Treatment with osteopontin inhibitors

Abstract

Disclosed herein are methods of treatment for diseases and disorders with inhibitor agents of full-length or thrombin-cleaved osteopontin. Particularly disclosed herein are methods comprising treatment of diseases and disorders including for example, granulomas or granulomatous diseases, disorders, or conditions (e.g., non-infectious granulomatous diseases, disorders, or conditions), with antibodies, or antigen-binding fragments thereof, that inhibit thrombin-cleavage of osteopontin or block the activity of thrombin cleavage fragments of osteopontin.

Description

25-10906 (103241.007521) PATENTTREATMENT WITH OSTEOPONTIN INHIBITORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Application No. 63 / 744,731, filed January 13, 2025, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure pertains to treatment of granulomas or granulomatous diseases, disorders, or conditions.BACKGROUND

[0003] Non-infectious granulomatous diseases are chronic inflammatory conditions characterized by the formation of organized immune cell aggregates, primarily macrophages and T cells, within affected tissues. Some diseases, such as granuloma annulare, are confined to the skin, whereas others, including sarcoidosis and Crohn’s disease, are systemic and involve multiple organs.

[0004] Corticosteroids remain the first-line therapy by broadly suppressing inflammation. However, their prolonged use is associated with serious side effects, including diabetes, osteoporotic fractures, and weight gain. For example, the current treatment is high dose prednisone sometimes with methotrexate for more than 1 year followed by tapering the dose of prednisone with major side effects including diabetes.

[0005] It is estimated there are currently over one million cases worldwide with greater than 50,000 new cases a year in the United States, but the current treatment is inadequate for the number of cases. These chronic diseases impose a substantial burden to patients and health care systems; although sarcoidosis may resolve spontaneously in some individuals, approximately 50% of patients receive corticosteroids for months to years (Ref.1). Thus, there remains a critical unmet need for corticosteroid-sparing, mechanism-based therapies that can specifically disrupt granuloma formation.25-10906 (103241.007521) PATENTSUMMARY

[0006] Disclosed herein are methods of treatment for diseases and disorders with inhibitors of full-length or thrombin-cleaved osteopontin. Particularly disclosed herein are methods comprising treatment of diseases and disorders including for example, granulomas or granulomatous diseases, disorders, or conditions (e.g., non-infectious granulomatous diseases, disorders, or conditions), with antibodies, or antigen-binding fragments thereof, that inhibit thrombin-cleavage of osteopontin or block the activity of thrombin cleavage fragments of osteopontin.

[0007] In one aspect, disclosed herein are methods of treatment for diseases and disorders with inhibitors of full-length or thrombin-cleaved osteopontin.

[0008] In some embodiments, the methods comprise administering to the subject an effective amount of an inhibitor of osteopontin. In some embodiments, the inhibitor of osteopontin is an anti-osteopontin antibody. In some embodiments, the anti- osteopontin antibody binds to full- length osteopontin or thrombin-cleaved osteopontin. In some embodiments, the anti-osteopontin antibody prevents thrombin cleavage of osteopontin. In some embodiments, the anti-osteopontin antibody blocks integrin binding of thrombin-cleaved fragments of osteopontin. In some embodiments, the methods further comprise administering to the subject an anticoagulant.

[0009] In some embodiments, methods disclosed herein treat or prevent granulomas or a granulomatous disease, disorder, or condition in a subject in need thereof. In some embodiments, the granuloma or granulomatous disease is a non-infectious granuloma or granulomatous disease, disorder, or condition. In some embodiments, the non-infectious granulomatous disease, disorder, or condition is selected from sarcoidosis, granuloma annulare, necrobiosis lipoidica, necrobiotic xanthogranuloma, annular elastolytic giant cell granuloma (AEGCG), cutaneous Crohn disease, interstitial granulomatous dermatitis, palisading neutrophilic and granulomatous dermatitis. In some embodiments, the non-mfectious granulomatous disease is sarcoidosis.

[0010] The present disclosure also provides compositions comprising an osteopontin inhibitor and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the compositions further comprise an anticoagulant.25-10906 (103241.007521) PATENT

[0011] Also disclosed herein are uses of an osteopontin inhibitor in a medicament for the treatment or prevention of granulomas or a granulomatous disease, disorder, or condition in a subject in need thereof.

[0012] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 illustrates increased recruitment of macrophages in skin granulomas.

[0014] FIG. 2 depicts how osteopontin is specifically expressed in macrophages in human skin granulomas.

[0015] FIG. 3 illustrates how osteopontin induces a pro-inflammatory profile in macrophages after skin injury.

[0016] FIG. 4 provides the results of an in vitro assay with bone marrow-derived macrophages, which demonstrated that thrombin-cleaved osteopontin induced macrophage aggregation.

[0017] FIG. 5 is a schematic of the mouse model of granuloma formation, top, and exemplary histological analysis, lower, to quantify granuloma formation for the entire lung.

[0018] FIGS. 6A-6E show thrombin-cleaved OPN facilitates granuloma formation in the mouse model. FIG. 6A & 6C show exemplary histological images of WT, OPN-KO and OPN-KI mice using the mouse model of granuloma. FIG. 6B & 6D are graphs of the granuloma index, and FIG. 6E is a graph providing quantification of granuloma by area. n=7 for WT and OPN-KO mice, n=3 for OPN-KI mice.

[0019] FIGS. 7A-7F show that anti-OPN antibody A6 suppresses granuloma formation in the mouse model. FIGS. 7A & 7D shows exemplary histological images of IgG control and A6 treated mice. FIGS. 7B and 7F show the quantification of granuloma by area, and FIGS. 7C and 7E show the quantification of granuloma index.

[0020] FIGS. 8A-8F illustrate how inflammatory macrophages induce osteopontin in non-infectious granulomatous diseases. (FIG. 8A) Cell chat signaling analysis from 28 patients with non-infectious skin granulomas reveals osteopontin (OPN, also known as SPP1) signaling as one of the top hits unique to affected tissue. (FIG. 8B) UMAP plot of all immune cells identified in the scRNA-seq data set of 28 patients with non-infectious skin granulomas.25-10906 (103241.007521) PATENT(FIG, 8C) Density UMAP plot of all cells demonstrating that SPP1 expression is specific to the myeloid cluster. (FIG. 8D) Dot plot demonstrating SPPl expression predominantly in myeloid cells. (FIG. 8E) Dot plot of immune cells subcluster demonstrating SPP1 expression in macrophages in affected skin (Macl). (FIG. 8F) Mac 1 and Mac 2 cells as a percent of total cells per sample. Student’s t-test two tailed and unpaired. *P<0.05, **P<0.01, ***P<0.001.

[0021] FIGS. 9A-9H show how SPP1 is specifically expressed in macrophages of non-infectious granulomatous diseases. Single-cell RNA-sequencing dataset of affected and unaffected skin from 28 patients with granulomatous skin diseases (Ref. 7). (FIG. 9A) Cell chat analysis reveals outgoing SPP1 signaling in myeloid cells. (FIG. 9B) Dot plot comparing SPP1 expression across all cell types in unaffected and affected skin samples. (FIG. 9C) Marker genes defining immune subsets. (FIG. 9D) UMAP plot of all immune cells. (FIG. 9E) Density UMAP plot of immune cells demonstrating that SPP1 expression is specific to the Macl cluster. (FIG. 9F) Dot plot comparing SPP1 expression across affected and unaffected skin of individual sarcoidosis patients. (FIG. 9G) Dot plot comparing SPP1 expression across affected and unaffected skin of individual non-sarcoidosis granuloma patients. (FIG. 9H) Bar plot showing relative contribution of immune cells in unaffected and affected granulomatous skin as percentage of total cells. For all dot plots, dot size reflects percent cells expressing the gene, and color illustrates level of gene expression. Data depicted as mean ± SEM. Student’s t-test two tailed and unpaired. *P<0.05, **P<0.01, ***P<0.001.

[0022] FIGS. 10A-10E illustrate how human non-infectious granulomatous diseases specifically induce osteopontin. (FIG. 10A) Spatial transcriptomics confirmed expression of SPPl within skin granulomas. Dot color illustrates level of gene expression. (FIG 10B) Dot plot demonstrating no expression of SPPl in circulating macrophage precursors. (FIG. 10C) Dot plot comparing Tissue factor (F3) expression across cell clusters. (FIG. 10D) Bar plot quantifying the percent of Tissue factor (F3+) positive macrophages expressing SPPl. (FIG.10E) Dot plot comparing SPPl expression in different inflammatory skin diseases. MC: molluscum contagiosum. BP: bullous pemphigoid. AA: alopecia areata. SLE: systemic lupus erythematosus. DLE: discoid lupus erythematosus. CTCL: Cutaneous T-cell lymphoma. HS: hidradenitis suppurativa. LP: lichen planus. AD: atopic dermatitis.25-10906 (103241.007521) PATENT

[0023] FIGS. 11A-11D pertain to the identification of TREM2 and F3 in non-infectious granulomatous diseases. (FIG. 11A) Dot plot comparing TREM2 expression across all immune cells in unaffected and affected skin samples. (FIG. 11B) Bar plot quantifying the percent of TREM2+ macrophages expressing SPP1. (FIG. 11C) Spatial transcnptomics confirmed expression of F3 within skin granulomas. (FIG. 11D) Dot plot comparing Tissue factor (F3) expression across all immune cell clusters between unaffected and affected skin. For all dot plots, dot size reflects percent cells expressing the gene, and color illustrates level of gene expression.

