Compositions and methods for the treatment of idiopathic pulmonary fibrosis

Abstract

Disclosed herein are various embodiments relating to the treatment of idiopathic pulmonary fibrosis in a subject in need thereof through the administration of composition comprising an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IDIOPATHIC PULMONARY FIBROSISCROSS-REFERENCES & RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 649,113 filed May 17, 2024, and entitled “USE OF IPILIMUMAB TO TREAT IDIOPATHIC PULMONARY FIBROSIS,” which is hereby incorporated by reference in its entirety under 35 U.S.C. § 119(e).STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under HL114470, HL139617, HL151702, and HL152246 awarded by the National Institutes of Health and BX003056 awarded by the Department of Veteran Affairs. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The disclosure relates to medicine generally and the treatment of idiopathic pulmonary fibrosis, specifically.BACKGROUND

[0004] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and typically fatal interstitial lung disease of unknown etiology. It is characterized histopathologically by usual interstitial pneumonia (UIP) and clinically by irreversible scarring (fibrosis) of the lung parenchyma, leading to a gradual decline in lung function. Patients with IPF typically experience worsening dyspnea, reduced exercise tolerance, and declining pulmonary function, most notably reflected by decreases in forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO). The median survival following diagnosis is approximately three to five years, underscoring the urgent need for effective therapies.

[0005] The pathogenesis of IPF involves a complex interplay of epithelial injury, aberrant wound healing, fibroblast activation, and excessive deposition of extracellular matrix (ECM) components such as collagen. Unlike classical inflammatory diseases, IPF appears to be driven more by abnormal tissue remodeling than by persistent inflammation. Current evidence suggests that profibrotic growth factors and cytokines — including transforming growth factor-beta (TGF-P), connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF) — play central roles in promoting fibroblast proliferation, myofibroblast differentiation, and ECM accumulation.

[0006] At present, treatment options for IPF are limited. The antifibrotic agents pirfenidone and nintedanib are approved for clinical use and have been shown to slow disease progression, as evidenced by reduced rates of FVC decline. However, these agents do not halt or reverse fibrosis, and many patients continue to experience functional deterioration. Furthermore, both drugs are associated with significant side effects, and neither has demonstrated a clear survival benefit. Lung transplantation remains the only curative option, though it is suitable for only a minority of patients due to age, comorbidities, and limited organ availability.

[0007] There remains an unmet medical need for safer and more effective therapies that can halt or reverse the fibrotic process in IPF. Recent advances in understanding the molecular and cellular mechanisms underlying fibrogenesis have opened new avenues for targeted interventions. Among the promising molecular targets is CTGF, a matricellular protein that functions downstream of TGF-P and is critically involved in fibroblast activation and ECM production.

[0008] Accordingly, there is a need in the art for novel therapeutic strategies that can more directly and selectively interfere with the fibrotic pathways implicated in IPF.BRIEF SUMMARY OF THE INVENTION

[0009] Humans have evolved highly specialized immune systems that rely on the activity of cytotoxic T cells to eliminate irreparably damaged cells from tissues / organs. This endogenous quality control mechanism is essential for maintaining organ structure and function. In recent years, this property of cytotoxic T cells has been leveraged to design anti-tumor strategies in which immune checkpoint inhibitors are used to reactivate exhausted T cells for the treatment of multiple cancers. Many opportunities to extend this endogenous immune- surveillance function beyond cancer remain unexplored.

[0010] Fibrosis of various organs in humans typically originates from regenerative failure or delayed repair of damaged epithelium that triggers immune and stromal tissue responses. Cellcell interactions within this dynamic tissue microenvironment determine the outcome of the repair-regenerative response. Recent studies have implicated the persistence of senescent cells innon-resolving fibrosis, while its transient emergence may be pro-regenerative. A failure in immune surveillance of senescent cells may result in the pathological conversion from pro- regenerative to pro-fibrotic outcomes. Persistence of senescent cells may, in turn, create an immunosuppressive microenvironment that leads to T cell exhaustion, a process that is well appreciated in cancer progression.

[0011] Despite interest in developing senolytic agents for treating persistent / progressive fibrotic disorders, the contributions of endogenous immune-mediated cytolysis and clearance of senescent cells to fibrosis resolution remain unclear.

[0012] Variant 1 relates to a method for treating pulmonary fibrosis in a subject in need thereof comprising: administering to the subject an effective amount of a composition, the composition comprising an anti-CTLA-4 antibody.

[0013] Variant 2 relates to the method of variants 1 and 3-14, wherein the pulmonary fibrosis is an idiopathic pulmonary fibrosis.

[0014] Variant 3 relates to the method of variants 1-2 and 4-14, wherein the anti-CTLA-4 antibody is ipilimumab.

[0015] Variant 4 relates to the method of variants 1-3 and 5-14, wherein the ipilimumab is administered at a dose of from about 1 mg / kg to about 10 mg / kg subject body weight.

[0016] Variant 5 relates to the method of variants 1-4 and 6-14, wherein the ipilimumab is administered at a dose of about 5 mg / kg subject body weight.

[0017] Variant 6 relates to the method of variants 1-5 and 7-14, wherein the ipilimumab is administered at repeated doses at predetermined intervals.

[0018] Variant 7 relates to the method of variants 1-6 and 10-14, wherein the ipilimumab is administered 1-4 times per week.

[0019] Variant 8 relates to the method of variants 1-6 and 10-14, wherein the ipilimumab is administered for a period of 2-8 weeks.

[0020] Variant 9 relates to the method of variants 1-6 and 10-14, wherein the ipilimumab is administered twice weekly for a period of 4 weeks.

[0021] Variant 10 relates to the method of variants 1-9, wherein the administration of the anti-CTLA-4 antibody to the subject results in an increase in the subject of one or more of vital capacity, forced vital capacity, residual volume, forced expiratory volume, forced inspiratoryflow, peak expiratory flow rate, inspiratory reserve volume, tidal volume, total lung capacity, expiratory reserve volume, and / or maximum voluntary ventilation.

[0022] Variant 11 relates to the method of variants 1-10, wherein the anti-CTLA-4 antibody is administered in conjunction with one or more second therapy or treatment.

[0023] Variant 12 relates to the method of variants 1-11, wherein the one or more therapy or treatment is an anti-fibrotic drug.

[0024] Variant 13 relates to a method for treating idiopathic pulmonary fibrosis in a subject in need thereof comprising administering to the subject an effective amount of a composition, the composition comprising Ipilimumab and a pharmaceutically acceptable carrier thereof.

[0025] Variant 14 relates to a method for treating idiopathic pulmonary fibrosis in a subject in need thereof comprising administering to the subject an effective amount of a composition, the composition comprising Ipilimumab at a dose of about 5 mg / kg subject body weight and a pharmaceutically acceptable carrier thereof.

[0026] While multiple embodiments are disclosed, still other embodiments of the disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. As will be realized, the disclosure is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1A is a diagram of a study design and workflow, according to one embodiment.

[0028] FIG. IB is a Cell Atlas image of the expression of CTLA4 in relevant T cells, according to one embodiment.

[0029] FIG. 1C is a plot of the principal component analysis of spatial transcriptomics data of subjects with IPF versus controls, according to one embodiment.

[0030] FIG. 2A is a digital spatial profiling image of stained lung tissue of a control subject, according to one embodiment.

[0031] FIG. 2B is a digital spatial profiling image of stained lung tissue of a subject with IPF, according to one embodiment.

[0032] FIG 3 A is a plot showing differential expressions of genes based on a log2 fold change, according to one embodiment.

[0033] FIG. 3B is a box and whisker plot showing counts of ACTA2, according to one embodiment.

[0034] FIG. 3C is a box and whisker plot showing counts of COL1A1, according to one embodiment.

[0035] FIG. 3D is a box and whisker plot showing counts of COL3A1, according to one embodiment.

[0036] FIG. 3E is a box and whisker plot showing counts of COL6A1, according to one embodiment.

[0037] FIG. 3F is a box and whisker plot showing counts of AGER, according to one embodiment.

[0038] FIG. 4A is a plot of enrichment score with varying IPF, according to one embodiment.

[0039] FIG. 4B is a bubble plot of IPA analysis on different expressed genes, according to one embodiment.

[0040] FIG. 4C is a bubble plot of IPA analysis on different expressed genes, according to one embodiment.

[0041] FIG. 4D is a plot of GSEA Hallmark pathway analysis of the differentially expressed genes, according to one embodiment.

[0042] FIG. 5 is an unsupervised hierarchically clustered heat map of protein counts, according to one embodiment.

[0043] FIG. 6A is a violin plot of read counts of a-SMA, according to one embodiment.

[0044] FIG. 6B is a violin plot of read counts of CTLA4, according to one embodiment.

[0045] FIG. 7A is a cell distribution map of Banovich / Kropski dataset of CTLA4 in subpopulations, according to one embodiment.

[0046] FIG. 7B is a cell distribution map of UMAP of CTLA4 in subpopulations, according to one embodiment.

[0047] FIG. 7C is a box and whisker plot of CTLA4 expression, according to one embodiment.

[0048] FIG. 7D is a cell distribution map of QuPAth quantitation of H&E staining, according to one embodiment.

[0049] FIG. 7E is a violin plot of quantitative Masson’s trichrome staining, according to one embodiment.

[0050] FIG. 7F is a violin plot of total lung hydroxyproline levels, according to one embodiment.

[0051] FIG. 8A shows immunostaining images of lung tissues of subjects, according to one embodiment.

