (or in the case of Nivolumab, isotype-treated) controls and IL4, Nivo or IL4+Nivo treated THP-1 cells. We observed that IL-4 induced greater CCL18 secretion in the presence of Nivolumab (FIG. 6B). We next examined the consequence of increased CCL18 on M2 polarization status. Flow cytometry of IL-4 treated differentiated THP1 cells revealed, as expected, increased expression of CD209 and CD206 (FIGS. 13A-13B). CCL18 treatment (either alone or in combination with IL-4) did not inducesignificant change in CD209 or CD206 expression, suggesting that secreted CCL18 is insufficient, by itself, to promote M2 skewing and IL-4 induced CCL18 secretion does further promote M2 skewing in an autocrine fashion. These data suggest that CCL18 secretion may be a consequence of, but not contributing to, M2 polarization, in vitro. Next, we questioned whether CCL18 by M2 macrophages could be affecting, in a paracrine fashion, the phenotype of Ml- polarized macrophages.

[0127] When we examined the “non-M2” population (i.e. CD209loCD16310) in our flow cytometric data, we noted that these cells expressed high levels of IL-ip (FIG. 14A). These Ml- like macrophages constituted a very small population of cells (<20% of all macrophages) thatwere not different (as a percentage of total macrophages) between control and CIP samples (FIG. 14B). Given our findings that CCL18 was increased in the BAL supernatant, we examined whether CCL18 may be skewing Ml macrophages, however small a population, in a pro- inflammatory fashion. For this question, we again turned to differentiated THP-1 cells. Expectedly, induction of Ml skewing in THP-1 cells (with IFNy-LPS) decreased CD14 expression, as reported previously (22). While CCL18 treatment alone did not affect CD 14 expression, CCL18 treatment attenuated IFN-LPS induced downregulation in CD14 (FIG. 6C). These data provide evidence that CCL18 by M2 macrophages may be acting on a small population of Ml -like macrophages to sustain CD 14 expression (and as a consequence, a pro- inflammatory state) in these cells.

[0128] We now reasoned that if an M2 -Ml signaling axis (via CCL18) existed, then we might observe differences in the receptor for CCL18, CCR8, in our Ml population. First, we observed that there were no differences in CCR8+ total macrophages or M2 macrophages in Control or CIP samples (FIGS. 15A-15B). However, among the small Ml-like macrophage population, the data suggest a small but significant increase in CCR8 expression (FIG. 6D). Notably CCR8+Ml macs were essentially absent in control samples. To further explore how CCL18 may be skewing the inflammatory phenotype of Ml macrophages, we examined the consequence of CCL18 on IFNy-LPS induced production of CXCL-10, a classical Ml-associated chemokine. Indeed, CCL18-treated cells produced a several log-fold increase CXCL-10 secretion in THP-1 cells treated with IFN-LPS compared to untreated cells controls also stimulated with IFN-LPS. CXCL10, once made, can bind with its ligand CXCR3 in T-cells to effect chemotaxis and pro-inflammatory signaling. Thus, we posited that if a CCL18 - Mlmacrophage - CXCL10 axis existed, we would observe CXCL10-CXCR3 ligand-receptor enrichment in our cell-cell interaction analysis of scRNAseq data. When we examined all CXCL ligand-receptor relationship, CXCL10-CXCR3 was one of only 3 ligand-receptor pairs identified as being overexpressed in the scRNAseq dataset. The ligands for CXCL 10, CXCR3, was only observed in T-cell clusters in our scRNAseq data. A ligand-receptor chord diagram (FIG. 6F) revealed communication between a macrophage cluster(Macs, C6) and T-cell clusters and a cluster of Treg cells, providing evidence that macrophage - lymphocyte crosstalk via the CXCL10-CXCR3 axis in CIP. To further validate CXCL10 as a) increased in CIP and b) associated with, and possibly downstream of, CCL18, we examined CXCL10 levels in human BALF. In fact, in an earlier study, we had already shown that CXCL-10 levels were increased in the BALF of CIP patients (10). However, these measurements were performed using a multiplex assay as part of a panel of cytokine measurements. Now, we sought to re-measureCXCL-10 in an additional cohort of human BALF samples, using individual ELISA assays. As shown in FIG. 6G, we again observed an increase in BALF CXCL-10 levels, a finding which we validated by measuring CXCL-10 levels in the BALF of control and CIP samples from the Leuven cohort (FIG. 6H). Lastly, we reasoned that if CCL18 did promote CXCL-10 secretion (via its effects on Ml macrophages), then CCL18 and CXCL-10 levels could be correlated in human samples. Indeed, regression of BALF CCL18 and CXCL-10 levels in CIP BALF samples revealed a moderate to high correlation (R2 = 0.89; p < 0.05 - FIG. 61). With this data in hand, we turned to associations between BALF CCL18 and BAL cell counts. We reasoned that if CCL18 promoted CXCL-10 production, and CXCL-10 is known to recruit lymphocytes, CCL18 may be correlated with BAL lymphocytosis, which we previously reported in CIP samples. Since steroids affect both CCL18 and BAL lymphocyte counts, we examined this relationship only in steroid-naive patients (i.e. patients who were not on steroids at the time of bronchoscopy). As shown in FIG. 61, we observe a strong and significant correlation between CCL18 levels and BAL lymphocytes (in the positive direction). Collectively, these data provide evidence that CCL18 a) is produced by M2 -like macrophages, via a PD-1 dependent mechanism and b) CCL18 acts on Ml macrophages to increase CXCL-10 production and lymphocytes to the alveolar space.

