Therapeutic Antibodies Against Osteopontin

Abstract

Therapeutic antibodies specific for osteopontin and methods of using them to treat osteopontin-related disorders are provided. In particular, antibodies or antigen-binding fragments thereof that inhibit thrombin cleavage of osteopontin or block the activity of thrombin-cleaved fragments of osteopontin are provided. In addition, antibody conjugates, pharmaceutical compositions or formulations containing the antibodies or antibody conjugates, and kits containing the antibodies, conjugates, or formulations are also provided.

Description

Background Art

[0001] Osteopontin (OPN) is a multifunctional protein of matrix cells that has a highly conserved RGD domain that binds to a wide range of integrins. Thrombin cleavage at Arg153 in mice (Arg168 in humans) generates OPN-Arg (OPN-R) and an OPN C-terminal fragment (OPN-CTF). OPN-R, which has SVVYGLR (SEQ ID NO: 3) at the C-terminus, binds to a subset of integrins (α4β1 and α9β1) to which full-length OPN does not bind. The physiological role of the OPN-R fragment is thought to include a role as an immunomodulatory substance that promotes cell adhesion, migration, and survival (Kahles F. et al. (2014) Mol. Metab. 3:384). The role of the OPN-CTF fragment has not been fully studied, but it has been shown to interact with dendritic cells in response to chemokines and promote their chemotaxis (Shao Z. et al. (2014) J. Biol. Chem. 289:27146). OPN-R has been involved in inflammatory disorders such as rheumatoid arthritis (Song JJ. et al. (2011) J. Clin. Invest. 121:3517), but its role in cancer remains unclear (Castello LM. et al. (2017) Mediators Inflamm. 2017:4049098). Carboxypeptidase B2 (CPB2) or carboxypeptidase N (CPN) removes the C-terminal arginine from OPN-R and converts it to OPN-Leu (OPN-L), suppressing integrin binding to the SVVYGLR (SEQ ID NO: 3) binding motif (Myles T. et al. (2003) J Biol Chem. 278(51):51059-51067, Shao Z. et al. (2014) J. Biol. Chem. 289:27146). Elevated levels of OPN-R and OPN-L were seen in synovial fluid samples from patients with rheumatoid arthritis, but not as much in patients with osteoarthritis or psoriatic arthritis (Sharif S. et al. (2009) Arthritis Rheum 60:2902).The role of OPN-CTF has also been demonstrated in a murine experimental autoimmune encephalomyelitis (EAE) model suggesting that antibodies against OPN-CTF have a protective effect (Clemente N. et al. (2017) Front. Immunol. 8:321).

Prior Art Documents

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Summary of the Invention

Means for Solving the Problems

[0003] There are provided therapeutic antibodies specific for osteopontin, and methods of using them for treating osteopontin-related disorders. In particular, antibodies that inhibit thrombin cleavage of osteopontin, or the interaction of thrombin-cleaved fragments of osteopontin with integrin and / or other cell receptors, are useful for treating osteopontin-related disorders such as inflammation, cardiac hypertrophy, myocardial fibrosis, and cancers that overexpress osteopontin such as melanoma, glioblastoma, ovarian cancer, breast cancer, and lung cancer. In one aspect, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to osteopontin or a thrombin cleavage fragment thereof, and the antibody inhibits thrombin cleavage of osteopontin or integrin binding to the thrombin cleavage fragment of osteopontin. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an OPN-R fragment or an OPN-CTF fragment. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising Arg168 of osteopontin. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an osteopontin peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 1-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47.

[0004] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the light chain.

[0005] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy-chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy-chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy-chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light-chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light-chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light-chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto).

[0006] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto).

[0007] In certain embodiments, the antibody is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, nanobody, bispecific antibody, bispecific T cell engager antibody, trispecific antibody, Fab fragment, Fab’ fragment, F(ab’) 2 fragment, F v fragment, or scFv fragment.

[0008] In another aspect, a composition for treating an osteopontin-related disorder is provided, the composition comprising an antibody or an antigen-binding fragment thereof described herein that specifically binds to osteopontin or a thrombin cleavage fragment thereof, wherein the antibody or antigen-binding fragment thereof inhibits thrombin cleavage of osteopontin or integrin binding to a thrombin cleavage fragment of osteopontin. In some embodiments, the osteopontin-related disorder is melanoma, glioblastoma, ovarian cancer, cardiac hypertrophy, myocardial fibrosis, or inflammation.

[0009] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles, and ointments.

[0010] In certain embodiments, the composition further comprises an anti-cancer therapeutic agent including, but not limited to, chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiation sensitizers, and radioisotopes, or combinations thereof.

[0011] In certain embodiments, the composition further comprises a B-Raf inhibitor, a MEK inhibitor, or a combination thereof. Exemplary B-Raf inhibitors include, but are not limited to, dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720. Exemplary MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901.

[0012] In another aspect, a method of treating an osteopontin-related disorder is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein that specifically binds to osteopontin at the thrombin cleavage site or to a thrombin cleavage fragment of osteopontin. In certain embodiments, the antibody specifically binds to an OPN-R fragment or an OPN-CTF fragment. In some embodiments, the antibody inhibits integrin binding to the OPN-R fragment. Preferably, the antibody does not interfere with the thrombin clot-promoting activity in the subject.

[0013] In certain embodiments, the osteopontin-related disorder is a cancer that overexpresses osteopontin. For example, cancers that overexpress osteopontin include, but are not limited to, melanoma, glioblastoma, ovarian cancer, breast cancer, and lung cancer. In some embodiments, the method further comprises administering at least one additional anti-cancer therapeutic agent including, but not limited to, chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiosensitizers, and radioisotopes, or combinations thereof.

[0014] In certain embodiments, the method further comprises administering a B-Raf inhibitor. Exemplary B-Raf inhibitors include, but are not limited to, dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720.

[0015] In certain embodiments, the method further comprises administering a mitogen-activated protein kinase (MEK) inhibitor. Exemplary MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof is administered according to a daily dosing schedule or intermittently. Multiple cycles of treatment can be administered to the subject for a period sufficient to result in at least a partial tumor response, or more preferably a complete tumor response. In some embodiments, the period is at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years or more.

[0017] In another aspect, a method for inhibiting the growth and / or proliferation of tumor cells in a subject is provided, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein that specifically binds to osteopontin or a thrombin cleavage fragment thereof, wherein the antibody or antigen-binding fragment thereof inhibits thrombin cleavage of osteopontin or integrin binding to the thrombin cleavage fragment of osteopontin.

[0018] In another aspect, a conjugate is provided that comprises an antibody or antigen-binding fragment thereof described herein and a drug selected from the group consisting of an anti-cancer therapeutic agent, a detectable label, and an imaging agent. Exemplary anti-cancer therapeutic agents include, but are not limited to, cytotoxic agents, drugs, toxins, nucleases, hormones, immunomodulators, apoptosis promoters, anti-angiogenic agents, boron compounds, photoactive agents, and radioisotopes.

[0019] In another aspect, a kit is provided that comprises a composition comprising an antibody or antigen-binding fragment thereof described herein and instructions for using the kit to treat an osteopontin-related disorder. The kit may further comprise means for administering the composition to the subject.

[0020] In another aspect, a method for producing an antibody is provided, the method comprising inducing an immune response in a subject against an immunogenic peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 5-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47.

[0021] In another aspect, an isolated nucleic acid is provided that comprises: a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 25; b) a nucleotide sequence encoding a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26; c) a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO: 27; d) a nucleotide sequence encoding a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28; e) a nucleotide sequence having 80-100% sequence identity (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) with the nucleotide sequences of a)-d); and f) the complements of a)-e).

[0022] In another aspect, a recombinant nucleic acid is provided that comprises a promoter operably linked to a nucleic acid described herein.

[0023] In another aspect, a vector system is provided that comprises one or more vectors encoding an antibody or an antigen-binding fragment thereof described herein.

[0024] In certain embodiments, the vector system comprises one or more vectors encoding an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof comprises heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20.

[0025] In certain embodiments, the vector system comprises one or more vectors encoding an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof comprises heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 22, and a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 24.

[0026] In certain embodiments, the vector system comprises one or more vectors encoding an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 28 or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 28.

[0027] In certain embodiments, the vector system described herein encodes an antibody or an antigen-binding fragment thereof that is chimerized or humanized.

[0028] In another aspect, a host cell comprising the vector system described herein is provided.

[0029] In another aspect, a method of producing an antibody or an antigen-binding fragment thereof is provided, the method comprising: a) culturing a host cell comprising the vector system described herein under conditions suitable for the production of the antibody or an antigen-binding fragment thereof; and b) isolating the antibody or an antigen-binding fragment thereof from the host cell.

[0030] In another aspect, a hybridoma producing the antibody described herein is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention is best understood when read in conjunction with the following detailed description and the accompanying drawings. It is emphasized that various features of the drawings are not to scale. Conversely, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures.

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DETAILED DESCRIPTION OF THE INVENTION

[0033] There are provided therapeutic antibodies specific for osteopontin and methods of using them for treating osteopontin-related disorders. In particular, antibodies that inhibit thrombin cleavage of osteopontin or block the activity of thrombin cleavage fragments of osteopontin, or antigen-binding fragments thereof, are provided. In addition, antibody conjugates, and pharmaceutical compositions or formulations containing the antibody or antibody conjugate, and kits containing the antibody, conjugate, or formulation are also provided.

[0034] Before describing the therapeutic antibodies and methods of using them in the treatment of osteopontin-related disorders, it should be understood that the present invention is not limited to the specific methods or compositions described, and that the invention itself may naturally vary. Also, since the scope of the present invention is limited only by the appended claims, it should be understood that the terminology used herein is for the purpose of merely describing particular embodiments only.

[0035] When ranges of values are provided, it should be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is specifically disclosed herein, unless the context clearly dictates otherwise. Each smaller range between any of the specified values or intervening values in the specified range and any other specified value or intervening value in that specified range is encompassed by the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range, and each range that includes any of the limits of the smaller range, neither of the limits of the smaller range, or both of the limits of the smaller range is also encompassed by the present invention, subject to any specifically excluded limits within the specified range. When the specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but some potential and preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials in connection with which the publication is cited. It should be understood that, in case of any conflict, the present disclosure prevails over any disclosure of the incorporated publications.

[0037] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the invention. Any method described can be performed in the order of the events described, or in any other order that is logically possible.

[0038] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, and reference to "the cancerous cell" includes reference to one or more cancerous cells and equivalents thereof, such as cancer cells, tumor cells, neoplastic cells, and malignant cells known to those skilled in the art.

[0039] The term "antibody" encompasses monoclonal antibodies, polyclonal antibodies, as well as hybrid antibodies, modified antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349:293-299; and U.S. Patent No. 4,816,567); bispecific antibodies, bispecific T cell engager antibodies (BiTE), trispecific antibodies, and other multispecific antibodies (see, e.g., Fan et al. (2015) J. Hematol. Oncol. 8:130, Krishnamurthy et al. (2018) Pharmacol Ther. 185:122-134), F(ab’) 2 and F(ab) fragments; F vMolecules (non-covalently linked heterodimers, e.g., see Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (e.g., see Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (e.g., see Wang et al. (2016) Int Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26); dimer and trimer antibody fragment constructs; minibodies (e.g., see Pack et al. (1992) Biochem 31:1579-1584; Cumber (1992) J Immunology 149:1209-126); humanized antibody molecules (e.g., see Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and any functional fragments obtained from such molecules, such fragments retaining the specific antigen-binding properties of the parental antibody molecule.

[0040] The phrase "specifically (or selectively) binds" with respect to the binding of an antibody to an antigen (e.g., osteopontin or a thrombin cleavage fragment thereof) refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological substances. Thus, under specified immunoassay conditions, a particular antibody binds to a particular antigen at least two times above background and does not substantially bind to other antigens present in the sample in significant amounts. Specific binding to an antigen under such conditions may require an antibody selected for its specificity for the particular antigen. For example, antibodies produced against an antigen from a particular species such as rat, mouse, or human can be selected to obtain only antibodies that specifically immunoreact with the antigen and do not react with other proteins, except for polymorphic variants and alleles. This selection can be achieved by removing antibodies that cross-react with molecules from other species. Various immunoassay formats can be used to select antibodies that specifically immunoreact with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (see, e.g., Harlow & Lane Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction is at least two-fold above background signal or noise, and more typically greater than 10- to 100-fold above background.

[0041] As used herein, "antibody fragment" and all its grammatical variants are defined as a part of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, and that part does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab’, Fab’-SH, F(ab’) 2and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), for example, (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain and having no associated heavy chain portion, or fragments thereof containing the three CDRs of the light chain variable domain, (3) single-chain polypeptides containing only one heavy chain variable region and having no associated heavy chain portion, or fragments thereof containing the three CDRs of the heavy chain variable region, and (4) nanobodies containing a single Ig domain from a non-human species or other specific single domain binding module; and multispecific or multivalent structures formed from antibody fragments, including but not limited to. In antibody fragments containing one or more heavy chains, the heavy chain(s) can contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and / or can contain any hinge region sequence found in an intact antibody, and / or can contain a leucine zipper sequence that is fused to or located in the hinge region sequence or the constant domain sequence of the heavy chain(s).

[0042] A "humanized antibody" is an immunoglobulin molecule containing a minimal sequence derived from a non-human immunoglobulin. A humanized antibody comprises a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species such as a mouse, rat, or rabbit (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human residues. A humanized antibody may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequences. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to regions of a non-human immunoglobulin and all or substantially all of the framework (FR) regions being regions of a human immunoglobulin consensus sequence. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0043] As used herein, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0044] When referring to a polypeptide, "isolated" means that the indicated molecule is separated from the whole organism in which it is found naturally and is distinct, or is present in the substantial absence of other biological macromolecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule lacking all or a portion of the sequences that are normally associated with it in nature, or a sequence that, while existing in nature, has heterologous sequences associated with it, or a molecule dissociated from a chromosome.

[0045] The term "conjugated" refers to the binding of two compounds or agents by covalent or non-covalent means.

[0046] Terms such as "treatment", "treating", "treat" are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease and / or its symptom(s), and / or therapeutic in terms of partially or completely stabilizing or curing a disease and / or an adverse effect caused by the disease. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the occurrence of a disease and / or symptom(s) in a subject who may have a predisposition to the disease or symptom but has not yet been diagnosed as having it, (b) inhibiting the disease and / or symptom(s), i.e., preventing their onset, or (c) alleviating the disease symptom(s), i.e., causing an antibody to the disease and / or symptom(s). Those in need of treatment include those in which the disease has already occurred (e.g., those having cancer), as well as those in which prevention is desired (e.g., those having an increased susceptibility to cancer, those suspected of having cancer, those at risk of recurrence, etc.).

[0047] The term "unit dosage form" as used herein refers to physically discrete units suitable as unit dosages for humans and animals, each unit containing a predetermined amount of a compound of the present disclosure calculated to be sufficient to produce the desired therapeutic effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specification of the novel unit dosage form depends on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0048] "Osteopontin-related disorders" include, but are not limited to, inflammation, cardiac hypertrophy, myocardial fibrosis, and any disease or disorder associated with an increase in the level of osteopontin or its thrombin cleavage fragment, including cancers such as melanoma, glioblastoma, ovarian cancer, breast cancer, and lung cancer that overexpress osteopontin.