[0052] FIG. 8B shows immunostaining images of lung tissues of subjects, according to one embodiment.

[0053] FIG. 9A is a Xenium spatial plot of fibrotic regions, according to one embodiment.

[0054] FIG. 9B is a Xenium spatial plot of fibrotic regions with regions of histopathological staining identified, according to one embodiment.

[0055] FIG. 9C is a bar graph with fibrotic regions and minimally fibrotic regions shown, according to one embodiment.

[0056] FIG. 9D is a Xenium spatial plot with overlay in an IPF lung, according to one embodiment.

[0057] FIG. 9E is a magnified Xenium spatial plot with overlay in an IPF lung, according to one embodiment.

[0058] FIG. 9F is a bar graph with CTLA4 transcript numbers in IPF lungs, according to one embodiment.

[0059] FIG. 10A is a diagram of a mouse model study design and workflow, according to one embodiment.

[0060] FIG. 10B is a diagram of a mouse model study experimental outline, according to one embodiment.

[0061] FIG. 11 shows representative images of H&E (top) and Masson’s trichrome (bottom) stained whole lung sections, according to one embodiment.

[0062] FIG. 12A is a plot of the quantification of Masson’s trichrome staining between test groups, according to one embodiment.

[0063] FIG. 12B is a bar graph of the gene expression of pro-fibrotic gene Acta2, according to one embodiment.

[0064] FIG. 12C is a bar graph of the gene expression of pro-fibrotic gene Collal, according to one embodiment.

[0065] FIG. 12D is a bar graph of the gene expression of pro-fibrotic gene Acta2, according to one embodiment.

[0066] FIG. 12E is a bar graph of the gene expression of pro-fibrotic gene Collal, according to one embodiment.

[0067] FIG. 13A is a UMAP image of whole lung cells from the IgGl isotype control and ipilimumab groups, according to one embodiment.

[0068] FIG. 13B is a UMAP image of whole lung cells from the IgGl isotype control and ipilimumab groups, according to one embodiment.

[0069] FIG. 14 shows a graph of the cell type annotation and relative proportions, according to one embodiment.

[0070] FIG. 15 is a volcano plot showing differential gene expression in T+ cell clusters, according to one embodiment.

[0071] FIG. 16A is a violin plot showing gene expression of Icos in the Cd35+ and Cds+ T cell clusters, according to one embodiment.

[0072] FIG. 16B shows representative flow cytometry plots of lung cells isolated from IgGl and ipilimumab treated groups, according to one embodiment.

[0073] FIG. 16C is a plot depicting the percentage of CTLA4 expressing CD8+ T cells from IgGl isotype control and ipilimumab treated groups, according to one embodiment.

[0074] FIG. 17A is a violin plot showing AT2 cell expression of sftpb, according to one embodiment.

[0075] FIG. 17B is a violin plot showing AT2 cell expression of sttpc, according to one embodiment.

[0076] FIG. 17C is a violin plot showing AT2 cell expression of sftpd, according to one embodiment.

[0077] FIG. 18 is a volcano plot showing differential gene expression in the sftpal+ cell cluster, according to one embodiment.

[0078] FIG. 19 is a high-plex single-cell spatial phenotyping image of pro-SPC+ cells in IgGl isotype control lung tissues, according to one implementation.

[0079] FIG. 20 is a high-plex single-cell spatial phenotyping image of ipilimumab lung tissues, according to one implementation.

[0080] FIG. 21A is a phenotyping of pro-SPC+ cells in whole lung, according to one implementation.

[0081] FIG. 21B is a bar graph showing the percentage of SPC and Ki67+ cells in the sample groups, according to one implementation.

[0082] FIG. 21C is a plot showing representative flow cytometry plots of Ki67+SPC cells in the sample groups, according to one implementation.

[0083] FIG. 22 is a spatial profiling image of IHC staining of lungs from IgGl control group, according to one embodiment.

[0084] FIG. 23 is a spatial profiling image of IHC staining of lungs from ipilimumab group, according to one embodiment.

[0085] FIG. 24A are images of immunohistochemistry staining in lung tissue sections of the IgGl isotype control group, according to one embodiment.

[0086] FIG. 24B are images of immunohistochemistry staining in lung tissue sections of the ipilimumab group, according to one embodiment.

[0087] FIG. 25 is a plot comparing the p 16 INK4a positive cells observed in high power field of the test groups, according to one embodiment.

[0088] FIG. 26 is a schematic representation of ex vivo studies performed, according to one embodiment.

[0089] FIG. 27 shows representative images of CD8+ T cells killing of SPiDER-0Gal positive cells, according to one embodiment.

[0090] FIG. 28 shows live cell percent confluency of CD8+ T cells and whole lung cells isolated from bleomycin mouse treated mice, according to one embodiment.DETAILED DESCRIPTION

[0091] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an active component” refers to one or mixtures of active components, and reference to “the method for” includes reference to equivalent steps and methods known to those skilled in the art, and so forth,

[0092] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0093] “Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, preferably orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0094] The term “monoclonal antibody” (“mAb”) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody.MAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.

[0095] An “antibody” (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0096] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain antibody.

[0097] A “human” antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms “human” antibodies and “fully human” antibodies and are used synonymously.

[0098] A “humanized antibody” refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an Ab, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A “humanized” antibody retains an antigenic specificity similar to that of the original antibody.

[0099] As used herein, the terms “idiopathic pulmonary fibrosis” and “IPF” describe a chronic, progressive fibrosing interstitial pneumonia of unknown cause, limited to the lungs and associated with the radiologic and / or histopathologic pattern of usual interstitial pneumonia (UIP).

[0100] “Cytotoxic T-Lymphocyte Antigen-4” (CTLA-4) refers to an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo, and binds to two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). The term “CTLA-4” as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385. A “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice,rats and guinea pigs. In some embodiments, the subject is a human. The terms, “subject” and “patient” are used interchangeably herein.

[0101] A “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in vitro assays.

[0102] As used herein, the terms “treating”, “treatment,” and “therapy,” in the context of the invention, mean the administration of an anti-CTLA-4 antibody to subjects with IPF or at risk for developing IPF. In some embodiments, the subjects with IPF are “unresponsive to conventional treatment,” i.e., unresponsive to conventional prior art treatments of IPF including corticosteroids, cyclophosphamide, and azathioprine. In further embodiments, the IPF subjects treated with anti- CTLA-4 antibodies have responded to conventional treatment and the anti- CTLA-4 antibodies are being administered after the cessation of conventional treatments or in addition to conventional treatments. In other embodiments, the IPF subjects treated with anti- CTLA-4 antibody are those subjects that are treatment naive and include newly diagnosed IPF subjects.

[0103] HuMAbs that bind specifically to CTLA-4 with high affinity have been disclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238. Other anti-CTLA-4 mAbs have been described in, for example, U.S. Pat. Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121. The anti-CTLA-4 HuMAbs disclosed in U.S. Pat. Nos. 6,984,720, 7,605,238 have been demonstrated to exhibit one or more of the following characteristics: (a) binds specifically to human CTLA-4 with a binding affinity reflected by an equilibrium association constant (Ka) of at least about 107M-1, or about 109M-1, or about 10nM-1to 10nM-1or higher, as determined by Biacore analysis; (b) a kinetic association constant (ka) of at least about 103, about 104, or about 105m-1s-1; (c) a kinetic disassociation constant (kd) of at least about 103, about 104, or about 105m-1s-1; and (d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodiesuseful for the present invention include mAbs that bind specifically to human CTLA-4 and exhibit at least one, at least two, or at least three of the preceding characteristics. An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat. No. 6,984,720.

[0104] An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat. No.6,984,720. Ipilimumab is an anti-CTLA-4 antibody for use in the methods disclosed herein. Ipilimumab is a fully human, IgGl monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands, thereby stimulating T cell activation.

[0105] The disclosed treatment methods and compositions relate the treatment of idiopathic pulmonary fibrosis (IPF) in a subject in need thereof by way of administration of a composition comprising an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0106] The composition may take the form of a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, powder, or lyophilized cake. Injectable forms include sterile aqueous solutions, dispersions, and sterile powders intended for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0107] Formulations containing an anti-CTLA-4 antibody for use according to the present disclosure can be prepared by combining the antibody with pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic at the dosages and concentrations administered. These formulations may include buffers such as phosphate, citrate, or other organic acids; antioxidants like ascorbic acid or methionine; and preservatives including octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, or benzyl alcohol. Additional components may include alkyl parabens such as methyl or propyl paraben; other stabilizers such as catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol; carriers; hydrophilic polymers like polyvinylpyrrolidone; carbohydrates such as glucose, mannose, or dextrins; chelating agents like EDTA; and sugars including sucrose, mannitol, trehalose, or sorbitol. Salt-forming counter-ions such as sodium, metal complexes, non-ionic surfactants, and polyethylene glycol may also be included.

[0108] In certain embodiments, formulations may additionally contain low molecular weight polypeptides, carriers such as serum albumin, gelatin, or immunoglobulins, and aminoacids like glycine, glutamine, asparagine, histidine, arginine, or lysine. Sustained-release preparations may also be formulated using polymers such as poly(lactic acid), poly(glycolic acid), or their copolymers, along with other sustained-release matrices commonly used in pharmaceutical applications.