[0129] CCL18 administration in mice is sufficient to recruit macrophage and T-cells subsets that are increased in CIP: While the abovementioned in vitro data shed some light macrophage phenotypes and CCL18 expression and paracrine CCL18 signaling in CIP, a morefundamental question remained. It remained unclear whether, in vivo, CC 18 was sufficient to initiate and sustain pro-inflammatory signal to increase the number of inflammatory cells in the lung, and whether these chemoattractant properties of CCL18 were PD-1 dependent. To determine this, we examined the effect of intra-tracheal (i t) CCL18 in 14-16 week old C56B6 / J mice. Since our overarching hypothesis was that CIP pathobiology requires multiple hits, we reasoned that CCL18 treatment, while sufficient to hone pro-inflammatory myeloid and lymphoid cells to the lung, would nevertheless be insufficient to induce overt lung injury. As shown in FIGS 16A-16C, CCL18 induced mild (approximately 4%) but significant weight loss in the first 48hafter instillation, but this was not significantly different between WT and PDF ' mice (FIGS. 16A-16B). This mild weight loss was not accompanied by evidence of acute lung injury, based on histology or BAL albumin measurements (FIG. 16C). This provided evidence that any differences in BAL immune cell changes between CCL18- treated WT and PDF ' mice that we may observe were not the consequence of widespread, non-specific lung injury due to CCL18 instillation.

[0130] We next performed flow cytometry on both BAL and lung homogenates in WT and PDF ’ mice under control (i.t. PBS) and experimental (i.t. CCL18) conditions at the 48h timepoint. At baseline, BAL cell percentages of AM, IM, CD4+or CD8+cells were not different between WT and PDF7' mice (FIGS. 7A-7H). In WT mice, CCL18 treatment decreased BAL AM but increased BAL IM, CD4+and CD8+percentages. Loss of PD1' ' did not significantly change the AM or CD8+response to CCL18. However, in PDF7' mice, CCL18 induced greater numbers of BAL IM (FIG. 6B) and CD4+cells (FIG. 7C). We now turned to the lung homogenate. Interestingly, unlike the BAL, baseline CD4+and CD8+cells were higher in PDF ' mice compared to WT controls. The differential response to CCL18 with regards to IM numbers was even more pronounced in the lung homogenate (FIG. 7F), with CCL18 treatment inducing a robust increased homogenate IM numbers compared to CCL18-treated WT mice.

[0131] In summary, our flow cytometry analysis of WT and PD1- / - mice treated with PBS or CCL18 revealed: a) baseline differences in CD4+and CD8+cells in the lung homogenate of PD1- / - mice; b) a CCL18 response in WT mice characterized by a decrease in AM, increase in IM, increase in BAL CD4+and CD8+and c) differential (i.e. higher) response to CCL18 in PDF / _mice with regards to BAL and lung homogenate IM percentages and BAL CD4+ cells.

[0132] Furthermore, we examined the effect of CCL18 over-expression (using Lentivirus) on macrophage function in vitro and in vivo. As shown in FIG. 19 (includes FIGS. 19A-19C), we observed that CCL18 over expression was sufficient to increase transcript levels of IL-lbeta, a key pro-inflammatory cytokine in macrophage. CCL18 over-expression in vivo increased CD4 and CD8 cells as well as alveolar macrophages, further supporting a causal, pathogenic role for CCL18 in promoting alveolar inflammation.

[0133] DISCUSSION

[0134] In this study, using human samples (spanning four different cohorts of patients), in vitro experiments and animal models, we examined the role of lung macrophages in patients with checkpoint inhibitor pneumonitis using orthogonal methods. We present data in support of increased production of the chemokine CCL18 in the BALF of CIP patients at the transcript and protein level, with external validation of our results using independent cohorts. In addition, we show that specific macrophage subpopulations (e.g. interstitial macrophages) are increased in CIP, again with external validation. We link these two observations by showing that M2 polarized macrophages may be a source of CCL18 in the lung. That CCL18 expression appears relatively restricted to the lung (and possibly to macrophages in the lung) increases the translational / therapeutic attractiveness of specifically targeting CCL18, due to lower probability of off-target effects given lack of expression in other tissues. Our data also suggest that CCL18 appears sufficient to induce infiltration of lymphocytes and IMs into the lung, but that these chemotactic functions are augmented in the absence of PD-1. As part of these data, we also observe that loss of PD1 appears to induce baseline changes in CD4 and CD8 cell numbers in the mouse lung. Overall, our data suggest an important role for CCL18 in the pathobiology of CIP.

