Methods of treatment with osteopontin modulators

Abstract

Disclosed herein are methods of treatment for diseases and disorders with modulators of full-length or thrombin-cleaved osteopontin. Particularly disclosed herein are methods comprising treatment of tissue fibrosis (e.g., pulmonary, cardiac, liver, kidney, skin) with antibodies, or antigen-binding fragments thereof, that inhibit thrombin-cleavage of osteopontin or block the activity of thrombin cleavage fragments of osteopontin.

Description

Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) METHODS OF TREATMENT WITH OSTEOPONTIN MODULATORS TECHNICAL FIELD

[0001] Disclosed herein are methods of treatment for diseases and disorders with modulators of full-length or thrombin-cleaved osteopontin. Particularly disclosed herein are methods comprising treatment of tissue fibrosis (e.g., pulmonary, cardiac, liver, kidney, skin) with antibodies, or antigen-binding fragments thereof, that inhibit thrombin-cleavage of osteopontin or block the activity of thrombin cleavage fragments of osteopontin.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 744,683, filed January 13, 2025, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled STDU2_44072_601_SequenceListing.xml (Size: 155,969 bytes; and Date of Creation: January 13, 2026) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Tissue fibrosis is characterized by excessive accumulation of fibrous connective tissue and organ dysfunction. Skin fibrosis can form as a result of keloids, wounds / trauma, reconstruction surgeries, surgical scars, cosmetic surgeries, whereas other tissue fibroses (e.g., cardiac, liver, kidney) can form during the progression of other disease and disorders, e.g., nonalcoholic steatohepatitis (NASH) in the liver. Global predominance of each type of fibroses is hard to define but overall tissue fibrosis presents a significant health problem with only surgical options being somewhat efficacious. Thus, new non-invasive therapies are needed to treat and prevent tissue fibrosis, as well as any underlying medical conditions which may be leading to the formation of fibrosis in the tissue.SUMMARY

[0005] In one aspect, disclosed herein are methods of treatment for diseases and disorders with modulators of full-length or thrombin-cleaved osteopontin.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0006] In some embodiments, the methods comprise administering to the subject an effective amount of a modulator of osteopontin. In some embodiments, the modulator of osteopontin is an anti-osteopontin antibody or an antigen-binding fragment thereof. In some embodiments, the anti-osteopontin antibody or antigen- binding fragment thereof binds to full-length osteopontin or thrombin-cleaved osteopontin. In some embodiments, the anti-osteopontin antibody or antigenbinding fragment thereof prevents thrombin cleavage of osteopontin. In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof blocks integrin binding of thrombin-cleaved fragments of osteopontin.

[0007] In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises one, two, or all three heavy chain variable region (VH) complementarity determining regions, HCDR1, HCDR2, and HCDR3. and optionally, also one, two, or all three light chain variable region (VL) complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, of any one of the exemplary antibodies the sequences provided in Table 1.

[0008] In some embodiments, the HCDR1 comprises an amino acid sequence of any of SEQ ID NOs: 34, 37, 39, or 166; the HCDR2 comprises an amino acid sequence of any of SEQ ID NOs: 35, 38, 40, or 167; and the HCDR3 comprises an amino acid sequence of any of SEQ ID NOs: 36, 41, or 168. In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 34 or 37; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35 or 38; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 39; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 166; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 167; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 168.

[0009] In some embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 42 or 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43 or 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 42; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID NO:Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

[0010] In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to any of SEQ ID NOs: 2, 6, 7, and 8. In some embodiments, the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3 or 9. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6, 7, or 8 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9.

[0011] In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 46; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 51: the LCDR2 comprises the amino acid sequence of SEQ ID NO: 53; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 55.

[0012] In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4. In some embodiments, the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5.

[0013] In some embodiments, the methods disclosed herein treat or prevent tissue fibrosis or a disease or disorder characterized by tissue fibrosis in a subject in need thereof. In some embodiments, the tissue fibrosis is pulmonary, cardiac, liver, kidney, skin fibrosis. In some embodiments, the tissue fibrosis is skin fibrosis. In some embodiments, the tissue fibrosis is liver fibrosis. In some embodiments, the disease or disorder characterized by tissue fibrosis is nonalcoholic steatohepatitis (NASH).

[0014] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1C show skin fibrosis in osteopontin (OPN)-KI, OPN-KO mice, and mice treated with anti-OPN antibodies, A6 and C6R, in a back wound model. FIGS. 1A and IB show suppression of skin fibrosis in OPN-KI and OPN-KO. FIG. 1C shows reduced fibrosis in a back wound model following treatment with anti-OPN antibodies.

[0016] FIGS. 2A and 2B show WT, OPN-KI and OPN-KO mice on NASH diet compared to mice with the same genotypes on control diet. FIG. 2 A is a graph of weight gain for WT, OPN-KI and OPN-KO mice on NASH diet compared to mice with the same genotypes on control diet. FIG. 2B is a graph of the area under the curve was calculated from the data in FIG. 2 A and compared by one way ANOVA with post hoc Bonferroni correction. * p<0.05, *** pcO.OOl, **** p<0.0001.