[0049] The terms "tumor," "cancer," and "neoplasm" are used interchangeably and refer to a mammalian cell or population of cells, such as a cell proliferative, hyperproliferative, or differentiative disorder, whose growth, proliferation, or survival exceeds that of normal counterpart cells. Typically, the growth is uncontrolled. The term "malignant tumor" refers to invasion of adjacent tissues. The terms "metastasis" or secondary, recurrent or recurrent tumor, cancer or neoplasm refer to the spread or dissemination of a tumor, cancer or neoplasm to other sites, locations, or regions within the subject, which are different from the primary tumor or cancer. Neoplasms, tumors, and cancers include benign, malignant, metastatic, and non-metastatic species, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of a neoplasm, tumor, or cancer, or a neoplasm, tumor, cancer, or metastasis that is progressive, worsening, stable, or in remission.In particular, the terms "tumor", "cancer", and "neoplasm" include cancers such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, and small cell carcinoma, and are not limited to, but include head and neck cancer, skin cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral nerve tumor, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, extracranial embryonal cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell cancer, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic cervical squamous cell carcinoma, multiple myeloma and other plasma cell tumors, mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, oropharyngeal cancer, osteosarcoma and bone cancer including fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, as well as Wilms' tumor and other pediatric kidney tumors.

[0050] In particular, the term "melanoma" includes any type of melanoma at any stage, including metastatic melanoma. For example, the term "melanoma" includes, but is not limited to, malignant nevus, melanoma derived from malignant nevus, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, and desmoplastic melanoma. Melanoma may contain changes (mutations) in genomic DNA sequences, meaning that the proteins encoded by melanoma cells are different from the proteins in other parts of the patient's body. As an example, B-Raf, a protein involved in growth signaling to cells, may have mutations. Thus, the term also includes B-RAF mutant melanomas, including, but not limited to, melanomas containing the V600E or V600K mutations.

[0051] "Antitumor activity" is intended to mean a decrease in the cell proliferation rate, and thus a decrease in the growth rate of an existing tumor or a tumor that develops during treatment, and / or the destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and thus a decrease in the overall size of the tumor during treatment. Such activity can be evaluated using animal models.

[0052] The "therapeutically effective dose or amount" of an antibody or antigen-binding fragment thereof that specifically binds to osteopontin or a thrombin-cleaved fragment thereof is intended to mean an amount that, when administered as described herein, results in a beneficial therapeutic response such as antitumor activity or anti-inflammatory activity. The exact amount required will vary for each subject depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular one or more drugs used, the mode of administration, etc. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0053] As used herein, the term "tumor response" means the reduction or disappearance of all measurable lesions. The criteria for tumor response are based on the WHO reporting criteria [WHO Offset Publication, 48 - World Health Organization, Geneva, Switzerland, (1979)]. Ideally, all one - or two - dimensionally measurable lesions should be measured at each evaluation. If multiple lesions are present in any organ, such measurement may not be possible, and in such situations, up to six representative lesions should be selected if possible.

[0054] As used herein, the term "complete response" (CR) means the complete disappearance of all clinically detectable malignant disease, determined by two evaluations at least four weeks apart.

[0055] As used herein, the term "partial response" (PR) means a greater than 50% decrease from baseline in the sum of the products of the longest perpendicular diameters of all measurable disease, without progression of evaluable disease and without evidence of any new lesions determined by at least two consecutive evaluations at least four weeks apart. The evaluation should show a partial decrease in the size of lytic lesions, re - calcification of lytic lesions, or a decrease in the density of blastemic lesions.

[0056] The terms "polypeptide", "peptide", and "protein" are used interchangeably to refer to polymers of amino acid residues. This term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding natural amino acids.

[0057] "Substantially purified" generally refers to the isolation of a substance (e.g., an antibody, conjugate, compound, drug, nucleic acid, polynucleotide, protein, polypeptide, or peptide), such that the substance constitutes a majority of the sample in which it is present and is defined as a percentage of the total sample. Typically, a substantially purified component in a sample comprises 50%, preferably 80% - 85%, more preferably 90% - 95% of the sample. Techniques for purifying the substance of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and precipitation according to density.

[0058] The terms "recipient", "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sport, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is a human.

[0059] "Pharmaceutically acceptable excipient or carrier" optionally may be included in the compositions of the present invention and refers to an excipient that does not cause a significant adverse toxic effect in a patient.

[0060] Examples of "pharmaceutically acceptable salts" include, but are not limited to, amino acid salts, salts prepared with inorganic acids such as chlorides, sulfates, phosphates, diphosphates, bromides, and nitrates, or salts prepared from the corresponding inorganic acid forms of any of the foregoing, such as hydrochlorides, etc., or salts prepared with organic acids such as malates, maleates, fumarates, tartrates, succinates, ethylsuccinates, citrates, acetates, lactates, methanesulfonates, benzoates, ascorbates, p-toluenesulfonates, palmitates, salicylates, and stearates, as well as estolates, gluceptates, and lactobionates. Similarly, examples of salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0061] As used herein, the terms "detectable label", "detecting agent", "imaging agent", "diagnostic agent", and "detectable moiety" are used interchangeably and include, but are not limited to, fluorescent agents, chemiluminescent substances, chromophores, bioluminescent proteins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, isotope labels, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin, or haptens), etc., and refer to detectable molecules or substances. The term "fluorescent agent" refers to a substance or a part thereof that can exhibit fluorescence within a detectable range. Specific examples of detectable labels that can be used in the practice of the present invention include 3 H、 2 H, 120 I、 123 I、 124 I、 125 I、 131 I、 35 S、 11 C、 13 C、 14 C、 32 P、 15 N、 13 N、 110 In、 111 In、 177 Lu、 18 F、 52 Fe、 62Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 15 O, 186 Re, 188 Re, 51 M, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82m Rb, and 83 Isotope labels including radioactive and non-radioactive isotopes such as Sr are included. In particular, detectable labels include, but are not limited to, 64 Cu, 89 Zr, 68 Ga, 177 Lu, 82 Rb, 11 C, 13 N, 15 O, and 18 Positron radionuclides suitable for PET imaging such as F, or include, but are not limited to, 67 Ga, 99m Tc, 123 I, and 131 γ-radionuclides suitable for single photon emission computed tomography (SPECT) imaging such as I may be included. Detectable labels also include, but are not limited to, Mn 2+ , Fe 3+ , Fe 2+ , Gd 3+ , Ti 2+ , Cr 3+ , Co 2+ , Ni 2+ , and Cu 2+It may contain non-radioactive paramagnetic metal ions suitable for MRI imaging such as those mentioned. Detectable labels also include CAL Fluor dyes such as SYBR Green, SYBR Gold, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, and CAL Fluor Red 635; Quasar dyes such as Quasar 570, Quasar 670, and Quasar 705; Alexa Fluor such as Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 784; cyan dyes such as Cy 3, Cy3.5, Cy5, Cy5.5, and Cy7; fluorescein, 2’,4’,5’,7’-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM), 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein (JOE), hexachlorofluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX), tetramethylrhodamine (TAMRA), FITC, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas Red, luminol, and quantum dots, including but not limited to these fluorophores, alkaline phosphatase (AP), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo r , G418 r) Enzymes such as dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase (lacZ), and xanthine-guanine phosphoribosyltransferase (XGPRT), β-glucuronidase (gus), placental alkaline phosphatase (PLAP), and secreted embryonic alkaline phosphatase (SEAP). Enzyme tags are used together with their cognate substrates. These terms also include chemiluminescent labels such as luminol, isoluminol, acridinium ester, and peroxyoxalate, as well as bioluminescent proteins such as firefly luciferase, bacterial luciferase, Renilla luciferase, and aequorin. These terms also include microspheres color-coded by known fluorescence intensity (see, e.g., microspheres using the xMAP technology provided by Luminex (Austin, TX)), microspheres containing quantum dot nanocrystals, e.g., microspheres containing different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals manufactured by Life Technologies (Carlsbad, CA)), glass-coated metal nanoparticles (see, e.g., SERS nanotags manufactured by Nanoplex Technologies, Inc. (Mountain View, CA)), barcode materials (e.g., submicron-sized striped metal rods, see, e.g., nanobarcodes manufactured by Nanoplex Technologies, Inc.), microparticles encoded with colored barcodes (see, e.g., CellCard manufactured by Vitra Bioscience (vitrabio.com)), glass microparticles having digital holographic code images (see, e.g., CyVera microbeads manufactured by Illumina (San Diego, CA)), near-infrared (NIR) probes, and nanoshells.These terms also include contrast agents such as ultrasonic contrast agents (e.g., SonoVue microbubbles containing sulfur hexafluoride, Optison microbubbles containing an albumin shell and an octafluoropropane gas core, Levovist microbubbles containing a lipid / galactose shell and an air core, Perflexane lipid microspheres containing perfluorocarbon microbubbles, and Perflutren lipid microspheres containing octafluoropropane encapsulated in an outer lipid shell), magnetic resonance imaging (MRI) contrast agents (e.g., gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset, gadobutrol, gadoxetic acid), and radiographic contrast agents for, e.g., computed tomography (CT), radiography, or fluoroscopy (e.g., diatrizoic acid, metrizoic acid, iothalamide, iotalamic acid, ioxitalamic acid, ioglycamic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, iohexol, ioxaglic acid, iopamidol, iopromide, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iodobitrol, ioxilan, ioxamic acid, iotroxate, ioglycamide, adipiodone, iobenzamic acid, iopanoic acid, iotetric acid, iopodate sodium, tyropanoic acid, and iopodate calcium). The detectable label or imaging agent may be indirectly or directly bound to the antibody, and the label or contrast agent facilitates the use of the antibody in imaging.

[0062] "Identity" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid or two polypeptide sequences are "substantially identical" to each other when the sequences show at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% - 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% - 98% sequence identity over the defined length of the molecules. As used herein, substantially identical also refers to a sequence that shows complete identity to a designated sequence. In general, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100 to directly compare the sequence information between the two molecules. For peptide analysis, ALIGN, which employs the local homology algorithm of Smith and Waterman, Advances in Appl. Math. 2:482 - 489, 1981, and programs such as those available in Dayhoff, M.O. Atlas of Protein Sequence and Structure, M.O. Dayhoff ed., 5 Suppl. 3:353 - 358, National Biomedical Research Foundation, Washington, DC, can be used to assist in the analysis. Programs for determining nucleotide sequence identity, such as BESTFIT, FASTA, and GAP programs that similarly rely on the Smith and Waterman algorithm, are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI).These programs are recommended by the manufacturer and are readily utilized using the default parameters described in the Wisconsin Sequence Analysis Package mentioned above. For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a gap penalty of six nucleotide positions.

[0063] Another way to establish percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, default parameters (e.g., gap start penalty 12, gap extension penalty 1, and gap 6) are used in the scoring table and the Smith-Waterman algorithm can be used. From the generated data, the "match" value reflects "sequence identity". Other programs suitable for calculating percent identity or similarity between sequences are generally known in the art. For example, another alignment program is BLAST, which is used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss Protein+Spupdate+PIR. Details of these programs are readily available.

[0064] Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-strand specific nuclease(s) and sizing of the digested fragments. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions defined for a particular system. Defining appropriate hybridization conditions is within the scope of the art. See, for example, Sambrook et al. (supra); DNA Cloning (supra); Nucleic Acid Hybridision (supra).

[0065] As used herein to describe a nucleic acid molecule, "recombinant" means a polynucleotide of genomic, cDNA, viral, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is naturally associated. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, a gene of interest is cloned and then expressed in a transformed organism as further described hereinafter. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0066] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for insertion. For example, direct uptake, transduction, or f-mating are included. The exogenous polynucleotide can be maintained as a non-integrated vector, for example, as a plasmid, or alternatively, can be integrated into the host genome.

[0067] The terms "recombinant host cell", "host cell", "cell", "cell line", "cell culture", and other such terms that denote a microorganism or higher eukaryotic cell line cultured as a single cell entity refer to a cell that can be or has been used as a recipient for a recombinant vector or other transfer DNA and includes the progeny of the original transfected cell.

[0068] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences (or "control elements"), is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo. The boundaries of the coding sequence can be determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. The coding sequence can include, but is not limited to, cDNA derived from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from viral or prokaryotic DNA, and even synthetic DNA sequences. Transcription termination sequences can be located 3' to the coding sequence.

[0069] Typical "control elements" include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), optimization sequences for translation initiation (located 5' to the coding sequence), and translation termination sequences.

[0070] "Operably linked" refers to the arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a coding sequence can affect the expression of the coding sequence if appropriate enzymes are present. The promoter need not be contiguous with the coding sequence so long as it functions to induce the expression of the coding sequence. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence and still the promoter sequence can be considered to be "operably linked" to the coding sequence.

[0071] An "expression cassette" or "expression construct" refers to an assembly that can induce the expression of a target sequence(s) or gene(s). An expression cassette generally includes, as described above, a control element such as a promoter operably linked to the target sequence(s) or gene(s) (to induce transcription), and often also includes a polyadenylation sequence. In certain embodiments of the present invention, the expression cassettes described herein can be contained within a donor polynucleotide, plasmid, or viral vector construct. In addition to the components of the expression cassette, the construct can also include one or more selectable markers, a signal that allows the construct to exist as single-stranded DNA (e.g., the M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., the SV40 or adenovirus origin of replication).

[0072] A "purified polynucleotide" refers to a polynucleotide or fragment thereof that is substantially free of proteins with which the polypeptide is naturally associated, e.g., containing less than about 50%, preferably less than about 70%, more preferably at least about 90% less of such proteins. Techniques for purifying a polynucleotide of interest are well known in the art and include, for example, disruption of cells containing the polynucleotide with chaotropic agents, and separation of the polynucleotide(s) and protein by ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0073] The term "transfection" is used to refer to the uptake of foreign DNA by cells. When exogenous DNA is introduced into the cell membrane, the cell is "transfected". Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell. This term refers to both stable and transient uptake of genetic material and includes the uptake of peptide- or antibody-conjugated DNA.

[0074] A "vector" can transfer a nucleic acid sequence into a target cell (e.g., viral vector, non-viral vector, particulate carrier, and liposome). Typically, "vector construct", "expression vector", and "gene delivery vector" mean any nucleic acid construct capable of inducing expression of a nucleic acid of interest and introducing the nucleic acid sequence into a target cell. Thus, this term includes cloning and expression vehicles, as well as plasmids and viral vectors.

[0075] The publications discussed herein are provided only for their disclosures prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates and may need to be independently confirmed.

[0076] It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention.

[0077] Anti-osteopontin antibody There is provided an anti-osteopontin antibody that specifically binds to one or more epitopes of osteopontin. In some embodiments, the anti-osteopontin antibody specifically binds to the thrombin cleavage site, inhibits thrombin cleavage of osteopontin, and / or specifically binds to the thrombin cleavage fragment of osteopontin (e.g., the OPN-R fragment or the OPN-CTF fragment) and reduces the biological activity of the thrombin cleavage fragment. In certain embodiments, the anti-osteopontin antibody specifically binds to an epitope containing Arg168 of osteopontin. The foregoing numbering is with respect to the reference amino acid sequence of human osteopontin (SEQ ID NO: 8), and it should be understood that the corresponding positions in osteopontin proteins obtained from other species are also intended to be encompassed by the present disclosure.