[0024] FIGS. 12A-12J demonstrate how thrombin-cleaved osteopontin is necessary for granuloma formation. (FIG. 12A) Left panel, H& E staining of whole mount, mid-coronal sections of lung tissue in WT, OPN-KO, and OPN-KI mice (n=12 WT, n=9 OPN KO, n=12 OPN-KI). WT mice exhibit more purple-staining immune cell aggregates than OPN-KO or OPN-KI mice. Right panel, immunohistology for macrophages (CD68+ or CD163+). Higher magnification panels as indicated. (FIG. 12B) Quantification of aggregates per high powered field (Granuloma index). (FIG. 12C) Quantification of aggregates per surface area (Granuloma area). (FIG. 12D) Quantification of macrophage immunohistology (Macrophage area). (FIG 12E) Macrophage content assessed by flow cytometry (FIG 12F) Levels of full-length and (FIG. 12G) thrombin-cleaved OPN within lung tissue. (FIG. 12H) H& E staining of whole mount, mid-coronal sections of lung tissue in Spp1fl / fl: LysMcre+ / +and littermate control mice (n=12 Spp1fl / fl: LysMcre+ / +, n=8 control). Control mice exhibit more purple aggregates than Spp1fl / fl: LysMcre+ / +mice. (FIG. 121) Quantification of aggregates per high powered field (Granuloma index). (FIG. 12J) Quantification of aggregates per surface area (Granuloma area). Mean±SEM. Student’s t-test two tailed and unpaired (12C-12G). One-way ANOVA (12B, 12I). *P<0.05, **P<0.01, ***P<0.001.

[0025] FIGS. 13A-13E show SPP1 upregulation within macrophages in cardiac sarcoidosis. SPP1 is upregulated within macrophages in cardiac sarcoidosis. Data from single-cell RNA-sequencing dataset of control and cardiac sarcoidosis heart tissue (Ref. 23). (FIG. 13A) UMAP plot of all cell types. (FIG. 13B) Density UMAP plot demonstrating that SPP1 expression is specific to the monocyte and macrophage clusters, (FIG. 13C) UMAP plot of all macrophages demonstrating elevated SPP1-expressing macrophages in affected cardiac25-10906 (103241.007521) PATENTsarcoidosis tissue, (FIG. 13D) Cell chat analysis of cardiac sarcoidosis reveals SPP1 signaling is strongest in macrophages. (FIG. 13E) Dot plot comparing SPP1 expression across all cell types in control and sarcoid cardiac tissue. Dot size reflects percent cells expressing the gene, and color illustrates level of gene expression.

[0026] FIGS. 14A-14C illustrate how thrombin-cleaved osteopontin promotes immune cell aggregation. (FIG. 14A, 14B) Using an in vitro granuloma formation assay, OPN-R induced a greater number of immune cell aggregates compared to OPN-FL. (14C) Representative image of immunohistology of immune cell aggregates confirming presence of macrophages and T cells. Mean±SEM. Student’s t-test two tailed and unpaired. *P<0.05.

[0027] FIGS. 15A-15C pertain to macrophage analysis in WT, OPN-KO, and OPN-KI mouse pulmonary granulomas. (FIG. 15A) Immunohistology demonstrating that granulomas consist of macrophages (CD 163+ or CD68+), B cells (CD45R+) and T cells (CD3E+). Red dotted line outlines the granuloma. (FIG. 15B) Left panel, H& E staining of whole mount, mid-coronal sections of lung tissue depicting that OPN-KO and OPN-KI mice have the capacity to form granulomatous aggregates. Right panel, immunohistology for macrophages (CD163+ or CD68+). Higher magnification panels as indicated. (FIG. 15C) Flow cytometry' gating scheme for analysis of macrophages in murine lung tissue.

[0028] FIGS. 16A-16H concern pharmacologic targeting of thrombin-cleaved osteopontin reduces in vivo granuloma formation. (FIG. 16A) Treatment with direct thrombin inhibitor (dabigatran etexilate, DE). H& E staining of whole mount, mid-coronal sections of lung tissue of QDOT-treated WT mice fed control chow or dabigatran chow (n=8 in control chow; n=6 in dabigatran chow). (FIG. 16B) Quantitation of granuloma formation by granuloma index (granulomas per high powered field) or (FIG. 16C) surface area involved. (FIG, 16D) Activated partial thromboplastin time test in plasma of mice fed DE-supplemented chow or control chow, (FIG, 16E) Treatment with antibody targeting thrombin cleavage of osteopontin (A6). Left panel: H& E staining of whole mount, mid-coronal sections of lung tissue of QDOT-treated WT mice treated with A6 or IgG control (n=10 for each group). Right panel: immunohistology for macrophages (CD163+ or CD68+). Higher magnification panels as indicated, (FIG. 16F) Quantitation of granuloma formation by granuloma index (granulomas per high powered field) or (FIG. 16G) surface area involved. (FIG. 16H)25-10906 (103241.007521) PATENTQuantification of macrophage immunohistology (Macrophage area). (FIG. 16A) Treatment with DE + A6. H& E staining of whole mount, mid-coronal sections of lung tissue of QDOT-treated WT mice fed control chow + IgG control, or DE chow + A6 (n=6 in both groups). (FIG. 16B) Quantitation of granuloma formation by granuloma index (granulomas per high powered field) or (FIG. 16C) surface area involved. (FIG. 16D) Activated partial thromboplastin time test in plasma of mice fed DE-supplemented chow + A6 or control chow + IgG control. Mean±SEM. Student’s t-test two tailed and unpaired. *P<0.05, **P<0.01, ***P<0.001.

[0029] FIG. 17 depicts a proposed model of OPN function in granuloma formation. Pharmacologic blockade of thrombin-cleaved OPN at three different points reduced granuloma formation.

[0030] FIGS. 18A and 18B provide the results of a test targeting full length or thrombin-cleaved osteopontin with respective antibodies.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031] As disclosed herein, a mouse model was created in which the thrombin cleavage site in the OPN gene has been mutated such that the produced OPN is no longer a substrate of thrombin and cannot be cleaved by thrombin (OPN-KI mice). These mice were used in a model of sarcoidosis in which the disease is induced by inhalation of a nanoparticle containing cadmium. In the OPN-KI and OPN-KO mice there were no granulomas whereas WT mice had significant numbers of granulomas.

[0032] Antibodies were used that inhibit thrombin cleavage of osteopontin or bind to the C-terminus of OPN-R and block its interactions with target integrins. These antibodies have shown no side effects in use in mice and have no findings after a single dose application in rats. They do not block coagulation or platelet aggregation despite preventing thrombin cleavage of osteopontin. In cancer models those antibodies phenocopy the OPN-KI mouse model and prevent and / or treat sarcoidosis.

[0033] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

[0034] Definitions25-10906 (103241.007521) PATENT

[0035] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.

[0036] The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. For example, a composition “consisting essentially of’ recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components.

[0037] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0038] As used herein, the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also di scloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. Other meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.25-10906 (103241.007521) PATENT

[0039] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0040] The term “ammo acid” or “any ammo acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids.

[0041] Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non- proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include [J-amino acids (P3and p2), homo- ammo acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids.Unnatural or non-natural amino acids also include modified amino acids, “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the ammo acid.According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other25-10906 (103241.007521) PATENTas compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state.

[0042] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0043] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, orDF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is.

[0044] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four- character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N- methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3- diaminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y-aminobutanoic acid), |3-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), phPro (P-homoproline), phPhe (P-homophenylalanine) and Bip (p, P diphenyl alanine), and Ida (Iminodiacetic acid).

[0045] ‘ ‘Antibody” and “antibodies” as used herein refer to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi¬ specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark,25-10906 (103241.007521) PATENTa whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described m Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology’ 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0046] Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding. “CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence.

[0047] There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted “CDR1,” “CDR2,” and “CDR3,” for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region, A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the25-10906 (103241.007521) PATENTarnino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0048] A whole antibody typically consists of four polypeptides: two identical copies of a heavy (II) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C -terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (X), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0049] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the P sheets that provide the structural framework of the variable region (see, e.g., C. A.Janeway et al, (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N. Y. (2001)).

[0050] “Humanized” forms of non-human (e g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable regi on of the recipi ent are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capability. In some instances,25-10906 (103241.007521) PATENTframework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

[0051] Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0052] As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 107M1(e.g., >107M'1, >108M'1, >109M'1, >1010M’1, >10nM" >1012M"1, >1013M"1, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

[0053] The terms “fragment of an antibody,” “antibody fragment,” and “antigenbinding fragment” of an antibody and the like are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9)'. 1126-1129 (2005)), Any antigen-bin ding fragment of the antibody described herein is within the scope of the present disclosure. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region25-10906 (103241.007521) PATENT(or portions thereof), the constant region (or portions thereof), or combinations thereof.Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (hi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

[0054] The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstem, Eur. J. Immunol., 5. 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U. S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0055] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0056] The terms “immunogen” and “antigen” are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An “immune response” can entail, for example, antibody production and / or the activation of immune effector cells. An antigen in the context of the disclosure can comprise any subunit, fragment, or epitope of any proteinaceous or non-protemaceous (e.g., carbohydrate or lipid) molecule that provokes an immune response in a mammal. The term “epitope” refers to a sequence of an antigen that is recognized by an antibody or an antigen25-10906 (103241.007521) PATENTreceptor. Epitopes also are referred to m the art as “antigenic determinants,” In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope may include chemically active surface groupings of molecules such as ammo acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three- dimensional structural characteristics (e.g., a “conformational” epitope) and / or specific charge characteristics. The antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity.

[0057] As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs m a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence of the present disclosure after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid or protein is longer than a reference sequence, additional nucleotides or amino acids that do not align with the reference sequence are not taken into account for determining sequence identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al,, Proc. Natl, Acad. Sci, USA, 106(10): 3770-3775 (2009), Durbin et al,, eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al,, Nucleic Acids Res,, 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).25-10906 (103241.007521) PATENT

[0058] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The polypeptides may be modified by the addition of sugars, lipids or other moieties not included in the ammo acid chain.