[0017] FIG. 3 is a graph of fibrosis score from picrosirius stained slides of liver compared by one way ANOVA with post hoc Bonferroni correction, ns: not significant, * p<0.05, ** p<0.01.DETAILED DESCRIPTION

[0018] 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). The mouse model was used as a comparison to wild type (WT) mice and mice in which the OPN gene has been knocked out (OPN-KO mice) in a NASH model. The OPN-KI and, to a lesser extent, the OPN-KO mice developed less fibrosis than WT mice as well as being less glucose intolerant on the NASH diet while they gained weight compared to mice on a control diet.

[0019] Antibodies were developed which 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 indications after a single dose application in rats. They do not block coagulation or platelet aggregation despite preventing thrombin cleavage of osteopontin. In cancer models, these antibodies phenocopy the OPN-KI mouse model and prevent and / or treat cancer.

[0020] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) Definitions

[0021] 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. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. 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.

[0022] 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, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). 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.

[0023] 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.

[0024] 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 discloses 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. OtherAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 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.

[0025] 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.

[0026] The term “amino acid” or “any amino 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. 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 P-amino acids (P3and p2). homo-amino 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 amino 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 other asAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 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.

[0027] 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.

[0028] 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 an amino acid, whether not part of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of an amino acid, whether or not part 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 retained.

[0029] 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), -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 diphenylalanine), and Ida (Iminodiacetic acid).

[0030] “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, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, aAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 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 in 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 camelids. 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.

[0031] 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.

[0032] 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 the amino 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 primarilyAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0033] A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) 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 (Cui. 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.

[0034] 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 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)).

[0035] “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 region of the recipient 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, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. 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,Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 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).

[0036] 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 107M (e.g., >107M4, >108M , >109M , >1O10M , >10nM‘ >1012M , >1013M , 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.

[0037] The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding 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-binding 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 region (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, (iii) 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 antibodyAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

[0038] 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 Milstein, 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.

[0039] 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.

[0040] 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-proteinaceous (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 antigen receptor. Epitopes also are referred to in 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 amino 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.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0041] As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in 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 EASTA programs (e.g., EASTA3x, LAS™, 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)).

[0042] 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 amino acid chain.

[0043] 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. Lor 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 associatedAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) with the condition. Tn 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.

[0044] 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.

[0045] 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., a modulator 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.

[0046] 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).Moreover, subject may mean any living organism, preferably a mammal (e.g., humans and nonhumans) that may benefit from the administration of compositions contemplated herein.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.Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0047] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, 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 beenAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) observed. Tn 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.

[0048] 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.

[0049] 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.Methods of Treatment

[0050] Disclosed herein are methods of treatment for diseases and disorders with modulators of full-length or thrombin-cleaved osteopontin.a. Modulators of osteopontin (OPN)

[0051] The methods comprise administering to a subject in need thereof an effective amount of a modulator 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-terminal fragment (OPN-CTF). OPN-R, which has SVVYGLR (SEQ ID NO: 1) at its C-terminus, binds to a subset of integrins (a4pi and a9pi) to which full-length OPN does not bind. Other proteases than thrombin can cleave full length osteopontin and thrombin cleaved osteopontin (OPN-R) generating fragments that contain the integrin binding sequences. If thrombin cleavage of osteopontin has occurred, these protease cleaved fragments will also contain the sequence, SVVYGLR (SEQ ID NO: 1), that binds to integrins (e.g., a4pi and a9pi) to which full-length osteopontin does not bind.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0052] Osteopontin modulators may disrupt the expression of osteopontin, prevent or lessen thrombin cleavage of osteopontin, and / or block or inhibit integrin binding. For example, the osteopontin modulators may bind at or near the integrin binding sites, thereby preventing access of integrins to the binding site by steric hinderance. In some embodiments, the osteopontin modulators block or inhibit integrin binding by thrombin cleaved osteopontin, thrombin cleaved full-length osteopontin or any fragments thereof. In some embodiments, the osteopontin modulators block or inhibit binding to integrins to which full-length osteopontin does not bind (e.g., a4pi and a9pi). In some embodiments, the osteopontin modulators bind to the sequence within osteopontin exposed by thrombin cleavage, SVVYGLR (SEQ ID NO: 1) or to sequences adjacent to it preventing integrin binding to osteopontin or its fragments.

[0053] Suitable modulators of osteopontin may bind to full-length or thrombin-cleaved osteopontin to prevent integrin binding and / or thrombin cleavage. Exemplary osteopontin modulators 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 and / or ligand binding)), a small molecule inhibitor of osteopontin, or combinations thereof.

[0054] In some embodiments, the osteopontin modulator is a small molecule osteopontin inhibitor. Exemplary osteopontin inhibitors, include, but are not limited to brefalamide, agelastatin A, and mesalazine.