[0109] A wide range of pharmaceutically acceptable carriers, excipients, and stabilizers is known in the field and listed in standard references, including the United States Pharmacopeia, Japanese Pharmacopeia, European Pharmacopeia, and British Pharmacopeia. Additional resources include Remington’s Pharmaceutical Sciences (Gennaro, ed., 2000), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition (Hardman, Limbird, and Gilman, eds., 2001, McGraw-Hill), the FDA's Inactive Ingredient Search database, and the Handbook of Pharmaceutical Additives (Ash, ed., Synapse Information Resources, Inc., 3rd Ed., 2007).

[0110] Parenteral compositions intended for injection are typically sterile and may be provided in unit dosage forms such as ampoules, syringes, or injection pens, or in multi-dose containers that often contain a preservative. In some cases — such as with lyophilized products or concentrates — reconstitution or dilution is required prior to administration.

[0111] The anti-CTLA-4 antibodies can be supplied or administered at a variety of concentrations. In some embodiments, concentrations may be at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, or 200 mg / ml. In other embodiments, concentrations may be no more than about 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml, or 300 mg / ml. Still further embodiments include concentration ranges such as 5 mg / ml to 20 mg / ml, 20 mg / ml to 50 mg / ml, 50 mg / ml to 100 mg / ml, 100 mg / ml to 200 mg / ml, or 200 mg / ml to 300 mg / ml.

[0112] Various pulmonary function parameters commonly used in the field can assist in determining an effective dose of an anti-CTLA-4 antibody. An effective dose is one that reduces, stabilizes, or reverses the pathological rate of decline in one or more pulmonary function measures, or serves to monitor a patient's response to anti-CTLA-4 antibody therapy. These parameters include the following:

[0113] Vital capacity (VC) refers to the total volume of air that can be inhaled and exhaled from the lungs. It is the sum of the inspiratory reserve volume, tidal volume, and expiratoryreserve volume. Forced vital capacity (FVC) is the amount of air exhaled during a maximal forced breath following full inhalation. FVC % predicted represents the subject's measured FVC expressed as a percentage of the predicted value for that individual; unless otherwise specified, these values are reported as absolute percentages.

[0114] Residual volume (RV) is the amount of air remaining in the lungs after a maximal exhalation. Forced expiratory volume (FEV) measures the volume of air exhaled during a forceful breath over a set period, such as one second (FEVi) or six seconds (FEVs). Forced inspiratory flow (FIF) refers to the volume of air inhaled during a forceful breath over a specific time frame, such as one second (FIFi) or six seconds (FIFe). Peak expiratory flow rate (PEFR) is the highest flow rate achieved during a forced exhalation.

[0115] Inspiratory reserve volume (IRV) is the maximum volume of air that can be inhaled following a normal inspiration. Tidal volume (TV) is the amount of air inhaled or exhaled during one normal respiratory cycle, typically at rest. Inspiratory capacity (IC) is calculated as the sum of the inspiratory reserve volume and tidal volume.

[0116] Functional residual capacity (FRC) is the sum of the expiratory reserve volume and residual volume, representing the amount of air left in the lungs at the end of a normal exhalation. Total lung capacity (TLC) is the total amount of air the lungs can hold, defined as the sum of vital capacity and residual volume. Expiratory reserve volume (ERV) is the maximum volume of air that can be exhaled following a normal expiration.

[0117] Maximum voluntary ventilation (MW) is the volume of air that can be moved in and out of the lungs within a specified period during repeated maximal effort. The FEVi / FVC ratio indicates the proportion of the forced vital capacity that is exhaled during the first second and is commonly used to evaluate airflow limitation.

[0118] Most of these pulmonary function parameters can be measured using a spirometer, a standard tool in clinical practice. Residual volume, however, is typically assessed using indirect techniques such as radiographic planimetry, body plethysmography, closed-circuit gas dilution (including helium dilution), or nitrogen washout.

[0119] In some embodiments, the patient is treated for a minimum of 2 weeks, 3 weeks, 4 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, 27 weeks, 30 weeks, 36 weeks, 40 weeks, 48 weeks, 1 year, or 2 years. In other embodiments, the patient is treated every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 10 weeks, or 12weeks as indicated by the patient's healthcare practitioner. In additional embodiments, the patient is treated for a maximum of 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, 27 weeks, 30 weeks, 36 weeks, 40 weeks, 48 weeks, 1 year, 2 years, 3 years, 4 years, or 5 years. In further embodiments, the treatment duration is between 1 week to 24 weeks, 24 weeks to 48 weeks, 48 weeks to 2 years, 3 weeks to 2 years or 3 weeks to 3 years.

[0120] In certain embodiments, the method for treating IPF in a subject in need thereof comprises administering at least 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or 60 mg / kg of an anti-CTLA-4 antibody to the subject per a one, two, or three week period. In particular embodiments, the anti-CTLA-4 antibody is administered at a dose of about 15 mg / kg or about 30 mg / kg every three weeks.

[0121] In certain embodiments, the anti-CTLA-4 antibody is administered at least twice, beginning with a loading dose followed by one or more maintenance doses. The term "loading dose" refers to an initial dose given within a defined time frame to rapidly establish a desired therapeutic antibody concentration or effect.

[0122] The loading dose may be at least 1 mg / kg, 5 mg / kg, 10 mg / kg, 12.5 mg / kg, 15 mg / kg, 20 mg / kg, 22.5 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 75 mg / kg, or 100 mg / kg. Alternatively, the loading dose may be defined as the amount of antibody sufficient to achieve a plasma concentration of at least 0.1 pg / ml, 1.0 pg / ml, 5 pg / ml, 10 pg / ml, 20 pg / ml, 25 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 75 pg / ml, 100 pg / ml, 125 pg / ml, 150 pg / ml, or 200 pg / ml, when measured approximately 21 days after administration (Cmin).

[0123] The term "maintenance dose" refers to a subsequent dose administered to sustain the therapeutic concentration or effect established by the loading dose. For instance, the maintenance dose may be used to continue a reduction, stabilization, or reversal in the pathological rate of decline in forced vital capacity (FVC) that was initially achieved by the loading dose. Typically, maintenance doses are lower than loading doses.

[0124] The maintenance dose may be administered at intervals of approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, or 24 weeks after the initial loading dose. Alternatively, it may be given no more than about 1 to 24 weeks after the loading dose. In further embodiments, administration may occur within about 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 6 weeks, 1 to 8 weeks, 2 to 10 weeks, 6 to 12 weeks, 10 to 20 weeks, or 12 to 25 weeks following the loading dose.

[0125] The anti-CTLA-4 antibody or a pharmaceutical composition containing it may be administered through a bolus intravenous injection. In other embodiments, it may be delivered via infusion lasting at least 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours. The antibody may also be administered subcutaneously in a concentrated formulation. Alternatively, it may be delivered as an aerosolized powder or a nebulized solution for inhalation.

[0126] In certain embodiments, the methods for treating IPF described herein involve administering an anti-CTLA-4 antibody alongside one or more additional therapies. The phrase “in combination” refers to administering the anti-CTLA-4 antibody either before, at the same time as, or after the administration of these additional therapies. The use of the term does not imply a specific sequence or timing and includes both simultaneous and sequential administration. These therapies may be delivered via the same or a different route of administration compared to the anti-CTLA-4 antibody.

[0127] Current treatments for IPF, approved by the United States FDA, include nintedanib and pirfenidone. Nintedanib is a triple angiokinase inhibitor that targets receptor tyrosine kinases, FGFR, PDGFR, and VEGFR. Pirfenidone is a small molecule inhibitor that targets pro- fibrotic cytokines such as TGF-beta.

[0128] In some embodiments, the anti-CTLA-4 antibody is administered in combination with currently FDA-approved drugs, notably nintedanib and pirfenidone.

[0129] Additional embodiments involve combining the anti-CTLA-4 antibody with one or more antioxidants, such as glutathione, taurine, niacin, or N-acetylcysteine (NAC). In certain cases, NAC is the chosen antioxidant for use alongside the antibody. The antibody may also be administered in conjunction with anti-fibrotic agents, which may include nintedanib and pirfenidone.

[0130] Further therapeutic combinations may involve administering the anti-CTLA-4 antibody with at least one additional agent selected from pirfenidone (Esbriet®), nintedanib (OFEV or Vargatef®), or other anti-fibrotic agents such as phosphodiesterase 4B (PDE4B). In some embodiments, the additional therapeutic agent may also be delivered in aerosolized form, for example, aerosolized interferon-y as described in U.S. patent application Ser. No. 12 / 319,851.

[0131] As disclosed herein, administration of ipilimumab has the unexpected result of accelerating lung epithelial regeneration and diminished fibrosis induced by lung- specific instillation of the chemotherapeutic drug, bleomycin. In various embodiments, the ipilimumab treatment results in the expansion of Cd3e+ T cells, diminished accumulation of senescent cells, and robust expansion of type 2 alveolar epithelial cells, facultative progenitor cells of the alveolar epithelium. Administration of the various compositions disclosed herein may spur upregulation of the immune checkpoint protein, cytotoxic T-lymphocyte associated protein 4 (CTLA4) on CD8+ T cells adjacent to regions of active fibrogenesis in human idiopathic pulmonary fibrosis (IPF).