[0059] An “effective amount” refers to an amount sufficient to elicit a desired biological response (e.g., treating a condition). As will be appreciated by those skilled in the art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, a “therapeutically effective amount” is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0060] A “pharmaceutically acceptable carrier” as used herein generally refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0061] The term “pharmaceutical formulation” as used herein generally refers to a preparation which is in such form as to permit the biological activity of an active ingredient (e.g,, an inhibitor of osteopontin) contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.25-10906 (103241.007521) PATENT

[0062] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children).

[0063] Moreover, subject may mean any living organism, preferably a mammal (e.g., humans and non- humans) that may benefit from the administration of compositions contemplated herein.

[0064] Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.

[0065] Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0066] As used herein, the terms “treatment,” “treat,” and “treating” can refer to reversing, alleviating, reducing, lessening, delaying the onset of, or inhibiting the progress of a disease or condition, or one or more signs or symptoms thereof. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease disorder or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence,

[0067] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the disclosed proteins, polypeptides, nucleic acids, polynucleotides, and small molecules into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. The administration can be by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.25-10906 (103241.007521) PATENT

[0068] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0069] Methods of Treatment

[0070] Disclosed herein are methods of treatment for diseases and disorders with inhibitors of full-length or thrombin-cleaved osteopontin.

[0071] The methods comprise administering to a subject in need thereof an effective amount of an inhibitor of osteopontin. Osteopontin is a matricellular multifunctional protein with a highly conserved RGD domain that binds to a wide range of integrins. Thrombin cleavage at Argl53 in mouse (Argl68 in humans) generates OPN-Arg (OPN-R) and OPN-C-termmal fragment (OPN-CTF). OPN-R, which has SVVYGLR (SEQ ID NO: 3) at its C-terminus, binds to a subset of integrins (a4pi and a9pi) that full-length OPN does not bind to.

[0072] As the phrase is used herein, an “inhibitor of osteopontin” can be any moiety or suite of moieties that, for example, function to disrupt or decrease the expression of osteopontin, prevent or lessen thrombin cleavage of osteopontin, or block integrin binding. Suitable inhibitors of osteopontin may bind to full-length or thrombin- cleaved osteopontin to prevent integrin binding and / or thrombin cleavage. Exemplary osteopontin inhibitors include, but are not limited to, gene silencing oligonucleotides (e.g., an siRNA, an antisense oligonucleotide, dominant-negative, a short-hairpin RNA, a miRNA, a dicer-substrate RNA, a DNAzyme, an guide RNA), protein configured to bind osteopontin or a substrate thereof (e.g,, an anti-osteopontin antibody (e.g., a monoclonal, polyclonal, murine, chimeric, humanized, or human antibody targeting an osteopontin epitope or osteopontin ligand, thus interfering with osteopontin activity or ligand binding)), a small molecule inhibitor of osteopontin, or combinations thereof,

[0073] In some embodiments, the inhibitor of osteopontin is an anti-osteopontin antibody. In some embodiments, the anti-osteopontin antibody prevents thrombin cleavage. In some embodiments, the anti-osteopontin antibody blocks integrin binding. In some25-10906 (103241.007521) PATENTembodiments, the anti-osteopontin antibody blocks integrin binding to the thrombin cleavage fragment of osteopontin.

[0074] In some embodiments, the anti-osteopontin antibody is selected from those described in International Patent Publication WO 2021030209, herein incorporated by¬ reference in its entirety. In some embodiments, the anti-osteopontin antibody is antibody; X6 as described in WO 2021030209. In certain embodiments, the anti-osteopontin antibody is an antibody having light chain and / or heavy chain sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to A6.

[0075] In some embodiments, the anti-osteopontin antibody is selected from those described m Zhou, Qin et al., Journal of Thrombosis and Haemostasis, Volume 23, Issue 11, 3691 - 3703 (November 2025), herein incorporated by reference in its entirety. In some embodiments, the anti-osteopontin antibody is antibody C6R as described in Zhou, et al. In certain embodiments, the anti-osteopontin antibody is an antibody having light chain and / or heavy' chain sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to C6R.

[0076] Provided herein are methods for treating or preventing granulomas or a granulomatous disease, disorder, or condition. As used herein, the term “granuloma” refers to small, granular inflammatory lesions and / or ball-like collection of immune cells formed as a result of inflammatory reaction, for instance, when the immune system attempts to eliminate substances (e.g., infectious organisms such as bacteria and fungi as well as other materials such as keratin, suture fragments and vegetable particles) that it perceives as foreign but is unable to eliminate, A “granulomatous disease, disorder, or condition” refers to any disease, disorder, or condition characterized by the presence of granuloma(s).

[0077] In some embodiments, the granuloma or granulomatous disease is a non-mfectious granuloma or granulomatous disease, disorder, or condition. Non-mfectious granulomatous disease, disorder, or conditions include, but are not limited to, sarcoidosis, granuloma annulare, necrobiosis lipoidica, necrobiotic xanthogranuloma, annular elastolytic giant cell granuloma (AEGCG), cutaneous Crohn’s disease, interstitial granulomatous dermatitis, palisading neutrophilic, and granulomatous dermatitis.25-10906 (103241.007521) PATENT

[0078] In select embodiments, the non-infectious granulomatous disease is sarcoidosis. Granulomas can appear in almost any organ, and most often appear in the lungs or the lymph nodes. Other common sites include the liver, spleen, skin and eyes. The involvement of a specific organ may be mild or severe, self-limited or chronic, and limited or wide-ranging in extent. Symptoms usually appear gradually but can occasionally appear suddenly. The common symptoms experienced by sarcoidosis patients comprise fatigue, shortness of breath, cough that will not go away, skin lesions or skin rashes on face, arms, or shins, inflammation of the eyes, weight loss, night sweats, dyspnea, cough, chest discomfort, crackles, malaise, weakness, anorexia, weight loss, or fever. Other symptoms include, for example, enlarged lymph glands (armpit lump), enlarged liver, enlarged spleen, dry mouth or nosebleed. Symptoms of different types of sarcoidosis are described below. The methods described herein may result in a reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of sarcoidosis, e.g., detectably reduces, the number of, or degree of severity of, or reduces the rate of increase in the number of, or degree of severity, one or more granulomas in said individual.

[0079] Sarcoidosis can be systemic (e.g., systemic sarcoidosis) or local (e.g., localized sarcoid-like reactions). In some embodiments, the sarcoidosis is selected from systemic sarcoidosis, cutaneous sarcoidosis, Lofgren’s syndrome, neurosarcoidosis, pulmonary sarcoidosis, cardiac sarcoidosis, ocular sarcoidosis, hepatic sarcoidosis, musculoskeletal sarcoidosis, renal sarcoidosis, and sarcoidosis with the involvement of other organs or tissues.

[0080] Systemic sarcoidosis is sarcoidosis with multiple organ involvement. In one embodiment, provided herein is a method of treating a subject having or suspected of having systemic sarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of systemic sarcoidosis, e.g., general symptoms such as weight loss, fatigue, loss of appetite, fever, chills, night sweats, formation of granulomas, fatigue, aches and pains, or specific symptoms related to a particular organ affected (e.g., dry eyes, swelling of the knees, blurry vision, shortness of breath, cough, skin lesions such as rashes etc.).25-10906 (103241.007521) PATENT

[0081] Lofgren's syndrome is an acute presentation of systemic sarcoidosis, which is typically characterized by the triad of erythema nodosum, bilateral hilar adenopathy and arthritis or arthralgias. It may also be accompanied by fever. In one embodiment, provided herein is a method of treating a subject having or suspected of having Lofgren's syndrome. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of Lofgren's syndrome.

[0082] Cutaneous sarcoidosis is a complication of sarcoidosis with skin involvement. In one embodiment, provided herein is a method of treating a subject having or suspected of having cutaneous sarcoidosis. In certain embodiments, the cutaneous sarcoidosis comprises annular sarcoidosis, erythrodermic sarcoidosis, ichthyosiform sarcoidosis, hypopigmented sarcoidosis, morpheaform sarcoidosis, mucosal sarcoidosis, papular sarcoid, scar sarcoid, subcutaneous sarcoidosis and ulcerative sarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of cutaneous sarcoidosis, e.g., skin lesions or conditions, such as papules (e.g., granulomatous rosacea, acne or benign appendageal tumors), skin plaques (e.g., psoriasis, lichen planus, nummular eczema, granuloma annulare, cutaneous T- cell lymphoma, Kaposi's sarcoma or secondary syphilis), lupus, erythema nodosum (e.g., raised, red, firm skin sores, cellulitis, furunculosis or other inflammatory panniculitis), and maculopapular eruptions, nodular lesions deeper in the skin or infiltration of old scars.

[0083] Neurosarcoidosis refers to sarcoidosis in which inflammation and abnormal deposits occur in the brain, spinal cord, and any other areas of the nervous system. In one embodiment, provided herein is a method of treating a subject having or suspected of having neurosarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of neurosarcoidosis, e.g., facial weakness (e.g., facial palsy), nerves in the eye or nerves that control taste, smell, or hearing, changes in menstrual periods, excessive tiredness (e.g,, fatigue), headache, visual changes, retinopathy, radicular pain, loss of bowel or bladder control, carpal tunnel syndrome, and / or paraplegia, excessive thirst or25-10906 (103241.007521) PATENThigh urine output, confusion, disorientation, decreased hearing, dementia or delirium, dizziness or vertigo (e.g., abnormal sensation of movement), double vision or other vision problems, facial palsy (weakness, drooping), headache, loss of sense of smell or taste, abnormal tastes, psychiatric disturbances, seizures or speech impairment, muscle weakness or sensory losses, or in some occasions, hypopituiarism.

[0084] Pulmonary sarcoidosis refers to sarcoidosis that affects pulmonary tissues or organs. In one embodiment, provided herein is a method of treating a subject having or suspected of having pulmonary sarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of pulmonary sarcoidosis, e.g., granulomas in alveolar septa, bronchiolar, and bronchial walls, shortness of breath, cough, loss of lung volume and abnormal lung stiffness, abnormal or deteriorating lung function, decrease in lung volume, decreased compliance, scarring of lung tissue, or bleeding from the lung tissue.