[0055] In some embodiments, the modulator of osteopontin is an anti-osteopontin antibody or an antigen-binding fragment thereof. In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof prevents or lessens thrombin cleavage. In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof blocks or inhibits integrin binding. For example, the anti-osteopontin antibodies or antigen-binding fragments thereof may bind at or near the integrin binding site, thereby preventing access of integrins to the binding site by steric hinderance. In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof blocks or inhibits integrin binding to a thrombin cleavage fragment of osteopontin, e.g., thrombin cleaved full-length osteopontin or any fragments thereof. In someAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) embodiments, the anti-osteopontin antibodies or antigen-binding fragments thereof block or inhibit binding to integrins to which full-length osteopontin does not bind (e.g., a4pi and a9pi). In some embodiments, the anti-osteopontin antibodies or antigen-binding fragments thereof bind to the sequence within osteopontin exposed by thrombin cleavage, SVVYGLR (SEQ ID NO: 1) or to sequences adjacent to it preventing integrin binding to osteopontin or its fragments.

[0056] In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof 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 or an antigen-binding fragment thereof is selected from those described in International Patent Application No. PCT / US2026 / 010601, herein incorporated by reference in its entirety.

[0057] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, specifically bind to full length osteopontin (OPN-FL) and, optionally, inhibit thrombin cleavage of osteopontin, specifically bind to a thrombin cleavage fragment of osteopontin (e.g., OPN-R fragment) and reduce biological activity of the thrombin cleavage fragment, and / or specifically bind to a carboxypeptidase thrombin cleavage fragment of osteopontin (e.g., OPN-L fragment) and reduce biological activity of the carboxypeptidase thrombin cleavage fragment.

[0058] In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises one, two, or all three HCDRs, and optionally, also one, two, or all three of the corresponding LCDRs of any one of the exemplary antibodies, the sequences of which are provided in Table 1. In some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises all six CDRs of any one of the exemplary antibodies, the sequences of which are provided in Table 1.

[0059] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises an HCDR1 of any of SEQ ID NOs: 34, 37, 39, or 166; an HCDR2 of any of SEQ ID NOs: 35, 38, 40, or 167; and an HCDR3 of any of SEQ ID NOs: 36. 41. or 168. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 34 or 37; the HCDR2 of SEQ ID NO: 35 or 38; and the HCDR3 of SEQ ID NO: 36. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 39; the HCDR2 of SEQ ID NO: 40; and the HCDR3 of SEQ ID NO: 41. In some embodiments, an anti-osteopontin antibody, or antigenAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) binding fragment thereof, comprises the HCDR1 of SEQ TD NO: 166; the HCDR2 of SEQ ID NO: 167; and the HCDR3 of SEQ ID NO: 168.

[0060] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the LCDR1 of SEQ ID NO: 42 or 169; the LCDR2 of SEQ ID NO: 43 or 170; and the LCDR3 of SEQ ID NO: 44.

[0061] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 34 or 37; the HCDR2 of SEQ ID NO: 35 or 38; the HCDR3 of SEQ ID NO: 36; the LCDR1 of SEQ ID NO: 42; the LCDR2 of SEQ ID NO: 43; and the LCDR3 of SEQ ID NO: 44.

[0062] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 39; the HCDR2 of SEQ ID NO: 40; the HCDR3 of SEQ ID NO: 41; the LCDR1 of SEQ ID NO: 42; the LCDR2 of SEQ ID NO: 43; and the LCDR3 of SEQ ID NO: 44.

[0063] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 166; the HCDR2 of SEQ ID NO: 167; the HCDR3 of SEQ ID NO: 168; the LCDR1 of SEQ ID NO: 169; the LCDR2 of SEQ ID NO: 170; and the LCDR3 of SEQ ID NO: 44.

[0064] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 46; the HCDR2 of SEQ ID NO: 47; and the HCDR3 of SEQ ID NO: 50. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 45; the HCDR2 of SEQ ID NO: 47; and the HCDR3 of SEQ ID NO: 49. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 46; the HCDR2 of SEQ ID NO: 48; and the HCDR3 of SEQ ID NO: 50.

[0065] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the LCDR1 of SEQ ID NO: 51; the LCDR2 of SEQ ID NO: 53; and the LCDR3 of SEQ ID NO: 55. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the LCDR1 of SEQ ID NO: 52; the LCDR2 of SEQ ID NO: 54; and the LCDR3 of SEQ ID NO: 55.

[0066] In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 46; the HCDR2 of SEQ ID NO: 47; the HCDR3Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) of SEQ ID NO: 50; the LCDR1 of SEQ ID NO: 51 ; the LCDR2 of SEQ ID NO: 53; and the LCDR3 of SEQ ID NO: 55. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 44; the HCDR2 of SEQ ID NO: 47; the HCDR3 of SEQ ID NO: 49; the LCDR1 of SEQ ID NO: 51; the LCDR2 of SEQ ID NO: 53; and the LCDR3 of SEQ ID NO: 55. In some embodiments, an anti-osteopontin antibody, or antigen binding fragment thereof, comprises the HCDR1 of SEQ ID NO: 46; the HCDR2 of SEQ ID NO: 48; the HCDR3 of SEQ ID NO: 50; the LCDR1 of SEQ ID NO: 51; the LCDR2 of SEQ ID NO: 53; and the LCDR3 of SEQ ID NO: 55.