[0132] Tissue regenerative responses in mammals are highly orchestrated processes, involving intricate cell-cell interactions that either result in restoration of normal tissue architecture and function, or in aberrant repair culminating in fibrosis. While fibrosis has traditionally been thought to represent the end-stage of aberrant tissue repair or failed tissue regeneration, it is now increasingly recognized to be a dynamic and reversible process. However, elucidation of mechanisms that resolve tissue fibrosis have lagged behind that of fibrosis development or initiation. Since most human fibrotic disorders that affect diverse organ systems come to clinical attention when they are either persistent or progressive, it is critical to understand biological mechanisms that promote fibrosis resolution vs. progression. This disclosure identifies a critical role for CD8+ cytotoxic T cells in immune- surveillance and elimination of senescent cells that emerge during the lung injury repair process. Furthermore, this disclosure identifies the CTLA4 immune checkpoint protein as a critical contributor to a persistent / progressive fibrosis in an animal model of repetitive genotoxic lung injury.

[0133] While single-cell transcriptomics of isolated cells from normal and diseased tissues have the potential to identify novel cell populations and diverse differentiation states / fates, they lack spatial context. By contrast, spatial transcriptomics / proteomics provide critical information of heterogenous cell populations within unique tissue niches despite reduced resolution at the single cell level. This allows for hypothesis-generating observations involving multiple cell types in diseased tissues. In this disclosure, certain genes and proteins were identified, which were differentially expressed within IPF lung tissues. In some embodiments, the differentially expressed genes and proteins were located within the fibroblastic foci.

[0134] The finding that cellular immune responses were reduced in IPF lungs was unexpected, as was the upregulated expression of the immune checkpoint protein, CTLA4. Additionally, CTLA4 was not expressed on (myo)fibroblasts or epithelial cells within fibroblastic foci, but rather on CD8+ T cells within this fibrotic niche. In addition to confirming that CTLA4 is primarily expressed in T cell subpopulations, a search of larger single-cell RNA transcriptomic datasets of human IPF served to validate the original DSP observation that CTLA4 is upregulated in IPF lungs. In some embodiments, the dataset of human IPF was the IPF Cell Atlas. Furthermore, immunofluorescence staining of IPF lungs confirmed the cell-cell proximity of CTLA4-expressing CD8+ T cells and epithelial / fibroblastic cells within fibroblastic foci which is known to be enriched for senescent cells.

[0135] In the work of this disclosure, the functional role of CTLA4 in the context of lung injury repair was explored using a mouse model of repetitive bleomycin injury that produces more durable fibrotic responses. The use of humanized CTLA4 knock-in mice allowed for the analysis of the physiological consequences of targeting CTLA4 with a monoclonal antibody, ipilimumab, FDA- approved for various human cancers. An expansion of the Cd3e+ subpopulation of T cells with increased expression of Icos and Tcf7 in lungs of ipilimumab treated mice was observed, supporting a de-repressive effect of targeting CTLA4 in these cells. Notably, activation of this cytotoxic T cell population was associated with a marked increase in the numbers of AT2 cells, facultative stem cells of the lung’s alveolar compartment. Both sc- RNAseq and immunofluorescence staining approaches confirmed the enhanced regenerative responses in association with diminished fibrosis in ipilimumab treated mice. This disclosure demonstrates a pro-regenerative effect of targeting CTLA4 and activating cytotoxic T cells during the fibrotic phase of lung injury repair. During this delayed phase and under the same conditions / context, this disclosure shows that CD8+ T cells isolated from ipilimumab treated mice are more efficient in ex vivo killing of senescent cells exposed to bleomycin in vivo.

[0136] This disclosure shows that activation of an endogenous T cell population can promote regeneration by targeting senescent cells which accumulate in fibrotic tissues. Additionally, the activation of endogenous immune-surveillance mechanisms to mediate pro- regenerative and anti-fibrotic effects has not been previously demonstrated.

[0137] Understanding of pro-regenerative roles of immune cells is critical in devising more effective therapies for chronic fibrotic disorders. Given the pervasive focus on the pro-inflammatory (and pro-fibrotic) effects of immune cells, patients with IPF have traditionally been treated with potent immunosuppressive drug regimens. Patients treated with the immunosuppressive regimen in this clinical trial demonstrated unexpectedly worse outcomes with higher mortality and increased hospitalization rates, suggesting that immune pathways may be protective in IPF patients with established fibrosis. Without being bound by theory, it is plausible that, based on the results of the findings reported in this disclosure, steroids and other immunosuppressive agents may confer unanticipated detrimental, pro-fibrotic effects through loss of immune- surveillance functions. Conversely, activating these endogenous immune mechanisms may promote pro-regenerative tissue repair responses.

[0138] EXAMPLES

[0139] Example 1 - The immune checkpoint inhibitor CTLA4 is upregulated on T cells within regions of active fibrogenesis in human idiopathic pulmonary fibrosis.

[0140] In various embodiments of the presently disclosed methods, systems, and devices involve immune checkpoint inhibitor CTLA4 being upregulated on T cells within regions of active fibrogenesis in human idiopathic pulmonary fibrosis (IPF). As would be understood by those skilled in the art, spatial transcriptomics and proteomics may provide new insights into cell-cell interactions within tissue niches of normal and diseased organs. In carrying out certain embodiments, spatial transcriptomics and proteomics in the lung tissues of human subjects with IPF were conducted. FIG. 1A shows a diagram of this process. FIG. IB shows the Q3 normalized counts of the CTLA4 gene in T cell subpopulations of subjects with IPF versus a control from scRNA-sequencing data. FIG. 1C shows the principal component analysis of spatial transcriptomes of subjects with IPF versus control subjects.

[0141] In the disease process of this Example, active fibrogenesis may occur with morphological lesions, referred to as “fibroblastic foci.” The fibroblastic foci may be marked by the presence of alpha- smooth muscle actin (a-SMA)-expressing myofibroblasts within alveolar regions that normally do not express the a-SMA marker in alveoli of non-fibrotic postnatal adult lung. The fibroblastic foci may also be restricted to smooth muscle cells surrounding more proximal airways and muscularized blood vessels. To accurately identify fibroblastic foci, IPF lung tissues for a-SMA, pan-cytokeratin (a marker of lung epithelium), and CD31 (a marker of vascular endothelium) may be stained. FIG. 2A shows the stained lung tissue of a normal subjectand FIG. 2B shows the stained lung tissue of a subject with IPF, both imaged with the above defined morphological markers.

[0142] In some embodiments, twelve regions of interest (ROIs) capturing fibroblastic foci within lung sections of two different subjects with IPF and 12 ROIs from two age-matched control subjects were analyzed using digital spatial profiling (DSP). In certain embodiments, the DSP was done using GeoMx, by nanoString. In these embodiments, 18,676 genes from the GeoMx transcriptomics atlas were assayed. Within those assays, 24 ROIs and 9,101 of these genes were detected in >1% of segments. FIG. 3 A shows differential expression of these genes based on a log2 fold change cutoff of 1.5 and p < 0.05. The differential expressions showed an upregulation of classical pro-fibrotic genes, including a-SMA (ACTA2), collagen 1A1 (COL1A1), collagen 3A1 (COL3A1) and collagen 6A2 (COL6A2), in IPF vs. control, as shown in FIGS. 3B-3F. In contrast, alveolar type 1 (ATI) epithelial marker gene, advanced glycosylation end product- specific receptor (AGER) was markedly downregulated in these embodiments. Other ATI markers, HOP homeobox (HOPX; 0.39 log2 fold, p = 0.052) and caveolin 1 (CAV1; 0.45 log2 fold, p = 0.054) were downregulated but did not reach statistical significance.

[0143] Gene set enrichment analysis (GSEA) revealed significant enrichment of the TGF-P signaling pathway in IPF subjects, such as is shown in FIG. 4A. This is consistent with a central role for this cytokine in fibrotic diseases. Ingenuity pathways analysis (IPA) of genes differentially expressed between control and IPF cells revealed a state of decreased “cellular immune response,” which can be seen in FIGS. 4B and 4C. FIGS. 4B and 4C show IPA analysis was performed on differentially expressed genes of healthy controls and patients with IPF. The cellular immune response that is downregulated is highlighted in red. Data are reported as mean + SEM; statistical differences in FIGs. 4B^4D were tested using a paired Student’s t-test (two- tailed). FIG. 4D shows the GSEA Hallmark pathway analysis of the differentially expressed genes, comparing IPF vs. control (n = 12 ROIs each; p < 0.05; NES > 1.5).

[0144] Interestingly, Ingenuity pathway analysis (IPA) of genes differentially expressed between control and IPF lungs revealed decreased “cellular immune response.” This response can be seen in FIG. 4D. A full list of genes in the ingenuity pathway analysis can be seen found in Yadav, S., ...Kolls, J., & Thannickal, V. J. (2025). Reactivation of CTLA4-expressing T cells accelerates resolution of lung fibrosis in a humanized mouse model. The Journal of ClinicalInvestigation, https: / / doi.org / 10-l 172 / JCI181775, which is hereby incorporated by reference in its entirety under 37 CFR § 1.57.

[0145] In some embodiments, a proteomics immune panel encompassing seventy-nine protein targets and seven protein modules was used to gain insights into immune dysfunction in IPF. The seven protein modules may be human immune cell profiling protein core, human immune activation status protein module, human immune cell typing protein module, human pan-tumor protein module, human PI3K / AKT signaling protein module, human nC cell death protein, and human MAPK signaling protein module. Spatial profiling of these proteins across the same IPF and control tissues employed for spatial transcriptomics confirmed a high expression of CTLA4 protein in ROIs enriched in a-SMA. FIG. 5 shows an unsupervised hierarchically clustered heat map of protein counts for IPF (n = 12 ROIs) and control (n = 12 ROIs). Protein profiling was performed with a 79-plex oligonucleotide-antibody cocktail for 24 ROIs.