[0085] Cardiac sarcoidosis refers to sarcoidosis with myocardial involvement. In one embodiment, provided herein is a method of treating a subject having or suspected of having cardiac sarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of cardiac sarcoidosis, e.g., myocardial infarction, cardiomyopathy, aneurysm, angina, aortic stenosis, aortitis, arrhythmias, arteriosclerosis, arteritis, atherosclerosis, atrial fibrillation and flutter, bradycardia, cardiomegaly, carditis, congestive heart failure, embolism, endocarditis, fibrillation, hypertension, hypotension, and the like.

[0086] Ocular sarcoidosis is sarcoidosis that affects the eye. In one embodiment, provided herein is a method of treating a subject having or suspected of having ocular sarcoidosis. In some embodiments, the methods described herein result in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, of one or more symptoms of ocular sarcoidosis, e.g., uveitis (e.g., granulomatous uveitis), uveoparotitis, retinal inflammation, loss of visual acuity, blindness red, watery eyes, iris nodules, retinochoroiditis, conjunctivitis, lacrimal gland involvement or proptosis.25-10906 (103241.007521) PATENT

[0087] In certain embodiments, sarcoidosis can involve muscle, hepatic, joint, hematologic, psychiatric, renal, splenic, nasal sinus, bone, oral gastric or intestinal, endocrine, pleural or reproductive system and present with respective symptoms.

[0088] In some embodiments, the methods described herein further comprise administration with one or more additional therapies to treat the disease or disorder, or one or more symptoms of the disease or disorder. The additional therapy may include administration of an additional therapeutic agent or a therapy not connected to administration of another agent including, for example, surgery.

[0089] The additional therapy may be administered at the same time as the initial therapy. For example, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the additional therapy may precede or follow the treatment of the initial therapy by time intervals ranging from hours to months.

[0090] In some embodiments, the additional therapeutic agent comprises an immune modulator, a steroid, an analgesic, an antimicrobial agent, an immunotherapy, an anticoagulant, or a combination thereof.

[0091] Administration

[0092] In the methods disclosed herein, administration may be by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to, oral (e.g., by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary’ (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose); rectal; vaginal; parenteral (e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal injection); or by implant of a depot, for example, subcutaneously or intramuscularly.

[0093] Any of the inhibitors of osteopontin (e.g., anti-osteopontin antibodies) may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the inhibitor of osteopontin (e.g., anti-osteopontin25-10906 (103241.007521) PATENTantibody) may be mixed with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure.

[0094] The phrase “pharmaceutically acceptable,” as used in connection with compositions and / or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human).Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and / or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0095] When utilized as a method of treatment, the effective amount and / or dosage may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.

[0096] In the context of the present discl osure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease.25-10906 (103241.007521) PATENT

[0097] Also provided herein are compositions comprising an osteopontin inhibitor according to any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the compositions comprise two or more different osteopontin inhibitors according to the present disclosure.

[0098] The osteopontin inhibitors according to the present disclosure may be provided in a composition that is formulated for any type of administration. For example, the compositions may be formulated for administration orally, topically, parenterally, enterally, or by inhalation (e.g., intranasally). The active agent may be formulated for neat administration, or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing / dispersing agent, tablet¬ disintegrating agent, encapsulating material, film former or coating, flavoring agent, or printing ink. Any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active agent may be incorporated into sustained-release preparations andformulations. Administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.

[0099] In powders, the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the active compound may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active agent(s) in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.25-10906 (103241.007521) PATENT

[0100] Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators.

[0101] Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.

[0102] Suitable examples of liquid carriers, diluents and excipients, for example, for oral, topical, or parenteral administration, include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.

[0103] For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration. Solutions of the active agents can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0104] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably25-10906 (103241.007521) PATENTsterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In some instances, the antimicrobial peptides themselves may be sufficient to prevent contamination by microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0105] Sterile injectable solutions may be prepared by incorporating the active agent in the pharmaceutically appropriate amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions may be prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and freeze drying techniques that yield a powder of the active ingredient or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0106] Thus, an osteopontin inhibitor may be in the present compositions and methods in an effective amount by any of the conventional techniques well-established in the medical field. For example, the administration may be in the amount of about 0.1 mg / day to about 500 mg per day. In some embodiments, the administration may be in the amount of about 250 mg / kg / day. Thus, administration may be in the amount of about 0.1 mg / day, about 0.5 mg / day, about 1.0 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 5025-10906 (103241.007521) PATENTmg / day, about 100 mg / day, about 200 mg / day, about 250 mg / day, about 300 mg / day, or about 500 mg / day.

[0107] It will be appreciated that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present disclosure. The selected dosage level will depend on a variety of factors including, but not limited to, the relative activity of the inhibitor of osteopontin (e.g., anti-osteopontin antibody), the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side¬ effects.

[0108] Administration in vivo can be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0109] The inhibitors of osteopontin (e.g., anti-osteopontin antibodies) may be administered once, on a continuous basis (e.g. by an intravenous drip), or on a periodic / intermittent basis, including about once per hour, about once per two hours, about once per four hours, about once per eight hours, about once per twelve hours, about once per day, about once per two days, about once per three days, about twice per week, about once per week, and about once per month. The composition may be administered until a desired reduction of symptoms is achieved.

[0110] Other therapies, as included in the above methods, may be used in combination with the inhibitors of osteopontin (e.g., anti-osteopontin antibodies).Administered “in combination,” as used herein, means that two (or more) different treatments25-10906 (103241.007521) PATENTare delivered to the subject during the course of the subject's affliction with the disorder, e.g,, the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0111] The following examples further illustrate aspects of the disclosure, but should not be construed as in any way limiting its scope.

[0112] Examples

[0113] Antibody Sequences

[0114] Monoclonal antibody A6 heavy chain 1 and light chain 4 are both disclosed in WO 2021030209, which is incorporated herein by reference in its entirety’.Example 1 — Mechanism of Disease

[0115] The present inventors leveraged their direct access to human diseased tissue to interrogate fundamental mechanisms of disease, 4mm skin biopsies were collected (affected and unaffected skin) from 18 sarcoidosis and 10 non-sarcoidosis skin granuloma patients with clinically active and histologically validated skin disease. Unaffected skin served as control samples. From 55 samples, 492,200 single-cell RNA-sequencing (scRNAseq) profiles were annotated. Identified were 19 types of immune cells. Both25-10906 (103241.007521) PATENTsarcoidosis and non-sarcoidosis affected skin recruited more macrophages and dendritic cells compared to unaffected skin (FIG. 1, left side).

[0116] Among all profiled cells, OPN expression was specific to myeloid cells and more specifically, the macrophage (Mac) I and 3 clusters (FIG. 2).Example 2 —Role of Thrombin-Cleaved Osteopontin

[0117] The present inventors performed single-cell RN A- sequencing of injured skin in WT, OPN-KO, and OPN-KI mice. It was discovered that macrophages in WT mice exhibit an inflammatory phenotype, and this activation is abrogated in OPN-KO and OPN-KI mice (FIG. 3). These results were confirmed in a separate experiment by real-time PCR (data not shown). Thus, it was concluded that thrombin-cleaved OPN may be responsible for activating macrophages to induce granuloma formation.

[0118] To understand how macrophages may be affected by thrombin-cleaved OPN, in vitro assays were performed with bone marrow derived macrophages (BMDM). BMDM was stimulated with recombinant full-length OPN (OPN-FL) and thrombin-cleaved OPN (OPN-R). Both stimuli induced similar amounts of macrophage migration compared to vehicle control (data not shown).

[0119] Notably, OPN-R specifically induced macrophage aggregation that was not seen with OPN-FL stimulation (FIG. 4). Thus, it was concluded that thrombin-cleaved OPN activates macrophages to promote granuloma formation.Example 3 - Granuloma

[0120] For an in vivo granuloma model, mice lungs were instilled with a single dose of Qdot (40 uL) (Q21361MP, Thermo Fisher) particles. 30 days later, mice were sacrificed, and lung tissues were harvested for analysis (FIG. 5). Lung tissues were fixed overnight at 4°C in 4% paraformaldehyde solution. Lung samples were processed for sectioning by standard methods.

[0121] Histology was performed on mid-sagittal sections of lungs. Histology slides were imaged using tiled imaging at lOx magnification on a Leica DM6B / DMC2900 imaging system (Leica Microsystems, Buffalo Grove, IL). Granulomas were counted for each 10X field and averaged over a total number of 10X fields per lung. The granuloma score for lung25-10906 (103241.007521) PATENTtissue was calculated as number of granulomas per 10X field. Each experiment was repeated twice independently.

[0122] As shown in FIGS. 6B-6E, OPN-K1 and OPN-KO mice have a lower granuloma index as compared to wild-type mice indicating the likelihood that thrombin-cleaved OPN facilitates granuloma formation.

[0123] A6 antibodies (24mg / kg) were given daily for five days before injury. After injury, mice were treated twice a week via intraperitoneal injection. As shown in FIGS. 7A-7F, antibody A6 (labeled “Antibody 1” in FIGS. 7D-7F) suppressed granuloma formation in wild¬ type mice, as compared to control mice treated with IgG.Example 4 — OPN As a Signaling Factor in Human Skin Granulomas

[0124] Skin (affected and unaffected) and blood for single-cell RN A- sequencing were collected from 28 patients diagnosed with different types of skin granulomas, including sarcoidosis, granuloma annulare, xanthogranuloma, annular elastolytic giant cell granuloma, or rubella-virus associated skin granulomatous dermatitis (Ref. 8). The Seurat object from this study was used and performed Cellchat, which measures ingoing and outgoing signals within single-cell datasets, to identify the major signaling pathways active among all cell types. Compared to control unaffected skin, OPN (SPP1) was identified as one of the most highly induced signaling pathways (Figure 8A). Notably, OPN outgoing interaction strength was highest in myeloid cells (Figure S1A). Consistent with this analysis, OPN transcript in lesional skin was largely restricted to the myeloid lineage with minimal to low levels of expression in other cell types and within non-lesional skin (Figure 8B-8D, 9B). Thus, OPN is among the strongest signaling pathways activated in granulomatous skin diseases and primarily expressed in the myeloid lineage.