[0067] In certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a deamidation sensitive (asparagine deamidation) sites (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}SIT) sensitive sites, oxidation sensitive sites, etc., or an undesired cysteine residue, to any residue that does not affect selectivity, affinity, avidity, and developability. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser / Thr residue to a different residue, for instance by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn- Ala. When such a site is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues. Accordingly, substitution in a CDR sequence to remove one of the implicated residues is also encompassed by the present disclosure.

[0068] In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprises a VH sequence having at least 70% identity (e.g.. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the VH sequence of any one of the exemplary antibodies, the sequences of which are provided in Table 1. In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprises a VH sequence having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%. at leastAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the VH sequence of any one of the exemplary antibodies, the sequences of which are provided in Table 1.

[0069] In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprises a VL sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, at least 99%) to the corresponding VL sequence of any one of the exemplary antibodies, the sequences of which are provided in Table 1. In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprises a VL sequence having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the corresponding VL sequence of any one of the exemplary antibodies, the sequences of which are provided in Table 1.

[0070] In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprise a VH sequence having at least 70% identity and VL sequence having at least 70% identity of any one of the exemplary antibodies the sequences of which are provided in Table 1. In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprise a VH sequence having at least 90% identity and VL sequence having at least 90% identity of any one of the exemplary antibodies the sequences of which are provided in Table 1. In some embodiments, the anti-osteopontin antibody or an antigen-binding fragment thereof comprise a VH sequence and VL sequence of any one of the exemplary antibodies the sequences of which are provided in Table 1.

[0071] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or completely) identity to any of SEQ ID NOs: 2, 6, 7, and 8. In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence of SEQ ID NO: 2, 6, 7, and 8. In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VL sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or completely) identity to SEQ ID NO: 3 or 9. In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VL sequence of SEQ ID NO: 3 or 9.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0072] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence at least 90% identical to SEQ ID NO: 2 and a VL sequence at least 90% identical to SEQ ID NO: 3.

[0073] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence at least 90% identical to SEQ ID NO: 6, 7, or 8 and a VL sequence at least 90% identical to SEQ ID NO: 9.

[0074] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or completely) identity to SEQ ID NO: 4. In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence of SEQ ID NO: 4.

[0075] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VL sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or completely) identity to SEQ ID NO: 5. In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VL sequence of SEQ ID NO: 5.

[0076] In some embodiments, the anti-osteopontin antibodies, or antigen binding fragments thereof, comprise a VH sequence at least 90% identical to SEQ ID NO: 4 and a VL sequence at least 90% identical to SEQ ID NO: 5.

[0077] As would be recognized by one of ordinary skill in the art based on the present disclosure, one or more amino acids of the aforementioned anti-osteopontin antibodies, or antigen fragments thereof, can be replaced or substituted with a different amino acid. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Ti-p). Non-aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q orAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) Gin), lysine (K or Lys), and arginine (R or Arg). Aliphatic amino acids may be sub-divided into four sub-groups. The “large aliphatic non-polar sub-group” consists of valine, leucine, and isoleucine. The “aliphatic slightly-polar sub-group” consists of methionine, serine, threonine, and cysteine. The “aliphatic polar / charged sub-group” consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. The “small-residue sub-group” consists of glycine and alanine. The group of charged / polar amino acids may be sub-divided into three sub-groups: the “positively-charged sub-group” consisting of lysine and arginine, the “negatively-charged sub-group” consisting of glutamic acid and aspartic acid, and the “polar sub-group” consisting of asparagine and glutamine. Aromatic amino acids may be sub-divided into two sub-groups: the “nitrogen ring sub-group” consisting of histidine and tryptophan and the “phenyl sub-group” consisting of phenylalanine and tyrosine.

[0078] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other and therefore resemble each other most in their impact on the overall protein structure. Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0079] In addition, one or more amino acids can be inserted into the anti-osteopontin antibodies, or antigen-binding fragments thereof (e.g., insertion into the heavy and / or light chain variable region amino acid sequence). Any number of suitable amino acids can be inserted into the amino acid sequence of the antibody or antigen-binding fragment thereof. In this respect, at least one amino acid (e.g.. 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the antibody or antigen-binding fragment thereof. For example, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7. 8, 9, or 10 amino acids) may be inserted into the amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof. In this respect, the amino acid(s) can be inserted into an antibody or antigen-binding fragment thereof in any suitable location. Preferably, the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the antibody or antigen-binding fragment thereof.