[0135] Our in vitro data unexpectedly suggest that PD1 inhibition alone is sufficient to increase CCL18 secretion in M2-skewed THP-1 cells. This argues for a cis- PD1 / PD-L1 interaction in macrophages, since these experiments did not involve co-culture with T-cells or other cell types Indeed, such within-cell interactions of checkpoints and their ligands has been recently reported (23, 24). Our in vitro and human data around CCL18, Ml skewing and CXCL- 10 suggest crosstalk between CCL18 secretion from M2 macrophages and CXCL-10 secretion by Ml macrophages. In particular, our data showing higher IFN-LPS induced CXCL-10 secretion in the presence of CCL18 suggests that, in addition to its recruitment activities, CCL18 also functions as a modulator of Ml macrophage phenotypes and induces the recruitment of pro-inflammatory T-cells in part via the induction of CXCL-10 in Ml macrophages. CXCL-10 is known to hone conventional T-cell subsets, specifically, central memory T-cells, which we have previously shown to be increased in CIP BAL. That said, CCR8 is expressed in T-cells, so it is possible that CCL18 may also have additional effects on lymphocytes that are independent of induction of CXCL10 production in Ml macrophages.

[0136] As noted earlier, our human BAL and mouse model data suggest that CCL18 increases number of lung macrophages, specifically IMs. However, our data does not indicate how much of the increased numbers in macrophages is due to CCL18-induced recruitment vs. CC118-induced changes in proliferation within existing macrophages. CCL18 may also be promoting monocyte to macrophage differentiation. Our data shows a tight correlation between increasing CCL18 and decreasing BALF monocyte levels, and the flow cytometric pattern on our AMs and IMs suggest that the majority of these cells are monocyte-derived macrophages. Additionally, CCL18 levels, in addition to being highly correlated in the positive direction with BAL lymphocytosis, were also observed to highly correlated in the negative direction with BAL monocyte counts (FIG. 17). Once macrophages are present in the alveolus, it is likely that additional cues, derived from other cell types in the alveolar space, drive continued proliferation / persistence of macrophages in CIP. Our ligand-receptor analyses suggest that paracrine secretion of pro-inflammatory mediators such as CCL5 and MIF may be playing a role in promoting macrophage dysfunction in CIP. However, whether production of factors like MIF and CCL5 occurs alongside CCL18 production, rather than as a consequence of CCL18 signaling, is unclear; one possibility is that CCL18 promotes, in addition to CXCL-10, the production of additional chemokines that service to sustain a pro-inflammatory macrophage phenotype; however, another explanation for these results is that other factors (e.g. ongoing antitumor response) contribute to the production of these chemokines like CCLs and MIF that in CIP patients, conspire with CCL18 to augment both the number and inflammatory state of lung macrophages.

[0137] Mice do not express CCL18, but do express the CCR8 receptor. A differential response between WT and PD F ' mice to CCL18 and lack of a non-specific neutrophilic injury suggests that our in vivo findings are not a non-specific injury response to the instillation of CCL18. Rather, CCL18 appears to participate in and be sufficient for chemoattraction of immune cell subsets, including IMs and CD4+cells, into the alveolus, in a PDl-dependentmanner. However, why loss of PD-1 impacts the cell types recruited by CCL18 is unclear. Lastly, whether CCL18 effects in the context of ICI use is via CCR8 is not yet fully elucidated. CCR8 has been previously credentialed as a receptor for CCL18 (21); however, whether other receptors are upregulated in CIP patients is not known. We view the lack of tumor or a second insult in our in vivo model as a strength, as it reveals the relative contribution of CCL18 to lung inflammation in the absence of other stimuli such as a tumor that is responding to ICI and / or a superimposed viral / bacterial infection. Future studies adding “hits” to this model, including the presence of tumor as well as viral infection are planned.

[0138] CCL18 has been previously been shown to be associated with fibrotic lung disease. Indeed, we wondered whether the M2 -like macrophages, some of which also appear to be CCL18hi are pro- fibrotic. We do note that fibronectin (FN1) appears to be upregulated in cluster 0, the most prominent cluster in CIP BAL, while ligand-receptor analyses revealed a variety of ligand- receptor interactions between fibronectin-1 originating from macrophage clusters 0 and 4 and a variety of ligands in on T-cells (FIGS. 18A-18D). Additional work, including functional assays assessing the ability to CIP macrophages to promote extracellular matrix deposit! on / fibrinogenesis and the role of macrophage-derived FN1 signaling on CD8 cell function would be useful in this regard. Clinically, we recently reported that a small percentage of CIP patients progress to develop chronic CIP, characterized by persistent interstitial, infiltrates, and some of these patients progress to frank fibrosis. However, the specific clinical (or tumor-related) characteristics that predispose some (but not most) patients to progress to fibrosis remain unclear. Once a larger cohort is available, examining CCL18 and / or fibronectin levels at the time of diagnosis and the risk of CIP progression will provide greater insight into this aspect of CIP.