[0078] As used herein, the term "anti-osteopontin antibody" includes full-length antibodies and antigen-binding fragments thereof that contain the antigen-binding region of the antibody, such as Fab, F(ab’) 2 , or F v fragments, and molecules containing the CDRs of anti-osteopontin antibodies such as single-chain variable fragments (scFv), or any other type of antibody fragment of interest (see the definition of the term "antibody" above). Anti-osteopontin antibodies that can be used in the practice of the subject methods include monoclonal antibodies, polyclonal antibodies, hybrid antibodies, modified antibodies, chimeric antibodies, and humanized antibodies, and: hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349:293-299 and U.S. Patent No. 4,816,567); F(ab’) 2 and F(ab) fragments; F vMolecules (non-covalently linked heterodimers, see, e.g., Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662, and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26); dimer and trimer antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and UK Patent Publication GB2,276,169 (published September 21, 1994)); and any functional fragments obtained from such molecules (such fragments retain the specific binding properties of the parent antibody molecule), including but not limited to these.

[0079] In certain embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof specifically binds to an osteopontin peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 1-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47.

[0080] In certain embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the light chain.

[0081] In certain embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto).

[0082] In certain embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 26, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto).

[0083] Antibodies that specifically bind to osteopontin at the thrombin cleavage site or to thrombin cleavage fragments of osteopontin (e.g., OPN-R fragment or OPN-CTF fragment) can be prepared using any suitable method known in the art. See, for example, Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). For example, an antigen comprising a peptide fragment of osteopontin containing the thrombin cleavage site of osteopontin or a thrombin cleavage fragment of osteopontin (e.g., OPN-R fragment or OPN-CTF fragment) can be used to induce an immune response in mammals such as mice, rats, rabbits, guinea pigs, monkeys, or humans to produce polyclonal antibodies. Exemplary peptides that can be used to induce an immune response to produce anti-osteopontin antibodies include, but are not limited to, osteopontin peptides comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 1-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47 (see also the Examples). If desired, the antigen can be conjugated to a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surfactants (e.g., lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, and dinitrophenol). Among the adjuvants used in humans, BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are particularly useful.

[0084] Monoclonal antibodies that specifically bind to osteopontin antigen can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma method, the human B cell hybridoma method, and the EBV hybridoma method (Kohler et al., Nature 256, 495-97, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-30, 1983; Cole et al., Mol. Cell Biol. 62, 109-20, 1984).

[0085] In addition, splicing of mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity, a technique developed for the production of "chimeric antibodies," can also be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-55, 1984; Neuberger et al., Nature 312, 604-08, 1984; Takeda et al., Nature 314, 452-54, 1985). Monoclonal antibodies and other antibodies can also be "humanized" to prevent the patient from initiating an immune response to the antibody when the antibody is used therapeutically. Such antibodies can have a sequence that is sufficiently similar to that of a human antibody used directly in therapy or may require modification of some key residues. Sequence differences between rodent antibodies and human sequences can be minimized by site-directed mutagenesis of individual residues or by grafting entire complementarity-determining regions to replace residues that differ from those in the human sequence.

[0086] Alternatively, as described below, humanized antibodies can be produced using recombinant methods. Antibodies that specifically bind to a particular antigen can contain partially or fully humanized antigen-binding sites, as disclosed in U.S. Patent No. 5,565,332. Human monoclonal antibodies can be prepared in vitro as described in Simmons et al., PLoS Medicine 4(5), 928-36, 2007.

[0087] Alternatively, techniques described for the production of single-chain antibodies can be adapted to produce single-chain antibodies that specifically bind to a particular antigen using methods known in the art. Antibodies with related specificities but distinct idiotype compositions can be generated by chain shuffling from a random combinatorial immunoglobulin library (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991). Single-chain antibodies can also be constructed using DNA amplification methods such as PCR, using hybridoma cDNA as a template (Thirion et al., Eur. J. Cancer Prev. 5, 507-11, 1996). Single-chain antibodies can be monospecific or bispecific and can be bivalent or tetravalent. The construction of tetravalent bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, Nat. Biotechnol. 15, 159-63, 1997. The construction of bivalent bispecific single-chain antibodies is taught in Mallender & Voss, J. Biol. Chem. 269, 199-206, 1994.

[0088] The nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into cells to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly, for example, using filamentous phage technology (Verhaar et al., Int. J Cancer 61, 497-501, 1995; Nicholls et al., J. Immunol. Meth. 165, 81-91, 1993).

[0089] Antibodies that specifically bind to the osteopontin antigen can be produced by inducing in vivo production in a lymphocyte population or by screening an immunoglobulin library or a panel of highly specific binding reagents, as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).

[0090] Chimeric antibodies can be constructed as disclosed in WO93 / 03151. Multivalent and multispecific binding proteins, such as the "diabody" described in WO94 / 13804, which are derived from immunoglobulins, can also be prepared.

[0091] In some embodiments, anti-osteopontin antibodies from B lymphocytes are obtained from blood donors and cloned. Nucleic acids encoding the light and heavy chains of an antibody or fragments thereof containing the variable domain complementarity determining regions (e.g., Fab) can be amplified by PCR and cloned into a vector. ScFv antibodies can be generated by cloning into a vector a construct that connects the light and heavy chains via a linker within one open reading frame. The blood donor can be a donor of any species. In some embodiments, human blood donors are used for the production of human antibodies. In other embodiments, camelid blood donors are used for the production of camelid antibodies. Camelid antibodies can be derived from, for example, dromedary camels, Bactrian camels, llamas, and alpacas. Such camelids produce a unique type of antibody lacking a light chain. Heavy chain antibodies (HCAb) or variable domain fragments thereof (e.g., single domain antibodies or nanobodies) can also be used in the subject methods (see, e.g., Vincke et al. (2012) Methods Mol. Biol. 911:15-26, Krah et al. (2016) Immunopharmacol. Immunotoxicol. 38(1):21-8, which are incorporated herein by reference).

[0092] Antibodies can be purified by methods well known in the art. For example, antibodies can be affinity purified by passing them over a column to which the relevant antigen binds. The bound antibody can then be eluted from the column using a buffer of high salt concentration.

[0093] Chimeric and humanized antibodies Suitable anti-osteopontin antibodies include fully humanized antibodies, humanized, or chimeric versions of such antibodies. For example, humanized antibodies are useful for in vivo applications in humans because they have low antigenicity. Similarly, antibodies that have been caninized, felinized, etc. are useful for applications in dogs, cats, and other species, respectively. Examples of the desired antibodies include humanized antibodies, or antibodies that have been caninized, felinized, equinized, bovinized, porcinized, etc., and variants thereof.

[0094] Chimeric monoclonal antibodies in which the variable Ig domains of non-human (e.g., mouse) monoclonal antibodies are fused to human constant Ig domains can be generated using standard procedures known in the art (see Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984), and Boulianne et al, Nature 312, 643-646, (1984)).

[0095] Humanized antibodies can be achieved by various methods including, for example: (1) grafting non-human complementarity determining regions (CDRs) onto human framework and constant regions (a process referred to in the art as humanization by "CDR grafting"); (2) transplanting the entire non-human variable domain but "covering" them with a human-like surface by replacement of surface residues (a process referred to in the art as "veneering"); or (3) substituting human amino acids at positions that are judged likely to have a low probability of having a deleterious effect on either antigen binding or protein folding but a high probability of reducing immunogenicity in a human environment (e.g., HUMAN ENGINEERING). In the present disclosure, humanized antibodies can include "humanized", "veneered", and / or "HUMAN ENGINEERED" antibodies. These methods are disclosed, for example, in Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immunol. 28:489-498 (1991); Padlan, Molec. Immunol. 31:169-217 (1994); Studnicka et al. U.S. Patent No. 5,766,886; Studnicka et al., Protein Engineering 7:805-814, 1994; Co et al., J. Immunol. 152, 2968-2976 (1994); Riechmann, et al., Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng. 4:773-783 (1991), each of which is incorporated herein by reference.

[0096] CDR grafting involves introducing one or more of the six CDRs of the mouse heavy and light chain variable Ig domains into the appropriate four framework regions of the human variable Ig domain. This technique (see Riechmann, et al., Nature 332:323-27 (1988)) utilizes the conserved framework regions (FR1-FR4) as scaffolds to support the CDR loops, which are the major contact points with the antigen. However, a drawback of CDR grafting is that it can result in humanized antibodies with significantly lower binding affinities than the original mouse antibodies because amino acids in the framework regions can contribute to antigen binding and amino acids in the CDR loops can affect the association of the two variable Ig domains. To maintain the affinity of humanized monoclonal antibodies, the CDR grafting technique can be improved by selecting human framework regions that most closely resemble those of the original mouse antibody and by site-directed mutagenesis of single amino acids within the framework or CDRs using computer modeling of the antigen-binding site (e.g., Co et al., J. Immunol. 152, 2968-2976 (1994)).

[0097] Antibody fragment The anti-osteopontin antibody can be in the form of an antibody fragment. An antibody fragment includes a portion of an intact full-length antibody and can include the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’) 2 , Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibody fragments such as bispecific, trispecific, and tetraspecific antibodies (e.g., diabodies, tribodies, tetrabodies), minibodies, chelate recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, and other polypeptides formed from antibody fragments. See, for example, Holliger & Hudson (Nat. Biotech. 23:1126-36 (2005)).

[0098] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH domains, each having a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields the F(ab’)2 fragment, a divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, which have two "single-chain Fv" or "scFv" antibody fragments that include the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. The Fv polypeptide can further include a polypeptide linker between the VH and VL domains that allows the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which the VL and VH regions pair to form a monovalent molecule via a synthetic linker, enabling them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The Fd fragment consists of the VH and CH1 domains.

[0099] Additional antibody fragments include domain antibody (dAb) fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989). Diabodies are antibodies in which the VH and VL domains are expressed on a single polypeptide chain, using a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites (see, for example, EP 404,097; WO 93 / 11161; Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994). Diabodies can be bispecific or monospecific.

[0100] Method for producing anti-osteopontin antibody As discussed above, the present disclosure provides antibodies that specifically bind to osteopontin or its thrombin cleavage fragment, and the antibodies inhibit thrombin cleavage of osteopontin or the interaction between the thrombin cleavage fragment of osteopontin and integrin and / or other cell receptors. Exemplary methods for making anti-osteopontin antibodies are shown below.

[0101] Antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, antibodies can be made and isolated using phage display methods. Antibodies can also be isolated from the sera of animal hosts immunized with an immunogenic composition comprising osteopontin, including the full protein and fragments thereof. Exemplary antibodies include isolated antibodies that can specifically bind to osteopontin (e.g., the thrombin cleavage site of osteopontin) or thrombin cleavage fragments of osteopontin.

[0102] The antigen for coating wells for phage display panning, or the immunogenic composition used to induce the antibodies of the present disclosure, may contain aggregates of one or more antigens. This method may include exposing the antigen to aggregation conditions to form aggregates. Thus, the production method described above may further include the step of forming aggregates of the isolated antigen. Examples of aggregation conditions include heating, addition of excipients that promote aggregation, and the like.

[0103] The antigen used for coating wells for phage panning or for inducing the antibodies of the present disclosure may be conjugated to another molecule. For example, the antigen may be conjugated to a second molecule such as a peptide, polypeptide, lipid, carbohydrate, etc. that helps with solubility, storage or other handling properties, cell permeability, half-life, release and / or distribution by targeting a specific cell (e.g., cancer cell, cardiomyocyte, etc.) or cell location (e.g., plasma membrane, lysosome, endosome, mitochondrion, etc.), tissue, or other body location (e.g., blood, a specific organ, tumor, etc.).

[0104] A particular embodiment of the antigen conjugated to a second molecule is when the second molecule is an immunomodulatory agent. An "immunomodulatory agent" is a molecule that directly or indirectly changes the immune response. Specific classes of immunomodulatory agents include those that stimulate or assist in stimulating the immune response. Examples include antigens and antigen carriers such as toxins or derivatives thereof including tetanus toxoid.

[0105] Phage display Phage display is used for high-throughput screening of protein interactions. Phages can be utilized to display antigen-binding domains expressed from repertoires or combinatorial antibody libraries (e.g., human or mouse). Phages expressing antigen-binding domains that bind to osteopontin or its thrombin cleavage fragments can be selected or identified, for example, using labeled osteopontin, or osteopontin bound or captured on a solid surface or beads. Phages used in these methods are typically filamentous phages containing fd and M13 binding domains expressed from phages having a disulfide-stabilized Fv antibody domain recombinantly fused to either Fab, Fv (individual Fv regions from the light or heavy chain), or phage gene III or gene VIII protein. Exemplary methods are described, for example, in EP 368 684 B1; U.S. Patent No. 5,969,108, Hoogenboom, H.R. and Chames, Immunol. Today 2000, 21:371; Nagy et al. Nat. Med. 2002, 8:801; Huie et al., Proc. Natl. Acad. Sci. USA 2001, 98:2682; Lui et al., J. Mol. Biol. 2002, 315:1063, which are each incorporated herein by reference. Some publications (e.g., Marks et al., Bio / Technology 1992, 10:779-783) describe the production of high-affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as strategies for constructing large phage libraries. In another embodiment, ribosome display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al., Nat. Biotechnol. 2000, 18:1287; Wilson et al., Proc. Natl. Acad. Sci. USA 2001, 98:3750; or Irving et al., J. Immunol. Methods 2001, 248:31).Cell surface libraries may be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 2000, 97:10701; Daugherty et al., J. Immunol. Methods 2000, 243:211). Such procedures provide an alternative to conventional hybridoma technology for the isolation and subsequent cloning of monoclonal antibodies.

[0106] In phage display methods, functional antibody domains are presented on the surface of phage particles that carry the polynucleotide sequences encoding them. For example, DNA sequences encoding the heavy chain variable (VH) and light chain variable (VL) regions are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of lymphoid tissue) or a synthetic cDNA library, or are otherwise isolated. The DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and can be cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated into E. coli and the E. coli is infected with helper phage. The VH or VL region is usually recombinantly fused to either phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to the antigen of interest (e.g., the serine protease domain of osteopontin) can be selected or identified with the antigen, for example, using a labeled antigen, or an antigen bound or captured on a solid surface or bead.

[0107] Further examples of phage display methods that can be used to generate antibodies include those disclosed in PCT Application No. PCT / GB91 / 01134, PCT Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, each of which is incorporated herein by reference in its entirety.

[0108] As described in the above references, after phage selection, the antibody coding region can be isolated from the phage and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, Fab, Fab’, and F(ab’) fragments can be recombinantly produced using techniques known in the art, such as those disclosed in PCT Publication WO92 / 22324; Mullinax et al., BioTechniques 1992, 12:864-869; and Sawai et al., AJRI 1995, 34:26-34; and Better et al., Science 1988, 240:1041-1043 (the above references are incorporated herein by reference in their entirety). 2 Techniques for recombinantly producing fragments can also be utilized.