[0080] In addition, one or more amino acids can be deleted from the anti-osteopontin antibodies, or antigen-binding fragments thereof (e.g., insertion into the heavy and / or light chain variable region amino acid sequence). Any number of suitable amino acids can be deleted from the amino acid sequence of the antibody or antigen-binding fragment thereof. In this respect, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids), can be deleted from the amino acid sequence of the antibody or antigen-binding fragment thereof. For example, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may be deleted from the amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof.b. Tissue Fibrosis

[0081] Provided herein are methods for treating or preventing tissue fibrosis, or fibrosis as a result of a disease or disorder (“fibrotic diseases”). Fibrosis is the formation of excessive connective tissue in an organ or tissue, commonly in response to damage or injury. A hallmark of fibrosis is the production of excessive extracellular matrix following local trauma. The normal physiological response to injury results in the deposition of connective tissue, but this initially beneficial reparative process may persist and become pathological, altering the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia. An influx of inflammatory cells, includingAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) macrophages and lymphocytes, results in cytokine release and amplifies the deposition of collagen, fibronectin, and other molecular markers of fibrosis.

[0082] The methods disclosed herein can treat any grade or stage of fibrosis or fibrotic diseases. The term “grade,” when used in the context of a fibrosis or a fibrotic disease, refers to the severity of the necrosis and inflammation (“necroinflammation”) associated with the particular disease or disorder. In some embodiments, the methods prevent or delay progression to the next grade of the of fibrosis or fibrotic disease. In some embodiments, the methods decrease the grade of fibrosis or fibrotic disease. The term “stage,” when used in the context of a fibrosis or a fibrotic disease, refers to the degree of scarring, with the end stage resulting in death or organ failure (e.g., cirrhosis in the case of liver fibrosis). In some embodiments, the methods prevent or delay progression to the next stage of the fibrosis or fibrotic disease. In some embodiments, the methods drop the stage of the fibrosis or fibrotic disease. The precise definition for each grade and / or stage depends on the particular disease or disorder and / or the scoring system used.

[0083] Fibrotic diseases or disorders are those which are characterized by having fibrosis in one or more tissues. Fibrotic disorders include, but are not limited to, systemic and local scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as a result of non-alcoholic steatohepatitis (NASH), fibrosis as a result of chronic hepatitis B or C infection, kidney disease, heart disease resulting from scar tissue, and eye diseases such as macular degeneration, and retinal and vitreal retinopathy. Additional fibrotic diseases include fibrosis resulting from chemotherapeutic drugs, radiation-induced fibrosis, and injuries and burns.

[0084] Fibrosis affects nearly all tissues and organ systems. In some embodiments, the tissue fibrosis is cardiac fibrosis, skin fibrosis, skeletal muscle fibrosis, hepatic fibrosis, kidney fibrosis, pulmonary fibrosis, or diabetic fibrosis.

[0085] Lung fibrosis, also referred to as pulmonary fibrosis, is a serious medical condition that involves scarring of the lung tissue. This condition occurs when the alveoli and interstitial tissue of the lungs become inflamed and develop scars on the tissue in an attempt to repair themselves. Pulmonary fibrosis involves gradual exchange of normal lung parenchyma with fibrotic tissue (fibrous scar). The replacement of normal lung with scar tissue causes irreversible decrease in oxygen diffusion capacity. Pulmonary fibrosis can be caused by many conditionsAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) including infections, environmental agents (e.g., asbestos, silica, exposure to certain gases), long term exposure to irritants (e.g., tobacco smoke as in COPD), exposure to ionizing radiation (e.g., such as radiation therapy to treat tumors of the chest), lupus, genetic conditions (e.g., as in cystic fibrosis), and certain medications (e.g. amiodarone, bleomycin, pingyangmycin, busulfan, methotrexate, and nitrofurantoin). Methods herein are suitable for use with lung fibrosis caused by any agent or disease or for lung fibrosis of unidentifiable cause. In one embodiment, provided herein is a method of treating a subject having or suspected of having lung fibrosis. In some embodiments, the method results in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, in fibrotic tissue.

[0086] Liver fibrosis or hepatic fibrosis is the excessive accumulation of extracellular matrix proteins (including collagen), and subsequent scarring process, that occurs in most chronic liver diseases. With time, advanced liver fibrosis results in cirrhosis of the liver. Fibrosis of the liver may be associated with alcohol abuse, viral hepatitis (B, C and D), non-alcoholic fatty liver disease (NAFLD) associated with obesity, non-alcoholic steatohepatitis (NASH), metabolic dysfunction-associated fatty liver disease (MAFLD), diabetes, protein malnutrition, coronary artery disease, corticosteroids, auto-immune hepatitis, inherited diseases (e.g., cystic fibrosis, alpha- 1 -antitrypsin deficiency, etc.), primary biliary cirrhosis, drug reaction and exposure to toxins. In one embodiment, provided herein is a method of treating a subject having or suspected of having liver fibrosis. In some embodiments, the method results in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, in fibrotic tissue. In some embodiments, the methods result in decreasing the likelihood and the speed of cirrhosis development. Methods herein are suitable for use with liver fibrosis caused by any agent or disease or for liver fibrosis of unidentifiable cause. In select embodiments, the disease or disorder characterized by tissue fibrosis is NASH. NASH refers to a combination of fatty liver (NAFLD) with inflammation and occurs in individuals who drink little or no alcohol.