[0139] We emphasize that our in vivo mouse experimental design is not a model of CIP per se. For one, this model lacks the presence of tumor and there is no overt evidence of lung injury in either WT or PDF7' mice. We designed the model with the purpose of addressing whether elevations of alveolar CCL18 concentrations were sufficient to elicit an inflammatory response, and if this was a PD-1 dependent phenomenon. Thus, we purposely chose a model of mild lung injury (weight loss of < 5% at peak injury) without an acute lung injury component to focus on the chemoattractant effects of CCL18 rather than non-specific effects related to widespread alveolar damage that might occur with more overwhelming lung insults. Thus, whileour in vivo model sheds light on the sufficiency of CCL18 to facilitate alveolar inflammation in vivo.

[0140] In conclusion, our data provide evidence for an important role for CCL18 as a biomarker for CIP as well as a potential significant component of CIP pathobiology.

[0141] EXAMPLE 2: METHODS

[0142] Approval: At all institutions, institutional IRB, ethical approval and consent was obtained for all participants. All human work was approved by the IRB at the respective hospitals. All procedures in mice were performed in accordance with NIH Guide for the care and use of laboratory animals. Animal protocols were approved by the Animal Care and Use Committee (Johns Hopkins University).

[0143] Study populations: 1. Hopkins cohort: Patients were eligible for enrollment if they were previously diagnosed with a malignancy and were on ICI therapy. The diagnosis of CIP was made via multi-disciplinary adjudication by the immune related Toxicity (irTox) team. Of note, members of the irTox team were blinded to the research study consent status of the patient. Similarly, research consent was performed and obtained by team members not involved in the adjudication process. In some cases, a bronchoscopy was not able to be performed before initiation of steroids due to the severity of presenting symptoms; thus, a subset of patient samples were obtained while patients were already on corticosteroid therapy for presumed CIP. A CIP diagnosis was made based on our previously published adjudication criteria. BAL was performed as follows. In control patients, the middle lobe was lavaged. In CIP patients, an area of the lung with new infdtrates (e.g. GGOs, consolidation) was lavaged. In NSCLC patients or patients with lung metastases, whenever possible, an area of inflammation distinct from tumor was chosen. Baseline characteristics of the study cohort are provided in Table 1.

[0144] 2. Leuven cohort: As described previously, fifteen pneumonitis patients and six observational trial. ICI-pneumonitis diagnosis was based on compatible clinical history and radiological findings, in the presence of a negative infectious work-up by bronchoscopy with BAL and after excluding other causes of pneumonitis (including, e.g., progressive disease and radiotherapy-induced pneumonitis), as assessed by the treating pulmonologist and confirmed by pulmonologist and researcher a dedicated thoracic radiologist. Four pneumonitis patients were excluded; two infectious pneumonia and two radiotherapy-induced pneumonitis cases, such that ICI-pneumonitis patients were included for analyses. The most affected lung lobe / segment (withnew infiltrates) was lavaged before initiation of corticosteroid therapy. Control patients were recruited at the time of bronchoscopy because of a newly clinically diagnosed lung tumor, and underwent bronchoalveolar lavage of an unaffected contralateral lobe in the absence of clinical, radiological or pathological arguments for a (concomitant) infectious or inflammatory process.

[0145] 1. MD Anderson cohort: From March 2017 to January 2018, three patients with acute myeloid leukemia who received ICI therapies and four control patients with acute myeloid leukemia who did not receive ICI therapy were recruited. These patients underwent BAL due to suspicion of bacterial or fungal pneumonia. Among the seven patients who received ICI therapies, four were found to have infectious pneumonia (and thus excluded from the control group) and three were found to have CIP after multi-disciplinary adjudication. BAL was performed as clinically indicated by the geographic distribution of opacities on radiological imaging.