[0109] Immunity and Antibody Production A method for inducing an antibody in a host animal involves administering an effective amount of osteopontin or a fragment thereof as an antigen to a host animal (i.e., a suitable mammal such as a mouse, rabbit or guinea pig, a suitable bird such as a chicken, or a camelid), thereby inducing the production of an antibody that specifically binds to osteopontin or a thrombin-cleaved fragment thereof. Exemplary immunogenic peptides that can be used to induce an immune response in a host animal to produce anti-osteopontin antibodies include, but are not limited to, immunogenic peptides comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 5-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47 (see also the Examples). Methods of immunizing animals, including the adjuvants used, booster schedules, injection sites, suitable animals, etc., are well understood in the art (e.g., Harlow et al. (Antibodies: Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.)), and the administration of live cells to animals has been described for several mammals and birds (e.g., McKenzie et al (Oncogene 4:543-8, 1989), Scuderi et al (Med. Oncol. Tumor Pharmacother 2:233-42, 1985), Roth et al (Surgery 96:264-72, 1984) and Drebin et al (Nature 312:545-8, 1984)). Next, a population of antibody-producing cells is generated. In one embodiment, the cell population is produced using the hybridoma method well known to those skilled in the art (see, e.g., Harlow Antibodies: A Laboratory Manual, First Edition (1988) Cold Spring Harbor, N.Y.). The cells are fused to immortalized cells such as myeloma cells or transformed cells that can replicate indefinitely in cell culture, thereby producing an immortal immunoglobulin-secreting cell line. The immortal cell line used can be selected to be deficient in an enzyme required for the utilization of a particular nutrient.Many such cell lines (such as myelomas) are known to those skilled in the art and include, for example, thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPRT). These deficiencies enable the selection of fused cells, for example, according to the ability to grow on hypoxanthine aminopterin thymidine medium (HAT). In alternative embodiments, a cell population expressing a monoclonal antibody can be generated using phage display methods.

[0110] Anti-osteopontin antibodies containing antigen-binding fragments of anti-osteopontin antibodies can also be produced by genetic engineering. In this technique, antibody-producing cells are sensitized to the desired antigen or immunogen, similar to standard hybridoma procedures. Messenger RNA isolated from immune spleen cells or hybridomas is used as a template for generating cDNA using PCR amplification. By inserting appropriate segments of the amplified immunoglobulin cDNA into an expression vector, a library of vectors is produced, each containing one heavy-chain gene and one light-chain gene that retain the initial antigen specificity. A combinatorial library can be constructed by combining the heavy-chain gene library and the light-chain gene library. This results in a library of clones that co-express heavy and light chains (similar to the Fab fragment or antigen-binding fragment of an antibody molecule). Vectors carrying these genes are co-transfected into a host (such as bacteria, insect cells, mammalian cells, or other suitable protein-producing host cells). Inducing antibody gene synthesis in the transfected host causes the heavy and light chain proteins to self-assemble and generate active antibodies that can be detected by screening with the antigen or immunogen.

[0111] Phage panning and screening Once a population of antibody-producing cells or phages is generated, the antibodies are screened using one or a combination of various assays. Generally, these assays can be grouped into functional assays, assays that detect the binding affinity or specificity of the antibody, and assays that detect the ability of the antibody to initiate or inhibit a process.

[0112] For example, the antigen is bound to beads or wells or other solid supports and incubated with phages presenting the antibody of interest. After washing, the bound phages are then recovered by inoculation of log-phase E. coli cells. The cells are grown and expanded with helper phage. The steps are repeated for amplification of the tightly bound phages. After several rounds of enrichment, phage-infected E. coli colonies are picked and Fab antibodies are purified from the periplasmic fraction. The purified antibodies are then analyzed according to methods known in the art. Certain specific exemplary examples are detailed below.

[0113] A population of antibodies isolated from phage-infected cells or hybridomas is further analyzed and / or screened for binding to a single antigen (i.e., an antigen not mixed with other antigens of the plurality of antigens) among a plurality of antigens, either in vitro or in situ (e.g., on cells). Immunospecific binding can be performed according to routine and known methods in the art. Examples of immunoassays that can be used include, but are not limited to, Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and competitive and non-competitive assay systems using techniques such as protein A immunoassay. See, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety.

[0114] The antibodies of the present disclosure can also be screened in vivo. This method involves administering an anti-osteopontin antibody to an animal model for a disease or condition (i.e., an osteopontin-related disorder) and determining the effect of the antibody on the disease or condition of the model animal. The in vivo assay of the present invention includes a control, and a suitable control includes a sample in the absence of the antibody. Generally, a plurality of assay mixtures are performed in parallel at different antibody concentrations to obtain differential responses to various concentrations. Typically, one of these concentrations functions as a negative control, i.e., at zero concentration or below the detection level.

[0115] The monoclonal antibody of interest is an antibody that modulates, i.e., reduces or increases, the symptoms of the disease or condition of the animal model by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or more when compared to the control in the absence of the antibody. Generally, the monoclonal antibody of interest renders the subject animal similar to an equivalent animal not suffering from the disease or condition. Antibodies having therapeutic value identified using the methods and compositions of the present invention are referred to as "therapeutic" antibodies.

[0116] The selected monoclonal antibody of interest can be grown in vitro using routine tissue culture methods or in vivo using a mammalian subject. For example, pristane-primed mice can be inoculated with log-phase hybridoma cells in PBS for ascites production. The ascites can be stored at -70°C prior to further purification.

[0117] Method of Screening The screening method provided by the present disclosure may involve the use of a phage library to screen for antibodies that specifically bind to osteopontin or its thrombin cleavage fragments, and optionally have any of the additional features described herein (e.g., inhibit thrombin cleavage of osteopontin or integrin binding to thrombin cleavage fragments of osteopontin). The binding agent may be selected for its strong inhibition of osteopontin and / or its specific binding affinity. The method may be carried out according to the phage display methods described above.

[0118] Briefly, osteopontin or its fragment can be immobilized on an ELISA plate or beads by covalent or non-covalent interactions such as hydrophobic adsorption, biotin-avidin interaction, and Ni 2+ -6×His interaction. The phage library is then incubated with the immobilized antigen / protease, washed, and recovered. During panning and selection, the bound phage is recovered and amplified in E. coli. Multiple consecutive rounds of selection ensure the selection of phages presenting polypeptides that act as antibodies specific for osteopontin or its thrombin cleavage fragments. The stringency of washing is increased over several rounds (e.g., 3 rounds). Many techniques well known in the art can be used to increase the specificity of the recovered phages. Examples include increasing the washing time, increasing the detergent concentration, increasing the salt concentration, and inclusion of known macromolecular inhibitors (e.g., small peptide substrates, BPTI, echistatin, and / or previously identified antibody inhibitors). Identification of inhibitory antibodies may include ELISA and inhibition assays. Details of the assays performed in the method of selecting and isolating anti-osteopontin antibodies have been described above.

[0119] Also contemplated by the present disclosure is a library of nucleic acid constructs encoding candidate anti-osteopontin antibodies. The library encodes a plurality of candidate anti-osteopontin antibodies that can have one or more common polypeptide regions (e.g., at least one heavy or light chain CDR) and at least one other polypeptide region that varies among the population.

[0120] Nucleic acids, vectors, and vector systems encoding anti-osteopontin antibodies In certain embodiments, anti-osteopontin antibody heavy and light chains, or antibody fragments thereof (e.g., Fab fragment, Fab’ fragment, F(ab’) 2 fragment, F v fragment, or scFv fragment) are produced from a vector. A “vector” is a composition that can be used to deliver a nucleic acid of interest into the interior of a cell. Polynucleotides encoding antibody light and / or heavy chains, or fragments thereof, can be introduced into cells using a single vector or separate vectors (i.e., a vector system). The ability of constructs to produce anti-osteopontin antibodies can be determined empirically.

[0121] A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like. Expression constructs can be replicated in living cells or produced synthetically. For the purposes of this application, the terms “expression construct,” “expression vector,” and “vector” are used interchangeably to indicate the application of the present invention in a general and illustrative sense and are not intended to limit the present invention.

[0122] In one embodiment, an expression vector for expressing an anti-osteopontin antibody comprises a promoter "operably linked" to a polynucleotide encoding an anti-osteopontin heavy and / or light chain, or a fragment thereof (e.g., including their variable domain CDRs). As used herein, the phrases "operably linked" or "under transcriptional control" mean that the promoter is in the correct position and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0123] In certain embodiments, the nucleic acid encoding the polynucleotide of interest is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by, or introduced into, the cellular synthetic machinery required to initiate specific transcription of a gene. The term "promoter" is used herein to refer to a group of transcriptional control modules clustered around the start site of RNA polymerase I, II, or III. Typical promoters for mammalian cell expression include, among others, the SV40 early promoter, CMV promoters such as the CMV immediate early promoter (see, e.g., U.S. Pat. Nos. 5,168,062 and 5,385,839, which are incorporated herein by reference in their entirety), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter. Other non-viral promoters, such as the promoter derived from the mouse metallothionein gene, are also used for mammalian expression. These and other promoters can be obtained from commercially available plasmids using techniques well known in the art. See, e.g., Sambrook et al. (supra). Enhancer elements can be used in conjunction with a promoter to increase the expression level of the construct. Examples include the SV40 early gene enhancer as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer / promoter derived from the terminal repeat sequence (LTR) of Rous sarcoma virus as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, and elements derived from human CMV as described in Boshart et al., Cell (1985) 41:521, such as the element contained in the CMV intron A sequence.

[0124] Typically, a transcription terminator / polyadenylation signal is also present in the expression construct. Examples of such sequences include, but are not limited to, those derived from SV40 as described in Sambrook et al. (supra), and bovine growth hormone terminal sequences (see, e.g., U.S. Patent No. 5,122,458). Further, 5'-UTR sequences can be placed adjacent to the coding sequence to enhance their expression. Such sequences can include UTRs that contain an internal ribosome entry site (IRES).

[0125] The inclusion of an IRES enables the translation of one or more open reading frames from a vector. The IRES element attracts the eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997) 22:150-161. A number of IRES sequences are known, including sequences derived from a variety of viruses, such as the leader sequences of picornaviruses such as encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), polio leader sequences, hepatitis A virus leader, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), the IRES element derived from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), the dialdiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc.Although not limited to, various non-viral IRES sequences including yeast-derived IRES sequences, as well as the type 1 receptor IRES of human angiotensin II (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRES (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6):1074-1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1):37-44) are also used herein. These elements are readily commercially available in plasmids commercially available, for example, by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA), and GeneCopoeia (Rockville, MD). Also, see IRESite (iresite.org), a database of experimentally verified IRES structures. The IRES sequence can be included in a vector, for example, to express an anti-osteopontin antibody heavy chain or a fragment thereof in combination with an anti-osteopontin antibody light chain or a fragment thereof from an expression cassette.

[0126] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to enable the production of multiple protein products (e.g., an anti-osteopontin antibody heavy chain or a fragment thereof in combination with an anti-osteopontin antibody light chain or a fragment thereof) from a single vector. The 2A linker peptide is inserted between the coding sequences of the multicistronic construct. The self-cleaving 2A peptide enables the co-expressed proteins from the multicistronic construct to be produced at equimolar levels. Various virus-derived 2A peptides can be used, including but not limited to those derived from foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus-1. For example, see Kim et al. (2011) PLoS One 6(4):e18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001) Gene Ther. 8(11):864-873, which are incorporated herein by reference in their entirety.

[0127] An expression vector or vector system containing one or more vectors can be used to transform isolated cells, cell lines, or cell populations, and the gene product of interest (e.g., antibody light and heavy chains, or fragments thereof) is selectively expressed by the single or multiple cells. In some embodiments, an anti-osteopontin antibody is produced and secreted. The transformed cells secrete the antibody into the surrounding medium. Certain regulatory sequences can be included in the vector to enhance secretion, for example, using the tissue plasminogen activator (TPA) leader sequence, interferon (γ or α) signal sequence, or other signal peptide sequences from known secreted proteins. The secreted antibody can then be isolated by various techniques described herein using standard purification techniques such as, but not limited to, hydroxyapatite resin, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunosorbent techniques, affinity chromatography, immunoprecipitation, etc.

[0128] Alternatively, the protein is not secreted and the transformed cells are disrupted using chemical, physical, or mechanical means that lyse the cells but keep the recombinant protein substantially intact. Intracellular proteins can also be obtained by removing components from the cell membrane, for example, by using detergents or organic solvents, such that leakage of the polypeptide occurs. Such methods are known to those skilled in the art and are described, for example, in Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0129] For example, methods of cell disruption include, but are not limited to, sonication (sonication or ultrasonication); agitation; liquid or solid extrusion; heat treatment; freeze-thaw; drying; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecyl sulfate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells will mainly be a matter of choice and will depend on the cell type in which the polypeptide is expressed, the culture conditions, and any pretreatment used.

[0130] Following cell disruption, cell debris is generally removed by centrifugation, and the polypeptide produced intracellularly is further purified using standard purification techniques including, but not limited to, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunosorption techniques, affinity chromatography, immunoprecipitation, etc.

[0131] Antibodies containing heavy and light chains, or fragments thereof, can be purified using affinity purification, for example, by protein A affinity chromatography, immunoaffinity chromatography using an antibody that binds to an anti-osteopontin heavy or light chain epitope), or antigen-specific affinity chromatography using immobilized osteopontin antigen. The selection of a suitable affinity resin is within the scope of the art. After affinity purification, the expressed antibody can be further purified using conventional techniques well known in the art, for example, by any of the techniques described above.

[0132] Antibody conjugate Anti-osteopontin antibodies are also used in therapeutic and diagnostic (e.g., in vivo imaging, etc.) applications. For example, such antibodies can be conjugated to a payload such as a therapeutic agent (e.g., a cytotoxic payload) or a labeling agent (e.g., an in vivo imaging agent), and when the antibody binds to osteopontin or its thrombin cleavage fragment, the therapeutic or labeling agent is selectively delivered to target cells (e.g., cancer cells that overexpress osteopontin) with elevated levels of osteopontin or its thrombin cleavage fragment. Selective targeting of therapeutic or imaging agents to cells that overexpress osteopontin can reduce unwanted exposure of non-target cells to the therapeutic agent and reduce toxicity upon administration. Further, in imaging applications (e.g., in vivo imaging for diagnostic, prognostic, and / or any other purpose), selective binding of the antibody to cells that overexpress osteopontin concentrates the imaging agent within such cells, thereby increasing the signal-to-noise ratio and diagnostic / prognostic value of the resulting image.

[0133] Accordingly, any anti-osteopontin antibody described herein may be in unconjugated form, or may be directly conjugated to an agent such as a therapeutic and / or imaging (e.g., diagnostic) agent, or may be indirectly conjugated to a carrier polymer containing such other therapeutic or imaging agent.