[0087] Skin fibrosis or dermal fibrosis is excessive scarring of the skin and is a result of a pathologic wound healing response. There is a wide spectrum of fibrotic skin diseases: scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, and eosinophilic fasciitis. Exposure to chemicals or physical agents (e.g., mechanical trauma, bum wounds) are also potential causes of fibrotic skin disease. Methods herein are suitable for use with skin fibrosis caused by any agent or of unidentifiable cause. Skin fibrosis is intended toAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) cover the fibrosis of any skin tissue and epithelial cells including, without limitation, blood vessels and veins, internal cavity of an organ or a gland such as ducts of submandibular, gallbladder, thyroid follicles, sweat gland ducts, ovaries, kidney; epithelial cells of gingival, tongue, palate, nose, larynx, esophagus, stomach, intestine, rectum, anus and vagina; derma, scar, skin and scalp. In one embodiment, provided herein is a method of treating a subject having or suspected of having skin fibrosis. In some embodiments, the method results in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, in skin tissue.

[0088] Cardiac fibrosis, a hallmark of heart disease, is thought to contribute to sudden cardiac death, ventricular tachyarrhythmia, left ventricular (LV) dysfunction, and heart failure. Cardiac fibrosis may also refer to an abnormal thickening of the heart valves due to inappropriate proliferation of cardiac fibroblasts but more commonly refers to the proliferation of fibroblasts in the cardiac muscle. Methods herein are suitable for use with cardiac fibrosis due to any cause. In one embodiment, provided herein is a method of treating a subject having or suspected of having cardiac fibrosis. In some embodiments, the method results in the detectable reduction of progression, detectable lessening of worsening, and / or detectable improvement, in cardiac tissue.

[0089] 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, oxygen therapy, dialysis, transplant of affected organ, .

[0090] In certain embodiments, the additional therapeutic agent is appropriate for particular symptoms associated with a fibrotic disease and / or inflammation. For example, immunosuppressive agents (e.g., methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil), agents designed to increase blood flow (e.g.. nifedipine, amlodipine, diltiazem, felodipine, or nicardipine), agents intended to decrease fibrosis, (e.g., d-penicillamine, colchicine, PUVA, Relaxin, cyclosporine, TGF beta blockers and / or p38 MAPK blockers), agents designed to increase heart function (e.g., ACE inhibitors and beta blockers) and / or steroids or broncho-dilators are suitable for use with the disclosed methods.

[0091] In some embodiments, the additional therapeutic agent is an anti-neoplastic and / or anti-fibrotic agent. For example, pantethine (D-bis-(N-pantothenyl-P-aminoethyl)-disulfide) hasAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) been reported to be effective for the inhibition of hepatic fibrosis; a hydrazine derivative, benzoic hydrazide, has been shown to be a powerful antifibrotic agent; angiotensin inhibitors in combination with nitric oxide stimulators or certain pyridoxal benzoyl hydrazones or their analogs has been show for use in inhibiting fibrosis or the progression of fibrosis; Ai adenosine receptor antagonists and / or P?x purinoce tor antagonists have been shown for use in treating or preventing fibrosis; somatostatin agonists, hepatocyte growth factors (HGFs), chymase inhibitors, and antagonists of IL-13 have all been reported to effectively inhibit fibrosis.

[0092] 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.

[0093] In some embodiments, the additional therapeutic agent comprises an immune modulator, a steroid, an analgesic, an antimicrobial agent, an immunotherapy, or a combination thereof.

[0094] The methods may further comprise diagnosing a subject as having fibrosis or a fibrotic disease and / or confirming a diagnosis of fibrosis or fibrotic disease. Currently, the most reliable method available for diagnosing a fibrosis is through tissue biopsy. Other means for detecting the presence of fibrosis or developing fibrosis include computerized axial tomography (CAT or CT) scan, ultrasound, magnetic resonance imaging (MRI), and monitoring the level of one or more serum markers known to be indicative of fibrosis (e.g., various types of collagens). The precise manner of diagnosing fibrosis also varies depending on the organ where the fibrotic process takes place. For instance, biopsies are generally effective for diagnosing fibrosis of most organs, whereas endoscopy involving a fiber optic instrument (e.g., a sigmoidoscope or a colonoscope) can be a less traumatic alternative to detect fibrosis of certain organs such as the intestine.

[0095] Because the method of the present invention is also effective for the prevention of the onset of fibrosis or the slowing of its progression after onset, patients with heightened risk of fibrosis fall within the patient population suitable for treatment using the method of the present invention. Such patients are identified based on prior diagnosis of certain diseases and conditions known to lead to fibrosis.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) Administration

[0096] 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.

[0097] Any of the modulators 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 modulator of osteopontin (e.g., anti-osteopontin antibody) may be mixed with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure.

[0098] 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.

[0099] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides;Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) di saccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0100] 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.

[0101] In the context of the present disclosure 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.

[0102] 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 modulator of osteopontin (e.g., anti-osteopontin antibody), the route of administration, the time of administration, the rate of excretion of the compound, the rate of inactivation 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.