[0125] Subclustering of immune cell populations in the dataset identified two dominant macrophage subpopulations, Macl and Mac2 (Figure 9C, 9D) (Ref 8), Mad exhibited an inflammatory phenotype, including expression of inflammatory marker genes: CCL3, CSTB, and CHTT1, and Mac2 exhibited an anti-inflammatory phenotype, including expression of anti-inflammatory marker genes: SELENOP, RNASEl, MS4A7, and INSIG 1 (Ref. 8). The majority of OPN expression occurred within the Macl macrophage population (Figure 8E, Figure 9D, 9E). These changes were present in most of the individual25-10906 (103241.007521) PATENTgranulomatous patient samples (Figure 9F, 9G). Within affected skin, the inflammatory Macl population was significantly increased, and the anti-inflammatory Mac 2 population was decreased compared to control (Figure 8F, Figure 9H). Lymphoid cells and B cells were also elevated in affected granulomatous skin (Figure 9H). TREM2-expressing macrophages were found in both Mac! and Mac2 populations as a minor subset (< 20%), and these cells rarely co-expressed OPN transcript (~<10%) (Figure 11 A, 1 IB).

[0126] Visium spatial transcriptomics from the affected skin of two granulomatous patients and one unaffected skin as a control, who were part of the single-cell sequencing cohort, revealed strong expression of OPN transcript in the dermis, specifically where granulomas were visualized by H& E staining (Figure 10A). In addition to skin, single-cell RNA-sequencing on blood from our skin granuloma patients was also performed. None of the circulating macrophage precursors (CD16+ or CD14+ monocytes) in granuloma patient blood expressed OPN transcript (Figure 10B). Thus, induction of OPN expression only occurred within the tissue.

[0127] Thrombin is known to cleave full-length OPN into smaller active fragments but measuring thrombin activation in tissues remains technically challenging. As a surrogate marker, it was found that Tissue factor (F3), a regulatory protein that activates thrombin to initiate the blood coagulation cascade, was also specifically expressed within infiltrating Macl macrophages in both the single-cell and spatial datasets (Figure 10C, 11C, 1 ID).Moreover, ~70% of Tissue factor-expressing macrophages also expressed OPN transcript (Figure 10D). Thus, OPN-secreting macrophages may also activate the thrombin pathway.

[0128] To broaden the present findings, published single-cell datasets of other skin infectious granulomatous and inflammatory diseases were interrogated, including, but not limited to, leprosy, atopic dermatitis, psoriasis, hidradenitis suppurativa, and vitiligo (Refs. 9-15). Strikingly, pseudobulk analyses of the immune cells revealed that OPN transcript induction was the highest in non-infectious granulomatous diseases and minimally activated in any other disease (Figure 10E).

[0129] It was asked whether OPN induction was elevated in sarcoidosis granulomas beyond the skin. The only publicly available single-cell dataset for sarcoidosis tissue, other than skin, is derived from cardiac sarcoidosis (Ref. 16). Compared with control25-10906 (103241.007521) PATENTheart tissue, macrophages in cardiac sarcoidosis exhibited higher OPN transcript levels, a greater abundance of OPN+ macrophages, and enhanced OPN signaling (Figure 13).Although single-cell analysis of sarcoidosis lung tissue remains limited, prior studies using immunohistochemistry identified OPN+ cells within lung sarcoidosis granulomas (Ref. 17). Taken together, OPN emerged as the predominant signaling pathway selectively induced within non-infectious tissue granulomas. This induction arose in inflammatory macrophages, was spatially localized within lesions, and coincided with evidence of potential thrombin activation.Example 5 — Mouse Model of Non-infectious Granuloma Formation

[0130] Inventors’ work and others have demonstrated that wild-type (WT) mice treated with cadmium nanoparticles (QDOT) developed pulmonary granulomas that were similar in immune cell composition to human sarcoidosis granulomas, including macrophages, T cells, B cells, and innate lymphoid cells (Ref. 8). 30 days after treating WT mice with QDOT, H& E staining of whole-mount mid-coronal sections revealed discrete purple-stained immune cell aggregates studded throughout the lung parenchyma and an overall dense staining tissue structure (Figure 12A). Immunohistochemistry revealed that, similar to in-vivo human sarcoid granulomas, macrophages (CD68+ or CD163+) and T cells (CD3E+) formed discrete immune cell aggregates with B cell aggregates (CD45R+) localized to the core edges (Figure 12A, right panels; Figure 15A).

[0131] Granuloma formation was quantified using three complementary metrics. First, counted were the number of purple aggregates on H& E sections per high powered field across the entire lung (granuloma index; Figure 12B). Second, the total surface area of lung involvement was measured (granuloma area; Figure 12C). Third, macrophage staining was quantified relative to the total surface area involved (macrophage area; Figure 12D). These analyses showed that QDOT-exposed WT mice generated granulomas in a reproducible and consistent manner. It was then asked whether OPN was necessary for non- infectious granuloma formation.Example 6 - Thrombin-cleaved OPN is necessary for in vivo granuloma formation

[0132] Mice genetically lacking OPN (OPN-KO) were treated with QDOT and harvested lungs 30 days later. H& E staining revealed significantly fewer aggregates in OPN-25-10906 (103241.007521) PATENTKO lungs compared to WT control mice, with almost complete absence of aggregates and a markedly less dense tissue structure (Figure 12A, middle panel, Figure I2B-D).Immunofluorescence for macrophages confirmed their lack of recruitment in the lung. Thus, OPN is necessary for pulmonary granuloma formation in mice.

[0133] To assess whether thrombin cleavage of OPN is necessary for pulmonary granuloma formation, mice engineered with a specific mutation in the OPN thrombin cleavage site (OPN-KI) were used. These mice retain full-length OPN protein, but it cannot be cleaved by thrombin (Ref. 18). OPN-KI mice treated with QDOT also developed significantly fewer granulomas compared to WT control mice and largely phenocopied OPN-KO mice (Figure 12A, bottom panel, Figure 12B-D). It is noted that while OPN-KO and OPN-KI mice exhibit substantially decreased granuloma formation, these mice are still capable of forming granulomas (Figure 15B). Flow cytometry of single-cell suspensions from QDOT-treated lung tissues revealed that OPN-KI lung tissue contained fewer macrophages compared to WT lung tissue (Figure 12E, gating scheme described in Figure 15C).

[0134] It was next confirmed that thrombin cleavage of OPN was occurring within our QDOT-induced mouse model of granuloma formation by performing an ELISA specific for thrombin-cleaved OPN products in lung tissue from QDOT-treated mice (Ref. 18). In WT mice, both full-length and thrombin-cleaved OPN products were detected in QDOT-treated lung tissue (Figure 12F, 12G). As expected, OPN-KO mice exhibited no signal. Not detected were significant amounts of full-length OPN or thrombin-cleaved OPN in OPN-KI mice, likely due to decreased macrophage infiltration. Taken together, thrombin-cleaved OPN, and not full-length OPN, is necessary for granuloma formation, with this activation occurring in affected tissue.

[0135] To test whether macrophages were the cellular source of OPN in granuloma formation, mice deficient for OPN specifically in macrophages (Spp1fl / fl: LysMcretd) were generated and used in the QDOT pulmonary granuloma model. Similar to OPN-KO mice and OPN-KI mice, QDOT-treated lungs of Spp1fl / fl: LysMCre mice exhibited reduced granuloma formation compared to litermate control mice (Figure 12H-I2J). Thus, macrophage-specific OPN is necessary for granuloma formation.25-10906 (103241.007521) PATENTExample 7 - Thrombin-cleaved OPN promotes macrophage-driven immune cell aggregation

[0136] To test whether OPN-R stimulation modulates macrophage behavior, a transwell migration assay was performed. Both OPN-FL or OPN-R-stimulated macrophages exhibited increased migration compared to untreated controls, with no difference observed between the two treatments, consistent with previous literature (Ref. 21). Next, a well- established in vitro granuloma formation assay using PPD-coated beads and human PBMCs was employed (Ref. 22). Compared to OPN-FL, OPN-R treatment induced the formation of more numerous and larger immune cell aggregates (Figure 14A). Immunostaining confirmed the presence of both macrophages (CD68+) and T cells (CD3E+) within these aggregates (Figure 14B). Taken together, these findings demonstrate that OPN-R induces more physical aggregation between macrophages and T-cells than OPN-FL.Example 8 - Therapeutic potential o f blocking thrombin-cleaved osteopontin

[0137] To explore whether pharmacological blockade of thrombin cleavage of OPN may serve as a targeted therapy for non-infectious granulomatous diseases, two distinct approaches were employed. First, the oral direct thrombin inhibitor dabigatran etexilate was administered via chow to WT mice. Mice were then exposed to QDOT to induce granuloma formation and continued on dabigatran or control chow for one month. Dabigatran etexilate-treated mice developed significantly fewer granulomas, as measured by both granuloma index and area involved, compared to controls (Figure 16A-C). Effective dosing was confirmed by prolonged partial thromboplastin time, despite some samples being hemolyzed (Figure 16D). These results demonstrate that direct thrombin inhibitors effectively block non-infectious granuloma formation in vivo.

[0138] Second, monoclonal antibody (A6) was used that was designed to bind to the thrombin-cleavage site on full-length human OPN, thereby preventing its cleavage by thrombin (Ref, 23). Mice were pre-treated for 5 days with A6 antibody before QDOT exposure, followed by biweekly A6 dosing. Compared to IgG-treated controls, A6-treated mice developed significantly fewer granulomas (Figure 16E, bottom panel, Figure 16F-G). Immunofluorescence confirmed decreased macrophage recruitment in A6-treated lungs25-10906 (103241.007521) PATENT(Figure 16E, 16H), Thus, pharmacologic inhibition of thrombin mediated cleavage of OPN reduces pulmonary granuloma formation and identifies two potential targeted therapies.