[0134] In some embodiments, the antibody is conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radioactive conjugate). Suitable chemotherapeutic agents include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Suitable toxins include bacterial toxins such as diphtheria toxin; plant toxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al J.Natl.Cancer Inst.92(19):1573-81(2000); Mandler et al., Bioorg.Med.Chem.Letters 10:1025-1028(2000); Mandler et al., Bioconjugate Chem.13.786-91(2002)), maytansinoids (EP 1391213; Liu et al., Proc.Natl.Acad.Sci.USA 93:8618-23(1996)), auristatins (Doronina et al., Nat.Biotech.21:778-84(2003) and calicheamicin Lode et al., Cancer Res.58:2928(1998); Hinman et al., Cancer Res.53:3336-3342(1993)).

[0135] Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzyme labels (such as horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels or luminescent labels or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for achieving such labeling are known. See, for example, (Sternberger, L.A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E.A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J.W. J. Immunol. Meth. 13:215 (1976)).

[0136] In certain embodiments, the imaging agent of the anti-osteopontin antibody conjugate of the present disclosure is an imaging agent used for in vivo imaging such as near-infrared (NIR) optical imaging, single photon emission computed tomography (SPECT) / CT imaging, etc. Labeling agents used for such applications include, but are not limited to, fluorescent labels and radioisotopes, etc. In certain embodiments, the labeling agent is a multimodal in vivo imaging agent that enables in vivo imaging using two or more imaging techniques (see, for example, Thorp-Greenwood and Coogan (2011) Dalton Trans. 40:6129-6143).

[0137] The conjugation of the antibody portion is described in U.S. Patent No. 6,306,393. General techniques are also described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Patent No. 5,057,313. This general method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine function to which a plurality of drugs, toxins, chelating agents, boron adducts, or other therapeutic agents have been added. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0138] The carrier polymer may be, for example, aminodextran or a polypeptide of at least 50 amino acid residues. Various techniques for conjugating drugs or other agents to carrier polymers are known in the art. Polypeptide carriers can be used in place of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain and can be from about 100 to 5000 amino acid residues. At least some of the amino acids should be lysine residues, or glutamic acid or aspartic acid residues. The pendant amines of lysine residues and the pendant carboxylates of glutamine and aspartic acid are convenient for attaching drugs, toxins, immunomodulators, chelating agents, boron adducts, or other therapeutic agents. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, their copolymers, and mixed polymers of these amino acids with others, such as serine, which impart desirable solubility characteristics to the resulting loaded carriers and conjugates. Examples of agents to which the antibody can be conjugated include any of the cytotoxic chemotherapeutic agents described herein.

[0139] Conjugated antibodies can be prepared by directly conjugating the antibody component with a therapeutic agent or a labeling agent. The general procedure is similar to the method of indirect conjugation, except that the therapeutic agent or the labeling agent is directly bound to the oxidized antibody component. For example, the carbohydrate portion of the antibody can be conjugated with polyethylene glycol to extend the half-life.

[0140] The therapeutic or labeling agent can bind to the hinge region of the reduced antibody component via disulfide bond formation or using a heterobifunctional cross-linking agent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods”, Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,”, Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). Various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene) are known in the art.

[0141] Pharmaceutical composition An anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate containing the same) can be formulated into a pharmaceutical composition optionally containing one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Suitable excipients for injectable compositions include water, alcohol, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, etc. The excipients can also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.

[0142] The composition can also include an antibacterial agent to prevent or inhibit microbial growth. Non-limiting examples of antibacterial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0143] Antioxidants can similarly be present in the composition. Antioxidants are used to prevent oxidation and thereby prevent the degradation of the antibody or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0144] Surfactants can be present as excipients. Exemplary surfactants include polysorbates such as "Tween® 20" and "Tween® 80", and pluronics® such as F68 and F88 (BASF, Mat Olive, New Jersey)); sorbitan esters; phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamine (preferably not in liposomal form), fatty acids, and lipids such as fatty esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc, and other such suitable cations.

[0145] Acids or bases can be present as excipients in the composition. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof, but are not limited thereto.

[0146] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and the specific requirements of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts (ranging from low to high amounts) of the excipient, examining stability and other parameters, and then determining the range in which optimal performance is achieved without significant side effects. However, generally, the excipient(s) are present in the composition in an amount of about 1% to about 99% by weight of the excipient, preferably about 5% to about 98% by weight, more preferably about 15% to about 95% by weight, and most preferably at a concentration of less than 30% by weight. These aforementioned pharmaceutical excipients are described together with other excipients in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), the “Physician’s Desk Reference”, 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0147] The composition encompasses all types of formulations, particularly those suitable for injection, such as powders or lyophilized products that can be reconstituted with a solvent prior to use, as well as ready-to-inject solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of diluents suitable for reconstituting the solid composition prior to injection include bacteriostatic water for injection, 5% aqueous glucose solution, phosphate buffered saline, Ringer's solution, physiological saline, sterile water, deionized water, and combinations thereof. For liquid pharmaceutical compositions, solutions and suspensions are contemplated. Additional preferred compositions include compositions for oral or topical delivery.

[0148] The pharmaceutical formulations herein can also be contained in syringes, implant devices, etc., depending on the intended mode of delivery and use. Preferably, the composition comprising the anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof) described herein is in unit dosage form, i.e., a metered or packaged form containing an amount of the anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof) suitable for a single administration.

[0149] The compositions herein can optionally contain one or more additional agents, e.g., a drug for treating osteopontin-related disorders, or other pharmaceuticals used for treating the condition or disease of a subject. For example, the formulated preparation can contain an anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof), and one or more drugs for treating cancer, including but not limited to chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiation sensitizers, and radioisotopes. For treating melanoma, the compound preparation can contain a B-Raf inhibitor, a MEK inhibitor, or a combination thereof. Exemplary B-Raf inhibitors include, but are not limited to, dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720. Exemplary MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901. Alternatively, such agents can be contained in a composition separate from the composition comprising the anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof), and can be co-administered simultaneously with, before, or after the composition comprising the anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof).

[0150] Method for treating osteopontin-related disorders The present disclosure provides methods for treating osteopontin-related disorders associated with the activity of osteopontin. Exemplary osteopontin-related diseases include, but are not limited to, inflammation, cardiac hypertrophy, myocardial fibrosis, and cancers that overexpress osteopontin such as melanoma, glioblastoma, ovarian cancer, breast cancer, and lung cancer, and any disease or disorder associated with an increase in the level of osteopontin or a thrombin cleavage fragment thereof.

[0151] This method generally involves administering to a subject in need thereof a therapeutically effective amount of any anti-osteopontin antibody, antigen-binding fragment, or conjugate described herein, alone (e.g., monotherapy) or in combination with one or more additional therapeutic agents (e.g., anti-cancer therapeutic agents, B-Raf inhibitors, and / or MEK inhibitors) (e.g., combination therapy). In certain embodiments, the method involves treating an osteopontin-related disorder by administering to a patient in need thereof a therapeutically effective amount of an anti-osteopontin antibody (e.g., an antibody of the present disclosure that specifically binds to osteopontin or a thrombin cleavage fragment thereof and inhibits thrombin cleavage of osteopontin or binding of integrin to a thrombin cleavage fragment of osteopontin).

[0152] A variety of hosts can be treated according to this method. Generally, such hosts are "mammals" or "mammalian," terms that are widely used to describe organisms belonging to the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host is a human.

[0153] Prodrugs of the antibody compositions of the present disclosure are also contemplated in the methods described herein. Such prodrugs are generally functional derivatives of a compound that is readily convertible in vivo to the required compound. Thus, in the methods of the present disclosure, the term "administering" includes administering the specifically disclosed compound or a compound that, although not specifically disclosed, converts in vivo to a particular compound after administration to a subject in need thereof. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Wermuth, “Designing Prodrugs and Bioprecursors” in Wermuth, ed. The Practice of Medicinal Chemistry, 2d Ed., pp. 561-586 (Academic Press 2003). Prodrugs include esters that hydrolyze in vivo (e.g., in the human body) to produce the compounds described herein. Suitable ester groups include, but are not limited to, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic acids, alkenoic acids, cycloalkanoic acids, and alkanedioic acids, in which each alkyl or alkenyl moiety has up to 6 carbon atoms. Exemplary esters include formates, acetates, propionates, butyrates, acrylates, citrates, succinates, and ethylsuccinates.

[0154] Dosage In the methods of the present disclosure, an effective amount of an anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate comprising the same) is administered to a subject in need thereof. The amount administered will vary depending on the goal of administration, the particular osteopontin-related disorder being treated, the health and physical condition of the individual being treated, age, the taxonomic group of the individual being treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired, the formulation of the anti-osteopontin antibody or conjugate, the assessment of the medical situation by the clinician performing the treatment, and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined through routine clinical trials. For example, the amount of an anti-osteopontin antibody or conjugate used to inhibit inflammation, cardiac hypertrophy, myocardial fibrosis, or cancer cell growth, metastasis and / or invasiveness is below an amount that would otherwise be irreversibly toxic to the subject (i.e., the maximum tolerated dose). In other cases, the amount is around or well below the toxicity threshold but still within the effective concentration range or as low as the threshold dose.

[0155] Individual doses are typically greater than the amount necessary to produce a measurable effect in the subject and can be determined based on pharmacokinetics and pharmacology for absorption, distribution, metabolism, and excretion ("ADME") of the antibody or conjugate and thus based on the in vivo behavior of the composition within the subject. This includes, for example, consideration of the route of administration and dose that can be adjusted for parenteral (applied by a route other than the gastrointestinal tract for systemic or local effects) application. For example, administration of an anti-osteopontin antibody or conjugate is typically either local or via injection (e.g., intravenous, intramuscular, or intratumoral) or a combination thereof.

[0156] The pharmacokinetics of an antibody or conjugate and the corresponding biological activity within a subject are typically measured against the fraction of antibody present at the target of interest. For example, once administered, an antibody can accumulate at a biological target (e.g., osteopontin) that concentrates substances in diseased cells with elevated levels of its osteopontin or thrombin cleavage fragments. Thus, a dosing regimen in which the antibody is administered to accumulate at the target of interest over time can be part of a strategy that allows for lower individual doses. This can also mean, for example, that the dose of an antibody that is more slowly cleared in vivo can be reduced compared to the effective concentration calculated from in vitro assays (e.g., the effective amount in vitro is close to mM concentration, whereas in vivo it is less than mM concentration).

[0157] As an example, the effective amount of a usage or dosing regimen can be measured from the IC50 of a given antibody for inhibiting thrombin cleavage of osteopontin or integrin binding to thrombin cleavage fragments of osteopontin. "IC50" is intended to mean the concentration required for 50% inhibition in vitro. Alternatively, the effective amount can be measured from the EC50 of a given antibody concentration. "EC50" is intended to mean the plasma concentration required to obtain 50% of the maximum effect in vivo.

[0158] Generally, for the anti-osteopontin antibodies or conjugates of the present disclosure, the effective amount is usually 200-fold or less of the calculated IC50. Typically, the amount of antibody or conjugate administered is less than about 200-fold, less than about 150-fold, less than about 100-fold of the calculated IC50, and in many embodiments, less than about 75-fold, less than about 60-fold, less than 50-fold, less than 45-fold, less than 40-fold, less than 35-fold, less than 30-fold, less than 25-fold, less than 20-fold, less than 15-fold, less than 10-fold, and even less than about 8-fold or 2-fold of the calculated IC50. In one embodiment, the effective amount is about 1-fold to 50-fold of the calculated IC50, and sometimes about 2-fold to 40-fold, about 3-fold to 30-fold, or about 4-fold to 20-fold of the calculated IC50. In other embodiments, the effective amount is the same as the calculated IC50, and in certain embodiments, the effective amount is an amount greater than the calculated IC50.

[0159] The effective amount can be 100-fold or less of the calculated EC50. For example, the amount of antibody or conjugate administered is less than about 100-fold, less than about 50-fold, less than about 40-fold, less than 35-fold, less than 30-fold, or less than 25-fold of the calculated EC50, and in many embodiments, less than about 20-fold, less than about 15-fold, and even less than about 10-fold, less than 9-fold, less than 9-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold of the calculated EC50. In one embodiment, the effective amount is about 1-fold to 30-fold of the calculated EC50, and sometimes about 1-fold to 20-fold, or about 1-fold to 10-fold of the calculated EC50. In other embodiments, the effective amount is the same as the calculated EC50, and in certain embodiments, the effective amount is an amount greater than the calculated EC50.

[0160] The effective amount can be readily determined empirically from assays, safety and escalation and dose range studies, the relationship of the individual clinician and patient, and in vitro and in vivo assays.

[0161] Administration At least one therapeutically effective dose of an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) that inhibits thrombin cleavage of osteopontin or integrin binding to a thrombin cleavage fragment of osteopontin is administered. The "therapeutically effective dose or amount" of an anti-osteopontin antibody or an antigen-binding fragment thereof is intended to be an amount that, when administered, results in a positive therapeutic response with respect to the treatment of an individual for an osteopontin-related disorder. Of particular interest is the amount of an anti-osteopontin antibody, or an antigen-binding fragment thereof, that provides an antitumor or anti-inflammatory effect, or reduces cardiac hypertrophy or myocardial fibrosis. A "positive therapeutic response" is intended to mean that an individual undergoing treatment according to the invention exhibits improvement in one or more symptoms of an osteopontin-related disorder for which the individual is being treated.

[0162] Thus, for example, a “positive treatment response” is an improvement in a disease associated with the therapy and / or an improvement in one or more symptoms of a disease associated with the therapy. For example, a positive treatment response regarding cancer treatment refers to one or more of the following improvements in the disease: (1) a decrease in tumor size, (2) a decrease in the number of cancer cells, (3) inhibition of tumor growth (i.e., delaying to some extent, preferably stopping), (4) inhibition of cancer cell invasion into peripheral organs (i.e., delaying to some extent, preferably stopping), (5) inhibition of tumor metastasis (i.e., delaying to some extent, preferably stopping), and (6) some alleviation of one or more symptoms associated with cancer. Such treatment responses can be further characterized as the degree of improvement. Thus, for example, the improvement can be characterized as a complete response. A “complete response” is evidence of the disappearance of all symptoms and signs of all measurable or evaluable diseases, including physical examinations, clinical tests, nuclear and radiation tests (i.e., CT (computed tomography) and / or MRI (magnetic resonance imaging)), and all other non-invasive procedures repeated for all initial abnormalities or sites that were positive at the time of enrollment in the study. Alternatively, the improvement of the disease can be classified as a partial response. A “partial response” is intended to be a decrease of more than 50% in the sum of the products of the perpendicular diameters of all measurable lesions when compared to the pre-treatment measurements (partial response is not applicable for patients with only evaluable responses).