[0146] FIGS. 6A and 6B are violin plots showing normalized read counts of a-SMA (FIG. 6A) and CTLA4 (FIG. 6B). Statistical comparisons are between reads from healthy controls (n = 12 ROIs) and patients with IPF (n = 12 ROIs). FIG. 6C is a plot showing correlation of CTLA4 and ACTA2, statistical comparisons are between reads from healthy controls (n = 12 ROIs) and patients with IPF (n = 12 ROIs).

[0147] Table 1 contains the complete list of proteins that were expressed in regions of interest in IPF vs. control.Table 1 - Differences in protein expression within regions of interest (ROIs) in IPF vs. control (n = 12 ROIs)

[0148] To determine if the finding of the upregulated expression of the CTLA4 immune checkpoint protein was more broadly applicable to human lung fibrotic disorders, a publiclyavailable single cell RNA-sequencing (scRNA-seq) dataset was queried. The dataset query confirmed that CTLA4 is highly expressed in pulmonary fibrosis, preferentially within the T cell population, in a cohort of 20 patients with pulmonary fibrosis, including 12 IPF, and 10 controls representing 114,396 total cells. FIG. 7A shows a cell distribution map in the Banovich / Kropski dataset of CTLA4 in subpopulations in scRNA-sequencing data. FIG. 7B shows a cell distribution map of UMAP of CTLA4 expression in immune cells. FIG. 7C shows a plot of CTLA4 expression in proliferating T cells in ILD patients compared to control / healthy subjects. FIG. 7D shows the QuPAth quantitation of H&E staining. FIGS. 7E and 7F show the quantitative Masson’s trichrome staining and total lung hydroxyproline levels, respectively. FIGS. 7C-7F illustrate the expression of CTLA4 on CD8+ or CD4+ positive cells in lung tissues from patients with IPF. CTLA4 (green), CD8+ T cell (red) CD4+T cell (red), and DAPI (blue). Scale bars, 50 pm. Data are reported as mean ± SEM. Statistical differences in FIGS. 7A-7F were tested using a paired Student’s t-test (two-tailed).

[0149] Based on the cell-specific expression of CTLA4, the spatial relationships of T cells within fibroblastic foci by immunofluorescence staining of human IPF and control subjects were explored. CTLA4+ cells were found to be localized near or adjacent to a-SMA expressing myofibroblasts in IPF, while these cells were relatively absent around normal a-SMA expressing smooth muscle cells lining blood vessels or airways in control subjects. FIG. 8A shows the representative immunostaining images of lung tissues from healthy controls and IPF patients for CTLA4 in green, a-SMA in red, and DAPI in blue, with the scale bars representing 50 pm.

[0150] Within fibroblastic foci of IPF lungs, CD8+ T cells were the predominant T cell subset expressing CTLA4 compared to CD4+ T cells. FIG. 8B shows the expression of CTLA4 on CD8 or CD4 positive cells in lung tissues from patients with IPF, with CTLA4 shown in green, CD8+T cell shown in red, and CD4+T cell shown in red, and DAPI shown in blue, with the scale bars representing 50 pm. Statistical differences between the images in row A of FIG. 8A and row B of FIG. 8B were tested using paired Student’s t-test (two-tailed). These data suggest a potential role for CTLA4+ expressing CD8+ T cells in cell-cell crosstalk within active fibrotic niches in human IPF.

[0151] Spatial transcriptomics using the lOx Genomics Xenium transcriptomics platform designed for the human lung were performed to explore spatial relationships of the expressive cells of IPF lungs. In three IPF lung samples, a total of 62,485 cells, containing 55,928,562transcripts from a 289 gene panel were identified. Using the Xenium annotated myofibroblast markers, COL5A2 and COL8A1 (shown in FIG. 9A), fibroblastic foci which localized to previously identified regions by histopathological staining were distinctly identified, which can be seen in FIG. 9B. FIG. 9 A shows the Xenium spatial plot showing expression of COL5A2, CTLA4, AGER and SFTPD in fibrotic regions in section A, and shows the Xenium spatial plot showing expression of C0L8A1, CTLA4, AGER and SFTPD in fibrotic regions in section B.

[0152] To determine the expression of CTLA4 at single-cell resolution in CD8+ T cells, transcript levels of CTLA4 in this cell population were quantitated and relative levels of expression were compared in fibrotic regions (enriched for COL5A2) compared to less fibrotic regions (enriched for AGER). This analysis showed significantly higher CTLA4 levels in CD8+ T cells localized to fibroblastic foci than in regions of the IPF lung without distinct foci, which can be seen in FIG. 9C. FIG. 9C is a bar graph displaying CTLA4 transcripts level in less fibrotic and fibrotic regions of interest (ROIs). Data are from three IPF lungs, with 5 fibrotic and less fibrotic regions analyzed per IPF lung. In FIG. 9C, the bar graph represents “fibrotic regions” vs. “minimally fibrotic regions” with 5 regions each from 3 different IPF lungs and where n = 15 per group.

[0153] The gene expression patterns of CTLA4 in specific T cell subpopulations using the Xenium-based spatial transcriptomics were characterized. It was found that fibroblastic foci appear to be markedly enriched for CD8 gene expression, while CD4 was more uniformly expressed throughout the IPF lung. This can be seen in FIG. 9D, which gives Xenium spatial plot showing expression of COL5A2, CD8, CD4 and overlay in IPF lung.

[0154] Importantly, within fibroblastic foci, CTLA4 was preferentially upregulated in CD8 expressing cells in comparison to CD4 cells. This can be seen in FIGS. 9E and 9F. FIG. 9E is a Xenium spatial plot showing expression of COL5A2, CD8, CD4 with a magnified view showing CTLA4 transcript expression on CD8+ T cells. FIG. 9F plots CTLA4 transcript numbers in CD4+ T cells vs. CD8+ T cells in IPF lungs, where the bar graph represents “fibrotic regions” vs. “minimally fibrotic regions” with 5 regions each from 3 different IPF lungs and where n = 15 per group. Data are reported as mean ± SEM; statistical differences were tested using a paired Student’s t-test (two-tailed). These data suggest a potential role for cell-cell crosstalk between CTLA4+ expressing CD8+ T cells and myofibroblasts within active fibrotic niches of human IPF lungs.

[0155] Example 2 - CTLA4 checkpoint blockade induces alveolar type 2 cell regeneration and resolves fibrosis.

[0156] CTLA4 has been linked to deficient T cell responses in cancer, but its role in fibrotic disorders remains unclear. To determine a potential role for this immune checkpoint protein in lung fibrosis, a humanized mouse model carrying exon 2 and 3 of the human CTLA4 gene (hCTLA4 mice; C57BL / 6 background) encoding an epitope containing its extracellular ligand binding and transmembrane domains was used. This epitope is known to bind the FDA-approved anti-CTLA4 monoclonal antibody, ipilimumab. FIG. 10A shows the schematic representation of the humanized CTLA4 knock-in mouse carrying exons 2 and 3 of the human CTLA4 gene. In this Example, mice were treated with two doses of bleomycin (1.25 U kg'1body weight) 14 days apart to induce a more sustained fibrotic response. To more specifically target the fibrotic (vs. inflammatory) response, we started treatment 21 days after the second bleomycin dose with either ipilimumab or control isotype IgGl (5 mg kg'1body weight twice per week for 9 doses). FIG. 10B shows the experimental outline of humanized CTLA4 mice subjected to repetitive bleomycin injury induced pulmonary fibrosis via oropharyngeal administration of bleomycin (1.25 U kg-1 body weight), two doses, 14 days apart - on day 21 after the 2nd dose. Mice were treated with intraperitoneally with either control IgGl isotype or the anti-CTLA4 monoclonal antibody ipilimumab at doses of 5 mg kg-1 body weight twice per week for 4.5 weeks (total of 9 doses).

[0157] Ipilimumab treated mice showed diminished fibrosis, as evidenced by hematoxylin and eosin staining (top panel of FIG. 11) and Masson’s trichrome staining (bottom panel of FIG. 11). FIG. 11 shows representative images of H&E (top) and Masson’s trichrome (bottom) stained whole lung sections from bleomycin-induced mice treated with either IgGl isotype control or ipilimumab. The scale bars are 800 pm.

[0158] The diminished fibrosis in the ipilimumab treated mice was also evidenced by quantitative Masson’s trichrome staining, such as is shown in FIG. 12A, and expression of the pro-fibrotic genes, a-SMA (shown in FIG. 12B) and Collal (shown in FIG. 12C). FIG. 12 A shows the quantification of Masson’s trichrome staining in the IgGl isotype control or ipilimumab groups. Collagen containing areas were identified with machine learning algorithms using inForm® automated image analysis software. Data represent IgGl isotype control and ipilimumab (n = 4 in each group). FIGS. 12B and 12C are bar graphs showing gene expressionof pro-fibrotic gene Acta2 (FIG. 12B) and Collal (FIG. 12C). The data represent IgGl isotype control and ipilimumab (n = 4 in each group).

[0159] FIGS. 12D and 12E are bar graphs showing more data regarding gene expression of Acta2 (FIG. 12D) and Collal (FIG. 12E) in lungs of mice treated with IgGl control or ipilimumab (n = 6 per group). Data are reported as mean + SEM; statistical differences were tested using a paired Student’s t-test (two-tailed).