[0139] Recognizing that patients present to the clinic with established granulomas, it was next tested whether thrombin-cleaved OPN contributes to granuloma maintenance. Mice were first exposed to QDOT and allowed one month for granuloma development before beginning concurrent treatment with dabigatran and A6 or vehicle control. Treatment with dabigatran plus A6 significantly reduced granuloma burden by both granuloma index and area compared to controls (Figure 161-16K). Active dabigatran treatment was confirmed by prolonged partial thromboplastin time (Figure 16L). These data suggest that pharmacologic blockade of thrombin cleavage of OPN not only prevents granuloma formation but also promotes resolution of established pulmonary granulomas.Example 9 — Pharmacologic Targeting of Thrombin-Cleaved Osteopontin

[0140] FIGS. 18A and 18B illustrate the results of experiments involving pharmacologic targeting of thrombin-cleaved osteopontin with an antibody, C6R, that directly binds to thrombin-cleaved osteopontin and does not bind full-length osteopontin. Antibody C6R is disclosed in Zhou, Qin et al., Journal of Thrombosis and Haemostasis, Volume 23, Issue 11, 3691-3703 (November 2025), the entire contents of which are incorporated herein by reference.

[0141] FIG. 18A shows tissue sections showing respective treatments with IgG, A6 antibody, and C6R antibody. The images represent H& E staining of whole mount, mid-coronal sections of lung tissue of QDOT-treated WT mice (n=9). FIG. 18B provides quantitation of granuloma formation by granuloma index (granulomas per high powered field) or surface area involved. Mean±SEM. Student’s t-testtwo tailed and unpaired. *P<0.05, **P<0.01, ****P<0.0001.Materials and Methods

[0142] Mouse Models. Mice were group-housed in the animal facility of the University of Pennsylvania on a 12-hour light / 12-hour dark cycle with ad libitum access to water and normal chow. For in vivo granuloma model, mice lungs were instilled with a single dose of QDOT (40 uL) (Q21361 MP, Thermo Fisher) particles (Ref. 45). 30 days later, mice were sacrificed, and lung tissues were harvested for analysis. Granuloma index was calculated25-10906 (103241.007521) PATENTas follows: number of granulomatous regions / number of stitched 1 OX images. Granuloma area was calculated using the trainable WEKA segmentation tool in FIJI as follows:(granulomatous area in lung / total lung area) x 100.

[0143] Antibody treatments. The murine antibody used to target the cleavage of OPN by thrombin ( A6) was generated and shared by Leung Lab at Stanford (Ref. 46). Mice were injected intraperitoneally with A6 or IgG control (24 mg / 'kg) every day for five days prior to QDOT treatment. After QDOT treatment, mice were dosed twice per week for the duration of the experiment. For the maintenance experiment, mice were treated with QDOT once, and granuloma were allowed to form for 23 days. Mice were then injected every day for 5 days, then dosed twice per week for 4 weeks.

[0144] Dabigatran-supplemented chow. Dabigatran etexilate (DE) (NDC 31722-622-60, Camber) was purchased from the Hospital of the University of Pennsylvania pharmacy. DE was compounded into AIN-93G Purified Rodent Diet at a concentration of 10 mg / g by Dyets Inc., Bethlehem, Pennsylvania. Mice were fed DE chow or control chow for 5 days prior to QDO T treatment. Mice had ad libitum access to DE chow or control chow for the duration of the experiment. For the maintenance experiment, DE chow was provided to mice after 23 days of QDOT exposure and mice had ad libitum access for the duration of the experiment.

[0145] Activated partial thromboplastin time (APTT) test. Mouse blood was collected in a ratio of 1 part sodium citrate to 9 parts whole blood and centrifuged at 2,000 g for 15 min at 4°C before freezing the plasma at -80°C. A fully automated Siemens coagulation analyzer CA-660 was used for the measurement of APTT. The test was performed by the Translational Core Laboratory at the Children’s Hospital of Philadelphia. Samples that were hemolyzed or did not generate a value were excluded from the analysis in Figures 16D and 16L.

[0146] Histology and immunohistochemistry. Standard histology and immunostaining protocols were followed, and investigators were blinded to tissue origin during histologic staining (Ref. 47). In brief, the fresh skin tissue was fixed overnight at 4°C in 4% paraformaldehyde (J19943-K2, Thermo Fisher Scientific). The tissue was dried in 70% ethanol, trimmed, placed into tissue cassettes, processed (VIP5b, Sakura) and embedded into25-10906 (103241.007521) PATENTwax (Leica Paraplast X-tra) blocks. Blocks were cut using disposable blades (D554P, Sturkey) on a rotary microtome (RM2235, Leica) set at 5 μm thickness. Sections were floated on a water bath (145702, Boekel) set at 43°C and collected onto positively charged glass slides (Fisherbrand Superfrost Plus). Following overnight drying at room temperature, slides were baked for 30 minutes at 60°C, followed by H& E staining using an automated Stainer (Leica Autostainer XL) Tissue embedding, sectioning, staining, and slide processing was performed in the Skin Biology and Disease Resource -Based Core (SBDRC) at the Department of Dermatology, University of Pennsylvania. H& E-stained sections were examined under bright-field microscopy and images acquired on a Keyence BZ-X700 microscope.

[0147] F or immunohistochemistry, the following antibodies were used: CD3E (78588T, Cell Signaling), CD45R (MCA1258GT, BioRad), CD163 (68922S, Cell Signaling), CD68 (97778S, Cell Signaling), anti-rabbit Alexa Fluor 594 conjugate (8889S, Cell Signaling), anti-rat IgG - Alexa Fluor 488 conjugate (A21208, Thermo Fisher).Immunofluorescent histology slides were imaged using tiled imaging at 10X magnification on a Keyence BZ-X700 microscope.

[0148] Full length and cleaved OPN ELISA. Lungs of WT, OPN KO, or OPN-KI mice were homogenized in IX RIP A buffer containing IX protease inhibitor cocktail (11836170001, Roche). Sandwich ELISAs were used to detect OPN-FL or cleaved OPN as previously described (Ref. 48). Briefly, ELISA plates were coated with anti-mouse OPN antibody AF808 (100 pL, 2 pg / mL) in PBS buffer at RT for 2 hours. Non-specific binding was blocked by adding 100 pL 1% BSA in PBS for 1 hour. Purified recombinant mouse OPN FL (0.156-10 ng / mL), mouse OPN-R (0.625-50 ng / mL), and mouse OPN-L (0.625-50 ng / ml) were used to construct calibration curves. Standards and samples were diluted in 1% BSA in PBS and incubated in the wells for 2 hours before washing with 100 pL 0.05% Tween 20 in PBS. Wells were incubated with the following specific antibodies (0.5-1 pg / mL) diluted in 1% BSA in PBS for 1 h.; for mOPN-FL use antibody BAF808 (0.5 pg / mL); for mOPN-R; biotinylated anti-OPN-R (0.5—1 pg / mL); for mOPN-L biotinylated anti-OPN-L (0.5—1 pg / mL). Wells were washed with 100 pL PBS and incubated with 100 pL, streptavidin in conjunction with peroxidase, 100 ng / mL in 1% BSA-PBS for 1 hour. Wells were washed25-10906 (103241.007521) PATENTtwice with 100 pL PBS and incubated with 100 pL tetramethylbenzidine substrate for 15 min. Stop solution was added and absorbance was measured at 450 nm on a plate reader.

[0149] Bone marrow-derived macrophages. Bone marrow-derived macrophages were generated as previously described (Ref. 49). Briefly, mouse monocytes were isolated from bone marrow through magnetic column separation using the mouse Monocyte Isolation Kit (Miltenyi Biotec, 130-100-629). Isolated monocytes were cultured with DMEM high glucose (11965092, Gibco) containing 10% FBS and differentiated to macrophages by the addition of M-CSF1 (1 ng / mL). Successful macrophage differentiation was verified by flow cytometry.

[0150] Flow cytometry analysis of macrophages. For mouse macrophage detection, single cell suspensions were prepared from mouse lung tissues or from bone marrow-derived monocytes. Cells were stained with Zombie Aqua cell viability dye (423101, BioLegend) for 15 minutes in the dark at room temperature. Cells were pretreated with Mouse TruStain FcX1MFc-blocking agent (101320, BioLegend) and subsequently stained with the following monoclonal antibodies: anti-CD45 (103137, BioLegend), anti-F4 / 80 (123149, BioLegend), anti-CD3 (100203, BioLegend). Samples were acquired on a five-laser BD LSRFortessa flow cytometer and all sample data was analyzed using FloJo software version 10.10 (BD).

[0151] Cell culture. HEK293T cells were grown in DMEM / F12 (11320033, Gibco) + 10% FBS (A5256701, Gibco) and IX anti / anti. Raw 264.7 cells were grown in DMEM high glucose with Glutamax (10566016, Gibco) + 10% FBS and IX anti / anti. All cell lines were split at sub-confluent density. The recombinant fragments of OPN used for treatments were produced as previously described (Ref. 50).

[0152] Western Blot. Standard western blot protocols were followed. Briefly, cells were lysed with R IP A buffer (Cell Signaling, 9806) and whole protein was quantified with the Pierce BCA protein assay (ThermoFisher, 23225). 40 pg of protein was run on gels (NP0335BOX, ThermoFisher) and transferred to a PVDF membrane using BioRad Trans Blot Turbo transfer system. The following antibodies were used: anti-ITGBl (4706S, Cell Signaling), anti-ITGA9 (AB140599, Abeam), anti-ITGA4 (8440S, Cell Signaling), anti-CD44 (15675-1 -AP, Proteintech), anti-ERK1 / 2 (9102S, Cell Signaling), anti-phospho-ERK1 / 225-10906 (103241.007521) PATENT(9101 S, Cell Signaling), anti-P-Actin (4967S, Cell Signaling), HRP-linked anti-rabbit IgG (7074S, Cell Signaling), HRP-linked anti-mouse IgG (7076S, Cell Signaling).Chemiluminescent images were acquired on a Chemi-Doc imaging system (BioRad).