[0163] In certain embodiments, a composition comprising an anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof), and / or one or more other therapeutic agents, e.g., other drugs for treating cancer, inflammation, cardiac hypertrophy, myocardial fibrosis, or other pharmaceuticals, are administered according to a daily dosing schedule or intermittently. For example, the therapeutically effective dose can be administered 1 day per week, 2 days per week, 3 days per week, 4 days per week, or 5 days per week, etc. "Intermittent" administration is intended to mean that the therapeutically effective dose can be administered, for example, every other day, every 2 days, every 3 days, etc. For example, in some embodiments, the anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof) is administered twice or three times a week over a long period of time, such as 1, 2, 3, 4, 5, 6, 7, 8... 10... 15... 24 weeks, etc. "Twice a week" or "two times per week" is intended to mean that two therapeutically effective doses of the agent are administered to the subject within a 7-day period, starting on the first day of the first week of administration, with a minimum dosing interval of 72 hours and a maximum dosing interval of 96 hours. "Three times a week" or "three times per week" is intended to mean that three therapeutically effective doses are administered to the subject within a 7-day period, allowing for a minimum dosing interval of 48 hours and a maximum dosing interval of 72 hours. For the purposes of the present invention, this type of administration is referred to as "intermittent" therapy. According to the methods of the present invention, a subject can receive intermittent therapy (i.e., twice or three times weekly administration of a therapeutically effective amount) during one or more weekly cycles until the desired therapeutic response is achieved. The agent can be administered by any of the acceptable routes of administration described hereinafter in this specification.

[0164] A composition comprising an anti-osteopontin antibody, or an antigen-binding fragment thereof (or a conjugate thereof) is typically administered, but not necessarily, orally, by injection (subcutaneous, intravenous, or intramuscular), by infusion, locally or topically. Additional modes of administration such as intraarterial, intraperitoneal, pulmonary, nasal, transdermal, intralesional, intrasternal, intrasubstantial, rectal, transdermal, transmucosal, intrathecal, pericardial, intraarterial, intraocular, etc. are also contemplated. When the anti-osteopontin antibody, or an antigen-binding fragment thereof (of the conjugate) is administered by injection, the administration may be by continuous infusion, or by single or multiple boluses.

[0165] For example, the anti-osteopontin antibody or conjugate can be administered by infusion or by local injection, for example, at a rate of about 50 mg / hour to about 400 mg / hour, about 75 mg / hour to about 375 mg / hour, about 100 mg / hour to about 350 mg / hour, about 150 mg / hour to about 350 mg / hour, about 200 mg / hour to about 300 mg / hour, about 225 mg / hour to about 275 mg / hour. Exemplary infusion rates are, for example, about 1 mg / m 2 / day to about 9 mg / m 2 / day, about 2 mg / m 2 / day to about 8 mg / m 2 / day, about 3 mg / m 2 / day to about 7 mg / m 2 / day, about 4 mg / m 2 / day to about 6 mg / m 2 / day, about 4.5 mg / m 2 / day to about 5.5 mg / m 2 / day, including about 0.5 mg / m 2 / day to about 10 mg / m 2 / day to achieve the desired therapeutic dose. Administration (e.g., by infusion) can be repeated over a desired period, for example, over a period of about 1 day to about 5 days, or once every few days, for example, over about 5 days, about 1 month, about 2 months, etc.

[0166] The preparation according to the present invention is also suitable for topical treatment. In certain embodiments, the composition is used for the topical delivery of an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) for the treatment of cancer. For example, the composition can be administered directly to a tumor or cancerous cell. Administration can be effected by perfusion via a local catheter or by direct injection into the lesion. For the treatment of melanoma, the composition can be administered topically, for example, in a cream or gel containing an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) applied to the skin at the site of the lesion.

[0167] The pharmaceutical formulation may be in the form of a liquid solution or suspension immediately prior to administration, but may take other forms such as syrups, creams, ointments, tablets, capsules, powders, gels, matrices, suppositories, etc. A pharmaceutical composition containing an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) and other agents can be administered using the same or different routes of administration according to any medically acceptable method known in the art.

[0168] In another embodiment, a pharmaceutical composition containing an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) and / or other agents is administered prophylactically, for example, to prevent the occurrence or progression of an osteopontin-related disorder. Such prophylactic use is particularly valuable for subjects at high risk of cancer progression or heart hypertrophy or cardiomyopathy.

[0169] In another embodiment of the present invention, a pharmaceutical composition containing an anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) and / or other agents is in a sustained-release formulation or a formulation administered using a sustained-release device. Such devices are well known in the art and include, for example, transdermal patches and small implantable pumps that can provide drug delivery over time in a continuous steady state at various doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0170] The present invention also provides a method for administering a conjugate comprising an anti-osteopontin antibody or an antigen-binding fragment thereof (e.g., conjugated to a therapeutic agent, a detectable label, or an imaging agent) provided herein to a patient having an osteopontin-related disorder. The method comprises administering a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition, by any of the modes described herein. The administration method can be used to treat any osteopontin-related disorder responsive to treatment with an anti-osteopontin antibody. More specifically, the compositions herein are effective for the treatment of inflammation, cardiac hypertrophy / cardiomyopathy, and cancer associated with increased expression of osteopontin.

[0171] One of ordinary skill in the art will understand the conditions that can be effectively treated with an anti-osteopontin antibody. The actual dosage administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the medical practitioner, and the conjugate being administered. A therapeutically effective amount can be determined by one of ordinary skill in the art and adjusted according to the specific requirements of each particular case.

[0172] Generally, a therapeutically effective amount is in the range of about 0.50 mg to 5 g of anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof) per day, more preferably about 5 mg to 2 g per day, even more preferably about 7 mg to 1.5 g per day. Preferably, such dosages are in the range of 10 - 600 mg four times a day (QID), 200 - 500 mg QID, 25 - 600 mg three times a day (TID), 25 - 50 mg TID, 50 - 100 mg TID, 50 - 200 mg TID, 300 - 600 mg TID, 200 - 400 mg TID, 200 - 600 mg TID, 100 - 700 mg twice a day (BID), 100 - 600 mg BID, 200 - 500 mg BID, or 200 - 300 mg BID. The amount of the compound administered depends on the potency of the particular anti-osteopontin antibody or an antigen-binding fragment thereof (or a conjugate thereof), as well as the desired magnitude or effect and the route of administration.

[0173] The purified anti-osteopontin antibody or its antigen-binding fragment or its conjugate (similarly in this case, preferably provided as part of a pharmaceutical formulation) can be administered alone or in combination with one or more other therapeutic agents for treating osteopontin-related disorders, such as anti-cancer therapy agents, for example, chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents or targeted therapy agents, or other pharmaceuticals used for treating specific conditions or diseases, according to various dosing schedules depending on the judgment of the clinician, the needs of the patient, etc. Specific dosing schedules are known to those skilled in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, but are not limited to, five times a day, four times a day, three times a day, twice a day, once a day, three times a week, twice a week, once a week, twice a month, once a month, once every month, and any combination thereof. Preferred compositions are those that require administration no more than once a day.

[0174] An anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof can be administered before, simultaneously with, or after other agents. When provided simultaneously with other agents, the anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof can be provided in the same composition or in different compositions. Thus, the anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof, and other agents can be presented to an individual by combination therapy. "Combination therapy" is intended to administer to a subject undergoing therapy such that a combined therapeutic effect of substances is brought about. For example, combination therapy can be achieved by administering, according to a specific dosing schedule, a dose of a pharmaceutical composition containing an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof and a dose of a pharmaceutical composition containing at least one other agent such as another drug for treating cancer, and these together constitute a therapeutically effective dose. Similarly, the anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof, and one or more other therapeutic agents can be administered at at least one therapeutic dose. As long as a combined therapeutic effect of these substances is brought about in the subject undergoing therapy, the administration of separate pharmaceutical compositions can be carried out simultaneously or at different times (i.e., on the same day or different days, in either order sequentially).

[0175] If a subject undergoing therapy according to the aforementioned dosing schedule shows a partial response or recurrence after a long period of remission, a subsequent series of combination therapies may be required to achieve a complete remission of the disease. Thus, after a period of rest for a certain period from the first treatment period, the subject may have one or more additional treatment periods using an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof. Such a period during the treatment period is referred to herein as a break period. It should be recognized that the length of the break period depends on the degree of tumor response (i.e., complete or partial) achieved by combination therapy during any previous treatment period with these therapeutic agents.

[0176] In cancer patients, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may, but is not limited to, be combined with any other medical treatment for cancer such as surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy, or molecular targeted therapy or biotherapy. Any combination of these other medical treatment methods and an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof can be used to effectively treat the cancer of the subject.

[0177] For example, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may, but is not limited to, be combined with chemotherapy using one or more chemotherapeutic agents such as abtrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracycline, azacitidine, azathioprine, BiCNU, brexanoxane, busulfan, bleomycin, camptosar, camptothecin, carboplatin, carmustine, celbidine, chlorambucil, cisplatin, cladribine, cosmege, cytarabine, cytosal, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, elence, elsapar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamptin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, IFEX, irinotecan, lamvis, leukeran, leustatin, matsulan, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, misramycin, mutamycin, myleran, milosar, navelbine, nipecot, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, printol, raltitrexed, taxotere, taxol, teniposide, thioguanine, tomudex, topotecan, valrubicin, verban, vepecid, vinblastine, vindesine, vincristine, vinorelbine, VP-16 and bumo.

[0178] In another example, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may include, but is not limited to, tyrosine kinase inhibitors such as imatinib mesylate (also known as Gleevec, STI-571), gefitinib (also known as Ilesa, ZD1839), erlotinib (commercially available as Tarceva), sorafenib (Nexavar), sunitinib (Sutent), dasatinib (Sprycel), lapatinib (Tykerb), nilotinib (Tasigina), and bortezomib (Velcade); Janus kinase inhibitors such as tofacitinib; ALK inhibitors such as crizotinib; Bcl-2 inhibitors such as obatoclax and gossypol; PARP inhibitors such as iniparib and olaparib; PI3K inhibitors such as perifosine; VEGF receptor 2 inhibitors such as apatinib; AN-152 (AEZS-108) doxorubicin conjugated with [D-Lys(6)]-LHRH; Braf inhibitors such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors such as trametinib; CDK inhibitors such as PD-033291 and LEE011; Hsp90 inhibitors such as salinomycin; small molecule drug conjugates such as vintafolide; serine / threonine kinase inhibitors such as temsirolimus (Torisel), everolimus (Afinitor), vemurafenib (Zelboraf), trametinib (Mekinist), and dabrafenib (Tafinlar); and may be combined with targeted therapy using one or more small molecule inhibitors or monoclonal antibodies such as rituximab (commercially available as MabThera or Rituxan), trastuzumab (Herceptin), alemtuzumab, cetuximab (commercially available as Erbitux), panitumumab, bevacizumab (commercially available as Avastin), and ipilimumab (Yervoy).

[0179] In a further example, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may, but is not limited to, be combined with an immunotherapy using any of the following: cancer vaccines (e.g., E75HER2-derived peptide vaccine, Nelipepimut-S (NeuVax), Sipuleucel-T), antibody therapies (e.g., trastuzumab, ado-trastuzumab emtansine, alemtuzumab, ipilimumab, ofatumumab, nivolumab, pembrolizumab, or rituximab), cytokine therapies (e.g., interferons including type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ), and interleukins including interleukin (IL-2)), adjuvant immunotherapies (e.g., polysaccharide-K), adoptive T cell therapies, and immune checkpoint blockade therapies.

[0180] In a further example, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may, but is not limited to, be combined with radiotherapy using radioisotopes including iodine-131, strontium-89, samarium-153, and radium-223. Further, radiotherapy may, but is not limited to, be combined with administration of radiosensitizers such as cisplatin, nimorazole, and cetuximab.

[0181] In the case of a patient having melanoma, treatment with an anti-osteopontin antibody or an antigen-binding fragment thereof or a conjugate thereof may be combined with administration of a B-Raf inhibitor, a MEK inhibitor, or a combination thereof. Exemplary B-Raf inhibitors include, but are not limited to, dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720. Exemplary MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901.

[0182] Kit Any of the compositions described herein may be included in a kit. For example, the kit may include an anti-osteopontin antibody, an antigen-binding fragment thereof, a conjugate of an anti-osteopontin antibody, a pharmaceutical formulation comprising an anti-osteopontin antibody and / or its conjugate, a recombinant nucleic acid or vector system encoding an anti-osteopontin antibody, and / or a composition comprising a host cell (transfected with a recombinant nucleic acid or vector system encoding an anti-osteopontin antibody or a separate one).

[0183] In some embodiments, the kit includes an anti-osteopontin antibody or an antigen-binding fragment thereof that specifically binds to osteopontin or a thrombin cleavage fragment thereof, and the antibody inhibits thrombin cleavage of osteopontin or integrin binding to the thrombin cleavage fragment of osteopontin. In certain embodiments, the kit includes an anti-osteopontin antibody conjugate or a formulation comprising the antibody and / or conjugate.

[0184] In certain embodiments, the antibody or an antigen-binding fragment thereof included in the kit specifically binds to an OPN-R fragment or an OPN-CTF fragment. In certain embodiments, the antibody or an antigen-binding fragment thereof specifically binds to an epitope comprising Arg168 of osteopontin. In certain embodiments, the antibody or an antigen-binding fragment thereof specifically binds to an osteopontin peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-7, SEQ ID NOs: 9-12, and SEQ ID NO: 47.

[0185] In certain embodiments, the antibody or antigen-binding fragment thereof included in the kit comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) and comprises a heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) and comprises a light chain.

[0186] In certain embodiments, the antibody or antigen-binding fragment thereof included in the kit comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto).

[0187] In certain embodiments, the antibody or antigen-binding fragment thereof included in the kit comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 26, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 28, or a sequence having at least about 80-100% sequence identity thereto (including any percent identity within this range, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) in the light chain.

[0188] In certain embodiments, the anti-osteopontin antibody included in the kit is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, nanobody, bispecific antibody, bispecific T cell engager antibody, trispecific antibody, Fab fragment, Fab’ fragment, F(ab’) 2 fragment, F v fragment, or scFv fragment.

[0189] The kit may include one or more pharmaceutical formulations containing the antibody compositions described herein. Thus, the kit may include a single pharmaceutical composition present as one or more unit dosage forms. In still other embodiments, the kit may include two or more separate pharmaceutical compositions. In some embodiments, the pharmaceutical composition is suitable for topical delivery (e.g., a gel or cream containing an antibody or conjugate for the treatment of melanoma).

[0190] In some embodiments, the kit may further include a composition comprising a B-Raf inhibitor, a MEK inhibitor, or a combination thereof.

[0191] In some embodiments, the kit may further include a composition comprising one or more anti-cancer therapeutic agents including, but not limited to, chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiation sensitizers, and radioisotopes, or combinations thereof.

[0192] The composition can be in liquid form or can be lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials including glass or plastic. The container can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle). The kit can further include a container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, or dextrose solution. The kit can also include other materials useful to the end user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles, and syringes, or other delivery devices. The kit can also provide a delivery device pre-filled with an anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof).

[0193] In addition to the above components, the kit of the subject matter may further include (in certain embodiments) instructions for carrying out the method of the subject matter (i.e., instructions for treating the osteopontin-related disorders described herein). These instructions can be present within the kit of the subject matter in various forms, one or more of which can be present within the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, such as in the package of the kit, in an accompanying document, etc., for example, one or more sheets of paper on which the information is printed. Yet another form of these instructions is a computer-readable medium on which the information is recorded, such as a floppy disk, a compact disk (CD), a DVD, a Blu-ray, a flash drive, etc. Still another form of these instructions that can be present is a website address that can be used via the Internet to access the information at a remote location.