[0160] To gain insights into the mechanisms by which CTLA4 immune checkpoint blockade mediates its anti-fibrotic effects, scRNA-seq on lungs of mice treated with ipilimumab and control isotype IgGl was performed. Consistent with its targeted mechanism of action, ipilimumab treatment resulted in the expansion of T cells, specifically Cd3e+T cells, such as is shown in FIGS. 13A and 13B. FIGS. 13A and 13B show UMAP of whole lung cells from the IgGl isotype control and ipilimumab groups. Cell clusters identified by scRNA-seq are colored by cell type. UMA indicating the sftpal+ cell cluster is highlighted with a hexagon.

[0161] The T cell activation / differentiation marker genes, Icos and Tcf7, were markedly increased in T cells, seen in FIG. 15, with preferential upregulation in Cd3e+ T cells, in comparison to Cd3d+ T cells. This comparison can be seen in FIG. 15. FIG. 15 is a volcano plot showing differential gene expression in T+ cell cluster of scRNA-seq IgGl isotype control and ipilimumab data set. A total of 99 DEGs [log2(FC) > 0.3 and FDR-adjusted P < 0.05]. FIG. 16A is a violin plot showing gene expression of Icos in the Cd38+ and Cds-i- T cell clusters of the scRNA-seq data for whole lung cells from the IgGl isotype control or ipilimumab groups.

[0162] Flow cytometric analyses of collagenase-digested lungs at the same time-point showed an increase in both the %CD8+ cells and the subpopulation expressing CTLA4 following ipilimumab treatment. FIG. 16B shows representative flow cytometry plots of lung cells isolated from IgGl and ipilimumab treated groups and stained for CD45, CD3, CTLA4, and CD8. FIG. 16C shows a bar graph depicting percentage of CTLA4 expressing CD8+ T cells from IgGl isotype control and ipilimumab treated groups from FIG. 16B. Data are reported as mean ± SEM, and statistical differences were tested using a paired Student’s t-test (two-tailed).

[0163] In concert with these changes, a remarkable increase in the number of alveolar epithelial type 2 (AT2; marked by the Sftpal gene) cells, facultative stem cells of the distal lung epithelium, was noted in the ipilimumab treated group, as shown in FIGS. 13A and 13B. FIG. 14shows a graph of the cell type annotation and relative proportions from scRNA-seq of IgGl and ipilimumab treated groups.

[0164] In the Sftpal+ AT2 cell cluster, increased gene expression of other surfactant protein (Sftp) coding genes, Sftpb, Sftpc, and Sftpd were confirmed. FIGS. 17A-17C and FIG. 18 show this data. FIGS. 17A-17C are violin plots showing AT2 cell expression of the marker genes sftpb (FIG. 17A), sftpc (FIG. 17B), sftpd (FIG. 17C) in the sftpal+ cell cluster. FIG. 18 is a volcano plot showing differential gene expression in the sftpal+ cell cluster of the scRNA-seq data set for the IgGl isotype control and ipilimumab groups. A total of 399 DEGs were identified [log2(FC) > 0.3, FDR-adjusted P < 0.05].

[0165] In this Example, a total of 399 genes were differentially expressed between the ipilimumab and control isotype IgGl group in the Sftpal+ AT2 cell cluster. Table 2 shows the complete list of genes expressed.Table 2 - Differentially expressed genes in the AT2 cell cluster, comparing ipilimumab and control IgGl groups of bleomycin injured mice.

[0166] The expansion of AT2 cells in the ipilimumab treated group versus the control isotype IgGl group was confirmed by high-plex single-cell spatial phenotyping of pro-SPC expressing cells in whole lung tissue sections. This can be seen in FIGS. 19, 20, and 21A-21C. FIGS. 19 and 20 are representative images of pro-SPC+ cells in IgGl isotype control and ipilimumab groups. The scale bar is 50 pm. FIG. 21 A is a phenotyping of pro-SPC+ cells in whole lung. The images were processed with machine learning algorithms in inForm® automated image analysis software. Spatial analysis of pro-SPC+ cells was done in IgGl isotype control and ipilimumab (n = 5 in each group). Together, these data indicate that targeting CTLA4-expressing T cell subsets mediates robust epithelial regenerative responses and enhances resolution following fibrogenic lung injury. FIG. 2 IB is a bar graph showing the percentage of SPC and Ki67+ cells in IgGl isotype control and ipilimumab groups (n = 3 mice per group). Data are reported as mean ± SEM; statistical differences in FIGS. 21A and 21B were tested using a paired Student’s t-test (two-tailed). FIG. 21C shows representative flow cytometry plots of Ki67+SPC cells in IgGl and ipilimumab groups.

[0167] The high-plex single-cell spatial phenotyping discussed above involved analyzing pro-SPC-expressing cells in whole lung tissue sections along with a-SMA, CD8, CTLA4 staining to visualize distribution of epithelial cells, myofibroblasts, CD8+ T cells and the expression of CTLA4. These studies confirmed the proximity of CTLA4-expressing CD8 T cells in regions of active fibrosis enriched in a-SMA expressing myofibroblasts and overall reduced population of pro-SPC expressing AT2 cells in bleomycin injured mice, while this association of “exhausted” CD8+ T cells with myofibroblasts was reduced in ipilimumab treated mice in association with an expansion of AT2 cells. FIGS. 22 and 23 show IHC staining of lungs from IgGl control and ipilimumab treated mice. FIG. 22 shows a representative multiplex IHCstaining with a-SMA (green), pro-SPC (yellow), CD8 (cyan), and CTLA4 (red) in IgGl group. FIG. 23 shows a representative multiplex IHC staining with a-SMA (green), pro-SPC (yellow), CD8 (cyan), and CTLA4 (red) in the ipilimumab treated group.

[0168] Example 3 - Ipilimumab reduces the accumulation of p 16I lk4expressing senescent cells in vivo and potentiates CD8 T cell cytotoxic responses ex vivo following fibrogenic lung injury.

[0169] Cellular senescence is increasingly being implicated in the etiology of age-related diseases, including organ fibrosis, p 16Illk4aexpressing cells, both epithelial and mesenchymal, have been found to be increased in lung samples of individuals with IPF, and senolytic clearance by pharmacologic approaches have been proposed as a potential treatment for IPF. To determine whether CTLA4 checkpoint blockade functions as an endogenous immune mechanism for clearance of senescent cells, the number ofexpressing cells in lung tissues of mice treated with ipilimumab and control IgGl. Significantly fewer p I6lllk4apositive cells were observed in lungs of ipilimumab-treated mice were compared, as shown in FIGS. 24A, 24B, and 25. FIGS. 24A and 24B show immunohistochemistry staining of p 16I K4in lung tissue sections from the two groups: receiving either IgGl isotype control or ipilimumab groups (n = 4 in each group). The scale bars are 100 pm. FIG. 25 shows the quantification of p 16 INK4a positive cells observed in high power field (hpf) of the IgGl isotype control and ipilimumab (n = 4 in each group).

[0170] The ex vivo cytotoxic effects of CD8+ T cells on lung cells isolated from bleomycin injured mice were assessed. Activation of these CD8+ T cells ex vivo resulted in decreased cell viability and cell attachment suggesting increased cell killing under conditions of CTLA4 blockade, as depicted by FIG. 26. FIG. 26 is a schematic representation of ex vivo studies of CD8+ T cell-mediated killing. Direct cytotoxicity of ipilimumab-treated CD8+ T cells on sorted b-GAL+ senescent cells was confirmed by co-incubation and live cell imaging, as shown in FIGS. 27 and 28. FIG. 27 shows representative images of CD8+ T cells killing of SPiDER-PGal positive cells. CD8+ T cells and SPiDER-0Gal positive cells were isolated from bleomycin mice and co-cultured in a 1:2 ratio for 12 h with or without ipilimumab. FIG. 28 shows live cell percent confluency measured using an Incucyte system, for both CD8+ T cells and whole lung cells isolated from bleomycin mouse treated mice. These cells were co-cultured for 12 hours in al:2 ratio with or without ipilimumab (0.05 pg ml'1). Together, these in vivo and ex vivo datasupport cytotoxic effects of CD8+ T cells on senescent cells, and their enhanced killing efficiency with CTLA4 blockade.

[0171] Example 4 - Methods - Human Lung Tissue Specimen

[0172] Biospecimens of idiopathic pulmonary fibrosis (IPF) patients and controls were obtained from a biorepository established through a Program Project Grant, P01 HL114470, supported by the National Institutes of Health, USA. The Institutional Review Board at Tulane University, New Orleans, Louisiana approved the use of deidentified patient specimens.

[0173] Example 5 - Methods - Spatial Transcript Profiling- nanoString GeoMx

[0174] For the nanoString GeoMx Transcriptomics Assay, tissue slides were prepared according to the protocol provided in the user manual (NanoString, MAN- 10100-02). In brief, 5 [im sections of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and healthy controls were examined for spatial proteomics. The Human Whole Transcriptome Atlas, comprising 18,676 RNAs, was utilized on the GeoMx Digital Spatial Profiler Platform (NanoString Technologies, Seattle, WA) to capture genetic data from transcribed mRNA. Morphological markers, including pan-cytokeratin (pan-CK; a marker for lung epithelium), CD31 (a marker for vascular endothelium), and a-smooth muscle actin (a- SMA; a marker for smooth muscle cells and myofibroblasts), were employed to visualize regions of interest. For IPF samples, regions of interest (ROIs) were selected to capture fibroblastic foci enriched in a-SMA within alveolar regions. In healthy control lung tissues, ROIs were randomly chosen to represent normal-appearing areas of airway epithelium, blood vessels, and alveoli. After incubation (mRNA probe) and hybridization (morphological markers), the slides were loaded into the GeoMx Digital Spatial Profiler (DSP) instrument, which utilizes a programmable digital micromirror device (DMD) to direct UV light at specific ROIs. This light activation releases photolabile indexing oligonucleotides in a region- specific manner. The released oligos were collected through microcapillary aspiration, dispensed into a microtiter plate, and quantified using next-generation sequencing (NGS) for digital counting. A total of 18,676 genes were assayed using the NanoString Human Whole Transcriptome Atlas. The DSP instrument was used to capture high-resolution wide-field scans of each tissue section, from which ROIs were selected for UV-induced oligo collection. The barcoded oligos were then collected and digitally quantified using an Illumina sequencer.