[0153] Quantitative Real-Time PCR. RAW 264.7 cells were plated at 30,000 cells / cm2in 6-well plates and treated at 90% confluence for 6 hours with vehicle control, OPN-FL (20 nM), or OPN-R (20 nM). Total RNA was isolated using the RNeasy Kit (74106, Qiagen). Total RNA was quantified by UV absorbance on a Nanodrop (ThermoFisher) and complementary DN A (cDNA) (2 pg) was synthesized using the QuantiTect Reverse Transcription Kit (205313, Qiagen). The cDNA was then subjected to analysis by quantitative real-time PCR (qPCR) using TaqMan Fast Gene Expression Master Mix (ThermoFisher) and a Life Technologies QuantStudio 7 Pro instrument. The following TaqMan probe-based assays were used: Actb (Mm02619580 gl), Nos2 (Mm00440502_ml), Tnf (Mm00443258 ml), Illb (Mm00434228 ml), Ptgs2 (Mm00478374 ml), CD80 (Mm00711660_ml), Sppl (Mm00008338_cn). mRNA expression was normalized to Actb and reported as relative expression using the comparative AACt method.

[0154] In vitro PBMC aggregation assay. The in vitro PBMC aggregation assay and PPD-coated bead generation were performed as previously described (Ref. 22). Briefly, 250,000 healthy donor PBMCs / cm2were grown for 4 days in RPMI 1640 medium containing 10% human serum in the presence of OPN-FL (20 nM), OPN-R (20 nM), or LPS (500 ng / mL) and PPD-coated beads (1 pL / 1 million PBMCs). The following antibodies were used to prevent aggregation at a final concentration of 2.5 pg / mL: anti-ITGBl (4706S, Cell Signaling), anti-ITGA9 (AB 140599, Abeam), anti-ITGA4 (8440S, Cell Signaling), anti-CD44 (15675-1-AP, Proteintech), IgG control (3900S, Cell Signaling). Aggregate images were acquired on a Keyence BZ-X700 microscope. Human PBMC-cleared supernatants, collected 4 days after treatment, were analyzed for IFN-y by ELISA (430116, BioLegend) according to the manufacturer’s recommendations. Cell aggregates were identified by manual visual inspection of acquired images. An aggregate was defined as a cluster of >4 cells in direct, contiguous contact. Marker files for all annotated images were archived to ensure reproducibility and are available from the corresponding author upon request. For each25-10906 (103241.007521) PATENTindependent experiment (n = 3), between 3 and 7 non- overlapping fields of view were randomly selected for analysis resulting in the total data points presented in the graphs.

[0155] Immunofluorescence. RAW 264.7 cells were plated on cell culture-treated glass coverslips at 30,000 cells / cm2in 6-well plates and treated at 90% confluence for 6 hours with vehicle control, OPN-FL (20 nM), or OPN-R (20 nM). Standard immunofluorescence protocols were followed. Briefly, cells were fixed with 4% PF A for 10 mm at RT, washed three times with DPBS (Gibco, 14190-136), then permeabilized with 0.5% Triton X-100 (Thermo Scientific, 28314) for 10 mm. After permeabilization, cells were washed three times with DPBS for 5 min, then blocked in 1% BSA (Sigma, A4503) in PBST (DPBS with 0.05% Tween 20, pH 7.4 (BioRad, 1706531)) for 60 min at RT. After block samples were incubated with anti-iNOS primary antibody (2982S, Cell Signaling) for 1 hour at RT and washed three times with PBS T for 5 min. Cells were then incubated with anti-rabbit Alexa Fluor 594 Conjugate (8889S, Cell Signaling) for 1 hour at RT. After incubation with secondary antibody, samples were washed 3 times with DPBS and mounted with Fluoromount-G Mounting Medium with DAPI (00-4959-52, ThermoFisher).

[0156] Human PBMC aggregates were grown on cell culture-treated glass coverslips at and immunostained as described above. The following antibodies were used: anti-CD68 (14-0688-82, Invitrogen), anti-CD3 (17617-1-AP, Proteintech), anti-mouse Alexa Fluor 488 Conjugate (4408, Cell Signaling), anti-rabbit Alexa Fluor 594 Conjugate (8889S, Cell Signaling).

[0157] Chemotaxis transwell assay. Chemotaxis assays were performed using 6.5 mm, 8 pm transwell inserts (Costar, 3422). 500,000 RAW 264,7s were washed and plated on the upper chamber of the transwell in 200 pL RAW 264.7 medium containing 0.5% serum (low serum media) and OPN-FL (20 nM) or OPN-R (20 nM). The bottom chamber contained 500 pL of media with 10% serum. After incubation for 2 hr at 37°C in a CO2 incubator, cells in the underside of the transwell were fixed with 70% ethanol, stained with crystal violet (94448, Sigma), imaged. Cells were quantified using FIJI and reported as number of cells per 4X field.

[0158] Data Analysis. scRNA-seq and spatial transcriptomics data were downloaded from (Ref, 8) and further analyzed with Seurat and Loupe Browser, No changes25-10906 (103241.007521) PATENTto the basic analysis parameters were made nor do we report any original code. Patient characteristics and clinical information are available in Supplemental table 1 from (Ref. 8).

[0159] scRNA-seq. The scRNA sequencing data was mapped to the GRCh38 reference genome to generate gene count and cell barcode matrices using the “cellranger count” function from the cellranger pipeline (version 5.0.1, 10X Genomics). All downstream analysis steps were performed using the R package Seurat (Ref. 51) (ver. 4.3.0, https: / / github.com / satijalab / seurat) unless otherwise noted. In brief, seurat functions ‘ReadlOX’ and ‘CreateSeuratObject’ were used to import and create a merged Seurat object from all filtered feature barcode matrices generated by the cellranger pipeline. Cells with less < 250 genes, < 500 UMI, < 0.80 log 10 Genes per UMI, and more than 20% mitochondrial reads were excluded from the merged Seurat object for further analysis. Genes that were detected in less than 10 cells were also discarded. DoubletFinder was used to identify potential cell doublets as a final quality control (Ref. 52). To determine and regress out the effect of cell cycle, each cell was given a cell cycle phase score using the Seurat function ‘CellCycleScoring’ (Ref. 53). The individual datasets were then log-normalized and scaled by linear regression against the number of reads. The FindVariableFeatures function followed by SelectIntegrationFeatures function (nfeatures = 3000) were used to identify variable genes from each individual Seurat object. For cross-tissue data integration and batch correction, ‘FindlntegrationAnchors’ and TntegrateData’ were applied to the individual sample Seurat object. Following sample integration, dimensionality reduction was performed using the RunPCA and RunUMAP function generated UMAP plots. Next, Louvain clustering was performed with the‘FmdC lusters’ function using the first 40 PCs and at resolution 1,4. The ElbowPlot function was used in Seurat, visual inspection of DimHeatmap plots at different dimensions and R package clustree to choose an optimum number of dimensions and resolution,

[0160] Cell type annotation. Three complementary approaches were used to annotate the identities of different cell clusters: (1) checking the expression of lineage-specific marker genes identified from previously published single-cell RNA-Seq studies in our query cluster marker genes list and in differentially expressed genes of the query cluster. (2) applying an unbiased cell type recognition method named deCS (R package) (Ref. 54), which leverages mapping of the top 100 genes from the query cluster to the reference transcriptomic25-10906 (103241.007521) PATENTdatasets of known cell types such as BlueprintEncode (Ref. 55), MonacoImmune reference (Ref. 56), and Database of Immune Cell Expression (DICE) data (Ref. 57). First was applied deCS to determine if the predicted annotations were consistent with findings and then assigned the identity to the cluster. (3) The methods to confirm skin and blood neutrophil identity are described supra. The sample statistics and marker gene dot plots were made by using dittoSeq (v 1.4.1). The t-distributed stochastic neighbor embedding (t-SNE) was applied to visualize the single cell transcriptional profile in 2D space based on the SNN graph described above (Ref. 58). Other bar plots, boxplots, violin plots and heatmaps were generated by customized R code through ggplot2 (v3.2.1, R package) (Ref. 59).

[0161] Ligand Receptor analysis. R package CellChat (1.5.0) was used to study the ligand-receptor interaction networks between different immune cell subclusters. The ligand receptor interaction analysis was performed on the immune subcluster from the scRNA-seq dataset. The analysis was performed on the paracrine signaling network. For the present analysis ligand-receptor interactions were considered that were expressed in at least 10 cells. The CellChat algorithm calculates an aggregated ligand-receptor interaction score based on a method called Trimean’. The CellChat algorithm has the added advantage of comparing two or more single-cell datasets and gives a comparative score for the given cell types. These scores represent the probability of interaction among the ligand-receptor pairs. The probability was then visualized using functions such asnetAnalysis_signalingRole_scatter, which visualizes the major sender and receiver across all cell types, and netAnalysis_signalingChanges_scatter, which identifies the major signaling networks acting within a given cell type.