[0194] Usefulness The anti-osteopontin antibody or antigen-binding fragment thereof (or conjugate thereof) described herein is useful for the treatment of osteopontin-related disorders, including inflammation, cardiac hypertrophy, myocardial fibrosis, and any disease or disorder associated with an elevated level of osteopontin or its thrombin cleavage fragment, such as melanoma, glioblastoma, ovarian cancer, breast cancer, and lung cancer.

[0195] In particular, the anti-osteopontin antibody or its antigen-binding fragment (or its conjugate) is useful for the treatment of melanoma, including but not limited to malignant melanoma, melanoma-derived melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, and desmoplastic melanoma. Melanoma cells may contain changes (mutations) in genomic DNA sequences, meaning that the proteins encoded by melanoma cells are different from proteins in other parts of the patient's body. As an example, B-Raf, a protein involved in growth signaling to cells, can have mutations. In particular, the anti-osteopontin antibody or its antigen-binding fragment (or its conjugate) can be used to treat B-RAF mutant melanomas, including but not limited to melanomas containing the V600E or V600K mutation. The anti-osteopontin antibody (or its antigen-binding fragment) can be used to treat melanoma at any stage, including metastatic melanoma.

[0196] Experiment The following examples are presented to provide a complete disclosure and description of the methods of making and using the invention to those skilled in the art and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to indicate that the following are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0197] All publications and patent applications cited herein are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0198] The present invention has been described with respect to specific embodiments found or proposed by the inventors so as to include preferred modes for carrying out the present invention. Those skilled in the art will recognize that, in light of the present disclosure, numerous modifications and variations can be made in the specific embodiments illustrated without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made to the underlying DNA sequence without affecting the protein sequence. Considerations of biological functional equivalence allow changes to be made to the protein structure without affecting the biological action in kind or amount. All such modifications are intended to be within the scope of the appended claims.

[0199] Example 1 Thrombin cleavage of osteopontin promotes melanoma growth and progression Osteopontin (OPN) is a multifunctional protein of matrix cells with a highly conserved RGD domain that binds to a wide range of integrins. Thrombin cleaves OPN into two fragments, OPN-R with a new integrin-binding site and a C-terminal fragment (CTF) that promotes chemotaxis (Figure 1). OPN-R with SVVYGLR (SEQ ID NO: 3) at the C-terminus binds to integrin α4β1 and α9β1. Mutation of arginine to alanine (R153A) at the thrombin cleavage site renders OPN resistant to thrombin cleavage. Nemoto et al. (J. Bone Mineral Res. 2001) reported that the growth of B16 melanoma is suppressed in mice lacking OPN (OPN knockout [KO] mice). However, despite extensive studies on the importance of OPN in cancer progression, the role of thrombin cleavage of OPN remains unclear.

[0200] To further understand the role of thrombin cleavage of OPN in the pathology of inflammatory disorders and cancer, in an experimental mouse model, the thrombin cleavage site of OPN was inactivated by introducing an Arg153Ala mutation into the OPN gene. Mice with thrombin cleavage-resistant OPN were generated by replacing Arg153 with Ala (OPNR153A; OPN knock-in [KI]). B16 melanoma cells were subcutaneously inoculated into the flanks of the mice, tumor growth was monitored, and tumor weights were measured after sacrifice. In a metastasis model, B16 melanoma cells were intravenously injected through the tail vein of the mice, and after sacrifice, the number of nodules was visually counted and melanoma in the lungs was quantified by determining the melanin content (melanin is a recognized measure of the amount of melanoma cells in the lungs). Some mice were fed a solid diet containing dabigatran etexilate, a thrombin inhibitor.

[0201] When monitored as volume over time or body weight at sacrifice, robust tumor growth was observed in wild-type (WT) mice, but tumor growth was suppressed to a similar extent in both OPN knockout (KO) mice and OPNR153A-KI mice (Figures 2A - 2B). In the metastasis model, WT mice had more tumor nodules in the lungs and more melanin in the lungs than OPN KI mice (Figures 3A - 3B).

[0202] The role of thrombin cleavage of OPN was confirmed by treating mice with dabigatran etexilate (DE), a specific oral thrombin inhibitor. In wild-type mice (WT) treated with DE, tumor growth in the flanks (Figures 4A - 4B) decreased to the same level as that seen in OPN-KI mice. Similarly, in the metastasis model, metastasis in WT mice decreased to the same level as that seen in OPN-KI and OPN-KO mice (Figures 5A - 5B).

[0203] To demonstrate that thrombin cleavage of OPN is important in other cancers, an ovarian cancer model with Id8 cells (ovarian cancer) injected intraperitoneally was used. To represent tumor growth, the body weight of the mice was measured as weight gain. The growth of Id8 ovarian cancer tumors in OPN-KI mice was significantly reduced compared to that in WT mice.

[0204] Our data provide the first evidence of the importance of thrombin cleavage of OPN in cancer biology. OPN KI mice showed a decrease in B16 tumor growth equivalent to the absence of OPN, suggesting that thrombin cleavage of host OPN plays an important role in melanoma growth in vivo. In addition, in OPN KI mice, the growth of Id8 ovarian cancer was suppressed compared to WT mice. Treatment with the thrombin inhibitor dabigatran etexilate reduced the growth of B16 melanoma in both subcutaneous and metastatic models to levels similar to those observed in mice lacking OPN or having OPN that could not be cleaved by thrombin.

[0205] Example 2 Therapeutic monoclonal antibodies The results of the melanoma and ovarian cancer tests (Example 1) using OPN-KI mice indicate that blockade of OPN cleavage and / or blocking of OPN fragments have the potential for therapeutic intervention in melanoma and other cancers. Monoclonal antibodies of different formats (mAB, bispecific, trispecific, nanobody, etc.) are prepared against the following targets. OPN-FL: Antibodies that block the cleavage of OPN: Antibodies against human OPN or the OPN sequence SVVGLR (SEQ ID NO: 1) or SKSKKF (SEQ ID NO: 2) that bind to the anion-binding exosites 1 and 2 of thrombin. OPN-R: Antibodies that block integrin binding to OPN-R using antibodies against the exposed SVVYGLR (SEQ ID NO: 3) of human OPN OPN-CTF: Antibodies that block the SKSKKFRR (SEQ ID NO: 4) sequence of human OPN that block CTF multimer formation

[0206] Use: Melanoma and other cancers can be treated with antibodies, either as monotherapy or in combination with standard therapies. The therapeutic effect is obtained by generating monoclonal antibodies against the OPN thrombin cleavage site (SVVYGLRSKSKKF, SEQ ID NO: 9), the OPN-R SVVYGLR (SEQ ID NO: 3) integrin binding motif, and the OPN-CTF region SKSKKFRRPD (SEQ ID NO: 10) involved in CTF multimer formation in cancer. Antibodies targeting the binding sites of OPN on thrombin, exosite 1 and 2, may also be useful for reducing OPN cleavage by thrombin. Peptides that specifically bind to thrombin binding exosite 1 and 2 but do not interfere with the procoagulant activity of thrombin may also be useful.

[0207] This antibody is useful for achieving the beneficial effect of preventing OPN cleavage without the side effect of bleeding risk when a thrombin inhibitor or other anticoagulant is used.

[0208] Example 3 Production of a monoclonal antibody that inhibits thrombin cleavage of osteopontin Our goal was to identify a monoclonal antibody (Mab) that binds to full-length osteopontin (OPN-FL) and blocks its cleavage by thrombin. Figure 7 shows an alignment of the OPN protein sequences around the thrombin cleavage sites of rabbit, rat, mouse, human, rhesus monkey, cynomolgus monkey, and pig. To develop rabbit monoclonal antibodies, a peptide antigen

Number

Number

[0209] Rabbit monoclonal clones were screened using direct ELISA against the OPN antigen peptide (Figure 8). 35 positives (shown) were identified. For the assay to identify clones producing anti-OPN IgG, 96-well plates were coated overnight with 2 μg / mL avidin, and then the OPN antigen peptide labeled with biotin was added for 30 minutes. Then, after diluting the supernatant from the clones 1:50, it was detected with a 10 3 fold-diluted horseradish peroxidase (HRP)-labeled anti-rabbit IgG Fc. The substrate for HRP was added and the color was measured.

[0210] To determine the concentration of IgG in the clone supernatant, 96-well plates were coated overnight with 2 μg / mL anti-rabbit IgG. Then, after diluting the supernatant from the clones 1:50, it was detected with a 10 3 fold-diluted horseradish peroxidase (HRP)-labeled anti-rabbit IgG Fc. The substrate for HRP was added and the color was measured. The concentration of IgG in the clone supernatant was calculated from the standard dilution curve of rabbit IgG.

[0211] Figure 9 shows the screening results of rabbit monoclonal antibodies in a direct ELISA against antigenic peptides, mouse OPN-FL, and human OPN-FL. Clones that bound to OPN-FL derived from human or mouse were identified. Four clones (A2, A6, E5, and G7) were selected for further investigation. In the peptide antigen binding assay, the EC 50 of these four clones was determined (Figure 10). To determine the EC 50 of the purified anti-OPN IgG, a 96-well plate was coated overnight with 2 μg / mL of avidin, and then an OPN antigen peptide labeled with biotin was added for 30 minutes. Next, the IgG in the supernatant from the selected clones was purified by binding to and eluting from protein A / G sepharose beads. After serially diluting the purified IgG, it was detected with a 10 3 -fold diluted horseradish peroxidase (HRP)-labeled anti-rabbit IgG Fc. The substrate for HRP was added, and the color was measured.

[0212] Figure 11 shows the EC 50 values of the four clones for binding to the OPN antigen peptide. Based on the data in Figure 10, the EC 50 values were calculated from a four-parameter fit equation using Prism Graphpad. The EC 50 values were 2.6 ng / mL for clone A2, 3.2 ng / mL for clone A6, 4.0 ng / mL for clone E5, and 93.2 ng / mL for clone G7.

[0213] The EC 50Values were determined (Figure 12), and it was found that all clones including A2, A6, and E5 bind to mouse OPN-FL. Goat polyclonal anti-mouse osteopontin IgG (2 μg / ml) (R&D Systems, Minneapolis, MN) in PBS buffer was coated onto 96-well ELISA plates, and non-specific binding sites were blocked with 1% BSA in PBS for 1 hour. Recombinant mouse osteopontin protein (R&D Systems, Minneapolis, MN) was diluted with 1% BSA in PBS (starting from 200 ng / ml and diluted), and incubated for 2 hours. After washing with 0.05% Tween® 20 in PBS, samples were incubated for 1 hour with purified antibodies A2, A6, E5, G7, or IgG control (500 ng / ml) in PBS containing 1% BSA. After washing with 0.05% Tween® 20 in PBS, samples were incubated for 1 hour with peroxidase-conjugated goat anti-rabbit IgG antibody (100 ng / ml) in PBS containing 1% BSA. After washing, tetramethylbenzidine substrate was incubated for 10 minutes, followed by the addition of stop solution, and absorbance was measured at 450 nm.

[0214] In addition, the EC of four clones in a direct binding ELISA against human OPN-FL 50Values were determined and all four clones were found to bind to human OPN-FL (Figure 13). Mouse monoclonal anti-human osteopontin antibody (4 μg / ml) in PBS buffer (R&D Systems, Minneapolis, MN) was coated onto 96-well ELISA plates and non-specific binding sites were blocked with 1% BSA in PBS for 1 hour. Recombinant human osteopontin protein (GST-OPN-his, purified in-house) was diluted with 1% BSA in PBS (starting dilution from 200 ng / ml) and incubated for 2 hours. After washing with 0.05% Tween® 20 in PBS, samples were incubated for 1 hour with purified antibodies A2, A6, E5, G7 or IgG control (500 ng / ml) in PBS containing 1% BSA. After washing with 0.05% Tween® 20 in PBS, samples were incubated for 1 hour with peroxidase-conjugated goat anti-rabbit IgG antibody (100 ng / ml) in PBS containing 1% BSA. After washing, tetramethylbenzidine substrate was incubated for 10 minutes, followed by addition of stop solution and measurement of absorbance at 450 nm.

[0215] Next, the ability of the monoclonal antibodies from the four clones to inhibit thrombin cleavage of human OPN-FL was determined by detecting inhibition of the generation of OPN-R, one of the cleavage products, in a specific ELISA for OPN-R. All four clones were found to inhibit thrombin cleavage of human OPN-FL (Figure 14). Recombinant human osteopontin protein (3.2 μM, purified in-house) was incubated at room temperature for 20 minutes with purified antibodies A2, A6, E5, G7 or IgG control, or without them. Human thrombin (32 nM) (R&D Systems) was added to the samples, incubated at 37 °C for 10 minutes, followed by addition of PPACK to 4.8 μM. Samples were diluted 1 / 300 with 1% BSA in PBS and the generated OPN-R was measured by OPN-R specific ELISA.

[0216] Human OPN-R ELISA: Mouse monoclonal anti-human osteopontin antibody (4 μg / ml) in PBS buffer (R&D Systems, Minneapolis, MN) was coated onto 96-well ELISA plates, and non-specific binding sites were blocked with 1% BSA in PBS for 1 hour. Recombinant human OPN-R protein (GST-OPN-R, Myles et al, 2003 JBC)) was diluted with 1% BSA in PBS (starting from 200 ng / ml and diluted). Standards and samples of thrombin cleavage products (starting from 1 / 300 dilution and further diluted) were incubated for 2 hours. After washing with 0.05% Tween® 20 in PBS, the samples were incubated for 1 hour with biotinylated rabbit polyclonal anti-OPN-R antibody (500 ng / ml) (Sharif et al, 2009 A+R) in PBS containing 1% BSA. After washing, the samples were incubated for 1 hour with peroxidase-conjugated streptavidin (100 ng / ml) in PBS containing 1% BSA. After washing, the tetramethylbenzidine substrate was incubated for 10 minutes, followed by the addition of stop solution, and absorbance was measured at 450 nm. The amount of OPN-R produced in each sample was calculated from the OPN-R standard in the OPN-R ELISA, and then the inhibition of thrombin cleavage of each antibody clone at different concentrations was calculated and graphed. [Figure 15] shows the inhibition percentages of four clones in the inhibition of cleavage of human OPN-FL. A2 is 82%, A6 is 95.4%, E5 is 77.2%, and G7 is 60.1%.

[0217] Also, in the specific ELISA for OPN-R, the inhibition of thrombin cleavage of mouse OPN-FL was evaluated for four clones by determining the production of OPN-R, one of the cleavage products. All four clones were found to inhibit thrombin cleavage of mouse OPN-FL (Figure 16). Recombinant mouse osteopontin protein (R&D systems) (2 μM) was incubated at room temperature for 20 minutes with purified antibodies A2, A6, E5, G7 or IgG control (2 μM), or without them. Human thrombin (20 nM) (R&D Systems) was added to the samples, incubated at 37 °C for 10 minutes, and subsequently PPACK was added up to 4.7 μM. The samples were diluted 1 / 100 or 1 / 30 with 1% BSA in PBS, and the generated OPN-R was measured by OPN-R specific ELISA.