[0146] Gustave-Roussy cohort: Patients with suspected CIP (n=6) underwent bronchoscopy as part of the diagnostic work-up. Similar to the other cohorts, all cases were adjudicated in a multi-disciplinary fashion, and infection / al ternative causes were excluded. The BAL was done in the context of routine clinical practice and residual sample was used for flow cytometry / mass cytometry.Human BAL sample processing:

[0147] 1. Hopkins: Sample was obtained from the bronchoscopy suite in a 15mL conical tube transported over ice to the laboratory where it was then spun at 500g for 5 minutes at 4° C. After centrifugation, the supernatant was carefully removed and stored in ImL aliquots at -80°C for further analysis. The cell pellet was resuspended in phosphate- buffered solution (PBS). In cases where an excessive amount of mucus was present, the sample was filtered through a 70pm cell strainer. If the pellet appeared bloody, it was treated with 3mL of Ammonium-Chloride- Potassium (ACK) lysing buffer (Cat A1049201; Gibco, NY) and incubated for 3-5 minutes before adding 3mL of PBS. The pellet was then counted using an automated cell counter TC20 (Cat Al 049201; Bio-Rad, CA). The pellet was then subjected to another round of centrifugation at 350g for 8 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in Bambanker cell freezing media (Cat CS-02-001; GC lymphotec, Japan) at a concentration of 2xl06cells / mL per tube. The sample was transferred to a cell freezing container (Cat CS-02- 001; GC lymphotec, Japan) and stored at -80°C for 24 hours before being transferred to liquidnitrogen for long-term storage. We did not observe significant differences in the % of live cells retrieved from control or CIP samples (FIG. 8A).

[0148] 2. Leuven, MD Anderson and Gustave-Roussy cohorts: Sample processing for samples was performed as described previously (11,12,25)

[0149] scRNAseq:

[0150] Hopkins: Aliquots of BALF cells were thawed and processed for single-cell RNA sequencing. Single cell RNA-seq libraries were prepared from viably frozen BALF samples using the 10X Chromium platform, and 5' DGE library preparation kits according to the manufacturer’s recommended protocols (10X Genomics, Pleasonton, CA). Briefly, frozen BALF samples were rapidly thawed at 37°C and were washed three times in DPBS to remove mucus and debris. Cells were counted manually with a hemocytometer and re-suspended in 0.04% BSA in DPBS to a final concentration of 1000 cells / pL. Cells and gel beads were partitioned using the 10X Genomics Chromium controller instrument aiming for recovery of 10,000 cells per sample followed by RNA capture and cell-barcoded cDNA synthesis using Chromium Next GEM Single Cell V(D)J Reagent Kits vl.l (CG000207). Libraries for gene expression were sequenced using NovaSeq 6000 system (Illumina, San Diego, CA) with targeted sequencing depth of 50,000 reads per cell. Cell Ranger v3.1.0 was used to demultiplex the FASTQ reads, align them to human transcriptome, and generate a digital gene expression (DGE) matrix for each sample. Analysis of scRNAseq datasets was performed using Seurat. Cutoffs for % mtDNA were set at 50%; this determination was made by examining the distribution of mtDNA reads in the concatenated dataset and observing that there was a clear separation of cell populations around this cutoff. However, to ensure that our results were not skewed by this cutoff choice, a separate analysis was performed using a more stringent (10%) cutoff, which produced similar results in terms of our readouts (i.e. increased macrophage clusters, including a large macrophage cluster expressing high levels of CCL18). Following normalization and scaling, clustering and UMAP projections were constructed using Seurat. Cell-cell interactions were calculated from the final Seurat object using CellChat26. The code used to generate the Hopkins scRNAseq dataset figures is available at github.com / suresh-lab / CCL18-CIP.

[0151] 2. Leuven: BALF processing and scRNAseq details for the Leuven cohort were described in detail previously (11).

[0152] CCL18 Assay: BALF fluid was obtained from preserved samples. CCL18 HumanELISA Kit (Invitrogen, Cat. EHCCL18) was used to quantify CCL18 levels according to the manufacturer’s instructions. Briefly, samples were thawed and allowed to reach room temperature. Assay components were also allowed to reach room temperature prior to use. BALF samples were diluted 5-100x using the diluent provided in the kit. A dilutional series was prepared for the CCL18 standard. Next, lOOpL of either the standard or the sample was added to a pretreated 96-well plate and incubated overnight at 4°C with gentle shaking. The plate was washed four times with the provided wash buffer before adding the biotin conjugate. After washing off the excess biotin conjugate, Streptavidin and TMB substrate were sequentially added to the plate, completing the sandwich ELISA assay. At the end of the incubation period, the stop solution was added, and the samples were analyzed using a plate reader (iMark™ Microplate Absorbance Reader, Bio-Rad) at 450nm. A 4-parameter algorithm was employed to draw the best standard curve fit, facilitating the determination of CCL18 levels in the BALF samples.

[0153] BALF Albumin Assay: Albumin levels in BAL supernatant from WT and PD F ' mice were used. Albumin levels were measured using an albumin assay kit (Abnova, KA1612) BALF CXCL-10 Assay: Stored BALF supernatants were thawed and allowed to reach room temperature. The Human CXCL10 / IP-10 DuoSet ELISA kit (Bio-techne, DY266 and DY008B) was employed for the assay. Plates were coated with capture antibody, washed and blocked per assay instructions. Samples were measured alongside a standard curve to determine CXCL-10 concentrations. Following incubation at room temperature (20 minutes), absorbanceat 450 nm was measured. A 4-parameter algorithm was used to fit the standard curve per assay instructions.