[0162] Functional enrichment analysis. The Gene Over-representation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using R package “clusterProfler” (3.18,1) and enrichplot (1.10.2),

[0163] Spatial transcriptomics. Following the mapping of spatial RNA-seq data, Seurat and Giotto software were used to analyze the data. In brief, Seurat was used to load the spatial data and the SpatialFeaturePlot function was used to plot the number of nUMI (n Count_S patial) and number of genes (nfeature_Spatial). The individual samples were then normalized using SCTransform. To generate gene plots of key genes, the interactive plotting25-10906 (103241.007521) PATENTfeature SpatialDimplot was used from the Seurat pipeline. To identify the differences in spatial distribution of immune cells in unaffected and affected sections, the cell-gene matrix and spatial coordinates were analyzed using Giotto. Briefly, the cell-gene matrix and cell spatial coordinates were processed to create a Giotto object. After image alignment, the Giotto object was filtered for genes detected in a minimum number of cells (cutoff ≥ 5) and minimum genes detected per cell (cutoff ≥ 100). The filtered object then underwent normalization, dimensional reduction, and clustering. Default parameters were used to find the spatial distribution of genes using a ranking method. For cell type annotation, dampened weighted least squares - based deconvolution were used where the signature matrix used for deconvolution was derived from the same patient’s scRNA-seq data set.

[0164] Statistics. Presented data combine all experiments, and unless noted, all experiments were repeated 2-3 times independently. Experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment, unless noted in the text. Comparisons between two groups were carried out using two-tailed unpaired Student’s t-test unless noted in the text. In all tests, a p-value of less than 0.05 is considered significant. Levels of significance are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 in the text. When appropriate, specific p values are provided.Data availability. All sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) and are publicly available as of publication, (a) GSE227041(https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE227041 Token: cpwbaucadbavdun). (b)GSE226896(https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE226896 Token: wfcpyekutpufjsr).

[0165] The preceding data reveals a fundamental molecular mechanism driving the formation and maintenance of non-infectious granulomas (Figure 17). The data invoke a model wherein a disease-specific stimulus recruits and activates tissue-infiltrating macrophages, triggering them to secrete full-length osteopontin (OPN). Chronic stimulation induces a subset of these macrophages to express tissue factor, triggering local thrombin activation and resulting in cleavage of full-length OPN into bioactive fragments. These fragments reinforce macrophage activation and drive immune cell aggregation, the hallmark25-10906 (103241.007521) PATENTof granuloma formation. In this way, OPN-positive macrophages generate both the substrate (OPN) and the enzymatic machinery (tissue factor / thrombin), establishing a self-sustaining circuit that stabilizes granulomas.

[0166] This mechanism situates granuloma biology at the intersection of coagulation and inflammation. Thrombin is classically viewed as a master regulator of coagulation, but it also exerts pro-inflammatory effects by activating protease-activated receptors and cleaving matricellular proteins such as OPN. Increased thrombin activity has been detected within sarcoidosis plasma, and our findings may explain the increased incidence of spontaneous blood clots in sarcoidosis patients (Refs. 24, 25). Importantly, it has been presently demonstrated that OPN’s granuloma-promoting activity requires thrombin-mediated cleavage, reframing prior associations between OPN expression and disease activity.

[0167] OPN is a matricellular protein involved in cell adhesion, migration, and immune regulation (Ref. 18). OPN has been linked to granulomatous inflammation through elevated tissue and plasma levels in sarcoidosis, yet its mechanistic contribution was undefined (Refs. 26-30). The present disclosure clarifies that thrombin-cleaved OPN, not full-length OPN, drives immune cell aggregation. This specificity may explain why OPN is broadly expressed in other inflammatory and neoplastic conditions without inducing granulomas: local thrombin activity is absent. In contrast to prior reports that T cells are the primary source of OPN, our findings identify macrophages as the key producers, aligning with recent work establishing OPN+ macrophages promoting tumor formation (Ref. 18). Secreted thrombin-cleaved OPN may also act on T cells. These insights begin to resolve the long-standing uncertainty surrounding OPN’s pleiotropic downstream biology across inflammation and cancer, where it ranks among the top 5% of expressed genes but has resisted mechanistic definition (Ref. 31).

[0168] The present findings identify at least two promising therapeutic strategies for granulomatous diseases, including resolution of established granulomas, an especially relevant goal for most sarcoidosis patients. First, direct thrombin inhibitors such as dabigatran etexilate, already in clinical use for anti coagulation, could be repurposed to disrupt the OPN-thrombin axis. However, their 3% annual risk of major bleeding is a significant concern for25-10906 (103241.007521) PATENTthe chronic treatment required in granulomatous diseases. The A6 antibody, by selectively targeting the pathogenic thrombin cleavage site within OPN, effectively prevented granuloma formation in our study and represents a compelling candidate for human trials.

[0169] The present discovery that elevated OPN expression in skin sarcoidosis macrophages extends to other organs, including the heart and lung, suggesting that the mechanisms driving systemic inflammatory diseases transcend organ boundaries. This finding has broader implications: the ability to directly interrogate accessible tissues like blood and skin may provide insights into systemic inflammation affecting less accessible organs. By defining the coagulation-immune signaling axis in non-infectious granulomatous inflammation and establishing thrombin-cleaved OPN as a critical regulatory checkpoint, now available new therapeutic avenues for diseases that have long lacked targeted treatments. These findings transform the understanding of granuloma biology and offer hope for patients with these chronic, debilitating conditions.

[0170] References. The following publications are referred to in the preceding disclosure:1. Sangani R, Bosch NA, Govender P, Scarpato B, Walkey AJ, Newman J, et al.Sarcoidosis Treatment Patterns in the United States: 2016-2022. Chest.2025; 167(4): 1099-106.2. Stanton LA, Fenhalls G, Lucas A, Gough P, Greaves DR, Mahoney JA, et al.Immunophenotyping of macrophages in human pulmonary’ tuberculosis and sarcoidosis. Int J Exp Pathol. 2003;84(6):289-304.3. Krausgruber T, Redl A, Barreca D, Doberer K, Romanovskaia D, Dobnikar L, et al.Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation. Immunity. 2023;56(2):289-306 e7.4. Nakamizo S, and Kabashima K, Cutaneous Granulomas: Mechanisms, Cellular Interactions, and Therapeutic Insights. Br J Dermatol. 2025.5. Nakamizo S, Sugiura Y, Ishida Y Ueki Y, Yonekura S, Tanizaki H, et al. 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[0171] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0172] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0173] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

25-10906 (103241.007521) PATENTWhat is Claimed:

1. A method of treating or preventing granulomas or a granulomatous disease, disorder, or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an osteopontin inhibitor.

2. The method of claim 1, wherein the granuloma or granulomatous disease is a non-mfectious granuloma or granulomatous disease, disorder, or condition.

3. The method of claim 1 or claim 2, wherein the non-infectious granulomatous disease, disorder, or condition is selected from sarcoidosis, granuloma annulare, necrobiosis lipoidica, necrobiotic xanthogranuloma, annular elastolytic giant cell granuloma (AEGCG), cutaneous Crohn disease, interstitial granulomatous dermatitis, palisading neutrophilic and granulomatous dermatitis.

4. The method of any preceding claim, wherein the non-infectiousgranulomatous disease is sarcoidosis.

5. The method of any preceding claim, wherein the osteopontin inhibitor is an anti-osteopontm antibody.

6. The method of claim 5, wherein the anti-osteopontin antibody is A6.

7. The method of claim 5, wherein the anti-osteopontin antibody is an antibody having light chain and / or heavy chain sequence that is at least 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to A6.

8. The method of claim 5, wherein the anti-osteopontin antibody is C6R.

9. The method of claim 5, wherein the anti-osteopontin antibody is an antibody having light chain and / or heavy chain sequence that is at least 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to C6R.

10. The method of claim 5, wherein the anti-osteopontin antibody binds to full- length osteopontin or thrombin-cleaved osteopontin.25-10906 (103241.007521) PATENT11. The method of claim 5, wherein the anti-osteopontin antibody preventsthrombin cleavage of osteopontin.

12. The method of claim 5, wherein the anti-osteopontin antibody blocks integrin binding of thrombin-cleaved fragments of osteopontin.

13. The method according to any preceding claim, further comprisingadministering to the subject an anticoagulant.

14. An osteopontin inhibitor for use in treating or preventing treating orpreventing granulomas or a granulomatous disease, disorder, or condition in asubject in need thereof.

15. The osteopontin inhibitor according to claim 13, wherein the osteopontin inhibitor is an anti-osteopontin antibody.

16. The osteopontin inhibitor according to claim 14, wherein the anti-osteopontin antibody is an antibody having light chain and / or heavy chain sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to antibody A6.

17. The osteopontin inhibitor according to claim 14, wherein the anti-osteopontin antibody is an antibody having light chain and / or heavy chain sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to antibody C6R18. The osteopontin inhibitor of claim 14, wherein the anti-osteopontin antibody binds to full-length osteopontin or thrombin-cleaved osteopontin.

19. The osteopontin inhibitor of claim 13, wherein the anti-osteopontin antibody inhibits thrombin cleavage of osteopontin.

20. The osteopontin inhibitor of claim 13, wherein the anti-osteopontin antibody blocks integrin binding of thrombin-cleaved fragments of osteopontin.25-10906 (103241.007521) PATENT21. A composition comprising an osteopontin inhibitor according to any one of claims 14-20 and a pharmaceutically acceptable carrier, diluent, or excipient.22, The composition according to claim 21, further comprising an anticoagulant.23, Use of an osteopontin inhibitor according to any one of claims 14-20 in a medicament, or of a composition according to claim 21 or claim 22, for the treatment or prevention of granulomas or a granulomatous disease, disorder, or condition in a subject in need thereof.24, The use according to claim 23, wherein the granuloma or granulomatous disease is a non- infectious granuloma or granulomatous disease, disorder, or condition.25, The use according to claim 23 or claim 24, wherein the non-infectious granulomatous disease, disorder, or condition is selected from sarcoidosis, granuloma annulare, necrobiosis lipoidica, necrobiotic xanthogranuloma, annular elastolytic giant cell granuloma (AEGCG), cutaneous Crohn disease, interstitial granulomatous dermatitis, palisading neutrophilic and granulomatous dermatitis.26, The use according to any one of claims 23-25, wherein the non-infectious granulomatous disease is sarcoidosis.

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