[0175] Example 5 - Methods - Immunofluorescence Staining

[0176] Consecutive sections from control and IPF lung tissues were processed for immunofluorescence staining. These unstained sections were incubated at 65 °C to deparaffinize and were then allowed to stand at room temperature for 45 minutes. The remaining paraffin was removed through immersion in xylene. The tissue sections were gradually hydrated by sequential immersion in a graded alcohol series (absolute ethanol, 98%, 95%, 70% ethanol) and, finally, in water. Antigens were unmasked by steaming in a coplin jar filled with Diva decloaker antigen retrieval solution (Biocare, V2004). The slides were cooled for 20 minutes and washed with deionized water. Non-specific antibody binding was blocked using a background blocking solution (Biocare, BS966). Lung sections were incubated with primary antibodies against CTLA4 (Biocare, API3211AA), CD8 (Biocare, ACI3160AA), CD4 (Biocare, API3209AA) and a-smooth muscle actin (Cell Signaling Technology, 19245). Primary antibodies were visualized with donkey anti-rabbit secondary antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A32754), and goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific, A32723). Sections were washed (PBS / Tween 0.01%) and mounted with ProLong™ Gold antifade mount with DAPI (Thermo Fisher Scientific, P36941). Images were captured using a fluorescence microscope (Keyence, BZ-X716) and representative images are presented.

[0177] Example 6 - Methods - Spatial Transcript Profiling- Xenium

[0178] Xenium In Situ Platform was utilized for subcellular mapping of 289 genes (Xenium VI) in lungs of three human subjects with IPF. Each probe in this system consists of two paired sequences that are complementary to the targeted mRNA, along with a unique gene- specific barcode. When these paired sequences bind to their target, they undergo ligation, forming a circular probe. This circular structure is then amplified through rolling circle amplification, which enhances the signal-to-noise ratio and improves target detection and decoding.

[0179] Example 7 - Methods - Sample Processing

[0180] Tissue samples were submitted to the COBRE Multiomics Core at Tulane University for processing. In brief, sample preparation involved rehydrating and sectioning FFPE blocks with a microtome (Leica RM2135) to obtain 5 pm sections, which were then mounted onto Xenium slides (lOx Genomics). After an overnight drying period, the slides were stored in a sealed desiccator at room temperature for up to 7 days before being placed in imaging cassettes for further processing. The deparaffinization and decrosslinking steps rendered sub-cellular RNA targets accessible. Gene panel probe hybridization was conducted overnight at 50°C using a Bio-Rad DNA Engine Tetrad 2. Following this, unbound probes were washed away. Ligase was then introduced to circularize the paired ends of the bound probes at 37 °C for 2 hours, followed by rolling circle amplification at 30°C for 2 hours. The prepared slides were stored in PBS-T in the dark at 4°C for up to 5 days before being analyzed with the Xenium Analyzer (please also refer manufacturer’s instructions CG000578 and CG000580).

[0181] Example 8 - Methods - Post-Xenium H&E

[0182] After the Xenium run, slides were removed from the Xenium Analyzer instrument and the quencher removed according to lOx Genomics’ Demonstrated Protocol CG000613. Immediately following, the slides were H&E-stained. H&E sections were scanned using the Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences).

[0183] Example 9 - Methods - Animal Studies

[0184] Our studies almost exclusively used male mice based on the higher preponderance of the idiopathic pulmonary fibrosis in males. Mice studies were conducted in accordance with protocols approved by Tulane University Institutional Animal Care and Use Committee (protocol #1196). Humanized CTLA4 mice (hCTLA4) were purchased from genOway and housed at Tulane University Vivarium. We have used 12 weeks old male mice for this study. hCTLA4 mice were anesthetized with isoflurane / oxygen and were administered bleomycin via the oropharyngeal route. Each mouse was administered 1.25 U kg-1 body weight Bleomycin in two doses, two weeks apart. On day 21, the control bleomycin group received IgGl isotype control (Bio X cell, BE0297) and the treatment bleomycin group was received ipilimumab (Bio X cell, SIM0004) at a concentration of 5 mg kg-1 body weight twice a week for 4.5 weeks by intraperitoneal injection. At the end of the treatments, the mice were euthanized, and the right lungs were collected for single-cell RNA sequencing and the left lungs for histologic analysis.

[0185] Example 10 - Methods - Single-cell RNA-seq

[0186] A million cells were collected from the right lung of the mice treated with either IgGl isotype control or ipilimumab. The lungs were minced into (~1 mmA3) using a scalpel prior to enzymatic dissociation. The minced sections were then transferred into a 50 ml conical tube containing 10 ml of enzymatic digestion mix (Collagenase 1 mg / ml with DNase I in 9 ml of serum free DMEM). The conical tubes were then placed on a 37° C rocking shaker (220 rpm) for 30 minutes. The digestion mixture was then filtered using a 70 Dm cell strainer. Cells were then washed 10% of complete DMEM (500 g for 5 minutes at 4°C). The resulting pellet wasresuspended in 1 ml of PBS (containing 0.04% BSA). Using a Cellometer, we validated cell numbers and viability counts before the preparation of the scRNA. A commercially available lOx single cell RNA seq technology kit (lOx Genomics Inc, Pleasanton, CA) was used to evaluate 5000 live cells from each sample. Single cell suspensions were partitioned into nanoliter-scale Gel beads-In-Emulsion (EMs). Full-length barcoded cDNAs were then generated and amplified by PCR to obtain sufficient mass for library construction. Single cell 3’ libraries with standard Illumina P5 and P7 paired-end constructs were prepared by enzymatic fragmentation, end-repair, polyA-Tailing and adaptor ligation. Library quality control tests were performed by using an Agilent High Sensitivity DNA kit with Agilent 2100 Bioanalyzer and quantified using a Qubit 2.0 fluorometer. Libraries were pooled to a final concentration of 1.8 pM and were sequenced with paired end single index configuration using Illumina NextSeq 2000. Cell Ranger version 6.1.2 (lOx Genomics) was used to aligned scRNA-seq data to mmlO mouse. SoupX v 1.6.2 was used to analyze 10X Cell Ranger output with default parameters to remove contaminating ambient RNA. The ambient RNA corrected SoupX gene matrix outputs were then output files were read into process raw sequencing Seurat suite version 4.2.0 (38) and cells with low quality were further excluded from the downstream analysis. For clustering, principal-component analysis was performed for dimension reduction. The top 10 principal components (PCs) were selected using a permutationbased test implemented in Seurat and passed to UMAP for clustering visualization. Differentially expressed genes were identified for each cluster with the ‘Find All Markers’ function and cell types were assigned using the cell markers.

[0187] Example 11 - Methods - Lung Hydroxyproline Measurements

[0188] A colorimetric assay kit from Sigma- Aldrich (CAT#MAK008) was used to measure hydroxyproline content in whole lungs of experimental mice. The reaction of oxidized hydroxyproline with 4-dimethyl amino benzaldehyde yields a calorimetric product proportionate to the amount of hydroxyproline present, which is used to calculate the hydroxyproline concentration using the standards provided. Whole lungs from mice were weighed and homogenized in water (10 mg tissue / 100 pl H2O). The homogenized samples were then hydrolyzed by incubating them for 3 hours at 120°C with 12N hydrochloric acid. The hydrolyzed sample was mixed and centrifuged at 10,000 g for 3 minutes, and 10 mL of clear supernatant was transferred to a 96- well plate and placed in a 60 °C oven to dry samples. Chloramine T / Oxidation Buffer Mixture Diluted DMAB Reagent freshly prepared according tomanufacturer’s instructions. After the addition of 100 pl of Chloramine T / Oxidation Buffer (5 min incubation at RT) and 100 pl diluted AM AB reagent to each well, the plate was incubated for 90 minutes at 60°C, and absorbance measured at 560 nm.

[0189] Example 12 - Methods - Mouse Histological Analysis

[0190] At the end of the animal experiments, mice were euthanized, and the lungs were perfused with PBS. After perfusion, the left lobe of the mouse lung was fixed in 10% neutral buffered (pH 7.4) formalin for 24 hours at room temperature. The lung tissues were paraffin- embedded and sectioned at 5 pm thickness. These sections were utilized for H&E staining, Masson's trichrome staining methods as described previously. Slides were scanned on Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Trichrome stained areas were quantified with machine learning algorithms using inForm® automated image analysis software inbuilt. H & E staining was quantified using QuPath software.