[0154] THP-1 cell culture: Passage 5-6 of THP-1, TIB-202 (ATCC) cell line was used in the experiments. We utilized Complete Growth Media (CGM) for all cell culture experiments. CGM consisted of RPML1640 (with L-glutamine, Gibco, 12633012) supplemented with 10% heat-inactivated Fetal Bovine Serum (Coming, 35-011-CV), 1% penicillin-Streptomycin (Millipore Sigma, A5955), and 2-Mercaptoethanol (2 -ME, Thermo Fisher Scientific, 21985023) at a concentration of 0.05mM. The THP-1 cells were stored in liquid nitrogen, and the vial was thawed prior to use by gently agitating it in a 37°C water bath for 2 minutes. Once thawed, the cells were transferred to a vial containing 9 mL of prewarmed media. A cell count was performed before centrifuging the cells at 130g for 8 minutes. The supernatant was discarded,and the cells were initially plated at a density of 2 x 105cells per mL and allowed to grow to 70- 80% confluency in the incubator at 37°C and 5% CO2. To differentiate the THP-1 cells into macrophages, cells were treated with 150 nM Phorbol 12-Myristate 13-Acetate (PMA, Millipore Sigma, 79346) for 24 hours. All stimulation media were freshly prepared 20 minutes prior to use. Throughout the experiments, unstimulated controls of THP-1 cells were maintained in fresh CGM for the duration of the stimulation timeframe. The stimulation parameters are outlined below.

[0155] 1. Interleukin-4 (IL-4): IL-4 (Gibco, PHC0044) was used at a concentration of20ng / mL and cells were incubated for a duration of 48h.

[0156] 2. LPS and Interferon- y (IFN- y): LPS at 20ng / mL in addition to 20ng / mL ofIFN- y (PeproTech, 300-02) was added to the media and cells were incubated for 48h.

[0157] 3. Chemokine Ligand-18 (CCL18): CCL-18 (R&D, 394-PA) was used at a concentration of 300ng / mL. Cells were treated with CCL18 for 24h. We chose a CCL18 concentration (300 ng / mL) that was within the standard deviation of values observed in human CIP BALF samples (mean±SD BALF CCL18 concentration in CIP patients: 128ng±241 ng / mL).

[0158] Flow cytometry (Hopkins)

[0159] 1. Flow cytometer machine type, configuration, voltages: We used the CytekAurora (Biosciences) spectral flow cytometer machine to perform multi-parametric flowcytometry. During BAL cell analysis, we set the forward scatter (FSC) gain at 15 and the side scatter (SSC) gain at 20. The laser area scaling factor and laser delay values were automatically set and updated upon completion of the daily quality control check. We captured the laser signal area and height for all samples, and in some cases, we also recorded the laser signal width. Prior to use, the voltage settings for the cytometer were determined through a daily quality check. To ensure the capture of all positive peaks, the gain for each laser was adjusted by -60%. A table of antibodies, clone and vendor is provided in Supplementary Tables 1-3.

[0160] 2. Staining details: Samples were thawed, washed and resuspended in 200 pL of PBS. We added up to 106 cells in 200 pL of PBS per well to a 96-well U-bottom plate for staining. After washing, a cocktail of Live / Dead and Fc block was prepared by combining 1 pL of Live / Dead blue (Invitrogen L34961) and 10 pL of Fc block (BD Biosciences, 564219) per 1 mL of PBS. 100 pL of this mixture was added to each sample prior to incubation (RT, 20 minutes). Samples were washed and then underwent, sequentially: surface staining,fixation / permeabilization, staining for antibodies requiring conjugation, followed by intracellular staining, as detailed next. Surface staining was accomplished by incubated samples with a master mix of surface antibodies (4°C, 30 minutes), followed by washing with 50 pL of fluorescence- activated cell sorting (FACS) buffer (0.5% bovine serum albumin in PBS) Fixation and permeabilization was performed by adding 100 pL of fix / perm kit (eBiosciences, 00-5523-00; 30 minutes, 4°C) followed by washing with permeation buffer (150 pL). In the case of CCL18 and FABP4 staining (which required conjugation), primary and secondary antibodies (30 minute incubation) were added sequentially to samples at room temperature. Lastly, for other intracellular staining, the intracellular master mix and incubated the samples for 1 hour at 4°C. Afterward, samples were washed and resuspended in 200 pL of FACS buffer prior to performing flow cytometry. All samples were analyzed within 24 hours of staining. Fluorescent minus one(FMO) controls were prepared in the same fashion as the remainder of the staining protocol except for the removal of the antibody of interest. Bead particles (BD Biosciences, 560497) were used to create compensation tubes. 2 drops of bead particles were added to each ImL of PBS. 200uL of the mixture was then used for each tube with the addition of 0.5uL of each antibody in the stain protocol. 50-500K of cells from each experiment were isolated at the beginning of the staining protocol in order to act as unstained controls. These cells were not subjected to any staining parameters but underwent fixation for the same length of time as the stained cells.