[0103] 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 ofAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 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.

[0104] The modulators 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.

[0105] Other therapies, as included in the above methods, may be used in combination with the modulators of osteopontin (e.g., anti-osteopontin antibodies). Administered “in combination,” as used herein, means that two (or more) different treatments are 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.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0106] The following examples further illustrate aspects of the disclosure but should not be construed as in any way limiting its scope.EXAMPLESExample 1Skin Fibrosis

[0107] For the skin fibrosis experiments, a stented dorsal back wound model was used.Briefly, a 6mm disposable biopsy punch (Acuderm) was used to make two circular full thickness wounds on the dorsal back skin of mice. Silicon wound splints (Grace Bio-Labs) were sutured with 4-0 Nylon to prevent skin contracture. Wounds were dressed with a sterile occlusive dressing and monitored daily. Borders were monitored frequently by application of permanent marker. After 30 days injured skin was harvested.

[0108] Fibrosis was assessed by using trichrome stain and picrosirius red staining. The paraffin sections were de-waxed. hydrated and the nuclei were stained with hematoxylin.Picrosirius red (Sigma- Aldrich, 365548) was then added for one hour. The slides were washed twice with acidified water (0.05% glacial acetic acid). The slides underwent dehydration in three changes of 100% ethanol, followed by clearing in xylene, and finally, mounting in a resinous medium. The PSR images were acquired on a Leica DM6B-Z microscope using a light polarizer equipped with a 32 mm quarter- wave plate and an ICT / P analyzer module. The acquired images were analyzed for fibrosis by quantifying the percent of collagen 1 (PSR) signal within the wounded tissue region.

[0109] The experiments were conducted on mice in which the thrombin cleavage site in the osteopontin gene was mutated such that the produced OPN protein is no longer a substrate of thrombin and cannot be cleaved by thrombin (OPN-KI) and mice in which the OPN gene has been knocked out (OPN-KO). As shown in FIGS. 1A and IB, in the OPN-KI and the OPN-KO mice, the percentage of fibrotic area was decreased as compared to wild-type mice.

[0110] Similar experiments were conducted on wild-type mice, but the mice were administered two different anti-osteopontin antibodies: A6 which blocks thrombin cleavage and C6R which blocks binding of thrombin-cleaved OPN (OPN-R) to a4[M & a9pi integrins. The A6 or C6R antibodies (24mg / kg) were administered daily for five days before injury. After injury, mice were treated twice a week via intraperitoneal injection. As shown in FIG. 1C, the percentAtty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) fibrosis was decreased, more significantly with the C6R, as compared to control mice treated with IgG.Example 2Non-alcoholic Steatohepatitis & Liver Fibrosis

[0111] Six to eight week old male mice were weighed and randomized to receive either normal diet or NASH diet from dyets (diet number 103298 GI Modified western diet with lOg / kg peanut butter added) and the water for the NASH mice is 15% com syrup. Mice were weighed once a week. After 12-16 weeks, mice were euthanized, livers were dissected out and placed in 10% neutral buffered formalin (v / v). After at least 24 hours, tissues were processed, place in paraffin wax and sectioned onto slides. The slides were stained with H&E, Picrosirius Red and Oil Red O before being read by a pathologist.

[0112] All genotypes gain weight on NASH diet (FIG. 2) but OPN-KI mice show reduced fibrosis (FIG. 3).Antibody SequencesTable 1.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)

[0113] 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.

[0114] 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.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0115] 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

Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) CLAIMSWhat is claimed is:

1. A method of treating or preventing tissue fibrosis or a disease or disorder characterized by tissue fibrosis in a subject in need thereof comprising administering to the subject an effective amount of an osteopontin modulator.

2. The method of claim 1, wherein the tissue fibrosis is pulmonary, cardiac, liver, kidney, skin fibrosis.

3. The method of claim 1 or 2, wherein the tissue fibrosis is skin fibrosis.

4. The method of claim 1 or 2, wherein the tissue fibrosis is liver fibrosis.

5. The method of any of claims 1-4, wherein the disease or disorder characterized by tissue fibrosis is non-alcoholic steatohepatitis (NASH).

6. The method of any of claims 1-4, wherein the disease or disorder characterized by tissue fibrosis is scleroderma.

7. The method of any of claims 1-6, wherein the osteopontin modulator is an anti-osteopontin antibody or an antigen-binding fragment thereof.

8. The method of claim 7, wherein the anti-osteopontin antibody or antigen-binding fragment thereof binds to full-length osteopontin or thrombin-cleaved osteopontin.

9. The method of claim 7 or 8, wherein the anti-osteopontin antibody or antigen-binding fragment thereof prevents thrombin cleavage of osteopontin.

10. The method of any of claims 7-9, wherein the anti-osteopontin antibody or antigen-binding fragment thereof blocks integrin binding of thrombin-cleaved fragments of osteopontin.

11. The method of any of claims 7-10, wherein the anti-osteopontin antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3. wherein, the anti-osteopontin antibody or antigen-binding fragment thereof comprises one, two,Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) or all three HCDRs, and optionally, also one, two, or all three of the corresponding LCDRs of any one of the exemplary antibodies the sequences provided in Table 1.