[0191] Example 13 - Methods - Multispectral Fluorescence Immunohistochemistry Pro- SPC

[0192] Multispectral fluorescence immunohistochemistry (mIHC) staining was performed using an Opal 7- Color Automation IHC Kit (Akoya Biosciences, Marlborough, MA), following the manufacturer's guidelines. The following primary antibodies were used: pro-SPC (Abeam, ab90716, 1:500), a-SMA (Cell Signaling Technology, 19245, 1:100), CD8 (Cell Signaling Technology, 98941, 1:100), and CTLA4 (Cell Signaling Technology, 53560, 1:100). The protocol involved deparaffinizing the slides, performing antigen retrieval, and blocking with antibody diluent (provided in the kit). The slides were incubated overnight with the primary antibodies, followed by a 10-minute incubation with HRP-conjugated secondary polymer (provided in the kit); slides were then incubated with HRP-reactive OPAL fluorescent reagents: Opal 480 (CD8), Opal 520 (for a-SMA) Opal 570 (for pro-SPC) Opal 690 (CTLA4) (staining with each Opal was performed as instructed by manufacturer, Akoya Biosciences). After incubation, the sections were counterstained with DAPI and mounted. Imaging was performed using the Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Spectrally unmixed images were generated using Akoya Biosciences vl.4.8 software, and further analysis was conducted using machine learning algorithms in the inForm software. Tissue regions were categorized into pro-SPC+ and "other" (cells negative for pro- SPC). The cell phenotype classification, implemented in the inForm software, was based onmultinomial logistic regression using image features derived from texture analysis and cell segmentation. The density of pro-SPC+ cells (cells / mm2) was calculated.

[0193] Example 14 - Methods - Multispectral fluorescence immunohistochemistry pie^

[0194] m-IHC staining was performed as described above. Akoya Biosciences,NEL840001KT was used for multiplexing. The following antibodies pro-SPC (abeam, ab90716, 1:500) pl6INK4A (Abeam, EP1551Y, 1:100) were used. The protocol involved deparaffinizing the slides, performing antigen retrieval, and blocking with the antibody diluent (provided in the kit). The slides were then incubated with the primary antibodies, followed by incubation with the HRP-conjugated secondary polymer (provided in the kit). Subsequently, the slides were incubated with HRP-reactive OPAL fluorescent reagents: Opal 520 (pro-SPC) and Opal 690 (pl6INK4A) Images of the tissue regions were captured using the inForm software package, and the pl6INK4A expression was analyzed.

[0195] Example 15 - Methods - Flow Cytometry

[0196] The lungs were minced into (~1 mm3) using a scalpel prior to enzymatic dissociation. The minced sections were then transferred into a 50 ml conical tube containing 10 ml of enzymatic digestion mix (1 mg / ml collagenase with DNase I in 9 ml of serum free DMEM). The conical tubes were then placed on a 37° C rocking shaker (220 rpm) for 30 minutes. The digestion mixture was then filtered using a 70 pm cell strainer. Cells were then washed 10% of complete DMEM (500 g for 5 minutes at 4°C). The resulting pellet was resuspended in 0.2 ml of PBS. Cells were then stained for flow cytometry. The antibodies used included anti-CD45 (BioLegend, 103115), CD3 (BioLegend, 100205), CD8 (BioLegend, 140403), CD4 (BioLegend, 100506), CD152 (BioLegend, 130-116-931), pro-SPC (Santa Cruz Biotechnology, sc-518029), EpCAM (Thermo Fisher Scientific, 17-5791-80), Ki67 (Santa Cruz Biotechnology, sc-23900).

[0197] Example 16 - Methods - T cell-Mediated Killing Assay

[0198] hCTLA4 mice were anesthetized with isoflurane / oxygen and exposed to two doses of bleomycin (1.25 U kg'1body wt, oropharyngeal route) administered two weeks apart. Five weeks after the first bleomycin dose, mice were euthanized, and lungs were harvested for cell isolation. In brief, lungs were minced into ~1 mm3pieces using a scalpel prior to enzymatic dissociation. The minced sections were then transferred into a 50 ml conical tube containing 10ml of enzymatic digestion mix (collagenase, 1 mg / ml with DNase I in 9 ml of serum free DMEM). The conical tubes were then placed on a 37°C rocking shaker (220 rpm) for 30 minutes. The digestion mixture was then filtered using a 70 pm cell strainer. Cells were then washed with 10% DMEM and centrifuged (500 g for 5 minutes at 4°C). The resulting pellet was resuspended in 1 ml of PBS containing 0.04% BSA. The major fraction of these cells (70%) was used for CD8+ T cell purification using negative selection with a commercial kit (STEMCELL Technologies). The remaining 30% of (whole lung) cells were stained with SPiDER-P-GAL (DoJindo, SG04-1) and plated into 48- well plate. Next these cells co-cultured with purified CD8+T cells in a 2:1 ratio (0.3 x 104 non-purified cells and 0.15 x 104 CD8+ T cells) in the presence and absence of ipilimumab (SIM0004, Bio X cell; 0.05 pg / ml) for 12 hours. The CD8+ T cell-mediated killing with resulting loss of green signals was assessed by IncuCyte system (IncuCyte® SX5 Live-Cell Analysis System). In parallel, we designed an assay to directly visualize killing of senescent fibroblasts by CD8+ T cells in the presence / absence of ipilimumab. Fibroblasts were isolated from bleomycin-injured lungs by adherence purification as described above. After two weeks, adherent fibroblasts were stained with SPiDER-P-GAL (DoJindo, SG04-1) and flow sorted to isolate senescence-associated pGal (SA- Gal) expressing fibroblasts. CD8+ T cells were stained with red (Biotium, 30089) dye for 15 minutes and washed with PBS to remove unbound stain. The isolated SA-P-GAL expressing fibroblasts were co-cultured with labelled CD8+ T cells in a 2: 1 ratio with / without ipilimumab treatment at the concentration of 0.05 pg / ml for 12 h (as described above for non-purified cells). Images of the co-cultured cells were acquired with the IncuCyte® SX5 Live-Cell Analysis System.

[0199] Example 17 - Methods - Statistics and Reproducibility

[0200] Statistical comparisons between groups were performed using Mann-Whitney test or by Student’s t-test. Statistical analysis was performed with GraphPad Prism 10.0.2 (GraphPad Software, Inc., San Diego, CA, USA).

[0201] Example 18 - Methods - Data Availability

[0202] Data related to the paper are available from the corresponding author on reasonable request. scRNA-seq data were deposited in the gene expression omnibus database as GEO submission number GSM7657238, release date March 31, 2025.

[0203] Although the disclosure has been described with reference to preferred embodiments, persons skilled in the art will recognize that changes may be made in form anddetail without departing from the spirit and scope of the disclosed apparatus, systems and methods.

Claims

CLAIMSWhat is claimed is:

1. A method for treating pulmonary fibrosis in a subject in need thereof comprising: administering to the subject an effective amount of a composition, the composition comprising an anti-CTLA-4 antibody.

2. The method of claim 1, wherein the pulmonary fibrosis is an idiopathic pulmonary fibrosis.

3. The method of claim 2, wherein the anti-CTLA-4 antibody is ipilimumab.

4. The method of claim 3, wherein the ipilimumab is administered at a dose of from about 1 mg / kg to about 10 mg / kg subject body weight.

5. The method of claim 4, wherein the ipilimumab is administered at a dose of about 5 mg / kg subject body weight.

6. The method of claim 4, wherein the ipilimumab is administered at repeated doses at predetermined intervals.

7. The method of claim 6, wherein the ipilimumab is administered 1-4 times per week.

8. The method of claim 7, wherein the ipilimumab is administered for a period of 2-8 weeks.

9. The method of claim 8, wherein the ipilimumab is administered twice weekly for a period of 4 weeks.

10. The method of claim 1, wherein the administration of the anti-CTLA-4 antibody to the subject results in an increase in the subject of one or more of vital capacity, forced vital capacity, residual volume, forced expiratory volume, forced inspiratory flow, peak expiratory flow rate, inspiratory reserve volume, tidal volume, total lung capacity, expiratory reserve volume, and / or maximum voluntary ventilation.

11. The method of claim 1 , wherein the anti-CTLA-4 antibody is administered in conjunction with one or more second therapy or treatment.

12. The method of claim 11, wherein the one or more therapy or treatment is an anti-fibrotic drug.

13. A method for treating idiopathic pulmonary fibrosis in a subject in need thereof comprising administering to the subject an effective amount of a composition, the composition comprising Ipilimumab and a pharmaceutically acceptable carrier thereof.

14. The method of claim 13, wherein the ipilimumab is administered for a period of 2-8 weeks.

15. The method of claim 14, wherein the ipilimumab is administered twice weekly for a period of 4 weeks.

16. The method of claim 15, wherein the administration of the composition to the subject results in an increase in the subject of one or more of vital capacity, forced vital capacity, residual volume, forced expiratory volume, forced inspiratory flow, peak expiratory flow rate, inspiratory reserve volume, tidal volume, total lung capacity, expiratory reserve volume, and / or maximum voluntary ventilation.

17. A method for treating idiopathic pulmonary fibrosis in a subject in need thereof comprising administering to the subject an effective amount of a composition, the composition comprising Ipilimumab at a dose of about 5 mg / kg subject body weight and a pharmaceuticallyacceptable carrier thereof.

18. The method of claim 17, wherein the ipilimumab is administered for a period of 2-8 weeks.

19. The method of claim 18, wherein the ipilimumab is administered twice weekly for a period of 4 weeks.

20. The method of claim 19, wherein the administration of the composition to the subject results in an increase in the subject of one or more of vital capacity, forced vital capacity, residual volume, forced expiratory volume, forced inspiratory flow, peak expiratory flow rate, inspiratory reserve volume, tidal volume, total lung capacity, expiratory reserve volume, and / or maximum voluntary ventilation.

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