[0161] 3. Gating strategy: Gating strategy used outlined in FIGS. 1, 8A-8C. These gates were used based on consensus statement on BALF flow cytometry by the American Thoracic Society. Debris was removed from the sample, followed by gating out any doublets and dead cells. This was followed by isolating the immune cells with CD45+, and removal of CD3+ cells. Macrophages were then selected by their CD206 positivity. This was confirmed by gating in CD1 lb+, CD 169+ cells as either alveolar macrophages or interstitial macrophages according to their CD169 positivity (low CD 169 were considered interstitial). Cells that lacked CD206 staining were further analyzed by removal of cells that expressed either CD 163 or CD209. Then, cells with low SCC and CD14 positive were separated according to their CD16 expression into different types of monocytes.

[0162] Flow cytometry (Gustave-Roussy): Several iterative generations of antibody panels were used for both flow cytometry and mass cytometry experiments. For the analyses presented herein, only antibodies consistently present on all panels (and therefore stained for inall samples) were used. Full antibody listing is provided in Supplementary Table 4 and 5. For CyTOF samples, macrophages were gated as follows: 7AAD-, CD45+, CD3-, CD206+. For flow cytometry, macrophages were gated as follows : Viable cells, CD45+, CD3-, CD 14+ / -, CD206+, CDl lb+.

[0163] AM / IM differentiation was performed based on CD169 status, similar to the Hopkins cohort. However, we could only measure IM and AM subsets in the flow cyometry samples (n=3) since the CyTOF panel did not contain the CD169 marker.

[0164] CCL-18 mouse model: All the animal protocols described in this study were approved by the Institutional Animal Care and Use Committee (ACUC) at the Johns Hopkins University, School of Medicine. 14-16 wk old C56B6 / J WT or PD1-Z- mice were used (Jackson Labs). Mice were anesthetized by injecting intraperitoneally 150 mg / kg of ketamine and 13.5 mg / kg of acetylpromazine. Depth of anesthesia was confirmed with paw pinch. A 20g cathether was used to intubate the trachea trans-orally, with direct visualization of the trachea made possible via a small 1cm neck incision. After intubation, the catheter was connected to a ventilator with 200uL stroke volume and 200 strokes per minute. After successful intubation, 50uL of either PBS or CCL18 (at desired mg / kg adjusted to mice weight) was instilled. After instillation mice were reconnected to the ventilator for 30-60 seconds. Midline neck incisions were closed and mice were monitored for recovery. Mice were weighed prior to the initiation of the experiment and at every 24h interval, including prior to harvest. Mice with weight loss greater than 20% or experiencing significant distress post-procedure such as lethargy, inability to reach water or food, labored breathing, impaired response to external stimuli or decreased mental alertness were euthanized. For harvest, mice were placed in a closed container containing 5% isoflurane. Isoflurane exposure was continued for 1 minute after the mice stopped breathing. Euthanasia was confirmed by visual and physical examination. A thoracotomy was done and the trachea was cannulated using a 20-guage catheter as described above. 1 mL of calcium-free PBS was added intratracheally, using a 1 mL syringe. Full lung expansion was achieved prior to aspirating the instilled fluid using the same syringe. This step was repeated for a total of 2 mL yield. The thoracic cavity was then opened, and the lungs were harvested by careful dissection. Macrophages were gated as follows : Viable cells, CD45+, CD3-, CD14+ / -, CD206+, CDl lb+.

[0165] AM / IM differentiation was performed based on CD 169 status, similar to the Hopkins cohort. However, we could only measure IM and AM subsets in the flow cyometry samples (n=3) since the CyTOF panel did not contain the CD169 marker.

[0166] CCL-18 mouse model: All the animal protocols described in this study were approved by the Institutional Animal Care and Use Committee (ACUC) at the Johns Hopkins University, School of Medicine. 14-16 wk old C56B6 / J WT or PD1- / - mice were used (Jackson Labs). Mice were anesthetized by injecting intraperitoneally 150 mg / kg of ketamine and 13.5 mg / kg of acetylpromazine. Depth of anesthesia was confirmed with paw pinch. A 20g cathether was used to intubate the trachea trans-orally, with direct visualization of the trachea made possible via a small 1cm neck incision. After intubation, the catheter was connected to a ventilator with 200uL stroke volume and 200 strokes per minute. After successful intubation, 50uL of either PBS or CCL18 (at desired mg / kg adjusted to mice weight) was instilled. After instillation mice were reconnected to the ventilator for 30-60 seconds. Midline neck incisions were closed and mice were monitored for recovery. Mice were weighed prior to the initiation of the experiment and at every 24h interval, including prior to harvest. Mice with weight loss greater than 20% or experiencing significant distress post-procedure such as lethargy, inability to reach water or food, labored breathing, impaired response to external stimuli or decreased mental alertness were euthanized. For harvest, mice were placed in a closed container containing 5% isoflurane. Isoflurane exposure was continued for 1 minute after the mice stopped breathing. Euthanasia was confirmed by visual and physical examination. A thoracotomy was done and the trachea was cannulated using a 20-guage catheter as described above. 1 mL of calcium-free PBS was added intratracheally, using a 1 mL syringe. Full lung expansion was achieved prior to aspirating the instilled fluid using the same syringe. This step was repeated for a total of 2 mL