[0218] Mouse OPN-R ELISA: Goat polyclonal anti-mouse osteopontin IgG (4 μg / ml) (R&D Systems, Minneapolis, MN) in PBS buffer was coated onto 96-well ELISA plates, and non-specific binding sites were blocked with 1% BSA in PBS for 1 hour. Samples of thrombin cleavage products (starting from 1 / 30 or 1 / 100 dilution and further diluted) were incubated for 2 hours. After washing with 0.05% Tween® 20 in PBS, the samples were incubated for 1 hour with biotinylated rabbit polyclonal anti-OPN-R antibody (2 μg / ml) (Sharif et al, 2009A+R) in PBS containing 1% BSA. After washing, the samples were incubated for 1 hour with peroxidase-conjugated streptavidin (100 ng / ml) in PBS containing 1% BSA. After washing, the tetramethylbenzidine substrate was incubated for 10 minutes, followed by addition of the stop solution, and absorbance was measured at 450 nm. The amount of OPN-R generated in each sample was calculated from the OPN-R standard in the OPN-R ELISA, and then the inhibition of thrombin cleavage for each antibody clone at different concentrations was calculated and graphed.

[0219] The clones were sequenced. The amino acid sequences of the heavy and light chains of the A2, A6, E5, and G7 monoclonal antibodies, as well as the nucleic acid sequences encoding them, and the sequences of their CDRs were determined and are shown in SEQ ID NOs: 13 to 46 in the Sequence Listing. The present invention provides, for example, the following items. (Item 1) An isolated antibody or an antigen-binding fragment thereof that specifically binds to osteopontin or a thrombin cleavage fragment thereof, and inhibits thrombin cleavage of osteopontin or integrin binding to the thrombin cleavage fragment of osteopontin. (Item 2) The antibody or antigen-binding fragment thereof according to Item 1, comprising a heavy chain complementarity-determining region 1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity-determining region 2 (CDR-H2) containing the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity-determining region 3 (CDR-H3) containing the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 32, a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 34. (Item 3) a) A heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and b) A light chain comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to Item 2. (Item 4) The antibody or antigen-binding fragment thereof according to Item 1, comprising a heavy chain complementarity-determining region 1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 35, a heavy chain complementarity-determining region 2 (CDR-H2) containing the amino acid sequence of SEQ ID NO: 36, a heavy chain complementarity-determining region 3 (CDR-H3) containing the amino acid sequence of SEQ ID NO: 37, a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 38, a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO: 39, and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 40. (Item 5) a) A heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22, and b) a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 24, and the antibody or antigen-binding fragment thereof according to item 4. (Item 6) A heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46, and the antibody or antigen-binding fragment thereof according to item 1. (Item 7) a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 26, b) a light chain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 28, and the antibody or antigen-binding fragment thereof according to item 6. (Item 8) The antibody or antigen-binding fragment thereof according to item 1, wherein the thrombin cleavage fragment of osteopontin is an OPN-R fragment or an OPN-CTF fragment. (Item 9) The antibody or antigen-binding fragment thereof according to item 8, wherein the antibody inhibits integrin binding to the OPN-R fragment. (Item 10) The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody specifically binds to an epitope containing Arg168 of osteopontin numbered with respect to the reference sequence of SEQ ID NO: 8. (Item 11) The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody specifically binds to an osteopontin peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 7, SEQ ID NOs: 9 to 12, and SEQ ID NO: 47, or consisting of the same. (Item 12) The antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, Fab fragments, Fab' fragments, F(ab') 2 fragments, F v fragments, and scFv fragments, and the antibody or antigen-binding fragment thereof according to any one of items 1 to 11. (Item 13) A composition for treating osteopontin-related disorders, comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 12. (Item 14) The composition according to item 13, further comprising a pharmaceutically acceptable excipient or carrier. (Item 15) The composition according to item 14, wherein the pharmaceutically acceptable carrier is selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles, and ointments. (Item 16) The composition according to item 14 or 15, further comprising an anti-cancer therapeutic agent. (Item 17) The composition according to item 16, wherein the anti-cancer therapeutic agent is selected from the group consisting of chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiation sensitizers, and radioisotopes. (Item 18) The composition according to any one of items 13 to 17, wherein the osteopontin-related disorder is inflammation, cardiac hypertrophy, myocardial fibrosis, melanoma, glioblastoma, ovarian cancer, breast cancer, or lung cancer. (Item 19) The composition according to item 18, further comprising a B-Raf inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, or a combination thereof. (Item 20) A method for treating an osteopontin-related disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the composition according to any one of items 13 to 19. (Item 21) The method according to item 20, wherein the antibody is a humanized antibody. (Item 22) The method according to item 20 or 21, further comprising administering at least one additional anti-cancer therapeutic agent. (Item 23) The method according to item 22, wherein the anti-cancer therapeutic agent is selected from the group consisting of chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, apoptosis promoters, angiogenesis inhibitors, photoactive agents, radiation sensitizers, and radioisotopes. (Item 24) The method according to any one of items 20 to 23, wherein the osteopontin-related disorder is inflammation or cancer associated with overexpression of osteopontin. (Item 25) The method according to any one of items 20 to 24, wherein the osteopontin-related disorder is cardiac hypertrophy, myocardial fibrosis, melanoma, glioblastoma, ovarian cancer, breast cancer, or lung cancer. (Item 26) The method according to item 24, further comprising administering a B-Raf inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, or a combination thereof. (Item 27) The method according to item 25, wherein the B-Raf inhibitor is selected from the group consisting of dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720. (Item 28) The method according to item 25, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901. (Item 29) The method according to any one of items 20 to 28, wherein the antibody is administered according to a daily dosing schedule or intermittently. (Item 30) The method according to any one of items 20 to 29, wherein multiple cycles of treatment are administered to the subject for a period sufficient to result in at least a partial tumor response. (Item 31) The method according to item 30, wherein the period is at least 6 months. (Item 32) The method according to item 31, wherein the period is at least 12 months. (Item 33) The method according to any one of items 20 to 32, wherein a complete tumor response is achieved. (Item 34) The method according to any one of items 20 to 33, wherein the antibody does not interfere with the procoagulant activity of thrombin in the subject. (Item 35) A method for inhibiting the growth and / or proliferation of tumor cells in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 12. (Item 36) A kit comprising the composition according to any one of items 13 to 19 and instructions for using the kit for treating cancer or inflammation. (Item 37) The kit according to item 36, further comprising means for administering the composition to a subject. (Item 38) A method for producing the antibody according to item 1, comprising inducing an immune response in a subject against an immunogenic peptide comprising a sequence selected from the group consisting of SEQ ID NO: 5-7, SEQ ID NO: 9-12, and SEQ ID NO: 47. (Item 39) A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 12 and a drug selected from the group consisting of an anti-cancer therapeutic agent, a detectable label, and an imaging agent. (Item 40) The conjugate according to item 39, wherein the anti-cancer therapeutic agent is selected from the group consisting of a cytotoxic agent, a drug, a toxin, a nuclease, a hormone, an immunomodulator, an apoptosis promoter, an anti-angiogenic agent, a boron compound, a photoactive agent, and a radioisotope. (Item 41) An isolated nucleic acid, a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 25, b) A nucleotide sequence encoding a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26, c) A nucleotide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO: 27, d) A nucleotide sequence encoding a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28, e) A nucleotide sequence having 90% sequence identity with the nucleotide sequences of a) to d), and f) The complement of a) to e), an isolated nucleic acid. (Item 42) A recombinant nucleic acid comprising a promoter operably linked to the nucleic acid according to Item 41. (Item 43) A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to Item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34. (Item 44) The vector system according to Item 43, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20. (Item 45) A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40. (Item 46) The vector system according to item 45, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22, and a light chain comprising the amino acid sequence of SEQ ID NO: 24 or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 24. (Item 47) A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46. (Item 48) The vector system according to item 47, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 28 or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 28. (Item 49) The vector system according to any one of items 43 to 48, wherein the antibody or antigen-binding fragment thereof is chimerized or humanized. (Item 50) A host cell comprising the vector system according to any one of items 43 to 49. (Item 51) A method for producing an antibody or an antigen-binding fragment thereof, comprising: a) culturing the host cell according to item 50 under conditions suitable for the production of the antibody or the antigen-binding fragment thereof; and b) isolating the antibody or the antigen-binding fragment thereof from the host cell.

Claims

**Claim 1** An isolated antibody or antigen-binding fragment thereof that specifically binds to osteopontin or a thrombin cleavage fragment thereof, which inhibits thrombin cleavage of osteopontin or integrin binding to the thrombin cleavage fragment of osteopontin, wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy-chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy-chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light-chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light-chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light-chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 34, or wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy-chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy-chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light-chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light-chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light-chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 40, or wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy-chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy-chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light-chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light-chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light-chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 46, an isolated antibody or antigen-binding fragment thereof. **Claim 2** a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and b) a light chain comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, and the antibody or antigen-binding fragment thereof according to claim 1.

3. a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 22, b) a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 24, and the antibody or antigen-binding fragment thereof according to claim 1.

4. a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 26, b) a light chain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 28, and the antibody or antigen-binding fragment thereof according to claim 1.

5. The thrombin cleavage fragment of osteopontin is an OPN-R fragment or an OPN-CTF fragment, and the antibody or antigen-binding fragment thereof according to claim 1.

6. The antibody inhibits integrin binding to the OPN-R fragment, and the antibody or antigen-binding fragment thereof according to claim 5.

7. The antibody specifically binds to an epitope containing Arg168 of osteopontin numbered relative to the reference sequence of SEQ ID NO: 8, and the antibody or antigen-binding fragment thereof according to claim 1.

8. The antibody specifically binds to an osteopontin peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 1 to 7, SEQ ID NOs: 9 to 12, and SEQ ID NO: 47, and the antibody or antigen-binding fragment thereof according to claim 1.

9. The antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, an F(ab') ２ fragment, an F ｖ fragment, and a scFv fragment, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A composition for treating an osteopontin-related disorder, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The composition according to claim 10, further comprising a pharmaceutically acceptable excipient or carrier.

12. The pharmaceutically acceptable carrier is selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles, and ointments, and the composition according to claim 11.

13. The composition according to claim 11 or 12, further comprising an anti-cancer therapeutic agent.

14. The composition according to claim 13, wherein the anti-cancer therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, a biotherapeutic agent, an apoptosis promoter, an angiogenesis inhibitor, a photoactive agent, a radiation sensitizer, and a radioisotope.

15. The composition according to any one of claims 10 to 14, wherein the osteopontin-related disorder is inflammation, cardiac hypertrophy, myocardial fibrosis, melanoma, glioblastoma, ovarian cancer, breast cancer, or lung cancer.

16. The composition according to claim 15, further comprising a B-Raf inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, or a combination thereof.

17. The composition according to any one of claims 10 to 16, wherein the antibody is a humanized antibody.

18. The composition according to any one of claims 10 to 17, wherein the composition is administered in combination with at least one additional anti-cancer therapeutic agent.

19. The composition according to claim 18, wherein the anti-cancer therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, a biotherapeutic agent, an apoptosis promoter, an angiogenesis inhibitor, a photoactive agent, a radiation sensitizer, and a radioisotope.

20. The composition according to any one of claims 10 to 19, wherein the osteopontin-related disorder is inflammation or cancer associated with overexpression of osteopontin.

21. The composition according to any one of claims 10 to 20, wherein the osteopontin-related disorder is cardiac hypertrophy, myocardial fibrosis, melanoma, glioblastoma, ovarian cancer, breast cancer, or lung cancer.

22. The composition according to claim 20, wherein the composition is administered in combination with a B-Raf inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, or a combination thereof.

23. The composition according to claim 16 or 22, wherein the B-Raf inhibitor is selected from the group consisting of dabrafenib, vemurafenib, sorafenib, LGX818, GDC-0879, and PLX-4720.

24. The composition according to claim 16 or 22, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, and PD-325901.

25. The composition according to any one of claims 10 to 24, wherein the antibody is administered according to a daily dosing schedule or intermittently.

26. The composition according to any one of claims 10 to 25, wherein multiple cycles of treatment are administered to a subject in need of treatment for osteopontin-related disorders for a period sufficient to result in at least a partial tumor response.

27. The composition according to claim 26, wherein the period is at least 6 months.

28. The composition according to claim 27, wherein the period is at least 12 months.

29. The composition according to any one of claims 10 to 28, which results in a complete tumor response.

30. The composition according to any one of claims 10 to 29, wherein the antibody does not interfere with the clotting-promoting activity of thrombin in a subject in need of treatment for osteopontin-related disorders.

31. A composition for inhibiting the growth and / or proliferation of tumor cells in a subject, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

32. A kit comprising the composition according to any one of claims 10 to 16 and instructions for using the kit for treating cancer or inflammation.

33. The kit according to claim 32, further comprising means for administering the composition to a subject.

34. A composition for use in the method of producing the antibody according to claim 1, comprising an immunogenic peptide comprising a sequence selected from the group consisting of SEQ ID NO: 5 to 7, SEQ ID NO: 9 to 12, and SEQ ID NO: 47, wherein the method comprises inducing an immune response of a subject to the composition.

35. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a drug selected from the group consisting of an anti-cancer therapeutic agent, a detectable label, and an imaging agent.

36. The conjugate according to claim 35, wherein the anti-cancer therapeutic agent is selected from the group consisting of a cytotoxic agent, a drug, a toxin, a nuclease, a hormone, an immunomodulator, an apoptosis promoter, an anti-angiogenic agent, a boron compound, a photoactive agent, and a radioisotope.

37. A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:

34.

38. The vector system according to claim 37, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:

20.

39. A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 37, a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 38, a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:

40.

40. The vector system according to claim 39, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 22, and a light chain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

24.

41. A vector system comprising one or more vectors encoding the antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 41, a heavy-chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 42, a heavy-chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 43, a light-chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 44, a light-chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 45, and a light-chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:

46.

42. The vector system according to claim 41, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 28 or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

28.

43. The vector system according to any one of claims 37 to 42, wherein the antibody or antigen-binding fragment thereof is chimerized or humanized.

44. A host cell comprising the vector system according to any one of claims 37 to 43.

45. A method for producing an antibody or antigen-binding fragment thereof, comprising: a) culturing the host cell according to claim 44 under conditions suitable for the production of the antibody or antigen-binding fragment thereof; and b) isolating the antibody or antigen-binding fragment thereof from the host cell.

46. The vector system is a) a vector comprising the nucleotide sequence of SEQ ID NO: 13 and a vector comprising the nucleotide sequence of SEQ ID NO: 15, or b) a vector comprising the nucleotide sequence of SEQ ID NO: 17 and a vector comprising the nucleotide sequence of SEQ ID NO: 19 The vector system according to claim 38.

47. The vector system according to claim 40, comprising a vector comprising the nucleotide sequence of SEQ ID NO: 21 and a vector comprising the nucleotide sequence of SEQ ID NO:

23.

48. The vector system according to claim 42, comprising a vector comprising the nucleotide sequence of SEQ ID NO: 25 and a vector comprising the nucleotide sequence of SEQ ID NO: 27.

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