12. The method of claim 11, wherein:the HCDR1 comprises an amino acid sequence of any of SEQ ID NOs: 34, 37. 39, or 166; the HCDR2 comprises an amino acid sequence of any of SEQ ID NOs: 35, 38, 40, or 167; and the HCDR3 comprises an amino acid sequence of any of SEQ ID NOs: 36, 41, or 168; and / orthe LCDR1 comprises an amino acid sequence of SEQ ID NO: 42 or 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43 or 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

13. The method of claim 12, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 34 or 37; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35 or 38; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 36.

14. The method of claim 12, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 39; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 41.

15. The method of claim 12, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 166; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 167; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 168.

16. The method of any of claims 12-15, wherein the LCDR1 comprises an amino acid sequence of SEQ ID NO: 42; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

17. The method of any of claims 12-15, wherein the LCDR1 comprises an amino acid sequence of SEQ ID NO: 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

18. The method of any of claims 12-17, wherein the VH comprises an amino acid sequence having at least 90% identity to any of SEQ ID NOs: 2, 6, 7, and 8.

19. The method of any of claims 12-18, wherein the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3 or 9.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 20. The method of any of claims 12-19, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3.

21. The method of any of claims 12-19, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6, 7, or 8 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9.

22. The method of claim 11, wherein:the HCDR1 comprises the amino acid sequence of SEQ ID NO: 46; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 50; and / orthe LCDR1 comprises the amino acid sequence of SEQ ID NO: 51; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 53; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 55.

23. The method of claim 22, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4.

24. The method of claim 22 or 23, wherein the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5.

25. The method of any of claims 22-24, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5.

26. An osteopontin modulator for use in treating or preventing tissue fibrosis, or a disease or disorder characterized by tissue fibrosis in a subject in need thereof.

27. The osteopontin modulator of claim 26, wherein the osteopontin modulator is an anti-osteopontin antibody or an antigen-binding fragment thereof.

28. The osteopontin modulator of claim 27, wherein the anti-osteopontin antibody or antigenbinding fragment thereof binds to full-length osteopontin or thrombin-cleaved osteopontin.

29. The osteopontin modulator of claim 27 or 28, wherein the anti-osteopontin antibody or antigen-binding fragment thereof inhibits thrombin cleavage of osteopontin.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 30. The osteopontin modulator of claim 27 or 28, wherein the anti-osteopontin antibody or antigen-binding fragment thereof blocks integrin binding of thrombin-cleaved fragments of osteopontin.

31. The osteopontin modulator of any of claims 26-30, wherein the anti-osteopontin antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein the anti-osteopontin antibody or antigen-binding fragment thereof comprises one, two, or all three HCDRs. and optionally, also one, two, or all three of the corresponding LCDRs of any one of the exemplary antibodies the sequences provided in Table 1.

32. The osteopontin modulator of claim 31, wherein:the HCDR1 comprises an amino acid sequence of any of SEQ ID NOs: 34, 37. 39, or 166; the HCDR2 comprises an amino acid sequence of any of SEQ ID NOs: 35, 38, 40, or 167; and the HCDR3 comprises an amino acid sequence of any of SEQ ID NOs: 36, 41, or 168; and / orthe LCDR1 comprises an amino acid sequence of SEQ ID NO: 42 or 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43 or 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

33. The osteopontin modulator of claim 32, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 34 or 37; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35 or 38; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 36.

34. The osteopontin modulator of claim 32, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 39; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 41.

35. The osteopontin modulator of claim 32, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 166; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 167; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 168.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432) 36. The osteopontin modulator of any of claims 32-35, wherein the LCDR1 comprises an amino acid sequence of SEQ ID NO: 42; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 43; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

37. The osteopontin modulator of any of claims 35-36, wherein the LCDR1 comprises an amino acid sequence of SEQ ID NO: 169; the LCDR2 comprises an amino acid sequence of SEQ ID NO: 170; and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 44.

38. The osteopontin modulator of any of claims 32-37, wherein the VH comprises an amino acid sequence having at least 90% identity to any of SEQ ID NOs: 2, 6, 7, and 8.

39. The osteopontin modulator of any of claims 32-38, wherein the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3 or 9.

40. The osteopontin modulator of any of claims 32-39, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3.

41. The osteopontin modulator of any of claims 32-39, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6, 7, or 8 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9.

42. The osteopontin modulator of claim 31, wherein the anti-osteopontin antibody or antigenbinding fragment thereof comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein:the HCDR1 comprises the amino acid sequence of SEQ ID NO: 46; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47; and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 50; and / orthe LCDR1 comprises the amino acid sequence of SEQ ID NO: 51; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 53; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 55.

43. The method of claim 42, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4.Atty. Docket: STDU2-44072.601(Client Ref: S22-084 / 432)44. The method of claim 42 or 43, wherein the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5.

45. The method of any of claims 26-44, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 4 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5.

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