[0167] yield. The thoracic cavity was then opened, and the lungs were harvested by careful dissection. No imputation was performed in situations where flow cytometric or cytokine values could not be measured for technical reasons (e.g. insufficient sample amount). Outliers were identified using robust regression and outlier removal (ROUT) method as previously described (28).

[0168] Supplementary Table 1

[0169] Supplementary Table 2

[0170] Supplementary Table 3OTHER EMBODIMENTS

[0171] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0172] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure.

Claims

What is claimed:

1. A method of preventing or treating a lung disease or disorder in a subject, comprising: administering to the subject a pharmaceutical composition comprising one or more agents in a therapeutically effective dose to modulate CCL18 chemokine levels; thereby treating the subject.

2. The method of claim 1, further comprising administering one or more agents which inhibit M2 macrophage secretion of chemokines.

3. The method of claim 2, wherein the one or more agents which selectively inhibit M2 macrophage secretion of chemokines inhibit CCR1 binding of CCR1 ligands or inhibit IL-4 levels or inhibit CXCL-10 levels or combinations thereof.

4. The method of claim 3, wherein CCR1 ligands comprise CCL3, CCL5, or CCL8.

5. The method of claim 1, wherein the lung disease or disorder comprises checkpoint inhibitor pneumonitis (CIP), asthma, pneumonia, chronic obstructive pulmonary disease (COPD), bronchitis, acute respiratory distress syndrome (ARDS), interstitial lung disease (ILD), inflammation or lung cancer.

6. The method of claim 4, wherein the lung disease or disorder is checkpoint inhibitor pneumonitis (CIP).

7. The method of claim 6, wherein macrophages comprising markers CD45+FSC-AhlCD3" CD206+are increased in subjects having CIP as compared to a healthy subject.

8. The method of claim 6, wherein distinct macrophage subsets are detected in subjects with CIP9. The method of claim 7, wherein the macrophage subsets comprise alveolar macrophages and interstitial macrophages.

10. The method of claim 8, wherein the alveolar macrophages express CD1 lb+CD169hl.

11. The method of claim 8, wherein the interstitial macrophages express CD1 lb+CD16910.

12. The method of claim 8, wherein the interstitial macrophages are increased in subjects having CIP as compared to a healthy subject.

13. The method of any one of claims 7-12, wherein macrophages expressing M2 markers comprising CD163 and CD206 and low levels of Ml marker CD80 are increased in subjects having CIP as compared to a healthy subject.

14. The method of any one of claims 1-13, wherein the one or more agents comprise small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds.

15. A method of treating a subject diagnosed with cancer, comprising: administering to the subject a pharmaceutical composition comprising one or more agents in a therapeutically effective dose to modulate CCL18 chemokine levels; thereby treating the subject.

16. The method of claim 15, wherein the subject is undergoing immunotherapy.

17. The method of claim 15, wherein the pharmaceutical composition comprising one or more agents decreases CCL18 chemokine levels.

18. The method of any one of claims 15-17, wherein the pharmaceutical composition is administered prior to, during the course of, after immunotherapy or combinations thereof, has been administered to the subject.

19. The method of claim 18, wherein the pharmaceutical composition is administered as an aerosol or nebulized formulation.

20. The method of claim 18, wherein the pharmaceutical composition is administered intra- subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intravitreal (i.v.i.), intra-cisterna magna (i.c.m.), or intrasternal injection.

21. A method of identifying candidate agents which inhibit CCL18 comprise: contacting a cell expressing CCL18, a substrate comprising CCL18 or fragments thereof, or a solution comprising CCL18 or fragments thereof; conducting assays to measure decreases in CCL18 expression or binding to CCL18 or fragments thereof;thereby identifying candidate agents.

22. The method of claim 21, wherein the assays comprise immunoassays, : immunoassays, Southern blots, Western blots, polymerase chain reaction (PCR), Northern blots, sequencing, reverse-transcriptase PCR, microarray technology, immunohistochemistry, enzyme-linked immunosorbent assay, flow cytometry mass spectrometry, Forster resonance energy transfer, time-resolved fluorescence energy transfer, amplified luminescent proximity homogeneous assay, fluorescence polarization, cell-based assays or combinations thereof.

23. The method of claim 22, wherein the assay is a high throughput screening (HTS) assay.

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